Influence of different crosslinking treatments on the physical properties of collagen membranes

The physical properties of collagen-based biomaterials are profoundly influenced by the method and extent of crosslinking. In this study, the influence of various crosslinking treatments on the physical properties of reconstituted collagen membranes was assessed. Five crosslinking agents viz., GTA, DMS, DTBP, a combination of DMS and GTA and acyl azide method were used to stabilize collagen matrices. Crosslinking density, swelling ratio, thermo-mechanical properties, stress-strain characteristics and resistance to collagenase digestion were determined to evaluate the physical properties of crosslinked matrices. GTA treatment induced the maximum number of crosslinks (13) while DMS treatment induced the minimum (7). Of the two diimidoesters (DMS and DTBP), DTBP was a more effective crosslinking agent due to the presence of disulphide bonds in the DTBP crosslinks. T(s) for DTBP and DMS crosslinked collagen were 80 degrees C and 70 degrees C, and their HIT values were 5.4 and 2.85MN/m(2), respectively. Low concentration of GTA (0.01%) increased the crosslinking density of an already crosslinked matrix (DMS treated matrix) from 7 to 12. Lowest fracture energy was observed for the acyl azide treated matrix (0.61MJ/m(3)) while the highest was observed for the GTA treated matrix (1.97MJ/m(3)). The tensile strength of GTA treated matrix was maximum (12.4MPa) and that of acyl azide treated matrix was minimum (7.2MPa). GTA, DTBP and acyl azide treated matrices were equally resistant to collagenase degradation with approximately 6% solubilization after 5h while the DMS treated was least stable with 52.4% solubilization after the same time period. The spatial orientation of amino acid side chain residues on collagen plays an important role in determining the crosslinking density and consequent physical properties of the collagen matrix.     

Effects of photochemical crosslinking on the microstructure of collagen and a feasibility study on controlled protein release

Photochemical crosslinking is an emerging technique able to modify the physicochemical properties of collagen. However, whether this technique can be used to modify collagen-based structures for drug delivery has not been studied. This study demonstrated that the microporous structure of photochemically crosslinked collagen was affected by rose Bengal and laser energy level. Using the optimized process parameters, the authors fabricated photochemically crosslinked collagen structures encapsulated with sample proteins and demonstrated that photochemical crosslinking reduced the initial burst effect and protein release without compromising the protein bioactivity. The fiber meshwork in collagen structures was also characterized, and it was found that photochemical crosslinking did not significantly alter the mesh size. This study reports the effects of photochemical crosslinking on the microstructure of collagen structures and suggests the feasibility of using photochemically crosslinked collagen structures for controlled protein release.     

Photomediated crosslinking of C6-cinnamate derivatized type I collagen

Synthesis and characterization of cinnamated Type I collagen and its related mechanical properties after photomediated crosslinking were investigated in detail. Using an EDC/NHS conjugation method, collagen was chemically modified to incorporate a photosensitive cinnamate moiety. The cinnamated collagen was fully characterized by 1H NMR, UV-vis, and circular dichroism (CD) spectroscopy, as well as by rheological and mechanical analyses. Cinnamated collagens with varying degrees of derivatization retained collagen triple helical structure. The rheological spectra of collagen solutions demonstrate that the storage modulus decreases with increasing cinnamate content, owing to a decrease in physical crosslinking. The kinetics of the crosslinking process in both hydrated gels and dry films were monitored by UV-vis spectroscopy and confirmed that crosslinking was complete within 60 min of irradiation. The uniaxial stress-strain behavior of crosslinked collagen films, including Young's modulus and ultimate tensile strength, was comparable to values reported for glutaraldehyde-crosslinked monomeric collagen films. These data demonstrate that derivatization of collagen with photosensitive cinnamate moieties provides a facile route for solid-state crosslinking, thereby improving the mechanical properties of collagen and enhancing the potential applicability of collagen-based materials in tissue engineering and drug delivery.     

Crosslinking of collagen gels by transglutaminase

Collagen is commonly used as a tissue-engineering scaffold, yet its in vivo applications are limited by a deficiency in mechanical strength. The purpose of this work was to explore the utilization of a unique enzymatic crosslinking procedure aimed at improving the mechanical properties of collagen-based scaffold materials. Type I bovine collagen gel was crosslinked by transglutaminase, which selectively mediates the chemical reaction between glutamine and lysine residues on adjacent protein fibers, thus providing covalent amide bonds that serve to reinforce the three-dimensional matrix. The degree of crosslinking was verified by thermal analysis and amine group content. The denaturation temperature of crosslinked collagen reached a maximum of 66 +/- 1 degrees C. The chemical reaction was confirmed to be noncytotoxic with respect to bone marrow stromal cells acquired from New Zealand White rabbits. Tube-shaped cellular constructs fashioned from crosslinked collagen and bone marrow stromal cells were found to have burst pressures significantly higher than their noncrosslinked analogs (71 +/- 4 mmHg vs. 46 +/- 3 mmHg; p < 0.01). Thus, the transglutaminase mediated reaction served to successfully strengthen collagen gels while remaining benign toward cells.     

Preparation and characterization of porous crosslinked collagenous matrices containing bioavailable chondroitin sulphate

Porous collagen matrices with defined physical, chemical and biological characteristics are interesting materials for tissue engineering. Attachment of glycosaminoglycans (GAGs) may add to these characteristics and valorize collagen. In this study, porous type I collagen matrices were crosslinked using dehydrothermal (DHT) treatment and/or 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC), in the presence and absence of chondroitin sulphate (CS). EDC covalently attaches CS to collagen. DHT crosslinking preserved a porous matrix structure. However, attachment of CS to DHT-treated matrices using EDC, resulted in collapsed surfaces, CS located only at the matrix exterior. EDC crosslinking resulted in a partial matrix collapse. This could be prevented if crosslinking was carried out in the presence of ethanol. Matrix porosity was then preserved. The presence of CS during EDC crosslinking resulted in covalent immobilization of CS throughout the matrix. The amount of CS incorporated was increased if crosslinking was performed in the presence of ethanol. EDC-crosslinked matrices, with and without CS, had increased denaturation temperatures and decreased free amine group contents. The susceptibility of these matrices towards degradation by proteolytic enzymes was diminished. Immobilized CS increased the water-binding capacity and decreased the denaturation temperature and tensile strength. Immobilized CS bound anti-CS antibodies and was susceptible to chondroitinase ABC digestion, demonstrating its bioavailability. The modified matrices were not cytotoxic as was established using human myoblast and fibroblast culture systems. It is concluded that the use of ethanol during EDC crosslinking, offers an elegant means for the preparation of defined porous collagenous matrices containing bioavailable, covalently attached CS.     

First steps towards tissue engineering of small-diameter blood vessels: preparation of flat scaffolds of collagen and elastin by means of freeze drying

Porous scaffolds composed of collagen or collagen and elastin were prepared by freeze drying at temperatures between -18 and -196 degrees C. All scaffolds had a porosity of 90-98% and a homogeneous distribution of pores. Freeze drying at -18 degrees C afforded collagen and collagen/elastin matrices with average pore sizes of 340 and 130 mum, respectively. After 20 successive cycles up to 10% of strain, collagen/elastin dense films had a total degree of strain recovery of 70% +/- 5%, which was higher than that of collagen films (42% +/- 6%). Crosslinking of collagen/elastin matrices either in water or ethanol/water (40% v/v) was carried out using a carbodiimide (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, EDC) in combination with a succinimide (N-hydroxysuccinimide, NHS) in the presence or absence of a diamine (J230) or by reaction with butanediol diglycidylether (BDGE), followed by EDC/NHS. Crosslinking with EDC/NHS or EDC/NHS/J230 resulted in matrices with increased stiffness as compared to noncrosslinked matrices, whereas sequential crosslinking with the diglycidylether and EDC/NHS yielded very brittle scaffolds. Ethanol/water was the preferred solvent in the crosslinking process because of its ability to preserve the open porous structure during crosslinking. Smooth muscle cells were seeded on the (crosslinked) scaffolds and could be expanded during 14 days of culturing.     

Crosslinked collagen hydrogels as corneal implants: Effects of sterically bulky vs. non-bulky carbodiimides as crosslinkers

We have previously shown that recombinant human collagen can be crosslinked with N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) to fabricate transparent hydrogels possessing the shape and dimensions of the human cornea. These corneal implants have been tested in a Phase I human clinical study. Although these hydrogels successfully promoted corneal tissue and nerve regeneration, the gelling kinetics were difficult to control during the manufacture of the implants. An alternative carbodiimide capable of producing hydrogels of similar characteristics as EDC in terms of strength and biocompatibility, but with a longer gelation time would be a desirable alternative. Here, we compared the crosslinking kinetics and properties of hydrogels crosslinked with a sterically bulky carbodiimide, N-Cyclohexyl-N′-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate (CMC), with that of EDC. CMC crosslinking was possible at ambient temperature whereas the EDC reaction was too rapid to control and had to be carried out at low temperatures. The highest tensile strength obtained using optimized formulations were equivalent, although CMC crosslinked hydrogels were found to be stiffer. The collagenase resistance of CMC crosslinked hydrogels was superior to that of EDC crosslinked hydrogels while biocompatibility was similar. We are also able to substitute porcine collagen with recombinant human collagen and show that the in vivo performance of both resulting hydrogels as full-thickness corneal implants is comparable in a mouse model of an orthotopic corneal graft. In conclusion, CMC is a viable alternative to EDC as a crosslinker for collagen-based biomaterials for use as corneal implants, and potentially for use in other tissue engineering applications.     

Enhanced biological stability of collagen with incorporation of PAMAM dendrimer

The crosslinking methods of collagen using glutaraldehyde (GTA) and 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) are frequently performed in biomedical applications, but both methods still have their own disadvantages, including the GTA cytotoxicity and low degree of EDC/NHS crosslinking. In this study, we incorporated polyamidoamine (PAMAM) dendrimer with surface amine groups into the two aforementioned crosslinking methods to improve the biostability and structural integrity of collagen. Fifty micromolar of dendrimer concentration was found to have negligible in vitro cytotoxicity and was used for EDC and GTA crosslinking of collagen. The collagenase digestion assay showed that the collagen scaffolds crosslinked in the presence of PAMAM exhibited a higher denature temperature and higher resistance against collagenase digestion compared with their counterparts without dendrimer. Cell proliferation with human conjunctival fibroblasts showed that the incorporation of PAMAM in EDC crosslinking significantly increased the proliferation. All the crosslinked scaffolds also exhibited higher structural stability than the noncrosslinked scaffold. Crosslinking with EDC and PAMAM together yielded substantially higher proliferation and may be a suitable collagen scaffold for biomedical applications.     

Photochemical crosslinking improves the physicochemical properties of collagen scaffolds

Collagen is a natural biomaterial with excellent biocompatibility. However, unprocessed collagen has low stability and weak mechanical strength, which limits its application in tissue engineering. The current study aimed to improve the physicochemical properties of collagen scaffolds by using photochemical crosslinking. Collagen gel was reconstituted and photochemically crosslinked by using laser irradiation in the presence of a photosensitizer. Scanning electron microscope was used to characterize the surface and cross-sectional morphology. Stress-strain relationship and other mechanical properties were determined by uniaxial tensile tests. Thermostability and water-binding capacities also were analyzed by using differential scanning calorimetry and swelling ratio measurements, respectively. Photochemically crosslinked porous structures showed fine microstructure with interconnected micron-sized pores, whereas uncrosslinked controls only showed macrosheet-like structures. The stabilizing effect of photochemical crosslinking also was revealed by retaining the three-dimensional lamellae-like structures after thermal analysis in crosslinked membranes but not in the controls. Photochemical crosslinking also significantly reduced the swelling ratio, improved the stress-strain relationship, peak load, ultimate stress, rupture strain, and tangent modulus of collagen membranes. The current study showed that an innovative photochemical crosslinking process was able to produce collagen scaffolds with fine microstructures; to strengthen, stiffen, and stabilize collagen membranes; and to modify their swelling ratio. This may broaden the use of collagen-based scaffolds in tissue engineering, particularly for weight-bearing tissues.     

Characterization of collagen gel solutions and collagen matrices for cell culture

The influence of glutaraldehyde as a crosslinking agent to increase the strength of collagen matrices for cell culture was examined in this study. Collagen solutions of 1% were treated with different concentrations (0-0.2%) of glutaraldehyde for 24 h. The viscoelasticity of the resulting collagen gel solution was measured using dynamic mechanical analysis (DMA), which demonstrated that all collagen gel solutions examined followed the same model pattern. The creep compliance model of Voigt-Kelvin satisfactorily described the change of viscoelasticity expressed by these collagen gel solutions. These crosslinked collagen gel solutions were freeze-dried to form a matrix with a thickness of about 0.2-0.3 mm. The break modulus of these collagen matrices measured by DMA revealed that the higher the degree of crosslinking. the higher the break modulus. The compatibility of fibroblasts isolated from nude mouse skin with these collagen matrices was found to be acceptable at a cell density of 3 x 10(5) cells/cm2 with no contraction, even when using a concentration of glutaraldehyde of up to 0.2%.     

Effect of crosslinking on the performance of a collagen-derived biomaterial as an implant for soft tissue repair: a rodent model

One of the main problems in healthcare is the loss of tissues resulting from diseases, post-surgery complications or trauma. As a result there is a need for biomaterials designed to promote tissue regeneration and improve wound healing. This study assessed the effect of crosslinking of a porcine dermal collagen matrix with regard to strength of implant/host tissue integration, implant biocompatibility and general healing in a rodent model. Permacol?, a crosslinked acellular collagenous biomaterial was compared with its noncrosslinked equivalent at 3, 6, and 12 months postsubcutaneous implantation. Both matrices were well tolerated and showed no evidence of inflammation or adverse responses either in the host tissue or implants. Progressive integration of the implants with the surrounding tissue was observed. Cellular response was similar for both collagenous matrices although, at 3 and 6 months, noncrosslinked implants showed a significantly higher level of cellular penetration than crosslinked implants. However, at 12 months crosslinked implants showed significantly higher levels of cellular density, neo-vascularisation and integration with host tissue. Additionally, at long term, noncrosslinked implants lost volume suggesting some absorption. The crosslinking process does not seem to be detrimental to cellular response and biocompatibility.     

Cross-linked demineralized dentin maintains its mechanical stability when challenged by bacterial collagenase

The molecular structure, weight loss, and mechanical properties of demineralized dentin of noncrosslinked/crosslinked by glutaraldehyde (GA) were investigated when being challenged by bacterial collagenase solution over time in this study. Raman spectra proved that crosslinking occurred in demineralized dentin matrices after being treated with GA. Meanwhile, the weight of the cross-linked demineralized dentin matrices did not change after being challenged by bacterial collagenase solution up to 1 week. However, the weight of noncross-linked dentin collagen fell by almost 45% after degradation for 5 h, and up to 100% after 19 h. The tensile strength of demineralized dentin matrices did not show a significant change after being crosslinked, while the stiffness of demineralized dentin matrices showed more improvement than that of noncross-linked collagen. The toughness of demineralized dentin matrices decreased slightly after being crosslinked. Importantly, neither the tensile strength of GA-cross-linked demineralized dentin nor its stiffness changed over time in either control buffer or collagenase solution compared with that of noncross-linked controls. These results suggested that improving the degree of crosslinking in dentin collagen could be one method to inhibit its biodegradation and further to increase the durability of dental restorations.     

Biomechanical properties of carbodiimide crosslinked collagen: influence of the formation of ester crosslinks

There is a growing interest in the use of collagen matrices for tissue engineering. To prevent rapid degradation and to improve their mechanical properties, collagen matrices have been modified using different crosslinking agents. Among the different agents used, water soluble carbodiimides (such as N'-(3-dimethylaminopropyl)-N-ethylcarbodiimide, EDC) in combination with N-hydroxysuccinimide (NHS) are attractive systems, because no additional chemical entities are incorporated in the matrix. EDC/NHS crosslinking leads to amide bond formation between activated carboxyl groups and amine groups. Recently, we proposed that in addition to amide bond formation, ester links are also formed between activated carboxyl groups and hydroxyl groups. This was based on observations we made after development of a new method to quantify concentrations of carboxyl groups of collagen materials before and after crosslinking. The current study is directed to the influence of ester bond crosslinks formed after crosslinking of collagen with EDC/NHS on its physical-chemical and biomechanical properties. Reconstituted dermal bovine collagen patches (RDBC) were used as model material and were crosslinked with EDC/NHS. In one RDBC group, collagen amine groups were blocked with propionaldehyde prior to crosslinking, while in the other group unprocessed RDBC was crosslinked without additional matrix modifications. It was shown that after activation of collagen carboxyl groups with EDC and NHS, amide crosslinks as well as ester crosslinks with collagen hydroxyl groups were formed. It was furthermore demonstrated that the ester crosslinks of EDC/NHS-crosslinked RDBC could be removed by mild hydrolysis affording collagen matrices with improved mechanical properties.     

Crosslinking and composition influence the surface properties, mechanical stiffness and cell reactivity of collagen-based films

This study focuses on determining the effect of varying the composition and crosslinking of collagen-based films on their physical properties and interaction with myoblasts. Films composed of collagen or gelatin and crosslinked with a carbodiimide were assessed for their surface roughness and stiffness. These samples are significant because they allow variation of physical properties as well as offering different recognition motifs for cell binding. Cell reactivity was determined by the ability of myoblastic C2C12 and C2C12-α2+ cell lines (with different integrin expression) to adhere to and spread on the films. Significantly, crosslinking reduced the cell reactivity of all films, irrespective of their initial composition, stiffness or roughness. Crosslinking resulted in a dramatic increase in the stiffness of the collagen film and also tended to reduce the roughness of the films (R(q) = 0.417 ± 0.035 μm, E = 31 ± 4.4 MPa). Gelatin films were generally smoother and more compliant than comparable collagen films (R(q) = 7.9 ± 1.5 nm, E = 15 ± 3.1 MPa). The adhesion of α2-positive cells was enhanced relative to the parental C2C12 cells on collagen compared with gelatin films. These results indicate that the detrimental effect of crosslinking on cell response may be due to the altered physical properties of the films as well as a reduction in the number of available cell binding sites. Hence, although crosslinking can be used to enhance the mechanical stiffness and reduce the roughness of films, it reduces their capacity to support cell activity and could potentially limit the effectiveness of the collagen-based films and scaffolds.     

Tethering a laminin peptide to a crosslinked collagen scaffold for biofunctionality

Cell adhesion peptide regulates various cellular functions like proliferation, attachment, and spreading. The cellular response to laminin peptide (PPFLMLLKGSTR), a motif of laminin-5 alpha3 chain, tethered to type I collagen, crosslinked using microbial transglutaminase (mTGase) was investigated. mTGase is an enzyme that initiates crosslinking by reacting with the glutamine and lysine residues on the collagen fibers stabilizing the molecular structure. In this study that tethering of the laminin peptide in a mTGase crosslinked collagen scaffold enhanced cell proliferation and attachment. Laminin peptide tethered crosslinked scaffold showed unaltered cell morphology of 3T3 fibroblasts when compared with collagen and crosslinked scaffold. The triple helical structure of collagen remained unaltered by the addition of laminin peptide. In addition a dose-dependent affinity of the laminin peptide towards collagen was seen. The degree of crosslinking was measured by amino acid analysis, differential scanning calorimeter and fourier transform infrared spectroscopy. Increased crosslinking was observed in mTGase crosslinked group. mTGase crosslinking showed higher shrinkage temperature. There was alteration in the fibrillar architecture due to the crosslinking activity of mTGase. Hence, the use of enzyme-mediated linking shows promise in tethering cell adhesive peptides through biodegradable scaffolds.     

Semi-synthetic collagen/poloxamine matrices for tissue engineering

Collagen-containing poloxamine hydrogels were produced with the aim of overcoming the low stiffness displayed by collagen gels that are not otherwise chemically crosslinked. Matrices were obtained by functionalization of a four-arm PEO-PPO block copolymer (poloxamine, Tetronic) with methcrylate groups and subsequent free radical polymerization of water solutions of the modified polymer in the presence of collagen. The resulting matrices had a sharp increase in stiffness, when compared to pure collagen gels. For example, whereas collagen had a storage modulus (G') around 70 Pa and a loss modulus (G') of 10 Pa, a crosslinked collagen/poloxamine system containing 8.3% crosslinked poloxamine had G' and G' values of 7400 and 1000 Pa, respectively. HepG2 cells were seeded within the gels before the crosslinking and the viability levels estimated by AlamarBlue assay were between 65% and 91% for systems containing 0.04-0.09 wt% photoinitiator. HepG2 and endothelial cells also adhered to and spread on the surface of the collagen-containing specimens, suggesting their potential utility in tissue engineering.     

Attachment of glycosaminoglycans to collagenous matrices modulates the tissue response in rats

Biocompatibility and tissue regenerating capacity are essential characteristics in the design of collagenous biomaterials for tissue engineering. Attachment of glycosaminoglycans (GAGs) to collagen may add to these characteristics by creating an appropriate micro-environment. In this study, porous type I collagen matrices were crosslinked using 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide, in the presence and absence of chondroitin sulfate and heparan sulfate. The tissue response to these matrices was evaluated after subcutaneous implantation in rats. Biocompatibility of the matrices was established by the induction of a transitional inflammatory response, and the generation of new host tissue. Non-crosslinked collagen was gradually resorbed and replaced by collagenous connective tissue. By contrast, crosslinked matrices, with and without GAGs. retained their scaffold integrity during implantation, and supported the interstitial deposition and organization of extracellular matrix. In addition, crosslinking decreased tissue reactions at late time intervals. No calcification in any of the implants was observed. The presence of GAGs preserved porous lamellar matrix structures. Heparan sulfate in particular promoted angiogenesis at weeks 2 and 4, predominantly at the matrix periphery. The almost complete absence of macrophages and giant cells associated with collagen-GAG matrices, after 10 weeks implantation, indicated a reduced foreign body reaction. It is concluded that attachment of GAGs to collagen matrices modulates the tissue response. The potential of these biocompatible scaffolds for tissue engineering is increased by preserving porous matrix integrity. promoting angiogenesis and reducing foreign body reactions.     

Cross-linked type I and type II collagenous matrices for the repair of full-thickness articular cartilage defects--a study in rabbits

The physico-chemical properties of collagenous matrices may determine the tissue response after insertion into full-thickness articular cartilage defects. In this study, cross-linked type I and type II collagen matrices, with and without attached chondroitin sulfate, were implanted into full-thickness defects in the femoral trochlea of adolescent rabbits. The tissue response was evaluated 4 and 12 weeks after implantation by general histology and two semi-quantitative histological grading systems. Four weeks after implantation, type I collagenous matrices were completely filled with cartilage-like tissue. By contrast, type II collagenous matrices revealed predominantly cartilaginous tissue only at the superficial zone and at the interface of the matrix with the subchondral bone, leaving large areas of the matrix devoid of tissue. Attachment of chondroitin sulfate appeared to promote cellular ingrowth and cartilaginous tissue formation in both types of collagen matrices. Twelve weeks after implantation, the differences between the matrices were less pronounced. The deep parts of the subchondral defects were largely replaced by new bone with a concomitant degradation of the matrices. The original cartilage contours in defects with type I collagen-based matrices were repaired with fibro-cartilaginous tissue. Defects containing type II matrices showed an increase in the amount of superficial cartilage-like tissue. The original contour, however, was not completely restored in all animals, occasionally leaving a central depression or fissure. It is concluded that different types of collagen matrices induce different tissue responses in full-thickness articular cartilage defects. Type I collagen-based matrices are superior to guide progenitor cells from a subchondral origin into the defect. In type II collagen-based matrices cell migration is less, but invading cells are directed into a chondrocyte phenotype. Based on these observations it is suggested that a composite matrix consisting of a deep layer of type I collagen and a more superficial layer of type II collagen may be the matrix of choice for cartilage regeneration.     

The kinetics of 1,4-butanediol diglycidyl ether crosslinking of dermal sheep collagen

Dermal sheep collagen was crosslinked with 1,4-butanediol diglycidyl ether (BDDGE) or modified with glycidyl isopropyl ether (PGE). The reduction in amine groups as a function of time was followed to study the overall reaction kinetics of collagen with either BDDGE or PGE. Linearization of the experimental data resulted in a reaction order of 2 with respect to the amine groups in the PGE masking reaction, whereas a reaction order of 2.5 was obtained in the BDDGE crosslinking reaction. The reaction orders were independent of the pH in the range of 8.5-10.5 and the reagent concentration (1-4 wt %). The reaction order with respect to epoxide groups was equal to 1 for both reagents. As expected, the reaction rate was favored by a higher reagent concentration and a higher solution pH. Because the BDDGE crosslinking reaction occurs via two distinct reaction steps, the content of pendant epoxide groups in the collagen matrix was determined by treating the collagen with either O-phosphoryl ethanolamine or lysine methyl ester. The increase in either phosphor or primary amine groups was related to the content of pendant groups. Crosslinking at pH 9.0 resulted in a low reaction rate but in a high crosslink efficacy, especially after prolonged reaction times. A maximum concentration of pendant epoxide groups was detected after 50 h. Reaction at pH 10.0 was faster, but a lower crosslinking efficacy was obtained. At pH 10.0, the ratio between pendant epoxide groups and crosslinks was almost equal to 1 during the course of the crosslinking reaction.     

Glyoxal crosslinking of cell-seeded chitosan/collagen hydrogels for bone regeneration

Chitosan and collagen are natural biomaterials that have been used extensively in tissue engineering, both separately and as composite materials. Most methods to fabricate chitosan/collagen composites use freeze drying and chemical crosslinking to create stable porous scaffolds, which subsequently can be seeded with cells. In this study, we directly embedded human bone marrow stem cells (hBMSC) in chitosan/collagen materials by initiating gelation using β-glycerophosphate at physiological temperature and pH. We further examined the use of glyoxal, a dialdehyde with relatively low toxicity, to crosslink these materials and characterized the resulting changes in matrix and cell properties. The cytocompatibility of glyoxal and the crosslinked gels were investigated in terms of hBMSC metabolic activity, viability, proliferation and osteogenic differentiation. These studies revealed that glyoxal was cytocompatible at concentrations below about 1mM for periods of exposure up to 15 h, though the degree of cell spreading and proliferation were dependent on matrix composition. Glyoxal-crosslinked matrices were stiffer and compacted less than uncrosslinked controls. It was further demonstrated that hBMSC can attach and proliferate in three-dimensional matrices composed of 50/50 chitosan/collagen, and that these materials supported osteogenic differentiation in response to stimulation. Such glyoxal-crosslinked chitosan/collagen composite materials may find utility as cell delivery vehicles for enhancing the repair of bone defects.