Synthetic neoglycopolymer-recombinant human collagen hybrids as biomimetic crosslinking agents in corneal tissue engineering

Biomaterials 30 (2009) 5403–5408

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Synthetic neoglycopolymer-recombinant human collagen hybrids as biomimetic crosslinking agents in corneal tissue engineering Kimberley Merrett a, Wenguang Liu a, Debbie Mitra b, Kenneth D. Camm b, Christopher R. McLaughlin a, c, Yuwen Liu c, Mitchell A. Watsky d, Fengfu Li c, May Griffith a, c,1, Deryn E. Fogg b, *,1 a

Department of Cellular and Molecular Medicine, University of Ottawa, 451 Smyth Road, Ottawa, ON K1S 8M5, Canada Department of Chemistry, Centre for Catalysis Research and Innovation, University of Ottawa, 10 Marie Curie, Ottawa, ON K1N 6N5, Canada University of Ottawa Eye Institute, 501 Smyth Road, Ottawa, ON K1H 8L6, Canada d Department of Physiology, University of Tennessee Health Sciences Centre, 894 University Ave. Memphis, TN 38163-0000, USA b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 April 2009 Accepted 10 June 2009 Available online 2 July 2009

Saturated neoglycopolymers, prepared via tandem ROMP-hydrogenation (ROMP ¼ ring-opening metathesis polymerization) of carbohydrate-functionalized norbornenes, are investigated as novel collagen crosslinking agents in corneal tissue engineering. The neoglycopolymers were incorporated into recombinant human collagen type III (RHC III) as collagen crosslinking agents and glycosaminoglycan (GAG) mimics. The purely synthetic nature of these composites is designed to reduce susceptibility to immunological and allergic reactions, and to circumvent the transmission of animal infectious diseases. The collagen-neoglycopolymer biomaterials exhibit higher stability to collagenase-induced biodegradation than the control materials, composites of RHC III crosslinked using EDC/NHS (EDC ¼ 1-ethyl-3-(3dimethyl aminopropyl) carbodiimide; NHS ¼ N-hydroxysuccinimide). Even at this proof of concept stage, the thermal stability, enzymatic resistance, and permeability of the neoglycopolymer hydrogels are comparable or superior to those of these fully optimized control materials, which have successfully been tested clinically. Tensile strength is adequate for transplantation, but lower than that of the optimized control materials. Ó 2009 Elsevier Ltd. All rights reserved.

Keywords: Biomimetic materials Crosslinking Collagen Cornea Tissue engineering Neoglycopolymer

1. Introduction Biologically derived extracellular matrix (ECM) macromolecules such as collagen provide a three-dimensional framework for connective tissues such as bone, cartilage, and tendons, as well as skin and the cornea [1,2]. Collagen matrices are of particular interest as highly biointeractive biomaterials, which can provide optimal microenvironments for cell differentiation during organogenesis or regeneration [3–5]. Nature’s ability to design materials that encompass a wide range of features, including structural control and biological activity, has inspired extensive research efforts in the field of tissue engineering. Here we explore the use of wholly synthetic, crosslinked collagen matrices in the fabrication of bio-functional materials relevant to corneal tissue engineering. Collagen is a triple helical coil of amino acid sequences comprised mainly of repeating residues of glycine, proline, and

* Corresponding author. E-mail address: [email protected] (D.E. Fogg). 1 Equal contribution. 0142-9612/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2009.06.016

hydroxyproline [1]. Collagen hydrogels evince high biocompatibility, and are conducive to activating progenitor cells that promote regeneration (as found in successful Phase 1 human clinical trials [6]). However, they are inherently biodegradable, and must be stabilized by crosslinking. Glutaraldehyde [7,8] and other bifunctional small molecules [9], including genipin and water-soluble carbodiimides [10] (e.g. N-ethyl-N0 -dimethylaminopropyl carbodiimide, EDC), are currently among the most widely used collagen crosslinking agents. Their limitations, however, include the susceptibility of the resulting materials to calcification [11,12], the potential for local cytotoxicity [9] or discoloration [10] (a drawback for corneal applications; vide infra), and, at worst, a retained susceptibility to rapid, uncontrolled in vivo biodegradation. In EDCcrosslinked collagen hydrogels, for example (in which the crosslinking agent is not incorporated into the final construct), the ‘‘stabilized’’ gels are in fact remodelled fairly rapidly by host enzymes [13]. Our most recent work strongly suggests that incorporation of synthetic components is required to increase the stability and enzyme resistance of collagen-based hydrogels. For example, PEG–phosphorylcholine composites create an interpenetrating network [14], in which the synthetic lipid component appears to block enzymatic attack from collagenase.

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Less explored are carbohydrate-derived crosslinkers, although natural glycation processes have long been known to result in stiffening of collagen [15], and glucose and ribose have been used as non-cytotoxic, non-calcifying collagen crosslinking agents [16], in some cases in conjunction with UV irradiation [17]. Inspired by these precedents, we explored the utility of carbohydrate-functionalized polymers as potentially biomimetic, non-cytotoxic collagen crosslinking agents, within the context of corneal tissue engineering. Our interest in polymeric crosslinking agents stems from their potential to simultaneously enhance binding through the multivalent effect, while maintaining the spacing of collagen fibrils, and hence free movement of water and nutrients. In transparent tissues such as the cornea, control over the spacing and fibril sizes of collagen is thought to be regulated by keratan sulphate type I glycosaminoglycans (GAGs) [18,19], macromolecules bearing repeating disaccharide units of galactose and N-acetylglucosamine. Ring-opening metathesis polymerization (ROMP) is a powerful, versatile synthetic methodology enabling the molecular-level design of macromolecular materials [20]. ROMP of functionalized norbornenes has been used to construct an enormous range of ‘‘designer’’ polymers, including biologically active macromolecules [21] explored in cell signaling [22–26], drug delivery [27–30], DNA diagnostic [31], and antibacterial [32,33] applications. Of particular interest in the present context are pioneering studies by Kiessling et al., which demonstrated that ROMP-derived neoglycopolymers interact with cell receptor sites, with a binding affinity enhanced by the polyvalent presentation of donor sites [26,34,35]. Use of such synthetic neoglycopolymers to crosslink recombinant collagen is highly attractive as a strategy for accessing a novel class of composites that – unlike animal-derived materials – do not entail potential exposure to infectious agents such as viruses or prions. Of added value is the batch-to-batch uniformity of recombinant collagens, relative to animal-extracted materials. A number of recent reports describe the use of recombinant collagens in drug delivery applications [36], and tissue engineering of bone, skin, and cartilage [5], as well as of the cornea [37]. Here we describe the use of ROMP neoglycopolymers as collagen crosslinking agents and GAG mimetics, within a wholly synthetic polymer-recombinant human collagen hybrid.

mixed. The final solution was dispensed as flat sheets into glass molds, cured at 100% humidity at room temperature for 48 h, post-cured at 37  C for 1 day, then washed extensively in PBS to extract any non-crosslinked neoglycopolymer remaining. 2.3. Optical property measurements Refractive indices (RIs) of fully hydrated neoglycopolymer-RHC III and control hydrogels were recorded using an Abbe refractometer (Model C10, VEE GEE, Scientific Inc., Kirkland, WA) at 21  C with bromonaphthalene as the calibration agent. Transmission and back-scattering measurements were made at 21  C, both for white light (quartz-halogen lamp source) and over narrow spectral regions (Dy1/2 of 40 nm, centered at 450, 500, 550, 600 and 650 nm), on a custom-built instrument [42]. 2.4. Mechanical property measurements The tensile strength, elongation at break, and elastic moduli of the hydrogels were determined on an Instron electromechanical universal tester (Model 3342) equipped with Series IX/S software, using a crosshead speed of 10 mm/min. Flat hydrogels, 0.44 mm thick, were equilibrated in PBS and cut into 10 mm  5 mm rectangular sheets. The actual gauge length of each specimen used for testing was 5 mm. Three specimens were measured for each hydrogel formulation. 2.5. Equilibrium water content measurement After removal from the molds, hydrogels were immersed in PBS for 7 days at 4  C. They were then removed, gently blotted dry with filter paper, and immediately weighed on a microbalance to record the wet weight of the sample. They were allowed to dry at room temperature under vacuum to constant weight (for 24 h or longer), following which the total equilibrated water content of the hydrogels (Wt) was calculated according to the following equation: Wt ¼ ðW  W0 Þ=W  100% where W and W0 denote weights of PBS-equilibrated and dried samples, respectively. 2.6. Thermal properties: differential scanning calorimetry (DSC) The thermal properties of the hydrogels were examined on a Perkin–Elmer DSCCellbase differential scanning calorimeter (Instrument Specialists Incorporated, Spring Grove, IL). Heating scans were recorded in the range of 8–70  C at a scan rate of 5  C/min. Pre-weighed samples of the PBS-equilibrated hydrogels (weights ranging from 5 to 10 mg) were surface-dried with filter paper and hermetically sealed in an aluminum pan to prevent water evaporation. PBS was used as a blank reference. The denaturation temperature (Td) was measured at the maximum of the endothermic peak. 2.7. Glucose permeability

2. Material and methods 2.1. Materials Recombinant human collagen type III (RHC III) produced in yeast cells (Pichia pastoris) was purchased from FibroGen Inc. (South San Francisco, CA), freeze-dried, and reconstituted to make a 13.7 w/w% solution. This solution was transferred into a plastic syringe and centrifuged at 4  C to completely remove entrapped air bubbles, affording a clear, viscous solution. The norbornene monomers 1a [38] and 1b [39], and the metathesis catalyst RuCl2(IMes)(C5H5N)2(]CHPh) 2 [40] were prepared by the literature methods. All other reagents were of analytical grade and used as received. 1-Ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and sodium cyanoborohydride (NaBH3CN) were supplied by Sigma–Aldrich (Oakville, Ontario), and N-hydroxysuccinimide (NHS) by Fluka (Buchs, Switzerland). Phosphate-buffered saline (PBS, pH 7.2) was prepared from the tablet form (Calbiochem Corp, Darmstadt, Germany). Milli-Q deionized water (Millipore, Billerica, MD) was used throughout. RHC III was crosslinked using EDC/NHS by the reported method [37,41], for use as a control material.

The glucose permeability of the hydrogels was determined as described previously [41] at the normal physiological temperature of the cornea (35  C) using a modified Ussing chamber (Warner Instruments, Hamden, CT) with air-lift mixing. Colorimetric measurements were made at 540 nm using a glucose assay kit (GAG020, Sigma–Aldrich) with a Shimadzu UV-1601 spectrophotometer. The values were fit to a regression line of standards of known concentration. Diffusion coefficients were calculated using these values. 2.8. In vitro biocompatibility and performance In vitro biocompatibility and performance were measured using immortalized human corneal epithelial cells (HCEC) to evaluate epithelial coverage. In a slight modification of the reported method [43], HCECs were seeded on top of 1.5 cm2 hydrogel pieces, supplemented with a serum-free medium containing epidermal growth factor (Keratinocyte Serum-Free Medium, Life Technologies, Burlington, ON), and grown until confluent. At 8 days, constructs were imaged using an inverted Nikon Eclipse TE2000-E. Time to confluence was compared to plasma-treated, tissue-culture plastic controls.

2.2. Preparation of collagen-neoglycopolymer hydrogels 2.9. In vitro biodegradation Neoglycopolymers were prepared by tandem ROMP-hydrogenation of 1 via initiator 2 as previously described [39], and deprotected by the standard method [38]. Following dialysis in water (SpectraPor 7 membrane; cut-off 2000 Da) for 7 days with daily exchanges of double-distilled H2O, neoglycopolymer-RHC III hydrogels were prepared by mixing 0.2 mL of the 13.7 w/w% RHC III solution, 0.1 mL phosphate-buffered saline (PBS) and 0.2 mL of neoglycopolymer solution (solid collagen content is 2:1 w/w vs neoglycopolymer in PBS). Air bubbles were eliminated as above. Following addition of 2 N NaOH to adjust the solution to pH 8, 25 mL of NaBH3CN solution (1 mg/mL in PBS) was added, and the solution thoroughly

In vitro biodegradation was assessed as we previously described [37]. Briefly, hydrated neoglycopolymer-RHC III hydrogels (5–8 mg) were placed in vials containing a broad-spectrum collagenase in PBS solution (5 mL of a 5 U/mL solution of Type I Collagenase from Clostridium histolyticum, 318 U/mg solid, Sigma–Aldrich, Oakville, Ontario). The vials were incubated in an oven at 37  C. The gels were weighed at different time intervals after blotting their surfaces dry of moisture. The residual mass of the neoglycopolymer-RHC III hydrogels was tracked as a function of time, relative to the initial hydrated weight.

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3. Results and discussion 3.1. Fabrication of neoglycopolymer-collagen hydrogels Synthesis of the required neoglycopolymers was undertaken by tandem ROMP-hydrogenation of norbornene monomers 1 (Fig. 1), which bear galactose groups native to the collagenous stroma of the cornea [18]. The hydrogenation step is important in eliminating the potential for degradation [44,45] at the unsaturated sites otherwise present in the polymer backbone [46–49]. Simple, unhindered ROMP polyoctenes can be hydrogenated by tandem catalysis under mild conditions (50–60  C, 1 atm H2) in the presence of methanol [49], but bulky polynorbornenes are considerably more demanding, requiring hydrogen pressures of 1000 psi [39]. The high pressures are preferable to use of higher temperatures, which can trigger competitive thermal crosslinking of the unsaturated polymers (as found in diimide reduction protocols [50,51]). Less satisfactory results have been reported using other catalysts, despite their often outstanding performance in reduction of unsaturated small molecules at relatively low H2 pressures [50,51]. As the saturated polymers derived from mono-galactose monomer 1a [38] proved incompletely soluble in water following reduction and deprotection, we turned to the di-galactose norbornene 1b. We earlier described the controlled ROMP-hydrogenation of this sterically demanding 5,6-disubstituted monomer using initiator 2, which combines high ROMP activity with precise control over polymer chain lengths [39]. In situ protection of the catalyst with PCy3, followed by hydrogenation in the presence of methanol and triethylamine base, gave efficient access to the saturated polymers, which on deprotection afforded the water-soluble neoglycopolymer. This material was isolated and purified by dialysis prior to crosslinking. Polymers containing 50 repeat units, on average, were chosen for experimental convenience, as preliminary studies indicated no increase in tensile strength at higher polymer chain lengths. The galactose unit can participate in crosslinking with appropriate nucleophiles via its ring-opened aldose form, which exists in

NaCNBH3

+

Fig. 2. Cartoon depicting reductive amination process resulting in collagen crosslinking by neoglycopolymer. Ring-opened aldose form of galactose represented by diamonds ( ) and dominant cyclic form by triangles ( ). Accessible primary amine sites on collagen represented by open triangles ( ).

equilibrium with the dominant cyclic hemiacetal (see Fig. 2). Thus, incubation of the neoglycopolymer with collagen effects condensation of aldehydic sugar sites with accessible primary amines present in collagenous lysine or hydroxylsine groups. A hydrolytically stable secondary amine can be generated by in situ reduction [52] of the resulting imine linkages with sodium cyanoborohydride. Solid-state 13C{1H} NMR analysis confirmed retention of the galactose unit in the dried collagenous hydrogels following crosslinking and extraction. Slow gelation is advantageous in improving the homogeneity of collagen crosslinking, and hence material properties. In our previously developed hydrogels, in which we used poly-N-isopropylacrylamideco-acrylic acid-co-acryloxysuccinimide to crosslink bovine collagen [43], the reaction kinetics were difficult to control at comparably high concentrations of collagen, resulting in inhomogeneous materials. Considerably slower, well-controlled gelation of collagen is found using the neoglycopolymer (48 vs 16 h for the control materials). 3.2. Mechanical properties of collagen-neoglycopolymer hydrogels

(i)-(iv) OO

O

O

O

R

O

HO

O

O

HO O

O

Human cornea

Optical properties Refractive index Transmission (%) Backscatter (%) Permeability Glucose (cm2/s)

O O

HO O

The mechanical properties of the neoglycopolymer-RHC III collagen hydrogels are compared with those of our EDC/NHScrosslinked control materials in Table 1. The latter were previously shown to afford constructs that were adequately robust for use in human transplantation [6], with a tensile strength of 1.7 MPa, an

Table 1 Key properties of collagen-neoglycopolymer hybrids, with comparison to human corneas and EDC/NHS-crosslinked collagen control; n  3 samples for each test.

poly(1a): R' = H poly(1b): R' =

O O

R' poly(1)

HO HO

1a: R = H 1b: R = O

50

HO O

1

O O

O

Ph

H

HO OH

Fig. 1. Synthesis of neoglycopolymer via tandem ROMP-hydrogenation. (i) ROMP: 2 mol% RuCl2(IMes)(C5H5N)2(]CHPh) (2; Ph ¼ C6H5, IMes ¼ N,N0 -bis(mesityl)imidazol2-ylidene), CH2Cl2, 2.5 h; (ii) þ1.2 PCy3 (Cy ¼ cyclohexyl); (iii) hydrogenation: 1000 psi H2, 10 N(CH2CH3)3, CH3OH, 60  C, 3 h; (iv) deprotection: aq. 90% CF3CO2H, 15 min.

Mechanical properties Tensile strength (MPa) Elongation at break (%) Young’s modulus (MPa) Water content Thermal properties Denaturation temperature (Td,  C)

RHC type III collagen EDC/NHScrosslinked [37]

Neoglycopolymercrosslinked

1.375 [55] >85 [54] 6–8 [54]

1.35 89.8  0.9 0.81

1.35 86.9  0.8 0.32

2.5–3.0  106 [56,57]

1.19  0.07  106

2.4  0.2  106

3.8–19 [58,59] – 3–13 [60,61] 80% [60]

1.7  0.2 13.9  0.7 20  2 90%

0.44  0.02 32.4  9 2.2  0.3 90%

65.1

58.6

63.6

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elongation at break of 13.9%, and an elastic modulus of 20 MPa [37,53]. The neoglycopolymer-crosslinked materials, in comparison, are considerably more elastic, as indicated by their lower tensile strength, but increased elongation at break, and reduced Young’s modulus. The elasticity of the hydrogels is important as an indicator of suturability. The high modulus of the EDC/NHS-crosslinked hydrogel corresponds to a harder, less elastic, and relatively brittle material. While the tensile strength of the neoglycopolymer composites is low in comparison to these fully optimized control materials, it should be noted that hydrogels with tensile strengths as low as ca. 0.3 MPa have proved adequately robust for implantation [6,41,54]. With further optimization, therefore, neoglycopolymer-crosslinked hydrogels have the potential to yield materials that can better withstand surgical handling than the more brittle EDC/NHS-crosslinked scaffolds. 3.3. Optical properties The neoglycopolymer–RHC III composite is a colourless hydrogel, the optical clarity of which is essential for use as a corneal replacement. Its refractive index of 1.35 (identical to that of water; vide infra, and to the value measured for the EDC/NHS control) is slightly lower than the value for the human cornea. White light transmission is comparable to the corresponding value for the human cornea, while backscatter is considerably lower; both metrics are higher for the control material. 3.4. Glucose permeability The avascular cornea is characterized by a high permeability toward nutrients such as glucose, and an important characteristic of corneal tissue engineering scaffolds is thus a level of porosity sufficient to support molecular diffusion. The glucose permeability of the neoglycopolymer-crosslinked RHC III was measured at 2.4  0.2  106 cm2, a value comparable to that for the native human cornea, and superior to that for the control materials. 3.5. Water content The neoglycopolymer-RHC III gels contained 90% water, a figure comparable with the EDC/NHS-crosslinked RHC III controls, and 10% higher than the human cornea (Table 1). The high water content is consistent with the measured refractive index of 1.35 noted above, which corresponds to the refractive index of water.

Fig. 3. Growth of human corneal epithelial cells. Neoglycopolymer-crosslinked RHC III hydrogel after 8 days.

materials support the adhesion and proliferation of corneal epithelial cells, which reach confluence over 8 days in an 8 mm diameter sample. Cell proliferation is slightly slower than in the control, EDC/NHS-crosslinked RHC III hydrogel, for which confluence is achieved after 5 days, but remains within an acceptable time range based on clinical studies [6]. Cellular morphologies were normal and comparable in both cases. The EDC/NHS-crosslinked gels have now been shown to successfully effect corneal epithelium regeneration in humans [6]. 3.8. In vitro biological stability The biological stability of the collagen hydrogels was studied by assessing their resistance to degradation by collagenase. The longer lifetime of the neoglycopolymer-RHC III gel, relative to the EDC/ NHS-crosslinked control, is evident from the biodegradation profiles shown in Fig. 4. Their improved enzymatic resistance points toward the potential for application of the neoglycopoymerRHC III hydrogels in a wider range of transplantation contexts,

3.6. Thermal properties Calorimetric analysis by DSC allows measurement of the stability of the triple helical structure of collagen molecules. The denaturation temperature of collagen-based hydrogels is increased by formation of covalent crosslinks that stabilize the triple helix [9,62]. Td for the neoglycopolymer-RHC III hydrogel was 63.6  C, a value comparable to that of the human cornea, and 5  C higher than for the EDC/NHS-crosslinked control. The stability of the new materials is notable given their early stage of development, and augurs well for future clinical applications. It is particularly significant given their elasticity: the restoring force exerted by the longrange neoglycopolymer crosslink is evidently sufficient to stabilize the helical collagen array. 3.7. In vitro biocompatibility The in vitro biocompatibility of the neoglycopolymer-RHC III hydrogels was assessed by examining the growth rate of human corneal epithelial cells seeded on their surface (Fig. 3). These

Fig. 4. Biodegradation of crosslinked hydrogels by collagenase. Neoglycopolymer-RHC III ( ); RHC III control ( ).

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particularly where production of host enzymes at high levels would rapidly degrade the EDC/NHS-crosslinked constructs. 4. Conclusions ROMP neoglycopolymers represent dual-function, biomimetic crosslinking agents, which serve both as collagen crosslinkers and as GAG mimetics. The foregoing demonstrates that the flexibility of these long-range crosslinking materials enables a level of porosity consistent with biological permeation. First-generation neoglycopolymer-crosslinked RHC III hydrogels possess mechanical and optical properties that are adequate for use in transplantation. Their superior resistance to enzymatic biodegradation and denaturation, relative to the fully optimized EDC/NHS-crosslinked control materials, points to an expanded clinical scope. The fully synthetic nature of these neoglycopolymer-recombinant collagen hybrids eliminates the risks of pathogen transmission and xenogeneic immunoresponse inherent to animal-derived materials. Exploration and modulation of protein–carbohydrate interactions is one of the most prominent biological applications of ROMPderived neoglycopolymers to date. Our results provide a proof of concept that such ROMP materials provide a viable alternative to conventional crosslinking agents, and indeed offer a number of advantages. We are now exploring the application of these materials and their derivatives as multi-functional agents in tissue engineering applications. Acknowledgements This work was supported by the Natural Sciences and Engineering Research Council of Canada (Strategic Program Grant no. 246418, to M.G. and D.E.F.) and the Canada Foundation for Innovation. References [1] Prockop DJ. Collagens. In: Lennarz WJ, Lane MD, editors. Encyclopedia of biological chemistry. Oxford, UK: Elsevier; 2004. p. 482–7. [2] Gelse K, Poschl E, Aigner T. Collagens – structure, function, and biosynthesis. Adv Drug Deliv Rev 2003;55(12):1531–46. [3] Ma PX. Biomimetic materials for tissue engineering. Adv Drug Deliv Rev 2008;60(2):184–98. [4] Hollister SJ. Porous scaffold design for tissue engineering. Nat Mater 2005;4(7):518–24. [5] Yang C, Hillas PJ, Baez JA, Nokelainen M, Balan J, Tang J, et al. The application of recombinant human collagen in tissue engineering. BioDrugs 2004;18(2): 103–19. [6] Fagerholm P, Lagali NS, Carlsson DJ, Merrett K, Griffith M. Corneal regeneration following implantation of a biomimetic tissue-engineered substitute. Clin Transl Sci 2009;2:162–4. [7] Olde Damink LHH, Dijkstra PJ, Van Luyn MJA, Van Wachem PB, Nieuwenhuis P, Feijen J. Glutaraldehyde as a crosslinking agent for collagen-based biomaterials. J Mater Sci Mater Med 1995;6(8):460–72. [8] Jayakrishnan A, Jameela SR. Glutaraldehyde as a fixative in bioprostheses and drug delivery matrices. Biomaterials 1996;17(5):471–84. [9] Rault I, Frei V, Herbage D, Abdul-Malak N, Huc A. Evaluation of different chemical methods for crosslinking collagen gel, films and sponges. J Mater Sci Mater Med 1996;7(4):215–21. [10] Sung H-W, Chang W-H, Ma C-Y, Lee M-H. Crosslinking of biological tissues using genipin and/or carbodiimide. J Biomed Mater Res, A 2003;64A(3): 427–38. [11] Van Luyn MJA, Van Wachem PB, Dijkstra PJ, Olde Damink LHH, Feijen J. Calcification of subcutaneously implanted collagens in relation to cytotoxicity, cellular interactions and crosslinking. J Mater Sci Mater Med 1995;6(5): 288–96. [12] Nimni ME, Cheung D, Strates B, Kodama M, Sheikh K. Chemically modified collagen: a natural biomaterial for tissue replacement. J Biomed Mater Res 1987;21(6):741–71. [13] McLaughlin CR, Fagerholm P, Muzakare L, Lagali N, Forrester JV, Kuffova L, et al. Regeneration of corneal cells and nerves in an implanted collagen corneal substitute. Cornea 2008;27:580–9. [14] Liu W, Deng C, McLaughlin CR, Fagerholm P, Watsky MA, Heyne B, et al. Collagen-phosphorylcholine interpenetrating network hydrogels as corneal substitutes. Biomaterials 2009;30:1551–9.

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