| The amino acid structure of fish collagen is like that of terrestrial animal collagen,and it has good water solubility,biosafety,biocompatibility,and biodegradability.It is easy to extract,high yield and low production cost.Its living environment is relatively clean,and it has obvious advantages over land animals in risk control of virus carrying and cross infection,so it is an important source of medical collagen replacement.However,at present,the medical application of fish collagen is still in its infancy,and the research mainly focuses on laboratory extraction,modification and compound,and efficacy verification in vivo and in vitro of animals,etc.There is no large-scale preparation method of medical-grade fish collagen,as well as data and theories on application direction and efficacy mechanism.Studying these problems can provide direction and theoretical support for the application of fish collagen in the field of medical materials.Therefore,this study first broke through the pilot scale rapid preparation technology of fish collagen,and verified its physical and chemical properties,biological properties,biosafety,and degradation properties.Based on the clinical pathogenesis,animal models were constructed,and relevant quality evaluation and efficacy verification were carried out for sponge and artificial bone materials prepared from fish collagen.At the same time,based on the results of efficacy verification,histology,immunology,molecular biology and other aspects of the technology,the metabolic direction and mechanism of fish collagen in soft and hard tissues were preliminarily explored.The main contents are as follows:(1)Using tilapia skin as raw material,a pilot extraction process was designed and established to extract fish collagen and test its type,quality,and safety.The results showed that the extracted collagen was type I collagen with high purity,and the extraction rate reached 15%.Detection showed that the obtained fish collagen did not have any hemolysis reaction and sensitization reaction,did not cause any damage to blood cells and skin,had good cellular compatibility,no immune rejection reaction,good biosafety,good degradation performance.It was preliminarily confirmed that the fish collagen produced by this extraction process met the standards of implantable medical devices.(2)The hemostatic repair sponge material was prepared with fish collagen as raw material,and its hemostatic efficacy was verified through in vitro and in vivo hemostatic experiments,and the hemostatic mechanism was explored.The results showed that fish collagen sponge showed excellent performance in coagulation rate,coagulation speed,coagulation degree and in vivo coagulation,indicated that fish collagen sponge has good coagulation ability and met the requirements of clinical hemostatic materials.Electron microscope results showed that some sites on the fish collagen peptide chain could bind to specific sites of blood cells and platelets,and then activated the exogenous coagulation pathway to achieve the coagulation goal in a short time.(3)Rat acute wound model was established,and fish collagen sponge was used to verify its efficacy and study the metabolic direction and repair mechanism.The results of scanning electron microscopy and live/dead cell staining showed that the sponges were homogeneous in texture,stable in spatial structure,convenient for cell adhesion,crawling and proliferation with porosity and pore size.After implantation at the wound site,blood vessels could grow into the wound,promoted the expression of VEGF,FGF and other factors,and promoted the rapid completion of collagen deposition,tissue filling and re-epithelialization inside the wound.Through collagen fluorescence labeling,in vivo fluorescence tracing,comparative in situ analysis of frozen section fluorescence and immunohistochemistry of type I and Type III collagen antibodies showed that fish collagen sponge participated in the healing process of wound surface and became part of new type I and III collagen in some form and remained in the position of new tissue.According to the results of the transcriptome sequencing and RTPCR,after the implantation of fish collagen into the wound,the feedback mechanism was stimulated and the signal was transmitted to the body,resulting in the expression level of type I and III collagen-related genes was significantly lower than that of selfhealing individuals,which proved that part of fish collagen was used in the wound for the synthesis of type I and III collagen.(4)Rat diabetes model was constructed and chronic wounds were induced.Fish collagen sponge was used to repair chronic wounds and observe the therapeutic effect.Meanwhile,the expression of type I and type III collagen-related genes after fish collagen implantation was observed again.The results showed that fish collagen sponge had the same therapeutic effect in chronic wounds as in acute wounds.The results of stem cell loading experiment showed that fish collagen sponge and rat bone marrow mesenchymal stem cells could be effectively loaded,and the stem cells could be fixed at the site of the wound,which significantly improved the therapeutic effect.RT-PCR results of type I and type III collagen were the same as those of acute wounds,with low expression levels in both the single fish collagen implant and the stem cell-loaded fish collagen implant.(5)The reaction device and reaction conditions were designed to induce fish collagen mineralization in vitro,and fish collagen-based artificial bone material was obtained.Its physical and chemical properties,biological properties and safety were tested,and the repair effect and direction were observed in the bone defect model.By scanning electron microscopy,mechanical properties,and infrared spectroscopy,it was found that the artificial bone material had similar structure and mechanical strength to human cancellous bone.Cytotoxicity and cell adhesion experiments proved that it had good cytocompatibility and could induce cells to crawl and grow inside it.Hemolysis and subcutaneous implantation confirmed that it had good biosafety and biodegradability in vivo.The experimental results of repairing large segment of tibial bone defect in rats showed that the prepared artificial bone material had a certain osteogenic ability,and the degradation rate in rats basically matched the repair rate of tibial bone in rats.It was preliminarily confirmed that this material could be used as a bone repair material.In vivo fluorescence assay further confirmed that fish collagen,as an exogenous extracellular matrix component,participated in bone repair and become a component of new tissue,which was like its role in skin defect repair.According to the above research results,the following conclusions can be drawn:fast extraction,separation,purification,and sterilization of fish collagen can be achieved by using reaction kettle and membrane filtration equipment at low temperature,and the fish collagen extracted by this method basically met the quality standards of implanted medical type I collagen.The fish collagen also has mineralization sites with terrestrial animal collagen,which can achieve in vitro mineralization.The artificial bone material obtained has excellent physical and chemical properties,biological properties,biocompatibility,degradation,and other aspects.The sponge prepared from fish collagen has a good hemostatic effect in vivo and in vitro,and acute and chronic wound healing effect.The fish collagen based artificial bone material prepared by mineralization has good ability of inducing bone regeneration and bone repair.Through exploration,it was preliminarily confirmed that fish collagen can bind to specific sites of platelets and blood cells to induce blood agglutination and promote the occurrence of physical hemostatic reaction.It was preliminarily determined that fish collagen was utilized in the wound site and bone defect site in some form,become part of the new collagen structure,and then fed back to the body through the reverse regulation mechanism,so that the body could reduce the expression of type I and type III collagenrelated genes.This conclusion provided certain data support for the formulation of relevant standards of fish collagen raw materials and derived products,as well as the application direction of fish collagen,and provided a certain reference for in-depth study of the metabolism and mechanism of exogenous collagen in the body. |
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