5th international symposium of surface and

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Prof Justin Cooper-White – University of Queensland. Prof Hans ..... Justin Gooding University of New South Wales, Australia ...... Cristobal G. dos Remedios. 3.
5TH INTERNATIONAL SYMPOSIUM OF SURFACE AND INTERFACE OF BIOMATIERIALS HELD IN CONJUCTION WITH THE

24TH ANNUAL CONFERENCE OF THE AUSTRALASIAN SOCIETY FOR BIOMATERIALS AND TISSUE ENGINEERING

7—10 APRIL, 2015

SYDNEY, AUSTRALIA

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5th International Symposium of Surface and Interface of Biomaterials th

held in conjunction with the

24 Annual Conference of the Australasian Society for Biomaterials and Tissue Engineering (ASBTE) 7 - 10 April 2015 Welcome from the conference chairs The series of International Symposia on Surface and Interface of Biomaterials (ISSIB) was conceived in 2005 and the first conference held in Chengdu, China, in 2007. Following the great success of ISSIB 2007, the ISSIB meeting was held in Hong Kong (2010), in Japan (2011) and in Italy (2013). We are excited to announce that the 5th ISSIB meeting will take place at Doltone House Sydney, Australia from 7 - 10 April, 2015 and will be held in conjunction with the Australasian Society for Biomaterials and Tissue Engineering (ASBTE) 24th Annual Conference. Sydney is home to beautiful beaches, iconic buildings, historic landmarks, award-winning restaurants, and a uniquely vibrant culture. From the breathtaking views of Sydney Harbour to the serene tranquility of Hyde Park, Sydney has something for everyone. This meeting features four Plenary speakers, 17 Keynotes and more than 120 Talks as well as Rapid Fire and Poster sessions. In addition, we have three workshops and a trade display featuring many of our Sponsors. The meeting will provide an international forum for scientists, engineers, clinicians and medical device manufacturers to present and discuss the latest scientific findings and technological developments in a very challenging context connected with surfaces and interfaces of biomaterials. Students and Early Career Researchers are again a focus of our meeting and a Student/Early Carreer Workshop will allow optimal networking opportunity. We are also greatly indebted to all our Conference sponsors for their generosity! On behalf of the Australasian Society for Biomaterials and Tissue Engineering and the organising committee for the 5th ISSIB we are pleased that you could join us in Sydney in 2015!

Lisbeth Grøndahl

Megan Lord

Conference Sponsors The Organising Committee extends its appreciation to the following sponsors for their invaluable commitment and support:

Conference Partner

Diamond Sponsor

Gold Sponsors

Conference Dinner Sponsor

Silver Sponsors

Student Awards Sponsor

Exhibitors

Welcome Reception Sponsor

Refreshment Sponsor

Poster Sponsor

Session Sponsor

Sponsor

Table of Contents Conference Information ………………………………………………………….…...……. 1 Social Program ………………………………………………………………………..….…… 5 Conference and Dinner Venues …………………………………………………….……… 6 General Information………………………………………………………………………...….7 Plenary Speakers ……………………………………………………………………………… 9 Keynote & Invited Speakers ……………………………………………………………..... 11 Workshops …………………………………………………………………………………....... 12 Conference Program ………………………………………………………………………..…. 14 Poster Listing ………………………………………………………………………………….. 25 Plenary Abstracts ……………………………………………………………………………..

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Oral Abstracts ………………………………………………………………………………….. 32 Poster Abstracts ……………………………………………………………………………… 168

Conference Information Local Organising Committee A/Prof Lisbeth Grøndahl, Conference Chair – University of Queensland Dr Megan Lord, Conference Co-Chair – University of New South Wales Prof Keith McLean – CSIRO Dr Helmut Thissen – CSIRO Prof Peter Kingshott – Swinburne University Prof Justin Cooper-White – University of Queensland Prof Hans Griesser – University of South Australia Prof Nico Voelcker – University of South Australia A/Prof Krasimir Vasilev – University of South Australia Prof Bruce Milthorpe – University of Technology Sydney A/Prof Chiara Neto – University of Sydney Dr Rylie Green – University of New South Wales Dr Wojciech Chrzanowski – University of Sydney

International Organising Committee Nan Huang (China, Chair) Kazuhiko Ishihara (Japan, Co-Chair) Hai Bang Lee (Korea, Co-Chair) Michael Grunze (Germany) Yang Leng (China) Pankaj Vadgama (UK) Erwin A Vogler (USA) Laura Poole-Warren (Australia)

ASBTE Committee Members

President: Dr Tim Woodfield Vice-President: A/Prof Lisbeth Grondahl Executive Officer: Dr Helmut Thissen Treasurer and Secretary: Dr Penny Martens Committee Members: Dr Bryan Coad, Prof Tony Weiss, Dr Travis Klein, Dr Veronica Glattauer Student Representatives: Robyn Aston (UQ, QLD), Ulises Alejandro Aregueta-Robles (UNSW,

NSW), Peter Koegler (Swinburne University, VIC), Morteza Kafshgari (Mawson Institute, UniSA, SA), Seamus Tredinnick (Uni Canterbury, NZ)

IUSBSE/ILC Delegates: Prof Laura Poole-Warren, Prof Justin Cooper-White STA Liaison Officer: Dr Kate Fox

Conference Venue Doltone House Darling Island Wharf, Ground Floor of Accenture Building, 48 Pirrama Road, Pyrmont NSW 2009. https://goo.gl/maps/cDWdd

Conference Managers

Small Talk Events

Clive McFarland Andrea McFarland 1

Registration The registration desk is located by the entrance to the exhibition area in the South Wharf. Registration desk operating Tuesday 7 April Wednesday 8 April Thursday 9 April Friday 10 April

hours: 11:00-18:00 07:30-17:00 07:30-15:00 08:00-12:00

Entitlements All registered delegates are entitled to the following:  Admission to Conference Sessions (on days of registration)  Admission to Conference Workshops (on days of registration)  Conference satchel and all official documentation, including program booklet and access to abstract proceedings  One Beverage Ticket for the Poster Session on Wednesday 8 April 2015  Ticket to Conference Dinner on Thursday 9 April 2014 (except single-day registrations)  Catering on all Conference days (on days of registration) All delegates registered for the ISSIB/ASBTE joint meeting or the ISSIB meeting are additionally entitled to:  Admission to Welcome Reception on Tuesday 7 April 2015

Name Badges Each delegate registered for the Conference will receive a name badge at the registration desk. This badge will be your official pass and must be worn to obtain entry to all sessions and social functions.

Speaker Preparation Speakers are asked to visit the Speaker Preparation Desk in their session room well in advance of their session to upload their presentations and make any final changes if required.

Poster Presentations

Poster Presentations will be displayed in the Exhibition Space. Poster Presenters are reminded to stand near their posters during the Poster Session on Wednesday 8 April, 2015.

Messages All messages received during the Conference will be placed on the Message Board in the registration area. To collect or leave messages please visit the registration desk.

Mobile Phones As a courtesy to fellow delegates and speakers, please ensure your mobile phones are switched off during Conference sessions.

Delegate Lists The delegate list contains name, company and country of all delegates. The Conference Organisers have excluded delegates who have withheld permission to publish their details, in accordance with the Privacy Act.

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Refreshments/Meal Breaks Morning and Afternoon teas and lunches will be served in the Exhibition Area in the South Wharf. Morning Tea Lunch Afternoon Tea Tuesday 7 April n/a n/a 15:30 - 16:00 Wednesday 8 April 10:20 - 11:00 12:00 - 13:30 15:15 - 15:45 Thursday 9 April 10:30 - 11:00 12:00 - 13:30 15:45 - 16:15 Friday 10 April 11:00 - 11:30 12:30 - 13:30 n/a

Special Dietary Requirements If you have notified the Conference Organisers of any special dietary requirements please be advised that this information has been supplied to the Conference venue and the venues for any social events you have registered to attend. It is requested that you make yourself known to the venue catering staff during meal breaks and social functions and advise them of your specific requirements.

Cloakroom The Cloakroom facilities are located to the rear of the Parkview rooms. Venue staff will be available at the registration desk to transfer items for storage. The Cloakroom is available for storage of personal items only and cannot be used for the storage of event-related material.

Privacy

Australia introduced the Privacy amendment (Private Sector) Act 2000 in 2001. The Conference Organisers comply with such legislation which is designed to protect the right of the individual to privacy of their information. Information collected in respect of proposed participation in any aspect of the Conference will be used for the purposes of planning and conduct of the Conference and may also be provided to the organising body or to the organisers of future ASBTE Conferences. All those participants included in the delegate list, which has been included in the Conference satchels, provided their permission upon registration.

Language The official language of the Conference is English. If you require assistance with additional languages please see staff at the registration desk.

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Floor Plan Floor plans of Doltone House Darling Island Wharf are provided below.

Green area: exhibition and posters, Red area: plenary and concurrent sessions, Blue area: concurrent sessions, Yellow area: concurrent sessions on Thursday 9 April 2015.

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Social Program The following events are included in the registration fee for delegates as indicated.

Welcome Reception* Date: Tuesday 7 April Dress: Smart Casual Venue: Doltone House Darling Island Wharf, Sydney Time: 18:00 - 20:00 Additional Tickets: $40 An invitation is extended to attend the Welcome Reception. This function will be a great opportunity to network with old friends and meet new acquaintances as we welcome you to Sydney and the Conference.

Proudly sponsored by

Poster Session** Date: Wednesday 8 April Dress: Smart Casual Venue: Doltone House Darling Island Wharf, Sydney Time: 17:45 - 19:30 An invitation is extended to attend the Poster Session. Following immediately after the Rapid Fire Session, this is where you will be able to discuss in details the most recent work of other delegates.

Proudly Sponsored by

Conference Dinner*** Date: Thursday 9 April Dress: Smart Casual Venue: Doltone House, Jones Bay Wharf Time: 19:30 - 22:00 Additional Tickets: $120.00 An invitation is extended to attend the Conference Dinner. Jones bay Wharf is a spectacular waterfront function centre on the foreshore of Sydney Harbour, with views of the Harbour Bridge, city skyline and the Darling Harbour precinct and an exclusive balcony and deck event space overlooking the harbour. This promises to be a truly memorable evening. Drinks will be served on the balcony from 7:00 pm, with dinner starting at 7:30 pm.

Proudly Sponsored by

*: ISSIB, ISSIB/ASBTE, Single-day Tuesday delegates. **: All delegates. ***: ISSIB, ASBTE, ISSIB/ASBTE delegtes. 5

Conference and Dinner Venues

Dinner venue

Conference venue

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General Information Airport Transfer The Kingsford Smith Airport is the main international and domestic airport. It is about 10 kilometres (6 miles) south-west of the Central Business District and Sydney Harbour. For details of direct bus transport from the airport to your hotel click this link, or you can catch the train for a 13 minute trip to Central Station.

Climate/Dress Recommendations April in Australia is mid-autumn. In Sydney the average temperature is in the 14.5°C (58°F) 21.5°C (71°F) range. Sydney-siders are friendly, casual and relaxed, and their dress code reflects this attitude. A number of Sydney restaurants do require you to dress appropriately (for example, no ‘thongs’ or flip-flops, shorts or singlets). For business, it is normal for suits to be worn by both men and women.

Credit Cards

Visa, Mastercard and AMEX will be accepted at the registration desk. Automatic teller machines (ATMs) are situated throughout Sydney.

Bank Facilities Banking hours are generally 09:30 to 16:00 hours and extended on Fridays to 17:00 hours. There is no ATM is available at the Conference Venue but the the closest is at the Star Casino on level 2 just opposite the venue.

Electricity Electrical current is 240/250 V, AC 50 Hz. The Australian three-pin power outlet is different from that in many countries, so you will need an adaptor. If your appliances are 110 V, check if there is a 110/240 V switch. If not, you will need a voltage converter. Universal outlets for 240 V or 110 V shavers are usually found in leading hotels.

Wireless facilities Wireless internet services are available within the main Conference facility. Login details will be provided at the registration desk.

Parking

The Star Casino Parking Station - Located on Pyrmont St, Pyrmont. For further information call (02) 9777 9000 Wilson Jones Bay Wharf Carpark - Located on 19-21 Pirrama Rd Pyrmont, 1800 727 5464. All day $16 Wilson Carpark Ticket Validation available: please see venue staff for more information.

Time Zones Australia is divided into three time zones: Western Standard: UTC/GMT + 8 hours - this applies to the whole of Western Australia including Perth, and is the same time zone as Singapore, Beijing, Shanghai, Hong Kong, Kuala Lumpur, Manila and Taipei. Central Standard: UTC/GMT + 9.5 hours - this applies to South Australia and Northern Territory. 7

Eastern Standard: UTC/GMT + 10 hours - this applies to Queensland. New South Wales. Australian Capital Territory, Victoria and Tasmania. Like most Australian states, daylight saving hours apply to New South Wales. Clocks advance forward by one hour between the first Sunday in October and the first Sunday in April the following year. Details of time zones can be obtained from www.australia.gov.au/aboutaustralia/our-country/time.

Goods and Services Tax A Goods and Services Tax (GST) of 10% is applied to the cost of all goods and services in Australia.

Smoking Doltone House Darling Island Wharf is a non-smoking venue. Federal law bans smoking in all Australian Commonwealth government buildings, public transport, airports and international and domestic flights. All Australian states and territories have banned smoking in enclosed public places, particularly workplaces and restaurants. There is no smoking in commercial outdoor eating or drinking areas and in outdoor public places such as patrolled beaches, children’s playground equipment, major sport stadiums and within 4 metres of non-residential building entrances. Smoking is permitted elsewhere outdoors, but littering is an offence.

Liability Insurance In the event of industrial disruption or natural disasters, the Australasian Society for Biomaterials and Tissue Engineering, the Organising Committee and Small Talk Events cannot accept responsibility for any financial or other losses incurred by the delegates. Nor can the Australasian Society for Biomaterials and Tissue Engineering, the Organising Committee or Small Talk Events take responsibility for injury or damage to persons or property occurring during the Conference. All insurance including medical cover and for expenses incurred in the event of the cancellation of the Conference is the individual delegate’s responsibility. The policy should include loss of fees/deposits through cancellation of your participation in the Conference or through the cancellation of the Conference itself, loss of airfares for any reason, medical expenses, loss or damage to personal property, additional expenses and repatriation should travel arrangements have to be altered. The conference secretariat will take no responsibility for any participant failing to insure.

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Plenary speakers Michael Grunze - Emeritus Chair Professor, University of Heidelberg, Germany Michael Grunze is Emeritus Chair Professor for Applied Physical Chemistry at the University of Heidelberg, Germany, and is now associated with the Institute of Functional Interfaces at the Karlsruhe Institute of Technology (KIT). He studied Chemistry and Physics at the Free University of Berlin and, as a Fulbright Fellow, at Knox College in Galesbury, Illinois. He received his Diploma in Chemistry in 1972 and his Ph.D. in Physical Chemistry in 1974 from the Free University of Berlin. During his Post-doc time in the laboratory of G. Ertl he investigated nitrogen adsorption and dissociation on iron single crystals, and extended this work later as staff-scientist at the Fritz-Haber-Institut der Max-PlanckGesellschaft to other adsorbate/substrate systems and the study of heterogeneous catalytic reactions on metal surfaces. From 1984 to 1988 he was Full Professor of Physics and Adjunct Professor of Chemistry at the University of Maine in Orono and Director of the Laboratory for Surface Science and Technology. During this tenure in Maine he started to work on polymer surfaces, which led into the ongoing investigation of ultrathin organic films and self-assembled monolayers in his group, including their applications. His present research focus is on the development of spectroscopic methods for in situ and in vivo investigations of biointerfaces and cells, in particular related to implant surfaces and marine biofouling. Michael Grunze received several scientific awards and served on national and international organizations and advisory boards, and was founding editor of the journal Biointerphases. Besides working on scientific and practical problems, he enjoys teaching, blue water sailing, and spending time with his grand children.

Nan Huang - Professor, Southwest Jiaotong University, China Nan Huang is the full professor of Biomaterials at the School of Materials Sciences and Engineering of Southwest Jiaotong University, China. He leads a group of fifteen professors, associate professors and engineers. The main research themes of the group are surface modification for anticoagulation, anti-infection, anti-restenosis, endothelialization, multifunctional surface modification, controlled drug release, biodegradable metals and polymers, functional nano particles, ion beam process, design and fabrication of implantable and interventional devices and regeneration medicine. He received B.Eng. and M.Eng degrees in Materials Science from Chongqing University and Southwest Jiaotong University, China in 1982 and 1985 respectively. He has worked at the Research Center of Biomedical Engineering, Erlangen University, Germany, the Rossendorf Research Center, Germany and the City University of Hong Kong. He has been Distinguished Professor and Dean of the School of Materials Science and Engineering, Southwest Jiaotong University since 1998 and 2007 respectively. He has published over 200 papers in SCI cited international journals, authored 7 book chapters, obtained over 30 patents and presented over 30 invited lectures. He is on the Editorial Boards of five journals. A drug eluting stent developed by his team has been implanted in over 20 thousand patients. He was elected as a Fellow of the International Union of Societies for Biomaterials Science & Engineering in 2008, and has been the Chairman of the International Committee of the International Symposium on Surfaces and the Interfaces of Biomaterials since 2007.

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Plenary speakers Milica Radisic - Professor and Canada Research Chair, University of Toronto, Canada Dr. Milica Radisic is Professor at the University of Toronto and Canada Research Chair (Tier 2) in Functional Cardiovascular Tissue Engineering. She obtained a B.Eng. from McMaster University in 1999, and Ph.D. from the Massachusetts Institute of Technology in 2004, both in Chemical Engineering. Dr. Radisic received numerous awards and fellowships, including MIT Technology Review Top 35 Innovators under 35. In 2010, she was named “The One to Watch” by the Scientist and the Toronto Star; she also received McMaster Arch Award. She was a recipient of the Professional Engineers Ontario-Young Engineer Medal in 2011, Engineers Canada Young Engineer Achievement Award in 2012, Queen Elizabeth II Diamond Jubilee Medal in 2013 and NSERC E.W.R Steacie Fellowship in 2014. In 2014 she was elected to the Royal Society of Canada, College of New Scholars, Artists and Scientists. The long term objective of Dr. Radisic’s research is to enable cardiovascular regeneration through tissue engineering and development of new biomaterials. Her research interests also include microfluidic cell separation and development of in vitro models for drug testing. Currently, Dr. Radisic holds research funding from CIHR, NSERC, CFI, ORF, NIH, and the Heart and Stroke Foundation. She is an Associate Editor for ACS Biomaterials Science & Engineering and a member of Editorial Board of Tissue Engineering. She serves on CIHR BME panel. She is actively involved with BMES (Cardiovascular Track Chair in 2013 and 2104) and TERMIS-AM (Council member, Chair of the Membership Committee). Her research findings were presented in over 100 research papers, reviews and book chapters with h-index of 38and over 4700 citations. She is a co-founder of a start-up company TARA Biosystems focused on the use of engineered tissues in drug development.

Martina Stenzel - Professor, University of New South Wales, Australia Martina Stenzel studied chemistry at the University of Bayreuth, Germany, before completing her PhD in 1999 at the Institute of Applied Macromolecular Chemistry, University of Stuttgart, Germany. She then moved to UNSW, Australia, as a postdoctoral fellow, sponsored by the DAAD and later was employed as lecturer at UNSW. She is now a full Professor and Co-director of the Centre for Advanced Macromolecular Design (CAMD), a UNSW research Centre dedicated to the synthesis and application of polymers. Her research interest is focused on the synthesis of functional polymers with complex architectures such as glycopolymers and other polymers for biomedical applications, especially polymers with in-build metal complexes for the delivery of metal-based anti-cancer drugs. The aim is to create nanoparticles for the treatment of cancer. One focal point is the formation of hybrid materials from biopolymers such as polysaccharides and proteins with synthetic polymers. Martina Stenzel has published more than 200 peer reviewed papers mainly on RAFT polymerization and their use in biomedical applications. Martina currently serves on eight editorial advisory boards as well as the ARC College of experts and is a scientific editor of Materials Horizons. Martina has received several awards for her work including the 2011 Le Fèvre Memorial Prize.

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Keynote speakers Marcela Bilek University of Sydney, Australia Allen Chen A*Star, Singapore Simon Cool A*STAR, Singapore Morten Foss Aarhus University, Denmark Justin Gooding University of New South Wales, Australia A. Jayakrishnan Indian Institute of Technology Madras, India Gilson Khang Chonbuk National University, Republic Of Korea Sally McArthur Swinburne University, Australia Jelena Rnjak-Kovacina University of New South Wales, Australia Luis Rodríguez-Lorenzo Higher Council for Research, Spain Mario Salwiczek CSIRO, Australia Mark Schembri University of Queensland, Australia R.K. Singh Raman Monash University, Australia Wei-Bor Tsai National Taiwan University Michael Yu University of Queensland, Australia Tomaso Zambelli ETH Zurich, Switzerland

Invited speakers Haifeng Chen Peking University, China James Chong University of Sydney, Australia Michal Dykas National University of Singapore, Singapore Majid Ebrahimi Warkiani University of NSW, Australia Lauren Kark University of New South Wales, Australia Melissa Knothe Tate University of NSW, Australia Melanie MacGregor University of South Australia, Australia Qing Li University of Sydney, Australia Yasuharu Oghoe Tokyo Denki University, Japan Olga Shimoni University of Technology Sydney, Australia Elise Stewart University of Wollongong, Australia Zhilian Yue The University of Wollongong, Australia

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Workshops Workshop I – Nano-manipulation and automated nanomechanical analysis with AFM

Wednesday 8 April 12:00 - 13:30

Nanosurf is an AFM manufacturer producing Next-Level nanotechnology tools. Originating from compact systems for the teaching market, Nanosurf now also provides comprehensive solutions for research and material characterization. In this workshop we will present two solutions based on the FlexAFM: FluidFM and our automated nanomechanical surface analysis tool. FluidFM by Cytosurge is an AFM based nano-manipulation tool using microfluidic cantilevers. The cantilevers are connected to a pressure controlling system that can either apply an overpressure for local liquid delivery or underpressure for local aspiration. The FluidFM system provides an overall software solution that integrating position, force, pressure and optical control. This opens a wide avenue of experiments [1,2] from cell adhesion, cell sorting, and cell injection to local spotting and nanolithography. Furthermore, FluidFM can be extended to electrochemistry and electrophysiology [3] with additional electronics. FlexANA is an Automated Nanomechanical Analysis tool originally designed for cancer diagnostics of clinical biopsies [4]. Biopsies show large height variations and may adhere strongly to a tip. In addition, it requires automation of measurement and analysis procedures to provide statistically relevant data. Other materials also benefit from the automation capability to measure the mechanical signature of material, like clinical tubing or scaffolds.

FluidFM: Nano-manipulation with a hollow cantilever under optical control by an inverted optical microscope. FlexANA: Mechanical characterization for e.g. cancer diagnostics

Workshop II – ASBTE Early Career Researcher Welcome

Wednesday 8 April 12:00 - 13:30

The workshop for early career researchers and students is designed to meet peers in a collegiate environment prior to the start of the ASBTE meeting. This workshop will include three speakers who will share their interesting career paths after completing their doctorates. There will also be time for a networking lunch. The speakers include:  Prof Martina Stenzel is a Professor of Chemistry at the University of New South Wales and a world leader in the synthesis of polymeric micelles for drug delivery. She will talk about her career both in Australia and abroad.  Dr Louis Tsai is a trainee patent and trademarks attorney at Phillips Ormonde Fitzpatrick. His presentation "Your Invention – Considerations for Protection" will discuss his path through research to the commercialisation sector and provide tips for navigating the commercialisation of your research. [See abstract page 63]

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Workshop III – The LifePod Project: A way forward for preterm infants born at the border of viability?

Thursday 9 April 13:00 - 13:30

Dr. Stephen Bird, Department of Obstetrics and Gynaecology, Melbourne Medical School Preterm birth is the delivery of infants at less than 37 weeks of a normal 40 week term, and is now the second major cause of death for infants under 5 years, globally. Preterm birth can occur without warning and has many causes. Extremely preterm infants are born on the border of viability, between 22 and 25 weeks. These infants fall through the cracks when it comes to active healthcare. This is because they have very poor short- and long-term outcomes. Approximately 70-80% will die shortly after birth and those that survive will have a short life, and suffer chronic ill health due to their prematurity. These babies often die because their lungs, hearts and brains are too immature to survive outside of the womb. As the result of these poor outcomes, most hospitals around the world will not treat infants born under 23 or 24 weeks of gestation. Instead, there are international guidelines indicating when, and under what circumstances to provide or withdraw active healthcare. Given this position on active treatment, it is particularly concerning that most of these babies are born healthy, and would develop normally if they could stay in the womb. The scale of the problem is concerning with an estimated 270,000 babies being born extremely preterm each year across the world and approximately 70% do not survive to leave hospital. For the surviving infants, it is estimated to cost $750 million in Australia alone, to raise these special needs infants to age 18. This cost is mostly absorbed by families and the Government. The pathway forward is to return these infants to a womb-like environment, so that they can continue on their normal growth trajectory, rather than to be subjected to an abnormal physiological environment for which they are not yet adapted. Myself and several passionate and concerned scientists and engineers want to create a groundswell of support around changing this situation. We want to create a new device that can perform all of the functions of the placenta & womb and therefore, return a pre-viable infant to a safe environment where it can continue to grow normally. The aim of this workshop is to describe the LifePod Project concept and to initiate a discussion and interest from scientists, engineers and material scientists, attending this conference.

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Program: Tuesday 7 April 2015 13:00 Welcome Address: Mid Wharf Megan Lord & Lisbeth Grøndahl Dr Armstrong - NSW Trade and Investment 13:15 Plenary 1: Mid Wharf

Chair: Bruce Milthorpe Michael Grunze (University of Heidelberg, Germany) A “stealth” coating for medical implants Concurrent Session 1: Mid Wharf Functional Coatings I

Chairs: Kate Fox and Simon Moulton

14:20 Keynote 1 Mario Salwiczek (CSIRO, Australia) Towards multifunctional biointerfaces: Exploring the chemical space of novel prebiotic polymer coatings 14:50 Zhilian Yue (The University of Wollongong,

Australia)

Responsive polymeric inks for bioprinting 15:05 Simon Moulton (Swinburne University of

Technology, Australia)

Medical Bionics, Cellular Interface and Bioprinting Research 15:20 Afternoon Tea: South Wharf Concurrent Session 3: Mid Wharf Functional Coatings II

Chairs: Kate Fox and Simon Moulton 16:00 Marcela Bilek (University of Sydney, Australia)

Plasma Processes for Covalent Attachment of Biomolecules on Surfaces: Utility of Ultra Thin Films and Atmospheric Pressure Plasma Treatments 16:15 Olga Shimoni (University of Technology

Sydney, Australia)

Self-Assembly Coating to Achieve Enhanced Photoluminescence from Nanodiamonds with a Single Nitrogen-Vacancy Defect Centre 16:30 Pengkai Qi (Southwest Jiaotong University,

China)

Immobilization of DNA Aptamers via Plasma Polymerized Allylamine Film to Construct an EPCCapture Surface 16:45 Marion Gaborieau (University of Western

Sydney, Australia)

Molecular Characterisation of Chitosan for Application in Cell Culture

Concurrent Session 2: North Wharf Biosensors and Bioelectronics I

Chairs: Nicolas Voelcker and Simon Corrie 14:20 Keynote 2 Justin Gooding (University of New South Wales,

Australia)

Molecularly Engineered Surfaces For Influencing Cells And Measuring Their Response To Stimuli 14:50 Beatriz Prieto-Simon (University of South

Australia, Australia)

Towards Cardiac Marker Point-of-Care Testing Devices 15:05 Simon Corrie (University of Queensland,

Australia)

Multiplexed capture of P falciparum HRP2 and total IgG from the skin of live mice using Microprojection Arrays Concurrent Session 4: North Wharf Biosensors and Bioelectronics II

Chairs: Nicolas Voelcker and Simon Corrie 16:00 Jacob Coffey (University of Queensland, Australia)

Wearable microprojection array patches for sampling biomarkers from skin: effect on skin capillaries and surface stability in vivo 16:15 Janusz Sadowski (Bionavis, New Zealand) MP-SPR new optical characterization method for molecular interaction and ultrathin films 16:30 Kye Robinson (University of Queensland,

Australia)

Surface Modification of Polycarbonate Surfaces for Biomedical Applications 16:45 Steven Spencer (CSIRO, Australia) Active Acoustic Estimation of Microbubble Properties

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17:00 Ying Yang (Southwest Jiaotong University,

17:00 Katrin-Stephanie Tuecking (University of

Heparinized Surface for Improving Hemocompatibility and Directing Vascular Cells Growth Behaviors

Infection Signaling with Bacterial Enzyme-Responsive Polymers in Vesicles and Nanoporous Silicon Membranes 17:15 Nicolas Voelcker (University of South

China)

17:15 Sara Kaabi Falaheih Asl (Nanyang

Technological University, Singapore)

Hydrothermal process is a promising method for surface modification of magnesium alloy for implant application 17:45 – 20:00 Welcome Reception: South Wharf

Siegen, Germany)

Australia, Australia)

Diagnostic and theranostic devices based on porous silicon for chronic wound care

Program: Wednesday 8 April 2015 08:30 Plenary 2: Mid Wharf

Chair: Laura Poole-Warren Nan Huang (Southwest Jiaotong University, China)

Surface Modification of Cardiovascular Materials – from Biocompatible to Surface Biomimetic Multifunction Concurrent Session 5: Mid Wharf Concurrent Session 6: North Wharf Patterned Biomaterials I Nanofabrication for biomaterial surfaces

Chairs: Chiara Neto and Sally McArthur

Chair: George Wang and Justin Dibbens

09:30 Keynote 3 Sally McArthur (Swinburne University, Australia) Patterning of Plasma Polymers: Challenges and Applications 10:00 Michael Higgins (University of Wollongong,

09:30 Keynote 4 Morten Foss (Aarhus University, Denmark) Screening of Biomaterials for guided cellular response

“Watching” Single Proteins in Action on Material Surfaces using High-Speed Atomic Force Microscopy 10:15 Chiara Neto (University of Sydney, Australia) Protein Micropatterns by PEG Grafting on Dewetted PLGA Films

Development of Micro/Nanofibrous Meshes as Smart Dressings for Chronic Wound Care 10:15 Thi Hong Vy Pham (Swinburne University,

Australia)

10:30 Morning Tea: South Wharf Concurrent Session 7: Mid Wharf Patterned Biomaterials II

Chairs: Chiara Neto and Sally McArthur 11:00 Elise Stewart (University of Wollongong, Australia)

Electrical Stimulation Using Conductive Polymer Polypyrrole Promotes Differentiation of Human Neural Stem Cells 11:15 Yu Suk Choi (University of Sydney, Australia) Patterned Mechanical Stiffening of PEG Hydrogels by NIR Laser 11:30 Chris Mallika Bhadra (Swinburne

University, Australia)

Antibacterial activity of of nano-wired titanium surfaces

10:50 Martina Abrigo (Swinburne University,

Australia)

Australia)

In-vitro interactions of eukaryotic cells with the complex nanopillar geometry of antibacterial surfaces Concurrent Session 8: North Wharf Bioreactors

Chair: Justin Cooper-White

11:00 Keynote 5 Allen Chen (A*Star, Singapore) Expansion of Human Pluripotent Stem Cells and Their Direct Differentiation to Cardiomyocytes in One Unit Operation Stirred Bioreactor

11:30 Veronica Glattauer (CSIRO, Australia) Platform Coating Approach for the Control of Biomolecule and Cell Material Interactions: Synthetic Surfaces for Cell Growth

15

11:45 Hitesh Pingle (Swinburne University,

11:45 Nick Glass (University of Queensland,

Binary colloidal crystal based plasma polymer patterning to study bacteria-surface interactions

Microbioreactor screening of soluble factors for differentiation of human Embryonic Stem Cells to renal lineages

Australia)

12:00 Lunch: South Wharf 12:30 Workshop 1: Mid Wharf

Chair: Wojciech Chrzanowski

Nano-manipulation and automated nanomechanical analysis with AFM Scitech Pty Ltd (Melbourne/Sydney)

Concurrent Session 9: Mid Wharf Analysis of Biomaterial Surfaces and Interfaces

Chairs: Wojciech Chrzanowski and Michael Higgins

13:45 Keynote 6 Tomaso Zambelli (ETH Zurich, Switzerland) FluidFM and biointerface: from cell adhesion to local patterning 14:15 Dipesh Khanal (University of Sydney,

Australia)

12:00 Workshop 2: North Wharf

Chairs: Robyn Aston and Ulises Aregueta Robles

ASBTE ECR/RHD Welcome Louis Tsai (POF, Australia) Martina Stenzel (University of New South Wales,

Australia)

13:30 ASBTE Opening Ceremony: North Wharf Megan Lord & Tim Woodfield Concurrent Session 10: North Wharf Stem Cell Therapies I

Chair: Robert Nordon

13:45 James Chong (University of Sydney,

Australia)

Cell Therapies to Repair and Regenerate the Injured Heart

Australia)

Nanoscale characterization and evaluation of nanotoxicity in 2D and 3D liver model 14:30 Vanessa Montaño-Machado (Laval

University, Canada)

ToF-SIMS Analysis to Elucidate the Interaction of Phosphorylcholine with Fibronectin Coatings 14:45 Andrey Shchukarev (Umeå University,

Sweden)

Surface characterization of insulin coated Ti6Al4V dental implant material conditioned in cell culture medium: XPS study 15:00 Kathryn Grandfield (McMaster University,

Canada)

4D Nanoscale Tomographies of Bone-Implant Interfaces 15:15 Afternoon Tea: South Wharf

14:10 Melissa Knothe Tate (University of New

South Wales, Australia)

Engineering Smart Surfaces and Interfaces to Promote Human Health Using Multiscale Mechanobiological Approaches 14:35 Majid Warkiani (University of New South

Wales, Australia)

The emerging role of inertial microfluidics for ultrahigh throughput, label-free cell sorting 14:55 Tony Simula (CRC for Cell Therapy

Manufacturing, Australia)

Rapid development and translation of affordable cell therapies

16

15:45 Rapid Fire: North Wharf

Chairs: Brooke Farrugia and Alexander Patton

Annabelle Chan (University of Sydney, Australia) Behnaz Aghaei-Ghareh-Bolagh (University of Sydney, Australia) Elena Kosobrodova (University of Sydney, Australia) Elizabeth Whitty (University of Western Sydney, Australia)

Fabian Obregon (University of Sydney, Australia) Hongrui Zhang (University of Wollongong, Australia) Innocent Macha (University of Technology, Sydney) Katharina Schirmer (University of Wollongong, Australia) Murat Gel (CSIRO, Australia) Robyn Aston (University of Queensland, Australia) Ryan Lee (Griffith University, Australia) Yaser Greish (United Arab Emirates University, United Arab Emirates) Zengxiao Cai (Deakin University, Australia) Zetao Chen (Queensland University of Technology, Australia) Yong Peng (CSIRO, Australia) 17:45 – 19:30 Poster Session: South Wharf

17

Program: Thursday 9 April 2015 Concurrent Session 11: Mid Wharf Plasma coatings

Concurrent Session 12: North Wharf Nanoparticles

Chairs: Megan Lord and Michael Yu

Concurrent Session 13: Parkview 1 Biomolecules and Cells at Surfaces and Interfaces

Concurrent Session 14: Parkview 2 Biomechanics & Mechanical Properties of Biomaterials

09:00 Keynote 7 Marcela Bilek (University of Sydney,

09:00 Keynote 8 Michael Yu (University of

09:00 Keynote 9 Wei-Bor Tsai (National Taiwan

09:00 Keynote 10 R.K. Singh Raman (Monash University,

Practical bioactive interfaces for biomedicine: Recent advances towards translation to applications in biomedical implants and microarrays 9:30 Yasuharu Ohgoe (Tokyo Denki

Tailoring Nano-materials for Healthcare Applications

Cell Responses to Nano-grooved Surfaces

Cracking of Magnesium Alloys for Bioimplant Applications

Chair: Krasimir Vasilev

Australia)

University, Japan)

Effect of oxygen plasma treatment for various a-C:H films 9:45 Thomas Michl (University of

South Australia, Australia)

Bacteriostatic plasma polymers that release nitric oxide

Queensland, Australia)

University)

Chair: Travis Klein Australia)

9:30 James Vassie (University of

9:30 Pen-Yuan Wang (Swinburne

9:30 Geoffrey Rodgers (University of

Cerium Oxide Nanoparticles: Functionalisation, Uptake and Therapeutic Effects in Human Cancer Cells 9:45 Kevin Jack (University of

Modulation of mesenchymal and pluripotent stem cell behaviour using ordered surface topographies and chemistries using binary colloidal crystals 9:45 Richard Williams (RMIT,

Acoustic Emission Monitoring and PostRevision Surface Roughness Assessment of Total Hip Replacement Implants

New South Wales, Australia)

Queensland, Australia)

The Modification of Nano-sized HAP Particles with Heparin and its Effect on Particle Distribution in PCL Scaffolds

10:00 Melanie MacGregor

10:00 Hongxu Lu (University of New

Plasma Polymerised PolyOxazoline Thin Films for Biomedical Applications

Enhanced Penetration and Drug Delivery by Crosslinked Polymeric Micelles into Multicellular Tumor Spheroids

(University of South Australia, Australia)

Chairs: Helmut Thissen and Wei-Bor Tsai

South Wales, Australia)

University, Australia)

Australia)

Multicomponent Hydrogel formed via Self-assembly Downregulates Pro Inflammatory Cytokines and induces Selective Apoptosis in Epithelial Cancer Cells 10:00 Berkay Ozcelik (CSIRO,

Australia)

Simple and effective low fouling surfaces for antimicrobial applications

18

Canterbury, New Zealand)

9:45 Zhexing Wang (University of

Melbourne, Australia)

The effect of impact load on the mechanical properties of chondrocytes and its association with cytoskeleton components 10:00 Qing Li (University of Sydney,

Australia)

Multiscale Remodelling and Topographical Optimization for Porous Implant Surface Morphological Design

10:15 Irina Kondyurina (University

10:15 Amy Phillips (Izon Science

10:15 Helmut Thissen (CSIRO,

10:15 Lauren Kark (University of New

Polyurethane medical implants improved by plasma immersion ion implantation

Sensitive analysis of surface properties of nanoparticles, and biomarker detection using TRPS

Peptide Based Copolymer Coatings for the Effective Control of Cell-Material Interactions

Comparison of Methods to Assess the Torsional Strength of the Stem-Neck Junction of Modular Hip Replacements

of Sydney, Australia)

Ltd., New Zealand)

Australia)

10:30 Morning Tea: South Wharf 11:00 Plenary 3: Mid Wharf

South Wales)

Chair: Tim Woodfield Milica Radisic (University of Toronto, Canada)

Human cardiac biowires and injectable cardiac tissues 12:00 Lunch: South Wharf 13:00 Workshop 3: North Wharf

Chair: Helmut Thissen

The LifePod Project: A way forward for preterm infants born at the border of viability? Stephen Bird (The University of Melbourne, Australia) Concurrent Session 15: Mid Wharf Concurrent Session 16: North Wharf Concurrent Session 17: Parkview 1 Biointerfaces I Antimicrobial coatings I Tissue Engineering

Chair: Krasimir Vasilev

Chairs: Hans Griesser and Lisbeth Grøndahl

Chair: Neil Cameron

Concurrent Session 18: Parkview 2 Scaffolds I

13:30 Keynote 11 Gilson Khang (Chonbuk National

13:30 Keynote 12 Mark Schembri (University of

13:30 Keynote 14 Jelena Rnjak-Kovacina (University of

Historical Aspect for the Gradient Surface and the Application for Biomedical Applications 14:00 Fang Wu (Sichuan University,

Uropathogenic Escherichia coli biofilms

13:30 Keynote 13 Simon Cool (A*STAR, Singapore) Using glycotherapeutic devices to regenerate body tissues

14:00 Michael Grunze (University of

14:00 Anthony Weiss (University of

14:00 Brooke Pereira (Monash

Chemistry, nano-structures, and SLIPS surfaces: no durable and non-toxic antifouling strategy in sight

Molecular positioning of nature’s elastic assembly modules to build complex multi-dimensional vascular and microvascular structures

Modelling the three-dimensional prostate cancer microenvironment in vitro using melt electrospun scaffolds

University, Republic Of Korea)

China)

Modulation of Cationicity of Chitosan for Tuning MSC Adhesion, Proliferation and Differentiation

Queensland, Australia)

Heidelberg, Germany)

Sydney, Australia)

19

Chair: Alan Ian Cassady

New South Wales, Australia)

Biomimetic approaches toward silk biomaterial vascularisation

University, Australia)

14:15 Haifeng Chen (Peking

14:15 Bryan Coad (University of

Directing cell behaviour by tailoring surface property of materials

Investigation of Natural Antifungal Compounds for Surface Coatings

University, China)

South Australia, Australia)

14:15 Ferdinand Wagner

14:15 David Nisbet (Australian Layer-By-Layer Functionalization of -Caprolactone) Nanofibre Scaffolds with Novel Polymer Influences Astrocyte Phenotype In Vitro 14:30 Jason Marroquin (Monash

(Queensland University of Technology, Australia)

14:30 Harish Padmanabhan

14:30 Renxun Chen (University of

Tissue engineering a humanized bone organ as a platform for primary bone tumour research 14:30 Javad Jafari (University of

Synergistic Effects of Canonical Wnt and BMP Signalling on the Differentiation of hMSCs to Osteoblasts 14:45 Michal Dykas (National

Inhibition of in vivo Microbial Colonisation of Peptide-Coated Biomaterials

Stimulation of cells with magnetic particles and magnetic fields for soft tissue engineering

(University of Queensland, Australia)

New South Wales, Australia)

Melbourne, Australia)

National University, Australia)

University, Australia)

A PEDOT microfibrous electrode for neural recordings

14:45 Tiziana Nardo (Politecnico di

14:45 Naveen Vijayan Mekhileri

14:45 Khoon Lim (University of Otago,

Integrated System for Automated 3D Assembly of Micro-tissues for Cartilage Regeneration

Oxygen Inhibition in 3D Printing of PhotoCurable Hydrogels

15:00 Peng-Yuan (George) Wang

Self-polymerizing DOPA as a functional layer for the development of antimicrobial coating on PTFE barrier membranes for GTR procedures 15:00 Kitty Ka Kit Ho (University of

High Throughput Fabrication of Colloidal Assemblies to Study Biointerfacial Interactions

Prevention of Bacterial Infections by Surface Immobilized Dihydropyrrolone Compounds

University of Singapore, Singapore)

Metal oxide material libraries as cell growth screening devices

(CSIRO, Australia)

Torino, Italy)

New South Wales, Australia)

15:15 Yin Xiao (Queensland

15:15 Debarun Dutta (University of

Biomaterials Immuno-OsteoModulation in the Development of Bone Substitute and Surface Coating 15:30 Diantao Zhang (Harbin

Immobilisation and Characterisation of Antimicrobial Peptides onto Biomaterial Surfaces

University of Technology, Australia)

Engineering University, China)

Preparation and Properties of Fluorinated Amorphous Carbon Films on Medical NiTi Alloys

New South Wales, Australia)

(University of Otago, New Zealand)

New Zealand)

15:00 Shuko Suzuki (Queensland Eye Institute, Australia) Modifications of Silk Fibroin Membranes to Enhance Human Corneal Limbal Epithelial Cell Growth for Ocular Surface Regeneration 15:15 Yinghong Zhou (Queensland

15:00 Caitlin Langford (Monash

Multifunctional Mesoporous Silica Nanospheres for Bone Tissue Engineering 15:30 Ellen Otte (CSIRO, Australia) The impact of heterotypic cell communication between MSCs and mature cells

Diffusion Tensor and Computed Tomography Microimaging Study of Scaffolds for Tissue Engineering 15:30 Nikola Ristovski (Queensland

University of Technology, Australia)

20

University, Australia)

Emulsion Templating: A Versatile Route to the Preparation of Biodegradable and Biocompatible Scaffolds for Tissue Engineering 15:15 Sean Powell (Queensland

University of Technology, Australia)

University of Technology, Australia) Melt Electrospinning as a Potential Additive Manufacturing Technique

15:45 Afternoon Tea: South Wharf Concurrent Session 19: Mid Wharf Biointerfaces II

Concurrent Session 20: North Wharf Antimicrobial coatings II

Concurrent Session 21: Parkview 1 Stem Cell Therapies II

Concurrent Session 22: Parkview 2 Scaffolds II

16:15 Fengjuan Jing (Southwest

16:15 Chiththaka Imihami Mudiyanselage (Queensland

16:15 Shanti Gurung (CSIRO,

16:15 Timothy Henderson (University Characterisation and in vitro analysis of HA cryogels for soft tissue regeneration

Chair: Bryan Coad

Jiaotong University, China)

The platelet behavior and NO releasing in vitro of Ti-Cu films by high power pulsed magnetron sputtering

Chairs: Hans Griesser and Lisbeth Grøndahl

University of Technology, Australia)

Chair: Anthony Weiss Australia)

16:30 Juan Wang (North Carolina A

16:30 Aditi Taunk (University of New

Inhibition of transforming growth factor beta receptor signaling promotes expansion of undifferentiated human endometrial MSCs 16:30 Sally Yunsun Kim (University

An ex vivo porcine artery model: Fluid convection and diffusion induced biodegradation behavior of magnesium metal 16:45 Kate Fox (RMIT, Australia) Diamonds are an implant’s best friend

Antibacterial biomaterials based on quorum sensing inhibitors

Cell-based therapy for targeted treatment of lung injury

& T State University, USA)

Visualising the interaction between nanopillars and bacteria membrane

South Wales, Australia)

of Sydney, Australia)

of Melbourne, Australia)

16:30 Ali Negahi Shirazi (University of

Sydney, Australia)

Stable micropatterned hydrogels from Gelatin

16:45 Israt Biva (University of South

16:45 Tania Banks (University of

16:45 Miina Bjorninen (University of

Identification of Plant-derived Antibacterial Compounds for Surface Coatings

The Effect of Electrical Stimulation on Adipose Stem Cells Cultured in Conductive Stereolithographic Scaffold Structures

Australia, Australia)

Queensland, Australia)

17:00 Krasimir Vasilev (University

17:00 Hans Griesser (University of

Investigations into the effects of electrical stimulation on human bone marrow-derived mesenchymal stem cells using a novel medium throughput microdevice platform 17:00 Jerran Santos (University of

The influence of material properties on cell fate

Dual-action surface coatings designed to combat polymicrobial mixed bacterial and fungal infections

Proteomic profiling of differentiating Adipose Derived Stem Cells for clinical therapy development

of South Australia, Australia)

Chair: Dietmar Hutmacher

South Australia, Australia)

Technology Sydney, Australia)

21

Wollongong, Australia)

17:00 Dhee Prakash Biswas

(University of Melbourne, Australia)

A combined gas templating and phase separation approach towards soft tissue engineering scaffolds

17:15 Bruce Milthorpe (University

17:15 Taryn Naidoo (University of

Interactions at Bio-material Interfaces

In vitro and in vivo Chondroinduction of Human Pluripotent Stem Sell-Derived Mesenchymal Stromal Clees in a PEGHA Based Hydrogel

of Technology, Sydney)

Queensland, Australia)

17:30 ASBTE AGM: North Wharf 19:00 for 19:30 Conference Dinner and ISSIB Close

22

17:15 Afeesh Rajan Unnithan

(Chonbuk National University, Republic of Korea) Simvastatin Loaded Biomimetic Graphene oxide-Chitosan-Hyaluronic acid 3D composite Scaffold for Bone Tissue Engineering

Program: Friday 10 April 2015 Concurrent Session 23: Mid Wharf Hydrogels I

Concurrent Session 24: North Wharf Drug Delivery I

9:00 Keynote 15 Luis Rodríguez-Lorenzo (Higher Council for

9:00 Keynote 16 A. Jayakrishnan (Indian Institute of Technology

Biodegradable and injectable hydroxyapatitehyaluronate hydrogels 9:30 Alexander Patton (University of New South

Polymeric Prodrugs of Amphotericin B and Primaquine

Chair: David Nisbet

Chair: Jelena Rnjak-Kovacina

Research, Spain)

Madras, India)

9:30 Gabriella Lindberg (University of Otago, New

Wales, Australia)

Zealand)

Fabrication of and cellular response to standalone PEDOT based conductive hydrogels 9:45 Aurelien Forget (University of South

Covalent Incorporation of Heparin in Gelatin Hydrogels for Improved Chondrocyte ReDifferentiation 9:45 Terence Hartnett (University of Melbourne,

From a 3D Vasculogenesis Model to Therapeutic Angiogenesis 10:00 Christoph Meinert (Queensland University of

Lamellar to cubic phase transitions: Tackling the bioactive frontier 10:00 Brooke Farrugia (University of New South

Australia, Australia)

Australia)

Technology, Australia)

Wales, Australia)

A hydrogel model to investigate the effects of varying cartilage tribological properties on chondrocyte mechanotransduction 10:15 Ulises Aregueta Robles (University of New

Chitosan Sulphate: engineering a biomimetic growth factor delivery system 10:15 Kiara Bruggeman (Australian National

South Wales, Australia)

University, Australia)

Survival and Function of Glial Cells in 3D Hydrogels

Temporally controlled release of multiple growth factors from a self-assembling peptide hydrogel tissue engineering scaffold 10:30 Alex Cavallaro (University of South Australia,

10:30 Penny Martens (University of New South

Wales, Australia)

Australia)

Poly(vinyl alcohol)/gellan gum Biosynthetis hydrogel for cartilage tissue engineering applications 10:45 Rui Li (Deakin University, Australia) Tuning the Physical and Biological Characteristics of Self-Assembled Peptide Scaffolds for Enhanced Cell Adhesion

Controlled and sustained release of pharmaceuticals via a single step solvent-free encapsulation 10:45 Srinivasa Reddy Telukutla (RMIT,

Australia)

Camptothecin conjugated to D-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS): conjugation chemistry, characterization and in vitro evaluation of anticancer activity

11:00 Morning Tea: South Wharf 11:30 Plenary 4: Mid Wharf

Chair: Keith McLean Martina Stenzel (University of New South Wales, Australia)

Design of functional micelles for enhanced cellular uptake and their movement in cancer spheroids 12:30 Lunch: South Wharf

23

Concurrent Session 25: Mid Wharf Hydrogels II

Concurrent Session 26: North Wharf Drug Delivery II

13:30 Steffen Cosson (CSIRO, Australia) Reflection at the Water/Oil-Surfactant Interface: Impacts for on chip UV Cross-linking of Cell-laden Hydrogel Micro-particles 13:45 Clementine Pradal (University of

13:30 Adrian Fuchs (University of Queensland,

Chair: Khoon Lim

Chair: Kevin Jack Australia)

Hyperbranched Polymers as Theranostic Platforms for Prostate Cancer 13:45 Neil Cameron (Monash University, Australia) Polypeptide Nanoparticles for Ocular Drug Delivery

Queensland, Australia)

Polyrotaxane hydrogels for tissue engineering applications 14:00 Justine Roberts (University of New South

14:00 Ted Chang (University of New South Wales,

Wales, Australia)

Australia)

Environmentally Responsive Biosynthetic, HeparinPVA Hydrogels for Biomedical Applications

Synthesis of Amphiphilic Block Copolymers for Encapsulation of Curcumin in the Study of Cellular Interaction and Uptake 14:15 Anitha A (University of Queensland,

14:15 John Forsythe (Monash University, Australia) Light Responsive Gelatin Hydrogels for Cell Culture Applications

Australia)

Combinatorial Approach of Curcumin and 5Fluorouracil Encapsulated Thiolated Chitosan Nanoparticles towards Colon Cancer Treatment 14:30 Ahmed Omar (Advanced Technology and

14:30 Tim Huber (University of Canterbury, New

Zealand)

New Materials Research Institute, Egypt)

Cross-linked all-cellulose composite hydrogels made from aqueous NaOH/urea solutions

Novel Amphoteric Alginate Coated Aminated Chitosan Microcapsules for Oral Drug Delivery 14:45 Patrice Castignolles (University of Western

Sydney, Australia)

Smart Poly(acrylic acid) for Anticancer Drug Delivery

15:15 ASBTE Close: Mid Wharf Tim Woodfield

24

Rapid Fire Poster Listing Poster Name No. 1 2 3

Annabelle Chan University of Sydney, Australia Behnaz Aghaei-Ghareh-Bolagh University of Sydney, Australia Elena Kosobrodova University of Sydney, Australia

Paper Title A Novel PCL-Hydrogel Composite for Intervertebral Disc Regeneration Tropoelastin-silk hybrid films for corneal tissue replacement Immobilization of tropoelastin on plasma immersion ion implanted polymer surface

Elizabeth Whitty 4 5 6 7 8 9 10 11 12 13 14 15

University of Western Sydney, Australia Fabian Obregon University of Sydney, Australia Hongrui Zhang University of Wollongong, Australia Innocent Macha University of Technology, Sydney Katharina Schirmer University of Wollongong, Australia

Cellular Response to Linear and Branched Poly(acrylic acid)

Zengxiao Cai Deakin University, Australia Zetao Chen Queensland University of Technology, Australia Yong Peng CSIRO, Australia

Novel Electrically Conductive Scaffolds for Muscle Tissue Engineering

New dentine derived hydrogel as an assisted scaffold Probing Molecular Interactions at the Cell-Polymer Electrode Interface using Single Cell Force Spectroscopy (SCFS) Drug delivery bioresorbable thin film composites for treatment of bone and implant-related infections

A Cytocompatible Method for Embedding Wires or Fibres into Hydrogels Highly Efficient Continuous Flow Biosensing with Murat Gel Bioluminescent Resonance Energy Transfer in Miniaturized CSIRO, Australia Reactors Robyn Aston Incorporation of polyelectrolyte complexes into alginate hydrogels and their effect on the alginate matrix University of Queensland, Australia Ryan Lee Effects of titanium surface modification on osseous healing in diabetes Griffith University, Australia Yaser Greish Towards the Regeneration of Gastric Epithelium Using a Tissue United Arab Emirates University, UAE Engineering Approach

Nutrient element-based bioceramic coating with high bonding strength and beneficial osteoimmunomodulatory properties Recombinant non-animal collagens as new biomaterials

25

Poster Listing Poster Name No. Guo-Chung Dong 16 National Health Research Institutes, Taiwan Jelena Rnjak-Kovacina 17 University of New South Wales, Australia 18

19 20 21 22 23

24 25 26 27 28 29 30 31 32 33 34

Paper Title Utilizing SPR to Study EGF- and BMP-Immobilizing on Scaffold Surface for Bone Tissue Engineering Lyophilized silk sponges: a versatile biomaterial platform for soft tissue engineering

Tamer Abd Elrazik City of Scientific Research and Technological Applications, Egypt Iman Manavi Tehrani University of Sydney, Australia

Experimental and Computational Analysis of a Novel Flow Channel to Assess the Adhesion Strength of Sessile Marine Organisms Antimicrobial polymeric membranes for wound dressing applications based on chitosan: Preparation, characterization and biomedical evaluation Starch-Poly(Propylene Carbonate) an Alternative to Poly(Lactic Acid) for Biomedical Applications

Matthew Park KAIST, Korea

Simultaneous Control of Neurite Length and Orientation Using Anisotripic Micropillar Arrays

Lewis Martin University of Sydney, Australia Sujay Prabakar Leather and Shoe Research Association of New Zealand, New Zealand Chris Mallika Bhadra Swinburne University of Technology, Australia Mohammed Altaf Hossain University of South Australia, Australia David Nisbet Australian National University, Australia Ying-Chih Liao National Taiwan University, Taiwan Jui-Yang Lai Chang Gung University, Taiwan Yu Suk Choi University of Sydney, Australia

Computer Simulations of Biomimetic Peptides for Immobilization on Plasma-Activated Surfaces

Simone Dimartino University of Canterbury, New Zealand

Conor Horgan Australian National University, Australia Geoffrey Rodgers University of Canterbury, New Zealand Guolong Meng Sichuan University, China Mibel Aguilar Monash University, Australia Nendar Herdianto Agency for The Assesment and Application of Technology, Indonesia

Immobilization of Quantum Dots in Collagen based Hydrogels and their Applications as Corneal Implants Dual functionality of of nano-wired titanium surfaces Investigation of Natural Antifungal Compounds for Surface Coatings Self-assembling peptide hydrogels for tissue repair in the central nervous system Three Dimensional Origami Structured Bio-Scaffold Carbodiimide Cross-Linked Amniotic Membranes as Limbal Epithelial Cell Scaffolds: Matrix Nanostructure Effect Stiffness does matter: ECM stiffness controls stem cell fate Development and Characterisation of Minimalist Co-Assembled Fmoc Self-Assembling Peptide (SAP) Systems for Tissue Engineering Multi-variable Optimization Routines for the Design of Porous Orthopaedic Scaffolds Surface characteristics and biological responses of Si, Mg and CO32- substituted HA coatings Supramolecular Sef-Assembly of β-Peptides: New Materials With Tunable Morphology And Chemical Function A Preliminary Study of Hydroxyapatite-Gelatin Biomedical Composite with Drug Release Function

26

Poster Listing Poster Name No.

Paper Title

35

Stephanie Lamont-Friedrich Mawson Institute, Australia

Micro-patterned arrays for investigating the contact-killing mechanism of antifungal surfaces

36

Athena Brunt Griffith University, Australia

Tocopherol and Ascorbic Acid Protect the Structural Properties of Bone Allograft During Gamma Irradiation

37

Elizabeth Whitty University of Western Sydney, Australia

38

Jingan Li Southwest Jiaotong University, China

39

Giada Barabaschi University of Sydney, Australia

Binding of a Fluorescent Tag Model and Cisplatin to Poly(acrylic acid) The effect of micro-patterned TiO2 nanotubes thin film on the deposition of endothelial cell extracellular matrix: for the purpose of enhancing surface biocompatibility Design and Fabrication of Hydrogel Scaffolds for Improving Oxygen Biotransportation

40

Jiajia Zhang Development of an artificial suprachoroidal fluid to model vision University of New South Wales, Australia prosthesis electrode behaviour in vitro

41 42 43 44 45

Yu-Te Liao National Taiwan University Rhys Walter James Cook University, Australia

Synthesis of Fe3O4 Nanoparticles contained Alginate Microparticle for Mass Production of Stem Cells

Lida Hou Harbin Engineering University, China Juan Wang North Carolina A & T State University, USA Aurelien Forget University of South Australia, Australia

Corrosion behaviour and hemolysis of biodegradable magnesium alloys Behavior of platelets on titanium oxide films synthesized by high power pulsed magnetron sputtering and DC magnetron sputtering Microwells as a Screening Platform for Matrix Interactions with Cell Spheroids

Electrochemical activity of magnesium alloys influencing calcium phosphate deposition

27

A “stealth” coating for medical implants M Grunze Institute for Functional Interfaces (IFG), Karlsruhe Institute of Technology, Karlsruhe, Germany and Applied Physical Chemistry, University of Heidelberg, Germany [email protected]

Needed for blood compatible medical devices are not surface coatings that resist protein and cell adsorption (“inert surfaces”), but surfaces that prevent the activation of the complement system and thus the immune response leading to inflammation. A solution to this problem appears to be the use of “stealth” surfaces, covered by a thick protein layer in which the macromolecules maintain their native conformation and thus “hide” the artificial device. In my talk I will discuss the characteristics of both “inert” and “stealth” surfaces. A commonly used prototype “inert “surface is coated with a layer of hydrated poly(ethylene oxide) (PEG) which acts as a steric barrier towards irreversible adsorption of proteins and cells. However, the limited stability of grafted PEG coatings in blood and the reported ability to activate an immune response limits its use for applications in medical implants. A “stealth” surface for implants can be made from poly(bis(trifluoroethoxy)phosphazene) (Polyzene®-F), a soft rubber like inorganic polymer with a -[P=N]n- backbone substituted by trifluoroethanol side groups, and of strictly linear structure and an extreme purity and high molecular weight. This inorganic polymer is used on cardiovascular devices (COBRA PzF™ Coronary Stent System) and to coat embolization particles (Embozenes®-). In my talk I will review the development of Polyzene®-F in the laboratories of the University of Heidelberg, the physical and chemical characteristics of Polyzene®-F films, and how these properties translate into the performance in biomedical assays, animal experiments and clinical trials.

28

Surface Modification of Cardiovascular Materials – from Biocompatible to Surface Biomimetic Multifunction N Huang Wang, J, Leng, YX, Yang P, Chen JY, Yang ZL Key Lab. of Advanced Materials Technology, Ministry of Education, Southwest Jiaotong University, Chengdu, Sichuan, China [email protected] Cardiovascular devices are extensively applied for saving the life of the patients of cardiovascular diseases. However complications such as thrombus formation, restenosis, infection etc are frequently occur and cause the implantation or intervention failure. The present applied surface modification on cardiovascular materials showed almost single function such as preventing blood coagulation, prohibiting restenosis, or anti-bacterium etc. However this surface state can not meet the need of the clinic requirement sufficiently. Such as drag release from a drag eluting stent can effective suppress restenosis but increase late thrombus which is even more dangerous for the patients. This paper reported the recent researches in author’s lab. to endow the materials surface with multifunction and how if play the role in the interaction with bioenvironment. A multilayer Ti-O film/biodegradable drug release coating on Co-alloy stent was prepared. And it showed the biodegradable drug release coating played the role suppressing restenosis in the acute period, and Ti-O film prevented blood coagulation and maintain longterm biocompatibility. Clinic trail of 300 patients for 4 years showed excellent clinic result that no late thrombus was found and restenosis was also low. Two years clinic application on over 10 thousands patients further proved this advantage. Based on the result a conception “time sequence functional stent” was proposed. For enhance the surface multifunction of vascular devices, several biomimetic functional surfaces were further developed. Our recent work revealed that heparin immobilized on plasma-polymerized allylamine (PPAam) film showed very special characteristics on which not only blood compatibility was improved, but the surface presented specifically ability to promote endothelial cell growth and inhibit smooth muscle cells proliferation simultaneously. In vivo investigation showed that this surface was effective endothelialized. A co-polymer coating was synthesized combine dopamine and selenocystamine, which can catalyze NO when contacting with blood. It played mimetic functions of native endothelium of vessel and showed that the NO-generating coating posses excellent hemocompatibility and simultaneously promote endothelial cells growth but prevent smooth muscle cells proliferation. And therefore the surface showed the multifunctions prevent restenosis and fast endothelialization in vivo. It is asserted that the multifunctional surface may become the new generation of the surface modification of biomaterials, on which the interaction between the artificial surface and biological environment can be better regulated and the novel surfaces should bring significant advantages for developing new generation of implantation and intervention devises. This work was supported by Key Basic Research Program 2011CB606204 and NSFC 81271701,81330031

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Human cardiac biowires and injectable cardiac tissues M Radisic Department of Chemical Engineering and Applied Chemistry / University of Toronto [email protected]

Engineering effective therapies for heart disease will require restoration of beating myocardium as well as revascularization of the injured or impaired area. Since human postnatal cardiomyocytes are terminally differentiated, it is not possible to obtain these cells and expand them from biopsies of primary tissue. Recent advances in stem cell biology and development of directed differentiation protocols enable derivation of cardiomyocytes from human pluripotent stem cells (hPSC). However, hPSC-derived cardiomyocytes are reflective of very early human development, limiting their utility in the generation of in vitro models of mature myocardium suitable for drug testing or restoration of adult hearts. We developed a new platform that combines three-dimensional cell cultivation in a microfabricated system with electrical stimulation to mature hPSC-derived cardiac tissues. We utilized quantitative structural, molecular and electrophysiological analyses to elucidate the responses of immature human myocardium to electrical stimulation and pacing. We demonstrated that the engineered platform allowed for the generation of 3-dimensional, aligned cardiac tissues (biowires) with frequent striations. Biowires submitted to electrical stimulation markedly increased myofibril ultrastructural organization, displayed elevated conduction velocity and altered both the electrophysiological and calcium handling properties versus non-stimulated controls. These changes were in agreement with cardiomyocyte maturation and were dependent on the stimulation rate. We will also discuss approaches to vascularizing cardiac tissue by development of new perfusable microfabricated scaffolds termed, AngioChips that are also suitable for engineering a human body on a chip. Lastly, placing a functional, beating, cardiac patch onto the heart currently requires opening of the chest. We will discuss shape-memory scaffolds that enable a minimally invasive delivery of engineered tissues in vivo.

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Design of functional micelles for enhanced cellular uptake and their movement in cancer spheroids M Stenzel Y Jiang, J Zhao, A Dag, K Babiuch, H Lu Centre for Advanced Macromolecular Design (CAMD), School of chemistry, University of New South Wales, Sydney, Australia [email protected] Background Micelles and vesicles have long been proposed as carriers for low molecular weight molecules including drugs. Especially micelles are found to be useful for the encapsulation of hydrophobic drugs in the core while maintaining the water solubility of the system with the hydrophilic shell. Latest developments in polymer synthesis allow the design of wellcontrolled architectures. However, synthetic polymers alone cannot provide the specificity and bioactivity of nature’s building blocks such as carbohydrates and protein. Aims Aim is the synthesis of nanoparticles based on synthetic polymers and proteins or carbohydrates for the design of effective drug carriers Results and Discussion Coating micelles with albumin is an effective strategy to ensure high cell uptake. Due to pathophysiological conditions in neoplastic tissue, high amounts of albumin accumulate in tumours, and metabolization by malignant cells is enhanced by specific receptor glycoproteins gp60 on the endothelial cell surface. Endfunctional polymers were prepared by RAFT polymerization and conjugated to albumin while simultaneously forming albuminbased micelles. The resulting albumin coated nanoparticles were selective in delivering their payload to cancerous cells while healthy cells were less affected. This drug delivery platform was developed for the delivery of common hydrophobic anti-cancer drugs, polymer-drug conjugates and for nucleic acids. Alternative strategies tested to enhance the cellular uptake by cancerous cells include the use of carbohydrates that can interact selectively interact with the appropriate surface receptor. We prepared a range of micellar system based on simple monosaccarides including glucose, mannose, fructose and fucose.

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Tuesday Session 1: Functional Coatings I

14:20-15:20

Towards multifunctional biointerfaces: Exploring the chemical space of novel prebiotic polymer coatings Salwiczek, M, Thissen H, Evans R.A. CSIRO Manufacturing Flagship, Clayton, VIC, Australia [email protected] [email protected] [email protected] Background: The requirement for biomaterials to include one or multiple specific biological functions is increasingly indicated in medical applications [1]. Implants are expected to perform several tasks in addition to their intended mechanical functions, for example preventing bacterial adhesion and providing a tissue compatible surface. Implementing several biological functions within one surface remains challenging and novel approaches that provide the flexibility to tailor coatings for different applications are greatly desired. We have developed a surface coating based on prebiotic polymers that can easily be established on a broad range of substrates and shapes [2]. The inherent chemical heterogeneity of these coatings provides different functional groups that can be exploited to perform in situ as well as secondary modifications. Objectives: Our aim is to explore how this coating interacts with a variety of functional groups using small molecules as model compounds. The most suitable candidates are then translated to actual biologically relevant molecules. Materials and Methods: Polystyrene served as the substrate for the coating using buffered aqueous solutions of aminomalononitrile as the starting material. Different electrophiles (aldehydes and epoxides), nucleophiles (amines and thiols) and metal ions (silver) were incorporated and their levels of incorporation evaluated (X-ray photoelectron spectroscopy and inductively coupled plasma optical emission spectroscopy). Biological responses such as bacterial and mammalian cell attachment were tested according to ISO standards. Results and Discussion: All of the tested model compounds are incorporated into the coatings to some extent. Electrophiles such as aldehydes as well as metal ions are the most reactive. Evaluations of the silver coated surfaces show that the intended biological function (bactericidal activity) is preserved upon incorporation into the coating. Furthermore, PEG functionalised coatings exhibit the expected reduced cell-adhesive properties. Conclusion: The results gained to date confirm our hypothesis and prove our concept that prebiotic polymer coatings provide the chemical flexibility to incorporate distinct biological functions. We are currently exploring possibilities to incorporate several functional molecules simultaneously to generate truly multifunctional coatings. [1] Busscher H.J. et al. Sci Transl. Med. 2012, 4, 153rv10 [2] Thissen H. et al. Hydrogen cyanide-based polymer surface coatings and hydrogels. Patent WO 2013/170308 A1.

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RESPONSIVE POLYMERIC INKS FOR BIOPRINTING Yue, Z, Liu, X, Chung, J, Wallace, GG ARC Centre of Excellence for Electromaterials Science, Intelligent Polymer Research Institute, University of Wollongong, Wollongong, NSW, 2522, Australia [email protected] Bioprinting is providing unprecedented opportunities for fabrication of complex tissue mimics in vitro. For its full potential to be reached, it is crucial to develop bio-inks that enable effective integration of printing technologies with technologies in tissue engineering and regenerative medicine, such as cell scaffolding, stem cell and controlled release technologies etc. A bio-ink should possess not only adequate fluidity to facilitate simultaneous deposition of cells and bio-factors with spatial precision, but also rapid and cyto-compatible sol-gel transition to render the printed structure stable in a biological environment. In addition, the printed structure should conform to the biological requirements for engineering/regeneration of targeted functional tissues, for instance, being conducive to specific cell-cell and cellmatrix interactions to promote phenotypic differentiation and functions, and biomechanical integrity etc. Herein, we provide an overview of the bio-inks developed for bioprinting, with a focus on our recent activities in cell printing. Responsive polymers that undergo phase transition upon a regional stimulus have gained increasing attention owing to their improved and stimulicontrolled printability. We discuss their applications in bioprinting, and highlight the need for further optimisation through engaging tissue engineering principles.

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Medical Bionics, Cellular Interface and Bioprinting Research Simon E Moulton Biomedical Engineering, Faculty of Science, Engineering and Technology, Swinburne University of Technology, Hawthorn, VIC 3122 ARC Centre of Excellence for Electromaterials Science & Australian Institute of Innovative Materials, University of Wollongong, Wollongong, NSW 2522, Australia. [email protected]

The recent advances in our ability to manipulate and characterise materials in the nanodomain may have brought us closer to creating more effective bionic interfaces. The nature of that interface is dependent upon the chemical, physical, morphological and mechanical properties of the implant. Research being undertaken with ACES Bionics Program continues to develop of a class of material, termed electromaterials that permits the on demand manipulation of the materials-biological interface. Additive Fabrication (bioprinting) enables multicomponent, multifunctional structure to be created in a single fabrication process. We are yet to feel the full force of the impact of this fabrication revolution on medical devices wherein personalisation and high level functionality is not just convenient but is mandatory for success. In our laboratories and in association with collaborators at St Vincent’s Hospital we are developing 3D printing and other additive fabrication tools to create conduits for nerve, muscle and bone/cartilage repair. Here we will demonstrate that through integrated advances in hardware, software, materials science and biology we are bringing 3D fabrication close to the clinic, closer to the surgery.

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Tuesday Session 2: Biosensors & Bioelectronics I

14:20-15:20

Molecularly Engineered Surfaces For Influencing Cells And Measuring Their Response To Stimuli Gooding, JJ1, Lu, X1, Gupta, B1, Zhu, Y1, Parviz, M1, Le Saux, G1, Ng, A1, Nicovich, P2, Reece, PJ3, Gaus, K2 1

Australian Centre of NanoMedicine, School of Chemistry and ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, University of New South Wales, Australia 2 Australian Centre for NanoMedicine and Centre for Vascular Research, University of New South Wales, Australia 3 School of Physics, University of New South Wales, Australia [email protected] Introduction Exploring cell-surface interactions are vitally important for both understanding cell –adhesion and for designing man made surfaces that interact with cells in a very well defined way. One strategy to perform such studies is to use model surfaces that contain cell adhesive ligands immobilised onto the surface of a self-assembled monolayer (SAM) modified surface. Such surfaces can mimic the extracellular matrix but with unambiguous presentation of cell adhesive ligands and control over the density of these ligands. Aims The aims of the presented study were 1) To develop a strategy for the preparation of well defined cell adhesive surfaces, using the cell adhesive peptide arginine-glycine-aspartic acid (RGD) as the cell adhesive ligand. 2) To develop methods for characterising these surfaces on the single molecule level 3) Show how make surfaces that can explore the impact of soluble stimuli on the response of the cells. Materials and Methods The modification of silicon with a SAM without an intervening oxide layer is achieved via hydrosilylation of alkenes or alkynes. Surfaces are modified with a base monolayer followed by an oligo(ethylene oxide) layer, to make the surfaces resistant to nonspecific adsorption of cells, and then RGD peptides. Results and Discussion The data shows that not only cell phenotype but cell migrant and cell outside-in signalling for bovine aortic endothelial cells is influences by the density of RGD ligands on a silicon surface. The optimal ligand density for signalling was an average spacing of 44 nm which is consistent with the density of RGD ligands in fibronectin. The amount of cell adhesion was sensitive to topography but for a given topography the optimal RGD density remained the same. It is shown that the accessibility of the RGD ligands can be controlled by using electrically switchable surfaces. In the case of macrophage cells, silicon photonic crystal surfaces can monitor the release of MMPs from these cells upon stimulation with LPS. Conclusion Cell response is shown to be highly sensitive to surface design. Acknowledgements The authors would like to thank the ARC and the NHMRC for funding

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Towards Cardiac Marker Point-of-Care Testing Devices Prieto-Simon, B1, Chew, DP2, Voelcker, NH1 1

Mawson Institute, University of South Australia, Mawson Lakes, SA, Australia Department of Cardiology, Flinders University/Flinders Medical Centre, Adelaide, SA Australia [email protected]

2

Background: Rapid and accurate assessment of cardiac markers elevation is required for proper diagnosis, prognosis and monitoring of cardiac injuries. The National Academy of Clinical Biochemistry establishes a diagnosis turn-around-time of less than 60 min once the patient is admitted to the hospital, recommendation failed by more than75% of hospitals. Cardiac marker point-of-care (POC) testing devices are becoming available as viable faster alternatives to meet clinical demands. Aims and Objectives: We aimed to develop cardiac marker diagnostic tools for troponin I (cTnI) and brain natriuretic peptide (BNP). cTnI is the cornerstone of myocardial damage, being the preferred cardiac marker. BNP is also a suitable biomarker due to the good correlation of its concentration with the severity of the cardiac episode. We expected electrochemical biosensors to fulfil the requirements of POC tests by providing rapid measurements, accuracy, ease of performance, cost-effectiveness and minimal or even absence of sample pretreatment. Nanoporous membranes were proposed to overcome the challenge of dealing with complex biological samples. Nanopores can perform as filters of certain interfering compounds and, if properly functionalised, minimise biofueling, while maximising the surface area and, therefore, the sensitivity. Materials and Methods: Cellulose acetate membranes coated with ZnO by atomic layer deposition were used as porous membranes. Gold electroless deposition was performed to facilitate the use of the membranes as electrochemical transducers, increase their robustness and provide functionalisation versatility. Anti-cTnI antibodies and BNP aptamers were used as biorecognition elements. To reduce the analysis time, working conditions of sandwich-based biosensors were optimised to perform only one-incubation step. Results and Discussion: Highly sensitive cTnI immunosensors were developed, showing limits of detection in the fg/mL level. Immunosensors prepared by site-directed immobilisation of antibodies onto hydrazide-modified surfaces, showed significant matrix effects when analysing cTnI spiked-serum samples. Non-oriented covalent binding of antibodies to carboxy-dendrimers self-assembled onto the porous membranes provided immunosensors able to detect cTnI in diluted serum samples at relevant clinical concentrations (100 pg/mL) without significant interfering effects. A BNP aptasensor was developed as a proof of concept that showed the feasibility to detect BNP in a sandwich approach involving two aptamers with affinity towards different BNP epitopes. Conclusion: Multimarker diagnostic devices able to detect clinical levels of cardiac markers directly in diluted samples in less than 45 min are envisaged as promising tools for the provision of early warnings of imminent heart failure and monitoring of patients’ evolution. Future work will combine the developed sensing approaches with label-free strategies to enable measurements in real time. The authors acknowledge financial support from the Australian Research Council’s Linkage Project Scheme 36

Multiplexed capture of P falciparum HRP2 and total IgG from the skin of live mice using Microprojection Arrays. Lee, KT1, Kendall, MAF1, Corrie, SR1 The University of Queensland, Australian Institute of Bioengineering and Nanotechnology, ARC Centre of Excellence in BioNano Science and Technology, Brisbane, Australia [email protected] Microprojection arrays (MPAs) are emerging biomedical devices that allow patients and healthcare workers to deliver or sample biomolecules via the epithelial layers of the skin. While most diagnostic tests rely on samples removed from the body (“in vitro diagnostics”), processing these samples to obtain biomarkers of interest represents a key technical bottleneck1. Significant research effort has focussed on transdermal delivery devices, but diagnostic applications are in the very early stages of development. A simple, inexpensive, painless and selective biomarker extraction/detection device could move some diagnostic procedures from the laboratory into the clinic, the GP office or the home. These devices also need careful design and evaluation, because by their nature they could be expected to cause specific and/or non-specific foreign body responses from the host. A key challenge in designing diagnostic MPAs is the development of surfaces that can efficiently and selectively capture proteins in the complex environment of the skin2. While comparative in vitro technologies (e.g. ELISA) can be optimised based on diluent composition, temperature, pH, ionic strength, etc., the only parameters available to optimize for these devices is the projection geometry, surface chemistry and application time. Recently we developed a multiplexed MPA device that could be applied to the skin of live mice to capture malaria-specific PfHRP2 protein and total IgG (skin penetration control) simultaneously. We optimized the solution-phase conditions of capture antibody immobilization (EDC/NHS chemistry) to COOH-PEG-modified MPAs, achieving an improvement in detection sensitivity of 100-fold in vitro. We applied these MPAs to the skin of live mice and were able to detect both analytes individually or in a multiplexed fashion, and confirmed our improvements in detection sensitivity. 1) 2)

Labuz SR, Takayama S, Lab Chip, 2014, 14, 3165-3171 Corrie SR, Fernando GJP, Crichton ML, Brunck MEG, Anderson CP and Kendall MAF, Lab Chip, 2010, 10, 2655-2658

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Tuesday Session 3: Functional Coatings II

16:00-17:30

Plasma Processes for Covalent Attachment of Biomolecules on Surfaces: Utility of Ultra Thin Films and Atmospheric Pressure Plasma Treatments Melnichuk, I1, Choukourov, A1, Kuzminova, A1, Bilek, MMM2, Vandrovcova, M3, Hanuš, J1, Kousal, J1, Shelemin, A1, Kylián, O1, Slavínská, D1, Bačáková, L2, Biederman, H1 1

Charles University in Prague, Faculty of Mathematics and Physics, Department of Macromolecular Physics, Prague, 18000, Czech Republic 2 School of Physics, University of Sydney, Sydney, NSW 2006, Australia 3 Institute of Physiology ASCR, Prague, 14220, Czech Republic [email protected]

Background Covalent immobilization of various biomolecules to surfaces is the key step in a wide range of applications in biomedicine and biotechnology, such as production of biosensors, protein microarrays or implantable biomedical devices. Surface embedded radicals in polymeric materials, produced by energetic ion bombardment of polymeric materials, have recently been shown to be highly effective in covalently immobilizing biomolecules directly from solution without the need for specific linker chemistry. Aims In this paper, we report and discuss two plasma-based strategies for preparation of surfaces that enable covalent biding of biomolecules. Results and discussion The first approach combines thermal vapour phase deposition of polyethylene and subsequent treatment of such created films by low-pressure Ar plasma. Both flat and nano-structured ultra-thin films produced in this way were shown to be able of covalently binding bovine serum albumin (BSA) via free radicals. It is demonstrated that at least one-day of incubation in BSA solution is needed to provide a sufficiently long time for protein binding to saturate. This suggests that radicals in such films are long-lived to be able to bind proteins covalently after some extended storage. The second strategy studied is a surface treatment process performed at atmospheric pressure in air that activates a polymeric surface to achieve linker-free covalent immobilization of biomolecules. Polytetrafluoroethylene (PTFE) foil was selected as a model polymer that was treated by a dielectric barrier discharge. It was confirmed by XPS analysis of protein incubated samples after their rigorous SDS washing at elevated temperature that PTFE after plasma activation is indeed capable of immobilizing proteins covalently. Moreover, it was shown in both cases studied that covalently attached biomolecules can retain their biological function. This was indicated by improved growth of MG63 cells on surfaces with immobilised recombinant human tropoelastin. Acknowledgements This work was performed under the COST Action MP1101 and was supported by the grant LD 12066 financed by the Ministry of Education, Youth and Sports of the Czech Republic.

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Self-Assembly Coating to Achieve Enhanced Photoluminescence from Nanodiamonds with a Single Nitrogen-Vacancy Defect Centre Shimoni, O.1, Bray, K.1, Previdi, R.1, Gibson, B.2, Aharonovich, I.1 1

School of Physics and Advanced Materials, University of Technology, Sydney, Ultimo, NSW, Australia 2 ARC Centre of Excellence for Nanoscale BioPhotonics, School of Applied Sciences, RMIT University, Melbourne, VIC, Australia [email protected] Over recent years nanodiamonds with fluorescent nitrogen-vacancy (NV) defect have attracted a profound interest due to applications in bio-sensing and bio-imaging.1,2 Moreover, nanodiamonds with single NV defects have utilized for high resolution magnetometry and single spin sensing owing to the optically detected magnetic resonance (ODMR) phenomenon of NV centres.3-5 Despite the recent progress in the area of single spin detection using NV centres, 6,7 optical detection of single fluorescent NV centres is still remaining challenging with a standard confocal microscope. This is due to a relatively long fluorescence lifetime (~ 11 – 22 ns) from nanodiamonds comparing with standard fluorescent molecular probes (1 – 5 ns) resulting in weak fluorescent signal. Here, we present simple, robust and fast approach to coat nanodiamonds with coordination complex consisting of iron ions and tannic acid.8 We determine that coating of individual nanodiamonds with complexes pronouncedly enhance photoluminescence of single NV defects embedded in 60 nm nanodiamonds. In addition, the enhancement of photoluminescence is accompanied by a reduction of fluorescence lifetime providing a new opportunity for high resolution sensing. More importantly, we demonstrate an excellent signal-to-noise ratio in the ODMR signal, which is an important characteristic for single spin sensing applications. Furthermore, as our method implies biocompatible and FDA approved materials, the coating does not interfere with nanodiamond biocompatibility and, therefore, can be used with a range of bioapplications, such as bio-imaging or bio-sensing. 1. 2. 3. 4. 5. 6.

7. 8.

Mochalin, V. N.; Shenderova, O.; Ho, D.; Gogotsi, Y. Nat. Nanotech. 2012, 7 (1), 1123. Schrand, A. M.; S. A. C. Hens; O. A. Shenderova . CRC Cr Rev Sol State 2009, 34 (1-2), 18-74. Ermakova, A.; Pramanik, G.; Cai, J. M.; Algara-Siller, G.; Kaiser, U.; Weil, T.; Tzeng, Y. K.; Chang, H. C.; McGuinness, L. P.; Plenio, M. B.; Naydenov, B.; Jelezko, F. Nano Letters 2013, 13, 3305. Horowitz, V. R.; Alemán, B. J.; Christle, D. J.; Cleland, A. N.; Awschalom, D. D. Proc. Natl. Acad. Sci. 2012, 109 (34), 13493-13497. Balasubramanian, G.; Chan, I. Y.; Kolesov, R.; Al-Hmoud, M.; Tisler, J.; Shin, C.; Kim, C.; Wojcik, A.; Hemmer, P. R.; Krueger, A.; Hanke, T.; Leitenstorfer, A.; Bratschitsch, R.; Jelezko, F.; Wrachtrup, J. Nature 2008, 455 (7213), 648-652. Kaufmann, S.; Simpson, D. A.; Hall, L. T.; Perunicic, V.; Senn, P.; Steinert, S.; McGuinness, L. P.; Johnson, B. C.; Ohshima, T.; Caruso, F.; Wrachtrup, J.; Scholten, R. E.; Mulvaney, P.; Hollenberg, L. Proc. Natl. Acad. Sci. 2013, 110 (27), 1089410898. McGuinness, L. P.; Yan, Y.; Stacey, A.; Simpson, D. A.; Hall, L. T.; Maclaurin, D.; Prawer, S.; Mulvaney, P.; Wrachtrup, J.; Caruso, F.; Scholten, R. E.; Hollenberg, L. C. L. Nat. Nanotech. 2011, 6(6), 358-363. Bray, K.; Previdi, R.; Gibson, B. C.; Shimoni, O.; Aharonovich, I. Nanoscale 2015, 7 (11), 4869-4874. 39

Immobilization of DNA Aptamers via Plasma Polymerized Allylamine Film to Construct an EPC-Capture Surface Qi, PK, Yang, ZL, Huang, N Southwest Jiaotong University, Chengdu, Sichuan, China [email protected] Background Currently, capturing endothelial progenitor cells (EPCs) and homing EPCs to the stent or vascular graft surfaces for realizing rapid in-situ endothelialization present one of the most promising strategies for the next generation of cardiovascular tissue engineering. Aims We explore a facile method to immobilize an EPC-specific aptamer onto a PPAam surface, aiming at constructing an EPC-capture surface for rapid endothelialization of implantable vascular materials. Materials and Methods EPC-aptamers were immobilized onto the PPAam coated SS by the electrostatic interaction between the -NH3+ and –PO4- groups in the aptamer. Surface morphology and the chemical structures were investigated by AFM, FTIR, XPS and QCM-D was applied for quantification of aptamer. Independent and co-culture of EPCs, ECs and SMCs were conducted to evaluation the interactions between these 3 types of cells and aptamer modified samples. Results FTIR peaks in the 1100 cm-1 to 1550 cm-1 region correspond to the phosphate stretching vibrations and base ring deformations coupled to sugar vibrations. The new peak of P on aptamer modified samples together with QCM-D technique confirms the conjugation amount is about 175 ng/cm2. Figure 1A shows the independent culture of EPCs, demonstrating that PPAam-DNA samples could capture more EPCs, Co-culture results of 3 type cells confirm that the aptamer could specifically bond EPCs rather than ECs and SMCs, suggesting the competitive adhesion advantage of EPCs to ECs and SMCs. (Figure 1B). Figure 1. A) DAPI and rhodamine123 staining of captured EPCs after 2 h, 6 h, 1 d and 3 d culture experiment under non-static condition. B) A) Immunofluorescence staining, cell counting and ritio of EPCs/ ECs and EPCs/ SMCs grown on different samples after co-culture for 2 h and 6 h. Discussion The cardiovascular tissue engineered materials are supposed to exhibit multiple functions. The electrostatic aptamer immobilization as a one-step dry process is performed in a short time, providing the combination of rapid endothelialization and other multifunctions. Conclusion EPC-binding aptamers are successfully immobilized onto the PPAam film through the electrostatic interaction. Evaluations of cell capture, adhesion and proliferation have demonstrated the specificity of the interaction with EPCs, the provided EC-friendly surfaces and no acceleration of the over proliferation of SMCs. Acknowledgement This work was supported by the National NSFC Key Program 81330031 References M. Avci-Adali, G. Ziemer, H. P. Wendel, Biotechnol. Adv., 28 (2010) 119−129. W. Tan, M. J. Donovan, J. Jiang, Chem. Rev., 113 (2013) 2842−2862.

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Molecular Characterisation of Chitosan for Application in Cell Culture Thevarajah, JJ1,2, Taylor, DL1,2, O'Connor, MD3, Wuhrer, R4, Graf, R5, Castignolles, P2, Gaborieau, M1 1

University of Western Sydney (UWS), Molecular Medicine Research group (MMRG), School of Science and Health (SSH), Parramatta, NSW, Australia 2 UWS, Australian Centre for Research on Separation Science (ACROSS), SSH, Parramatta, NSW, Australia 3 UWS, MMRG, School of Medicine, Campbelltown, NSW, Australia 4 UWS, Advanced Materials Characterisation Facility, Parramatta, NSW, Australia 5 Max Planck Institute for Polymer Research, Mainz, Germany [email protected]

Chitosan is derived from chitin, the second most abundant polysaccharide in the world, the main component of discarded crab and shrimp shells. Chitosan being antimicrobial and biocompatible is attractive as a cell growth substrate. To improve cell attachment RGDS peptides were grafted to chitosan films (Figure 1). The grafting reaction was monitored in real time using free-solution capillary electrophoresis (CE). This enabled fast separation and determination of the peptide and all other reactants in one separation with no sample preparation. Covalent grafting onto the chitosan film was demonstrated using solid-state NMR of swollen films. CE indicated that oligomers of RGDS, not simply RGDS, were grafted on the film, with a likely hyperbranched structure. To assess the functional properties of the grafted films cell growth was compared on control and peptide-grafted chitosan films: cell attachment was greatly improved to RGDS-grafted chitosan films.

Figure 1: Grafting peptides onto chitosan to enable cell culture. The cell attachment was not homogeneous across the film surface. This is surmised to originate in the heterogeneity of chitosan's chemical composition. Chitosan is a copolymer: it is composed of two different monomer units, glucosamine and N-acetyl-glucosamine. The average composition of a sample is called degree of acetylation (DA), it is the fraction of monomer units which are N-acetyl-glucosamine. In a given sample, there is a variety of chitosan molecules with different DAs. Solid-state NMR spectroscopy enables the determination of the average DA of chitosan.[1] Capillary electrophoresis in the critical conditions [2] allows the separation of chains with different compositions, and thus the assessment of the distribution of DAs within samples.[3] [1] C Gartner, BL Lopez, L Sierra, R Graf, HW Spiess, M Gaborieau, Interplay between structure and dynamics in chitosan films investigated with solid-state NMR, dynamic mechanical analysis and X-ray diffraction. Biomacromolecules, 12, 1380-1386 (2011). [2] JJ Thevarajah, M Gaborieau, P Castignolles, Separation and characterization of synthetic polyelectrolytes and polysaccharides with capillary electrophoresis. Advances in Chemistry, 2014, Article ID 798503 (2014). [3] M Mnatsakanyan, JJ Thevarajah, RS Roi, A Lauto, M Gaborieau, P Castignolles, Separation of chitosan by degree of acetylation using simple free solution capillary electrophoresis. Analytical and Bioanalytical Chemistry, 405, 6873-6877 (2013). 41

Heparinized Surface for Improving Hemocompatibility and Directing Vascular Cells Growth Behaviors Yang, Y, Yang, ZL, Huang, N Southwest Jiaotong University, Chengdu, Sichuan, China [email protected] Background As an important bio-polysaccharide, heparin has been widely used in cardiovascular devices due to its multiple biological functions including anticoagulation, inhibition of intima hyperplasia, anti-inflammation and interactions with specific proteins. However, few reports focus on how a heparinized surface influence the competitive growth behaviors between vascular endothelial cells (ECs) and smooth muscle cells (SMCs). Aims This work was aimed to develop a heparinized surface and investigate the resulting effect on hemocompatibility,growth behavior of vascular cells and tissue response. Materials and Methods An amine-rich coating was prepared for immobilizing heparin by dip-coating of the target materials(316L SS) in an aqueous solution of dopamine(PDAM) and hexamethylendiamine (HD). The amine groups of the PDAM/HD coating were used to immobilize heparin. Then, hemocompatibility evaluation, vascular cells growth behaviors and host response of heparinfunctionalized PDAM/HD coating were further investigated. Results The amine-rich coating was successfully prepared and the amine groups could be used to conjugate heparin (~900 ng/cm2). Acute thrombogenicity tested by human blood revealed an excellent antithrombotic performance of the heparinized surface. The Hep-PDAM/HD surface also created a favorable microenvironment for selectively promoting HUVECs competitive adhesion over HUASMCs. It was represented as significant adhesion, proliferation and migration of HUVECs, but remarkable inhibition in HUASMC proliferation and migration. When implanted subcutaneously, the Hep-PDAM/HD exhibited mitigated tissue response, with thinner fibrous capsule and less granulation formation compared to 316L SS. Discussion The multifunctional properties of heparin are generally attributed to its interactions with various proteins; to date, more than 100 heparin-binding proteins have been identified. The different action of heparin on directing vascular cell behaviors may be possibly due to the special interaction with heparin-binding cytokines that existed in culture medium or released from cultured cells. Conclusion We reported a facile method to prepare a heparinized coating, which exhibited excellence antithrombotic performance, selectively promoted HUVECs growth over HUASMCs and attenuated tissue response. These attractive unique multiple functions suggested an alternative strategy for designing a desirable new generation of stent. Acknowledgement This work was supported by the National NSFC Key Program 81330031. References [1] Clowes, A. E.; Karnowsky, M. J. Nature 1977, 265, 625–626. [2] Capila, I.; Linhardt, R. J. Angew. Chem. Int. Ed. 2002, 41, 390–412. 42

Hydrothermal process is a promising method for surface modification of magnesium alloy for implant application Sara Kaabi Falahieh Asl, S1,2, Nemeth, S2 , Tan, MJ1 1

School of Mechanical & Aerospace Engineering, Nanyang Technological University, 639708 Singapore 2 Singapore Institute of Manufacturing Technology, 638075 Singapore [email protected]

Biocompatible and bioresorbable magnesium and its alloys could be ideal alternatives for currently used implants (Ti, Co-Cr alloys) to avoid or minimise stress shielding effect which is the main drawback of currently used implants. However, the high corrosion rate of magnesium and its alloys limit their practical application. Thus, in this study, a hydrothermal coating process was developed to provide protective biocompatible and bioresorbable coatings. X-ray diffraction patterns (XRD) indicated sharp and well-defined peaks corresponding to monetite at low deposition temperature and a mixture of monetite and tricalcium phosphate at higher deposition temperature. Scanning electron microscopy (SEM) study of the morphology showed increasingly denser coating with higher deposition temperature. Coating adhesion properties were evaluated by pull-out test indicating cohesive failure at 5.2 to 5.8 MPa stress. The current coatings showed high hardness which decreased by increasing the deposition temperature. Raman spectroscopy studies showed that the coatings consisted of two distinct layers: tricalcium phosphate and monetite. Electrochemical impedance spectroscopy (EIS) confirmed that the coatings provided varying levels of corrosion protection. The corrosion results showed that the size of capacitance loops and the absolute impedance value (|Z|) increased by increasing the deposition temperature and corresponding growth in coating thickness. It was found that coating was partially converted to hydroxyapatite after 28 days immersion in simulated body fluid, confirming the bioactivity of the coatings. The results confirmed that the obtained coatings could be a promising candidate for surface modification of Mg for implant application.

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Tuesday Session 4: Biosensors & Bioelectronics II 16:00-17:30 Wearable microprojection array patches for sampling biomarkers from skin: effect on skin capillaries and surface stability in vivo Coffey JW, Corrie SR, Kendall MAF University of Queensland, Brisbane, Qld, Australia [email protected] Most clinical blood biomarker assays rely on needle sampling for in vitro diagnostic assays. This approach limits the frequency of diagnostic tests and is unsuitable for continuous monitoring. The skin, with its abundance of superficial capillary vessels, offers an alternative minimally invasive route to collect circulating biomarkers, enabling more frequent or continuous monitoring. The skin’s diagnostic potential, however, has been largely unrealised due to the lack of standardised methods to conveniently sample biomarkers. To address this challenge we proposed Microprojection array patches (MPAs) to capture circulating biomarkers from the skin (Fig. 1) [1, 2]. The surface of the MPAs is functionalised with low fouling polymers and capture probes with binding affinity to selectively capture circulating proteins in vivo, avoiding bulk fluid sampling. The device can then be removed and analysed by in vitro diagnostic assays or potentially integrated with portable biosensors. Accessing the biomarker of interest from the complex skin microenvironment remains a key challenge and is critical to improve the MPA detection sensitivity, particularly for low concentration biomarkers. Furthermore, the effects of degradative and/or fouling species within the skin on the functionality of the capture surface in vivo requires investigation. We evaluated the effect of the MPA design (length, density, array size) on the extravasation of circulating plasma proteins from the capillaries into the skin tissue and the concentration of target biomarker. MPA application increased the turnover and concentration of circulating proteins in the skin, facilitating access to circulating biomarkers, although this did not vary significantly with MPA design. Improved capture and detection sensitivity, however, was obtained by increasing the density, length and total array size of MPAs (over 10 min application), underlining the importance of contact area between skin and MPA in accessing target biomarkers. The functionality of the MPA surface (following application times 10 min 24 hours) was then assessed, revealing a significant (> 50 %) decrease in biomarker capture over this time, suggesting significant surface degradation. A key remaining challenge is to confirm the primary causes of this loss and improve surface stability during long term applications. Figure 1 a) Cryogenic scanning electron microscopy micrograph of MPA penetration/contact with skin layers (epidermis and dermis) b) Confocal microscopy compressed z-stack showing co-localisation of fluorescent dye deposited by MPA projections (green) with blood vessels (red) and dermal collagen

1. Corrie, S.R., et al., Surface-modified microprojection arrays for intradermal biomarker capture, with low non-specific protein binding. Lab on a Chip, 2010. 10(20): p. 2655-2658 2.Coffey, J.W., S.R. Corrie, and M.A.F. Kendall, Early circulating biomarker detection using a wearable microprojection array skin patch. Biomaterials, 2013. 34(37): p. 9572-958 44

MP-SPR new optical characterization method for molecular interaction and ultrathin films Jokinen, AJ, Granqvist, NM, Albers, WM, Sadowski, JW BioNavis Ltd, Elopellontie 3C, Ylöjärvi, Finland [email protected] Introduction Surface Plasmon Resonance (SPR) has been used few decades for label-free detection and characterization of biochemical kinetics and affinities for many different types of analysts. The physical phenomenon is not limited to biochemistry, but is applicable to other nanoscale characterization [1]. Results and discussion Aside of the traditional interactions, Multi-Parametric Surface Plasmon Resonance (MP-SPR) can be utilized to determine unique refractive index (RI) and thickness (d) of ultrathin films (d 0.5-100 nm) without knowledge of the RI of the material. These are important properties for many thin film coating industries and applications, and important knowledge in biomaterials also. The new method allows measurement of these properties for both dielectric layers, but also for metals and metal-like coatings that are difficult to measure with other optical methods. Two new methods utilizing MP-SPR have recently been introduced, either measuring in two different media (2M) with high RI difference, such as air and water [1-3], or at two or more different wavelengths (2W) of light [2,3] in order to characterize properties of ultrathin films. The MP-SPR method can also be used in conjunction with cellulose model surfaces in order to study properties and interactions of cellulose with other components [4,5]. Conclusions With the ability to characterize both kinetics and nanoscale layer properties, MP-SPR is an effective tool for nanomaterial, biomaterial and biochemical interactions research. This makes the MP-SPR a powerful tool for multidisciplinary research, where both material physical- and interaction properties are characterized. References [1] Albers, Vikholm-Lundin, Nano-Bio-Sensing, 1st, Springer 2010 [2] Liang, et al., Sens.Act.B,149(1), 2010, 212-220 [3] Granqvist et al. Langmuir 29 (27), 2013, 8561-8571 [4] Orelma et al., 12 (12), 2011, 4311–4318 [5] Kontturi et al., J.Mater. Chem. A, 2013, DOI: 10.1039/C3TA12998E

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Surface Modification of Polycarbonate Surfaces for Biomedical Applications Robinson, KJ1, Grøndahl, L2, Thurecht, KJ1,3, Kendall MAF1, Corrie SR1,2 1

The University of Queensland, Australian Institute for Bioengineering and Nanotechnology, Delivery of Drugs and Genes Group (D2G2), Brisbane, QLD, Australia 2 The University of Queensland, School of Chemistry and Molecular Biosciences, Brisbane, QLD, Australia 3 The University of Queensland, Centre for Advanced Imaging, Brisbane, QLD, Australia [email protected]

Early detection of disease ensures that appropriate treatment can be administered to the right people at the right time1. Proteins are most often detected using affinity-based ELISA assays combined with a catalytic optical or electrochemical reporter. To detect diseaserelated biomarkers early, these assays must be highly sensitive to the low concentration of biomarkers associated with disease, while at the same time being resistant to non-specific protein adsorption associated with complex biological samples. Current approaches are able to detect some biomarkers with modest sensitivity, but further advances in surface chemistry are required to achieve the lower antigen detection limits necessary for early detection. Specific areas of investigation include substrate selection and surface modification, and incorporation of antifouling polymer films that both reduce non-specific adsorption while providing covalent attachment sites for antibodies and related affinity probes. Polycarbonate is a potential substrate of interest as a polymeric material for production of bio-analytical devices such as microprojection arrays2. This interest stems from the high fidelity of 3-D feature replication on the micro-scale in polymer processing operations, (e.g. hot embossing). Recently we investigated electrophilic aromatic substitution reactions as a means to produce a modified PC surface rich in reactive primary amines, however a reduction step with poor efficiency has limited the applications of this strategy. Herein we show an improvement in the modification strategy for polycarbonate put forward by Yeow et al. with the use of tin chloride for specific and efficient aromatic nitro group reduction. It was found that the SnCl2 reduction deposited a layer of SnO2 which could be removed upon exposure to basic solutions. Removal of this layer allowed increased specific immobilisation of polymer on to the surface. Cloud point conditions were also found to be effective for maximising the immobilisation of polymers on to the surface. Commercial PEGs as RAFT polymers were attached via an NHS/maleimide linker as a functional antifouling layer for biomolecule conjugation and found to be capable of specific protein capture. 1). G. Sandler, J. Fry, Early clinical diagnosis, Lancaster, MTP Press: Boston, 1986. 2). B. Yeow, J. W. Coffey, D. A. Muller, L. Grondahl, M. A. F. Kendall, S. R. Corrie, Analytical Chemistry, 2013, 85 (1), 10196-10204

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Active Acoustic Estimation of Microbubble Properties Spencer, SJ Mineral Resources Flagship, CSIRO, NSW, Australia [email protected] Background Micron-sized gas bubbles, encapsulated in a stabilising shell, have been used for many years as ultrasound contrast agent (UCA) in clinical applications spanning radiology and cardiology. The targeted delivery of therapeutic agents loaded onto microbubbles is currently under investigation for clinical use in anti-cancer and gene-based therapies. Aims A method of acoustic resonance interference spectroscopy is described for estimating encapsulated microbubble properties. The technique is based on acoustic theory that relates the properties of a solids-loaded, encapsulated microbubble to the frequencies of active acoustic response resonance and interference features in the combined source and (bubble) scattered beams. Methods A non-linear equation for forced radial oscillation of an encapsulated microbubble with attached solids is formulated. The average pressure power at a receiver is found as a function of the frequency of (mild) acoustic excitation. Closed-form analytical solutions are found for the frequencies of the bubble fundamental resonance maximum, interference minimum and second harmonic resonance maximum at the receiver as a function of properties of the bubble, liquid-like medium and system geometry. Unique solutions to the inverse problem for estimation of microbubble equilibrium radius, attached solids mass and encapsulating layer dilatational viscosity are found in terms of these frequencies. Results Models of the average pressure power at a receiver indicate strong differences in the acoustic response of several different types of commercial UCA. The sensitivity of microbubble average pressure power response to solids loading is demonstrated. Example contour plots of microbubble equilibrium radius and attached solids mass loading as a function of fundamental resonance maximum and interference minimum frequencies are shown. Discussion \ Conclusion Acoustic resonance interference spectroscopy based on broadband acoustic excitation could be a strategy for estimating the size, solids mass loading and surface layer dilatational viscosity of populations of encapsulated microbubbles. Model results suggest the method is suitable for characterising micron sized bubbles loaded with picograms of solids. This method could be used to detect the progressive delivery of therapeutic agents loaded on microbubbles. It could also be used to detect the existence of chemical markers, antibodies or viral loads in a liquid by selective attachment on microbubbles

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Infection Signaling with Bacterial Enzyme-Responsive Polymers in Vesicles and Nanoporous Silicon Membranes Tuecking, K-S1, Voelcker, NH2, Schoenherr, H1 1

University of Siegen, Siegen, Germany University of South Australia, Adelaide, SA, Australia [email protected]

2

Bacterial wound infections caused by pathogenic bacteria may pose serious threats for the affected patients. For instance, they tend to slow down the healing process and lead to increased morbidity. State-of-the-art wound dressings decrease the risk of bacterial infections, however, for adequate healing they must not be removed before the tissue is regrown underneath. Despite the good barrier properties of the dressings, bacteria may still infect the wound, which unfortunately cannot be detected without removal of the dressing, sampling of wound material followed by elaborate time consuming tests. In this context, we develop novel wound dressings, which facilitate the direct detection of pathogenic bacteria like Staphylococcus aureus and Pseudomonas aeruginosa without any further tests. The infection signaling approach relies on enzyme-labile polymers like poly(lactic acid) (PLA), hyaluronic acid (HYA) and poly(L-lysine) (PLL). The selective degradation of the polymer triggers a colorimetric response. In a first approach reporter dye-loaded block copolymer vesicles made of poly(ethylene glycol)-block-poly(lactic acid) (PEG-b-PLA) and hyaluronic acid-block-poly(lactic acid) (HYA-b-PLA) were assembled, which release the signaling dye only in the presence of bacterial enzymes like proteinase K and hyaluronidase. The enzymatic degradation of vesicles filled with self-quenched dyes like fluorescein causes a light-up upon release of the dye, whereas the breakdown of vesicles filled with the hydrophobic dyes Nile Red results in a light-off signal. In the second approach, polymer-filled photonic porous silicon membranes (pSi) were developed, which respond to bacterial enzymes by a direct colour signal. This colour change is caused by a decrease in refractive index, when the polymeric filling is degraded. PLL and PLA were shown to enable the detection of proteinase K, which is a mimic of proteases of Pseudomonas aeruginosa. HYAfilled porous membranes signaled the presence of hyaluronidase, which is excreted by >90% of all known strains of Staphylococcus aureus. Finally pSi functionalized with HYA-b-PLA was tested as dual enzyme-responsive system to detect both enzymes. The two signaling systems developed hence comprise an attractive strategy for the autonomous detection of bacterial infections in wounds exploiting selective enzyme cleavage of polymers.

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Diagnostic and theranostic devices based on porous silicon for chronic wound care Voelcker, NH ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Mawson Institute, University of South Australia, Adelaide, SA, Australia [email protected] Chronic wounds are defined as ulcers that fail to heal over the course of several months. These ulcers affect about 1% of the population and can account for over 50% of community health care budgets. In the US, the annual cost of chronic wound care is estimated at over $25 billion per year. There is a clear and unmet need for improved prevention, treatment and management of chronic wounds. One particular opportunity consists in detection of markers associated with wound healing and infection, utilising devices imbedded within smart wound dressings or as point-of-care techniques to allow for continual or rapid wound assessment and monitoring[1]. This talk will explore the application of porous silicon as interferometric or photonic biosensor componentry [2] to underpin diagnostic and theranostics in chronic wound therapy. The first example relates to the detection of changes in temperature or pH in the wound environment as indicators of bacterial infection. This is achieved by grafting stimulussensitive polymers from the porous silicon matrix [3]. The changes in conformation of the polymer component at a critical temperature or pH value translate into changes in the optical properties of the porous silicon. These sensors can be integrated into existing wound dressings and can also release therapeutic drugs in response to changes in the wound environment, therefore having a theranostic function [4]. A second group of porous silicon biosensors was aimed at detecting matrix metalloproteinase (MMP) wound biomarkers. This was achieved either by immobilising a peptidomimetic MMP inhibitor in the porous layer [5] or by fluorescence enhancement effects of immobilised fluorogenic MMP substrates in porous silicon resonant microcavities. In both cases, MMP detection was achieved in human wound fluid at physiologically relevant concentrations. These types of biosensor may find application as a point-of-care device that is prognostic of the healing trajectory of chronic wounds. References: [1] T. Dargaville, B.L. Farrugia, J. Broadbent, S. Pace, Z. Upton, N.H. Voelcker, Biosensors and Bioelectronics, 41 (2013), 30-42. [2] A. Jane, R. Dronov, A. Hodges, N.H. Voelcker, Trends in Biotechnology, 27 (2009), 230-239. [3] R. Vasani, S. McInnes, M. Cole, A. Jani, A. Ellis, N.H. Voelcker, Langmuir, 27 (2011), 7843–7853. [4] S. Pace, R.B. Vasani, F. Cunin, N.H. Voelcker, New Journal of Chemistry, 37 (2013), 228-235. [5] F. Krismastuti, S. Pace, E. Melville, A. Cowin, T.R. Dargaville, N.H. Voelcker, Australian Journal of Chemistry, 66 (2013) 1428–1434.

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Wednesday Session 5: Patterned Biomaterials I

9:30-10:30

Patterning of Plasma Polymers: Challenges and Applications McArthur, SL ANFF-Vic Biointerface Engineering Hub, Swinburne University of Technology, Hawthorn, VIC, Australia [email protected] Plasma polymerization has been shown to be an attractive method for coating biomaterials surfaces with a wide variety of different chemistries (acids, glymes, anhydrides, amines and epoxies). A major advantage in the use of the plasma polymers is that they can be translated to a variety of substrate materials commonly used for biomedical devices including a range of polymers and inorganic supstrates. As a result the surface charge, hydrophobicity and ultimately protein adsorption characteristics of the channel can be tailored for the specific application requirements. Ultimately, many applications require the spatial control of surface chemistry. There are a number of approaches that have been utilised to pattern plasma polymers over a wide variety of scale lengths. This talk will review the range of application areas where patterning has been utilised and explore the challenges involved with producing and characterising micro- and nano-scale plasma polymer patterns.

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“Watching” Single Proteins in Action on Material Surfaces using High-Speed Atomic Force Microscopy Higgins, MJ University of Wollongong, NSW, Australia [email protected] We are currently undertaking research on protein- and cell-material interfaces, particularly the understanding of nanoscale and molecular interactions for enabling cell growth, development and repair. In this research, the use of conventional approaches for studying the dynamics of molecular interactions with materials and surfaces is challenging. For example, the often rough, opaque and fluorescent quenching properties of real-word materials such as polymers or ceramics are not amenable to optical and fluorescence techniques for tracking single molecule dynamics. This is where some of the emerging nanoscale and molecular characterization techniques, such as High-Speed Atomic Force Microscopy, present exciting opportunities for revealing the molecular-level dynamics on material surfaces. This presentation will highlight research on the use of High-Speed Atomic Force Microscopy (HS-AFM), which massively surpasses the capabilities of conventional AFM systems by enabling acquisition times of 50-100 milliseconds per image (10 - 20 frames/second). This takes AFM into the realm of video rate imaging that is defined as achieving speeds of  12 frames/sec; the human eye needs to visualize a sequence of images at this speed in order to perceive motion. Coupled with the ability to achieve 1-2 nanometer lateral image resolution in liquid, the HS-AFM has the unique ability to provide significant insight into both the nanoscale structural and interaction dynamics of single molecules and molecular assembly processes on surfaces in real-time. We will show results on the dynamics of single fibronectin proteins on mica, gold and polymer substrates with temporal resolution ranging from 2 – 10 frames/sec (i.e. 2 – 10 AFM images per second). The sequence of AFM images, which effectively produce a movie clip, reveal the single proteins as they, diffuse, rotate and interact with neighbouring proteins on the surface. After visualizing the dynamics of single protein – surface interactions, we also observe the two-state adsorption processes, including protein saturation and formation of a protein layer.

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Protein Micropatterns by PEG Grafting on Dewetted PLGA Films Manuel Ghezzi,1 Stuart C. Thickett,2 Andrew M. Telford,1 Christopher D. Easton,3 Laurence Meagher,3 and Chiara Neto1 1

School of Chemistry, The University of Sydney, Sydney, NSW 2006, Australia Centre for Advanced Macromolecular Design, The University of New South Wales, Sydney, NSW 2052, Australia 3 CSIRO Materials Science and Engineering, Clayton South, VIC 3169, Australia [email protected]

2

Background The ability to control protein and cell positioning on a microscopic scale is crucial in many biomedical applications, such as single cell studies. Aims In this paper, we report the preparation of surfaces that present protein patterns obtained by the dewetting of a protein-repellent polymer on a protein-adhesive polymer. Results and discussion We have developed and investigated the grafting of poly(ethylene glycol) (PEG) brushes onto poly(D ,L -lactide-co -glycolide) (PLGA) thin films, which were micropatterned by exploiting their spontaneous dewetting on top of polystyrene (PS) films. Dense PEG brushes with excellent protein repellence were achieved on PLGA by using cloud point grafting conditions, and selective adsorption of proteins on the micropatterned substrates was achieved by exploiting the different affinity for protein adsorption onto the PEG brushes and the PS holes. PEG-grafted PLGA films showed better resistance against spontaneous degradation in buffer than bare PLGA films, due to passivation by the thin PEG coating. Conclusions The simplicity of dewetting and subsequent grafting approaches, coupled with the ability to coat and pattern non-planar substrates give rise to possible applications of PEG-grafted PLGA films in single cell studies and cell cultures for tissue engineering.

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Wednesday Session 6: Nanofabrication

9:30-10:30

Screening of Biomaterials for guided cellular response Foss, M1, Ogaki, R1, Bennetsen, D1, Amin Y1, Abildtrup, LA1, Kraft, D1, Alexander M2 1

iNANO, Aarhus University, Aarhus, Denmark The University of Nottingham, Nottingham, UK [email protected]

2

Understanding the interaction between artificial materials and biological systems is of crucial importance in the design of tailored biomedical devices. A multitude of cues are known to influence the biological response. Topography, chemistry, mechanical properties and controlled release of biologically active agents are routes being explored. Often a reductionist approach is employed providing important fundamental insight. However, a complementary approach has evolved the later years where platforms for largescale screening of specific properties of biomaterial surfaces have been developed. A few of these screening routes for understanding and ultimately controlling the bio-interface will be addressed: cellular response to topographic micro- and nanostructures as well as to chemical patterns. The topographical arrays are produced by photolithography both alone and in combination with physical vapour deposition (PVD)/colloidal crystal templating. The chemical arrays are produced by Nanoimprint Lithography (NILT) combined with protein immobilization. As for cellular model systems the examples will include preosteoblasts, primary human fibroblasts, and human dental pulp stem cells (hDPSCs).

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Development of Micro/Nanofibrous Meshes as Smart Dressings for Chronic Wound Care Abrigo, M, Kingshott, P, McArthur, SL Swinburne University of technology, Melbourne, VIC, Australia [email protected] Diabetic, pressure, venous and arterial ulcers are a large social, economic and healthcare burden. These chronic non-healing wounds show delayed and incomplete healing processes exposing patients to high risk of infection. The design of wound dressings that combine the necessary morphological and physical requirements for wound healing with the value-added capability to address optimal cell responses and impair bacterial proliferation represents a major challenge in chronic wound care. Polymeric nanofibrous meshes fabricated through the electrospinning process are promising candidates as wound dressings due to their high surface area, micro-porosity and non-woven structure. Electrospun materials have been used as scaffolds for tissue engineering for a number of years, but there is surprisingly little literature on the interactions of fibers with bacteria. In this study, polystyrene (M.W. 250,000) meshes were electrospun. In order to understand microbial infiltration and control in wound dressings a number of microbiological assays (MTT, MTS and live/dead) were completed using E. coli, P. aeruginosa, and S. aureus in an effort to understand how the morphological properties and the surface chemistry of the electrospun meshes influence bacterial attachment, proliferation and growth. Fiber diameter was found to affect the capacity of wound bacteria to adhere onto the fibers and spread within the fibrous network. Bacterial size and shape also resulted to play a key role in regulating the interaction of bacteria with the fibers. Fiber surface chemistry was modified through the deposition of plasma polymerized thin films using three monomers: octadiene; acrylic acid; and allylamine. The latter was found to highly encourage bacterial attachment and proliferation when exposed to a E. coli biofilm, while the octadiene and acrylic acid coatings showed few cells adhered onto the fibers. Results suggest the possibility of tuning and combining fiber morphological properties and surface chemistry to control bacterial colonization of non-healing wounds

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In-vitro interactions of eukaryotic cells with the complex nanopillar geometry of antibacterial surfaces Pham, VTH1, Truong, VK1, Fulcher, A2, Bhadra, CM1, Mainwaring, DE1, Juodkazis, S1, Crawford, RJ1, Ivanova, EP*1 1

School of Science, Swinburne University of Technology, Hawthorn, Victoria, 3122, Australia 2 Monash Micro Imaging, Monash University, Victoria, 3800, Australia [email protected]

Over the last decade, nanostructured materials have been the focus of intensive research for the development of powerful tools for biological applications. These applications range from guiding cells, probing biomolecules, measurement of cellular forces, development of biosensors and antibacterial surfaces to drug delivery. It was recently shown that the high aspect ratio nanostructured surfaces of black silicon (bSi), a synthetic analogue of dragonfly wing surfaces, is bactericidal to different types of bacteria and also bacterial spores. The bacterial cells interacting with the surface appeared to be shredded by the nanopillar arrays present on these surfaces, leading to cell death. This effect appeared to be independent of the surface chemical composition. It was also shown that bSi surfaces caused spontaneous stress-induced erythrocyte deformation, leading to erythrocyte rupture and lysis. It remained unclear, however, whether bSi surfaces were able to support the growth of eukaryotic cells whilst still being able to exhibit antibacterial behaviour towards pathogenic bacteria. In this study, we investigated the attachment and proliferation of eukaryotic cells on the surface of bSi that had been infected with two types of bacteria. Infective doses (according to FDA (USA)) of Staphylococcus aureus CIP 65.8T and Pseudomonas aeruginosa ATCC 9027 were employed. The bSi surfaces were inoculated with the bacteria and left for 6 hours, after which time the surfaces were seeded with COS-7 cells and allowed to culture for 1, 3 and 7 days. The competitive colonisation of the bacterial and COS-7 cells on the bSi surface was assessed using scanning electron microscopy and confocal laser scanning microscopy. The real time interaction and attachment behaviour of COS-7 with bSi surface were visualized using sequential time lapse confocal microscopy. The results indicated that after 6 hours of contact with the nanopillars on bSi surface, both types of bacteria appeared to be deactivated, whilst COS-7 cells were able to successfully attach onto the infected nanostructured surfaces. The COS-7 cells maintained their viability and metabolic activity during the course of the experiment, reaching 95% confluency after 7 days. In contrast, nonstructured silicon surfaces remained significantly contaminated with bacteria and exhibited poor signs of COS-7 growth.

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Wednesday Session 7: Patterned Biomaterials II

11:00-12:00

Electrical Stimulation Using Conductive Polymer Polypyrrole Promotes Differentiation of Human Neural Stem Cells Stewart, EM1, Kobayashi, NR1, Higgins, MJ1, Quigley, AF1, Jamali, S1, Moulton, SE1,4, Kapsa, RMI1,2, Wallace, GG1, Crook JM1,3,5 1

ARC Centre of Excellence for Electromaterials Science, Intelligent Polymer Research Institute, AIIM Facility, Innovation Campus, University of Wollongong, Wollongong, NSW, Australia. Departments of 2 Medicine and 3 Surgery, St Vincent’s Hospital, The University of Melbourne, Fitzroy, VIC, Australia. 4 Biomedical Engineering, Faculty of Science, Engineering and Technology, Swinburne University of Technology, Hawthorn, VIC, Australia 5 Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW Australia. [email protected] Background Conducting polymers are carbon based organic materials able to be manipulated to form many different structures. Additionally, their conducting properties can be utilised to provide stimulation cues. Potential applications of these polymers include in vitro or implantable electrodes for excitable cell recording and stimulation and conductive scaffolds for cell support and tissue engineering. Aims In this study, we examined the effect of different dopants and inherent material properties on the support of human neural stem cell (hNSCs) growth and differentiation. We subsequently chose polypyrrole (PPy) doped with the anionic molecule dodecylbenzenesulfonate (DBS) to deliver a clinically relevant electrical stimulation paradigm and investigated the effects on differentiating hNSCs. Results Electrical stimulation of PPy(DBS) induced the hNSCs to predominantly β-III Tubulin (Tuj1) expressing neurons, with lower induction of glial fibrillary acidic protein (GFAP) expressing glial cells. In addition, stimulated cultures comprised nodes or clusters of neurons with longer neurites and greater branching than unstimulated cultures. Cell clusters showed a similar spatial distribution to regions of higher conductivity on the film surface. Conclusion Our findings support the use of electrical stimulation to promote neuronal induction and the biocompatibility of PPy(DBS) with hNSCs and opens up the possibility of identifying novel mechanisms of fate determination of differentiating human stem cells for advanced in vitro modelling, translational drug discovery, and regenerative medicine.

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Patterned Mechanical Stiffening of PEG Hydrogels by NIR Laser Choi, YS1, Hribar, KC2, Engler, AJ3, Chen S2 1

Cardiac Technology Centre, Kolling Institute of Medical Research, University of Sydney, St Leonards, NSW, Australia 2 Dept. of Nanoengineering, 3Bioengineering, University of California, San Diego, La Jolla, CA, USA [email protected]

Background The extracellular matrix (ECM) stiffness has been demonstrated to play a pivotal role in governing cell fate. For instance, soft (~1 kPa) versus stiff (>30 kPa) materials can dictate stem cells into neurogenic or osteogenic lineages, respectively. It has also been shown that stiff materials provide enhanced traction for cell motility, compared to soft substrates. Aims or Objectives Thus the design of biomaterials with tunable mechanical properties is central to tissue engineering, and can facilitate cell migration, proliferation, and differentiation. Materials and Methods Gold nanorods within a loosely crosslinked poly(ethylene glycol) (PEG) hydrogel matrix by UV photopolymerization absorb a focused laser to generate heat and thermally crosslink the network further. A femtosecond near-infrared (NIR) laser beam (pulse width = 100 femtoseconds, wavelength = 800 nm, 80 MHz) passes through an objective lens onto a motorized microscope stage and the stage moves according to a digital (computergenerated) pattern. Stiffness and topographical changes of the hydrogel was confirmed by atomic force microscopy (AFM; MFP3D, Asylum Research). A7R5 smooth muscle cells were cultured on patterned hydrogel for migration and alignment. Results and Discussion Hydrogels were polymerized with a baseline stiffness of 17kPa for mechanical patterning. Both writing speed and laser intensity dictated the degree to which the hydrogel stiffened. Laser writing criteria of 80mW:1.0 mm/s, 80mW:1.5mm/s, 80mW:2.0mm/s and 100mW:2.0 mm/s produced stiffness peaks of 350 ± 12 kPa, 65 ± 6 kPa, 35 ± 3 kPa, and 79 ± 11 kPa. We used 2.5 mM PEG-RGDS on 17 vs. 350kPa gel (80mW:1.0mm/s) to investigate the DPP’s utility in directing cell responses in vitro. A7R5 smooth muscle cells showed durotaxis (stiffness driven cell migration) over 5 days. Patterns with different spacing (50μm, 100μm, and 150μm) also showed migration and alignment. Conclusion Previously, other fabrication techniques have been used to pattern mechanical changes in hydrogels, such as photolithography, which required multiple materials including hard masks in addition to clean room facility. Here, this technique can modify stiffness in a highly tunable fashion without those requirements. This platform represents a potentially useful tool in locally tuning the mechanical properties of hydrogels for biomedical applications.

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Antibacterial activity of of nano-wired titanium surfaces Bhadra, CM, Pham THV, Truong, VK, Wang, JY, Jodkazis,S, Crawford, RJ. , Ivanova, EP. * Faculty of Science, Engineering and Technology, Swinburne University of Technology, PO Box 218, Hawthorn, Victoria, 3122 Australia [email protected] Bacterial infections associated with biomaterials or devices have been intensively studied for the past several decades. In the case of metallic implants including titanium and its alloys, it is critical to ensure that the surface of the implant is effectively integrated with the tissue surrounding the implant prior to appreciable bacterial adhesion taking place. Unfortunately often bacterial adhesion takes place before the tissue integration has been established. Decreasing the adhesion of common biofilm forming pathogenic bacteria can be a feasible approach towards reducing bacterial infection and simultaneous formation of a surface-tissue interface favourable for eukaryotic cells. In this study, we investigated the attachment response of primary human fibroblasts (pHF) cell lines on nano-wired titanium surfaces Characteristic nano-patterns on commercially pure ASTM Grade-2 titanium surfaces were hydrothermally etched in a highly alkaline solution. The chemical composition, wettability, surface topography and morphology of the titanium surfaces were characterized using scanning electron microscopy, X-ray photoelectron spectroscopy, X-ray diffractometry and water contact angle measurements. The pHF were found to be confluent on the nano-wired titanium surfaces without any evident cell damage and lysis as confirmed by scanning electron microscopy and confocal microscopy. The results of this study showed that the nano-wired titanium surfaces exhibited dual functionality being antibacterial towards two pathogenic bacteria, Staphylococcus aureus CIP 65.8T and Pseudomonas aeruginosa ATCC 9027, and at the same time promoting the pHF cells growth and differentiation.

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Binary colloidal crystal based plasma polymer patterning to study bacteria-surface interactions Pingle,H1, 2, Wang, PY 1, 2, 4, McArthur,S2,3 and Kingshott,P1, 2 1. Department of Chemistry and Biotechnology, Faculty of Science, Engineering and Technology, Swinburne University of Technology, Hawthorn, VIC 3122, Australia 2. Industrial Research Institute Swinburne (IRIS), Faculty of Science, Engineering and Technology, Swinburne University of Technology, Hawthorn, Victoria 3122, Australia 3. Department of Telecommunications, Electrical, Robotics and Biomedical Engineering, Faculty of Science, Engineering and Technology, Swinburne University of Technology, Hawthorn, Victoria 3122, Australia 4. CSIRO Materials Science and Engineering Clayton VIC 3168 Australia [email protected] A tremendous amount of research has been focused on the creation of surface chemical patterns on the micro- and nanoscale for use in the biomedical area. Researchers have shown that micro- and nanopatterns on surfaces influence biomolecule adsorption, and subsequent cell and bacteria adhesion. However, less work has been done related to precisely controlled patterning to study the initial attachment mechanisms of bacteria to surfaces, and subsequently how the patterning can influence the role played by biomolecular adsorption on biofilm formation. In this study we have taken advantage of binary colloidal self-assembly in a confined area to pattern surfaces by using the colloidal crystals as a mask in order to generate highly ordered allylamine plasma polymer patterns from the micro- to the nanoscale. PEG-aldehyde was attached to the plasma regions via cloud point grafting to prevent the nonspecific attachment of bacteria on the plasma patterned surface regions thereby controlling the adhesive sites by choice of the colloidal crystal morphology. Pseudomonas aeruginosa was chosen to study the bacterial interactions with these chemically patterned surfaces. Scanning electron microscope (SEM), x-ray photoelectron spectroscopy (XPS), zeta potentials, water contact angle measurements, and confocal fluorescence microscopy were used for pattern characterization, surface chemical analysis, and particle surface charge determination and live/dead imaging of attached bacteria. This study aims to successfully create chemical contrast on surfaces to be able to selective immobilise P. aeruginosa in a precise manner for studying of bacteria-DNA interactions, the mechanisms of biofilm formation, and further applications in biomedical research. .

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Wednesday Session 8: Bioreactors

11:00-12:00

EXPANSION OF HUMAN PLURIPOTENT STEM CELLS AND THEIR DIRECT DIFFERENTIATION TO CARDIOMYOCYTE IN ONE UNIT OPERATION STIRRED BIOREACTOR Chen, A, Lam, A, Ting, S, Reuveny, S, Oh, S Stem Cell Group, Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Singapore [email protected] Human pluripotent stem cells (hPSCs) are attractive cell sources for cellular replacement therapy due to their ability of self-renewal and differentiation to multiple lineages within the three germ layers. In particular, hPSC derived cardiomyocytes can be applied to treat myocardial infarcts. It was estimated that more than one billion cardiomyocytes are required to treat a patient with myocardial infarcts. To achieve an economically viable production of cardiomyocyte, there is a need to develop a scalable process for industrial scale bioreactors. Conventionally, the cultivation of hPSC is based on 2D plates with limited surface area and requires repetitive passaging for expansion. The subsequent cardiac differentiation may require several manual culture manipulations that limit the throughput of the process and is impractical for scaling up. To overcome this, we have developed a microcarrier based process for the expansion of hPSCs, and cardiomyocyte differentiation in one continuous and homogenous unit operation. Microcarriers are small particles where hPSCs can attach and grow on while suspended in culture medium by agitation. The microcarriers should be coated with extracellular matrix proteins which support cell attachment spreading and growth. To this end, several microcarriers and extracellular matrix coatings were tested for hPSC expansion. To achieve a defined and xeno-free microcarrier platform, we have demonstrated that the expansion of hPSC on recombinant xeno-free human laminin-521 coated microcarriers in serum free medium. Up to 3.5×106 cells/ml with 17 fold expansion in a spinner flask was achieved in comparison to 2D plate with 9 fold expansion, while maintaining high levels of pluripotency and stable karyotype. Differentiation of hPSC expanded on microcarriers can be done by a simple change of the growth medium to cardiac induction medium. Stirred bioreactors with gentle agitation generated consistent hPSCmicrocarrier aggregates and resulted in 4 times more cardiomyocyte (1.9106 cells/ml) than 2D culture (0.5106 cells/ml). Structure characterization showed extensive striated sarcomeres confirming cardiac ontogeny. Functionality was demonstrated by monitoring the cellular responses to well-known cardiovascular drugs. In conclusion, the microcarrier based hPSC expansion and differentiation is robust, scalable and can be further developed to meet the requirement in cell therapy, disease studies, drug screening, and tissue engineering.

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Platform Coating Approach for the Control of Biomolecule and Cell Material Interactions: Synthetic Surfaces for Cell Growth Glattauer, V1,2, Styan, K1,2, Ameringer, T1,2, Pasic, P1,2, Tsang, K1,2, White, JF1,2, Be, CL1,2, Haylock, D1,2, Thissen, H1,2, Meagher, L1,2 1

CSIRO Manufacturing Flagship, Bayview Avenue, Clayton 3168 VIC, Australia 2 CRC for Polymers, 8 Redwood Drive, Notting Hill 3168 VIC, Australia [email protected]

Background and Aims: The design of synthetic materials onto which cells can attach and grow in a controlled manner, particularly in chemically defined and serum-free medias is becoming of increasing importance in, for example, cell production for therapeutic applications. A key design feature for these materials for the expansion of cells is that the materials should have both very low non-specific protein adsorption and covalently attached ligands to mediate cell attachment. For cell therapy applications, these materials should be able to function effectively in cell culture media which is chemically defined and animal product free (i.e. serum-free). We have developed a platform coating approach, which in one step, results in coatings with very low non-specific protein adsorption, i.e. no initial chemical functionalisation or priming steps are required. In addition, the coatings also contain functional groups onto which cell attachment ligands such as peptides can be chemically attached. The approach can be used to produce coatings on many different formats of interest, such as multiwell plates, tissue culture flasks and microcarrier particles. Materials and Methods: In this study we have prepared a number of synthetic polymer coatings using a platform grafting from approach to produce materials for the culture of cells. Coatings were formed using a grafting from approach from a monomer feed comprising 10 mole percent acrylic acid and 90 mole percent acrylamide. Coupled to these coatings was a cyclic peptide (cRGDfK) which interacts specifically with αvβ3 integrins only. Results and Discussion: These surfaces were found to be suitable for the attachment and growth of murine L929 fibroblasts and human mesenchymal stem cells (hMSCs). Furthermore, in the case of hMSCs the surfaces were used to expand the cells over three passages in three different media (two were chemically defined and serum free). The hMSCs were characterised by their ability to differentiate into adipocytes, osteocytes and chondrocytes as well as maintenance of cell surface markers typically used to define hMSCs.

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Microbioreactor screening of soluble factors for differentiation of human Embryonic Stem Cells to renal lineages Glass, N1, Takasato, M2, Xuan Er, P2, Little, M2, Wolvetang, EW1, Cooper-White, J1,3,4 1

Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, QLD, Australia 2 IMB Kidney Research, Institute for Molecular Bioscience, The University of Queensland, QLD, Australia 3 CSIRO Manufacturing Flagship, VIC, Australia 4 Chemical Engineering, The University of Queensland, QLD, Australia [email protected]

Background The differentiation of human pluripotent stem cells (hPSCs) towards renal is of great interests for areas of health science from basic science to toxicology and regenerative medicine. Recently, processes for directing human embryonic stem cells to produce cell populations of the kidney such as nephrons and uretic bud have been demonstrated by the Little lab [1] and others [2]. Materials and Methods Here we have investigated the effect of several soluble factor perturbations using a 3 by 3 factorial microbioreactor previously develop in the Cooper-White lab [3]. This allows for 27 unique chemical inputs, with every combination of 3 factors at three combinations each. Furthermore, each condition is applied to 10 serial wells to allow for insight into paracrine or autocrine cell signalling. This system allows for 270 unique chemical states within the device. Results and Discussion Initial experiments examined the effect of known signalling pathways on the differentiation of hPSCs towards renal cell populations at day 9 and day 12 time points in the differentiation protocol. Results confirmed the essential factors for the differentiation and condensation of uretic epithelium cells (GATA3+, ECAD+) cells. This effect was synergistically enhanced in the presence of some of the factors investigated. Aggregates of nephrons (WT1+, ECAD+) have additionally been observed growing off condensed uretic epithelium structures. Image cytometry was then preformed across the entire device to provide FACs like data for the population of each well, showing that approximately 15% of the cells were nephrons.

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Wednesday Workshop 2: ECR/ASBTE Welcome

12:00-13:30

Your Invention – Considerations for Protection Tsai, LM Phillips Ormonde Fitzpatrick Patent and Trade Mark Attorneys, 367 Collins Street, Melbourne, VIC, 3000, Australia [email protected] Much of the research outcome in the field of biomaterials has intrinsic commercial potential such as tissue adhesives, contact lenses, stents, and orthopaedic implants. Without commercial support, inventions like these would not have progressed from the bench to the market place. A commercial partner will see little benefit in investing funds to develop the research if the work is not at least protected by some form of IP protection such as a patent. But what can be patented and what constitutes patentable subject matter in the light of the recent Court decisions on this subject? In Australia, one of the criteria for patentability is that an invention must qualify as a “manner of manufacture”. In other words, the invention must be considered to be of appropriate patentable subject matter. But what does this actually mean? How do compounds, mixtures, new uses of known compounds, methods, processes, etc commonly encountered in the field of biomaterials fall within the definition of a “manner of manufacture”? This presentation will discuss what constitutes patentable subject matter in Australia. It will also touch on recent Court decisions in this area, including the highly controversial gene patenting issue, and how these decisions could affect scientific fields of endeavour.

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Wednesday Session 9: Analysis of Biomaterial

13:45-15:15

FluidFM and biointerface: from cell adhesion to local patterning Zambelli, Tomaso Laboratory of Biosensors and Bioelectronics, ETH Zurich, Switzerland [email protected] Glass micropipettes are the typical instrument for intracellular injection, patch clamping or extracellular deposition of liquids into viable cells. The micro pipette is thereby slowly approached to the cell by using micro manipulators and visual control through an optical microscope. During this process, however, the cell is often mechanically injured which leads to cell death and failure of the experiment. To overcome these challenges and limitations of this conventional method we developed the FluidFM technology, an evolution of standard AFM microscopy combining nanofluidics via cantilevers with integrated microfluidic channel [1]. The channel ends at a well-defined aperture at the apex of the AFM tip while the other extremity is connected to a reservoir. The instrument can therefore be regarded as a multifunctional micropipette with force feedback working in liquid environment. We are focussing on three applications for “force-controlled” single-cell biology [2]: i) cytosolic and intranuclear injection, ii) cell adhesion, and iii) electrophysiology. At the same time we are using the FluidFM as lithography tool in liquid [3]. In this presentation, I will focus on two aspects: On the one hand, how surface functionalization influences the adhesion of mammalian cells and bacteria on an inorganic substrate. On the other hand, how local functionalization of a substrate with appropriate polymers tailors the neuron growth. [1] A. Meister et al, Nano Lett (2009) 9:2501 [2] O. Guillaume-Gentil et al, Trends Biotech (2014) 32:381 [3] R.R. Grüter et al, Nanoscale (2013) 5:1097

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Nanoscale characterization and evaluation of nanotoxicity in 2D and 3D liver model Khanal, D, Chrzanowski, W, Fu, D, Ramzan, I Faculty of Pharmacy, The University of Sydney, Sydney, NSW, Australia [email protected] Safety of nanomaterials, which are virtually everywhere including cosmetics, toiletries, food products including sweets, chewing gum etc., vitamins, and drugs, is of a major concern. There is already evidence that nanomaterials may induce serious health problems and their effects on living organisms are largely unexplored. In the light of alarming reports suggesting significant impact of nanoparticles on health there is an urgent need to develop new robust models for evaluation of their toxicity. Current approach for nanotoxicity study relies on the use of two-dimensional cell culture models which have severe limitation due to lack of “biomimmicry” and does not provide robust scientific evidence of nanoparticle effects on the human body. 3D Scaffold free liver models based on novel magnetic levitation technology can mimic complexity and functionality of tissues and will serve as a platform to study effect of nanomaterials on healthy and diseased tissues. Here we demonstrate the applicability of the model and some novel nanocharacterization techniques as Lorentz Contact Resonance (LCR), NanoIR, and Atomic Force Microscopy (AFM) based nanoindentation for probing nanotoxicity at individual cellular level and in the fabricated model. Complex tissue-like features were found to be formed within 24 hour of levitation with the hetereogeneity in morphology and nanomechanical stiffness across different regions when probed by LCR suggesting localization of nanoparticle around the edge of the spheroid structure. Nanodiamond was found to be nontoxic and was taken up by the cells when characterized by AFM & NanoIR techniques. These results confirm the applicability of model for high throughput screening for nanotoxicity and these nontoxic nanoparticles may be used for targeted therapy for treating liver-specific diseases in the future. LCR, NanoIR and AFM are very useful nanosensors for evaluating the nanomechanical properties and nanoscale morphology of cells and tissue models for identifying uptake and effect of nanomaterials

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ToF-SIMS Analysis to Elucidate the Interaction of Phosphorylcholine with Fibronectin Coatings Montaño-Machado, V. Chevallier, P. Noël, C. Turgeon, S. Houssiau, L. Pireaux, JJ. Pauthe, E. Mantovani, D. 1

Laboratory for Biomaterials and Bioengineering, Dept. of Min-Met-Materials Eng., & University Hospital Research Center, Laval University. 10 rue de l’Espinay, Québec, Québec, G1L3L5, Canada. 2 ERRMECe, University of Cergy-Pontoise, Site Saint-Martin, 2 Avenue Adolphe Chauvin, 95302 Cergy-Pontoise Cedex, France. 3 Laboratoire Interdisciplinaire de Spectroscopie Electronique, FUNDP, 61 rue de Bruxelles, B-5000 Namur, Belgium. [email protected] Introduction Studying coatings conformed by several biomolecules is an interesting approach to target different clinical complications of biomaterials. In this work, coatings containing fibronectin (FN) and phosphorylcholine (PRC) were studied for having complementary properties for cardiovascular applications (FN for endothelialization and PRC for hemocompatibility). FN was firstly grafted to a fluorocarbon polymer deposited by plasma process on stainless steel substrate. Subsequently, PRC was added in two different ways: adsorption or grafted process. The study of the interaction of PRC with FN to understand further biological performances was required and assessed by a highly sensitive technique: Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS). Materials and Methods Cleaned stainless steel was first coated by a plasma deposition process, leading to a thin film of a fluorocarbon compound (CFx). This film was therefore activated to further graft FN through glutaric anhydride. Afterwards PRC was adsorbed (30 min, 37 ºC) or grafted (via EDC-Imidazole). The distribution of characteristics ions of PRC was studied by static and profilometry tests through ToF-SIMS analyses. Clotting time measurements were performed to assess hemocompatibility properties. Results and Discussion The distribution of the PRC ion C5H15PNO4+ showed a high homogeneity on PRC adsorbed sample whereas an inhomogeneous one was observed for PRC grafted sample. Furthermore, profilometry analyses have evidenced that PRC was mostly distributed at the top most layer whatever the PRC process used. No ion of the covalent binding between PRC and FN was detected meaning that the grafting process was not efficient and PRC detected was just adsorbed. After measurements of clotting time, the adsorption process showed better hemocompatibility properties. Conclusion ToF-SIMS analysis was a powerful technique to study the interaction of PRC with FN. It allowed us to clearly observe the different interactions between molecules depending on the process used, adsorption or grafting, as well as the non-efficiency of the PRC grafting process. Profilometry measurements have clearly exhibited that the distribution of PRC was mainly at the topmost layer, and thus PRC can play its hemocompatible role as observed after clotting time measurements. This key information was impossible to assess before with other usual surfaces characterization techniques. 66

Surface characterization of insulin coated Ti6Al4V dental implant material conditioned in cell culture medium: XPS study Shchukarev, A1, Tengvall, P2, Ransjö, M2, Westerlund, A2 1

Umeå University, Umeå, Sweden Salgrenska Academy at Göteborg University, Göteborg, Sweden [email protected]

2

Background. Understanding of interface phenomena and corresponding surface modification are crucial for improving biocompatibility of osseointegrated implants. Recently, local delivery of exogenous insulin from an implant surface was found to enhance bone formation in non-diabetic rats [1]. The objective of this study is to follow insulin behaviour at the implant surface in biologically relevant media. Material and methods. Non-coated and insulin coated Ti6Al4V discs were conditioned in αminimum essential medium (α-MEM), with and without addition of 10% fetal bovine serum (FBS). Surface chemical composition of the discs before immersion and after interaction with cell culture media for 1 and 10 days was monitored by X-Ray photoelectron spectroscopy (XPS). Sample preparation protocol for XPS analysis is described in detail. Results. A continuous TiO2 layer dominates surface composition of non-coated discs. Preadsorption of insulin results in stable and reproducible surface coating. Analysis of C 1s spectra and C/N atomic ratio indicates structural re-arrangement of insulin molecules upon conditioning the coated discs in original α-MEM. This time-dependent process leads to exposing hydrophobic parts of insulin molecules towards the medium. Same interface phenomenon also occurs for serum protein adsorbed at the surface of nontreated discs from α-MEM with FBS. Conditioning of insulin coated discs in α-MEM with FBS results in additional protein adsorption. Restoration of C/N atomic ratio close to its original value (5:1) and corresponding C 1s spectra shed light on possible adsorption mechanism of FBS due to the hydrophobic moieties interaction between serum and insulin. Moreover, the C/N atomic ratio does not change with a time indicating stable structure of protein layer formed at the interface. Noticeable amount of calcium and phosphate ions incorporated into the layer is another characteristic interface feature. Conclusion. Similar to bone graft substitute materials [2], structurally stable protein layer at the interface of dental implant-biological tissue seems to be an indispensable prerequisite ensuring the biocompatibility. Pre-adsorbed insulin layer demonstrates a direct surface effect stabilizing the structure of secondary-adsorbed FBS and promoting accumulation of calcium and phosphate ions, thus creating a favourable interface environment for bone cells recognition – a condition important to initiate bone formation. [1] B. Malekzadeh, P. Tengvall , L.-O. Ohrnell, A. Wennerberg, A. Westerlund, Journal of biomedical materials research Part A. 2013, 101(1), 132-137. [2] A.Shchukarev, Z. Mladenovic, M. Ransjö, Surf. Interface Anal. 2012, 44, 919-923

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4D Nanoscale Tomographies of Bone-Implant Interfaces Wang, X, Langelier, B, Grandfield, K McMaster University, Hamilton, ON, Canada [email protected] Background and Objectives There is a fundamental challenge associated with understanding the junction between biomaterials and tissues due to the complex hierarchical structure of mineralized tissues. However, resolving the spatial and chemical structure of bone tissue at biomaterials interfaces presents the possibility to uncover the mechanisms for bone apposition to nanostructured implant surfaces. Here, we present an innovative set of correlative microscopy techniques to elucidate the structure and chemistry of bone-biomaterial interfaces with nanoscale and four-dimensional (spatial plus chemical) clarity using atom probe tomography (APT) and Electron Energy Loss Spectroscopy (EELS) tomography. Materials and Methods A laser-modified titanium implant and bone was retrieved from the human maxilla after 4 years. Using a focused ion beam, specimens from the bone-titanium interface and bone were sharpened into 100 nm diameter needle-shaped specimens. In the Titan 80-300 scanning transmission electron microscope operated at 300kV, specimens were rotated through ± 70° using an on-axis tomography holder and EELS spectrum images were recorded every 2°. Chemical maps were extracted for elements Ca, C, Ti and O and visualized using Amira software to identify the organic and inorganic motifs at the interface. APT was performed on the same sharpened tip using a CAMECA LEAP 4000UHR atom probe microscope. Results High-resolution spatial (x,y,z) plus chemical information was obtained. The location of organic and mineral structures at the interface was detected with a 3D resolution on the order of 6-9 nm. EELS mapping highlights the location of collagen fibrils and surrounding mineral. APT, with sub-nanometer resolution, detected fine chemical changes at the implant surface due to laser-modification, and localized gradients in bone structure with 1 ppm sensitivity. Discussion and Conclusion This study highlights the implications for understanding hierarchical structures interfacing to materials with electron and atom probe tomographies. The structure and chemical composition of the organic and inorganic components at bone-interface has been show at the nanoscale in 4D. In the future, these advanced correlative microscopy techniques provide a possibility to elucidate the role of locally or systemically delivered drugs on bone surrounding implants, an important concern in the treatment of bone pathologies

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Wednesday Session 10: Stem Cell Therapies I

13:45-15:15

Cell Therapies to Repair and Regenerate the Injured Heart James JH Chong1,2,3 1

School of Medicine, University of Sydney, Sydney, Australia Department of Cardiology, Westmead Hospital, Sydney, Australia 3 Centre for Heart Research, Westmead Millennium Institute for Medical Research, Sydney, Australia [email protected] 2

Cell therapies specifically targeting heart failure could greatly decrease morbidity, mortality and burgeoning health care costs worldwide. These novel therapies can be broadly grouped into two categories. The first, Adult Stem/Progenitor Cells (ASCs) have a limited ability to form down-stream differentiated cells (termed plasticity). Nevertheless, ASCs have already been used in many clinical trials investigating cardiac repair. Results have shown a favourable safety profile but inconsistent results regarding efficacy. The second category, Pluripotent Stem Cells (PSCs) have an unquestionable ability to form bonafide, spontaneously contracting, cardiomyocytes. However, as a cardiac regenerative strategy PSCs currently remain in the preclinical arena. This presentation will discuss recent work on a Platelet Derived Growth Factor Receptor-Alpha expressing ASC population and on human PSC derived cardiomyocytes (hPSC-CM). Particular focus will be made on recent nonhuman primate experiments demonstrating feasibility of human hPSC-CM as a potentially viable strategy for cardiac regeneration.

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Engineering Smart Surfaces and Interfaces to Promote Human Health Using Multiscale Mechanobiological Approaches Knothe Tate, ML University of New South Wales, Sydney, NSW, Australia [email protected] Background: Bounding membranes or interfaces between disparate tissues of the body exhibit smart properties that enable active compartmentalisation of tissues concomitant to integrated tissue function. The MechBio Team has invested significant efforts to understand the basic building blocks of such smart structures and, ultimately, to tune the dynamic function of engineered smart surfaces and interfaces that promote human health. Aims or Objectives: The aim of this talk is to give several examples of smart tissue interfaces [1-4], as well as multimodal, paired imaging and predictive computational modelling studies [5-9], that have enabled elucidation of such natural behaviour's underpinnings as well as the creation of new intellectual property including disruptive technologies and clinical tranalational outcomes. References: [1] Knothe Tate ML and Knothe U (1 Feb 2011) Composition and method for inducing bone growth and healing, US Patent Trade Office (PTO) 7,879,107. [2] Knothe Tate ML and Anderson EJ (24 Aug 2013) Flow directing materials and systems, US PTO 8,609,132. [3] Knothe Tate ML and Knothe U (17 Mar 2015) Multilayer surgical membrane, US PTO 8,9799,942. [4] Knothe Tate ML (published 19 Feb 2015) A Substrate, Application WO/2015/021503. [5] Knothe Tate ML et al. (2009) Biomaterials Bone as an inspiration for a novel class of biomaterials 30:133-140. [6] Knothe Tate ML et al. (2009) Pairing computational and scaled physical models to determine permeability as a measure of cellular communication in micro- and nano-scale pericellular spaces Microfluidics and Nanofluidics 4: 193-204. [7] Knothe Tate ML (2011) Smart body armor inspired by flow in bone. Smart Structures & Systems, 7(3): 223-228. [8] Knothe Tate ML et al. (2011) Surgical Membranes as Directional Delivery Devices to Generate Tissue in Critical Sized Defects, PLoS one, 6(12): e28702. [9] Knothe Tate et al. (2015) Grand challenge at the intersection of tissue engineering and next generation implants: Engineering and commercialization of tissueimplant interfaces using examples from the musculoskeletal and nervous systems, Science Translational Medicine, in press.

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The emerging role of inertial microfluidics for ultra-high throughput, label-free cell sorting Majid Warkiani University of New South Wales, Sydney, NSW, Australia [email protected] Cell sorting is critical for many applications ranging from stem cell research to cancer therapy. Isolation and fractionation of cells using microfluidic platforms have been flourishing areas of development in recent years. The need for efficient and high-throughput cell enrichment, which is an essential preparatory step in many chemical and biological assays, has led to the recent development of numerous microscale separation techniques. Sizebased passive particle filtration using inertial microfluidics have recently received great attention as a promising approach for particle focusing, filtration and fractionation due to its robustness and high rates of operation. The main advantage of inertial-based microfluidics approaches is that continues-flow separation without clogging can be realized using relatively large microchannels with relatively high resolution. In this seminar, I will describe our recent efforts in development of ultra-high throughput microfluidics systems for separation and fractionation of stem cells. Further, I will show that that high-throughput inertial microfluidics enables efficient sorting of Mesenchymal stem cells (MSCs) as a function of cell diameter, and show that this enables selection and sorting of osteoprogenitor cells from marrow for applications such as bone regeneration. Finally, I will present some of our efforts for largescale manufacturing and enrichment of MSCs inside perfusion bioreactores.

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Rapid development and translation of affordable cell therapies Simula T Cooperative Research Centre for Cell Therapy Manufacturing [email protected] The cell/tissue therapy industry is on the cusp of some very exciting times. The interest in engineered T cell therapies for the treatment of cancer is one example of an area that has captured the imagination of the venture capital and pharma/biopharma industries in the last 12-24 months. It is still a young industry and many challenges must be met, before it can truly claim to be long-term viable. Challenges include the need to close the gap between research and commercialisation, identifying suitable business models, reducing cost of goods (COGS) and logistical issues in delivery of cell therapies to the patient. The CRC for Cell Therapy Manufacturing is 18 months into its first term and from the outset the CRC’s main objectives were to tackle the issue of COGS and to create a framework for the rapid translation of CTs to the clinic. The CRC is investigating COGS by bringing together experts in biomaterials and the application of advanced surface coatings together with cell biologists working with a range of proven cell therapies, to improve the efficiency and lower costs in the isolation, expansion and delivery of therapeutic cells. Moreover, the experience within the CRC of process scale-up and preclinical testing and knowledge of the regulatory framework for cell therapies, places it in a unique position to rapidly translate cell therapies to early human clinical trials. The presentation will discuss the vision for the CRC and describe some very exciting projects being conducted in the centre.

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Thursday Session 11: Plasma Coatings

9:00-10:30

Practical bioactive interfaces for biomedicine: Recent advances towards translation to applications in biomedical implants and microarrays Marcela MM Bilek*1, Giselle Yeo1,2, Lewis Martin1, Elena Kosobrodova1, Steven Wise4, Anna Waterhouse2, Alexey Kondyurin1, Neil J Nosworthy1,3, Cristobal G. dos Remedios3, David R McKenzie1, Anthony S Weiss2 1

School of Physics, A28, University of Sydney, NSW 2006, Australia School of Molecular Biosciences, G08, University of Sydney, NSW 2006, Australia 3 School of Medical Sciences, F13, University of Sydney, NSW 2006, Australia 4 Heart Research Institute, Sydney, NSW 2042, Australia [email protected]

2

Although the scientific literature abounds with reports of wet-chemical approaches enabling the functionalisation of surfaces with functional biomolecules, few of these have been translated into applications due to complexity and cost of implementing the multistage reaction protocols involved. In this paper, we will describe an alternative approaches based on treatment with ions from plasma that overcome these problems. Radicals embedded in nanoscale carbon rich surface layers by energetic ion bombardment have been shown to covalently immobilize bioactive proteins [Proc. Nat. Acad. Sci 108(35) pp.14405-14410 (2011)] onto the surfaces of a wide range of materials, including polymers, metals, semiconductors and ceramics. This new approach delivers the strength and stability of covalent coupling without the need for chemical linker molecules and multi-step wet chemistry. Immobilization occurs in a single step directly from solution and the hydrophilic nature of the surface ensures that the bioactive 3D shapes of the biological molecules are minimally disturbed. This presentation will describe the fundamental science that underpins these new biointerfaces and report on current research that focuses on the development of applications. The emerging applications include antibody microarrays for highly multiplexed, analysis of cell surface markers and engineered bioactive surfaces for implantable biomedical devices that have the potential to overcome the major limitations of contemporary implantable biomedical devices, including rejection, severe immune responses and poor host tissue integration. Recent developments involving the use of designer molecules and peptides as well as progress in achieving multifunctional bioactivation of implant surfaces will be reviewed. Promising resolutions for practical application relevant issues such as postfabrication sterilization, regulatory approval and highly robust adhesion will also be discussed.

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Effect of oxygen plasma treatment for various a-C:H films Ohgoe, Y1, Kawachi, U1, Hirakuri, K2, Homma, A1 1

Tokyo Denki University, Saitama, Japan. 2 Tokyo Denki University, Tokyo, Japan [email protected]

Hydrogenated amorphous carbon (a-C:H) films have a variety of attractive properties in physical and chemical stabilities and promising for various applications in advanced technologies. Especially, biocompatible type of a-C:H films have been expected as a surface improvement technique for medical devices. However, characteristics of a-C:H films strongly depend on deposition methods, and there is many kinds of a-C:H film. It is not easy to select a-C:H film to achieve best performance. A mismatch between a-C:H film type and each application causes failure as a biological response. In our previous work, relationship between a-C:H film surface condition and NIH 3T3 cell proliferation was investigated and it was observed that C=O bond of a-C:H film strongly depends on cell adhesion with regardless deposition techniques. In this article, three different types of a-C:H film were deposited on each polystyrene dish (PS-dish) by radio frequency plasma chemical vapor deposition equipment. The a-C:H films deposition was carried out as the following conditions (r.f. power at 100 W, CH4 gas at 10 Pa, 50 Pa, and 100 Pa respectively). After the film deposition, the a-C:H film surfaces were treated by oxygen plasma (r.f. power at 100 W, O2 gas at 10 Pa). Before and after the O2 plasma treatment, cell adhesion of the a-C:H films were investigated. In the cell culture, NIH-3T3 cells were seeded on each a-C:H film surface for 72 hour. The cell adhesion was estimated by the Cell Titer-Blue™ Cell Viability Assay. Additionally, surface conditions of the a-C:H films were investigated by using X-ray Photoelectron Spectroscopy (XPS). As the C=O ratio is getting higher cell adhesion rate is getting greater as well. After the O 2 plasma treatment, wide range of C=O bond ratio at the a-C:H films was converged to around 20 % and cell adhesion level was equal to PS-dish. It means that the three different type aC:H films surfaces with regardless deposition methods were modified to quality of uniformity surface conditions. For variety of attractive properties of a-C:H film, the O2 plasma treatment is useful for standardization of surface modification for cell adhesion.

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Bacteriostatic plasma polymers that release nitric oxide Michl TD1, Doran M3, Osiezki M3, Bryan Coad2, Vasilev K2, Griesser HJ2 1

Ian Wark Research Institute, University of South Australia, Mawson Lakes, SA 5095, Australia 2Mawson Institute, University of South Australia, Mawson Lakes, SA 5095, Australia 3Institute of Health and Biomedical Innovation, Queensland University of Technology, 60 Musk Avenue, Kelvin Grove, QLD 4059, Australia

Implant related infections are on the increase, spurring research across numerous fields towards coatings that aim to counter this development. However, many of these coatings either lack selectivity between human cells and bacteria, are prone to induce resistance, and/or employ commercially and environmentally problematic chemicals and processes. A commercially and environmentally viable process for applying coatings onto biomedical devices is plasma polymerization, which offers unique advantages for producing thin coatings on a wide range of substrate materials on an industrial scale. Plasma polymerization and plasma methods have been used for a variety of purposes in the biomedical field.1However, so far plasma polymers have not achieved sufficient resistance to bacterial colonisation; while for example a heptylamine plasma polymer does reduce bacterial attachment to some extent, the reduction is insufficient. Good results have only been achieved when grafting antibacterial molecules onto plasma polymer surfaces.2 In contrast, we here report a novel type of plasma polymer, which stops the growth of bacteria on its surface for up to 12 hours while leaving human mesenchymal stem cells unharmed. These coatings release nitric oxide, a molecule used by bacteria for intercellular communication. This release interferes with the bacterial signalling and deactivates their cooperative behaviour; thus beating the bacteria with their own biomolecular tools. Such temporary protection may be sufficient to delay bacterial biofilm formation and ensuing infection after trauma until the body’s defense system is able to combat bacteria in a wound. Moreover, in contrast to antibacterial strategies that kill bacteria, this approach prevents the release of bacterial endotoxins that can cause adverse reactions such as anaphylactic shock. This novel class of coatings holds promise for leading to large-scale commercial products that could lower the severity of bacterial infections on medical devices and implants. Advantages of these novel coatings, besides their rapid and facile generation, are their biocompatibility with human cells and that their mode of action, usurping a bacterial signalling mechanism, is considered unlikely to lead to the development of bacterial resistance.

Figure 1: Plasma deposition of NOx releasing plasma polymer

(1) (2)

Figure 2: Bacterial testing using Staphyloccocus epidermis Siow, K. S.et al. Plasma Processes and Polymers 2006, 3, 392. Vasilev, K.; et al Plasma Processes and Polymers 2011, 8, 1010. 75

Plasma Polymerised PolyOxazoline Thin Films for Biomedical Applications MacGregor-Ramiasa, MN1; Cavallaro, AA1; Mierczynska, A2; Christo, SN3; Gleadle, JM4; Hayball, JD3 and Vasilev, K1 1

Mawson Institute, University of South Australia, Mawson Lakes, SA 5095, Australia. The Australian Wine Research Institute, Hartley Grove Cnr Paratoo Road, Urrbrae SA 5064, Australia 3 School of Pharmacy and Medical Sciences, University of South Australia, SA 5000, Australia 4 Renal Department, Flinders Medical Centre, Flinders University School of Medicine, Bedford Park, Adelaide, SA 5042, Australia [email protected] 2

Background Poly(2-oxazoline)s (POx) are emerging as new, potentially revolutionary polymers for the next generation of biomaterials, showing comparable or superior properties to well-established polymers such as Polyethylenglycol (PEG) and Polyvinylpyrrolidone (PVP). Yet, the development of POx based biomaterials is somewhat hindered by the complex wet synthesis methods used for oxazoline polymerisation and their subsequent surface grafting. Approach Herein, we demonstrate that an alternative environmentally friendly process can be used to produce thin Polyoxazoline films featuring a rich diversity of useful properties 1. Plasma polymerisation was utilized to create robust nanometer-thin Polyoxazoline coatings that can be placed on any type of solid substrates. Results Using this rapid, solvent and waste free method, chemically reactive and biocompatible oxazoline based materials were formed. In vitro studies and complementary QCM analysis revealed that these materials allow for control of protein and cell adhesion. Furthermore, the coatings considerably reduced biofilm formation from clinically-significant bacteria (i.e. Staphylococcus epidermidis). Conclusions Being a simple and scalable technique, we anticipate that the use of plasma polymerisation for Polyoxazoline coating deposition will enable development of novel biomedical applications.

1. Vasilev, K; Ramiasa, MN; Cavallaro, AA; “PLASMA POLYMERISED OXAZOLINE COATINGS AND USES THEREOF” Australian provisional patent application No. 2014903447

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Polyurethane medical implants improved by plasma immersion ion implantation Kondyurina I, Bao S, Kondyurin A, Bao R, Bilek M 1

University of Sydney, Sydney, NSW, Australia [email protected]

Polyurethane is widely used in medicine for permanently implanted devices due to its elasticity, mechanical strength, biostability, biocompatibility and hemocompatibility. However, the immune system recognises polyurethane as foreign and initiates an immune response that can result in a range of negative consequences including foreign body rejection, inflammation, bacterial infection, pain and dysfunction of the implant. Such responses, particularly bacterial colonisation as biofilms and severe pain and dysfunction, often necessitate expensive and high-risk revision surgery. In this paper, we describe the use of plasma immersion ion implantation for activation of the polyurethane surface to facilitate covalent binding of a biologically active protein layer and report on the results in application relevant assays. A polyurethane composition with mechanical properties adjusted to soft tissue was developed to match the mechanical properties of the vasculature and ensure suitability for surgical suture. Plasma immersion ion implantation in nitrogen plasma with 20 kV pulse bias and fluences in the range 1014-1016 ions/cm2 was performed to active the surfaces of films and tubes of small diameter made from this material. Mechanical properties of the structures were characterised with tensile testing whilst the chemistry and morphology of the surfaces were characterised by AFM, FTIR and XPS spectroscopy. Surface energy changes were assessed using the sessile drop method and contact angle measurement. The activated surface was then used to covalently immobilise bioactive protein molecules directly from solution. The immobilised protein layers were characterised with ELISA. The effects on cell adhesion in-vitro and on cellular responses in a mouse model of the ion treated materials both with and without protein immobilised will be reported.

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Thursday Session 12: Nanoparticles

9:00-10:30

Tailoring Nano-materials for Healthcare Applications Zhang, J, Ahmad NY, Zhang, HW, Song, H, Yu, M, Yu, CZ Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD 4072, Australia [email protected] Innovative nanomaterials that can deliver active compounds, including low molecular weight drugs, genetic molecules and therapeutic proteins, provide new opportunities in improving human /animal healthcare. Application-driven structure design of novel functional nanomaterials and fundamental understanding on structure-property relationship are key challenges in this exciting research area. In this presentation our recent progresses on functional nanomaterials for healthcare applications are presented. Queensland’s beef and lamb business is the State’s second largest industry with a direct turnover of $5 billion, however some pestivirus such as Bovine Viral Diarrhoea Virus 1 (BVDV-1) have caused significant economic losses to this critical industry. To address the challenges, we are developing nanoporous materials as selfadjuvants to deliver antigen proteins. The latest results will be communicated in the first part. In the second example, we will introduce a nanotechnology solution for pesticide delivery. Arthropod pests pose a serious threat to the Australian livestock industry, which is one of the world's largest producers and contributes ~1% to Australian’s gross domestic product (GDP). Spinosad is a naturally derived pesticide with low environmental impact and low mammalian toxicity. However, its use against ectoparasites of cattle is currently limited by its UV instability, low water solubility and higher cost. We are developing a nano-spinosad formulation to enhance photostability and the duration of insecticidal activity, through the use of a state-of-the-art nanofabrication technology. Cellular delivery of genetic molecules / protein / antibodies is at the forefront of nanomedicine with widespread applications. In the last part we will summarise several strategies we have developed for the synthesis of robust, nanoparticle based delivery platforms. Tailored functional nanoparticles with controlled structures and compositions have shown promising potential in biomolecules delivery.

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Cerium Oxide Nanoparticles: Functionalisation, Uptake and Therapeutic Effects in Human Cancer Cells Vassie, JA, Whitelock, JM, Lord, MS Graduate School of Biomedical Engineering, The University of New South Wales, Sydney, NSW, Australia [email protected]

Caveolin-1

Clathrin

ROS signal relative to untreated cells

Background: Cerium oxide nanoparticles (CNPs) are potent catalytic antioxidants that are capable of scavenging reactive oxygen species (ROS). ROS promote oxidative stress in a number of inflammatory pathologies, including cancer. ROS have also been implicated in the upregulation of angiogenic markers in cancer. Angiogenesis is one of the primary mechanisms of cancer cell proliferation and metastasis. Aims, Materials and Methods: In this study the endocytosis, intracellular localisation and therapeutic effects of rhombohedral-shaped CNPs synthesised via flame spray pyrolysis in the size range of 3–94 nm were analysed in the human cancer cell lines WiDr (colon carcinoma) and SKOV3 (ovarian adenocarcinoma) using laser scanning confocal fluorescence microscopy, flow cytometry and qPCR. The surfaces of the CNPs were functionalised with an aminosilane linker (APTES), which allowed for the attachment of the fluorophore, fluorescein isothiocyanate (FITC), and the cancer-targeting ligand, folic acid (FA). Results and Discussion: The uptake of CNPs in both cell lines was mediated by passive and active pathways, including clathrin-mediated, caveolae-mediated and macropinocytosis, and the active uptake pathways utilised by CNPs varied according to particle size, surface chemistry and cell type (Figure 1A). The attachment of FA to the surfaces of the CNPs resulted in a fourfold increase in their uptake in both cell lines. Unconjugated (bare) CNPs had a modulating effect on ROS in both cell lines which was most significant after 48 h of treatment (Figure 1B). 7 nm CNPs also appeared to modulate the expression of perlecan, basic fibroblast growth factor and vascular endothelial growth factor A on the protein level but not on the RNA level. Conclusion: A B CNPs have shown FITC-CNPs FA-FITC-CNPs potential for use as 120% an anticancer 100% therapeutic. Their effects are partly 80% governed by their 60% intracellular fate. Future studies 40% should examine 20% the effect larger0% sized CNPs have on angiogenesis. CNP diameter

Figure 1: Colocalisation of 94 nm FITC-CNPs and FA-FITC-CNPs (green) with vesicles (red) coated with clathrin or caveolin-1 in SKOV3 cells (A) after 4 h of treatment. Scale bare: 10 μm. Modulation of ROS in WiDr cells (B) after 24 (blue), 48 (green) and 72 h (red) of treatment with bare CNPs (3–94 nm) relative to cells only.

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The Modification of Nano-sized HAP Particles with Heparin and its Effect on Particle Distribution in PCL Scaffolds Jack, KS1; Goonasekera, CS1,2,3; Luong-Van, E4; Rai, B5; Bhakta, G5; Cool, SM5; Cooper-White, J3,6 and Grøndahl, L2 1

Centre for Microscopy and Microanalysis, The University of Queensland, St Lucia, QLD, Australia 2School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, QLD, Australia 3Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, QLD, Australia 4Institute of Materials Research and Engineering, A*STAR, Singapore 5Glycotherapeutics Group, Institute of Medical Biology, A*STAR, Singapore 6CSIRO, Manufacturing Flagship, Clayton, VIC, Australia [email protected] Nano-sized hydroxyapatite particles (nHAP) are of interest for use in artificial bone replacement materials due to their similarities to bone minerals in terms of size, chemical composition and crystallinity. They have been used in composite scaffolds in order to improve bioactivity as well as the mechanical properties of the scaffolds. To achieve these properties requires both a good degree of dispersion of the particles within the matrix as well as a high degree of presentation at the surface of the scaffold [1]. Additionally, nHAP can be used as a carrier of growth factors allowing fabrication of scaffolds that are both osteoconductive and osteoinductive. Recently, we have developed methodologies for chemically modifying nHAP particles with polymers and heparin and have investigated how these modifications effect the dispersion of the particles in PCL composite scaffolds as well as the release of BMP-2 from their surface [2]. Here we present our findings highlighting that the dispersion of the nHAP particles in a PCL scaffold (see Figure 1) results in enhanced bioactivity while mechanical properties are dependent on the % loading with a threshold concentration required. A mechanism is proposed to explain the effect of filler loading on mechanical properties of composite nHAP polyester materials. In addition, it is shown that the mode of heparin attachment to nHAP impacts on the subsequent release rate of BMP-2 from the nHAP.

Figure 1: An SEM/EDX image of a composite scaffold showing the distribution of HAP (green) [1] L Grøndahl, K Jack, CS Goonasekera (2014) Inorganic polymer composites for bone regeneration and repair In Bone Substitute Biomaterials Edited by Prof. Kajal Mallick, Woodhead Publishing; [2] CS Goonasekera, K Jack, J Cooper-White, L Grøndahl (2013) Attachment of poly(acrylic acid) to 3-aminopropyltriethoxysilane surface-modified hydroxyapatite. J Mater Chem B 1, 5842-5852 80

Enhanced Penetration and Drug Delivery by Crosslinked Polymeric Micelles into Multicellular Tumor Spheroids Lu, HX, Stenzel, MH Centre for Advanced Macromolecular Design, School of Chemistry, University of New South Wales, Sydney, NSW, Australia [email protected] Block copolymer micelles are water-soluble, biocompatible nanocarriers, which have frequently been used to carry, protect and deliver potential therapeutic molecules to solid tumours. Both pre-clinical and clinical studies have demonstrated that micelles prepared from block copolymers incorporating doxorubicin (DOX), paclitaxel, or cisplatin drugs can reduce the toxic side effects of the loaded drug while maintaining appreciable antitumor efficacy. Two-dimensionally (2D) cultured tumour cells have been used as the standard model for the studies of the uptaking of micelles and releasing of drugs in vitro. However, the 2D cell models are different from the real tumours, among which are the three-dimensional (3D) organization, drug and radio resistance, limited drug penetration and altered gene expression. Multicellular tumor spheroid (MCTS) is an established 3D model of solid tumours. Most MCTS exhibit higher similarity to in vivo tumour tissues than monolayer cells, and therefore have recently gained increasing recognition in biomedical research. More attempts have been made in using MCTS to investigate biological responses to polymeric micelles as nano carriers for therapeutic applications. However, the micelle penetration pathways in MCTS are yet unknown. In this study, micelles (uncrosslinked, UCM) were prepared by self-assembly of block copolymer poly(N-(2-hydroxypropyl methacrylamide-co-methacrylic acid)-block-poly(methyl methacrylate) (P(HPMA-co-MAA)-b-PMMA). Subsequently, the shells were crosslinked to form relatively stable micelles (CKM). Both UCM and CKM penetrated deeper and delivered more DOX into MCTS than the diffusion of the free DOX. Additionally, CKM revealed higher delivery efficiency than UCM. The inhibition of caveolae-mediated endocytosis, by Filipin treatment, decreased the uptake and penetration of the micelles into MCTS. Treatment with Exol, an exocytosis inhibitor, produced the same effect. Furthermore, movement of the micelles through the extracellular matrices (ECM), as modelled using collagen microspheroids, appeared to be limited to the peripheral layer of the collagen spheroids. Those results indicate that penetration of P(HPMA-co-MAA)-b-PMMA micelles depended more on transcellular transport than on diffusion through ECM between the cells. DOX-loaded CKM inhibited MCTS growth more than the UCM counterpart, due to possible cessation of endocytosis and exocytosis in the apoptotic peripheral cells, caused by faster release of DOX from UCM.

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Sensitive analysis of surface properties of nanoparticles, and biomarker detection using TRPS Phillips, A. J. Izon Science Ltd., Christchurch, New Zealand [email protected]

Liposomes have been used in medical applications for the past few decades, and more recently, extracellular vesicles are becoming recognised for their potential in therapeutic applications. Despite huge advances, challenges in the measurement and detailed characterisation of these structures are limiting progress. As well as size and concentration measurements, the analysis of the surface properties of these vesicles is important in order to analyse biological vesicles, and to precisely control the effects of synthetic vesicles for drug delivery and similar applications. We have used tunable resistive pulse sensing (TRPS) to study the surfaces of liposomes and extracellular vesicles, including in novel assays to detect surface markers. Passive targeting of liposomes for drug delivery is achieved through precise control over the surface charge, but the measurement of this surface charge is not trivial. Using a unique system beginning with negatively charged liposomes, we have developed assays using TRPS to monitor the change in surface charge upon coating with neutral molecules. This sensitive technique could be applied to the development of drug delivery liposomes, and the single particle resolution which is achieved could increase confidence in quality control. Analysis of surface charge was also used to develop a novel method of biomarker detection. Molecules on the surfaces of extracellular vesicles are becoming well known as indicators of the state of health of the organism or tissue from which they are released. Detection of these markers typically involves indirect techniques, which are inherently inaccurate and rarely quantitative. We have used single-particle measurement of surface charge to detect the presence of membrane-bound markers, through the change in charge which occurs when aptamers bind to the markers.

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Thursday Session 13: Biomolecules and Cells at Surfaces and Interfaces 9:00-10:30 Cell Responses to Nano-grooved Surfaces Tsai, WB National Taiwan University, Taipei, Taiwan [email protected] Anchorage-dependent mammalian cells are attached to the extracellular matrix (ECM), whose biochemical and physical cues dictate the fates of the cells. A substratum with a micro- or nanometre-scale 3-dimensional feature is a powerful tool for investigating cell responses to the ECM topographic structure. We fabricated a series of nano-grooved substrates containing grooves/ridges of 90 – 900 nm wide and 50 – 500 nm deep in order to investigate cell responses to nano-grooved surfaces. A general cell response to such anisotropic topography is cellular alignment and elongation along the grooves/ridges. Cells are more responsive to groove depth than groove width. Furthermore, cell nuclei also elongate and align on nano-grooves. Besides cellular morphology, cellular functions were also affected by nano-grooved topography. For example, albumin synthesis was enhanced in the hepatocytes that were cultured on the nano-grooved silicon substrates compared to those on the flat surface. We also found that gene transfection via polyethyleneimine carriers was decreased when the cells were cultured on nano-grooved surfaces. Grooved architecture is particularly suitable for investigation of tissues with cellular anisotropic arrangement, such as skeletal muscle, cardiac muscle, tendon, ligament, and nerve. We found that the differentiation of myoblasts into myotubes was enhanced by nanogrooves/ridges. Myogenesis of mesenchymal stem cells was also improved on grooved substrates. Furthermore, by combination of grooved structure with biochemical signals or elastic properties, we could investigate the contribution of different signals on cell behavior. We found that synthesis of type I collagen by anterior cruciate ligament cells was enhanced on the grooved structure that was conjugated with RGD. Similarly, RGD conjugation enhanced the myogenesis of myoblasts on nano-grooved surfaces. By culturing cardiomyocytes on grooved polystyrene or polyurethane, we found that the morphology and orientation of cardiomyocytes were affected mainly by the nanogrooved structures, while the contractile ability of the cells was regulated by a coupled effect of surface topography and stiffness. In conclusion, nano-grooved topography exerts a profound impact on cell behaviour, and would benefit the development of advanced biomaterials for biomedical applications.

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Modulation of mesenchymal and pluripotent stem cell behaviour using ordered surface topographies and chemistries using binary colloidal crystals Wang PY1,2, Thissen H2 and Kingshott P1 1. Industrial Research Institute Swinburne (IRIS) and Department of Chemistry and Biotechnology, Swinburne University of Technology, Hawthorn, 3122 VIC, Australia. 2. CSIRO Manufacturing Flagship, Bayview Avenue, Clayton, 3168 VIC, Australia [email protected] The control of cell behaviour on biomaterial surfaces is the key to a broad range of biomedical applications. Biomaterial surfaces with ordered surface topographies and chemistries can profoundly influence attached stem cells morphology, cell-cell communication, and differentiation, which is a deterministic factor in development of advanced biomaterials used in tissue engineering and regenerative medicine. Recently, we have established an elaborate surface to display ordered topographies with tuneable chemistry called self-assembled monolayer binary colloidal crystals (SAMBCCs). This approach circumvents the intrinsic issues of using traditional nanofabrication approaches for ordered nanotopography fabrication such as photolithography and electron beam lithography which can be time-consuming and costly. The ordered SAMBCCs display topographies ranging from tens of nanometers to a few microns as well as multiple chemistries, which are used for modulation of stem cell behaviour. SAMBCCs with four combinations, i.e. Si5PMMA, Si5PS, Si2PMMA, and Si2PS, plus flat Si, flat PS, and TCPS were selected for stem cell culture. The morphology, growth, and differentiation of primary human adiposederived stem cells (hADSCs), mouse embryonic stem cells (mESCs), and mouse induced pluripotent stem cells (miPSCs) were investigated on these novel substrates. The results showed that the morphological change of these cells is one of the most obvious changes on these substrates, i.e. the spreading area is smaller on the SAMBCCs than TCPS. This change resulted in the subsequent changes of the growth and phenotype/differentiation. This study demonstrate that SAMBCCs are an alternative option to present ordered surface topographies with chemical decoration abilities for stem cell culture, and the fate of stem cells can be modified using an optimised surface that potentially benefits the development of biomaterial, tissue engineering, and stem cell biology.

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Multicomponent Hydrogel formed via Self-assembly Downregulates Pro Inflammatory Cytokines and induces Selective Apoptosis in Epithelial Cancer Cells Li, R1, Bruggeman, K2, Nisbet, DR2, Williams, RJ1,3* 1 Centre for Chemistry and Biotechnology, School of Life and Environmental Sciences, Deakin University, Waurn Ponds; 2 Research School of Engineering, College of Engineering & Computer Science, The Australian National University, Canberra; 3 School of Aerospace, Mechanical and Manufacturing Engineering, RMIT University, Melbourne * [email protected] The link between the inflammatory response and the promotion of cancers is well established, particularly in the formation of endothelial cancer. Epidemiological studies have shown that chronic inflammation is a significant causative factor for these cancers; mediated by the clonal expansion of malignant cells due to the increased proliferation and reduced apoptosis. This is the stage where the inflammatory environment plays a key role as a tumour promoter through chronic and continuous up regulation of inflammatory genes. However, a significant challenge lies in achieving a spatially confined, sustained, nonsteroidal and selective suppression of the immune system. The spontaneous organisation of Self-assembled peptides (SAPs) into nanofibrillar hydrogels has been shown to produce inherently biocompatible and biodegradable materials that can be used as versatile scaffolds for the in vitro and in vivo applications [1_ENREF_1]. Here we utilise the inherent ability of biological molecules to undergo supramolecular organisation to include a powerful non-steroidal anti-inflammatory polysaccharide within the matrix itself. We chose the synergistic epitope RGD from fibronectin to produce a SAP using solid phase peptide synthesis, Fmoc-FRGDF [2]. Then, by allowing this SAP to assemble in the presence of a polyanionic polysaccharide we form a supramoleculary stabilised hydrogel. We show that the material consists of SAP fibrils decorated with the polysaccharide via a thorough investigation of the mechanism of assembly and the structures formed. We will discuss using a suite of characterisation techniques, including: 1H, 13C Nuclear magnetic resonance spectroscopy; Small angle neutron scattering; Atomic Force Microscopy; Transmission Electron Microscopy; Dynamic Scanning and Isothermal Titration Calorimetry; Fourier Transform-Infra Red, fluorescence and Circular dichroism spectroscopy; and parallel plate rheometry. To probe the utility of this material, we show that the hydrogels formed via this SAP are biocompatible for the for in vitro three-dimensional cell culture of primary human mammary fibroblast cells (hMFCs), but induce apoptosis in an oral squamous cancer cell line. Using proteomic and genomic analysis, we show that the hydrogel acts as an anti-mitotic agent through powerful and continuous down-regulation of cytokinesis promoters responsible for the G2/M transition. Investigating further, we show that the material acts via a significant downregulation of the NFκβ proinflammatory pathway, by monitoring its expression and the transcription of a range of inflammatory cytokines. References [1] Nisbet DR, Williams RJ. Self-assembled peptides: characterisation and in vivo In conclusion, weBiointerphases will demonstrate a simple and efficient way to produce a powerful antiresponse. 2012;7:1-14. cancer material with a range of applications. [2] Modepalli, V. N. et al. In vitro response to functionalized self-assembled peptide scaffolds for three-dimensional cell culture. Peptide Science 197-205 (2014) 85

Simple and effective low fouling surfaces for antimicrobial applications Ozcelik, B, Glattauer, V, Fairbanks, B, Thissen, H CSIRO, Clayton, VIC, Australia [email protected] Bacterial biofilm formation on material surfaces is a significant problem in the clinical space, especially in regard to medical implants. Biofilm presence can lead to serious complications, following both short and long term implantation of devices. Various methods utilising antibiotics, antimicrobial peptides, low-fouling coatings and other surface treatments have been utilised to battle or prevent biofilms. Individually these treatments can be effective in the short term against planktonic bacteria, but often lack the ability to resist biofilm formation. Thus it is necessary to produce multifunctional antimicrobial coatings that combine various strategies to successfully combat biofilm growth. In this study we describe the simple synthesis and preparation of polyethylene glycol (PEG) and/or polyhydroxyacrylamide (PHA) based surfaces as low-fouling base coatings for antimicrobial applications. X-Ray Photoelectron Spectroscopy (XPS) analysis of the surfaces showed successful coating with PEG/PHA. In vitro cell attachment studies using L929 cells showed minimal attachment on these surfaces after 24 hours as a result of the low-fouling property of PEG and PHA. A biofilm assay using S.epidermidis demonstrated that the PEG/PHA coated surfaces completely prevented the formation of the biofilm even after 24 hours. To prepare a multifunctional surface, the base coating also needs to allow facile immobilisation of bioactive molecules such as peptides or other antimicrobial agents. Incubation of the PEG/PHA surfaces with the model compound 2,2,2-trifluoroethylamine demonstrated effective incorporation during coating formation via XPS analysis, indicating that amine containing molecules can easily be bound onto surfaces. To further investigate this, the cyclic peptide cRGDfK was successfully incorporated into the crosslinked coating in a one-pot manner. The bioactivity of this peptide at the surface was demonstrated by promoting the attachment of L929 cells, while control experiments carried out with the non-bioactive peptide cRADfK did not lead to enhanced cell attachment. PEG/PHA surfaces can be produced via a robust and simple one-pot technique and show low-fouling and biofilm prevention properties while allowing incorporation of bioactive molecules. Hence, this platform method shows promising features for the fabrication of multifunctional antimicrobial surfaces while being suitable for a wide range of substrate materials and devices.

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Peptide Based Copolymer Coatings for the Effective Control of Cell-Material Interactions Koegler, P1,2, Wang, P.-Y.1,2, P. Pasic P1, Glattauer, V1, Gardiner, J1, Kingshott, P2, Thissen, H1 1

2

CSIRO Manufacturing Flagship, Clayton, VIC, Australia Swinburne University of Technology, Hawthorn, VIC, Australia [email protected]

Background: The effective control over cell-material interactions is essential for a broad range of biomedical applications in vitro and in vivo such as biosensors, cell culture tools and implantable medical devices. To achieve this, material surfaces or coatings have to provide control over non-specific responses originating from the adsorption of biomolecules such as proteins and simultaneously display bioactive signals that can be recognised by cell surface receptors. Here, our aim was to develop a copolymer based coating approach where one component provides low non-specific biointerfacial interactions while the other provides the bioactive signals. This approach was enabled by the synthesis of multiple acrylamideterminated peptides mediating cell attachment [1]. Materials and Methods: Amine-functionalised polymer surfaces were modified by covalent immobilisation of a macroinitiator carrying iniferter as well as carboxylic acid functional groups. Subsequently, copolymers with different molar ratios of acrylamide and acrylamide-terminated peptides containing sequences such as GRGDS or IKVAV were grafted via surface initiated free radical polymerization. X-ray photoelectron spectroscopy (XPS) was used to confirm the success of each coating step. The cellular response to these coatings was evaluated over a period of 24 hours. Results and Discussion: Surface analytical data confirmed the successful deposition of copolymer coatings. Cell attachment was low on acrylamide homopolymer coatings and copolymer control coatings representing acrylamide as well as polymerisable peptides containing non-bioactive sequences such as GRADS. In contrast, cell attachment was increased by the presence of bioactive peptides in the copolymer coatings. Cell attachment was also dependent on the molar ratio of peptide present in the coatings. Conclusion: We have demonstrated the effectiveness of a copolymer based coating approach relying on the synthesis and incorporation of radically polymerisable peptides for the effective control of cell-material interactions. This approach can easily be adapted in high-throughput screening approaches resulting in tailored coatings for specific applications. Our approach provides multiple advantages over strategies that rely on the immobilisation of bioactive signals from solution and is expected to find applications in biomedical devices. [1] P. Koegler et al, Biomacromolecules 15 (2014) 2265-2273.

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Thursday Session 14: Biomechanics & Mechanical Properties of Biomaterials 9:00-10:30 Cracking of Magnesium Alloys for Bioimplant Applications Raman Singh, RK Department of Mechanical & Aerospace Engineering and Department of Chemical Engineering, Monash University (Melbourne), VIC 3800 [email protected] Magnesium (Mg) alloys have recently attracted great attention as potential materials for temporary implant applications (such as pins, wires, screws, plates, stents). Usage of Mg alloys will completely avoid the cumbersome procedure of second surgery (which increases health care cost and inconvenience to the patients when such implants are constructed out of commonly used traditional materials such as titanium alloys, stainless steels and cobaltchromium alloys). However, there are some limitations in of Mg as a temporary implant. First, the high corrosion rates of Mg alloys in physiological environment may lead to loss in mechanical integrity of the implants. Secondly, the simultaneous action of the corrosive human-body-fluid (HBF) and the mechanical loading put the treat of stress corrosion cracking (SCC) and corrosion fatigue (CF). The latter is a vastly unexplored research area in use of Mg alloys as potential implant material. This presentation provides an overview of the experimental results on corrosion, SCC and CF of different Mg alloys in corrosive environments including the simulated body fluid (SBF), and discusses the associated fracture mechanisms as well as some mitigation measures.

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Acoustic Emission Monitoring and Post-Revision Surface Roughness Assessment of Total Hip Replacement Implants Rodgers, GW1, FitzPatrick, A1, Pedofsky, L1, White, D1, Kim, KT1, Hooper, G2, Woodfield, TBF2. 1

Dept. of Mechanical Engineering, University of Canterbury, Christchurch, New Zealand 2 Dept. of Orthopaedic Surgery, University of Otago Christchurch, New Zealand [email protected]

Background: Demographic ageing is creating an epidemic of degenerative joint disease and an increasing need to develop new diagnostic methods for detecting early wear or failure of Total Hip Replacement (THR) implants. Early diagnosis of implant deterioration can save significant time and cost by allowing orthopaedic surgeons to manage revision surgeries. Acoustic Emission (AE) monitoring of THR implants uses passive ultrasonic receivers to provide insight into wear mechanisms by relating observed vibrations to known failure modes. Aims or Objectives: This study compares in-vivo and in-vitro acoustic measurements of a range of THR implants and characterizes the squeaking of hard-on-hard bearing combinations. The presence of an audible implant squeak can cause significant embarrassment and discomfort to patients, but there the underlying cause is not fully understood. This study also seeks to determine the tribology of the THR articulation surfaces to provide insight into the underlying mechanics of implant squeaking. Materials and Methods: In-vivo AE monitoring of patients with squeaking implants is undertaken using an array of passive ultrasonic sensors to establish the frequency and location of vibrations within the soft tissue of the patient. Implants components retrieved during subsequent revision surgery are then manipulated during in-vitro testing to relate motion types to observed in-vivo signal characteristics. High magnification Scanning-Electron Microscopy (SEM) examination of the surface of the femoral head and acetabular liner was undertaken to provide insight into the wear mechanisms. Results and Discussion: Results indicate that implant squeaking is a multi-factor problem. The fundamental frequency of THR squeaking is observed to vary with the applied load and implant type. In-vitro testing indicates squeaking only occurs when there is a loss of lubrication between the bearing components, indicating that point loads or stripe wear that break the thin-film lubrication layer may contribute to implant squeaking in-vivo. Detailed examination of the femoral head bearing surface indicates evidence of inter-granular failure of the ceramic material. Conclusion: Overall, implant squeaking is a complex, multi-variable problem. This study indicates that localised loss of thin-film lubrication can contribute to implant squeaking of ceramic-on-ceramic bearing combinations and the occurrence of inter-granular failure of the ceramic bearing material.

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The effect of impact load on the mechanical properties of chondrocytes and its association with cytoskeleton components 1

Wang, Z1, Stevens, GW1, Lee, PVS2 , O’Connor, AJ1

Department of Chemical & Biomolecular Engineering, Particulate Fluids Processing Centre, University of Melbourne, Victoria, 3010, Australia 2 Department of Mechanical Engineering, University of Melbourne, Victoria, 3010, Australia [email protected]

Background: As a connective tissue that locates at the ends of bones in diarthrodial joints, articular cartilage suffers diverse mechanical loadings and friction. The mechanical properties and behaviour of articular chondrocytes play important roles in responding to mechanical loadings of the tissue. The cytoskeleton network within the chondrocytes is believed to make a major contribution to cells’ mechanical properties. Aims: The aims of this study were to investigate (1) whether the mechanical properties of chondrocytes can be altered by impact damage on articular cartilage, (2) if increased impact load can cause greater influence, and (3) its association with the cytoskeleton components. Methods: Osteochondral explants from porcine knee joints were subjected to different impact loads up to 7.5J/cm2 using a drop tower device. The impact energy density was estimated as the dropping potential energy/explant area. The chondrocytes were then isolated from the explants and the mechanical properties of the chondrocytes were tested by micropipette aspiration. The density of the cytoskeleton in the impacted and non-impacted chondrocytes was assessed by confocal fluorescence microscopy and ImageJ software by staining for F-actin. Results: Chondrocytes from both 3.75J/cm2 and 7.5J/cm2 impacted specimens exhibited a significantly higher Young’s modulus (p < 0.05) and higher apparent viscosity (p < 0.05) compared with the control specimens. No significant difference was found in these parameters between the specimens impacted at different energies (p > 0.05). Moreover, we found the F-actin fluorescence intensity was significantly different between the impacted and control specimens. Discussion: Impact injury of articular cartilage is a potential cause of post-traumatic osteoarthritis and it was shown in this study that a single impact loading can influence chondrocytes’ mechanical properties and their intracellular F-actin components. Moreover, the cytoskeleton is known to transmit forces to the nucleus, therefore the impact loadings might not only affect cell properties, but also cell functions. Conclusion: The results showed that the mechanical properties of articular chondrocytes can be altered by impact injury, but an increased impact loading did not have a measurably greater influence. The changes in mechanical properties of post-impact chondrocytes are associated with alterations in their cytoskeleton.

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Multiscale Remodelling and Topographical Optimization for Porous Implant Surface Morphological Design 1 1

Chen, J, 2Rungsiyakull, C, 1Li, W, 3Swain, MV, 1Li, Q

School of Aerospace, Mechanical and Mechatronic Engineering, The University of Sydney, NSW 2006, Australia 2 Department of Mechanical Engineering, Chiang Mai University, Muang 50200, Thailand 3 Faculty of Dentistry, The University of Sydney, NSW 2006, Australia [email protected]

Background Rapid and stable osseointegration remains a major concern in design of implantable prosthesis, which stimulates continuous interest in development of new implant materials and structures. Titanium is favoured by osseointegration for its biocompatibility; however, there are still some biomechanical drawbacks, such as stiffness mismatch to host bone and limited tissue growth onto the implant surface. Aims or Objectives The study aims to design a fully porous implant surface morphology formed by titanium bead/particle coating with both uniform and gradient patterns, based on a multiscale finite element modelling framework through time-dependent remodelling simulation to predict the micromechanical behaviours and mechanotransduction responses of bone. Materials and Methods A typical dental implantation setting is exemplified for investigation into surface topological parameters (e.g. particle size, volume fraction, and gradient) based on four popular indicators, namely density, uniformity, bone-implant-contact (BIC) ratio, and Tresca shear stress. The remodelling algorithm based on tissue strain energy density (SED) is adopted, and the microscopic models are bridged by the displacement field to the macroscopic models over the regions of interest. The response surface method is utilised to relate the morphological parameters to bone’s osseointegration measures. Results The bone remodeling responses of the microscopic models are evaluated with specific surface morphologies, in terms of different indicators in the peri-implant region. The osseointegration patterns are significantly affected by the surface topological parameters. Some specific graded surface morphologies have the potential in further improving osseointegration than the uniform ones. Discussion The topological parameters have contradictory effects on these four osseointegration indicators. Multiobjective optimisation is introduced to enhance the outcome, by providing a Pareto set of optimal solutions for different design requirements and patient-specific needs. Conclusion This study provides a novel surface design optimisation methodology for individual patients, allowing optimizing topographical parameters for a desirable patient-specific biomechanical environment to promote osseointegration.

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Comparison of Methods to Assess the Torsional Strength of the Stem-Neck Junction of Modular Hip Replacements Kark, L, Gorring, NM, Knothe Tate, ML, Simmons A University of New South Wales, Sydney, NSW, Australia [email protected] Background: Recent high profile recalls have placed modular hip replacements under increased scrutiny. Tribocorrosion at the tapered interference joints of these replacements has been associated with adverse local tissue reactions as well as systemic reactions due to increased serum metal ion concentrations [1-2]. Considerable attention has been directed toward the head-stem interface, but the stem-neck junction is infrequently examined despite postulations that it is the most likely source of these ions [3]. Our approach is to develop modelling methods to better understand the torsional stability of the stem-neck junction. Aims or Objectives: To assess the torsional strength of tapered interference joints typical of the stem-neck junction in modular hip replacements using: 1) experimental; 2) finite element; and 3) analytical methods. Materials and Methods: Experimental: Male and female Morse-type tapers were manufactured for specific use in this study. Three different material combinations were tested. Two impaction strategies were compared for each of the material combinations. Overall length of the specimen was measured both before and after impaction. An Avery-Denison torque-testing machine was used to determine loosening angle and loosening torque. A ramp rate of 10 degrees per minute was applied. All tests were conducted at room temperature and in a dry state. A sample size of five was used for each material combination and each impaction strategy. FE: CAD models of the male and female Morse-type tapers were imported into ANSYS 16, and meshed using tetrahedral elements. The joints were assembled by displacing the male relative to the female, after which a rotational displacement was applied to the male at a rate matching the experimental conditions. Eight insertion depths (from 0.1 to 0.8mm) were investigated, and loosening torque and angle extracted. Analytical: A closed-form equation derived from cylindrical interference fit mechanics was developed to relate insertion depth to loosening torque. Data Analysis: The FE and analytical methods were compared for each insertion depth using paired t-tests. The experimental results were compared to the other methods via graphical means. Results and Discussion: The loosening torques as determined using experimental methods were substantially lower than those derived from analytical and FE methods, which produced similar results to one another. References: [1] Bergmann et al. (2001) J Biomech 34: 859-71; [2] Effenberger et al. (2001) Arch Orthop Traum Su 121: 60-4; [3] Levine et al. (2013) J Bone Joint Surg Am 95(6): 512-8

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Thursday Session 15: Biointerfaces I

13:30-15:45

Historical Aspect for the Gradient Surface and the Application for Biomedical Applications. Gilson Khang Dept of BIN Fusion Tech and PolymerNano Sci & Tech, Chonbuk National University, 567 Baekje-daero, Deokjin, Jeonju, 561-756, Korea The importance of biomaterials has been recognized in biomedical research for over three decades. Their practical utilization in biomedical applications depends on the appropriate physical and biological responses collectively referred to as biocompatibility. The response of biomaterials in a biological environment is characteristically associated with their surface properties. The modification of biomaterials by various surface treatments has recently become an active topic in surface engineering. A number of research groups have focused on the preparation of surfaces with a gradually varying chemical composition along one dimension. Such a ‘‘gradient surface’’ is of particular interest for basic and applied studies of the interactions between biological species and surfaces as the dependence of a selected property, such as wettability, on composition, can be examined in a single experiment on one surface. In this lecture, we describe the preparation and characterization of gradient polymer surfaces, presenting an organized and detailed overview of the state-of-the-art on the base of our works during the last 30 years. First, the preparation of gradient surfaces and their characterization on the basis of historical aspect during last 30 years will be discussed. The introduction and characterizations of biofunctional groups on gradient surfaces will be then presented. Finally, we concentrate on the interactions of these surfaces with biological species, such as proteins and cells, important in the understanding of the basic science for biomedical application. Acknowledgement: This work was supported by NRF and MIFAFF and Global BK-21 PLUS.

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Modulation of Cationicity of Chitosan for Tuning MSC Adhesion, Proliferation and Differentiation Wu, F 1, Wu, F1 National Engineering Research Center for Biomaterials, Sichuan University, Chengdu, P.R China [email protected] Background: Chitosan and its derivatives have drawn wide attention in various areas of biomedical applications, however, its biocompatibility has remained a critical issue for its clinical success. The parameters of chitosan including molecular weight, degree of deacetylation, and the concentration of chitosan solution has been reported to influence its antibacterial efficacy, while there is still limited information about how to tune its biocompatibility. Current study demonstrates that the cationicity is the critical factor that not only modulates the antibacterial but also affects the biocompatibility. Objectives: The aim of study was to modulate the available positive charged sites of chitosan with the genipin concentration to influence MSC response in terms of cell adhesion, proliferation and differentiation. Materials and Methods: In this study, we selected water-soluble chitosan derivative (carboxymethyl chitosan) to form various crosslinking degrees of carboxymethyl chitosan (CM-chitosan) hydrogels by using different genipin content (0.5%, 1%, 2.5%, 5%, and 10%). In addition, we have evaluated the effect of the prepared chitosan hydrogels on MSC response in terms of cell adhesion, cytoskeleton organization, proliferation and differentiation. Results: The results showed that the cationicity played an important role in affecting the MSC responses. The cell proliferation increased as the genipin concentration increased. The genipin concentration directed the stem cell differentiation toward osteogenic lineages, as evidenced by the clear upward trend of osteogenic marker protein (ALP and RunX2) as the increase of genipin concentration, while it had no significant effect on chondrocytic lineages of MSC differentiation. Meanwhile, the chitosan sample with high genipin concentrations (10%) exhibited the best cell proliferation and promoted MSC differentiation into osteogenic lineages. Different from the results of the cell proliferation and differentiation, the MSCs had the best cell aggregation and adhesion-related protein expression and cell-cell interactions (Sox9, ICAM-1 and N-Cadhesion) on the chitosan-5% hydrogel. Discussion: The biocompatibility of chitosan could be tuned with the genipin concentrations, which control the available positive charged sites of chitosan. Conclusion: Overall, the results suggest that modulation of cationicity (amino content) by the crosslinking agent is an effective and simple approach to achieve excellent, which has great potential in orthopedic applications.

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Directing cell behaviour by tailoring surface property of materials Chen, H Department of Biomedical Engineering, College of Engineering, Peking University, Beijing 100871, China [email protected] The surfaces and interfaces are always a big issue when we address the biocompatibility of biomaterials. How to achieve an optimal surface structure which allows cells to invade the implant construct and guide the cells to proliferate, differentiate and develop into a specific tissue is of great importance in implants and scaffolds design for tissue repair and regeneration. In this presentation, the surface properties of typical polymeric, ceramic and metallic materials are tailored to guide cell behaviour and achieve better biocompatibility. The polymeric scaffold surfaces are functionalized by grafting peptides and growth factors to achieve better cell attachment and differentiation. Commonly there is no need to functionalize ceramic and metallic surface with peptides to enhance the biocompatibility, however the physical parameters of ceramic and metallic materials, such as 3D or 2D surface morphology, microstructure or nanostructure, hierarchical surface roughness and crystal phase can effectively affect cell behaviour. These results indicate that cell response is sensitive to the surface properties of materials and scaffold structural cues alone can be used to drive cell differentiation and achieve better biocompatibility. This investigation is supported by the National Basic Research Program of China (2012CB933903).

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SYNERGISTIC EFFECTS OF CANONICAL Wnt AND BMP SIGNALLING ON THE DIFFERENTIATION OF hMSCs TO OSTEOBLASTS Padmanabhan, H1, Frith, JE1, Titmarsh, DM1, Mehlman, MD1, CooperWhite, JJ123 1. Tissue Engineering and Microfluidics Laboratory, Australian Institute of Bioengineering and Nanotechnology, The University of Queensland, Australia; 2. The School of Chemical Engineering, The University of Queensland, Australia; 3. CSIRO, Manufacturing Flagship, Clayton, Victoria, Australia [email protected] Background: Mesenchymal stem cells (MSCs) are a rare population of non-hematopoietic stem cells present in the bone marrow, which are capable of differentiating into, as well as contributing to the regeneration of mesenchymal tissues such as bone, cartilage, muscle and adipose tissue. However, the pathways that are involved in the differentiation of MSCs into the respective lineages are still poorly understood. Objectives: 1.) To investigate the synergistic effects of Wnt and BMP signalling on osteogenesis; 2.) To study the impact of paracrine signalling during osteogenesis and identify the factors secreted during osteogenesis. Results & Discussion: To explore the impact of Wnt signalling on the osteogenic differentiation of MSCs, we firstly utilised a full factorial microbioreactor array (MBA) platform to rapidly and combinatorially screen several Wnt modulatory compounds (CHIR99021, IWP-4 and IWR-1) and characterise their effects on osteogenic differentiation potential. The results obtained from this experiment were contrary to our expectations as CHIR99021 (a Wnt agonist) had a profoundly inhibitory effect on osteogenesis, whereas previous reports from in vivo as well as in vitro studies have indicated that Wnt signalling is necessary for osteogenesis. Intriguingly, the Wnt antagonists (IWP-4 and IWR-1) inhibited osteogenesis, which was in-line with previously published reports. From gene expression analysis, it was shown that CHIR did indeed activate the canonical Wnt signalling pathway and upregulate other osteogenic markers (such as RUNX2), but it appeared to down-regulate alkaline phosphatase and osteopontin levels. These results correlate with the previous reports that suggest that, in the presence of dexamethasone, Wnt agonisation (using Wnt proteins) can result in a decrease in alkaline phosphatase levels and mineralisation. In an effort to remove dexamethasone, a synthetic glucocorticoid, and develop an in vitro assay that correlates with osteogenesis in vivo, we thereafter sort to probe further Wnt and BMP signalling, without Dexamethasone being present. Our preliminary experiments have shown that activating the Wnt signalling alone is not sufficient to induce osteogenesis, but when Wnt and BMP pathways are simultaneously activated (in the absence of dexamethasone), we can improve the differentiation outcome of hMSCs in vitro by at least 6fold, when compared to standard osteogenic conditions. Conclusion: The MBA platform has provided new insights into the relative importance of known pathways regulating osteogenesis, as well as permitting the study of paracrine signalling impacts during osteogenesis in hMSCs.

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Metal oxide material libraries as cell growth screening devices Dykas, MM1,2, Poddar, K1, Kutty, VK1,3, Desai, SK4, Patra, A1,2, Motapothula, MR1, Rana, A1, Bigliardi, M3, Bigliardi, PL3, Venkatesan, T1 1

NUSNNI – Nanocore, National University of Singapore. 2Graduate School for Integrative Sciences and Engineering, National University of Singapore. 3Institute of Medical Biology, AStar, Biopolis, Singapore. 4Mechanobiology Institute, National University of Singapore, Singapore, Singapore [email protected] Background For many years metal oxides as deposited by the pulsed laser deposition (PLD) technique have been under the lens for their interesting electrical and magnetic properties. In the recent years these metal oxides have spawned a great amount of interest due to their queer biological responses. While this field of research is still very much unexplored, it has been proven that not all metal oxides behave similarly in terms of their interaction with living cells. For example, ZnO is a known anti-bacterial agent while TiO2 is excellent in terms of biocompatibility. Hence it was thought to be the logical next step to set about comparing the biological response of a host of oxides. Objectives The objective of the study is to develop efficient screening device for biological growth on metal oxide surfaces. Moreover, determination of the cell adhesion mechanism to the atomically flat surface was the big question. Materials and Methods The chemistry of the surface is varied by depositing films of varying metal oxides by the Continuous Composition Spread by Pulsed Laser Deposition technique. Surface roughness is studied by AFM. Particle Induced X-Ray Emission measurement was used to chemically characterise the substrate, and contact angle measurement for surface wettability. Results and Discussion The material library on 4 inch wafer consisting of few metal oxides, where each spot on the wafer has different chemical composition affects the cells behaviour. Depending on the location on the wafer, the cell respond in different ways. Some of the cells migrate to more favourable locations, some adhere (but weekly) and some adhere with great force. The adhesion, proliferation and differentiation (in case of stem cells) of cells depends strongly on the substrate chemistry, it can be accelerated or retarded. Additionally, the use of growth supporting protein coatings, such as laminin or fibronectin can be restricted or full eliminated. The cell adhesion to the surface strongly depends on the ECM adhesion proteins and theirs adhesion to the surface, and can be easily controlled. Conclusion It has been demonstrated that not only by changing the topology of substrate, mainly roughness and patterning, but by tuning the surface chemistry the cell behaviour can be easily controlled.

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High Throughput Fabrication of Colloidal Assemblies to Study Biointerfacial Interactions Koegler, P1,2,3, Dunn, M2,3, Wang, PY1,2,3, Ozcelik B1, Kingshott, P2,3, Thissen, H1 1

CSIRO Manufacturing Flagship, Clayton VIC 3168 Industrial Research Institute Swinburne, Swinburne University of Technology, Hawthorn VIC 3122 3 Faculty of Science, Engineering and Technology, Swinburne University of Technology, Hawthorn VIC 3122 [email protected]

2

Controlling biointerfacial interactions is of great importance to a number of in vivo and in vitro biomedical applications such as implantable devices, biosensors, and tissue engineering scaffolds. Prevention of non-specific protein adsorption and the associated effective reduction in cell attachment whilst presenting specific biological cues such as peptides represents a promising route to gain superior control over cellular responses. The density with which these biological signals are displayed as well as the spacing between them has been proven to greatly influence the cellular response. In this context colloidal self assembly presents a promising and cost efficient way to generate a range of single and binary arrays with varying topography and chemistry with high spatial resolution. The combination of this assembly process with sophisticated surface modification strategies such as grafting-to or grafting-from can be used to study and control cell-surface interactions. In the present study we developed a high throughput method for the generation of colloidal assemblies by using a microarray printer. This allows for the rapid fabrication of a vast number of different assemblies with different topographies and chemistries. In addition to the high throughput fabrication of colloidal assemblies a mathematical approach was developed to characterise the order within the binary assemblies. Scanning electron microscopy (SEM) was used to confirm the successful deposition of colloidal assemblies on the substrate material. Assemblies were prepared on acrylamide-coated polystyrene slides to minimise protein adsorption and therefore cell attachment to the background. Initial cell culture experiments were conducted using L929 mouse fibroblasts in serum containing conditions for up to 24h. We believe this novel high-throughput approach will find broad application in the field of biomaterials and regenerative medicine to study the influence of different factors on the cellular response and to design devices with improved in vivo and in vitro performance.

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Biomaterials Immuno-Osteo-Modulation in the Development of Bone Substitute and Surface Coating Xiao, Y1, Chen, ZT1, Wu, CT1,2 1

Institute of Health and Biomedical Innovation, Queensland University of Technology, QLD, Australia 2 Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai, China [email protected]

As direct effector cells for osteogenesis, osteoblastic lineage cells are commonly used for evaluating the in vitro osteogenic capacity of bone biomaterials. This strategy achieves certain success in developing novel bone biomaterials. However, inconsistent results between in vitro and in vivo studies are not uncommon. Many potential bone biomaterials developed from the traditional strategy are found to behave undesirably in vivo in term of bone regeneration, indicating the mechanisms that govern the material’s capacity to mediate osteogenesis is still not well understood. Immuno-Osteo-Modulation (IOM) has revealed the important role of immune cells in the regulation of bone formation and remodelling. This has informed a paradigm shift in developing bone biomaterials from an inert to an IOM material, highlighting the importance of immune cells in the material-mediated osteogenesis. Neglecting the importance of the immune response is a major shortcoming in materials assessment, which may be the cause of the inconsistence between in vitro and in vivo conditions. Here we assessed the osteogenic property of β-tricalcium phosphate (β-TCP) in the presence of macrophages. The macrophage phenotype switched to M2 extreme in response to β-TCP extracts, which was related to the activation of calcium-sensing receptor (CaSR) pathway. Bone morphogenetic protein 2 (BMP2) was also significantly upregulated by the β-TCP stimulation, indicating that macrophage may participate in the β-TCP stimulated osteogenesis. We further coated Mg Scaffolds using β-TCP and revealed that this coating could change macrophage phenotype via the inhibition of the toll like receptor (TLR), leading to the macropahe-mediated osteogenic differentiation of bone marrow stromal cells. We also demonstrated that bioactive elements (Sr and Si) coating on biomaterials changed the immune reaction leading to reduced inflammation and enhanced osteogenesis. Our study demonstrated that biomaterials and surface coating can regulate immuneOsteogenesis. Therefore, a novel systematic evaluation for the development of bone biomaterials has been proposed for the importance of IOM.

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Preparation and Properties of Fluorinated Amorphous Carbon Films on Medical NiTi Alloys Zhang, DT 1, Li, Z1, Du, L1, Zhao, H2, Tong, YX1, Zheng, YF1,3, Li, L1 1

Harbin Engineering University, Harbin, HL, China Heilongjiang Provincial Hospital, Harbin, HL, China 3 Peking University, Beijing, BJ, China [email protected]

2

Fluorinated amorphous carbon films (a-C:F) have been proposed as extremely potential biomedical coatings due to their moderate biocompatibility and good properties. In this paper, the a-C:F films were deposited by radio-frequency plasma magnetron sputtering on NiTi alloys surface. Sputtered films composition, structure, mechanical properties and corrosion resistance properties are characterized by X-ray diffraction (XRD), Raman scattering spectroscopy (Raman), nano-indentation tests and electrochemical measurement. The results revealed that the a-C:F film was amorphous structure by magnetron sputtering; The films prepared at RF power 75W possess better mechanical properties. The corrosion resistance properties of a-C:F films reached a stable level with increasing immersion time and a-C:F film prevented the release of Ni ions effectively.

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Session 16: Antimicrobial Coatings I 13:30-15:45 Uropathogenic Escherichia coli biofilms Lo, AW1, Paxman, JJ2, Ong, CLY1, Heras, B2, Schembri, MA1 1

School of Chemistry & Molecular Biosciences, University of Queensland, Brisbane, Queensland, Australia 2 Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia. [email protected] Background: Uropathogenic Escherichia coli (UPEC) are a major cause of catheterassociated urinary tract infection (CAUTI), one of the most common nosocomial infections of humans. UPEC utilize a wide array of virulence factors including fimbrial and non-fimbrial adhesins to form biofilms on catheter surfaces. Aim: The aim of this study was to examine the function of different UPEC cell surfacelocated adhesins in the formation of biofilms. Materials and Methods: Biofilms were examined using static- and dynamic-based in vitro assays. UPEC were tagged using fluorescent reporter genes (e.g. the gene encoding green fluorescent protein) and biofilms were monitored over time by scanning confocal laser microscopy. Results and Discussion: UPEC fimbriae are long surface-located polymeric organelles that extend from the cell and mediate specific attachment to surfaces. Cloning and expression of the genes encoding several types of UPEC fimbriae (e.g. type 1 and type 3 fimbriae) has revealed they contribute to biofilm formation. In the case of type 3 fimbriae, the genes encoding these organelles in UPEC are located on a conjugative plasmid. We demonstrated that the expression of type 3 fimbriae by UPEC mediates significant biofilm growth, and that the transfer of the plasmid encoding type 3 fimbrial genes to recipient non-biofilm forming strains via conjugation represents a molecular mechanism to drive the formation of mixed biofilms. We have also investigated the contribution of multiple non-fimbrial UPEC adhesins for their role in biofilm formation. In particular, we studied the autotransporter group of proteins, which in contrast to fimbriae are expressed on the UPEC cell surface as single protein adhesins. Some UPEC autotransporters, such as the antigen 43 (Ag43) protein, mediate strong biofilm growth when over-expressed by UPEC. We have elucidated the structure of Ag43 and characterised the interface of the protein that mediates intercellular interactions within mixed UPEC aggregates and biofilms. Current work is aimed at the identification of molecules that block these interactions and thus inhibit biofilm growth. Conclusion: UPEC biofilm formation on abiotic surfaces is strongly associated with the expression of specific cell-surface-located adhesins. Inhibition of these interactions may lead to the development of new approaches to prevent biofilm growth.

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Chemistry, nano-structures, and SLIPS surfaces: no durable and non-toxic anti-fouling strategy in sight M.Grunze Institute for Functional Interfaces (IFG), Karlsruhe Institute of Technology, Karlsruhe, Germany and Applied Physical Chemistry, University of Heidelberg, Germany [email protected] The settlement and colonization of marine organisms on submerged man-made surfaces is a major economic and environmental problem for many industries. The most apparent detrimental effects of marine biofouling are increased fuel consumption of ships, clogging of membranes and heat exchangers, disabled underwater sensors, and growth of toxic biofoulers in aquaculture systems. The presently common—but environmentally extremely problematic—way to deal with marine biofouling is to incorporate biocides to kill the colonizing organisms. This talk summarizes our research to understand the mechanisms of biofouling and to find environmentally benign non-fouling strategies for marine applications. The aqueous biofouling environment consists of multiple and often cooperatively interacting species of various sizes. Significant differences in the initial settlement behavior of marine bacteria, spores, barnacle cyprids and diatoms are observed on different chemical surface compositions, but the continuous deposition of dissolved macromolecules and polymers leads to a “conditioning film” which soon renders any chemical modification of the surface useless. Topographic structures on surfaces change the macroscopic properties such as their wetting behavior, but also have a pronounced effect on how single cells and organisms attach, settle, and proliferate on the substrate. Both the enhancement of settlement, such as in cell cultures, but also the suppression of settlement can be the outcome of surface structuring. However, although correlations between organism size and topography can be established for individual species, the underlying mechanism by which topography effects attachment remains anything but clear. Finally, we investigated slippery liquid-infused porous surfaces (SLIPSs) for their antifouling properties in a marine environment. Micro-porous butyl methacrylate− ethylene dimethacrylate (BMA−EDMA) surfaces infused with perfluorinated liquids exhibited remarkable initial inhibition of settlement, comparable to that of a poly(ethylene glycol) (PEG) terminated surfaces. However, the long term stability against marine fouling in the field experiments was poor, due to unavoidable defects in the SLIPS surface, and the slow deposition of a conditioning film. In summary, all approaches tested so far, including super hydrophobic surfaces and the SLIPS surfaces, failed to resist biofouling when exposed to the environment over days and weeks rather than hours.

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Surface coatings with covalently attached echinocandins are highly effective in eliminating human fungal pathogens Coad, BR1, Griesser, SS1, Jasieniak, M1, Peleg, A2,3,4, Griesser, HJ1 1

Mawson Institute, Mawson Lakes, SA, Australia Department of Microbiology, Monash University, Clayton, VIC, Australia 3 Division of Infectious Diseases, Beth Israel Deaconess Medical Center, Boston, MA, United States 4 Department of Infectious Diseases, The Alfred Hospital, Melbourne, VIC, Australia [email protected] 2

Human fungal infections have been called the hidden killers – missed during diagnosis and poorly understood. On the surfaces of implanted biomaterials, pathogens such as Candida albicans are nearly always identified revealing their surprising ubiquity and ability to form or participate communally in biofilms. Compared to bacterial infections, invasive fungal infections are underdiagnosed, and consequently treatment with large doses of systemic antifungal agents are used only after they have established a mature biofilm and spread invasively. This intervention is recognized as being almost completely ineffective for eliminating biofilms. Especially for immunocompromised and paediatric patients, treatment often comes too late resulting in unacceptably low survival. New and approved antifungal drugs are our only hope for combating fungal infections. We believe that delivery of antifungal drugs from surface coatings could be an effective strategy for combating fungal biofilms. Fungi are susceptible to attack by the drug class of echinocandins by interfering with the integrity of the fungal cell wall – a thick protective matrix of glucans not present in mammalian cells. Our hypothesis is that surfaces coated with covalently attached echinocandins may be one way to eliminate the first fungal pathogens that attempt to settle on surfaces either pre- or post-implantation before they can take hold and form biofilms Our results show that echinocandins attached to surfaces are effective at eliminating 98 % of C. albicans colonies. Our methods for immobilisation, and washing confirm by surface analysis that we can rule out physisorption (reversible immobilisation through weak, nonspecific forces) as a possible explanation for surface mediated killing. This establishes a convincing basis for proving that the mechanism of killing must be due to only molecules presented from surface coatings with covalent bonds. Importantly, this provides new insight into the mechanism of action of echinocandins and a promising lead in combating fungal biofilms.

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INHIBITION OF IN VIVO MICROBIAL COLONISATION OF PEPTIDECOATED BIOMATERIALS Chen, R1, Willcox, MDP2, Ho, KKK1, Smyth, D3, Dutta, D2, Cole, N4, Kumar N1 1

School of Chemistry, University of New South Wales, Sydney NSW, Australia School of Optometry and Vision Science, University of New South Wales, Sydney NSW, Australia 3 Cochlear Ltd, Sydney, NSW, Australia 4 School of Chemistry and Forensic Science, University of Technology, Sydney, NSW, Australia [email protected]

2

Background The use of biomaterial implants and medical devices is an increasingly common and often life-saving procedure. However bacterial infections on biomaterials have emerged as a major problem. Implanted devices account for approximately 45% of all hospital-acquired infections and consequently represent a public health issue of major concern. We have developed a cationic peptide “melimine”, with excellent broad-spectrum antimicrobial activity, is not cytotoxic at active concentrations and is readily sterilisable. Furthermore, melimine is unusual compared to other AMPs in its ability to retain its activity when covalently attached to surfaces, making it ideal for development as potent antimicrobial coatings and therapies. Aims In this study we explored the ability of melimine to prevent in vivo bacterial adhesion and colonisation in rodent subcutaneous models when covalently tethered on titanium. Materials and Methods The in vitro antimicrobial efficacy of the melimine-modified surfaces against model pathogens (P. aeruginosa and S. aureus) was first determined by fluorescence confocal microscopy using live/dead staining. For the mice model of biomaterial infection, the disks were implanted subcutaneously in the flank of mice and 105 to 107 colony forming units of Staphylococcus aureus were injected into the pocket. Clinical responses including wound area, swelling, and redness were recorded each day for 5 days. On the 5th day, the total viable bacteria on the disks ex vivo were enumerated. Results Up to 6-fold reductions in in vitro bacterial adhesion were observed. In vivo efficacy of melimine-modified surfaces was demonstrated by up to 1 log reduction in viable bacteria compared with the controls. Discussion The starting concentration of melimine correlated positively with reduction in bacterial numbers, with the highest reduction seen in animals with the highest concentration of inoculum at 107. Further evaluations with animal models using Pseudomonas are being examined and melimine-coated prototype devices will be tested at longer endpoints at 7 and 21 days. Conclusion Melimine-coated biomaterials retain their activity in vivo and could be used to prevent device related infections.

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Self-polymerizing DOPA as a functional layer for the development of antimicrobial coating on PTFE barrier membranes for GTR procedures Nardo, T1,2, Chiono, V1, Fracchia, L3, Ceresa, C3, Tabrizian, M2, Ciardelli, G1 1

Department of Mechanical and Aerospace Engineering, Politecnico di Torino, Torino, Italy 2 Department of Biomedical Engineering, McGill University, Montreal (QC), Canada 3 Department of Pharmaceutical Sciences, Università degli Studi del Piemonte Orientale "A. Avogadro", Novara, Italy [email protected] Background Periodontal diseases are multifactorial and caused mainly by pathogenic bacterial species located in the subgingival niche (Listgarten, 1986). Regenerative approaches to treat periodontitis lesions offer exciting possibilities; however, they frequently fail to produce the desired clinical outcome due to infectious complications (Nowzari, 1995). In this context, long-term evaluation studies indicated that guided tissue regeneration (GTR) procedures, with currently available membranes, exhibit large variability in the surgical outcome due in part to bacterial infection following placement of the barrier membrane (Karring, 1993). Aims of the work In this study, a mussel-inspired approach was applied to develop an antimicrobial coating on polytetrafluoroethylene (PTFE) barrier membrane. The technique involves the application of a thin film coating of polymerised 3,4-dihydroxy-DL-phenylalanine (DOPA) on PTFE substrate, followed by a metallization process in silver nitrate (AgNO3). Materials and Methods PTFE films were grafted with poly(DOPA) at room temperature for 48 h (PTFE-DOPA). Then they were coated with Ag nanoparticles (AgNPs) by incubation in aqueous AgNO3 solution for 24 h (PTFE-DOPA-Ag). The films were characterized in terms of chemical composition (XPS analysis), morphology (SEM analysis) and topography (AFM analysis), and stability behavior of the coating. The antimicrobial efficacy and the toxicity of the coating were also evaluated. All quantitative data were presented as mean ± SD. Results and Discussion XPS analysis evidenced nitrogen and Ag presence after polyDOPA grafting and AgNPs deposition on PTFE-DOPA films. Both morphological and topographical analyses demonstrated the successful formation and deposition of AgNPs on PTFE-DOPA substrates, with uniform and mono-disperse spherical shape of about 68 nm size (Fig. 1a). The stability tests showed a complete release of AgNPs within 14 days. PTFE-DOPA-Ag films were biocompatible towards NIH 3T3 mouse fibroblasts after 1-3d culture time. SEM images showed that cells spread on functionalized films (Fig. 1b). (a)

(b)

Fig. (b) 1 SEM micrographs of (a) PTFE-DOPA-Ag films; (b) NIH 3T3 cells cultured for 3d on PTFE-DOPA-Ag films.

Conclusion.Physicochemical characterisations showed changes in surface chemical composition attributed to the grafting/polymerisation of a poly(DOPA) layer on the PTFE film surface. Moreover, the poly(DOPA) layer was effective in promoting the formation of an Ag NP-based coating (metallisation).

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Prevention of Bacterial Infections by Surface Immobilized Dihydropyrrolone Compounds Ho, KKK1, Chen, R1, Cole, N3, Willcox, MDP2, Kumar, N1 1

School of Chemistry; 2School of Optometry and Vision Science, University of New South Wales, Sydney, Australia 3School of Chemistry and Forensic Science, University of Technology, Sydney, Australia [email protected]

Background: Bacterial infection associated with the use of biomedical devices is one of the major complications in hospitals. At present, no safe, effective biomaterial coatings are available to reduce device-related infections. The effective treatment often requires the removal of implant itself, which results in high morbidity and mortality and large public health costs. Therefore, there is a clear need to develop biomaterials which reduce the risk of such infections. Halogenated furanones isolated from the red marine alga Delisea pulchra have been shown to interfere with the bacterial communication systems. Synthetic furanone analogues based on the dihydropyrrol-2-one (DHP) nucleus exhibit low cytotoxicity towards mammalian cells while retaining antimicrobial efficacy, making them potential therapies for preventing and treating infections on devices. Methods: In this study, DHPs were immobilized onto surfaces via click chemistry (coppercatalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC)). The resulting substrates were evaluated for their in vitro activity against two common pathogens Pseudomonas aeruginosa and Staphylococcus aureus.

Results: The results demonstrated that DHP attachment via click chemistry yield twice the surface concentration of DHP compared to surfaces developed in earlier studies. These DHP coatings exhibit a significant improvement in reducing the bacterial adhesion and biofilm formation of the target organisms P. aeruginosa and S. aureus, with reductions up to 97.3 and 96.8% for P. aeruginosa and S. aureus respectively. Conclusion: DHPs show great potential for use as novel antimicrobial coatings to combat device-related bacterial infections.

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Immobilisation and Characterisation of Antimicrobial Peptides onto Biomaterial Surfaces Dutta, D1; Kumar, N2; Willcox, MD1 1

School of Optometry and Vision Science, University of New South Wales, Sydney, Australia 2 School of Chemistry, University of New South Wales, Sydney, Australia [email protected]

Background: The development of infection associated with biomaterials results in high patient morbidity and mortality as well as high medical cost to the community. Antimicrobial peptides (AMPs) have broad-spectrum activity and are non-cytotoxic at concentrations required for antimicrobial activity. Biomaterial coatings with immobilised AMPs could be an attractive measure to reduce the incidence of device-related infection. Aims and Objectives: Immobilise AMPs on biomaterial surfaces by covalent attachment, characterise the surface coating and evaluate their antimicrobial activity. Materials and Methods: Four different AMPs, melimine, LL-37, bovine lactoferricin (LFc) and Mel4, were covalently immobilised onto hydroxyethyl methacrylate (HEMA) surfaces via EDC coupling using concentrations ranging from 0.026 µmol/ml to 1.3 µmol/ml. The surface attachment of AMPs was characterised by X-ray photo electron spectroscopy (XPS). Antimicrobial activity against the Staphylococcus aureus 31 and Pseudomonas aeruginosa 6294 was determined by measuring the reduction of viable bacterial adhesion to control surfaces with no AMPs. Results: XPS revealed increased amide nitrogen percentage indicating the presence of AMPs on the surfaces immobilised with LL-37 (3.5 %N) and LFc (2.0 %N) compared with Mel4 (1.5 %N) and melimine (1.3 %N). However, LFc coated surfaces did not show any antimicrobial activity at the tested concentrations, whereas LL-37 coated surfaces showed >2 log inhibition at the starting concentration ≥ 0.792 µmol/ml against P. aeruginosa, but no activity against S. aureus. At 1.3 µmol/ml, Mel-4 showed 1 log and >2 log inhibition against P. aeruginosa and S. aureus respectively. Similarly, > 3 log inhibition was observed for melimine immobilised contact lenses against S. aureus and P. aeruginosa at 0.792 µmol/ml and 1.3 µmol/ml. Discussion: Melimine and Mel4 immobilised biomaterial surfaces showed high levels of inhibition against the Gram positive and Gram negative bacteria. The amount of AMPs on the biomaterial surface did not correlate with the observed antimicrobial activity, which possibly indicates that the secondary and tertiary structure of the AMPs following surface immobilisation is crucial to deliver the bactericidal effect. Conclusion: Immobilisation of certain AMPs is an effective technique in producing antimicrobial biomaterial surfaces.

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Thursday Session 17: Tissue Engineering

13:30-15:45

Using glycotherapeutic devices to regenerate body tissues Cool, SM1,2, JH Hui2, M Raghunath3, K Bhakoo2,5, V Nurcombe1,4 1

Glycotherapeutics Group, Institute of Medical Biology, Agency for Science, Technology and Research (A*STAR), Singapore 2 Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 3 Department of Biomedical Engineering, National University of Singapore, Singapore 4

Lee Kong Chian School of Medicine, Nanyang Technological University-Imperial College London, Singapore
 5 Translational Molecular Imaging Group, Singapore Bioimaging Consortium, A*STAR, Singapore [email protected]

Serious adverse events associated with tissue repair strategies incorporating growth factors are denying patients of suitable alternatives to standard-of-care tissue grafts. Glycosaminoglycan sugars and heparan sulfate (HS) in particular, are an emerging therapeutic option because on their ability to mediate the bioactivity of pro-healing factors present at injury sites. In developing a library of unique HS variants that target such tissue growth factors, our lab has amassed significant strengths in promoting healing cascades without the need for exogenous applications of such growth factors. We have exemplified the efficacy of these HS variants in a range of cell therapy, orthopaedic, dermatological and vascular injury models. We show that HS engineered with high affinity for VEGF165 increases endothelial cell proliferation and tube formation in a VEGF165-dependent manner, and enhances angiogenesis in embryonic chorioallantoic membrane assays without the need for exogenous VEGF165. Notably, increased blood vessel formation is also observed in rodent models of vessel ischemia. Generating HS variants with increased affinity for particular growth factors is also proving successful for the treatment of bone trauma when combined with a range of FDA-approved bone void fillers in small and large animals. The success of this program highlights the considerable potential of HS strategies that serve as devices that enhance the body’s own healing cascades.

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Molecular positioning of nature’s elastic assembly modules to build complex multi-dimensional vascular and microvascular structures Weiss, A.S. 1,2,3,4 1

School of Molecular Bioscience, University of Sydney, NSW, Australia. 2 Bosch Institute, University of Sydney, NSW, Australia. 3 Charles Perkins Centre, University of Sydney, NSW, Australia. 4 Royal Prince Alfred Hospital [email protected] Background Mammalian tissue comprises an ensemble of molecular and cellular components that reflect its blended physical and biological makeup 1. This interplay means that the component construction biopolymers need to not only deliver durable tensile and compression performance, but they also need to participate in crosstalk with other molecular components and with cells 2. That harmony encompasses the flow of blood that courses through vasculature to exchange gases and nutrients, the nurtured functionality of resident and visiting cells, and local physical integrity. Elastin is a natural choice to make a reliable, tissue engineered, three-dimensional vasculature, given its essential role in the arterial wall and cell interactions 3. Elastin is predominantly made of the one protein, tropoelastin 4. Aims We utilised elastin’s biological rules of assembly to fabricate detailed, 2D and 3D elastic structures. Results and Discussion We made massive molecular protein assemblies and found that these are organised facilitators of endothelial progenitor and subsequent endothelial attachment, spreading and proliferation. We discovered that tropoelastin is a unique molecule that achieves the desired physiological response of increasing endothelialisation while conferring low thrombogenicity, so is exquisitely suited as a coating for flexible polymers and construction material for vascular conduits. We adapted the structural asymmetry and integrin binding polarity of the elastin monomer, tropoelastin, to decorate polymers for dermal and vascular applications to confer beneficial cell interactive and low thrombogenic properties. We covalently transferred these benefits to a panel of polymers. We combined tropoelastin with both synthetic and natural polymers to expand the uses of this versatile biomolecule. We found that these treatments enhance human dermal fibroblast and human endothelial cell attachment, cytoskeletal assembly and viability, combined with elevated cell junction PECAM-1 staining. Conclusion This process is yielding sophisticated elastic biomaterials that are tailored to match the elastin-rich tissues they are intended replace. Acknowledgments Funding from the ARC DP120103911, ARC DP130103693, ARC LP110200316, CDIP 12213, NHMRC APP1033079, NHMRC APP1039072, NIH R01 EB014283 and Wellcome Trust. References 1 Pampaloni, F., Reynaud, E. G. & Stelzer, E. H. The third dimension bridges the gap between cell culture and live tissue. Nat Rev Mol Cell Biol 8, 839-845 (2007). 2 Shamir, E. R. & Ewald, A. J. Three-dimensional organotypic culture: experimental models of mammalian biology and disease. Nat Rev Mol Cell Biol 15, 647-664 (2014). 3 Li, D. Y. et al. Elastin is an essential determinant of arterial morphogenesis. Nature 393, 276-280 (1998). 4 Holst, J. et al. Substrate elasticity provides mechanical signals for the expansion of hemopoietic stem and progenitor cells. Nat Biotechnol 28, 1123-1128 (2010).

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Tissue engineering a humanized bone organ as a platform for primary bone tumour research Wagner, F1,2, Holzapfel, BM1,3, Thibaudeau, L1, Levesque, JP4, Loessner, D1, Hutmacher, DW 1 1

Regenerative Medicine, Institute of Health and Biomedical Innovation, Queensland University of Technology, QLD, Brisbane, Australia. 2Department of Orthopaedic Surgery, University of Regensburg, Germany. 3Department of Orthopaedic Surgery, University of Wuerzburg, Germany. 4Stem Cell Biology Group – Blood and Bone Diseases Program, Mater Research Institute, Translational Research Institute, Brisbane, Australia [email protected] Background: The poor results of recent clinical investigations on survival of osteosarcoma (OS) patients urge for new preclinical models to minimize the risk of misleading experimental results due to interspecies-specific cross-talk. Objectives: We attempted to tissue engineer a humanized bone organ utilizing human pelvic bone which was implanted subcutaneously along with recombinant human bone morphogenic protein (rhBMP) and fibrin glue into NOD-scid mice as a platform for primary bone tumour research. Materials and Methods: 36 humanized bone constructs were allowed to form in vivo. 12 ossicles were harvested and analysed for bone properties and the presence of human bone marrow stem cells via µCT, immunohistochemistry and flow cytometry. 24 ossicles were injected with human osteosarcoma cells. In vivo tumour growth was monitored via bioluminescence imaging for 5 weeks. Results were compared to OS inoculated at the mouse tibia and to a humanized OS group which received the chemotherapeutic agent doxorubicin. Results: Viable human hematopoietic and mesenchymal stem cells as well as leucocytic cells were found after in vivo growth. Extracellular matrix components were in part human, still viable and forming novel bone. We reproducibly established humanized OS growth within these ossicles. Lung metastases were present in 72.7% of cases after 5 weeks. Histological evaluation determined the most common osteoblastic subtype of OS. Growth patterns of OS differed when cells were injected intratibially as depicted by a decreased tumour take rate and production of extracellular matrix. Typical clinical prognostic markers like VEGF were expressed by the humanized tumours. Intratibial OS growth showed directly opposite expression patterns. Treatment with doxorubicin reduced tumour proliferation and showed bone marrow depletion within the humanized ossicles. Discussion and conclusion: We have tissue engineered a humanized OS in vivo model for the first time and showed its superiority to conventional OS models. We could maintain a vital human hematopoietic stem cell niche and created an OS with a marker expression very similar to the human disease. We also proved the response to therapeutic agents, making this model relevant for preclinical testing.

110

Stimulation of cells with magnetic particles and magnetic fields for soft tissue engineering Jafari, J, Tran, PA, O`Connor, AJ Department of Chemical and Biomolecular Engineering, The Particulate Fluids Processing Centre, University of Melbourne, 3010, Victoria, Australia [email protected] Background Mechanical stimulation of cells can affect their morphology and behaviour. However, challenges can arise in the application of regular methods of imparting force to tissues due to issues including risk of tissue damage and infection, as well as difficulties in measurement of the exact forces applied to cells. Recently, some studies aimed to label cells with magnetic particles (MPs) and then apply force directly to them by using an external magnetic field (MF). While adipocyte and preadipocyte cells show mechanosensitive properties, their response to this method has not been reported clearly and most of the studies in the literature are limited to hard tissues. Aims or Objectives This study aims to examine the effect of MF and MPs on proliferation and differentiation of 3T3-L1 preadipocytes, as well as their efficacy in manipulating the cells into tissue aggregates. Materials and Methods 3T3-L1 preadipocyte cells were labelled with functionalized magnetic microparticles. For proliferation studies, cells were exposed to one- and two-sided magnetic fields by placing magnets next to the culture plates and their viability relative to controls was measured after 3 days. Differentiation studies, on the other hand, were performed by placing magnets on top of the plates, with and without adipogenic culture media. Oil RedO staining and image analysis were utilised to quantify the formation lipid droplets after 2 weeks. Results and Discussion Introducing the MPs to the 3T3-L1 cells in the absence of a MF led to a degree of reduction in cell viability. This indicated that these cells are relatively sensitive to the presence of MPs compared to 3T3 fibroblast cells, which did not show reduced viability in comparable tests. However, in the presence of a MF, the proliferation rate was significantly increased and viability returned to the level of the control. Changing the direction of the MF, on the other hand, did not have a significant effect on the cells’ proliferation rate. Preliminary differentiation studies showed that the use of MPs with or without MF did not inhibit differentiation of preadipocytes toward mature adipocytes. Conclusion Magnetic particles and fields were used to manipulate 3T3-L1 preadipocyte cells in proliferation and differentiation studies. Results suggested that the cell proliferation rate could be maintained in the presence of magnetic particles when a suitable magnetic field was applied and differentiation of the cells towards adipocytes could still be achieved.

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Integrated System for Automated 3D Assembly of Micro-tissues for Cartilage Regeneration Mekhileri, NV1, Lang, M2, Hooper, G1, Woodfield, TBF 1 1

Dept. of Orthopaedic Surgery, Centre for Bioengineering & Nanomedicine, University of Otago, Christchurch, New Zealand; 2Department of Mechanical Engineering, University of Canterbury, Christchurch, New Zealand [email protected] Background: Long term repair of damaged articular cartilage represents a major challenge [1]. The combination of high-throughput cell-aggregate or micro-tissue fabrication methods with 3D printed scaffolds as bottom-up approaches for tissue engineering of cartilage constructs are emerging strategies. However, fabrication of large, complex 3D constructs with precise arrangement of micro-tissue and scaffold components has been limited. Furthermore, no technology has been developed to automate the fabrication and delivery of 3D micro-tissues with 3D Printing [2]. Aims: To develop an automated 3D micro-tissue assembly system for fabricating tissueengineered constructs efficiently without adversely affecting cell viability. Methods: An automated 3D micro-tissue assembly system consisting of a fluidic based singularisation and injection module was designed and incorporated into a commercial 3D Bioprinter. The singularisation module is able to process multiple micro-tissues and deliver them one-at-a-time to an injection module (via custom LabView control software), which then inserts the micro-tissue to a specific location in a 3D printed scaffold. Human chondrocytes were isolated and Ø1mm micro-tissues were formed using a 96-well plate format [3]. The efficiency was determined by successful micro-tissue singularisation (n=100). Bright-field microscopy was used to determine any physical deformation and live/dead and trypan blue exclusion assay to quantify cell viability (n=4). Results: Singularisation efficiency was determined to be 97%±6.6. Comparison of brightfield microscope images of micro-tissues before and after singularisation revealed no noticeable physical deformation. Live/dead (Fig. 1a) and trypan blue exclusion (Fig. 1b) assay revealed no significant difference in viability between control and micro-tissues put through the device. Furthermore, a bi-layered tissue construct containing 18 micro-tissues was successfully assembled into a 3D printed scaffold (1mm fibre spacing) using the system (Fig. 1c). Micro-tissue 100

(b)80

(c)

% Viable

(a)

60 40 20 0

Control

Singularised and injected

Polymer fibre

Figure 1 (a) Live/dead staining of micro-tissues, (b) percentage of viable cells determined using trypan blue exclusion assay and (c) schematic illustration and photograph of a microtissue assembled construct. Conclusion: We demonstrated a novel and efficient system for the automated assembly of micro-tissues in 3D printed scaffolds without deforming or significantly affecting the viability of the micro-tissues. This technology paves a pathway for biofabrication of large assembled tissues with complex 3D architecture and of clinically relevant size and shape. References [1] Woodfield, T.B. et al. Biomaterials, 2004. 25(18): p. 4149-61. [2] Mironov, V. et al. Regen Med, 2008. 3(1): p. 93-103. [3] Schon, B.S. et al. Cell Tissue Res, 2012. 112

Modifications of Silk Fibroin Membranes to Enhance Human Corneal Limbal Epithelial Cell Growth for Ocular Surface Regeneration Suzuki, S1, Dawson, RA1,2, Shadforth, AM1,2, Bray, LJ1,2,3, Harkin, DG1,2, Chirila TV1,2,4 1

Queensland Eye Institute, Brisbane, QLD, Australia. 2Queensland University of Technology, Brisbane, QLD, Australia. 3Max Bergmann Center of Biomaterials, Dresden, Saxony, Germany. 4Univeristy of Queensland, Brisbane, QLD, Australia [email protected]

Background Bombyx mori silk fibroin (BMSF) has been investigated as a substratum for the cultivation of ocular cell types including human corneal limbal epithelial (HLE) cells. The micron-thickness membranes made from regenerated BMSF are mechanically strong, flexible, transparent, biocompatible and biodegradable, and present opportunities for further optimization. Aims To assess cellular responses of four types of modified BMSF membranes: 1. blended with A. Pernyi silk fibroin (APSF) which contains RGD domain, 2. surface functionalized with GRGDSPC peptides, 3. blended with BM silk sericin (BMSS), and 4. treated with poly(ethylene glycol) (PEG) to induce pore formation. Materials and Methods Fibroin and sericin were isolated and regenerated from BM or AP silk cocoons by aqueous procedures. Various types of BMSF membranes including standard and blends were manufactured by casting methods. The PEG treated membrane was washed thoroughly to create pores. The standard BMSF membrane was surface modified by chemically attaching GRGDSPC peptides. Their mechanical properties, transparency, surface characteristics and secondary structures were investigated. In vitro experiments for the attachment and growth of HLE cells on these membranes were carried out. Results and Discussion Neither grafting of RGD domains onto the surface of BMSF, nor blending with the RGDcontaining APSF was found to significantly improve the attachment of HLE cells. Although BMSS membranes had superior cell attachment ability compared to that of BMSF, their blend membranes did not show significant improvements. Mechanical properties of both APSF and BMSS blend membranes were dramatically reduced. The free-standing PEGinduced porous membrane was unstable to handle, but was stabilized by crosslinking with genipin. Growth of HLE cells on either standard and genipin crosslinked porous membrane was improved by co-cultivation of irradiated 3T3 cells on the opposite side, indicating both membranes have high permeability of soluble factors. The mechanical properties of genipin crosslinked PEG-treated membrane were considerably lower than those of the other membrane due to the porous structure. Conclusion We have investigated various modification approaches of BMSF membranes aiming to achieve improved cellular responses, however none of the methods significantly enhanced cellular responses of BMSF membranes without affecting mechanical properties.

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Multifunctional Mesoporous Silica Nanospheres for Bone Tissue Engineering Zhou, Y1, Shi M2, Shao J1, Wu C2, Chen Z1, Song B2, Chang J2, Xiao Y1 1

Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, Australia. 2 State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai, China. [email protected]

Background: Multifunctional bioactive materials for the treatment of bone defects have attracted much attention in the past several years. Since bone regeneration requires a coordinated cascade of cellular events involving angiogenesis and osteogenesis, it is imperative to design a biomaterial system which can simultaneously stimulate blood vessel formation and bone formation in order to repair and regenerate large bone defects. Aims: The overall goal of the present study is to develop a multifunctional biomaterial system for scaffolding and delivery of therapeutic ions and functional drugs to promote bone formation and vascularization. Materials and Methods: Mesoporous silica nanospheres (MSNs) were prepared with uniform sphere size and mesopores, which could release therapeutic silicon ions (Si). Dimethyloxaloylglycine (DMOG), a hypoxia-inducing therapeutic drug, was loaded in the mesopores of MSNs (D-MSNs). The effects of Si ions and DMOG on the osteogenic and angiogenic differentiation of bone marrow stromal cells (BMSCs) were systematically investigated in gene and protein levels. Results: The sustained release of DMOG from D-MSNs could mimic a hypoxic microenvironment by stabilizing HIF-1α stabilization, which greatly enhanced the VEGF secretion from BMSCs. Furthermore, the Si ions released from the prepared nanospheres significantly induced the expression of osteogenic markers (OCN, RUNX2 and OPN) of BMSCs. Discussion: D-MSN, serving as both Si ion reservoir and drug carrier, has the capacity for both osteostimulation (promoting new bone formation) and angiostimulation (inducing vascularization). This suggests that D-MSN has great potential as a multifunctional biomaterial system for bone tissue engineering application. Conclusion: The concept of delivering both therapeutic ions and functional drugs may offer a new strategy to construct multifunctional biomaterial system for bone tissue repair and regeneration.

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The impact of heterotypic cell communication between MSCs and mature cells Otte, EA1,3 and Cooper-White, JJ1,2,3 1

University of Queensland/AIBN, St Lucia, QLD, Australia University of Queensland/School of Chemical Engineering, St Lucia, QSLD, Australia 3 CSIRO/Manufacturing, Clayton, VIC, Australia [email protected]

2

Background An important and under-examined aspect of the microenvironment driving cell fate is the presence of different cell types. There are well known relationships between functional cells (such as cardiomyocytes or neurons) and supporting cells (such as cardiac fibroblasts or oligodendrocytes) but the nature, extent and specific effects of communication between cell types is not well defined, particularly with respect to differentiation. Objectives Presented here is a novel microfluidic device that allows the impact of cell-cell contact and paracrine signalling on cell behaviour to be examined. Methods Standard photolithographic techniques were used to create the simple and highly reproducible device from PDMS and glass, and microPIV was utilised to validate the flow design concept. As the device is designed to run under perfusion, cultures can be maintained for extended periods and easily assayed for end-point analysis using standard stains or antibodies to cell surface antigens. The device has been applied to co-cultures of mesenchymal stem cells (MSCs) with a number of differentiated cell types including endothelial cells and osteoblasts. Results and Discussion Results show the importance of heterotypic communication in MSC differentiation as well as preliminary experiments characterising the connections MSCs make with a number of different mature cell types including endothelial cells and osteoblasts. MSCs are able to form junctions with other cells that are permeable to proteins which have impacts on MSC behaviour. Heterotypic interactions impact both MSCs and endothelial cells in co-culture, with improved outcomes from a tissue engineering perspective. Conclusion Stem cell behaviour is strongly impacted by interactions with differentiated cells and understanding the mechanism of these interactions could lead to improved practices in tissue engineering.

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Thursday Session 18: Scaffolds

13:30-15:45

Biomimetic approaches toward silk biomaterial vascularisation Rnjak-Kovacina, J1,2, Wray, LS2, Coburn, JM2, Lord, MS1, Whitelock, JM1 and Kaplan, DL2 1

UNSW Australia, Sydney, NSW, Australia 2 Tufts University, Boston, MA, USA [email protected]

Biomaterials play a central role in modern regenerative medicine and tissue engineering strategies, where they serve as tuneable biophysical and biochemical milieus that direct cellular behaviour and function. However, one of the biggest obstacles in translating the advances in biomaterials research to clinical applications has been the lack of sufficient vascular tissue regeneration in current synthetic and natural biomaterials. We utilised silk biomaterials with tuneable physical properties as a platform to develop a range of biomaterial vascularisation strategies, including: 1) engineering of vascular-like hollow channels in 3D porous silk scaffolds, 2) in vitro pre-vascularisation of silk scaffolds with endothelial cells, 3) delivery of pro-angiogenic growth factor VEGF from a slow release depot engineered in the silk scaffold centre and 4) scaffold functionalisation with the pro-angiogenic extracellular molecule perlecan. Vascular-like hollow channels played an essential role in enhancing cell infiltration and delivering oxygen and nutrients to the scaffold bulk, and promoted enhanced host tissue integration (cell infiltration & matrix deposition) and vascularisation in vivo. Combinatorial approaches involving hollow channels and VEGF delivery or in vitro prevascularisation further enhanced biomaterial vascularisation compared to any single strategy. Perlecan, through its N-terminal heparan sulphate (HS) chains, supports angiogenesis via binding and signalling key vascular growth factors, while the C-terminal region of the protein core supports α2β1 integrin-mediated cell binding. Recombinantly expressed C-terminal Domain V of human perlecan was found to support endothelial cell interactions to the same extent as full-length perlecan and was explored as an ECM-based biological cue for endothelial cell recruitment and silk scaffold vascularisation.

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Modelling the three-dimensional prostate cancer microenvironment in vitro using melt electrospun scaffolds Pereira, BA1, De-Juan-Pardo, EM2, Lister, NL1, Hashimoto, K1, Lawrence, MG1, Ellem, SJ1, Hutmacher, DW2 & Risbridger, GP1 1

Monash University, Clayton, VIC, Australia Queensland University of Technology, Kelvin Grove, QLD, Australia [email protected]

2

Background Prostate cancer (PCa) is the second most frequently diagnosed malignancy in men, with 1.1 million cases worldwide annually. Progression of PCa is slow and most men are diagnosed with localised disease, with a subset of patients progressing to advanced, metastatic disease. It is acknowledged that the tumour microenvironment (stroma, extracellular matrix, vasculature, immune cells and hormones) plays a critical role in this disease progression; however there is a paucity of physiologically relevant in vitro models to investigate its contribution further. Aim Current in vitro models to examine the PCa microenvironment are inherently limited and do not allow for the study of discrete cell interactions. We have developed a modular threedimensional (3D) in vitro model to study the synergistic effects of the tumour microenvironment on localised PCa progression, with an initial focus on stromal-epithelial interactions. Materials and Methods Melt-electrospun scaffolds were formatted from poly(ε-caprolactone) (PCL) polymer spun in a 0/90⁰ pattern, forming flexible 15x15mm meshes with a pore size of ~100µm. Scaffolds were either etched with 0.1M sodium hydroxide (NaOH) or plasma treated to increase hydrophilicity and thus cell attachment. Validated patient-derived cancer-associated fibroblasts (CAFs) or non-malignant prostatic fibroblasts (NPFs) were incorporated into the treated scaffolds. Once confluent, tagged initiated benign epithelial cells (BPH-1) were cocultured on the fibroblast constructs. At day 1, scaffolds were fixed and the epithelial morphology (sphericity, volume, orientation, migration) analysed using confocal microscopy and Imaris software. Results and Discussion CAFs and NPFs proliferate and deposit extracellular matrix to form a 3D stromal network within the scaffolds. Cell penetrance is ~90µm (6-8 cell layers) with the plasma treatment significantly increasing cell attachment and eliminating cell clumping compared to NaOH etching. Preliminary data indicates that CAFs, but not NPFs, induce an invasive epithelial morphology in the benign epithelial, recapitulating the in vivo biology. Ongoing work includes the incorporation of mast cells, a resident population that expands peri-tumourally in PCa and is believed to contribute to early disease progression. Conclusion This model provides a physiologically relevant in vitro platform to study the prostate tumour microenvironment, with data highlighting the fundamental contribution of stromal-epithelial interactions on localised disease progression.

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Layer-By-Layer Functionalization of Poly(-Caprolactone) Nanofibre Scaffolds with Novel Polymer Influences Astrocyte Phenotype In Vitro Maclean, FL1, Lau, CL2, Horne, MK2,3, Beart, PM2 and Nisbet, DR1 1

Research School of Engineering, The Australian National University, Canberra, Australia. 2 Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, Australia 3 Department of Neurology, St Vincent’s Hospital, Fitzroy, Australia [email protected]

Astrocytes play an important role after traumatic brain or spinal cord injury, sealing the lesion site, preventing secondary degeneration and arresting growth in the acute phase after injury through the production of pro-inflammatory molecules such as chondroitin sulphate proteoglycans (CSPGs) [1]. However, if this astrocytic response is persistent, the chemical and physical barriers prevent functional recovery. Therefore, an important aspect for therapeutic strategies addressing central nervous system (CNS) inflammation is controlling the temporally significant phenotype change of astrocytes. Current 2D cell culture systems used to study cell behaviour do not recapitulate the 3D in vivo environment, and hence, current results are limited in their translation to in vivo experiments. We have previously shown that astrocytes cultured on 3D PCL nanofibres resulted in astrocyte infiltration within the scaffold, as well as reduced GFAP expression and the upregulation of BDNF and SLC1A2, indicating a cytotrophic phenotype [2]. As such, we -caprolactone) (PCL) nanofibre scaffolds with biologically relevant molecules to determine the effect on astrocyte phenotype in vitro. We have functionalised PCL nanofibres with heparin, poly-L-lysine (PLL) and a novel polymer, poly-llysine conjugated with lactobionic acid (PLL-LBA) to provide astrocytes with antiinflammatory and cell adhesive cues, as well as galactose moieties, respectively. PCL nanofibres were functionalised using the layer-by-layer (LbL) method, which is dependent on the formation (and subsequent crosslinking) of polyelectrolyte multilayers (PEMs) due to the electrostatic interactions between the polyanion (heparin) and the polycation (PLL or PLL-LBA). The average diameter of the PCL nanofibre diameters was 460 ± 130 nm, and was found to be unaffected by functionalization, with contact angle measurements showing a distinct difference in surface chemistry after the third bilayer of heparin + PLL or heparin + PLL-LBA. We have shown that providing astrocytes with a 3D environment as well as biochemical cues in vitro induces a cytotrophic phenotype. This is an important finding that we hope to implement in vivo in an attempt to change the astrocyte phenotype after traumatic injury, encouraging regeneration after the initial growth arrest to achieve functional recovery.

1. 2.

Rolls, A., R. Shechter, and M. Schwartz, The bright side of the glial scar in CNS repair. Nature Reviews Neuroscience, 2009. 10(3): p. 235-241. Lau, C.L., et al., 3D Electrospun scaffolds promote a cytotrophic phenotype of cultured primary astrocytes. Journal of Neurochemistry, 2014. 130(2): p. 215-226.

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A PEDOT microfibrous electrode for neural recordings. Marroquin, JB1, Zhou, K1, Parkington, HC2 Coleman, HA2, and Forsythe, JS1 1

Department of Materials Engineering and Department of Physiology, Monash University, Clayton, VIC 3800, Australia [email protected]

2

Current electrodes used for in vivo biomedical applications (e.g. prostheses, bionic implants) present serious limitations in obtaining stable, consistent and long-term stimulation or recordings. A major problem is unwanted glial invasion which leads to the formation of a nonexcitable “scar” around the stimulating site [1]. This scar increases resistance to current flow and also increases the distance between the neurons and the current source. In time, this unwanted glial response leads to the need for higher electrical currents and loss of specificity of stimulus. The use of higher voltages/currents results in damage to neural tissue due to Faradaic reactions. Also, proposed electrodes with a 2D surface have a limited proximity to neurons, which is greatly affected when a glial response occurs [2]. In the present study, a macroporous 3D neural electrode was developed using a PLLA electrospun fibrous template. The fibrous scaffold was imbued with electroactive properties by coating with a conductive polymer (PEDOT) via vapour phase polymerization. The electrical properties of the PEDOT coated substrates were studied in terms of sheet resistance measured by a 4-point probe, and scaffold morphology was investigated by scanning electron microscopy. Scaffold biocompatibility was assessed through in vitro assay using patch clamp electrophysiology. The scaffolds presented an average sheet resistance of 50 Ω/□, while fibres had a diameter between 0.5-1.5 µm. Cell cultures showed the scaffolds supported hippocampal neuron cell adhesion and neurite outgrowth within the fibrous structure of the scaffold, with no appreciable cell toxicity. Electrophysiology confirmed that substrate composition did not alter neural functional development, in terms of action potential generation and neural network formation, indicated by the occurrence of spontaneous excitatory post synaptic potentials. We have demonstrated successful fabrication of a PEDOT 3D neural electrode that provides a novel alternative to current technologies.

1. 2.

Vadim, S.P., A.T. Patrick, and M.R. William, Response of brain tissue to chronically implanted neural electrodes. Journal of Neuroscience Methods, 2005. 148(1): p. 118. Warren, M.G., E.N. Sharon, and V.B. Ravi, Implanted Neural Interfaces: Biochallenges and Engineered Solutions. Biomedical Engineering, 2009. 11(1): p. 124.

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Oxygen Inhibition in 3D Printing of Photo-Curable Hydrogels Lim, KS, Lindberg, GCJ, Schon, BS, Hooper, GJ, Woodfield,TBF CReaTE group, Department of Orthopaedic Surgery and Musculoskeletal Medicine, University of Otago, Christchurch. New Zealand. [email protected] Background: 3D printing of hydrogels has emerged as a new technology to fabricate tissue engineering scaffolds of designed and tailored architecture. The current state-of-art involves 3D printing the hydrogel precursor solution followed by crosslinking to maintain its structural integrity [1]. In particular, photo-initiated radical crosslinking has good spatial and temporal control, and is therefore widely explored. Additionally, 3D printing and crosslinking of cell-laden hydrogel constructs requires the presence of oxygen to maintain cell survival and viability. However, oxygen is known to quench the radicals causing incomplete crosslinking, which then leads to physical deformation of the scaffold [2]. Aims: This study aims to minimise the effect of oxygen inhibition in photo-curable 3D printed hydrogel constructs. Materials and Methods: 10wt% methacrylated gelatin (Gel-MA) hydrogels were fabricated in moulds (Ø5mm x 1mm). The concentration of the initiator (Irgacure2959) was varied between 0.05 and 0.5wt%. All samples were exposed to 3mW/cm2 of UV light with the macromer surface exposed to an oxygen rich atmosphere. The thickness of the gels before and after equilibrium swelling was measured. For cell encapsulation studies, expanded human articular chondrocytes (P2) were encapsulated at a density of 1x106cells/ml and their viability were assessed using live/dead assay at 1day. Results and discussion: At a concentration of 0.05wt% initiator as per literature, a significant reduction (~45%) in construct thickness post equilibrium swelling was observed (Fig1A). This result indicates that as the hydrogel surface was exposed to oxygen during crosslinking, oxygen was able to inhibit the crosslinking process by quenching the radicals. Increasing the initiator concentration successfully minimised the oxygen inhibition as observed by the limited reduction (~5%) in construct thickness (Fig1B). However, higher initiator concentrations also resulted in a significant decrease in cell viability (Fig1C).

Conclusion: Oxygen inhibition is a significant issue for 3D printing of hydrogel constructs. Future work will focus on developing new methods to combat this issue in order to fabricate scaffolds of good structural integrity without affecting the cell survival. References: [1] Malda, J. et al. Advanced Materials 2013, 25 (36), 5011-5028. [2] Melchels, F. P. W. et al. Journal of Materials Chemistry B 2014, 2 (16), 2282-2289 120

Melt Electrospinning as a Potential Additive Manufacturing Technique Nikola Ristovski1; Sam Liao1; Nathalie Bock1, Sean Powell1, Giles T.S. Kirby1, Keith A. Blackwood1, Maria Ann 1

Injury Prevention and Trauma Management Theme, Institute of Health & Biomedical Innovation, Queensland University of Technology, Brisbane, QLD [email protected]

Background: Melt electrospinning (MES) is the process of using an electrical charge to create micro scale fibres from a molten polymer [1] in a manner similar to fuse deposition modelling. Due to the small fibre size, the process is sensitive to forces and deposition is often disrupted. When depositing fibres, charge collection on the surface of the polymer creates an electrostatic repulsion between neighbouring strands. This creates alignment problems when fibres are too far away from the collector plate to diffuse this charge [2]. A technique must be produced to reduce such charge build-up. Objectives: The paper aims to produce a technique for quantifying fibre deposition accuracy as well as assessing the effectiveness of altering the application of potential between the spinneret and the collector. Materials and Methods: The following variables were kept constant throughout the experiment: Needle tip = 18 G; Tip to Collector (TTC) distance = 10 mm; Temperature = 700C; Feed rate = 40 µL/hr; Potential Difference = 10.5 kV; Polymer = Polycaprolactone (PCL, Mw = 43 000, Perstop, UK). The voltage across the tip and collector was varied at increments of 2 kV. Each scaffold was spun to a height between 4-6 mm. The scaffold was then cut vertically and SEM images were taken to quantify the level of alignment at each layer of the scaffold. Figure 1 shows a cross-sectional view for a 10x10mm scaffold, illustrating fibre alignment beyond the 1mm mark. Results: Fig. 1: Fibre stacking of a scaffold with 500 µm fibre spacing illustrating consistent stacking up to 1mm. Past this point, columbic forces become dominant. Within the disordered region, the columbic forces have completely overcome other forces and large amounts of disorder are observed. Discussion: Due to the process of MES, charge is stored on the surface of the polymer after deposition. Such charge creates a repulsive force between fibres and deposition accuracy is mitigated. Charge build-up is reduced by applying a positive and negative potential across the spinneret and the collector, respectively, and hence the columbic force is reduced. Conclusion: The dual voltage MES technique produced scaffolds with high levels of order for over 200 layers (equivalent to 8mm). The addition of the two power supplies reduced charge stored in the polymer, subsequently, fibre deposition accuracy increased and highly ordered scaffolds were produced. References: [1] Darrel H. Reneker, A.L.Y. (2008) Electrospinning Jets and Polymer Nanofibers. Polymer, 2387-2425; [2] Brown, T.D., P.D. Dalton (2011) Direct writing by way of melt electrospinning, Adv. Mater, 5651; 121

Diffusion Tensor and Computed Tomography Microimaging Study of Scaffolds for Tissue Engineering. Powell, SK, Ristovski, N, Liao, S, Blackwood, KA, Woodruff, MA, Momot, KI Injury Prevention and Trauma Management Theme, Institute of Health & Biomedical Innovation, Queensland University of Technology, Brisbane, QLD [email protected] Background Biofabrication has enormous potential to revolutionise the clinical treatment of serious injury and trauma. It involves the application of 3D printing principles to regenerative medicine, producing morphologically accurate and biologically functional tissue substitutes in a layer-by-layer manner. Direct-writing melt-electrospinning (DME) is one technique for fabricating biodegradable polymer supporting scaffolds with well-defined internal micro-architectures using micron-thick fibres. Fibre networks at this scale have desirable characteristics for cell attachment and proliferation, however, cell infiltration restricts fluid flow causing hypoxic cell death. A comprehensive understanding of molecular transport within DME scaffolds is, therefore, vital for the design of microarchitectures that maintain oxygen and metabolite exchange throughout the entire tissue growth phase. Aims In this study, we combined diffusion tensor (μDTI) and computed tomography (μCT) micro-imaging to investigate the relationship between internal micro-structure and molecular transport within DME scaffolds. Materials and Methods Two DME scaffolds were produced with 90° cross-hatched internal micro-architectures, average fibre spacing of 100μm and fibre diameters of 10μm and 50μm, respectively. Characterisation of the internal structure order and morphology was performed using the combination of T2-weighted magnetic resonance imaging and μCT. Time dependent diffusivity and diffusion anisotropy were obtained using μDTI for diffusion times (Δ) of 50ms, 200ms and 250ms. Results Both the μCT mean intercept length tensors and the μDTI diffusion tensors indicate anisotropy for both scaffolds, with their principal vectors closely aligned with the z-axis of the scaffolds (vertically through the layers). The MIL tensor was found to describe an oblate ellipsoid and the DT tensor described a prolate ellipsoid. Discussion and Conclusion The diffusion tensors were anisotropic with diffusion least restricted in the direction perpendicular to the fibre layers. This demonstrates that the cross-hatched scaffold micro-architecture restricts diffusion along the direction of the fibre layers and preferentially promotes molecular transport vertically through the layers. We also found that diffusivity in the direction of the fibre layers was invariant to the thickness of the fibres. μDTI appears to be a highly promising technique for the real-time monitoring of tissue regeneration with applications in tissue cultures and in vivo.

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Emulsion Templating: A Versatile Route to the Preparation of Biodegradable and Biocompatible Scaffolds for Tissue Engineering Langford, CR, Cameron, NR Department of Materials Engineering, Monash University, Clayton, VIC, Australia [email protected] The emulsion templating process offers a route to highly porous polymers with well-defined morphologies[1]. The process, whereby the continuous phase of a high internal phase emulsion (HIPE) is polymerized, results in materials referred to as polyHIPEs. These porous materials have found application in a wide range of areas including catalysis[2], organic synthesis[3], and in biotechnology[4, 5]. Recent developments in the photo-initiated polymerization of HIPEs have allowed for the preparation of polyHIPE polymers from monomers which form highly unstable emulsions, such as multifunctional thiol and alkene (typically acrylate) monomers[6]. The resulting polymers are biodegradable and have been found to be biocompatible in initial cell culture studies[5]. The surface chemistry of these materials can be altered by a variety of postpolymerization functionalization techniques[7, 8]. The preparation of biodegradable polyHIPEs from multifunctional thiols and acrylates will be described together with the methods by which the physical properties, morphology and surface chemistry can be varied. The preparation of layered materials with asymmetric properties and morphologies will also be described. Subsequently, the use of polyHIPE materials as scaffolds for tissue engineering will also be discussed.

[1] [2] [3] [4] [5] [6] [7] [8]

N. R. Cameron, Polymer 2005, 46, 1439. M. Ottens, G. Leene, A. Beenackers, N. Cameron, D. C. Sherrington, Industrial & Engineering Chemistry Research 2000, 39, 259. P. Krajnc, D. Stefanec, J. F. Brown, N. R. Cameron, Journal of Polymer Science Part a-Polymer Chemistry 2005, 43, 296. M. W. Hayman, K. H. Smith, N. R. Cameron, S. A. Przyborski, Journal of Biochemical and Biophysical Methods 2005, 62, 231. S. Caldwell, D. W. Johnson, M. P. Didsbury, B. A. Murray, J. J. Wu, S. A. Przyborski, N. R. Cameron, Soft Matter 2012, 8, 10344. E. Lovelady, S. D. Kimmins, J. J. Wu, N. R. Cameron, Polymer Chemistry 2011, 2, 559. L. Kircher, P. Theato, N. R. Cameron, Polymer 2013, 54, 1755. C. R. Langford, D. W. Johnson, N. R. Cameron, Polymer Chemistry 2014, 5, 6200.

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Thursday Session 19: Biointerfaces II

16:15-17:30

The platelet behavior and NO releasing in vitro of Ti-Cu films by high power pulsed magnetron sputtering Y.D.Tai1, T.Chen2, L.F.Yao , Long Li, F.J.Jing*, H.Sun,Y.X.Leng, N.Huang* 1

2

Key Lab for Advanced Technologies of Materials, Ministry of Education, China School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu, China [email protected]

Surface-induced thrombogenesis is known to be one of the reasons for failure of artificial cardiovascular prostheses. It is well known that Cu2+/Cu+ ions can catalyze the generation of NO from NO donor species(RSNO) . NO is a natural potent anti-thrombogenic agent, serving in one of its many functions to prevent activation and aggregation of platelets in blood vessels . Copper (Cu) is an essential trace element and titanium has chemical inertness and good biocompatibility. In the work described here, Ti-Cu films were fabricated on silicon wafers using a copper-titanium combined target by high power pulsed magnetron sputtering (HPPMS). The characteristics and copper release properties of Ti-Cu films were investigated. The NO release catalyzed by Ti-Cu films were examined and the antithrombotic properties of the samples were evaluated by in vitro blood platelet adhesion tests. The EDS results showed that the films achieved different atomic ratio of Cu to Ti: (i) 20:80 for the HPPMS(600V) (ii) 29:71 for the HPPMS(800V) technique respectively. XRD results exhibited that Cu is partially incorporated in the Ti lattice of the HPPMS films and partially formed new Ti-Cu phase. The wettability of Ti-Cu films was between that of Ti films and Cu films. The films with more Ti-Cu phase were detected higher Cu2+ concentration in phosphate buffered solution (PBS) in the first two weeks. It is estimated that the copper releasing properties were influenced mostly by the structures of Ti-Cu films. Compared with the pure Ti film, NO concentration increased significantly from the Ti-Cu films. The NO concentration of HPPMS600V was higher than the HPPMS-800v samples, consistenting with the release quantity of Cu. The platelet number of Ti/Cu films decreased significantly than the surface of films without the SNAP donor. The Ti-Cu film revealed the ability of catalytically decomposing NO donor(SNAP), generating NO; thus the surface of Ti-Cu films by HPPMS displayed the ability to inhibit platelet activation and aggregation, demonstrating good blood compatibility potential.

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An ex vivo porcine artery model: Fluid convection and diffusion induced biodegradation behavior of magnesium metal Wang, J1,2, Liu, L1, Koo, Y1,Sankar, J1, Huang, N2, Yun, Y1 1 North Carolina A & T State University, Greensboro, NC, USA 2 Southwest Jiaotong University, Chengdu, Sichuan, China [email protected] Backgound Absorbable metals have been widely tested in various in vitro environments to evaluate their possible suitability as a vascular stent material. However, there exists a gap between in vivo and in vitro test results. A key step is to identify and test the relevant microenvironments and parameters in test-systems. To effectively study, model, and optimize absorbable magnesium-based stents, characterizations need to be performed in hydrodynamic conditions that are found within vessel wall. Aims • Mimicking fluid convection and diffusion induced biodegradation behaviour of magnesium before and after endothelialization. • Establishing the relationship between fluid dynamics, mass transport and degradation reactions occurring on the Mg materials. Materials and Methods The porcine abdominal aortas were obtained from Matkinsmeat processors. Two as-drawn magnesium wires of 99.9% purity and a diameter of 250 ± 25 μm were put into the lumen and intima of an aorta. Each aorta was mounted into a chamber of the Lume GenBioreactor under the cellculture conditions for 5days. The flow rate of the circulated medium was 100 ml/min. A pulsed pressure oscillated in a range from 80 to 120 mmHg. Samples were analysed using X-ray computed tomography and scanning electron microscope. Results and Discussion The results indicated that the order of corrosion rates is dynamic convection> dynamic diffusion > static conditions (Figure 1). The volume losses at the static, dynamics convection, and dynamic diffusion were 19.1%, 52.2% and 30.3% for 3 days, as well as 36.1%, 100% and 40.1% for 5 days. Mg wire fractured on the fluidic convection environment for 3 days. A good design of stent must avoid a risk of fast fracture in fluidic convection environment.
 Figure 1. Cross-section CT images of corroded Mg wires at the static condition without the aorta (a and b), at the dynamic convection (c and d) and diffusion (e and f) conditions within the aorta for 3 days.Magnesium (red), corrosion products (yellow), and tissue (green).

Conclusion Convection and diffusion of the hydrodynamic microenvironments significantly affect on the different degradation behavior of magnesium materials. The ex vivo porcine artery model can provide a deeper and more accurate understanding of the biodegradation behavior.

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Diamonds are an implant’s best friend Fox, K., Tong, W., Aramesh, M., Tran, P. School of Aerospace, Mechanical and Manufacturing Engineering, RMIT University, Melbourne, VIC, Australia; School of Physics, University of Melbourne, Melbourne, VIC, Australia; Department of Chemical and Biomolecular Engineering, University of Melbourne, Melbourne, VIC, Australia [email protected] Diamond has been treated as an ideal material in advanced biomedical applications due to its outstanding biocompatibility, biochemical stability, chemical inertness and mechanical stability. More attractively for many biomedical applications, diamond has been selectively doped to become electrically conductive to deliver current to surrounding tissue. In the studies reported, nitrogen doped ultrananocrystalline diamond UNCD (N-UNCD) with a conductivity of about 46 S/cm was deposited on silicon by microwave plasma-enhanced chemical vapour deposition (MPCVD). In vitro cell studies were undertaken using fibroblast and primary cortical neuron cultures and in vivo studies for the Bionic Eye Project using feline models. Bioactivity was assessed using simulated body fluid. Here, we present the results of a number of in-house studies into the biomedical applications of diamond. We will discuss its use in a variety of bioapplications such as: 1. as the stimulating and recording electrode in the Australian Bionic Eye Project [1-3]; 2. as a direct cortical neural interfacing material [4, 5]; 3. as a hybrid material [6]; 4. as an orthopaedic scaffold [7]; 5. the appropriate sterilisation techniques for such a high surface area material;

1. 2. 3.

4.

5. 6. 7.

Ganesan, K., et al., An all-diamond, hermetic electrical feedthrough array for a retinal prosthesis. Biomaterials, 2014. 35(3): p. 908-915. Garrett, D.J., et al., Ultra-nanocrystalline diamond electrodes: optimization towards neural stimulation applications. Journal of neural engineering, 2012. 9(1): p. 016002. Hadjinicolaou, A.E., et al., Electrical stimulation of retinal ganglion cells with diamond and the development of an all diamond retinal prosthesis. Biomaterials, 2012. 33(24): p. 5812-5820. Fox, K. and Prawer, S., Neural circuits and in vivo monitoring using diamond. Quantum Information Processing with Diamond: Principles and Applications, 2014: p. 291. Tong, W., et al., Fabrication of planarised conductively patterned diamond for bioapplications. Materials Science and Engineering: C, 2014. 43: p. 135-144. Aramesh, M., et al., Multifunctional three-dimensional nanodiamond-nanoporous alumina nanoarchitectures. Carbon, 2014. 75: p. 452-464. Fox, K., et al., Diamond as a scaffold for bone growth. Journal of Materials Science: Materials in Medicine, 2013. 24(4): p. 849-861.

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The influence of material properties on cell fate Vasilev, K Mawson institute, University of South Australia, Adelaide, Australia [email protected] Materials mechanical properties are known to be an important regulator of cellular processes such as proliferation, differentiation and migration, and have seen increase in attention in recent years. In this talk, I will focus on our recent work on understanding the effect of surface stiffness on cell adhesion and proliferation. At present, there are only few approaches where the mechanical properties of thin films can be controllably varied across an entire surface. In this work, we present a technique for controlled generation of gradients of surface elastic moduli involving a weak polyelectrolyte multilayer (PEM) system of approximately100 nm thickness and time dependent immersion in a solution of 1-ethyl-3-(3dimethylaminopropyl) carbodiimide (EDC) as a crosslinking agent. Uniform surface chemistry across the gradient and wettability was provided by the addition of a 10 nm thick plasma polymer layer deposited from vapour of eitherallylamine or acrylic acid. We used the resultant stiffness gradients (0.5-110 MPa in hydrated state) to investigate the adhesion, morphology and proliferation on human dermal fibroblasts (HDFs). We show that substrate mechanical properties strongly influence HDF cell fate. We also found that in the experimental range of surface properties used in this study, the surface stiffness was a stronger driving force to cells fate compared to chemistry and wettability. I will also briefly talk about research efforts in our group directed to studying the influences of surface properties such as nanotopography, ligand density and chemistry of cell adhesion, proliferation and differentiation.

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Interactions at Bio-material Interfaces Milthorpe, BK1, Lord MS2 1

University of Technology Sydney, Sydney NSW, Australia 2 University of New South Wales, NSW Australia [email protected]

The interactions that occur at the surface of any biomaterial construct on introduction into a body are many and complex. For devices that do not penetrate an epithelial layer, eg urinary catheters, the potential interactions may be tractable by current methods. For devices that are implanted, even for relatively short period, the interactions are not. By the act of implantation the device, whether a simple piece of metal or a complex tissueengineered construct, is placed into a wound-site. From then on the time-course of events is dependent on a number of factors. We deal with this complexity by using simplified systems (eg simulated body fluids, single protein adsorption, and even cell culture) and concepts (eg water contact angle as a descriptor of surface chemistry). Whilst simplifying the system under investigation is a reasonable, it is timely to review the simplifications used and assess the impact of the inherent assumptions on our understanding and interpretation of behaviour in more complex situations. I will give some examples from past and current research, as well as ways for defining boundaries for the interpretation of data from simplified systems.

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Session 20: Antimicrobial coatings II 16:15-17:30 Visualising the interaction between nanopillars and bacteria membrane Bandara, IMCCD1, Oloyede, A1, Singh, S2, Tesfamicheal, T3, Chirutha, J4 1

School of Chemistry, Physics and Mechanical Engineering, Science and Engineering Faculty, Queensland University of Technology, Brisbane, Australia 2 Central Analytical Research Facility (CARF), Queensland University of Technology, Brisbane, QLD, Australia 3 Institute for Future Environments (IFE), Science and Engineering Faculty, Queensland University of Technology, Brisbane, QLD, Australia 4 Faculty of Health, Queensland University of Technology, Brisbane, QLD, Australia [email protected] The study of the interaction between cellular membrane and nanopillar structures is of significant interest today in material science because of the knowledge that can be gained towards manufacturing bactericidal surfaces and interfaces . Nanopillar structures have have been shown to have a range of effects on different types of cell interacting with them ; most notably the pillar-like nanostructures present on dragonfly wings are capable of killing bacterial, and alter the adhesion of stem cells to the surface. Though numerical model has been used in literature to explore and test the validity of killing effect by nanopillar structures, visualisation of interface is not yet available. In this paper, we hypothesize that the destructive effect of these nanopillars on bacteria is mostly due to the mechanical interaction between them and can be visualised by transmission electron microscopy (TEM). While it is clear in scanning electron microscopy and confocal spectroscopy images obtained to date that bacterial membrane rupture occurs the underlying mechanism for this occurrence remains unclear because it is not yet possible to visualise the interface between bacteria and nanopillars by current available imaging or spectroscopic methods, thereby limiting the complete understanding of this bactericidal action of these specific surface topology. In this study we have explored the application of TEM in resolving the nature of the interaction between Escherichia coli bacterium and the nano-pillar-structure on the wings of the dragonfly . Bacterium on dragonfly was serially sectioned across membrane and visualised under TEM at high resolution. TEM images obtained by this method provide a means of visualising the interface between cell membrane the nanotopological structure of this very interesting surface characteristic. We argue that this method can be further adapted to visualise and study interfacial interactions between various cells including bacteria, stem cells with nano pillar-like structures and for determining the mechanism of physical rupture of bacteria on nano pillars and the fate of stem cells on various nano surfaces.

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ANTIBACTERIAL BIOMATERIALS BASED ON QUORUM SENSING INHIBITORS Taunk, A1, Ho, KKK1, Chen, R1, Willcox, MDP2, Kumar, N1 1

School of Chemistry, University of New South Wales, Sydney, NSW, Australia School of Optometry and Vision Science, University of New South Wales, Sydney, NSW, Australia [email protected]

2

Background Infection of implanted medical device is one of the major causes of nosocomial infections. In spite of advances in biomaterial technologies, a significant proportion of the devices become colonized by bacterial biofilms, thus resulting in high risk of mortality. At present, rapid development and proliferation of bacterial resistance and also lack of new antibacterial drugs have made device related microbial infections extremely difficult to treat. Aims or Objectives The main aim of this study was to develop antibacterial biomaterial surfaces using compounds that do not kill the bacteria but instead are able to disrupt the bacterial communication pathways, in order to reduce the incidence of bacterial infection. Materials and Methods This study focuses on covalent attachment of potent quorum sensing (QS) inhibiting compounds, fimbrolides (FUs) and dihydropyrrol-2-ones (DHPs), onto glass surfaces by azide/nitrene chemistry. The modified surfaces were tested in vitro against pathogenic organisms such as Staphylococcus aureus using confocal laser scanning microscopy (CLSM). Results Successful attachment of compounds on the substrates was confirmed by X-ray photoelectron spectroscopy (XPS). The antibacterial efficacy was assessed, and significant reduction in bacterial adhesion and biofilm formation was observed on the FU and DHP coated surfaces. The results were found to be comparable to DHP coated surfaces prepared in earlier study via Michael addition reaction. Conclusion FUs and DHPs were able to retain their in vitro antibacterial efficacy after covalent attachment via azide chemistry. This approach is a promising strategy to develop efficient antibacterial biomaterials to reduce device related infections.

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Identification of Plant-derived Antibacterial Compounds for Surface Coatings Biva, IJ1, Ndi, CP2, Semple, S2 and Griesser, HJ3 1

Ian Wark Research Institute, University of South Australia, Adelaide, SA, Australia. 2Sansom Institute, School of pharmacy and Medical Sciences, University of South Australia, Adelaide, SA, Australia. 3Mawson Institute, University of South Australia, Adelaide, SA, Australia [email protected] While plant-derived antimicrobial compounds might also be of interest per se for the treatment of infectious diseases, our focus is on their potential use for the fabrication of antibacterial surface coatings to combat infections on surfaces of biomedical devices. Two types of approaches can be envisaged for using antibacterial compounds. One approach is diffusive release of antibiotics from a biomedical device; the other approach comprises the covalent coupling of molecular layers of antibacterial compounds to solid surfaces. 1 This investigation concentrates on identifying anti-bacterial compounds from traditionally used Australian plants and close relatives, seeking high activity as well as suitable chemical groups for covalent linking onto materials surfaces. Among Australia’s unique and distinct flora, Eremophila plants are prominent in traditional medicinal recipes. For example, a few species were used to treat skin disorders and sore throat.2 Studies with Eremophila extracts showed antibacterial activity and established a scientific basis for the anecdotal use of these species by native Australians.3 However, only a few species in the Eremophila genus have been studied for identifying antibacterial compounds. In the present study, we aim to isolate and identify compounds from additional Eremophila species that may provide stronger antibiotics than those found earlier. In particular, we are investigating E. alternifolia, which has not been studied to date even though in Aboriginal medicine it was the most highly valued plant among all Eremophilas. Acetone leaf-resin extracts of E. gibbosa and E. alternifolia have led to the isolation and structural identification of several novel active compounds of different classes; namely, diterpenes and flavonoids (Figure1). Most of these compounds were active against Gram-positive bacteria. Moreover, a flavanone isolated from E. alternifolia has shown promise as it eradicates even methicillin resistant Staphylococcus aureus (mRSA) and has substantial activity against biofilm producing bacteria. Consequently, all these active compounds might be useful as antibacterial coating ingredients on biomedical devices and would be a potential means to lessen implant-related infections.

(1)

(2)

(3)

(4)

(5)

Figure 1: Antibacterial compounds (1-5) isolated from E.gibbosa and E. alternifolia References: 1. Griesser et al, 2008. Chemistry in Australia 75, 5. 2. Ghisalberti, E. L., 1994. Journal of Ethnopharmacology 44, 1-9. 3. Palombo, E. A. and Semple, S. J., 2001. Journal of Ethnopharmacology 77, 151-157. 131

Dual-action surface coatings designed to combat polymicrobial mixed bacterial and fungal infections Griesser, SS, Saboohi, S, Jasieniak, M, Coad, BR, Griesser, HJ Mawson Institute, University of South Australia, Mawson Lakes, SA 5095, Australia [email protected] Recent clinical studies, for example at Monash University by Peleg et al., have shown that it has been under-appreciated, due to insufficient diagnosis techniques, how often biofilm infections on biomedical devices and implants can be polymicrobial not only with mixed bacterial populations but also containing fungal pathogens such as the particularly dangerous Candida albicans, which if liberated from the infection site can cause serious and at times fatal blood infections. There are many examples in the literature of coatings that have been successful in preventing bacterial colonisation in vitro but have not performed anywhere near as well in vivo. Our hypothesis is that a possible contributing factor to this is that fungal species co-existed and, after the bacteria were eliminated by bacteria-specific antimicrobials, had the field/surface to themselves to progress the infection, with bacteria then perhaps re-colonising the site as well after the antibiotic had worn off. In principle, antiseptics such as silver ions and chlorhexidine, as well as a number of membrane-destroying compounds, can eradicate bacterial and fungal pathogens simultaneously, but they possess the same lethality against human cells and tissue. Thus, specific drugs must be used that have minimal side effects on the human host. Due to their eukaryotic nature, fungal pathogens are much harder to combat with specific antibiotics; only a few clinically approved and effective specific antifungals exist. Thus, in theory a promising strategy against mixed bacterial and fungal infections involves delivery of at least two distinctly different drugs. We are exploring this scenario, using plasma polymer layers onto which antibiotics can be covalently grafted to prevent settlement of pathogens. As an alternative strategy, we are also using suitable plasma polymers for diffusive delivery. Matching the hydrophilicity/hydrophobicity of drugs and plasma polymers, we can achieve relatively high loadings even into coatings < 200 nm thick, as presented previously for the antibacterial compound levofloxacin. We are also exploring a “mix-andmatch approach of using both grafted and diffusive dual drugs systems.

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Session 21: Stem Cell Therapies II 16:15-17:30 Inhibition of transforming growth factor beta receptor signaling promotes expansion of undifferentiated human endometrial MSCs Shanti Gurung1, Jerome A Werkmeister2,3, Caroline E Gargett1 1

Monash University MIMR-PHI, Clayton, VIC 3168, Australia CSIRO Manufacturing Flagship, Bayview Avenue, Clayton, VIC 3168, Australia 3 Monash University, Department of Obstetrics and Gynaecology, Clayton, VIC 3168, Australia [email protected] 2

Background: One of the major hurdles in the use of mesenchymal stem cells for cell based and tissue engineering therapies is the need for culture expansion with retention of stem cell properties. Endometrial MSCs are an easily accessible alternate source of MSC that can be purified by bead sorting using the W5C5 antibody and culture expanded in an in-houseserum-free-medium (SFM) in hypoxic environment. However, eMSCs like other MSCs undergo spontaneous differentiation upon in vitro culture expansion into fibroblasts decreasing their purity. The aim of this study was to determine if a small molecule, targeting transforming growth factor beta receptor (TGFβR) could prevent spontaneous differentiation of eMSCs in culture. Materials and Methods: W5C5+ eMSCs were cultured in SFM with bFGF and EGF in 5% O2 and 5%CO2 until passage 5 (P5). At P6, they were divided into two groups, one treated with the TGFβR inhibitor, A83-01 (1µM) and other with 0.01% DMSO vehicle and cultured for 7 days, then analysed for expression of MSC markers, colony-forming ability (CFU) and in-vitro mesodermal lineage differentiation. qTR-PCR was used to determine differential expression of pluripotency genes. Flow cytometry was used to quantify autofluorescence, cell cycle status and apoptosis using Annexin V. Results: Cultured eMSCs from older women (>40 years) rapidly lose W5C5 marker expression compared to those from younger women (