Influence of different collagen species on physico-chemical properties of crosslinked collagen matrices
Introduction
The repair of musculoskeletal tissue defects, e.g. cartilage, bone, tendon, muscle, nerve, skin, is still a challenging clinical problem. With the development of tissue engineering and its promising repair strategies, there is an increasing interest in developing biocompatible and biodegradable materials for tissue regeneration. As well as absorbable and non-absorbable synthetics, biomaterials with chondroconductive properties based on natural polymers, particularly collagen have been developed [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14]. Collagen substrates are known to influence the growth characteristics of cells and also to modulate various aspects of cell behavior like cell-adhesion, proliferation and differentiation [3], [9], [10], [15], [16], [17], [18], [19], [20], [21].
The disadvantages of using collagen as a biomaterial for tissue repair are its low biomechanical stiffness and rapid biodegradation [22]. The high enzymatic turnover rate of natural collagen in vivo makes stabilization of collagen-based biomaterials necessary. This can be achieved by chemical crosslinking methods, which provide biomaterials with desired mechanical properties for implantation and defect repair [23], [24], [25]. Several chemical agents have been used to achieve this goal. Glutaraldehyde (GA), a bifunctional reagent for bridging amino groups, is the most widely used reagent for crosslinking collagen. However, GA is associated with cytotoxicity in vitro and in vivo, caused by the presence of unreacted functional groups or by the release of those groups during enzymatic degradation of the crosslinked biomaterials [26], [27].
Methods have been developed that allow the crosslinking of collagen materials directly without incorporation of the crosslinking reagent. For example, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) or acylazide were used to generate peptide-like bonds in biomaterials [28], [29]. Use of EDC and N-hydroxysuccinimide (NHS) to crosslink collagen seems to yield biomaterials with good biocompatibility, higher cellular differentiation potential [20], [30], [31] and with increased resistance against enzymatic degradation [28], [32]. The physicochemical and biological properties after crosslinking have been described extensively [29], [33], [34], [35]. For EDC/NHS-crosslinking, collagen of bovine [20], [25], [31], [32], [36], [37], [38], ovine [24], [28], [39], [40], [41] or porcine [30], [42] origin has been used.
Potential risks with the use of bovine collagen, such as the possibility of transmitting mad cow disease, has proven a significant drawback for manufacturing biomaterials based on this material. The use of equine collagen would serve as an alternative, free of the potential risk of this infection. However systematic comparative studies on the species-related properties of bovine and equine collagen are missing. The principal goal of this study was to assess the differences between the properties of bovine and equine-based collagen as tissue engineering scaffolds. The physico-chemical and ultrastructural differences were evaluated with and without crosslinking with EDC and NHS.
Section snippets
Substrate materials
Non-soluble, native equine and bovine fibrillar type I collagen was separated from Achilles tendons and purified by acidic and basic hydrolysis as well as controlled enzymatic treatment with pepsin, according to a standardized industrial protocol, which is also used for the production of the commercially available collagen-based hemostyptika Collatamp™, CollatampE™, and for commercial collagen-based drug delivery systems by Innocoll GmbH, Saal/Donau, Germany (Medical Grade Collagen, ISO 9001).
Amino acid composition
Differences between bovine and equine collagen could be detected in the amino acid composition. Equine collagen contained a higher amount of hydroxylysine and lysine, whereas bovine collagen had a slightly higher amount of proline and hydroxyproline residues. Cysteine was absent in both samples and both contained small amounts of tyrosine (Table 1).
Characterization of collagen crosslinking
Crosslinking of the collagen matrices could be controlled by variation of EDC/NHS concentration. The degree of crosslinking is inversely
Discussion
Collagen-based biomaterials are widely used for tissue engineering. Because of the high degradation rate of natural collagen in vivo, crosslinking is necessary to reduce biodegradation of the biomaterial for sufficient tissue repair. EDC/NHS-collagen crosslinking results in biomaterials with good biocompatibility, with high cellular differentiation potential [20], [30], [31] and good resistance against enzymatic degradation [28], [32]. In this study, matrices of different collagen origin and
Acknowledgments
The authors thank Andrea Havasi, Tanja Weinfurtner, Tom Böttner and Martina Kreuzer for excellent technical assistance. We are furthermore thankful to Prof. Dr. Joachim Hammer and his coworkers Ronny Mai and Johann Fierlbeck regarding the mechanical tests. This work was supported by grants of DFG (Ne 734/2 and He 378/29), ForBioMat, HighTechOffensive Bavaria and NIH-NIAMS (1 R01 AR-48132-01).
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