Preparation And Characterization Of Fish Collagen-based Composite Materials Containing BFGF-loaded Microspheres

The repair and regeneration of skin defect is still an urgent problem to solve in clinical,and the therapeutic method most commonly used is autologous skin transplantation.However,autologous transplantation has its limitation such as the lack of donor and second injury in the donor site.Skin tissue engineering provides a creative approach for skin tissue repair.Nowadays,the products of collagen-based skin tissue engineering have been developed.Nevertheless,the sources of collagen are mainly limited to bovine and porcine skins and bones,which could carry a high risk of religion problem and zoonoses,such as bovine spongiform encephalopathy and foot and mouth disease.Fish collagen,with good biocompatibility,biodegradability,low immunogenicity and so on,has similar amino acids to that of mammal collagen,and does not have the previously discussed infectious diseases and religion problem.Therefore,the fish collagen has potential applications as scaffold for tissue engineering,but this issue has seldom been done so far.In this study,the fish collagen-based composite materials containing bFGF-loaded microspheres were prepared and the potential to be used as tissue engineering scaffold was evaluated.The main contents are as follows:(1)The Col/Chi/CS films mixed with fish collagen,chitosan and chondroitin-6-sulphate were fabricated by crosslinking with DHT and EDC/NHS.SEM results indicated that the films had porous structure and the aperture was about 110±110 ?m.With the increase of chitosan,the swelling fold and degradation rate decreased,and mechanical strength enhanced.The optimal proportion of collagen to chitosan was 1:1.MTT results showed the films exhibited noncytotoxicity and good biocompatibility.(2)bFGF-loaded PLGA microspheres were prepared by double emusion method.SEM results showed that the microspheres were porous spherical shape,and the mean size were 6.2±1.0?23.3±1.9 and 41.2±4.2 ?m respectively.With the size increasing,the drug loading and encapsulation efficiency decreased at first and then increased,and the release rate decreased.CD and bioactivity assay implied that the protein remained its secondary structure and bioactivity after the prepapation of the microspheres.(3)The fish collagen-based composite scaffolds were fabricated incorporated with bFGF-loading PLGA microspheres.The results suggested that the microspheres were distributed onto the scaffold evenly.bFGF remained its secondary structure and bioactivity after the fabrication of the scaffold.The release rate of protein could be controlled by the proportion of collagen to chitosan and the size of the microsphere.Cell assay revealed that the composite scaffold had good biocompatibility and the ability to promote the proliferation of MRC-5 cells.After implanting the scaffold to the rats,there was no obvious immunological rejection.HE staining results showed that the scaffold could promote the cell proliferation and skin tissue repair.In conclusion,the Col/Chi/CS composites containing bF GF-loaded microspheres have good biocompatibility,and the ability to promote cell proliferation and skin tissue repair.Therefore,the composites have a potential to be used as the scaffold of skin tissue engineering.