Surface modification and characterisation of natural ...

Int. J. Biomedical Engineering and Technology, Vol. 9, No. 2, 2012

101

Surface modification and characterisation of natural polymers for orthopaedic tissue engineering: a review Mahesh Kumar Sah and Krishna Pramanik* Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela, Rourkela 769008, Orissa, India E-mail: [email protected] E-mail: [email protected] *Corresponding author Abstract: Tissue engineering has emerged as permanent solution to treat people suffering from severe pain because of tissue loss and damage. In this technique, the key challenge is the development of scaffold with desirable characteristics like mechanical strength, biomimic properties and surface characteristics. Natural polymers are more biocompatible and induce mineralisation of bone/cartilage tissue as compared to the commonly used synthetic polymers (e.g., PLA and PLGA). This paper describes the various types of natural biopolymers for the development of tissue engineered scaffolds, mode of applications, surface modification and characterisation. A combination of modification and analysis techniques ensures better interpretation of results. Keywords: surface modification; engineering; surface analysis.

natural

polymers;

scaffold;

tissue

Reference to this paper should be made as follows: Sah, M.K. and Pramanik, K. (2012) ‘Surface modification and characterisation of natural polymers for orthopaedic tissue engineering: a review’, Int. J. Biomedical Engineering and Technology, Vol. 9, No. 2, pp.101–121. Biographical notes: Mahesh K. Sah is a PhD candidate in the Department of Biotechnology and Medical Engineering at National Institute of Technology Rourkela, India. He received his BTech (Biotechnology) in 2007 from IET, Bundelkhand University, Jhansi (India) and joined NIT Rourkela. His interests include biomaterials processing and application for bone and cartilage tissue engineering, nanotechnology and stem cell research for efficient and improved tissue regeneration. He has won a number of prizes and awards and more than ten publications (journals and conferences) goes to his credit. He is member of Society for Biomaterials and Artificial Organs (India). Krishna Pramanik is a Professor in the Department of Biotechnology and Medical Engineering at NIT Rourkela. She received her PhD in Reaction Engineering from the Calcutta University in 1995. Her research interests in biomaterial and tissue engineering, cryopreservation of cells and cell-scaffold constructs, nanobiotechnology, topology of biopolymers. He has 20 years of experience in teaching, research and industry and a large number of publications (about 50nos.) in journals and conferences both at national and

Copyright © 2012 Inderscience Enterprises Ltd.

102

M.K. Sah and K. Pramanik international level goes to her credit. She has been associated with a number of prestigious professional bodies.

1

Introduction

Most frequent, traumatic and expensive ailments in human health care are caused due to the loss or failure of an organ or tissue. Severe damage of tissue or organ due to developmental abnormalities, trauma, infection or aging-related degeneration results in disability and extensive pain (e.g., osteoarthritis). Millions of people every year suffer from these tissue-related diseases like bone, cartilage, skin, burn, etc., worldwide (Goldstein and Bonadio, 1998; Ana, 2000). Current clinical treatments such as auto grafting or allografting and the use of synthetic materials such as metals and bone cements for bone and cartilage defects have several limitations such as improper and incomplete defect healing (Mikos and Temenoff, 2000). Though autograft transplantation is the preferred treatment but suffers limited supply and donor site morbidity (Younger and Chapman, 1989; Yaszemski et al., 1996). Whereas allografting introduces the risk of immunorejection that may cause lessening or complete loss of the bone inductive factors (Muschler et al., 1993; Bostrom and Mikos, 1997). Metals or bone cements have often resulted in complications such as stress, shielding-induced resorption of the surrounding bone and fatigue of the implant (Masuda et al., 1993). Therefore, in recent years research work has been directed towards the development of celluralised scaffolds for the regeneration of various tissues including bone and cartilage to treat the patient suffering from these diseases by the tissue engineering approach (Kvitas et al., 2002; Sittinger et al., 1996). However, there is need to develop suitable implants and their filler materials for improved reconstruction of large orthopaedic defects mechanically more suitable to their microenvironment (Brown and Cruess, 1982). This paper describes the various types of natural biopolymers, techniques to achieve better morphology of scaffold, Surface modification and techniques to analyse the surface characteristics of the tissue engineered scaffold. It is notable that polymer surface science has got broad spectrum of techniques and approaches, full of controversies, thus unmanageable to give a complete review at a place. This review is by no means includes all previous works, rather the aim of the paper is to make better understanding of the polymer application in tissue engineering area.

2

Scaffold materials for bone tissue engineering

The potential tissue engineered scaffold materials for bone defects are osteoinductive and osteoconductive. Osteoinduction causes pluripotent cells to differentiate into new bone tissue formation (Urist et al., 1967; Urist, 1994). These materials are necessary for bone repair in a location that would normally not heal itself if left untreated (Bostrom and Mikos, 1997). Whereas osteoconductive materials give support to host body for ingrowth of three dimensional structure for bone formation in which healing can occur without treatment. Researchers have found that osteoconductive properties of biopolymers depend on their location and the structure of the polymers (Kulkarni et al., 1971). The absorbable polymers for long bone defects promote bone growth by excluding

Surface modification and characterisation of natural polymers

103

surrounding soft tissue and undesired cell components from the defect location by maintaining an osteogenic-rich environment and allowing bone growth onto the polymer skeleton (Kulkarni et al., 1971). Cellular phenotype is affected by their relative hydrophobicity and percent crystallinity whereas Variations in surface charge affects cellular spreading or affinity for the surface, causing changes in phenotypic expression (Hollinger and Schmitz, 1997). There are several methods of processing the polymeric matrices including the most common method, solvent casting, particulate-leaching developed by Mikos (Burg et al., 1999). In order to refine this method, it is quite possible to modulate the pore topography and size to suit a particular cell type, e.g., osteoblasts. Since bone has very different structures depending on its function and location, pore size and porosity must be carefully modulated to control the release of a material complexed to the polymer (Whang et al., 1998; Holmes, 1979). Osteoblast proliferation is sensitive to surface topography (Mikos et al., 1994), strain or other mechanical stimuli. The adhesive, proliferative, and phenotypic properties of cells is affected by particle size, shape, and surface roughness. Cells can change topography, and they are most obviously sensitive to chemistry, topography, and surface energy. Particularly for an absorbable material, such surface features become interesting since this is a dynamic material, always presenting a new surface. The desirable characteristics of bone tissue-engineering scaffold (Levine et al., 1997; Peter et al., 1998) are as follows: •

suitable mechanical and physical properties for application

•

absorbs in predictable manner in concert with bone growth

•

does not induce soft tissue growth at bone/implant interface

•

average pore sizes approximately 200–400 µm

•

maximal bone growth through osteoinduction and/or osteoconduction

•

support to mineralisation

•

adaptability for irregular wound sites

•

no detrimental effects to surrounding tissue due to processing

•

sterilisable without loss of properties.

2.1 Types of natural polymers The biomaterial used for the development of scaffolds should be biocompatible, biodegradable and bioresorbable, non-toxic supporting cellular interactions and in vivo tissue formation with required physical and mechanical properties. Animal or plant derived proteins have been shown to be used as scaffold material for tissue engineering applications. An important aspect of natural materials is the induction of objectionable immune reaction due to the presence of endotoxins and impurities depending upon the source of material. Also, their quality may differ batch wise during large scale isolation and processing. Polymers from natural sources include •

proteins

•

polysaccharides

•

polyesters are considered to be potential candidates for scaffold preparation.

104

M.K. Sah and K. Pramanik

2.1.1 Protein-derived polymers The most important protein derived polymers used for scaffold preparation are Collagen and Silk proteins. Type I Collagen being the most abundant and investigated for tissue engineering applications (Hayashi, 1994). Heat treatments and/or Chemical glycation procedures are used to fabricate matrices with adequate mechanical properties. Collagen microsponges into synthetic polymeric scaffolds increase their mechanical performance (Chen et al., 2000). Collagen-based scaffolds, combined with active agents like growth factors have more therapeutic influence on tissue engineering approaches (Wallace and Rosenblatt, 2003). Silk proteins contain a highly repetitive primary sequence of simple amino acids that leads to a high content of β-sheets and responsible for good mechanical properties. Research have shown the utilisation of silk fibroin in tissue engineering applications, particularly where high mechanical strength and slow biodegradation is required (Altman et al., 2003). Experimental studies of MSCs seeded highly porous silk scaffolds for in vitro cartilage (Wang et al., 2005) and bone (Kim et al., 2005) regeneration have shown positive results.

2.1.2 Polysaccharides Polysaccharides have wide applications in tissue engineering particularly for the enhancement of mechanical properties. These biopolymers can broadly be divided into four major groups according to the source from which these are obtained. The polysaccharides are subdivided, based on their chemical structure. Starch and cellulose are the most important plant saccharides having special application in tissue engineering. The cohesive and hydrogen-bonded structure of cellulose fibres makes it exceptionally water insoluble with great strength exhibiting poor degradation in vivo. Alginate, an algal polysaccharide has potential to combine with calcium (Percival and McDowell, 1990) and this property can be utilised for mineralisation for orthopaedic tissue engineering. Galactose are also obtained from algae. Chitin and its derivative chitosan obtained from animal exoskeleton have been studied for bone regeneration and have been found with excellent biodegradability and cell-adhesive property. Hyaluronic acid, an important GAG component has been used for finishing of scaffold surface for improving its biocompatibility. Non-toxicity of Pullulan and unique rheological properties of xantan gum from microbial culture also finds application in vivo. Bacterial Cellulose (BC) has high water- holding capacity, crystallinity and biocompatibility giving it high tensile strength in wet state.

2.1.3 Naturally derived polyesters Polyhydroxyalkanoates (PHAs) are thermoplastic polyesters showing good biodegradability and biocompatibility is obtained from micro-organisms. Among these, PH(Butyrate) and PH(Butyrate- co-Valerate) are commonly used for tissue engineering applications. Materials more flexible, less crystalline and easy to process can be produced by copolymerisation from pure PHB. These materials can be used as support material for tissue engineering application.


105

2.2 Bone regeneration The major challenge concerned with polymeric scaffolds for application in bone tissue engineering is due to low mechanical strength and shape retention failure. It has been shown that silk-based scaffolds support human MSC (hMSC) adherence and proliferation in vitro. However, different responses were observed for bone-like tissue formation depending on materials and processing conditions. Due to fast degradation of collagen scaffolds, isomorphous replacement with a newly formed bone does not take place. In comparison with collagen scaffolds, silk and RGD–silk scaffolds were more supportive to osteogenesis due to their stable macroporous structure and slow degradation (Meinel et al., 2004). Other studies have proved that the crosslinking (Meinel et al., 2004) or the composite of collagen with materials having improved mechanical properties (Kose et al., 2004) forms scaffold robust enough to support bone regeneration. Due to high degree of swelling, some chitosan scaffolds are mechanically weak and instable. The increase in mechanical strength can be obtained not only by changing the process (Seol et al., 2004), but also by chemical bonding with other polymers such as alginate (Li and Zhang, 2005). A nano- and microfibre combined starch-based scaffold (Tuzlakoglu et al., 2005) showed unique architecture, being able to support cells as well as to provide supply of nutrient and gas to cells for bone regeneration.

3

Mode of application of naturally derived polymers for bone tissue engineering

The following approaches are recently evolved are being studied and employed presently by the researchers for bone tissue engineering applications as shown in Figure 1. Figure 1

Different approaches generally employed for bone tissue engineering applications (see online version for colours)

106


3.1 Acellular systems Acellular system was classified as materials for which no additional cellular component is cultured. Urist and several co-workers (Urist, 1994) reported the demineralised bone consistently induced bone formation in ectopic tissues of experimental animals. A hydrochloric acid extraction process decalcified the bone matrix, producing Demineralised Bone Matrix (DBM), a compound that was found to possess inherent osteoconductive and osteoinductive properties.

3.2 BMP systems Yasko et al. (1992) created 5 mm defects in the femora of 45 adult rats. By showing a 100% union rate using a combination of rhBMP 2 and DBM as a carrier, they concluded that BMP might prove to be a bone graft substitute of unlimited quantity. Reddi and Levine (Reddi, 1998) reported both cite insoluble collagen as a potential carrier for BMP, but the data are limited for this matrix. The implantation of purified, hydrophilic BMP promotes their dispersion shortly after implantation and before osteoinduction occurs (Nakahara et al., 1989). They are normally present in complex with carriers to prevent the dispersion and to deliver the BMPs slowly to the desired sites.

3.3 Cellular systems The collagen materials can be applied as cellular scaffolding systems. Since collagen possesses no inherent structural mechanical properties, engineering modifications of the material can form a stiffer polymer to enhance load bearing capacity of bone during the regenerative phase of healing. When treated with calcium solution, deposition of calcium phosphate improves mechanical integrity (Yaylaoglu et al., 1999). This technique has shown great promise in culture of chondrocyte and bone tissue engineering as well. Collagen sheets used to fabricate composite bone tissue-engineering scaffolds have already been reported (Du et al., 1999).

4

Surface nano-patterning of scaffolds

The types of nanotopographical features that can be created on materials are of two categories: unordered topographies and ordered topographies. Unordered topographies are typically those that spontaneously occur during processing. The techniques include – polymer demixing, colloidal lithography and chemical etching. These techniques are usually simpler, quicker, and less costly than the more complex equipments and processes needed to create ordered topographies. Ordered topographies are those that can be created with techniques like photolithography and electron beam lithography. Table 1 compares different nanoscale topography fabrication methods.

Surface modification and characterisation of natural polymers Table 1

107

Comparison of different nanoscale topography fabrication methods

Fabrication method Advantages

Disadvantages

Electron beam lithography

Precise geometries and patterns is computer-controlled

Expensive equipment, time consuming, lowers resolution

Electro-spinning

Can be used with biological polymers such as collagen

Can only create fibres

Colloidal lithography

Easier to pattern larger areas than with EBL

Specific feature geometries not possible

Chemical etching

Simple, fast, inexpensive

Specific feature geometries not possible Only sample features can be created

Polymer demixing

Simple, inexpensive and fast

Phase separation

Good for creating porous scaffolds, No organised pattern possible Porosity is easily controlled

Self-assembly

Relatively easy fabrication, inexpensive, precise control over pore size and distribution

Strength of materials can be insufficient for in vivo application

4.1 Electrospinning Electrospinning can be used to create a simple ordered topography of aligned fibre bundles. Controlled fabrication of model substrates allow for a systematic study of surface topographies and their effects on a variety of growth parameters. Study by Sepideh et al. reveals that no additional surface modifications is required for electrospun fibres since they support attachment and proliferation of mesenchymasl stem cells (Hagvall et al., 2008).

4.2 Electron beam lithography Electron beam lithography is used to create surface topographies at the nanometer scale for studying cellular growth and behaviour on these surfaces (Vieu et al., 2000). This method involves the use of high-energy electrons to expose an electron-sensitive resist (Madou, 2002) and has the ability to create single surface features down to about 3–5 nm (Vieu et al., 2000).

4.3 Photolithography This method can create precise geometries and patterns. It allows for investigators to observe a large population of cells in order to gain a significant understanding of the mechanisms of single-cell surface interactions. Dalby et al. (2004) investigated fibroblast response to arrays of nanopits created using EBL. Fabrication using EBL is time consuming and costly. To overcome this, nanometer patterns can be replicated in polymeric materials, making the ability to mass reproduce the patterns by a much faster and inexpensive process (Abrams et al., 2000). This can dramatically cut the time and methods for fabrication of nanoscale topography on bone/cartilage tissue engineering scaffolds.

108


4.4 Polymer demixing A unique method for creating nanoscale topographical features for use as cell substrate is through the use of polymer demixing. It involves the spontaneous phase separation of polymer blends which occurs under conditions such as spin casting onto silicon wafers (Affrosman et al., 1996). Polymer demixing can be used to create topographies similar to those commonly used to study cell growth on nanostructured surfaces. Polymer demixing may not be ideal for creating model surfaces to study cellular interactions with nanoscale features. Despite this limitation, nanotextured surfaces created by polymer demixing have been used to study the interactions of human foetal osteoblastic cells and other fibroblast cells to nanotopographies of various heights (Lim et al., 2005; Dalby et al., 2002).

4.5 Colloidal lithography Colloidal lithography is an inexpensive method for creating nanoscale topographies. This technique allows for the production of surfaces with controlled heights and diameters. Colloidal lithography involves the use of nanocolloids as an etch mask. These nanocolloids are dispersed as a monolayer and are electrostatically self assembled over a surface. To vary the surface structure, the coverage of the monolayer of colloid and the size of the colloid can be varied. The spacing between particles can be controlled by changing the ionic strength of colloid solution. Patterning with particle sizes of 20 nm has been demonstrated (Wood et al., 2002). With this technique, large surface areas can be patterned (∼cm2), making it a suitable technique for creating functional biomaterials for cell studies (Hanarp et al., 1999). Repeated adsorption of particles and charging of the substrate results in multilayer formation, finally fabrication of controlled 3D nanoporous particle films. Research is under way for in vitro cell studies of how nanotopography influences osteoblast (bone forming cell) adhesion and morphology.

4.6 Chemical etching This method produces nanoscale features on the surface of a material by soaking it in an etchant. Typical etchants are Hydrofluoric Acid (HF) and sodium hydroxide (NaOH). As the material is etched away, the surface is roughened creating pits and protrusions at the nanometer scale. This process is essentially a surface treatment and cannot create structures with any prescribed geometry or organisation. It can, however, provide a very quick, easy, and inexpensive means of creating a nanostructured surface by changing the scale of the roughness on the material surface. Three dimensional scaffolds with nanoscale features can be made by this method. The nanoroughened surface not only increase adhesion but provides more room for cell adhesion and population to grow into. Increased porosity also allows for greater infiltration of the scaffold by cells as well as increased nutrient and waste diffusion. Sun et al. (2007) investigated the osteoconductivity of PSi produced by electrochemical etching using nano, meso and macro scale pores in vitro.


5

109

Surface modification methods

The success of tissue growth depends inevitably on the cellular interaction with scaffold material as the nature of surface directly affects the cellular behaviour. Cell adhesion on the surface is determined by surface chemistry as well as its topography. The natural polymers provide the ideal environment for cell-material interactions. Therefore, various surface modification methods for natural biopolymers are being studied and is an active area of research. Two main strategies in surface engineering of biomaterials are often employed based on the understanding of the dominance of the biorecognition process on cell behaviours. The normal bioactivities of the adsorbed proteins is maintained by surface properties such as chemical composition, hydrophilicity/hydrophobicity, surface charge and roughness. Such nonspecific protein absorption does not induce specific cell behaviour. So the strategy to directly immobilise certain biomolecules on the biomaterial surfaces is more effective to induce specific cellular response (Zuwei et al., 2007). For bone tissue engineering common methods include abrasion and sand blasting, chemical treatment and surface activation. Several surface activation techniques employed are atom bombardment, plasma treatment, ion implantation, laser treatment, electron beam and welding depending on the type of biomaterial. The various methods of surface activation have been categorised in Table 2 whereas Table 3 compares the surface modification methods in terms of various topographical, cell/proteins and interfacial properties. Table 2

Methods of surface activation

Physical

Chemical

Radiation

Physical irradiation

Chemisorption

Plasma treatment

Adsorption

Oxidation (by strong acids)

Corona discharge

Langmuir-blodgett film

Ozone treatment

Photo activation (UV)

Flame treatment

Laser ion beam Electron beam

Table 3

The comparison of various popular surface modification processes

Biocompatibility

Plasma

UV-activation

Ion-beam

Corona

Hydrogel

+

+

Limited

+

+

Dry lubricity

Limited

Limited

+

–

–

Wet lubricity

+

+

Limited

Limited

+

Blood compatibility

+

+

Limited

–

+

Reduce bacterial adherence

+

+

+

–

+

Reduce protein adsorption

+

+

–

–

Limited

Immobilise biomolecules

+

+

–

Limited

–

Improve cell adhesion

+

+

Limited

+

–

Improve tissue integration

+

+

+

Limited

–

Improve adhesion

+

+

–

+

–

Hydrophilicity

110


5.1 Chemical modification Surface chemistry of biomaterial can influence surface adhesion as well as physiological pathways regulating cellular proliferation, differentiation, and survival. Improved surface chemistry of a biomaterial also aid in construction of an artificial tissue mimicking with native tissue in physical properties (Hutmacher, 2000). For bone tissue engineering, polymeric scaffolds modified with charged groups that are conducive to mineralisation can facilitate premineralisation of the scaffold with hydroxyapatite (Song et al., 2003) and induce differentiation of stem cells into an osteogenic lineage (Mauney et al., 2004). Likewise, cartilage tissue formation can be induced by use of sulphated polymers (Chen et al., 2007). For silk, the derivatisation of the carboxylic acid residues through carbodiimide coupling with primary amines is the most commonly used chemical modification method (Sofia et al., 2001). A simpler and effective method to tailor the structure and hydrophilicity of silk fibroin protein was developed using diazonium coupling chemistry (Amanda et al., 2008). Introducing hydrophobic functional groups cause rapid conversion of the protein from a random coil to a β-sheet structure, while addition of hydrophilic groups inhibits the process. Novel surface chemical modification approaches are under way, including (Chen et al., 1997): •

plasma polymerisation technology

•

tailoring di-peptide monolayers to induce surface wettability

•

self-assembly of nanoscale templates to induce biofunctionality of surface.

5.1.1 Gamma irradiation Gamma irradiation of surface enhance its surface properties by cross linking long chains. Yang et al. (2002) did experiment on the chitosan membranes irradiated by gamma rays when processed using infrared spectroscopy and mechanical properties was found to be improved. The infrared spectra of the chitosan membranes with and without irradiation were compared to investigate the mechanism for modification of the chitosan membranes induced by the gamma irradiation (Yang et al., 2002). The experimental results show that the gamma irradiation in the specified dose range can improve the mechanical properties of chitosan membranes while maintaining their excellent biocompatibility. Generally, two factors induce changes of mechanical properties in polymeric membranes: crosslinking between molecules and crystallinity (Yang et al., 2002; Wang, 1995). According to previous reports (Wang, 1995; Carlosand and Suarez, 1999), gamma irradiation affects the geometric regularity of the structure and the material crystallinity is closely related to its mechanical properties. Therefore, the gamma radiation effect on the crystallinity is one factor which improves the chitosan mechanical properties. Free groups, such as amido, alkyl, hydroxyl etc. in this polymeric saccharide long chain produce radicals. So crosslinking between radicals is another possible factor inducing the mechanical property improvements. The crystallinity reaches a maximum value for the gamma irradiation dose in an appropriate range. For macromolecular materials, enhanced crystallinity improves their rupture intensity, breaking elongation, Young’s modulus and other mechanical properties (Li and Zhang, 1996).


111

5.2 Plasma surface modification Plasma processes have been developed to attain specific surface properties of biomaterials. Plasma treatment offers flexibility, effectiveness, safety and environmental friendliness. The plasma is effective at near-ambient temperature without damage for most heat-sensitive biomaterials. Plasma treatment modifies only the near surface of treated substrates and does not change the bulk material properties. Plasma surface treatment improves interfacial adhesion by creating chemically active functional groups, such as amine, carbonyl, hydroxyl, and carboxyl groups. The process can also be used to alter surface energies according to the application. Polymers interaction with gas plasma can create hydrophilic and hydrophobic surfaces whereas the wettability of the surface will be increased by creating hydroxyl functionality using oxygen. In a similar way, surfaces can be specifically engineered to modify protein binding and improve blood compatibility. Plasma processes have been employed to modify the surface of metal implants for adhesion promotion to bone cements, or enhancing cell attachment and growth. The degradation rate of bioabsorbable polymer has been shown to be regulated by Plasma crosslinking at surface (Belcourt, 1999).

5.2.1 Atomic oxygen surface modification Atomic oxygen surface modification and texturing technology is being evaluated for use in a number of biomedical applications. It is useful for changing the wetting characteristics of surfaces, improving cell growth and adhesion and removing biologically active contaminants such as endotoxins, from the surfaces of orthopaedic implants. Benefits include surface texturing of polymers for cell growth and endotoxin removal. Researchers at the NASA Glenn Research Center also have used this process as an innovative approach to solve the problem of endotoxin contamination on the surfaces of orthopaedic implants. NASA’s approach to remove endotoxin is an atomic oxygen treatment process that occurs in a low-pressure (4–100 microns) air plasma and at a relatively low temperature (