Verbesserung der Biokompatibilität metallischer Implantate durch kovalente Anbindung einer quervernetzten Kollagenschicht

Ziel dieser Arbeit war die Verbesserung der Biokompatibilität von metallischen Implantaten durch die kovalente Anbindung einer quervernetzten Kollagenschicht. Als metallische Substrate wurden Reintitan, Ti6Al4V, Ti6Al7Nb und tantalbeschichteter Edelstahl verwendet. Als Beschichtungsmaterial diente fibrilläres und lösliches Typ I Kollagen. Die Biologisierung sollte durch eine optimierte oxidative Vorbehandlung der Oberflächen, die Anbindung von Silanhaftvermittlern und die kovalente Immobilisierung von bioaktivem Kollagen mit anschließender Quervernetzung erreicht werden. Die Anbindung der Haftvermittler konnte durch IR-Spektroskopie, XPS und einen kolorimetrischen Assay qualitativ und quantitativ nachgewiesen werden. Ein Einfluss der oxidativen Vorbehandlung auf die Anbindung wurde festgestellt. Durch die Quervernetzung von Kollagen mit einem wasserlöslichen Carbodiimid (EDC) konnte eine stark verbesserte Stabilität gegenüber enzymatischer Degradation erreicht werden, ohne daß negative Auswirkungen auf das zelluläre Verhalten von mesenchymalen Stammzellen auftraten. Die kovalente Bindung zwischen Protein und Implantatoberfläche konnte mit Hilfe des Enzyms Peroxidase entwickelt und bewiesen werden und wurde auf die Anbindung von Kollagen übertragen. Kovalent gebundenes Kollagen zeigte im Vergleich zu rein adsorptiv gebundenen Schichten eine verbesserte enzymatische und mechanische Stabilität. Eine verbesserte Biokompatibilität, angezeigt durch eine erhöhte Zellproliferation, konnte eindeutig durch die Immobilisierung von fibrillärem Kollagen auf tantalisierten Stahloberflächen nachgewiesen werden. Allerdings ergaben sich hier vergleichbare Ergebnisse für kovalent und adsorptiv beschichtete Implantatoberflächen. Gerade wegen der vielversprechenden Ergebnisse der auf Metalloberflächen durchgeführten Kollagenaseversuche und der mechanischen Tests könnten an dieser Stelle längerfristige in vitro- und in vivo Untersuchungen den eindeutigen Vorteil der kovalenten Anbindung noch bestätigen

Influence of surface pretreatment of titanium- and cobalt-based biomaterials on covalent immobilization of fibrillar collagen

Collagen type-I is a major component of the extracellular matrix of most tissues and it is increasingly utilized for surface engineering of biomaterials to accelerate receptor-mediated cell adhesion. In the present study, coatings with layers of fibrillar type-I collagen were prepd. on titanium, titanium alloy, and cobalt alloy to improve initial osteoblast adhesion and implant-tissue integration. To suppress the quick in vivo degrdn. rate of collagen the deposited layers were covalently immobilized at the metal surfaces as well as chem. cross-linked. The application of different oxidn. techniques to the metallic substrates resulted in surfaces with varying hydroxyl group contents, which directly influenced the amt. of immobilized silane coupling agents. It was found that a high d. of surface-bound coupling agents increased the stability of the covalently linked collagen layers. After coating of metallic biomaterials with a cross-linked collagen layer, an improved cellular response of human osteoblast-like cells (MG-63) in vitro could be recognized

Surface engineering of stainless steel materials by covalent collagen immobilization to improve implant biocompatibility

It was shown recently that the deposition of thin films of tantalum and tantalum oxide enhanced the long-term biocompatibility of stainless steel biomaterials due to an increase in their corrosion resistance. In this study, the authors used this tantalum oxide coating as a basis for covalent immobilization of a collagen layer, which should result in a further improvement of implant tissue integration. Because of the high degrdn. rate of natural collagen in vivo, covalent immobilization as well as carbodiimide induced crosslinking of the protein was performed. It was found that the combination of the silane-coupling agent aminopropyl triethoxysilane and the linker mol. N,N'-disulfosuccinimidyl suberate was a very effective system for collagen immobilizing. Mech. and enzymic stability testing revealed a higher stability of covalent bound collagen layers compared to phys. adsorbed collagen layers. The biol. response induced by the surface modifications was evaluated by in vitro cell culture with human mesenchymal stem cells as well as by in vivo s.c. implantation into nude mice. The presence of collagen clearly improved the cytocompatibility of the stainless steel implants which, nevertheless, significantly depended on the crosslinking degree of the collagen layer

Influence of different collagen species on physico-chemical properties of crosslinked collagen matrices

Collagen-based scaffolds are appealing products for the repair of cartilage defects using tissue engineering strategies. The present study investigated the species-related differences of collagen scaffolds with and without 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide/N-hydroxysuccinimide-crosslinking. Resistance against collagenase digestion, swelling ratio, amino acid sequence, shrinkage temp., ultrastructural matrix morphol., crosslinking d. and stress-strain characteristics were detd. to evaluate the physico-chem. properties of equine- and bovine-collagen-based scaffolds. Three-factor ANOVA anal. revealed a highly significant effect of collagen type, crosslinking and time on degrdn. of the collagen samples by collagenase treatment. Crosslinked equine collagen samples showed a significantly reduced swelling ratio compared to bovine collagen samples. The amino acid compn. of equine collagen revealed a higher amt. of hydroxylysine and lysine. Shrinkage temps. of non-crosslinked samples showed a significant difference between equine (60°) and bovine collagen (57°). Three-factor ANOVA anal. revealed a highly significant effect of collagen type, crosslinking and matrix condition on rupture strength measured by stress-strain anal. The ultrastructure, the crosslinking d. and the strain at rupture between collagen matrixes of both species showed no significant differences. For tissue engineering purposes, the higher enzymic stability, the higher form stability, as well as the lower risk of transmissible disease make the case for considering equine-based collagen. This study also indicates that results obtained for scaffolds based on a certain collagen species may not be transferable to scaffolds based on another, because of the differing physico-chem. properties