Development And Characterization Of Metal-mediated Cross-linking Of Collagen

As a key protein in the extracellular matrix,collagen provides an essential structural framework for human organs and tissues,and participates in important physiological processes such as cell adhesion,tissue remodeling and organ morphogenesis.Due to its superior biocompatibility,biodegradability and low immunogenicity,collagen is one of the most widely used biomaterials in the field of tissue engineering.However,the mechanical properties of natural collagen affect its clinical application.Therefore,the development of novel cross-linking strategies for collagen to achieve high mechanical strength and suitable enzymatic degradation rate without destroying its triple helix structure and biological activity,has become a hot target in the field of collagen.Unfortunately,the current physical and biological cross-linking methods has not yet given satisfactory outcomes,while the chemical cross-linking methods still can not eliminate the potential risks of cytotoxicity.This thesis aims to develop biocompatible and robust methods to cross-link recombinant and animal-derived collagen utilizing silver and iron ion-mediated biomineralization.The main contents are as follows:Chapter 1:We have given a brief introduction to the definition and classification,structure and in-vivo synthesis of collagen,summarized the research progress of collagen preparation methods,collagen-based biomaterials,and collagen cross-linking strategies.Chapter 2:We have discovered that the mixture of recombinant collagen and silver nitrate would quickly self-assemble to form exquisite and well-ordered collagen fibers under visible light irradiations at room temperature for the first time.By systematic investigation of the conditions such as recombinant collagen concentration,silver ion concentration,pH and irradiation time,the simple system of recombinant collagen and Ag~+has been demonstrated as a stable and efficient method to cross-link recombinant collagen under a wide range of experimental conditions to create fine collagen fibers.We have further extended this system to other proteins such as bovine serum albumin,casein,human serum albumin,pepsin and papain,and it revealed that silver ion is a green and broad-spectrum cross-linking agent for all the tested proteins under light irradiations.Silver ion-mediated biomineralization provides a novel,safe and efficient cross-linking method,showing promising applications in the preparation of recombinant collagen-based medical materials.Chapter 3:We have extended the silver ion-mediated biomineralization method to create cross-linked hydrogels of animal-derived type I collagen.By simply mixing bovine tendon type I collagen and silver nitrate solutions and incubating under visible light irradiation,collagen quickly self-assembled to form a hydrogel,and silver nanoparticles were produced at the same time.By optimizing the reaction conditions such as collagen concentration and anion concentration,we have successfully produced type I collagen hydrogel with higher mechanical strength and lower water swelling rate.The hybrid hydrogel displays low cytotoxicity and superior antibacterial ability.Silver ion-mediated biomineralization provides a simple and safe cross-linking method for generating type I collagen hydrogels.Chapter 4:We have discovered by screening experiments that the presence of iron ions could trigger type I collagen to form hydrogels.The gentle mixing of type I collagen and ferric chloride(or ferrous sulfate)solutions at room temperature quickly resulted in a hydrogel without the need of light irradiations.By optimizing the experimental conditions such as collagen concentration and iron ion(ferrous iron)concentration,we have successfully prepared type I collagen hydrogels with greater mechanical strength.The hydrogel displayed low cytotoxicity and superior cell adhesion.No nanoparticles were observed during the reaction,and the mechanism studies revealed that the iron ions induced the collagen self-assembly to form a hydrogel by the coordination with the side chain groups of collagen.The addition of iron ions provides a green and safe cross-linking method for generating type I collagen hydrogels with improved mechanical properties,and it has great potential in tissue engineering applications.