Thermoresponsive Hydrogels for Cartilage Tissue

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Thermoresponsive Hydrogels for Cartilage Tissue Engineering - Design, Synthesis and Material Properties Master of Science Thesis

MARKUS ANDERSSON Department of Chemical and Biological Engineering Biopolymer Technology CHALMERS UNIVERSITY OF TECHNOLOGY Göteborg, Sweden, 2007 Report No. 343

DIPLOMA THESIS NR 343 Department of Chemical and Biological Engineering / Biopolymer Technology

Thermoresponsive Hydrogels for Cartilage Tissue Engineering - Design, Synthesis and Material Properties

Termoresponsiva hydrogeler för Brosk Tissue Engineering - Design, Syntes och Material Egenskaper

Front page pictures; Thermo association of graft copolymer hydrogel. Above; Solution before and after grafting reaction Below; Schematic drawing of the polymers in solution. Left side; below association temperature. Right side; above association temperature.

Conducted by: Markus Andersson Supervisors: Mats Andersson Paul Gatenholm Examiner: Paul Gatenholm

Approved date:

__________________ Examiner´s name

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Sammanfattning

Sammanfattning Det är en stor efterfrågan på kliniska strategier för reparation av broskskador och olika procedurer har utvecklats de senaste decennierna. Ett flertal tekniker anammar användandet av solida scaffolds inom vilka chondrocyter prolifereras och tillverkar ny vävnad. Ett betydande problem avseende dessa strategier är bristfällig integrering mellan den nybildade och den nativa broskvävnaden. Ett sätt att överkomma denna problematik är nyttjandet av vätskeformiga polymerlösningar med in situ gelande egenskaper. Denna senaste innovation inom området för tissue engineering har möjligheten att fylla alla slags defekter med material. Termoresponsiva polymerlösningar är tänkbara kandidater för en sådan applikation. Termotjockande polymerer designades, bestående av en vattenlöslig hydrofil polysackaridhuvudkedja (WHB) med LCST-polymerer som sidokedjor. Natrium hyaluronat valdes som polysackarid-huvudkedja på grund av dess stora förekomst och betydelse i broskvävnad. Dessutom användes även karboxymetylcellulosa som huvudkedja på grund av dess intressanta egenskaper i vattenlösning. Polyetrar bestående av statistiska kopolymerer av etylenoxid (EO) och propylenoxid (PO) användes som LCST polymerer. Fasbeteendet hos polyetern bestäms av monomersammansättningen och de relaterade block-kopolymererna av EO och PO har blivit godkända av FDA för in vivo bruk. Grumlingspunktskurvor för olika polyetrar upprättades för att kunna skräddarsy gelbeteendet hos graft kopolymererna. LCST polymerens uppgift är att mikrofasseparera som funktion av en temperaturhöjning med en följande coil to globule fastransition. Ett gelnätverk upprättas när LCST sidokedjorna deltar i intermolekylära mikrofasseparerade aggregat. Makroskopisk fasseparation förhindras av den starkt hydrofila och vattenlösliga polysackarid huvudkedjan som sammanbinder de termiskt inducerad mikrodomänerna. Graft kopolymeren syntetiserades via EDC/NHS medierad amid formation mellan amino ändgruppen på polyetern och karboxylaten på polysackarid huvudkedjan. Syntes produkten renades upp med dialys följt av frystorkning och karaktäriserades med FTIR, NMR, SEC, elementar analys samt grumlingspunkts observationer och lutande vial metoden. Reaktionen var näst intill kvantitativ, men formation av EDC N-acylurea produkt på polysackariden kunde ej uteslutas. En steriliserings metodik av materialet upprättades baserat på filtrering av graft kopolymeren i utspädd vattenlösning. Proceduren möjliggjorde en effektiv och harmlös steriliseringsmetod med högt utbyte. Graft kopolymerer bestående av hydrofoba polyetrar besatt utomordentliga termogelande egenskaper inom rimligt temperatur intervall. Viskositeten samt förlust modulen och speciellt lagringsmodulen ökade med flertal magnituder som en funktion av temperaturen. Associationens begynnande var i direkt förbindelse med fas beteendet hos polyeter sidokedjan och den termotjockande effekten var kontinuerlig. Vid höga temperaturer uppträdde hydrogelen dessutom som ett fast viskoelastiskt material inom det tekniska frekvensområdet, med formation av en styv gel struktur. Ytterligare tänkbara förbättringar av graft kopolymerens struktur och egenskaper kan utföras. Mest signifikant är nyttjandet av längre polyetersidokedjor vilket möjliggör större lätthet att styra begynnandet av associationen med polyeterns fasbeteende. Längre polysackarid huvudkedjor gör det tänkbart att skapa hydrogeler vid mycket lägre koncentrationer. Ett pågående arbete utförs för att undersöka livsdugligheten av prolifererande chondrocyter i dessa nya biomaterial, samt påföljande formation av broskspecifik ECM vävnad (kollagen II samt proteoglykaner). Förekomst av specifik interaktion mellan kondrocyterna och det hyaluronat baserade biomaterialet måste analyseras. För att specifikt adressera kondrocyterna och i synnerhet en subpopulation av kondrocytprogenitor celler i den ytliga ledbrosk zonen, är det plausibelt att inkorporera RGD-peptid sekvenser i biomaterialet vilket kan åstadkommas med analog EDC/NHS medierad reaktionsmetodik.

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Abstract

Abstract As a consequence of the immense clinical demand for repair strategies concerning articular cartilage defects, different approaches have been developed during the last decades. Many techniques applies chondrocytes proliferated in solid scaffolds. These repair strategies render insufficient integration properties between the repair tissue and the native tissue. In order to subdue this obstacle, the latest innovation, liquid polymer solutions, with the possibility to fill any defect, with subsequent in situ gelling properties may be applied. Thermoresponsive polymer aqueous solutions are plausible candidates for in situ gelling systems. Thermothickening polymers were designed, comprising a polysaccharide WHB (Water soluble Hydrophilic Backbone) with LCST (Lower Critical Solution Temperature) polymers as sidechains. Sodium hyaluronate was chosen as polysaccharide backbone due to its abundance and importance in cartilage tissue. Carboxymethylcellulose was employed as well, ascribable to the intriguing solution properties of the cellulose derivate. As LCST polymer, polyethers constituting random copolymers of ethylene oxide (EO) and propylene oxide (PO) were employed. The phase behaviour is regulated by monomer composition and the related block copolymers of EO and PO have been FDA approved for in vivo use. In order to tailor the gelling properties of the graft copolymer, cloud point curves of different polyethers were conducted. It was established that the cloud point was reduced as a function of molecular weight and/or PO composition. The function of the LCST grafts is to commence in a microphase separation with a subsequent coil to globule phase transition as the temperature is increased. A gel network is created as the LCST grafts participate in intermolecular microphase separated aggregates. Macroscopic phase separation is prevented by the strong hydrophilicity of the WHB, connecting the thermally induced microdomains. The graft copolymers were synthesized by EDC/NHS mediated amide formation between the terminal amine group of the polyether and the carboxylate of the polysaccharide backbone. The product was purified by dialysis against distilled water followed by lyophilization and characterized with FTIR, NMR, SEC, elementary analysis as well as cloud point behaviour and vial tilting method of the solution. The reaction was almost quantitative but some formation of EDC N-acylurea adducts could not be excluded. A sterilization methodology of the material was established based on sterilization by filtration of the graft copolymer in dilute aqueous conditions. The procedure provides an efficient and gentle manner for sterilization with high yields. Graft copolymers consisting of hydrophobic polyethers possessed excellent thermogelling properties in the appropriate temperature interval. The viscocity as well as the loss modulus and in particular the storage modulus increased by several magnitudes as a function of temperature. The onset of association was directly connected to the phase behaviour of the polyether graft and the thermothickening effect was continuous. The hydrogel behaved as a solid viscoelastic material at elevated temperature in the frequency window of observation with subsequent formation of stiff gel structures. Further feasible improvements of the graft copolymer structures may be addressed. Most importantly, longer polyether grafts grants the onset of association to be tailored by the phase behaviour of the polyether with more ease. Longer WHB:s would enable hydrogel formation at much lower concentrations. There’s an ongoing work to investigate the viability of proliferating chondrocytes in this novel biomaterial as well as the formation of cartilaginous ECM (collagen II and proteoglycans). Specific interaction between the chondrocytes and the hyaluronan based biomaterial needs to be analysed. In order to specifically address the chondrocytes and especially a chondrocyte progenitor subpopulation residing in the superficial layer of articular cartilage, it’s conceivable to incorporate supplementary RGD peptides onto the graft copolymer which may be achieved by the same EDC/NHS reaction pathway. ii

Table of Contents

Table of Contents Sammanfattning ....................................................................................................................................................... i Abstract ................................................................................................................................................................... ii Table of Contents ................................................................................................................................................... iii List of Abbreviations ............................................................................................................................................. vi List of Figures and Schemes ................................................................................................................................ viii List of Tables ......................................................................................................................................................... xi 1 Introduction .......................................................................................................................................................... 1 2 Theory .................................................................................................................................................................. 2 2.1 Cartilage ....................................................................................................................................................... 2 2.1.1 Cartilage structure................................................................................................................................. 2 2.1.2 Cell morphology and zonal architecture ............................................................................................... 3 2.1.2.1 Superficial zone ............................................................................................................................ 3 2.1.2.2 Transitional zone........................................................................................................................... 4 2.1.2.3 Radial zone ................................................................................................................................... 4 2.1.2.4 Calcified zone ............................................................................................................................... 4 2.1.3 Extra cellular matrix ............................................................................................................................. 4 2.1.3.1 Collagen ........................................................................................................................................ 5 2.1.3.2 Proteoglycans ................................................................................................................................ 6 2.1.3.3 Miscellaneous ............................................................................................................................... 6 2.1.3.4 Cell to matrix organization ........................................................................................................... 7 2.2 Gels .............................................................................................................................................................. 8 2.3.1 Physical gels ......................................................................................................................................... 8 2.3.1.1 Thermoreversible gels ................................................................................................................... 8 2.3.1.1.1 Thermothinning, crystallization ............................................................................................... 8 2.3.1.1.2 Thermothickening, hydrophobic interaction ............................................................................ 9 2.3.1.1.3 Thermothinning, hydrophobic interaction .............................................................................. 11 2.3.1.2 pH dependent gels ....................................................................................................................... 12 2.3.1.3 Ionically crosslinked gels ............................................................................................................ 13 2.3.1.4 H-bond crosslinked gels .............................................................................................................. 14 2.3.1.5 Shearthinning gels....................................................................................................................... 14 2.3.1.6 Gels crosslinked via protein interactions .................................................................................... 15 2.3 Thermothickening Polymers ...................................................................................................................... 16 2.3.1 Poloxamer block copolymers ............................................................................................................. 16 2.3.1.1. The hydrophobic hydrophilic balance ........................................................................................ 18 2.3.2 WHB:s with LCST grafts ................................................................................................................... 20 2.3.2.1 LCST grafts................................................................................................................................. 21 2.3.2.1.1 Poly(EO-stat-PO) copolymers solution theory ....................................................................... 25 2.3.2.1.1.1 Enthalpy and Entropy ...................................................................................................... 27 2.3.2.1.1.2 Effect of additives ........................................................................................................... 32 2.3.2.1.1.2.1 Salt............................................................................................................................ 32 2.3.2.1.1.2.2 Neutral molecules ..................................................................................................... 34 2.3.2.1.1.2.3 Surfactants ................................................................................................................ 37 2.3.2.1.1.2.4 Solvent-solute-cosolute interaction .......................................................................... 38 2.3.2.2 Water soluble hydrophilic backbone........................................................................................... 40 2.3.2.2.1 Polymer solution theory and concentration regimes .............................................................. 41 2.3.2.2.1.1 Dilute regime ................................................................................................................... 43 2.3.2.2.1.2 Semidilute unentangled regime and c*............................................................................ 44 2.3.2.2.1.3 Semidilute entangled regime and c e ................................................................................ 48 2.3.2.2.2 Hyaluronic acid ...................................................................................................................... 49 2.3.2.2.3 Carboxymethylcellulose ......................................................................................................... 55 2.3.3 The microaggregates........................................................................................................................... 56 2.3.3.1 Applied micellar theory .............................................................................................................. 58 2.3.3.1.1 Polyether aggregates............................................................................................................... 58 2.3.3.1.2 C 12 aggregates ........................................................................................................................ 61 2.3.3.1.3 PNIPAAm aggregates ............................................................................................................ 63 2.4 Transient Network Theory ......................................................................................................................... 64 2.4.1 Temperature domains ......................................................................................................................... 67 2.4.2 Enthalpy of demicellization ................................................................................................................ 68 iii

Table of Contents 2.5 Coupling Reaction ...................................................................................................................................... 70 2.5.1 EDC .................................................................................................................................................... 70 2.5.2 NHS/sulfo-NHS .................................................................................................................................. 71 3 Materials and Methods ....................................................................................................................................... 73 3.1 Materials ..................................................................................................................................................... 73 3.2 Cloud Point Measurements......................................................................................................................... 74 3.3 Characterization of Prepolymers ................................................................................................................ 74 3.3.1 NMR ................................................................................................................................................... 75 3.3.2 MALDI-TOF ...................................................................................................................................... 75 3.3.3 SEC ..................................................................................................................................................... 75 3.3.3.1 Refractive index increment ......................................................................................................... 76 3.4 Synthesis .................................................................................................................................................... 77 3.4.1 Purification ......................................................................................................................................... 78 3.5 Characterization of graft copolymers ......................................................................................................... 79 3.5.1 IR ........................................................................................................................................................ 79 3.5.2 NMR ................................................................................................................................................... 79 3.5.3 SEC ..................................................................................................................................................... 79 3.6 Rheology .................................................................................................................................................... 79 3.7 Sterilization ................................................................................................................................................ 80 4 Results and Discussion....................................................................................................................................... 82 4.1 Cloud Point Measurements......................................................................................................................... 82 4.2 Characterization of Prepolymers ................................................................................................................ 84 4.2.1 Polyethers ........................................................................................................................................... 84 4.2.1.1 M-2070 ....................................................................................................................................... 84 4.2.1.1.1 NMR....................................................................................................................................... 84 4.2.1.1.2 MALDI-TOF .......................................................................................................................... 86 4.2.1.2 M-600 ......................................................................................................................................... 89 4.2.1.2.1 NMR....................................................................................................................................... 89 4.2.1.2.2 MALDI-TOF .......................................................................................................................... 89 4.2.1.3 M-2005 ....................................................................................................................................... 89 4.2.1.3.1 NMR....................................................................................................................................... 89 4.2.1.3.2 MALDI-TOF .......................................................................................................................... 89 4.2.2 Polysaccharides .................................................................................................................................. 91 4.2.2.1 Refractive Index Increment......................................................................................................... 91 4.2.2.2 SEC ............................................................................................................................................. 92 4.3 Synthesis .................................................................................................................................................... 92 4.4 Characterization of Graft Copolymers ....................................................................................................... 93 4.4.1 Cloud Point observation and vial tilting method ................................................................................ 93 4.4.2 SEC analysis ....................................................................................................................................... 94 4.4.3 IR ........................................................................................................................................................ 95 4.4.4 NMR ................................................................................................................................................. 101 4.4.5 Elementary analysis .......................................................................................................................... 102 4.5 Rheology .................................................................................................................................................. 103 4.5.1 Steady Shear Viscosimetry ............................................................................................................... 103 4.5.1.1 Temperature sweeps and the thermothickening effect .............................................................. 104 4.5.1.2 Flow curves and temperature regimes....................................................................................... 110 4.5.2. Oscillatory Rheometry ..................................................................................................................... 113 4.5.2.1 Threshold gelpoint .................................................................................................................... 113 4.5.2.2 Frequency sweeps and viscoelastical properties ....................................................................... 115 4.5.2.3 Amplitude sweeps and non linear network properties .............................................................. 117 5 Conclusions ...................................................................................................................................................... 122 6 Future Work ..................................................................................................................................................... 123 7 Acknowledgements .......................................................................................................................................... 124 8 References ........................................................................................................................................................ 126 9 Appendix .......................................................................................................................................................... 131 9.1 Synthesis Procedures ................................................................................................................................ 131 9.1.1 Appendix A ...................................................................................................................................... 131 9.1.2 Appendix B ....................................................................................................................................... 132 9.1.3 Appendix C ....................................................................................................................................... 133 9.1.4 Appendix D ...................................................................................................................................... 134 9.1.5 Appendix E ....................................................................................................................................... 135 iv

Table of Contents 9.1.6 Appendix F ....................................................................................................................................... 136 9.1.7 Appendix G ...................................................................................................................................... 137 9.1.8 Appendix H ...................................................................................................................................... 138 9.1.9 Appendix I ........................................................................................................................................ 139 9.1.10 Appendix J ...................................................................................................................................... 140 9.1.11 Appendix K .................................................................................................................................... 141 9.1.12 Appendix L ..................................................................................................................................... 142 9.1.13 Appendix M .................................................................................................................................... 143 9.1.14 Appendix N .................................................................................................................................... 144 9.1.15 Appendix O .................................................................................................................................... 145

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List of Abbrevations

List of Abbreviations 3D AAc ACT BCC c c* ce C 12 CAC CMC CMC CMT DME DMP DP DSC ECM EDC EHEC EO FCC G GAG GlcA GlcNAc H-bond HA HAc HLB HMSC HPC IPDI LCST LS M MALDI-TOF MALLS MC MW MWCO NIPAAm NHS NMR P(EO-b-PO) P(EO-stat-EO) PAA PAAc PAAm PAMD PBO PE PEA PEO PEOz PESc PHA PL PLA vi

three dimensional Acrylic acid autologous chondrocyte transplantation Body-Centred Cubic concentration overlap concentration entanglement concentration dodecyl Critical Association Concentration sodium carboxymethylcellulose Critical Micelle Concentration Critical Micelle Temperature dimethoxyethane dimethoxypropane Degree of Polymerization Differential Scanning Calorimetry Extra Cellular Matrix N-dimethylaminopropyl N-ethyl carbodiimide hydrochloride Ethyl Hydroxy Ethyl Cellulose ethylene oxide Face Centred Cubic guluronate/guluronic acid glycosaminoglycan D-glucuronic acid N-acetyl-D-glucosamine hydrogen bond sodium hyaluronate hyaluronic acid Hydrophiliv/Lipophilic Balance Human Mesenchymal Stem Cells Hydroxy Propyl Cellulose Isophoronediisocyanate Lower Critical Solutions Temperature Light Scattering mannurate/mannuronic acid Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mulit Angle Laser Light Scattering Methyl Cellulose Molecular Weight Molecular Weight Cut Off N-isopropylacrylamide N-hydroxysuccinimde Nuclear Magnetic Resonance poly(ethylene oxide-co-propylene oxide) block copolymer poly(ethylene oxide-co-propylene oxide) random/statistical copolymer poly(sodium acrylate) poly(acrylic acid) poly(acrylamide) poly(2,4,4-trimethylhexamethylene terephthalamide) poly(bythylene oxide) poly(ethylene) poly(ethylene adipate) poly(ethylene oxide) poly(2-ethyl-2-oxazoline) poly (ethylene succinate) poly(hexamethylene adipate) poly(lysine) poly(lactic acid)

List of Abbrevations PLGA PMA PNIPAAm PO PPO PS PVCL PVA PVIa RG RH RI SAXS SANS SC SDS SEC Sol Sulfo-NHS T T cp T ass TGT THF TTT TTS UCST VdW WHB

η0

τ

poly(lactic-co-glycolic acid) poly(methacrylic acid) poly(isopropylacrylamide) propylene oxide poly(propylene oxide) poly(styrene) poly(vinyl caprolactam) poly(vinyl alcohol) poly(vinyl imidazol) radius of gyration hydrodynamic radius Refractive Index Small Angle X-ray Scattering Small Angle Neutron Scattering Simple Cubic Sodium dodecyl sulphate Size Exclusion Chromatography solution N-hydroxysulfosuccinimide temperature cloud point association temperature. trans-gauche-trans tetrahydrofuran trans-trans-trans Time-Temperature Superposition Upper Critical Solution Temperature Van der Waals Water soluble Hydrophilic Backbone zero shear viscosity/plateau viscosity graft ratio

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List of Figures and Schemes

List of Figures and Schemes Figure 1 H&E stained cross sectional views of articular cartilage. ......................................................................... 4 Figure 2 Thermogelling polymeric tensides. ........................................................................................................... 9 Figure 3 Various block copolymers for in situ gelling systems. ............................................................................. 9 Figure 4 Chondrocytes in PNIPAAm gels. ........................................................................................................... 10 Figure 5 Ceramic Chitosan composite for tissue engineering. .............................................................................. 12 Figure 6 Thermogelling and phase diagrams for various poloxamers. ................................................................. 17 Figure 7 Micellar volume fraction and sol to gel transition. ................................................................................. 18 Figure 8 Effect of HLB on phase behaviour. ........................................................................................................ 19 Figure 9 Cloud point as a function of pH. ............................................................................................................. 21 Figure 10 Dewatering endoterms and NMR measurements of aggregation. ......................................................... 22 Figure 11 Pyrene detection of thermally induced hydrophobic aggregates........................................................... 23 Figure 12 PEO closed loop behaviour................................................................................................................... 26 Figure 13 Different solution behaviour of block and statistical P(EO-co-PO) copolymers. ................................. 27 Figure 14 Solution properties and interaction parameters for P(EO-stat-PO). ...................................................... 28 Figure 15 Excess volume ∆VE as a function of temperature at two concentrations A and B[49]. ........................ 30 Figure 16 Theoretical closed loop phase diagram for PEO and P(EO-stat-PO).................................................... 31 Figure 17 Amount of polar conformations as a function of temperature[62]........................................................ 32 Figure 18 Salting out and effect on thermothickening properties. ........................................................................ 33 Figure 19 Salting out effect on a poloxamer F68 phase diagram[36]. .................................................................. 34 Figure 20 Effect of organic additives on P(EO-stat-PO) solution properties[64]. ................................................ 35 Figure 21 Ternary phase diagram, P(EO-stat-PO)-water-organic carboxylic acid/hydrophobic additive[61]. ..... 36 Figure 22 Effect of surfactant on solution properties and thermothickening behaviour [43]. ............................... 38 Figure 23 Different mechanism of LCST reduction[43]. ...................................................................................... 39 Figure 24 LCST as a function of the interaction parameters[61]. ......................................................................... 40 Figure 25 Associative behaviour above c*............................................................................................................ 41 Figure 26 Concentration dependence on the osmotic pressure[71]. ...................................................................... 46 Figure 27 Reduced screening lengths ξ R vs. c c * . ............................................................................................ 47 Figure 28 Effect of entanglements on the viscosity concentration dependence. ................................................... 48 Figure 29 Molecular properties of HA. ................................................................................................................. 51 Figure 30 Solution properties of HA. .................................................................................................................... 52 Figure 31 Shearthinning behaviour of HA and concentration regimes. ................................................................ 52 Figure 32 Molecular conformation of HA in solution........................................................................................... 53 Figure 33 Investigation of the intermolecular PEO aggregates by SANS............................................................. 57 Figure 34 Thermothickening vs. Thermothinning[30]. ......................................................................................... 69 Figure 35 Thermoregulator ................................................................................................................................... 74 Figure 36 Postnova Analytics instrument. ............................................................................................................ 76 Figure 37 Material and chemical setup for synthesis. ........................................................................................... 77 Figure 38 Three necked flask cooled in icebath. ................................................................................................... 78 Figure 39 Dialysis purification procedure. ............................................................................................................ 78 Figure 40 Paar Physica, MCR 300 Universal Rheometer ..................................................................................... 80 Figure 41 Sterilization by filtration ....................................................................................................................... 80 Figure 42 Cell culturing flask. .............................................................................................................................. 81 Figure 43 Cloud point-/coexistence curve for B11/150, EO:PO=1:1, MW=2500 g/mol ...................................... 82 Figure 44 Cloud Point curve for various polyethers of different composition and molecular weights. ................ 83 Figure 45 Reduction of the cloud point curve in media compared to water.......................................................... 84 Figure 46 1H-NMR spectra of M-2070................................................................................................................. 85 Figure 47 Close up on peak sequence ................................................................................................................... 86 Figure 48 Mass distribution with baseline ............................................................................................................ 87 Figure 49 Mass distribution with baseline correction ........................................................................................... 87 Figure 50 MALDI-TOF spectra ............................................................................................................................ 89 Figure 51 M-2005 MALDI -TOF results .............................................................................................................. 90 Figure 52 Refractive index increment results........................................................................................................ 91 Figure 53 SEC-MALLS results of CMC, Cekol 30 .............................................................................................. 92 Figure 54 Cloud point behaviour and vial tilting method of synthesis 5............................................................... 93 Figure 55 RI signal of the polyether peak. ............................................................................................................ 94 Figure 56 LS signal of the polysaccharide backbone and graft copolymer. .......................................................... 95 Figure 57 IR spectra of CMC (green curve) and CMC derivate Synthesis 4 (red curve)...................................... 96 Figure 58 Resolved Peaks of CMC and CMC derivate Synthesis 4...................................................................... 97 viii

List of Figures and Schemes Figure 59 IR spectra of HA (blue curve) and HA derivate synthesis 7 (green curve) ........................................... 99 Figure 60 Resolved peaks of HA and HA derivate Synthesis 7. ......................................................................... 101 Figure 61 NMR spectrum and quantification methodology for HA and its derivates synthesis 13. ................... 102 Figure 62 Thermothickening behaviour of Synthesis 5. ..................................................................................... 104 Figure 63 Comparison of the temperature dependence of the viscosity between CMC and the CMC derivate Synthesis 5. ......................................................................................................................................................... 105 Figure 64 Comparison of the temperature dependence of the viscosity between HA and the HA derivates Synthesis 9 and Synthesis 13. ............................................................................................................................. 106 Figure 65 Concentration dependence of the thermothickening effect ................................................................. 107 Figure 66 T ass vs. T cp . ......................................................................................................................................... 107 Figure 67 Temperature dependence on the viscosity regarding synthesis 12 with small oligomeric LCST-grafts. ............................................................................................................................................................................ 108 Figure 68 Solvent effect on the thermothickening effect. ................................................................................... 109 Figure 69 Effect of the WHB on the thermothickening properties ..................................................................... 110 Figure 70 Flow curves of Synthesis 13 at different temperatures. ...................................................................... 111 Figure 71 Visualization of the shearthickening behaviour. ................................................................................. 112 Figure 72 The zero shear viscosity as a function of temperature ........................................................................ 113 Figure 73 Temperature dependence of the storage and loss moduli for the CMC derivate, Synthesis 11. ......... 114 Figure 74 Temperature dependence of the storage and loss moduli for the HA derivate, Synthesis 13. ............ 115 Figure 75 Frequency dependence of the storage modulus at different temperatures for the CMC derivate Synthesis 11. ....................................................................................................................................................... 116 Figure 76 Frequency dependence of the storage modulus at different temperatures for the CMC derivate Synthesis 5. ......................................................................................................................................................... 116 Figure 77 Frequency dependence of the storage modulus at different temperatures for the HA derivate Synthesis 13. ....................................................................................................................................................................... 117 Figure 78 Non linear behaviour of G ′′ as a function of temperature regarding synthesis 11. ............................ 118 Figure 79 Non linear behaviour of G ′ as a function of temperature regarding synthesis 11. ............................ 118 Figure 80 Visualization of the strain hardening behaviour of synthesis 11. ....................................................... 119 Figure 81 The strain hardening behaviour visualized by the complex viscosity η * . ......................................... 119 Figure 82 Non linear behaviour of G ′′ as a function of temperature regarding synthesis 13. ............................ 120 Figure 83 Non linear behaviour of G ′ as a function of temperature regarding synthesis 13. ............................ 120 Figure 84 Visualization of the strain hardening behaviour regarding the HA derivate Synthesis 13.................. 121 Figure 85 The strain hardening behaviour visualized by the complex viscosity η * regarding the HA derivate synthesis 13. ........................................................................................................................................................ 121 Scheme 1 The synovial joint[11]. ........................................................................................................................... 2 Scheme 2 Cross sectional view of articular cartilage[14]. ...................................................................................... 3 Scheme 3 ECM components in cartilage[20]. ......................................................................................................... 5 Scheme 4 Collagen composition[16]. ..................................................................................................................... 6 Scheme 5 Aggrecan composition [21]. ................................................................................................................... 6 Scheme 6 Swelling Pressure[21]. ............................................................................................................................ 7 Scheme 7 Semicrystalline polymer solutions. ......................................................................................................... 8 Scheme 8 Coil to globule transition of poly-N, N dialkylacrylamides.................................................................. 11 Scheme 9 Alginate gels. ........................................................................................................................................ 13 Scheme 10 H-bonding hydrogels. ......................................................................................................................... 14 Scheme 11 Biotin avidin crosslinking[34]. ........................................................................................................... 15 Scheme 12 Intermicellar interaction. .................................................................................................................... 18 Scheme 13 Molecular structure of A) PLA, B) PESc, C) PEA and D) PHA. ....................................................... 19 Scheme 14 Schematic picture of the association process for a WHB grafted with LCST sidechains[9]. ............. 20 Scheme 15 Schematic figures of different possible polymer phase diagrams. ...................................................... 25 Scheme 16 The most important polar and nonpolar of DME and DMP. .............................................................. 29 Scheme 17 Interaction between SDS and PPO[66]............................................................................................... 37 Scheme 18 Schematic figure of concentration regimes associated with c*. ......................................................... 44 Scheme 19 Schematic figure of the blob model. ................................................................................................... 46 Scheme 20 Molecular models of HA. ................................................................................................................... 49 Scheme 21 Molecular structures of GAG:s. .......................................................................................................... 50 Scheme 22 Schematic drawing of the interaction between HA and aggrecan. ..................................................... 54 Scheme 23 Schematic drawing of CMC. .............................................................................................................. 55 Scheme 24 Schematic picture of the self assembly process. ................................................................................. 56 Scheme 25 Schematic picture of the segregation process according to the phase diagram[35]. ........................... 58 ix

List of Figures and Schemes Scheme 26 Schematic picture of sticky Rouse dynamics of the self assembly process. ....................................... 61 Scheme 27 PAA-g-C 12 micellar model[30]. ......................................................................................................... 61 Scheme 28 Core corona micelle model for PAAm-g-PNIPAAm[68]................................................................... 63 Scheme 29 Different models of associating polymers. ......................................................................................... 64 Scheme 30 Characteristics of the junctions. .......................................................................................................... 66 Scheme 31 Model of the transient network and subsequent reorganization induced by macroscopic deformation[85]. ................................................................................................................................................... 67 Scheme 32 Relationship between the material properties and the transient network parameters[9]. .................... 68 Scheme 33 Structure of EDC ................................................................................................................................ 70 Scheme 34 Structure of NHS and sulfo-NHS ....................................................................................................... 71 Scheme 35 EDC/sulfo-NHS reaction pathway ..................................................................................................... 71 Scheme 36 Molecular models of polyethers and polysaccharides; ....................................................................... 73 Scheme 37 Schematic drawing of different chemical protons of a Jeffamine polyether....................................... 84 Scheme 38 Possible reaction pathways. ................................................................................................................ 98 Scheme 39 Possible carboxylate linkages. .......................................................................................................... 100

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List of Tables

List of Tables Table 1 Jeffamines ................................................................................................................................................ 73 Table 2 Dulbeccos Modified Eagle Medium ........................................................................................................ 74 Table 3 Sample preparation; ................................................................................................................................. 76 Table 4 Filters ....................................................................................................................................................... 81 Table 5 PEO parameters........................................................................................................................................ 88 Table 6 PPO parameters ........................................................................................................................................ 88 Table 7 MALDI-TOF parameters and results based on PEO................................................................................ 88 Table 8 MALDI-TOF parameters and results based on PPO ................................................................................ 88 Table 9 Solutions for refractive index increment measurements .......................................................................... 91 Table 10 Synthesis results displayed as the graft ratio τ. .................................................................................... 103 Table 11 Materials of synthesis 1........................................................................................................................ 131 Table 12 Materials of synthesis 2........................................................................................................................ 132 Table 13 Materials of synthesis 3........................................................................................................................ 133 Table 14 Materials of synthesis 4........................................................................................................................ 134 Table 15 Materials of synthesis 5........................................................................................................................ 135 Table 16 Materials of synthesis 6........................................................................................................................ 136 Table 17 Materials of synthesis 7........................................................................................................................ 137 Table 18 Materials of synthesis 8........................................................................................................................ 138 Table 19 Materials of synthesis 9........................................................................................................................ 139 Table 20 Materials of synthesis 10...................................................................................................................... 140 Table 21 Materials of synthesis 11...................................................................................................................... 141 Table 22 Materials of synthesis 12...................................................................................................................... 142 Table 23 Materials of synthesis 13...................................................................................................................... 143 Table 24 Materials of synthesis 13.1................................................................................................................... 144 Table 25 Materials of synthesis 14 ...................................................................................................................... 145

xi

Introduction

1 Introduction There is a vast clinical need for repair strategies when it comes to articular cartilage defects. Even small superficial defects can lead to osteoarthritis and loss of joint function. Osteoarthritis, considered by WHO to be one of the ten diseases causing the largest global disease burden, is the most common joint disorder and affects large fractions of the population with considerable suffering and functional impairment for the individual and huge costs for the society[1]. Since the articular cartilage tissue is avascular, aneural, alymphatic and due to lack of reparative cells, humoral factors and a low cell/matrix ratio, tissue regeneration in defects is strictly limited[2]. Injuries that penetrate the subchondral bone (full thickness) induce an intrinsic repair through bleeding that yields a fibrous cartilaginous tissue that is a poor substitute for hyaline articular cartilage, whereas more superficial injuries show even less or no repair with degeneration over time. The problems to overcome in cartilage tissue repair are to fill the defect with tissue of the same properties as healthy hyaline articular cartilage and to provide integration between the tissue repair and the native tissue. Another problem arises from the zonal organisation in articular cartilage where the chondrocytes differ phenotypically throughout the depth. Studies have shown that the best effect on mechanical properties of articular cartilage is achieved through a combination of different zonal chondrocytes[3]. The limited number of cells available and their restricted ability to proliferate, differentiate and regenerate tissue remains a problem for chondrocytes derived from articular cartilage biopsies. It is also important that the expanded cells retain their phenotypic function. Chondrocytes dedifferentiate upon repeating passages [4, 5]. Current cartilage repair strategies used today are arthroscopic repair (removal of injured tissue followed with induced full thickness bleeding), soft tissue grafts (transplantation of periosteum/perichondrium with chondrogenic potential to full thickness defects in order to obtain a hyaline-similar tissue repair), osteochondral transfer (allogenic in large defects, autogenic in small where difficulties arising from hindrance in lateral integration due to inevitable spaces) and ACT, first described (1994) and later reviewed (1999) by Brittberg et al. ACT involves excision of a healthy biopsy from a nonload bearing region, enzymatic digestion, cell expansion in vitro, saturation of periosteal graft/collagen over the defect and injection of chondrocytes under the periosteal/collagen flap[3]. A recent innovation in cartilage tissue repair is to utilize biodegradable biomaterials which prevent formation of scar tissue. Many studies have been carried out on implanted cells in premanufactured scaffolds, for instance liver cells and chondrocytes. Regarding solid scaffolds, the surface chemistry, surface microstructure/topology and porosity are of immense concern since all these parameters affect cell attachment, proliferation and differentiation.[6] The most recent innovation in tissue engineering approaches is injectable in situ gelling systems. These materials have the advantage to fill every form or shape and are thus ideal for cartilage tissue engineering where integration between the native tissue and the repair tissue is a major issue. It is possible to incorporate therapeutical agents directly in the biomaterial and there is no residual solvent, a possible contamination when it comes to solid scaffolds. The procedure does not require any surgical operation for placement. In situ gelling systems are proficient candidates as cell delivery techniques, but one must consider the gelling kinetics, matrix resorption rate, toxicity of degradation products and their metabolites as well as accumulation of high molecular weight products etc[6]. Thermothickening polymers are plausible aspirants for in situ gelling system. These polymer solutions have found their use in various industrial applications where there is requirement for viscosity control. The most common behaviour for polymer solution is gelling on decreasing the temperature (gelatine)[7] and the viscosity is usually a decreasing function of temperature because of the solvent’s thermal behaviour[7, 8]. This might be an obstacle in various applications such as aqueous suspensions, coatings, drilling fluids, cement, slurries etc[9]. Polyelectrolytes frequently employed as thickeners regress their efficiency at elevated temperatures, high ionic strength or excessive mechanical deformation and the control is lost[7, 9]. In addition, physical crosslinks are enviable in contrast to macromolecules or chemical crosslinks since junctions disrupted by mechanical stress, may be regenerated whereas large polymers are degraded and lose their effect as thickeners[9, 10]. The aim of this project is consequently to synthesize thermoresponsive polymers with physical crosslinks in solution, where the temperature dependent behaviour may be tailored through the polymer structure. The copolymers will be based on sodium hyaluronate, since it’s abundant in cartilage tissue and interacts with the chondrocytes, as well as carboxymethylcellulose as a polysaccharide reference material. Random copolymers of ethylene oxide and propylene oxide were chosen as the other part of the copolymer because of their controllable solution properties and since EO-PO copolymers have been FDA approved for in vivo use.

1

Theory - Cartilage

2 Theory 2.1 Cartilage Articular cartilage is the name of the hyaline cartilage covering the bone in a synovial joint (diarthrodial joints), that is the flexible joint with a joint cavity filled with synovial fluid which is secreted from the synovial membrane, covering all the non-cartilaginous surfaces inside the articular capsule, see Scheme 1. Scheme 1 The synovial joint[11].

2.1.1 Cartilage structure The function of the articular cartilage is to provide an almost frictionless surface (which lubricated with synovial fluid attains a friction coefficient of 0.001-0.06) and adsorbing and transmitting compressive, tensile and shear forces [12]. Hyaline articular cartilage is a porous tissue with high water content (65-80%) and a low cell/matrix ratio. The chondrocytes are embedded in an extra cellular matrix consisting mostly of collagen fibres (50–90% of the dry weight) II, VI, IX, X, XI and proteoglycans (5–10%) but also hyaluronic acid (the matrix composition will be discussed in detail in section 2.1.3 Extra cellular matrix on page 4. The amount and organization vary throughout the depth in order to respond to varying load distribution [4, 12]. The cells and the ECM form a layered structure, with different cell phenotypes, comprised of the superficial layer (flattened discoid cells), the transitional layer (rounded cells arranged in perpendicular columns), the radial layer and the calcified layer (larger chondrocytes), discussed in detail in section 2.1.2 Cell morphology and zonal architecture on page 2[4, 13]. Apart from different appearance, the chondrocytes also express different kinds of collagen which forms a parallel network at the surface and is perpendicular further down to the subchondral bone in order to resist shear at the surface and compression in the depth[13]. The proteoglycans also vary throughout the depth, with lubricin at the surface and large, highly negatively-charged, aggregating proteoglycans in the depth in order to draw in water (creates a swelling pressure that resists compression force) [12]. See Scheme 2 and Figure 1 envisaging the different layers of articular cartilage.

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Theory - Cartilage Scheme 2 Cross sectional view of articular cartilage[14].

2.1.2 Cell morphology and zonal architecture 2.1.2.1 Superficial zone In the superficial zone, the cells are small flattened discoid/ellipsoid and run parallel to the surface. The superficial chondrocytes express more collagen, and less proteoglycans compared to the other zones[15-17]. The numerous, very fine collagen fibres of the superficial zone are running parallel to the joint-surface in order to resist shear and tensile forces, thus making this section very stiff and strong [15, 17]. The amount of water, fibronectin, decorin and biglycan are the highest in the superficial zone and the unique proteoglycan (sometimes referred to as glycoprotein in the literature) lubricin[16], which is believed to lubricate the surface and inhibit synovial cell overgrowth, is expressed only in the superficial layer[18]. At the very top, a dense cell-devoid skin, called the lamina splenders, of thin, tightly packed collagen fibrils are preventing any non-cartilaginous macromolecules such as antibodies to enter and preventing leakage of cartilaginous macromolecules, such as proteoglycans. Small molecules such as nourishment substances and water from the synovial fluid can diffuse through this barrier though[15, 16]. This is of great importance since the avascular and alymphatic nature of this tissue renders it dependent on gradient diffusion of synovial fluid from the synovial capsule rather than vascular supply for nourishment and lymphatic drainage[19]. As will be described later, a chondrocyte progenitor subpopulation is residing in the superficial zone of cartilage.

3

Theory - Cartilage

Figure 1 H&E stained cross sectional views of articular cartilage. A) Cross section of articular cartilage. B) Large chondrocytes organized radial in lacunae. C) Flat disc shaped superficial chondrocytes arranged parallel to the surface. D) Spherical chondrocytes randomly arranged in the transitional zone. Hematoxylin and eosin (abbreviated H&E) are the most commonly used stains in histology and histopathology. Hematoxylin colours nuclei blue, eosin colours the cytoplasm pink[17].

2.1.2.2 Transitional zone In the transitional zone the cell density is lower than the superficial zone and the chondrocyte, occurring single or clumped in isogenous groups called lacunae, have a more spheroidal shape and are slightly larger than the superficial chondrocytes. The chondrocytes in this zone contain a higher concentration of synthesising organelles such as endoplasmatic reticulum and Golgi apparatus. This zone contains the highest amount of aggrecans and the collagen fibres have larger diameter than the superficial ones, taking an oblique course through the depth[15-17].

2.1.2.3 Radial zone The radial zone is the broadest one and decides the thickness of the articular cartilage, normally making up for 2/3 of the total articular cartilage thickness. The aggrecan and water content as well as the cell-density are the lowest with fairly large chondrocytes forming radial columns of lacunae. The collagen fibrils of the radial zone have the largest diameter and are running perpendicular to the surface in the orientation of the chondrocyte columns.

2.1.2.4 Calcified zone The calcified zone has transitional mechanical properties between the cartilage and the subchondral bone and resists vascular invasion, thus restricting nutritional flow from the subchondral bone. The chondrocytes dwelling in this zone are few and slightly smaller then the ones in the radial zone. The chondrocytes are here arranged in rows and are sometimes totally buried in calcified sepulchres suggesting that they have an overall lower metabolically level than other zonal chondrocytes. They have a hypertrophic phenotype, expressing collagen X, as in the hypertrophic zone of the growth plate[15, 16, 18]. The calcified zone anchors the articular cartilage to the subchondral bone by anchoring the collagen fibres to the bone thus reinforcing the overall structural integrity [19].

2.1.3 Extra cellular matrix The ECM in articular cartilage is, apart from tissue fluid (water, gases, small proteins metabolites and cations to balance the large negatively charged proteoglycans), mainly composed of collagen fibres, proteoglycans and hyaluronic acid[15, 20].

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Theory - Cartilage Scheme 3 ECM components in cartilage[20]. Collagen fibres (see 2.1.3.1 Collagen) interconnected in a HA matrix. The HA mesh is decorated by aggrecan, described in detail in 2.3.2.2.2 Hyaluronic acid on page 49. Abbreviations CS and KS are chondroitin sulphate and keratin sulphate respectively. G1, G2 and G3 are domains of the proteoglycan core of the aggrecan complex.

2.1.3.1 Collagen The collagen fibres found in cartilage comprises collagen II, III, V, VI, IX, X, XI, XII and XIV but the tissue is predominantly composed of collagen II, VI, IX and XI where collagen II, a homo trimer of α1(II) chains of procollagen fibrilles (compare collagen I which is composed of two α1(I) and one α2(I), see Scheme 4) constitutes 90–95 % of overall collagen composition [15, 19, 20]. Collagen II together with collagen IX and XI, thought to act as spacer molecules, form intercrossed meshes throughout the tissue. Collagen V and XI are believed to regulate fibre size. Amorphous collagen VI is only found in the pericellular region discussed below and collagen X is only found in the calcified layer [15, 16, 20].

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Theory - Cartilage Scheme 4 Collagen composition[16].

2.1.3.2 Proteoglycans Proteoglycans are dominated by large aggregating macromolecules called aggrecans. These molecules, schematically drawn in Scheme 5, are composed of a protein core, decorated with glycosaminoglycans, mainly chondroitin-6 sulphate, keratan sulphate and small portions of dermatan sulphate (note that hyaluronic acid is also a GAG and will be discussed more in detail later). These highly negatively charged molecules draw water into the tissue through osmotic potential thus swelling the tissue, see Scheme 6. This is restricted by the stiff crosslinked collagen network, thereby creating a swelling pressure which counteracts compression forces of the tissue[15, 16, 19, 21]. In addition to the larger proteoglycans, there are also smaller proteoglycans, mainly decorin, biglycan and the keratin sulphate containing fibromodulin. These small proteoglycans are believed to have an organizing function in the matrix[16]. Scheme 5 Aggrecan composition [21].

2.1.3.3 Miscellaneous There are also a number of important noncollageneous, nonproteoglycan glycoproteins (note the that glycoproteins contain only few mono or oligosaccharides, compared to the numerous polysaccharides on proteoglycans) such as anchorin C-II which anchors the chondrocyte to the collagen fibril, cartilage oligomeric protein which anchors the chondrocyte to the matrix in the territorial region, link protein which link the proteoglycans to the hyaluronan, and chondronectinI. There are also other proteins with similar functions as the glycoproteins such as fibronectin and tenascin [15, 20].

6

Theory - Cartilage Scheme 6 Swelling Pressure[21].

2.1.3.4 Cell to matrix organization In addition to the zonal matrix–variability, there is also variability in the matrix regarding the cellular level. The amorphous 2 μm thick pericellular matrix is just covering the cell surface and contains the nonfibrillar collagen VI, anchorin C–II, decorin and is rich in proteoglycans. The glycosaminoglycans of the proteoglycans differ from the rest of the tissue. The collagen is arranged in thin filamentous networks, interacting with hyaluronan and decorin. The chondrocytes have cytoplasmic extensions out in this region and the cell–associated pericellular region and the chondrocytes residing within are often termed emphchondron [15-17]. These probes enables the chondrocyte to interact with its environment, mainly through the CD 44 receptor that binds to hyaluronan and anchorin C-II and integrin receptor α1β1 and α2β2 which binds to collagen II thus ensuring tissue homeostasis. The chondrocytes produce all the ECM components in articular cartilage and as a cause of the hypocellular nature of the tissue the turnover of matrix is slow[16, 20]. Therefore, alteration in the tissue composition will affect the chondrocyte appearance. And since the chondrocyte morphology is important (through control of cellular volume and cytoskeletal arrangement) for the production of ECM, an altered appearance will result in a changed ECM production which will in its turn alter the mechanical properties of the tissue. Since the mechanical integrity of articular cartilage is relying on its delicate structural architecture, such a change will further alter the properties of the tissue. Surrounding the chondron is the territorial region. This region encloses individual or clusters of chondrocytes, called lacunae, providing them protection. The collagen fibrilles are thinner and randomly orientated compared to the aligned fibrilles in the interterritorial region. The chondrocyte cytoplasmic probes extend also into the territorial region, thus enabling the chondrocytes to interact with the ECM in the interterritorial matrix as well as with other chondrocytes in the same lacunae. The interterritorial region is characterized with an increase in collagen fibril thickness and orientation of the fibres [22].

7

Theory - Gels

2.2 Gels A gel may be defined as a 3D network swollen by solvent but where the network is still insoluble in any good solvent due to the crosslinks[6, 23]. A polymer gel may be considered to be a solid material but since the gel may absorb large amounts of solvent, the swollen gel has many viscous liquid-like properties and can be considered to be an intermediate material between liquids and solids[23, 24]. Normally the solvent constitutes the major part of the gel[6]. Gels may be classified into two subcategories, chemical and physical gels. A chemical gel, also termed permanent gel consists of covalent crosslinks whereas secondary interactions connect a physical gel[6, 23, 24]. It also must be stated that uncrosslinked polymer solutions may be considered as gel systems with entanglements as physically crosslinking junctions. Polymers solutions are yet homogenous systems compared to the nonergodic nature of crosslinked gels[23].

2.3.1 Physical gels The gelling of a physical gel may be ascribed to several different mechanism; temperature- or pHdependence, micellar packing, H-bonds, ionic crosslinking, solvent exchange, crystallisation and thickening after removal of shear (shear thinning polymers). In order to receive a physically crosslinked response from the system it is also important that the crosslinking is of a cooperative nature. As the first junction is formed, the energy barrier for the next is reduced[25]. In the following subsections, different categories of physical gels will be discussed with succeeding examples of in situ gelling experiments.

2.3.1.1 Thermoreversible gels Physical gels that are heat reversible are often labelled thermoreversible gels[6, 25]. These systems solidify upon cooling and melt upon heating (gelatine) or vice versa (concentrated Pluronics® solutions) with a variety of gelling mechanisms[23, 24]. Thermothickening polymer theory will be further discussed in chapter 2.3 Thermothickening Polymers on page 16.

2.3.1.1.1 Thermothinning, crystallization A number of natural thermoreversible gels exist, for instance gelatine (partially hydrolyzed collagen), agarose (from red seaweed, alternating copolymer of α-(1→4)-3,6-anhydro-L-galactose and β-(1→3)-Dgalactose), amylose and amylopectin (starch). These polymers in aqueous solution gel on decreasing the temperature due to renaturation of a triple helix (gelatine) or double helix (polysaccharides) crystallization[6]. Hydrogels composed of semicrystalline systems may also involve synthetic polymers for instance isotactic PVA or isotactic PMA [25]. Scheme 7 Semicrystalline polymer solutions. A) Helix formation and subsequent aggregation upon cooling[6]. B) Schematic picture of a semicrystalline crosslinked network[25].

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8

B

Theory - Gels

2.3.1.1.2 Thermothickening, hydrophobic interaction The thermal behaviour of cellulose derivates are exceptions when it comes to thermoreversible polysaccharide gels. Cellulose hydrophobically modified on the hydroxyl groups gel due to an increased polymer-polymer interaction with increasing temperature[6, 26]. Poloxamer (PEO-PPO-PEO non-ionic block copolymer tenside), commercially branded Pluronics®, aqueous solutions are classical thermoreversible gelling systems (see Figure 2).

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Figure 2 Thermogelling polymeric tensides. A) Poloxamer structure. B) Schematic picture of poloxamer (PEO=blue, PPO=violet) cubic micellar liquid crystalline phase [25]. C) Schematic model of surfactant monoolein and membrane protein bicontinuous cubic liquid crystalline phase[25]. D) 5 mm hydrogel of Pluronics F-127 formed by a Sciperio printer[25]. Above a certain concentration, the solution solidifies with increasing temperature. At further elevated temperature the gel melts. This behaviour will be discussed in more detail in chapter 2.3.1 Poloxamer block copolymers but arises from the dehydration of the PPO block which subsequently phase separates and aggregates through hydrophobic interaction into micellar structures (B, C). Above a critical micelle volume fraction, φ ≥ 0,53 , the micelles freeze into a micellar cubic liquid crystalline phase (B, D)[6, 25].

Poloxamer 407 gels at 37°C and at a concentration, c>20 wt%. Chondrocytes were revealed to produce more cartilage when injected as a cell suspension in Poloxamer 407 aqueous solution subcutaneously in athymic mice compared to a cell suspension in saline. Systems similar to Poloxamer copolymers exist; PPO-PEO-PPO, PLGA-PEG-PLGA, PEO-PLGA-PEO and PBO-PEO-PBO block copolymers see Figure 3 B-F. PEO-PLGA-PEO block copolymers display a sol-gel transition at room temperature and c=33 wt%. The gelling mechanism was studied subcutaneously in mice[6, 25]. Non ionic surfactants (alkyl-PEO, see Figure 3D) reminiscent of poloxamers, gel on increasing temperature. The surfactants form worm like micelles, forming entanglements similar to polymer entangled networks. Surfactants may also form various types of liquid crystalline phases see Figure 2C. A

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Figure 3 Various block copolymers for in situ gelling systems. A) PEO-PLGA-PEO block copolymer gel subcutaneously injected in mice[6]. B) Inversed poloxamer structure. C) PBO-PEO-PBO block copolymer. D) Non ionic surfactant. E) PEO-PLGA-PEO block copolymer. F) PLGA-PEO-PLGA block copolymer.

A nonresorbable P(NIPAAm-co-AAc) gel was subject for chondrocyte studies. The PNIPAAm phase separates upon a temperature increment, due to dehydration of a frozen water shell around the propyl group. 9

Theory - Gels

Chondrocytes from articular cartilage were proliferated in the gel and maintained their cartilage phenotype and produced collagen II (see Figure 4)[6]. Chitosan derivates were prepared by grafting PluronicsF127 (poloxamer 407) and PNIPAAm onto chitosan. The two separate derivates gel around room temperature but with different gelling mechanisms. HMSC were proliferated in vitro and the in situ gelling scaffolds induced chondrogenic differentiation. Both materials revealed the same good quality (90%) viability as an alginate control gel. The cell cultures expressed both aggrecan as well as collagen II and X[27]. Poloxamer 407 has also been grafted onto hyaluronic acid for drug release application of ciproflaxin[28].

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Figure 4 Chondrocytes in PNIPAAm gels. A) chondrocytes expanded in monolayer[6]. B) Frozen ice cluster around the propyl group of PNIPAAm[25]. C) Chondrocytes proliferated in a PNIPAAm-co-PAA gel[6]. D) Structure of PNIPAAm-co-PAAc. Chondrocytes proliferated in PNIPAAm-co-PAA retained their chondrogenic phenotype (C) in contrast to the fibroblastic appearance of chondrocyte proliferated in monoculture (A).

The above mentioned polysaccharide hydrogels involve sidechains of thermosensitive polymers grafted onto a WHB. The opposite relation is also possible where a hydrophilic polymer is grafted onto a LCST-backbone. PNIPAAm-g-PEO copolymer collapses into nanoparticles above 36°C and at c>8 % w/v the viscosity was found to increase with temperature[29].

10

Theory - Gels

2.3.1.1.3 Thermothinning, hydrophobic interaction The thermothickening behaviour of hydrophobically associated hydrogels is generally due to LCST nature (see chapter 2.3.2.1 LCST grafts) of one or more compartments of the polymer hydrogel. This is in contrast with for instance hydrophobically modified PAA, PAA-g-C 12 [30], where the viscosity of the hydrogel is a decreasing function of temperature (see chapter 2.3 Thermothickening Polymers and chapter 2.4 Transient Network Theory). There are though examples of LCST polymer with a reversible thermothinning character for instance Poly-N, N dialkylacrylamides[29] (see Scheme 8). Scheme 8 Coil to globule transition of poly-N, N dialkylacrylamides. A) Coil volume collapse of single LCST chain[29]. B) Loss of entanglements after coil to volume collapse[29]. C) Shrinkage of crosslinked LCST hydrogel[29]. D) Structure of the poly-N,N-dialkylacrylamide, PDEAAm-co-PDMAAm. An entangled polymer solution may undergo the volume transition as singlechains or entangled with further coalescence into microdomains of colloidal dimensions. Thus the entangled polymer network is lost followed by clouding of the solution. A chemical gel undergoes the volume transition as a whole entity[29].

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Theory - Gels

2.3.1.2 pH dependent gels Solutions of chitosan (β-(1→4) linked D-glucoseamine and N-acetyl-D-glucoseamine) may gel as the environment changes from acidic to neutral due to increased polymer-polymer hydrogen bonding. Chitosan is a biodegradable cationic polysaccharide produced from partial deacetylation of chitin and is soluble in water in its protonated form. Composites consisting of chitosan mixed with a ceramic calcium phosphate possessed a paste-like appearance at slightly acidic conditions. At physiological pH the suspensioned gelled, entrapping the ceramic phase (see Figure 5).

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Figure 5 Ceramic Chitosan composite for tissue engineering. A) Field emission SEM picture of the chitosan composite[6]. B) Structure of Chitosan. The SEM picture displays individual chitosan strands binding together the ceramic phase[6].

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a

Theory - Gels

2.3.1.3 Ionically crosslinked gels Di or trivalent counter ions may crosslink alginate aqueous solutions through ionic interaction. Alginate, (1→4)-linked β-D-mannurate (M) and α-L-guluronate (G) is a biodegradable polysaccharide extracted from the cell wall of brown algae. Once calcium salt is added to an aqueous alginate solution the systems gels which is frequently employed as a way of encapsulating cells. It was demonstrated that alginate gels support chondrocyte proliferation in vivo and in vitro[6]. The injectable biomaterial has though proven to exhibit immunogenicity. It is also feasible to mix alginate with polycations, e.g. chitosan or PL, a customary method for cell encapsulation in tissue engineering applications[25]. Akin to alginate, it is possible to obtain ionically crosslinked hydrogels with CMC and divalent cations, e.g. Ca2+[31]. Scheme 9 Alginate gels. A) Structure of alginate. B) MG alternating block. C) Ionically crosslinking via divalent cation or polycation[25]. D) G block. E) Schematic picture of alginate ionically crosslinking via the egg box model[32]. F) M block. G) Egg box structure. Alginate consists of blocks of G (D) or M (F) or strictly alternating M G blocks (B). It is the G blocks that may form the so called egg boxes (G and E). The calcium ion interacts with six oxygen atoms (G) and the structure is stabilized via intermolecular H-bonds[32]. OH

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Theory - Gels

2.3.1.4 H-bond crosslinked gels Hydrogen bonds are typically formed between H and C, N, O and F. PVA and blends of PVA and PEO may form H-bonded gels. Another example is blends of PAMD and PEOz see Scheme 10[25]. Scheme 10 H-bonding hydrogels. A) Hydrogen bonding between PAMD and PEOz. B) Chemical structure of PAMD (red) and PEOz (blue). C) Hydrogen bonding inside a water insoluble junction. D) Gel network of PAMD and PEOz blends. The blend consist of small water insoluble PAMD chains and long water soluble PEOz chains. The water soluble PEOz is crosslinked via H-bonds with PAMD inside the water insoluble junctions[33].

A

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2.3.1.5 Shearthinning gels One might label shearthinning of profoundly entangled polymers solutions as systems that gel upon removal of shear. When the entangled network is subjected to shear upon injection the solution viscosity drops due to loss of entanglements. This is the case for high molecular weight HAc frequently used in biomedical application. The HAc may also be crosslinked. Gels of high molecular weight PEO (20-100 kD) have been utilized to immobilize chondrocytes with formation of cartilage tissue. PEO is noninteractive with proteins and other biological molecules[6]. Aqueous solutions of hydrophilic polymers possess generally higher zero shear viscosity due to their swollen state compared to hydrophobic polymers. It must be stated that the shearthinning character is more pronounced for hydrophilic polymers[29].

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Theory - Gels

2.3.1.6 Gels crosslinked via protein interactions Protein interaction is actually a combination of the above discussed physical interaction mechanism as well as structural dispersion forces. An example is avidin crosslinked particle networks see Scheme 11[25]. Scheme 11 Biotin avidin crosslinking[34].

15

Theory – Thermothickening Polymers

2.3 Thermothickening Polymers Prior to the 90ies, there were scarce reports of thermothickening polymer solutions apart from occasional investigations of P(EO-b-PO) copolymers, cellulose ether derivates (MC, HPC and EHEC) as well as non-ionic cellulose ether along with ionic surfactant or hydrophobically modified WHB together with non ionic surfactant[7-9]. Except for the last case, the gelling in these systems occurs as a result of hydrophobic interaction. Regarding hydrophobically modified WHB non-ionic surfactant systems, the gelling arises when the non-ionic surfactant phase separates[8, 9, 35]. The polymer solutions are crosslinked through intermolecular aggregates. Many plausible polymer conformations might be considered to possess thermoreversible gelling behaviour, WHB grafted with hydrophobic moieties or LCST polymer side chains as well as block copolymers consisting of various combinations of water soluble hydrophilic-, hydrophobic and LCST-polymers. Regardless of the precise gelling mechanism, the aggregating effect is due to hydrophobic solvation. That is a heat exchange between the solute and solvent, ensuing in a rearrangement of H-bonds and VdW forces. Another very significant factor is the reduction of the water entropy. The water organizes into frozen ice clusters around the nonpolar group. This formation is unfavoured at elevated temperatures, resulting in aggregation of the hydrophobic segments[7]. This issue will be further discussed in chapter 2.3.2.1 LCST grafts. In order to augment the effect of hydrophobic association it is conceivable to incorporate supplementary hydrophobic moieties with the thermosensitive polymer. The hydrophobic additive could be another macromolecule (e.g. a protein or a synthetic polymer), a surfactant or a hydrophobic ceramic. The blend would consequently contain a mixture of association between the thermosensitive polymer and the hydrophobic component with plausible synergy effects. It is cognized that hydrophobically modified WHB:s may adsorb strongly on hydrophobic surfaces and hence stabilize hydrophobic particle dispersions, emulsions, organic solvents or membrane proteins in aqueous solution[7].

2.3.1 Poloxamer block copolymers Even though the copolymers in this project consists of P(EO-stat-PO) copolymers it is still significant to address the related P(EO-b-PO) copolymers from a perspective of solution properties. Poloxamer, commercially labelled Pluronic (BASF), have been FDA approved for in vivo use. The block copolymers arrange themselves in micellar aggregates above a CMC or CMT. The CMT is dependent on the concentration and the CMC on temperature[25]. Pluronic with the structure (EO) m (PO) n (EO) m are often labelled X ab with X= L(iquid), P(aste), F(lake) describing the physical state of the copolymer. The indexes a and b depict the content of EO and PO respectively; M wPO ≈ a ⋅ 300 and 10 ⋅ b is the wt% EO in the copolymer[36]. Above the LCST of PPO, the block copolymers organize in micelles with a PPO core and a PEO corona[37]. Unimers exist at low temperatures since cold water is good solvent for both PPO and PEO at cc*, see chapter 2.3.2.2.1 Polymer solution theory and concentration regimes on page 41 for further theory on concentration domains) when the copolymers overlap, the LCST chains may aggregate in interchain microdomains, and accordingly physically crosslink the system[8, 9, 40]. The copolymers in this project will have a graft copolymer structure, based on a WHB with LCST side chains. Scheme 14 Schematic picture of the association process for a WHB grafted with LCST sidechains[9]. A) The entire system is water soluble at low temperatures. B) When the temperature is raised above the LCST of the sidechains, the grafts may associate in thermal induced microdomains under semidilute conditions.

A

B

The thermothickening effect may be tailored by a diversity of variables; polymer concentration, nature and concentration of added salt, pH, temperature, shear stress, radiation, solvation strength, structure/design of the copolymer (size and nature of WHB as well as LCST grafts and the graft ratio “τ”). As previously discussed, it’s plausible with synergetic effects from other amphiphilic moieties, e.g. surfactants or polymers[8, 9, 35].

20

Theory – Thermothickening Polymers

2.3.2.1 LCST grafts The function of the LCST graft is to commence in a microphase segregation upon a temperature increment. The segregation is not only restricted to aqueous solution, but organic solvents and melts are also plausible[8, 41]. The thermosensitive LCST grafts have the most significant influence on the thermothickening behaviour. The chemical nature, molecular weight, grafting ratio as well as distribution of the LCST grafts has a direct impact on the solution properties. Hence, the association temperature, T ass , is directly coupled to the phase diagram of the free LCST-graft in aqueous solution and may be further affected by external parameters, e.g. salts and additives. Regarding the distribution of grafts, a heterogeneous distribution is unfavourable since it may lead to macroscopic phase separation[8]. Several studies have been attempted to relate the onset of association, T ass , to the cloud point of the free LCST graft in aqueous solution, T cp . T ass is obviously completely governed by the LCST of the graft. It has been reported that for a semidilute solution and a copolymer concentration >5 wt%, the thermodynamics of grafted polyether LCST chains are equivalent to free polyethers in aqueous solution[8, 9]. This argument holds especially if the grafts are long enough[42] and in this case T ass ≈T cp . Shorter grafts experience a electrostatic repulsion from the WHB, raising T ass relative T cp . Furthermore, the mere strong hydrophilicity of the WHB imparts a penalty (WHB corona formation) on micro phase separation. This is to be compared with the solution behaviour of free LCST- and WHB-chains in aqueous solution. The T cp of the LCST polymer is generally reduced in the presence of WHB polyelectrolyte, due to salting out (see chapter 2.3.2.1.1 Poly(EO-stat-PO) copolymers solution theory on page25) of the counter ion and a general incompatibility between the LCST polymer and the WHB. This leads to a subsequent segregation of polymers and an ensuing reduction of T cp [8, 42, 43]. As a cause of this incompatibility, the WHB has to be taken into account at higher copolymer concentration since T ass is described by the ternary diagram; solvent, LCST graft and WHB[8]. The LCST behaviour may be governed by other parameters, e.g. pH. Poly(dialkylaminoethylmethacrylates) are distinguished LCST polymers with pH dependence. It is also feasible to incorporate pH sensitive groups, e.g. PVIa. The cloud point is hence a function of pH (see Figure 9)[44-46].

A

B

C

D n

n

N

N N

n

N + N

N

Figure 9 Cloud point as a function of pH. A) Cloud point for P(NIPAAm-co-Via) copolymers with different compositions (PNIPAAm:PVIa; ●1:0,05, ▲1:0,5, ■ 1:1) as a function of pH at a copolymer concentration of 4 wt%[45]. B) Molecular structure of PVIa. C) Alternative molecular structure of PVIa. D) Protonated PVIa. A method in an attempt to control the LCST behaviour of PNIPAAm is to incorporate hydrophobic or hydrophilic comonomers in order to alter the HLB of the polymer. The LCST is strongly affected already at small additions of comonomer and the cloud point becomes more dependent on concentration. The interaction between the LCST graft and the WHB may be altered by incorporation of pH sensitive groups, e.g. PVIa (B, C). The interaction is attractive at low pH when to comonomer is protonated (D), in the case of anionic WHB, and repulsive at high pH[42].

21

Theory – Thermothickening Polymers

A large number of LCST polymers exist frequently utilized in different applications. Polyethers are a considerable subdivision of LCST polymers. PEO, PPO and P(EO-co-PO) have the advantage of being available monofunctionalized in various molecular weights and with differing compositions[8]. PNIPAAm is a popular, frequently employed LCST polymer due to its peculiar behaviour in water[7, 9, 42]. Different subdivisions of cellulose ether possess LCST behaviour, e.g. hydroxyalkylcellulose (EHEC, HPC) and MC[9, 26]. PVCL and polyphosphazenes are other examples of LCST polymers[44, 45].

A1

B1

A2

B2

C1

Figure 10 Dewatering endoterms and NMR measurements of aggregation. A1) Comparison of association between P(EO-stat-PO) and PNIPAAm grafts measured by DSC[7]. A2) Comparison of DSC measurements of free PNIPAAm and grafted PNIPAAm[30]. B1) 1H-NMR spectrum of PAA-g-PNIPAAm in D 2 O, 25°C. B2) 1H-NMR spectrum of PAAg-PNIPAAm in D 2 O at different temperatures. C2) Comparison between the thermothickening viscosity behaviour and the fraction, F, of dehydrated PNIPAAm grafts[47]. It is possible to conclude the percentage of phase separation at a certain T’>T ass by comparing ΔH T’ with ΔH max (A1). It is also possible to evaluate the fraction of dehydrated grafts by comparing ΔH max of the grafted and free LCST polymer (A2). It could be concluded that the dehydration was never quantitative for neither P(EO-stat-PO) nor PNIPAAm. The NMR signal of PNIPAAm (B1) disappears at elevated temperatures as a cause of signal broadening(B2). The water PNIPAAm interaction is replaced at high temperatures by PNIPAAm-PNIPAAm interaction. Since PNIPAAm solidify, the signal is no longer detectable[47].

The phase transition may be studied by differential scanning calorimetry (see Figure 10 A1 and A2)[7, 30, 41]. The association of PNIPAAm may also be studied by 1H- or 13C-NMR due to the formation of vitreous (glassy, quasi solid) aggregates (see Figure 10 B1-B3)[30, 47]. Evaluation of DSC and 1H/13C-NMR measurements of the phase transition have confirmed results from SANS measurements (see chapter 2.3.3 The microaggregates on page56). When the copolymer concentration is increased, the fraction of grafts participating in the microdomains increases. This behaviour is ascribed to an increased ionic strength, due to the counter ion of the WHB polyelectrolyte. The phase separation is never complete in any case though (see Figure 10A1, A2) since the hydrophilicity of the WHB prevents complete phase separation. It is interesting to compare DSC or NMR results with thermothickening viscosity results (see Figure 10 C1). Whereas DSC/NMR investigate one aggregate, viscosity measurements investigate the connectivity between aggregates. A peculiar notation is that when DSC or NMR results approaches a plateau value, the viscosity continue to increase. This phenomenon is explained by the disengagement rate of the microphase separated grafts which decrease upon increasing temperature[47]. These properties will be further discussed in chapter 2.4 Transient Network Theory on page 64. Fluorescence is a frequently employed method for the detection of hydrophobic aggregates. The fluorescent hydrophobic molecule disappears inside the hydrophobic micelles. The fluorescence is altered as the polarity of the surroundings is changed. Pyrene has been employed for detection of hydrophobic aggregates (see Figure 11)[43, 47, 48].

22

Theory – Thermothickening Polymers

A

B

C

D

Figure 11 Pyrene detection of thermally induced hydrophobic aggregates. A) Variation of the fluorescence intensity I 1 /I 3 ratio as a function of temperature for PNIPAAm ●, CMC ○, CMC-g-PNIPAAm27 (27 wt% PNIPAAm) ◊, CMC-g-PNIPAAm47 (47 wt% PNIPAAm) ∇ [48]. B) Variation of the fluorescence intensity I 1 /I 3 ratio as a function of temperature for 0,1 wt% PAA-g-PNIPAAm ●, 0,07 wt% PAA ◊ and 0,03 wt% PNIPAAm ○ in 0,1 M NaCl, [pyrene]=6∙10-7 M[43]. C) Variation of the I 1 /I 3 ratio as a function of temperature for CMC-g-PNIPAAm27 in pure water ◊, in buffer solution of pH=3 ♦, or pH=2 □[48]. D) Structure of pyrene.

The phase transition of the LCST grafted WHB may be studied with viscosimetry in dilute solutions in order to evaluate the hydrodynamic radius R H , videlicet the swelling/collapse of the LCST graft Equation 2

η red =

η sp c

=

η −η0 n0 c

=

t − t0 = [η ] + k H [η ]2 c t0 c

where [η ] is the intrinsic viscosity and k H is the Huggins coefficient, describing the quality of the solvent; 0,3 ≤ k H ≤ 0,5 for polymers in good or ordinary solvents. When polymer-polymer interactions start to prevail, k H > 0,5 characterizing for associating polymers. The corresponding equation for polyelectrolytes in dilute solution must be rectified by the so called Rabins relation; Equation 3

η sp c



c 3

I 2 where I is the ionic strength. Hydrophobically associating polymers may be described by the Fedors relation, which is suitable for macromolecular systems where the hydrodynamic properties are strongly dependent on concentration. The relation may be applied both on polyelectrolytes as well as associating polymer where the intra/inter chains association is strongly concentration dependent; Equation 4

1

=

1 1 1  − [η ]  c cm

  

2η rel2 − 1   where cm ≈ φ m which is the maximal packing fraction for particles since Fedors relation was developed for suspensions of rigid particles. The Huggins relation is however most suitable to describe the swelling/collapse of a macromolecule as a response to its chemical structure or its environment. The intrinsic viscosity provides an direct insight in the swelling/collapse behaviour of the polymer; v [η ] ~ where M 4π 3 v= RG and 3 [η ]M RG3 = 3 2 n and 6 Φ 1

23

Theory – Thermothickening Polymers

M n = xn M eq Generally, it was established that LCST polymer grafted onto a PAA WHB, resulted in a reduction of the intrinsic viscosity of the copolymer complex compared to the solitary WHB. The effect was obtained both according to the Huggins and Fedors relation, with a drastic reduction above the LCST of the graft. Although branched polymers of a given molecular weight possess a higher density and hence smaller volume than the corresponding linear polymers, it is still conceivable to compare the LCST grafted WHB with the WHB alone because of the significant lower molecular weight of the WHB compared to the graft copolymer. The Huggins coefficient changed for PAA-PPO from k H = 0,42 (T=20°C, NaCl=0,05M) below the LCST of PPO to k H = 1,2 (T=30°C, K 2 CO 3 =0,3M) above the LCST of PPO[7]. The intrinsic viscosity of CMC-g-PNIPAAm, was reported to be the weight average summation of the intrinsic viscosities of the homopolymers CMC and PNIPAAm[48].

24

Theory – Thermothickening Polymers

2.3.2.1.1 Poly(EO-stat-PO) copolymers solution theory Regarding small molecules, LCST solution behaviour, that is, a subsequent phase separation and segregation of solvent and solute upon an augmenting temperature is rare, but nevertheless exist, as for instance for nicotine in aqueous solutions. Concerning polymers, this solution property is more common, although the opposite, UCST is the normal, more intuitive behaviour that a solute dissolves more effortlessly at higher temperatures. The effect is mostly governed by the enthalpy of mixing, where a small difference in solvent monomer interaction may induce a large effect on the polymer[45]. Scheme 15 Schematic figures of different possible polymer phase diagrams. A) Schematic figure of phase diagram of an LCST solute. B) Schematic figure of phase diagram of an UCST polymer. C) Typical phase diagram for a water soluble polymer, enclosing a two-phase region with a hypothetical low UCST (usually below the freezing point of water. D) Phase diagram for a nonpolar polymer in organic solvent.

B One-phase region

Two-phase region

T [°C]

T [°C]

A

UCST

LCST Two-phase region

One-phase region

c [wt%]

C

UCST

c [wt%]

D Two-phase region

T [°C]

T [°C]

LCST Closed loop Two-phase region

LCST

One-phase region

UCST

One-phase region

UCST

Two-phase region

c [wt%]

Two-phase region

c [wt%]

25

Theory – Thermothickening Polymers

Regarding PEO, PPO, P(EO-co-PO) or PNIPAAm, the LCST is situated below the UCST (see Scheme 15C) which is typical for LCST polymers interacting via dipole-dipole bonds/H-bonds with the solvent. It is not to be confounded with nonpolar polymer in organic solvents, e.g. PS in benzene or PE in hexane. In these nonpolar systems, the LCST is positioned above the UCST. The phenomena arises because of a solvent expansion as the temperature is augmented and a subsequent entropy gain upon phase separation[49]. The phase diagram for PEO is strongly dependent on molecular weight (see Figure 12) Short chains are completely water soluble. Chains with a MW >2000 g/mol experience a closed loop two phase region (see Figure 12 and Scheme 15C). As the MW increases, so does the UCST and the LCST is reduced below 100°C. Simultaneously the two-phase region expands[9].

Figure 12 PEO closed loop behaviour. Phase diagram for PEO with different molecular weights aqueous solutions. M=1 020 000 g/mol ■, M=21 200g/mol ○, M=14 000 g/mol ▲, M= 8 000 g/mol ∇ , M= 2290 g/mol ●, M=2270 g/mol ∆, M=2180 g/mol▼[9].

In contrast to P(EO-b-PO) block copolymers, e.g. Pluronics, P(EO-stat-PO) random copolymers are not surface active[50]. The dissolution rate of P(EO-co-PO) copolymers decrease as the MW increases due to intramolecular H-bonds. The physical appearance is liquid for low MW and/or low EO/PO ratio and solid for high MW and/or high EO/PO ratio[51]. The phase diagram for P(EO-stat-PO) copolymers is identical to that of PEO. P(EO-stat-PO) does not crystallize and hence dissolve effectively, in contrast to PEO which may form aggregates in aqueous solution[49]. The HLB of the P(EO-stat-PO) regulates the LCST in aqueous solutions. PEO is very hydrophilic and possess a LCST >95°C. As described above, the phase diagram of PEO is dependent on the MW. PPO is rather hydrophobic due to the pendant methyl group. Only low molecular weight PPO oligomers are water soluble (>T cp concerning the same concentration/mass fraction of polyether. This discrepancy is ascribed to the low molecular weight of the graft, precisely discussed in chapter 2.3.2.1 LCST grafts on page 21. The electrostatic repulsion of the WHB backbone inflicts a penalty of microphase separation of the polyether graft. Furthermore, the hydrophilicity of the WHB polysaccharide imposes an enthalpic penalty on corona formation around the micellar LCST graft core (see chapter 2.3.3.1.1 Polyether aggregates on page 58). These effects are especially profound regarding smaller grafts. This is visualized by the temperature dependence on the viscosity concerning copolymer, synthesis 12 (CMC (Cekol 30)-g-M600, τ≈30%). The microphase separation of the small oligomeric M-600 is strongly restricted and no thermothickening effect is observed in the temperature interval of observation with only a feasible association at extremely high temperatures (see Figure 67). It is inconceivable to draw any conclusions of association at these temperatures since solvent evaporation is profound with induction of plausible concentration increments. 106

Results and Discussion

Synthesis 13, [strain rate=10s-1]

1000 0,5% 1% 2% 3% 4% 6% 10%

100

Viscosity [Pa·s]

10

1

0,1

0,01

0,001

0,0001 0

10

20

30

40

50

60

70

T [°C] Figure 65 Concentration dependence of the thermothickening effect

Tass vs Tcp 40 Cloud Point M-2005 Association Temperature Synthesis 13

35 30

T [°C]

25 20 15 10 5 0 0

5

10

15

20

25

cpolyether [% w/w] Figure 66 T ass vs. T cp .

107

Results and Discussion

Synthesis 12, T-sweep [strain rate=10s-1]

1

Viscosity [Pa·s]

Synthesis 12

0,1

0,01 10

20

30

40

50

60

70

80

90

Temperature [°C] Figure 67 Temperature dependence on the viscosity regarding synthesis 12 with small oligomeric LCST-grafts.

A comparison of solvents was conducted on the thermothickening behaviour. In Figure 68, the effect of solvent on the thermothickening effect of copolymer, synthesis 13 at a concentration of 6% was investigated with pure water, cell growth media (components listed in Table 2) or cell growth media with 10% w/v fetal calf serum as solvent. Below T ass , the viscosity of the copolymer is reduced as the solvent is changed from water to cell growth media. This is due to electrostatic shielding of the polyelectrolyte WHB. The polymer coil is contracted with a subsequent viscosity reduction (data not shown). It is also observed that the association temperature T ass is reduced compared to water. This is the result of the reduced solvent quality in the media, equivalent to the phase behaviour of the free polyether (see Figure 45). Incorporation of serum in the cell growth media did not alter the thermothickening properties of the graft copolymer solution. It may be concluded that the thermothickening effect is not especially altered upon change of solvent from water to cell growth media. The most important divergence is the reduction of T ass due to salting out and solvent quality reduction of the polyether graft. Thus, the thermothickening and subsequent thermogelling effect of cells grown in the graft copolymer hydrogel is expected to be similar to the behaviour in pure water.

108

Results and Discussion

Synthesis 13, 6%, T-sweep

100

water Cell growth media Cell growth media + 10% fetal calf serum

Viscosity [Pa·s]

10

1

0,1

0,01 0

10

20

30

Temperature [°C]

40

50

60 .

Figure 68 Solvent effect on the thermothickening effect.

The effect of the chemical nature of the backbone is investigated in Figure 69. Synthesis 5 and Synthesis 9 are graft copolymers of similar molecular weight and graft ratio but with differing polysaccharide WHB:s. There’s an immense discrepancy between the two graft copolymers. The absolute value of the viscosity is far lower regarding the HA derivate. Furthermore, the thermothickening effect of the HA derivate is inhibited compared to the CMC derivate. The divergence between the two graft copolymers dwells in the solution properties and behaviour of the respective WHB:s. The higher viscosity of the CMC derivate is consistent with the viscosity behaviour of pure CMC compared with pure HA of equivalent molecular weight. It is cognized that heterogeneity of the DS of CMC may induce aggregation of unsubstituted polysaccharide chains in a similar manner as the solution properties of cellulose. Above c*, such polymer-polymer interactions may crosslink the CMC solution to form a aggregated gel network in aqueous solution[80]. This aggregating property was observed in oscillatory measurements where higher concentrations of CMC or CMC derivate solution possessed a rather high elastic modulus ( G ′ > G ′′ over the entire temperature interval), even below T ass (data not shown). In addition the cellulose derivate CMC is a very stiff, rigid and extended polysaccharide in aqueous solution equivalent to the chain conformation of cellulose. The difference in the thermothickening effect (the slope of the curve) is more elusive. The explanation may lie in the polysaccharide corona formation around the micellar core. It’s conceivable that a higher intrinsic hydrophilicity of the HA WHB compared to the CMC WHB imposes a greater penalty of microphase separation. This is not to be confused with electrostatic repulsion which in the case of the CMC derivates should be higher since the CMC WHB has a higher charge density than HA. HA is furthermore a more dynamic and flexible polymer than CMC, shifting between two energetically stable conformations which is a possible explanation for its high water solubility[114]. It’s therefore plausible that the conformational entropy loss upon corona formation is greater regarding HA compared to CMC.

109

Results and Discussion

T-sweep

1000 Synthesis 9, 3% Syntheis 5, 3%

Viscosity [Pa·s]

100

10

1

0,1

0,01

0,001 10

20

30

40

50

60

70

c[wt%] Figure 69 Effect of the WHB on the thermothickening properties

4.5.1.2 Flow curves and temperature regimes A number of different situations may be envisaged as the associating graft copolymer aqueous solution is subjected to shear rate sweeps; 1. Disruption of intermolecular bonds due to stretching and subsequent deformation of the networks structure. This condition would result in a shearthinning behaviour as the strain rate increases. 2. The supramolecular structures are forced to move relative to each other, resulting in an augmentation of contacts between the polyether stickers with successive more intermolecular bonds. This situation would result in shearthickening effect. 3. The supramolecular aggregates are stretched to such a level that a linear relationship between the stretching and the imposed force to maintain the deformation (compare with the Hookian spring constant). As the shear rate increases, the force imposed on the physical crosslinks continues to increase, resulting also in a shearthickening behaviour. The obtained shear stress is thus a summary of all physical intermolecular bonds. Shearthinning is usually the normal behaviour regarding polymer melts or polymer in solution. Shearthickening exists, for instance pertaining to block or graft copolymers with the addition of surfactants in solution. The particular situation, polymethacrylic acid in solution displays a shearthickening behaviour explained by point 2[70]. All graft copolymers revealed a shearthinning character at elevated temperatures, where the shearthinning behaviour occurred at lower and lower shear rates as the temperature increased (see Figure 70). This onset of non Newtonian behaviour at lower and lower shear rate as the temperature increases is an indication of network formation[48, 91] and may be explained by transient network theory, see chapter 2.4.1 Temperature domains on page 67. The number of elastically active chains v is predicted to decrease as a function of shear rate upon shear flow at an arbitrary disengagement rate β with a subsequent viscosity reduction directly connected to v [89]. As the temperature increases, so does the hydrophobicity of the polyether graft, characterized by the relaxation time τ x . This indicates that the polyether grafts will snap form the junctions faster than they may reengage at lower and lower shear rates according to τ x > τ exp = 1 γ . At very high temperatures, a shearthickening behaviour was observed before the inception of the shearthinning behaviour. This is more legible in a linear plot of the viscosity vs. shear rate (see Figure 71). It’s 110

Results and Discussion

observed that the magnitude of the shearthickening behaviour increases as the temperature is augmented. This is a rather solid implication that the phenomenon is related to the LCST polyether grafts. The shear thickening behaviour might be an effect of an initial elastic response from the hydrophobic junctions, which at high shear rates are disrupted, that is, an initial yield stress. This explanation is coherent with the increment of the shearthickening magnitude as the temperature is augmented since the hydrophobicity, characterized by τ x , of the polyether grafts increases with temperature, henceforth strengthening the junctions. It’s also plausible that the size, shape or aggregation number microdomains are altered upon shear. Shear thickening has been reported in the literature regarding colloidal gels. Large shear induced structures are formed at low shear rates increasing the strength of the crosslinks connecting the network[115]. A similar explanation may be applied to the shearthickening behaviour of polysaccharide-g-polyether graft copolymers. At low shear rates, the polysaccharide backbones become more elongated in the direction of the flow as the shear rate increases. This leads to a subsequent exposure of more polyether grafts, antecedently being concealed inside the polymer coil, for interchain hydrophobic association. Thus, the number of elastically active chains v increases temporarily before complete destruction of the network and subsequent shearthinning. An interesting notation is that according to transient network theory, the number of elastically active chains are expected to approach a maximum value before an ensuing decrease upon elongational flow (compare with the expected behaviour concerning shear flow discussed above). The elongational viscosity follows the same trend[89]. Since steady shear flow induce a stretching of the polymer coil, this is an equitable comparison and the phenomenon observed on elongational flow my be applied on shear flow at low shear rates. But if low shear rates induce larger, more numerous or more concentrated polyether aggregates is not known.

Synthesis 13, 3%, Flow curves

1000

Viscosity [Pa·s]

100

10

1

0,1

10°C 15°C 20°C 25°C 27,5°C 30°C 32,5°C 35°C 37,5°C 40°C 42,5°C 45°C 50°C 55°C 60°C 65°C

0,01

0,001 0,0001

0,001

0,01

0,1

1

10

100

1000

Shear Rate [1/s] Figure 70 Flow curves of Synthesis 13 at different temperatures.

111

Results and Discussion

Synthesis 13, 3%, Flow curves

500

Viscosity [Pa·s]

400

300

200

100

10°C 15°C 20°C 25°C 27,5°C 30°C 32,5°C 35°C 37,5°C 40°C 42,5°C 45°C 50°C 55°C 60°C 65°C

0

-100 0,0001

0,001

0,01

0,1

1

10

100

1000

Shear Rate [1/s] Figure 71 Visualization of the shearthickening behaviour.

The plateau viscosity before the onset of the shear dependent behaviour, also labelled η 0 , the zero shear viscosity may be plotted against temperature, see Figure 72. The increment of the η 0 gives a more realistic insight in the material properties than a simple temperature sweep at constant shear rate. Two distinct temperature regimes are observed. A low temperature regime with a strong thermothickening effect and a high temperature regime with a flatter slope of the curve. This phenomenon has been observed for a variety of WHBg-LCST polymers, e.g. PAA-g-PNIPAAm and CMC-g-PNIPAAm[30, 91] and is discussed in detail in chapter 2.4.2 Enthalpy of demicellization on page 68. In the low temperature regime, the segregation is weak and the number of polyether grafts participating in the junctions increases with temperature according to the phase diagram of the polyether. Consequently, both parameters of the transient network theory, v and τ x increases. Furthermore, above T ass , a non Newtonian shearthinning behaviour appears and the increment of the thermothickening curve (log(η 0 ) vs. T) was perceived to be independent of concentration[91]. At a certain temperature, in the strong segregation, the phase separation is complete since the number of LCST chains in the water phase approaches zero according to the phase diagram. The hydrophobicity, τ x of the polyether chains is still an increasing function of temperature. Hence, the viscosity continues to increase, although with a reduced slope, due to an increment of the interaction between the polyether grafts in the junctions. The shearthickening character was ascribed to emerge in the high temperature regime followed by drastic shearthinning behaviour according to Aubrey et al. A dramatic viscosity drop above a certain shear stress may be a signature for a yield stress, indicating a crosslinked network structure. The slope of the thermothickening curve was, in the high temperature regime, found to be dependent of concentration with diminishing increment as the concentration increases[30, 91].

112

Results and Discussion

Synthesis 13, 3% [strain rate=10s-1]

1000

Synthesis 13, 3%

Zero Shear Viscosity [Pa·s]

100

10

1

0,1

0,01

0,001 0

10

20

30

40

50

60

70

T [°C] Figure 72 The zero shear viscosity as a function of temperature

4.5.2. Oscillatory Rheometry The thermogelling properties of the graft copolymer aqueous solutions were investigated by oscillatory rheometry. Temperature sweeps at constant frequency and strain were conducted in order to determine when the elastic response of the solution commences dominating the viscous response ( G ′ > G ′′ ). The temperature corresponding to this point was designated as the threshold gel point. Frequency sweeps at constant strain were performed in order to analyse the precise viscoelastical properties of the polymer solution as a function of temperature. Especially the transition between liquid polymer solution, gel as well as viscoelastic solid material. The non linear viscoelastic regime were analysed by strain sweeps, henceforth labelled amplitude sweeps. These analyses may provide an insight of the character and strength of the gel network.

4.5.2.1 Threshold gelpoint The thermothickening or thermogelling effect may be followed by oscillatory rheometry at constant strain and frequency. In Figure 73 and Figure 74 the storage modulus, G ′ and loss modulus, G ′′ are plotted as functions of temperature. Both moduli follow more or less the same behaviour. Below the association temperature, both moduli decrease as the temperature increases. Regarding HA derivates, G ′ is hardly measurable below T ass . At higher concentrations, CMC derivates possessed an elastic response greater than the viscous response G ′ > G ′′ , even below T ass , an indication of a gel network with longer relaxation times (data not shown). Both moduli deviated from the normal behaviour above an association temperature T ass in equivalence with viscosity measurements with a subsequent increment of both moduli. At a certain temperature, G ′ crosses G ′′ and the elastic response of the system commence dominating, see Figure 73 and Figure 74. This temperature may be described as a threshold gel point[30]. Below T ass , the solutions of the graft copolymers are viscous similar to the polysaccharide aqueous solution. Above the threshold gel point, the graft copolymer aqueous solutions possess a rather strong elastic behaviour. It must be stated that this gel point is somewhat frequency dependent. Furthermore, the two distinct temperature regimes observed concerning the temperature dependence of η 0 was also observed regarding the temperature dependence of the moduli. Equivalent to the temperature behaviour of η 0 . The increment of material properties was most eminent in the low temperature regime. The strength of the 113

Results and Discussion network in the respective temperature regimes were investigated by stress relaxation ( G (t ) = t − ∆ ) measurements by Aubry et al. It was found that the low temperature regime corresponds as a soft critical gel with a high network specific exponent ∆ , whereas the high temperature regime corresponds to a stiff critical gel with a low network specific exponent ∆ [91]. It was detected that the threshold gel point was always in the low temperature regime regarding CMC derivates and shifted toward the onset of the high temperature regime regarding the HA derivates. The discrepancy between the two polysaccharide derivates is simply explained by the intrinsic elastically properties of the CMC backbone. Thus, the curve of the storage modulus is simply shifted vertically to higher values regarding CMC compared to HA with a subsequent lower gel point temperature. There is no reason to discuss in terms of differing gelling mechanisms.

Synthesis 11, after reaction [γ=10%]

100 G' G''

G', High Segregation Regime 10

G', Low Segregation Regime

G', G'' [Pa]

G'', High Segregation Regime G'', Low Segregation Regime 1

0,1

0,01 0

10

20

30

40

T [°C] Figure 73 Temperature dependence of the storage and loss moduli for the CMC derivate, Synthesis 11.

114

50

60

Results and Discussion

Synthesis 13, 1% [f=1Hz]

10 G' G''

G', G'' [Pa]

1

0,1

0,01

0,001

0,0001 10

20

30

40

50

60

70

T [°C] Figure 74 Temperature dependence of the storage and loss moduli for the HA derivate, Synthesis 13.

4.5.2.2 Frequency sweeps and viscoelastical properties Frequency sweeps may be conducted in order to establish an insight in the viscoelastical properties of the material. In Figure 75, Figure 76 and Figure 77, the frequency behaviour of the storage modulus is plotted at different temperatures in the linear regime. Two important notations may be depicted. First of all, the absolute value of the modulus increases with temperature above T ass . Secondly, the slope of the curve commences flattening out at elevated temperature, becoming more or less frequency independent. The same behaviour was observed concerning the loss modulus but with a smaller increment of the value of the modulus as the temperature increased. Regarding G ′ , a liquid normally increases as ω 2 whereas polymer solutions usually increases as ω 1 in the frequency window of observation[88, 116-119]. At high temperatures, G ′ is about an order of magnitude larger than G ′′ and very weakly or non dependent on the frequency which is consistent with the formation of a solid gel structure[119]. Essentially, a frequency independence of the moduli indicates that the gel structure behaves as a viscoelastic solid material in the frequency window of observation. Since a gel may be considered as a material with properties somewhat in between a liquid and a solid, a gel point may be established when G ′ increases as

ω 1 2 . The transition from viscoelastic liquid to viscoelastic solid is continuous and at the gel point it is expected that G ′(ω ) = G ′′(ω ) [118].

115

Results and Discussion

Synthesis 11, after reaction [γ=1%]

1e+2

10°C 15°C 20°C 25°C 30°C 35°C 40°C 45°C 50°C 55°C

1e+1

G' [Pa]

1e+0

1e-1

1e-2

1e-3

1e-4 0,01

0,1

1

10

100

f [Hz] Figure 75 Frequency dependence of the storage modulus at different temperatures for the CMC derivate Synthesis 11.

Synthesis 5, 3% [γ=1%]

10000

1000

G' [Pa]

100

20°C 25°C 30°C 35°C 40°C 45°C 50°C 55°C 60°C

10

1

0,1 0,01

0,1

1

10

100

T [°C] Figure 76 Frequency dependence of the storage modulus at different temperatures for the CMC derivate Synthesis 5.

116

1000

Results and Discussion

Synthesis 13, 3% [γ=1%]

1e+3 1e+2 1e+1 1e+0

G' [Pa]

1e-1

20°C 30°C 40°C 45°C 50°C 55°C 60°C 35°C

1e-2 1e-3 1e-4 1e-5 1e-6 1e-7 1e-8 1e-9 0,01

0,1

1

10

100

1000

f [Hz] Figure 77 Frequency dependence of the storage modulus at different temperatures for the HA derivate Synthesis 13.

4.5.2.3 Amplitude sweeps and non linear network properties When the graft copolymer solution was subjected to strain sweeps (amplitude sweeps) a strain hardening phenomenon was observed at the onset of the non linear viscoelastic regime. At low temperature and high strains, G ′ decreases monotonically as ~ ω −2 in the nonlinear regime and G ′′ displays the typical peak and decreases thereafter as ~ ω −1 . The magnitude of the loss modulus peak increases as the temperature is augmented above T ass , see Figure 78. Aubry et al ascribed this extra viscous dissipation to stretching of the LCST stickers with a subsequent intensification of the hydrophobic interaction. As the temperature increases, so does the hydrophobic interaction. This explanation might be far fetched since this behaviour is a general feature of soft materials. Nevertheless, at deformation corresponding to the peak of the loss modulus, stress relaxation measurements on small time scales displayed a constant relaxation modulus. A constant relaxation modulus demands strong enough junctions and the phenomena was subsequently designated to the formation of extraordinary strong junctions in the weakly nonlinear domain of the strong segregation regime[91]. But it’s observed that at elevated temperature, G ′ also displays a peak before at the onset of the nonlinear viscoelastic regime, see Figure 79. The magnitude of the G ′ peak is more decipherable in a linear plot, see Figure 80. A clear indication of the strain hardening phenomena may be visualized by the complex viscosity η * plotted as a function of strain, see Figure 81. Both the peak of G ′ as well as the peak of η * increases in magnitude as the temperature is augmented prior to the collapse of the system. The strain hardening behaviour is not necessarily connected to the shear thickening behaviour since the strain hardening occur at a strain rate (~shear rate); γ 0 = ω ⋅ γ 0 where ω is the angular frequency;

ω = 2π ⋅ f differing from the shear rate corresponding to the shearthickening behaviour. The physically crosslinked gel is rather tenuous structure. The graft copolymer chains and their junctions are contorted. At low strains, the backbone of the gel structure will be stretched until a critical strain value where the intrinsic rigidity of the backbone stiffens the structure and a strain hardening behaviour is observed. At higher strains, the network is disrupted and the system collapses. This kind of strain hardening behaviour has been observed in colloidal gels[119].

117

Results and Discussion

It must be stated that the onset of the nonlinear viscoelastic regime instigated at lower and lower strains as the temperature increased.

Synthesis 11, after reaction [f=1Hz]

10

G'' [Pa]

1

15°C 25°C 26°C 27°C 28°C 29°C 30°C 35°C

0,1

0,01 0,01

0,1

1

10

100

1000

10000

Strain [%] Figure 78 Non linear behaviour of G ′′ as a function of temperature regarding synthesis 11.

Synthesis 11, after reaction [f=1Hz]

10

G' [Pa]

1

15°C 25°C 26°C 27°C 28°C 29°C 30°C 35°C

0,1

0,01 0,01

0,1

1

10

strain [%] Figure 79 Non linear behaviour of G′ as a function of temperature regarding synthesis 11.

118

100

1000

10000

Results and Discussion

Synthesis 11, after reaction [f=1Hz]

1,2

1,0

G' [Pa]

0,8

15°C 25°C 26°C 27°C 28°C 29°C 30°C

0,6

0,4

0,2

0,0

0,01

0,1

1

10

100

1000

10000

strain [%] Figure 80 Visualization of the strain hardening behaviour of synthesis 11.

Synthesis 11, after reaction [f=1Hz]

Complex Viscosity [Pa·s]

1 15°C 25°C 26°C 27°C 28°C 29°C 30°C 35°C 0,1

0,01 0,01

0,1

1

10

100

1000

10000

Strain [%] Figure 81 The strain hardening behaviour visualized by the complex viscosity η * .

The strain hardening behaviour was also observed in the HA-based hydrogels (see Figure 82, Figure 83, Figure 84 and Figure 85) but the effect was less profound as indicated by a small increment in the linear plot of the complex viscosity (Figure 85). 119

Results and Discussion

Synthesis 13, 3% [f=1Hz]

100

G'' [Pa]

10

1

10°C 20°C 25°C 27,5°C 30°C 35°C 40°C 45°C 50°C 55°C 60°C 65°C

0,1

0,01 0,01

0,1

1

10

100

1000

10000

Strain [%] Figure 82 Non linear behaviour of G ′′ as a function of temperature regarding synthesis 13.

Synthesis 13, 3% [f=1Hz]

1000

100

G' [Pa]

10

1

0,1

10°C 20°C 25°C 27,5°C 30°C 35°C 40°C 45°C 50°C 55°C 60°C 65°C

0,01

0,001

0,0001 0,01

0,1

1

10

Strain [%] Figure 83 Non linear behaviour of G′ as a function of temperature regarding synthesis 13.

120

100

1000

10000

Results and Discussion

Synthesis 13, 3% [f=1Hz]

80

60

G' [Pa]

40

10°C 20°C 25°C 27,5°C 30°C 35°C 40°C 45°C 50°C 55°C 60°C 65°C

20

0

0,01

0,1

1

10

100

1000

10000

Strain [%] Figure 84 Visualization of the strain hardening behaviour regarding the HA derivate Synthesis 13.

14

Complex Viscosity [Pas]

12 10 8 6

Synthesis 13, 3%, Amplitude sweep [f=1Hz] 10°C 20°C 25°C 27,5°C 30°C 35°C 40°C 45°C 50°C 55°C 60°C 65°C

4 2 0

0,01

0,1

1

10

100

1000

10000

strain [%] Figure 85 The strain hardening behaviour visualized by the complex viscosity η * regarding the HA derivate synthesis 13.

121

Conclusions

5 Conclusions A graft copolymer with in situ gelling properties due to a temperature increment was designed, synthesized and characterized. The copolymer consists of hydrophilic water soluble polysaccharides as backbone (CMC and HA) with polyether side chains (P(EO-stat-PO)). The function of the WHB is to maintain solubility of the structure and prevent macroscopic phase separation. The gelling properties are governed by phase behaviour of the polyether grafts. The phase behaviour of the polyether side chains was investigated by cloud point measurements. The LCST could be tailored by the EO:PO composition, molecular weight and the precise temperature of phase separation could be regulated by concentration of the polyether in aqueous solution. The polyethers were grafted onto the polysaccharide backbone via the EDC/NHS mediated amide formation between the terminal amine group of the polyether and the carboxylate of the polysaccharide backbone. It was determined with FTIR and NMR that the reaction results in the formation of an amide bond between the polysaccharide and the polyether and that the grafting reaction is almost quantitative. Furthermore, dialysis is an effective purification method and purges the reaction solution from unreacted polyether. Some O-N migration of the polysaccharide-EDC active intermediate with subsequent formation of Nacylurea could not be excluded, indicated by elementary analysis. CMC based derivates with high graft ratio rendered the graft copolymer insoluble in water. Regarding future cell studies, a sterilization protocol of the material was conducted. It was established that filtration provides an efficient and gentle sterilization method, harmless to the material and with high yields. Graft copolymers consisting of the hydrophobic, relatively long polyether labelled M-2005 possessed excellent thermothickening/thermogelling properties in the right temperature interval relevant concerning in situ gelling under physiological conditions. The onset of association, T ass , is directly connected to the LCST behaviour of the polyether graft and may consequently be tailored by the monomer composition and molecular weight of the polyether copolymer as well as grafting ratio and graft copolymer concentration. The solution properties of the WHB was found to affect the precise rheological material behaviour to a rather high extent and the association temperature was much higher then the cloud point of the binary system water-polyether (T ass >>T cp ). It was further ascertain that the hydrogel behaves as a viscoelastic solid material in the frequency window of observation at elevated temperatures.

122

Future Work

6 Future Work On basis on the thermothickening/thermogelling and viscoelastic material properties, a number of plausible improvements may be addressed. Most importantly, longer polyether grafts of high molecular weight (~10 000-20 000 g/mol) should be utilized. The onset of association would thenceforth approach the cloud point of the binary system polyetherwater (T ass ~T cp ) and the thermothickening behaviour may be tailored with more ease. Furthermore, the segregation and hydrophobic interaction in the microphase separated aggregates will be intensified, thus strengthening the network structures. In order to obtain equivalent mass fractions of grafts in the copolymer, smaller graft ratios are required regarding larger polyethers, thus reducing the alteration of water solubility and properties of the WHB. It’s conceivable to incorporate supplementary hydrophobic substances, e.g. proteins, synthetic polymers, surfactants or hydrophobic ceramics. Synergistic effects of association is to be expected with a subsequent reinforcement of the hydrophobic interaction, intensifying the strength of the network. If longer WHB:s are utilized, thermothickening properties may be achieved at reduced concentrations. The absolute value of the material properties will be improved. Imputable to the excellent thermogelling properties of the hydrogels as well as efficiency and ease of sterilization, the materials will be subjected to cell studies. The viability of chondrocytes inside the gel structure must be investigated as well as production of cartilaginous ECM (collagen II and proteoglycans). It is further of immense interestingness if the HA based biomaterials are able to specifically interact with the chondrocytes. In order to specifically address the chondrocytes, and more precisely the chondrocyte progenitor subpopulation, it’s feasible to incorporate supplementary RGD peptides into the polysaccharide WHB. This may be achieved, utilizing the same EDC/NHS mediated amide coupling between the terminal amine of the peptide and the carboxylate of the polysaccharide backbone.

123

Acknowledgements

7 Acknowledgements Since I have been rather solitary in this project, there are a number of people I would like to express my deep gratitude to. Without your collaboration and help, this diploma work would not have been realizable; • First of all I would like to thank my supervisor during the synthesis procedure, Professor Mats Andersson. All helpful discussions and guidance during the synthesis of the graft copolymers made it possible to attain this novel biomaterial. • I also want to thank my co-supervisor and examiner, Professor Paul Gatenholm for giving me the opportunity to do this diploma work and for believing that I possessed the capacity to achieve this. Your enthusiasm and strategic ideas were decisive for the project. • Without the guidance, helpful discussions as well as technical supervision of senior lecturer Johan Bergenholtz, the most significant part of the project, the investigation of the material properties would not have been possible. • For the last two years, Anders Mårtensson has played a vital part in probably every project I’ve attended. I’d like to thank you especially for our fruitful discussion regarding the IR characterization as well as technical support during the freeze drying procedure. I would further like to thank the people a the Applied Surface Chemistry department for helpful discussions regarding in particular the peculiar phase behaviour of polyether polymers as well as general polymer solution theory; • Krister Holmberg for thoroughgoing discussion of the polyether phase behaviour in aqueous solution as well as preliminary design of suitable copolymers. • Magnus Nydén for fruitful discussions regarding general polymer solution and gel properties as well as alternative characterization methods. • The refractive index increment measurements were performed on a Postnova Analytics instrument belonging to Jan-Erik Löfroth. I would consequently thank you for facilitating the analysis as well as helpful discussions regarding gels and other non ergodic systems. • For introducing me to the exciting world of bioconjugate techniques, I’d like to thank PhD student Anna Bergstrand. From our discussions concerning the EDC/NHS mediated amide formation, I was able to decipher the critical parts of the reaction with subsequent final grafting success. Apart from the above mentioned people from the Polymer Technology, I’d explicitly like to thank the following persons; • PhD student Lars Lindgren for technical assistance and helpful discussions regarding NMR analysis. • PhD student Stefan Hellström for technical guidance and methodology concerning MALDI-TOF measurements • PhD student Peter Westby for help with SEC measurements as well as fruitful discussions. • PhD student Tobias Köhnke for support concerning dialysis and filtration as well as discussion about the intriguing solution behaviour of carboxymethylcellose. • PhD student Anders Höije for assistance and support during the dialysis procedure and freeze drying procedure as well as many technical and administrative matters. Whether you liked it or not, I virtually became your second diploma worker. • Professor Thomas Hjertberg for fruitful theoretical discussion about polymer solution theory as well as generic polymeric material properties… • …and I must of course thank each and everyone at the polymer department for making this final year of Chalmers studies such a fun and interesting period. In addition to the personnel assisting me in the laboratory work, the suppliers of essential chemicals ought to be addressed; • Göran Kloow (Kelco) for supplying me with carboxymethylcellulose and many helpful discussion regarding the solution behaviour of CMC and alteration of the same upon chemical modification. • Dr. Kristoffer Tømmeraas (Novozymes Biopolymers) for contributing sodium hyaluronate as well as clarifications and discussions of the hyaluronan appearance in aqueous solution. • Dr. Katty Darragas for bestowing me with polyether Jeffamines in addition to administration and technical support. • Klaus Raab (Clariant) for providing me with monofunctionalized polyethers (polyoxy alkylene glycols) as well as technical support and administration

124

Acknowledgement •

Dr. Wolfgang Spiegler (BASF) for supplying me with monofunctionalized polyethers (Pluriol A) in addition to technical support and administration

From Brosklab at Sahlgrenska Universitetssjukhus and Kungsbacka Sjukhus, I’d like to thank the following persons for helpful discussions, enthusiasm concerning my work, intriguing dialogue concerning future cell studies, and many laughs; • Sebastian Concaro and Hannah Stenhamre for helpful discussions and technical support regarding the filtration by sterilization procedure, your enthusiasm and many laughs • Marie Leander for technical assistance concerning the arrangement of the cell study initiation as well as many laughs. • Mats Brittberg, who in collaboration with Paul Gatenholm assembled the vision of an in situ gelling material for articular cartilage repair and thus making the ultimate goal clear and legible.

I’d like to thank my roommate and old student friend, all the way back to primary school, Erik Sternemalm for encouragement in progress as well as relapse. One must add that sometimes, neither of us got so much done, but all our stupid jokes and laughs certainly made this period even more enjoyable. Finally I’d like to thank my beautiful Austrian fiancé Verena for accommodating me during this period. Without your love and support it would have been so much harder to pull this through. I of course also need to thank you as an art director for help regarding layout as well as photographs and picture modifications.

125

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Appendix

9 Appendix 9.1 Synthesis Procedures 9.1.1 Appendix A Synthesis 1

Test av kopplingskemi Table 11 Materials of synthesis 1 Beteckning Namn HANa Natrium hyaluronate EDC 1-ethyl-3-[3dimethylamino)propyl]carbodiimide sulfoNHS N-hydroxysulfosuccinimid amin 1,6-hexandiamin dihydroclorid

Molekylvikt (M) g/mol 1-3 *106 191,7

Monomervikt (M 0 ) g/mol 401

217,13 189,13

Synthesis 1. 2. 3. 4. 5. 6. 7.

HANa (100 mg, 025mmol –COO- grupper) upplöses i 33,3….. ml (c=3mg/ml) H 2 O (ca 1 dygn) 30ggr så mycket 1,6-hexandiamin dihydroclorid tillsätts (7,5 mmol, 1,42 g) pH justeras till 7,5 med 0,1 M NaOH/0,1 M HCl EDC (192 mg, 1mmol) vägs upp och hålls under kvävgas sulfoNHS (217 mg, 1mmol) löses upp i 1ml H 2 O och tillsätts. Uppvägd EDC under kvävgas upplöses i 1ml H 2 O och tillsätts omedelbart (inom sekunder). pH vidhålls vid 7,5 under omblandning genom addition av 0,1 M NaOH/(0,1 M HCl). Reaktionen fortgår under natten.

Upprening 8. pH återjusteras till 7 med 0,1M HCl 9. HA-derivatet dialyseras (MW cut off 12,000-14,000) mot destillerat vatten. 10. NaCl tillsätts tills en 5% w/v lösning erhålls och HA-derivatet fälls ut genom tillsatts av 3 volymsekvivalenter etanol, steget repeteras. 11. Utfällningen återlöses i H2O till en koncentration av 5 mg/mL och den upprenade produkten frystorkas och hålls vid 4°C under kvävgas (N 2 ) Karaktäriseing sker m.h.a. 1H-NMR i D 2 O på reagens samt slutprodukt. Signalen (för aminen) vid δ 2,9 (4H, CO NH CH 2 , CH 2 NH 2 ) och δ 1,6-1,1 (6H, CH 2 CH 2 CH 2 ) jämförs med signalen (för HANa) δ 1,9 (3H, HA-NHCO CH 3 ). (se även källa New strategy for chemical modification of………….., Paul Bulpitt et al)

131

Appendix

9.1.2 Appendix B Synthesis 2

CMC-graftadM-2070 (τ=5%) Table 12 Materials of synthesis 2 Beteckning Namn CMC Carboxymetylcellulosa EDC 1-ethyl-3-[3dimethylamino)propyl]carbodiimide NHS N-hydroxysuccinimid amin M-2070

Molekylvikt (M) g/mol 1*105 191,7

Monomervikt (M 0 ) g/mol 222 (0,25*162+0,75*242)

DS 0,75

115,09 2000

Synthesis 1. 2. 3. 4. 5. 6. 7.

CMC (1 g, M 0 =4,505*10-3 mol monomer) upplöses i 100ml (c=1% w/w) H 2 O 0,45g M-2070 (5% av M 0 =2,25*10-4 mol ) upplöses i ca 10ml H 2 O (både M-2070 samt CMC låtes upplösas under ca 1 dygn) pH justeras till 8 med 0,1 M NaOH/0,1 M HCl 1,29g EDC (67,5*10-4 mol, fast [EDC]:[NHS]:[amin]=30:7,5:1) väges upp och hålls under kvävgas 0,19g NHS (16,875*10-4 mol, fast) tillsätts till reaktionsblandningen under omrörning (ca 15 min). Uppvägd EDC (67,5*10-4 mol, fast [EDC]:[NHS]:[amin]=30:7,5:1) under kvävgas upplöses i 30 ml H 2 O) och tillsätts omedelbart (inom sekunder) till reaktionsblandningen under omrörning. pH vidhålls vid 8 under omblandning genom addition av 0,1 M NaOH/(0,1 M HCl). Reaktionen fortgår under natten.

Upprening 8. 9. 10. 11.

132

pH återjusteras till 7 med 0,1M HCl CMC-derivatet dialyseras (MW cut off 12,000-14,000) mot destillerat vatten. CMC-derivatet fälls ut genom tillsatts av 3 volymsekvivalenter etanol, steget repeteras. Utfällningen återlöses i H 2 O till en koncentration av 5 mg/mL och den upprenade produkten frystorkas/vakuumtorkas.

Appendix

9.1.3 Appendix C Syntes 3

Test av kopplingskemi (ethylenediamine) Material; Table 13 Materials of synthesis 3 Beteckning Namn HANa Natrium hyaluronate EDC 1-ethyl-3-[3dimethylamino)propyl]carbodiimide sulfoNHS N-hydroxysulfosuccinimid amin 1,6-hexandiamin dihydroclorid

Molekylvikt (M) g/mol 1-3 *106 191,7

Monomervikt (M 0 ) g/mol 401

217,13 189,13

Synthesis 1. 2. 3. 4. 5. 6. 7.

HANa (100 mg, 025mmol –COO- grupper) upplöses i 33,3….. ml (c=3mg/ml) H 2 O (ca 1 dygn) 30ggr så mycket 1,6-etylendiamin dihydroclorid tillsätts (7,5 mmol, 998 mg) pH justeras till 7,5 med 0,1 M NaOH/0,1 M HCl EDC (192 mg, 1mmol) vägs upp och hålls under kvävgas sulfoNHS (217 mg, 1mmol) löses upp i 1ml H 2 O och tillsätts. Uppvägd EDC under kvävgas upplöses i 1ml H 2 O och tillsätts omedelbart (inom sekunder). pH vidhålls vid 7,5 under omblandning genom addition av 0,1 M NaOH/(0,1 M HCl). Reaktionen fortgår under natten.

Upprening 8. pH återjusteras till 7 med 0,1M HCl 9. HA-derivatet dialyseras (MW cut off 12,000-14,000) mot destillerat vatten. 10. NaCl tillsätts tills en 5% w/v lösning erhålls och HA-derivatet fälls ut genom tillsatts av 3 volymsekvivalenter etanol, steget repeteras. 11. Utfällningen återlöses i H2O till en koncentration av 5 mg/mL och den upprenade produkten frystorkas och hålls vid 4°C under kvävgas (N 2 ) Karaktäriseing sker m.h.a. 1H-NMR i D 2 O på reagens samt slutprodukt. Signalen (för aminen) vid δ 2,9 (4H, CO NH CH 2 , CH 2 NH 2 ) och δ 1,6-1,1 (6H, CH 2 CH 2 CH 2 ) jämförs med signalen (för HANa) δ 1,9 (3H, HA-NHCO CH 3 ). (se även källa New strategy for chemical modification of………….., Paul Bulpitt et al)

133

Appendix

9.1.4 Appendix D Synthesis 4

CMC-graftadM-600 (τ=16,67%) Table 14 Materials of synthesis 4 Beteckning Namn

Molekylvikt (M) g/mol

Monomervikt (M 0 ) g/mol

DS

CMC

Carboxymetylcellulosa Cekol30

1*105

222 (0,25*162+0,75*242)

0,75

EDC

1-ethyl-3-[3dimethylamino)propyl]carbodiimide

191,7

NHS

N-hydroxysuccinimid

115,09

amin

M-600

600

Synthesis 1. 2. 3. 4. 5. 6. 7.

CMC (1 g, M 0 =4,505*10-3 mol monomer) upplöses i 100ml (c=1% w/w) H 2 O 0,45g M-600(5% av M 0 =7,5*10-4 mol ) upplöses i ca 10ml H 2 O. (både M-2005 samt CMC låtes upplösas under ca 1 dygn) Upplöst M-600 tillsätts till CMC lösningen. Låt stå 15 min. 5 ml av blandningen tas för SECanalys. pH justeras till 8 med 0,1 M NaOH/0,1 M HCl 0,647g NHS (5,625*10-3 mol, fast) tillsätts till reaktionsblandningen under omrörning (ca 15 min). 4,31g EDC (2,25*10-2 mol, fast [EDC]:[NHS]:[amin]=30:7,5:1) tillsätts omedelbart till reaktionsblandningen under omrörning. pH vidhålls vid 8 under omblandning genom addition av 0,1 M NaOH/(0,1 M HCl). Reaktionen fortgår under natten.

Upprening 8. 9. 10. 11. 12. 13. 14.

12

pH återjusteras till 7 med 0,1M HCl Totala reaktionsvolymen mäts noggrant 5ml av blandningen tas för SECanalys CMC-derivatet dialyseras (MW cut off 12,000-14,000) mot destillerat vatten. 1ml av dialysatet tas för SEC-analys. (CMC-derivatet fälls ut genom tillsatts av 3 volymsekvivalenter aceton, steget repeteras.) 12 Utfällningen återlöses i H 2 O till en koncentration av 5 mg/mL och den upprenade produkten frystorkas.

Utförs endast om SEC-analysen påvisar rester av oreagerad M-2070

134

Appendix

9.1.5 Appendix E Syntes 5

CMC-graftadM-2005 (τ=5%) Table 15 Materials of synthesis 5 Beteckning Namn

Molekylvikt (M) g/mol

Monomervikt (M 0 ) g/mol

DS

CMC

Carboxymetylcellulosa Cekol30

1*105

222 (0,25*162+0,75*242)

0,75

EDC

1-ethyl-3-[3dimethylamino)propyl]carbodiimide

191,7

NHS

N-hydroxysuccinimid

115,09

amin

M-2005

2000

Synthesis 1. 2. 3. 4. 5. 6. 7.

CMC (1 g, M 0 =4,505*10-3 mol monomer) upplöses i 100ml (c=1% w/w) H 2 O 0,45g M-2005 (5% av M 0 =2,25*10-4 mol ) upplöses i ca 10ml H 2 O i isbad 13. (både M-2005 samt CMC låtes upplösas under ca 1 dygn) Upplöst M-2005 tillsätts till CMC lösningen 14. Låt stå 15 min. 5 ml av blandningen tas för SECanalys. pH justeras till 8 med 0,1 M NaOH/0,1 M HCl 0,194g NHS (16,875*10-4 mol, fast) tillsätts till reaktionsblandningen under omrörning (ca 15 min). 1,295g EDC (67,5*10-4 mol, fast [EDC]:[NHS]:[amin]=30:7,5:1) tillsätts omedelbart till reaktionsblandningen under omrörning. pH vidhålls vid 8 under omblandning genom addition av 0,1 M NaOH/(0,1 M HCl). Reaktionen fortgår under natten.

Upprening 8. 9. 10. 11. 12. 13. 14.

pH återjusteras till 7 med 0,1M HCl Totala reaktionsvolymen mäts noggrant 5ml av blandningen tas för SECanalys CMC-derivatet dialyseras (MW cut off 12,000-14,000) mot destillerat vatten. 1ml av dialysatet tas för SEC-analys. (CMC-derivatet fälls ut genom tillsatts av 3 volymsekvivalenter aceton, steget repeteras.) 15 Utfällningen återlöses i H 2 O till en koncentration av 5 mg/mL och den upprenade produkten frystorkas.

13

4 % w/w för M-2005 i vatten har en cloud point på ~10°C Kylning är ej längre lika viktigt. Cloud Point för 0,4 % w/w lösning M-2005 i vatten har en cloud point på ~17-18°C 15 Utförs endast om SEC-analysen påvisar rester av oreagerad M-2070 14

135

Appendix

9.1.6 Appendix F Synthesis 6

CMC-graftadM-2005 (τ=10%) Table 16 Materials of synthesis 6 Beteckning Namn

Molekylvikt (M) g/mol

Monomervikt (M 0 ) g/mol

DS

CMC

Carboxymetylcellulosa Cekol30

1*105

222 (0,25*162+0,75*242)

0,75

EDC

1-ethyl-3-[3dimethylamino)propyl]carbodiimide

191,7

NHS amin

N-hydroxysuccinimid M-2005

115,09 2000

Synthesis 1. 2. 3. 4. 5. 6. 7.

CMC (1 g, M 0 =4,505*10-3 mol monomer) upplöses i 100ml (c=1% w/w) H 2 O 0,9g M-2005 (10% av n 0 =2,25*10-4 mol ) upplöses i ca 10ml H 2 O i isbad 16. (både M-2005 samt CMC låtes upplösas under ca 1 dygn) Upplöst M-2005 tillsätts till CMC lösningen 17. Låt stå 15 min. 5 ml av blandningen tas för SECanalys. pH justeras till 8 med 0,1 M NaOH/0,1 M HCl 0,388g NHS (33,75*10-4 mol, fast) tillsätts till reaktionsblandningen under omrörning (ca 15 min). 2,59g EDC (135*10-4 mol, fast [EDC]:[NHS]:[amin]=30:7,5:1) tillsätts omedelbart till reaktionsblandningen under omrörning. pH vidhålls vid 8 under omblandning genom addition av 0,1 M NaOH/(0,1 M HCl). Reaktionen fortgår under natten.

Upprening 8. 9. 10. 11. 12. 13. 14.

16

pH återjusteras till 7 med 0,1M HCl Totala reaktionsvolymen mäts noggrant 5ml av blandningen tas för SECanalys CMC-derivatet dialyseras (MW cut off 12,000-14,000) mot destillerat vatten. 1ml av dialysatet tas för SEC-analys. (CMC-derivatet fälls ut genom tillsatts av 3 volymsekvivalenter aceton, steget repeteras.) 18 Utfällningen återlöses i H 2 O till en koncentration av 5 mg/mL och den upprenade produkten frystorkas.

4 % w/w för M-2005 i vatten har en cloud point på ~10°C Kylning är ej längre lika viktigt. Cloud Point för 0,4 % w/w lösning M-2005 i vatten har en cloud point på ~17-18°C 18 Utförs endast om SEC-analysen påvisar rester av oreagerad M-2070 17

136

Appendix

9.1.7 Appendix G Syntes 7

HA-graftadM-600 (τ=16,67%) Table 17 Materials of synthesis 7 Beteckning Namn HA Sodium Hyaluronate Novozymes EDC 1-ethyl-3-[3dimethylamino)propyl]carbodiimide NHS N-hydroxysuccinimid amin M-600

Molekylvikt (M) g/mol 1*105

Monomervikt (M 0 ) g/mol 200

191,7

115,09 600

Synthesis 1. 2. 3. 4. 5. 6. 7.

HA (1 g, n 0 =5,0*10-3 mol monomer) upplöses i 100ml (c=1% w/w) H 2 O 0,5g M-600(5% av n 0 =8,3*10-4 mol ) upplöses i ca 10ml H 2 O. (både M-2005 samt CMC låtes upplösas under ca 1 dygn) Upplöst M-600 tillsätts till CMC lösningen. Låt stå 15 min. 5 ml av blandningen tas för SECanalys. pH justeras till 8 med 0,1 M NaOH/0,1 M HCl 0,72g NHS (6,25*10-3 mol, fast) tillsätts till reaktionsblandningen under omrörning (ca 15 min). 4,79g EDC (2,5*10-2 mol, fast [EDC]:[NHS]:[amin]=30:7,5:1) tillsätts omedelbart till reaktionsblandningen under omrörning. pH vidhålls vid 8 under omblandning genom addition av 0,1 M NaOH/(0,1 M HCl). Reaktionen fortgår under natten.

Upprening 1. 2. 3. 4. 5.

19

pH återjusteras till 7 med 0,1M HCl HA-derivatet dialyseras (MW cut off 12,000-14,000) mot destillerat vatten. 1ml av dialysatet tas för SEC-analys. (HA-derivatet fälls ut genom tillsatts av 3 volymsekvivalenter aceton, steget repeteras.) 19 Utfällningen återlöses i H 2 O till en koncentration av 5 mg/mL och den upprenade produkten frystorkas.

Utförs endast om SEC-analysen påvisar rester av oreagerad M-2070

137

Appendix

9.1.8 Appendix H Synthesis 8

CMC-graftadM-2005 (τ=2,5%) Table 18 Materials of synthesis 8 Beteckning Namn

Molekylvikt (M) g/mol

Monomervikt (M 0 ) g/mol

DS

CMC

Carboxymetylcellulosa Cekol30

1*105

222 (0,25*162+0,75*242)

0,75

EDC

1-ethyl-3-[3dimethylamino)propyl]carbodiimide

191,7

NHS

N-hydroxysuccinimid

115,09

amin

M-2005

2000

Synthesis 1. 2. 3. 4. 5. 6. 7.

CMC (1 g, M 0 =4,505*10-3 mol monomer) upplöses i 100ml (c=1% w/w) H 2 O 0,225g M-2005 (2,5% av n 0 =1,13*10-4 mol ) upplöses i ca 10ml H 2 O i isbad 20. (både M-2005 samt CMC låtes upplösas under ca 1 dygn) Upplöst M-2005 tillsätts till CMC lösningen 21. Låt stå 15 min. 5 ml av blandningen tas för SECanalys. pH justeras till 8 med 0,1 M NaOH/0,1 M HCl 0,097g NHS (8,45*10-4 mol, fast) tillsätts till reaktionsblandningen under omrörning (ca 15 min). 0,648g EDC (33,78*10-4 mol, fast [EDC]:[NHS]:[amin]=30:7,5:1) tillsätts omedelbart till reaktionsblandningen under omrörning. pH vidhålls vid 8 under omblandning genom addition av 0,1 M NaOH/(0,1 M HCl). Reaktionen fortgår under natten.

Upprening 8. 9. 10. 11. 12. 13. 14.

20

pH återjusteras till 7 med 0,1M HCl Totala reaktionsvolymen mäts noggrant 5ml av blandningen tas för SECanalys CMC-derivatet dialyseras (MW cut off 12,000-14,000) mot destillerat vatten. 1ml av dialysatet tas för SEC-analys. (CMC-derivatet fälls ut genom tillsatts av 3 volymsekvivalenter aceton, steget repeteras.) 22 Utfällningen återlöses i H 2 O till en koncentration av 5 mg/mL och den upprenade produkten frystorkas.

4 % w/w för M-2005 i vatten har en cloud point på ~10°C Kylning är ej längre lika viktigt. Cloud Point för 0,4 % w/w lösning M-2005 i vatten har en cloud point på ~17-18°C 22 Utförs endast om SEC-analysen påvisar rester av oreagerad M-2070 21

138

Appendix

9.1.9 Appendix I Syntes 9

HA-graftadM-2005 (τ=5%) Table 19 Materials of synthesis 9 Beteckning Namn HA Sodium Hyaluronate Novozymes EDC 1-ethyl-3-[3dimethylamino)propyl]carbodiimide NHS N-hydroxysuccinimid amin M-2005

Molekylvikt (M) g/mol 1*105

Monomervikt (M 0 ) g/mol 200

191,7

115,09 2000

Synthesis 1. 2. 3. 4. 5. 6. 7.

HA (1 g, n 0 =5*10-3 mol monomer) upplöses i 100ml (c=1% w/w) H 2 O 1g M-2005 (10% av n 0 =5*10-4 mol ) upplöses i ca 10ml H 2 O i isbad 23. (både M-2005 samt HA låtes upplösas under ca 1 dygn) Upplöst M-2005 tillsätts till HA lösningen 24. Låt stå 15 min. 5 ml av blandningen tas för SECanalys. pH justeras till 8 med 0,1 M NaOH/0,1 M HCl 0,43g NHS (3,75*10-3 mol, fast) tillsätts till reaktionsblandningen under omrörning (ca 15 min). 2,88g EDC (15*10-3 mol, fast [EDC]:[NHS]:[amin]=30:7,5:1) tillsätts omedelbart till reaktionsblandningen under omrörning. pH vidhålls vid 8 under omblandning genom addition av 0,1 M NaOH/(0,1 M HCl). Reaktionen fortgår under natten.

Upprening 8. 9. 10. 11. 12.

pH återjusteras till 7 med 0,1M HCl HA-derivatet dialyseras (MW cut off 12,000-14,000) mot destillerat vatten. 1ml av dialysatet tas för SEC-analys. (HA-derivatet fälls ut genom tillsatts av 3 volymsekvivalenter aceton, steget repeteras.) 25 Utfällningen återlöses i H 2 O till en koncentration av 5 mg/mL och den upprenade produkten frystorkas.

23

4 % w/w för M-2005 i vatten har en cloud point på ~10°C Kylning är ej längre lika viktigt. Cloud Point för 0,4 % w/w lösning M-2005 i vatten har en cloud point på ~17-18°C 25 Utförs endast om SEC-analysen påvisar rester av oreagerad M-2070 24

139

Appendix

9.1.10 Appendix J Synthesis 10

CMC(Cekol150)-graftadM-600 (τ=8,33%) Table 20 Materials of synthesis 10 Beteckning Namn

Molekylvikt (M) g/mol

Monomervikt (M 0 ) g/mol

DS

CMC

Carboxymetylcellulosa Cekol150

1*105

220,4 (0,27*162+0,73*242)

0,73

EDC

1-ethyl-3-[3dimethylamino)propyl]carbodiimide

191,7

NHS

N-hydroxysuccinimid

115,09

amin

M-600

600

Synthesis 1. 2. 3. 4. 5. 6. 7.

CMC (1 g, M 0 =4,537*10-3 mol monomer) upplöses i 100ml (c=1% w/w) H 2 O 0,227g M-600(8,33% av n 0 =3,78*10-4 mol ) upplöses i ca 10ml H 2 O. (både M-2005 samt CMC låtes upplösas under ca 1 dygn) Upplöst M-600 tillsätts till CMC lösningen. Låt stå 15 min. 5 ml av blandningen tas för SECanalys. pH justeras till 8 med 0,1 M NaOH/0,1 M HCl 0,326g NHS (2,8358*10-3 mol, fast) tillsätts till reaktionsblandningen under omrörning (ca 15 min). 2,174g EDC (1,134*10-2 mol, fast [EDC]:[NHS]:[amin]=30:7,5:1) tillsätts omedelbart till reaktionsblandningen under omrörning. pH vidhålls vid 8 under omblandning genom addition av 0,1 M NaOH/(0,1 M HCl). Reaktionen fortgår under natten.

Upprening 8. 9. 10. 11. 12. 13. 14.

26

pH återjusteras till 7 med 0,1M HCl Totala reaktionsvolymen mäts noggrant 5ml av blandningen tas för SECanalys CMC-derivatet dialyseras (MW cut off 12,000-14,000) mot destillerat vatten. 1ml av dialysatet tas för SEC-analys. (CMC-derivatet fälls ut genom tillsatts av 3 volymsekvivalenter aceton, steget repeteras.) 26 Utfällningen återlöses i H 2 O till en koncentration av 5 mg/mL och den upprenade produkten frystorkas.

Utförs endast om SEC-analysen påvisar rester av oreagerad M-2070

140

Appendix

9.1.11 Appendix K Synthesis 11

CMC(Cekol150)-graftadM-2005 (τ=10%) Table 21 Materials of synthesis 11 Beteckning Namn

Molekylvikt (M) g/mol

Monomervikt (M 0 ) g/mol

DC

CMC

Carboxymetylcellulosa Cekol150

1*105

220,4 (0,27*162+0,73*242)

0,73

EDC

1-ethyl-3-[3dimethylamino)propyl]carbodiimide

191,7

NHS amin

N-hydroxysuccinimid M-2005

115,09 2000

Synthesis 1. 2. 3. 4. 5. 6. 7.

CMC (1 g, M 0 =4,537*10-3 mol monomer) upplöses i 100ml (c=1% w/w) H 2 O 0,907g M-2005 (10% av n 0 =4,537*10-4 mol ) upplöses i ca 10ml H 2 O i isbad 27. (både M-2005 samt CMC låtes upplösas under ca 1 dygn) Upplöst M-2005 tillsätts till CMC lösningen 28. Låt stå 15 min. 5 ml av blandningen tas för SECanalys. pH justeras till 8 med 0,1 M NaOH/0,1 M HCl 0,392g NHS (34,03*10-4 mol, fast) tillsätts till reaktionsblandningen under omrörning (ca 15 min). 2,61g EDC (136*10-4 mol, fast [EDC]:[NHS]:[amin]=30:7,5:1) tillsätts omedelbart till reaktionsblandningen under omrörning. pH vidhålls vid 8 under omblandning genom addition av 0,1 M NaOH/(0,1 M HCl). Reaktionen fortgår under natten.

Upprening 8. 9. 10. 11. 12. 13.

pH återjusteras till 7 med 0,1M HCl Totala reaktionsvolymen mäts noggrant 5ml av blandningen tas för SECanalys CMC-derivatet dialyseras (MW cut off 12,000-14,000) mot destillerat vatten. 1ml av dialysatet tas för SEC-analys. (CMC-derivatet fälls ut genom tillsatts av 3 volymsekvivalenter aceton, steget repeteras.) 29

27

4 % w/w för M-2005 i vatten har en cloud point på ~10°C Kylning är ej längre lika viktigt. Cloud Point för 0,4 % w/w lösning M-2005 i vatten har en cloud point på ~17-18°C 29 Utförs endast om SEC-analysen påvisar rester av oreagerad M-2070 28

141

Appendix

9.1.12 Appendix L Synthesis 12

CMC-graftadM-600 (τ=33,33%) Table 22 Materials of synthesis 12 Beteckning Namn

Molekylvikt (M) g/mol

Monomervikt (M 0 ) g/mol

DC

CMC

Carboxymetylcellulosa Cekol30

1*105

222 (0,25*162+0,75*242)

0,75

EDC

1-ethyl-3-[3dimethylamino)propyl]carbodiimide

191,7

NHS

N-hydroxysuccinimid

115,09

amin

M-600

600

Synthesis 1. 2. 3. 4. 5. 6. 7.

CMC (1 g, M 0 =4,505*10-3 mol monomer) upplöses i 100ml (c=1% w/w) H 2 O 0,9g M-600(33,33% av n 0 =7,5*10-4 mol ) upplöses i ca 10ml H 2 O. (både M-2005 samt CMC låtes upplösas under ca 1 dygn) Upplöst M-600 tillsätts till CMC lösningen. Låt stå 15 min. 5 ml av blandningen tas för SECanalys. pH justeras till 8 med 0,1 M NaOH/0,1 M HCl 1,294g NHS (11,25*10-3 mol, fast) tillsätts till reaktionsblandningen under omrörning (ca 15 min). 8,62g EDC (4,5*10-2 mol, fast [EDC]:[NHS]:[amin]=30:7,5:1) tillsätts omedelbart till reaktionsblandningen under omrörning. pH vidhålls vid 8 under omblandning genom addition av 0,1 M NaOH/(0,1 M HCl). Reaktionen fortgår under natten.

Upprening 8. 9. 10. 11. 12. 13. 14.

30

pH återjusteras till 7 med 0,1M HCl Totala reaktionsvolymen mäts noggrant 5ml av blandningen tas för SECanalys CMC-derivatet dialyseras (MW cut off 12,000-14,000) mot destillerat vatten. 1ml av dialysatet tas för SEC-analys. (CMC-derivatet fälls ut genom tillsatts av 3 volymsekvivalenter aceton, steget repeteras.) 30 Utfällningen återlöses i H 2 O till en koncentration av 5 mg/mL och den upprenade produkten frystorkas.

Utförs endast om SEC-analysen påvisar rester av oreagerad M-2070

142

Appendix

9.1.13 Appendix M Syntes 13

HA-graftadM-2005 (τ=10%) Table 23 Materials of synthesis 13 Beteckning Namn HA Sodium Hyaluronate Novozymes EDC 1-ethyl-3-[3dimethylamino)propyl]carbodiimide NHS N-hydroxysuccinimid amin M-2005

Molekylvikt (M) g/mol 1*105

Monomervikt (M 0 ) g/mol 200

191,7

115,09 2000

Synthesis 1. 2. 3. 4. 5. 6. 7.

HA (1 g, n 0 =5*10-3 mol monomer) upplöses i 100ml (c=1% w/w) H 2 O 1g M-2005 (10% av n 0 =5*10-4 mol ) upplöses i ca 10ml H 2 O i isbad 31. (både M-2005 samt HA låtes upplösas under ca 1 dygn) Upplöst M-2005 tillsätts till HA lösningen 32. Låt stå 15 min. 5 ml av blandningen tas för SECanalys. pH justeras till 8 med 0,1 M NaOH/0,1 M HCl 0,43g NHS (3,75*10-3 mol, fast) tillsätts till reaktionsblandningen under omrörning (ca 15 min). 2,88g EDC (15*10-3 mol, fast [EDC]:[NHS]:[amin]=30:7,5:1) tillsätts omedelbart till reaktionsblandningen under omrörning. pH vidhålls vid 8 under omblandning genom addition av 0,1 M NaOH/(0,1 M HCl). Reaktionen fortgår under natten.

Upprening 8. 9. 10. 11. 12.

pH återjusteras till 7 med 0,1M HCl HA-derivatet dialyseras (MW cut off 12,000-14,000) mot destillerat vatten. 1ml av dialysatet tas för SEC-analys. (HA-derivatet fälls ut genom tillsatts av 3 volymsekvivalenter aceton, steget repeteras.) 33 Utfällningen återlöses i H 2 O till en koncentration av 5 mg/mL och den upprenade produkten frystorkas.

31

4 % w/w för M-2005 i vatten har en cloud point på ~10°C Kylning är ej längre lika viktigt. Cloud Point för 0,4 % w/w lösning M-2005 i vatten har en cloud point på ~17-18°C 33 Utförs endast om SEC-analysen påvisar rester av oreagerad M-2070 32

143

Appendix

9.1.14 Appendix N Syntes 13.1

HA-graftadM-2005 (τ=10%) (koncentrerad) Table 24 Materials of synthesis 13.1 Beteckning Namn HA Sodium Hyaluronate Novozymes EDC 1-ethyl-3-[3dimethylamino)propyl]carbodiimide NHS N-hydroxysuccinimid amin M-2005

Molekylvikt (M) g/mol 1*105

Monomervikt (M 0 ) g/mol 200

191,7

115,09 2000

Synthesis 1. 2. 3. 4. 5. 6. 7.

HA (3 g, n 0 =1,5*10-2 mol monomer) upplöses i 100ml (c=4% w/w) H 2 O 3g M-2005 (10% av n 0 =1,5*10-3 mol ) upplöses i ca 10ml H 2 O i isbad 34. (både M-2005 samt HA låtes upplösas under ca 1 dygn) Upplöst M-2005 tillsätts till HA lösningen 35. Låt stå 15 min. 5 ml av blandningen tas för SECanalys. pH justeras till 8 med 0,1 M NaOH/0,1 M HCl 1,29g NHS (1,125*10-2 mol, fast) tillsätts till reaktionsblandningen under omrörning (ca 15 min). 8,63g EDC (4,5*10-2 mol, fast [EDC]:[NHS]:[amin]=30:7,5:1) tillsätts omedelbart till reaktionsblandningen under omrörning. pH vidhålls vid 8 under omblandning genom addition av 0,1 M NaOH/(0,1 M HCl). Reaktionen fortgår under natten.

Upprening 8. 9. 10. 11. 12.

34

pH återjusteras till 7 med 0,1M HCl HA-derivatet dialyseras (MW cut off 12,000-14,000) mot destillerat vatten. 1ml av dialysatet tas för SEC-analys. (HA-derivatet fälls ut genom tillsatts av 3 volymsekvivalenter aceton, steget repeteras.) 36 Utfällningen återlöses i H 2 O till en koncentration av 5 mg/mL och den upprenade produkten frystorkas.

4 % w/w för M-2005 i vatten har en cloud point på ~10°C Kylning är ej längre lika viktigt. Cloud Point för 0,4 % w/w lösning M-2005 i vatten har en cloud point på ~17-18°C 36 Utförs endast om SEC-analysen påvisar rester av oreagerad M-2070 35

144

Appendix

9.1.15 Appendix O Syntes 14

HA-graftadM-2005 (τ=15%) Table 25 Materials of synthesis 14 Beteckning Namn HA Sodium Hyaluronate Novozymes EDC 1-ethyl-3-[3dimethylamino)propyl]carbodiimide NHS N-hydroxysuccinimid amin M-2005

Molekylvikt (M) g/mol 1*105

Monomervikt (M 0 ) g/mol 200

191,7

115,09 2000

Synthesis 1. 2. 3. 4. 5. 6. 7.

HA (1 g, n 0 =5*10-3 mol monomer) upplöses i 100ml (c=1% w/w) H 2 O 1,5g M-2005 (10% av n 0 =7,5*10-4 mol ) upplöses i ca 10ml H 2 O i isbad 37. (både M-2005 samt HA låtes upplösas under ca 1 dygn) Upplöst M-2005 tillsätts till HA lösningen 38. Låt stå 15 min. 5 ml av blandningen tas för SECanalys. pH justeras till 8 med 0,1 M NaOH/0,1 M HCl 0,65g NHS (5,625*10-3 mol, fast) tillsätts till reaktionsblandningen under omrörning (ca 15 min). 4,31g EDC (2,25*10-2 mol, fast [EDC]:[NHS]:[amin]=30:7,5:1) tillsätts omedelbart till reaktionsblandningen under omrörning. pH vidhålls vid 8 under omblandning genom addition av 0,1 M NaOH/(0,1 M HCl). Reaktionen fortgår under natten.

Upprening 8. 9. 10. 11. 12.

pH återjusteras till 7 med 0,1M HCl HA-derivatet dialyseras (MW cut off 12,000-14,000) mot destillerat vatten. 1ml av dialysatet tas för SEC-analys. (HA-derivatet fälls ut genom tillsatts av 3 volymsekvivalenter aceton, steget repeteras.) 39 Utfällningen återlöses i H 2 O till en koncentration av 5 mg/mL och den upprenade produkten frystorkas.

37

4 % w/w för M-2005 i vatten har en cloud point på ~10°C Kylning är ej längre lika viktigt. Cloud Point för 0,4 % w/w lösning M-2005 i vatten har en cloud point på ~17-18°C 39 Utförs endast om SEC-analysen påvisar rester av oreagerad M-2070 38

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