Development Of Biomimetic Fish Collagen Membranes And Study The Effects And Mechanisms For Inducing Tissue Regeneration

Objectives: Development of biomaterials for repairing skin and periodontal tissue defects have been an important direction in the field of trauma and regenerative medicine.Although mammal collagen has good biological activity,they still have the possibility to transmitting animal diseases and may be limited in application because of high price and religious belief.In order to avoid these problems and development biomaterials with biocompatibility,functionality and security,three kinds of novel electrospun biomimetic tilapia collagen nanofiber membranes,tilapia collagen/ bioactive glass composite nanofiber membranes and tilapia collagen/ bioactive glass/ chitosan composite nanofiber membranes were prepared in the present study.By discussing the physicochemical properties,the antibacterial properties,the biological effects and the molecular mechanism of these nanofiber membranes in inducing tissue regeneration,scientific evidences can be provided for the development,evaluation and application of new generation of biomaterials in repairing skin and periodontal tissue defects.Materials and Methods: Tilapia skin collagen were extracted by combining acid dissolution and pepsin digestion and its immunogenicity was preliminarily discussed by both in vitro and in vivo experiments.Three kinds of biomimetic tilapia collagen nanofiber membranes were fabricated by electrospinning.The morphological structures and physicochemical properties of these nanofiber membranes were characterized by a series of experiments.Antibacterial properties of these nanofiber membranes were discussed by colony counting assays.The biological effects of adhesion,proliferation,migration and differentiation of human keratinocytes(HaCaTs),human dermal fibroblasts(HDFs),human umbilical vein endothelial cells(HUVECs)or human periodontal ligament cells(HPDLCs),the pathway and mechanisms of inducing skin or periodontal regeneration from these nanofiber membranes were investigated by cellular and molecular studies.Finally,rat skin defect models and dog periodontal defect models were designed to testify the healing effects of these three kinds of biomimetic tilapia collagen nanofiber membranes and illustrate the mechanisms for inducing tissue regeneration in vivo.Results:(1)Tilapia skin collagen was successfully extracted with high purity and it was preliminarily suggested the collagen had no immunogenicity.(2)Three kinds of novel biomimetic electrospun tilapia collagen nanofiber membranes,tilapia collagen/ bioactive glass composite nanofiber membranes and tilapia collagen/ bioactive glass/ chitosan composite nanofiber membranes were successfully developed.(3)These three kinds of tilapia collagen membranes had good thermal stability and hydrophilicity.The mechanical strength of tilapia collagen/ bioactive glass composite nanofiber membranes were(21.87±0.21)Mpa,which met the requirements of skin.(4)The expression of differentiation-related genes(Involucrin,TGase1 and Filaggrin)of human keratinocytes(HaCaTs)and COL-I of human dermal fibroblasts were promoted by tilapia collagen nanofiber membranes.(5)The adhesion and proliferation of HaCaTs,HDFs and human umbilical vein endothelial cells(HUVECs)were promoted by tilapia collagen and collagen/ bioactive glass nanofiber membranes,which also increased the expression of MMP-9 and TGF-?1 in HaCaTs with the activation of TGF-?1/ERK1/2/MMP-9 signal pathway for accelerating HaCaTs migration.(6)It was confirmed that both tilapia collagen nanofiber membranes and collagen/ bioactive glass composite nanofiber membranes significantly promoted the wound healing of rat dorsal skin and induced the expression of EGF,COL-I and VEGF from cells of skin.(7)The adhesion,proliferation and expression of osteogenesis-related genes(RUNX-2?OPN?OCN and ALP)of HPDLCs were promoted by tilapia collagen/ bioactive glass/ chitosan composite nanofiber membranes with the activation of ERK1/2/RUNX-2/OPN pathway,which accelerated the healing of dog periodontal defects.(8)Tilapia collagen/ bioactive glass and tilapia collagen/ bioactive glass/ chitosan composite nanofiber membranes had some antibacterial effects to staphylococcus aureus and streptococcus mutans,respectively.Conclusion:(1)Biomimetic electrospun tilapia collagen nanofiber membranes with the capability of inducing tissue regeneration were successfully fabricated and two kinds of novel electrospun tilapia collagen/ bioactive glass and tilapia collagen/ bioactive glass/ chitosan composite nanofiber membranes with some antibacterial abilities were also developed for repairing skin wound or periodontal tissue defects.(2)The biological behaviors of human skin cells could be effectively regulated by tilapia collagen nanofiber membranes and tilapia collagen/ bioactive glass composite nanofiber membranes.TGF-?1/ ERK1/2/MMP-9 signal pathway may played an critical role in inducing HaCaTs migration.(3)Both tilapia collagen nanofiber membranes and tilapia collagen/ bioactive glass composite nanofiber membranes could significantly promote rat skin wound healing,which were related to the migration and differentiation of HaCaTs,the collagen secretion of HDFs,the proliferation of HUVECs and the expression of EGF,COL-I and VEGF from skin cells promoted by these membranes.(4)The biomimetic nanostructures,amino acid composition,amino acid sequence and hydrophilicity of tilapia collagen/ bioactive glass/ chitosan nanofiber composite nanofiber membranes could effectively promote the biological effects of HPDLCs and healing of dog periodontal defects.ERK1/2/RUNX-2/OPN may be the key signal pathway in the process of osteogenesis.These three kinds of novel electrospun tilapia collagen nanofiber membranes developed in this study exhibited excellent biological activity and healing effects in vivo,which had potential for skin and periodontal tissue regeneration.