| Collagen is a structural protein in the extracellular matrix(ECM)and is a high biosafety material.Collagen has excellent biological functions such as binding,protection,support,and repair.Collagen can participate in cell migration,differentiation and proliferation.Its application research is very extensive.The purpose of this study was to prepare collagen products from bovine Achilles tendon collagen and evaluated their biological properties.This study was explored as follows: Collagen solution was extracted from bovine Achilles tendon by pretreatment,degreasing,removal of heavy metal,removal of hybrid-protein,enzymatic hydrolysis and so on.And its characterization was identified.Cross-linked collagen membranes were prepared and evaluated for appearance and biological functions.Then,collagen freeze-dried products in bottle bottled was prepared and evaluated for its appearance and biological properties.The results of this study are as follows:(1)The fresh bovine tendon was subjected to a series of pretreatments such as removing the surface fascia,removing fat,removing heavy metals,and removing miscellaneous proteins.Bovine tendon precipitates were dissolved in acetic acid solution and enzymatically digested with pepsin.Collagen was purified by salting out and dialyzing to obtain a collagen solution.The results of optimization of collagen extraction conditions showed that the best time for removing impurity was 6 h,the optimal enzyme dosage was 2.5%,and the optimal time of enzymatic hydrolysis was 4 d.The characterization of the extracted collagen solution was verified by protein content determination,SDS-PAGE gel electrophoresis and amino acid composition analysis.The results showed that the extracted collagen had high protein content,good purity,and maintained a complete triple helix structure,which was consistent with the characteristics of type I collagen.This indicates that the extracted collagen is type I collagen.(2)A collagen solution with a solid content of 0.6% was poured into a 304 stainless steel tray model.It was freeze-dried at once for the first time and crosslinked with EDC-NHS.The cross-linked product was freeze-dried for the second time and stored under pressure.By optimizing the cross-linking conditions,it was found that the cross-linker concentration was best at 50 mM EDC and 25 mM NHS.By observing the morphology and microstructure of the collagen membrane,it was found that the collagen membrane was white and loose,with a fiber network,uniform internal pores,and good water permeability,which was conducive to cell growth.The biological evaluation of macrophage phagocytosis experiments,cytotoxicity assays,and immunological factors detection of collagen membranes showed that the collagen membranes are non-cytotoxic,non-stimulating immunogenic,and have good biocompatibility.(3)A collagen solution with a solid content of 0.2% was loaded into a neutral borosilicate vial.By vacuum freeze-drying,it was prepared into a pure collagen freeze-dried product with good biocompatibility and easy reconstitution.Optimizing the pre-freezing conditions of collagen freeze-dried products showed that different pre-freezing temperatures have a significant impact on the lyophilized product’s molding and reconstitution effects.Pre-frozen at-20℃,the prepared collagen freezedried product has the best appearance and reconstitution effect.The cytotoxicity evaluation of the collagen freeze-dried product showed that the freeze-dried collagen product has non-cytotoxic and has good biocompatibility.Through the above experiments,we extracted type I collagen by acid-enzymatic binding method using bovine tendon as raw material.Then a collagen membrane with white loose fiber mesh,smooth surface,stable microstructure,regular pore,flexibility and biocompatibility and a kind of high purity collagen freeze-dried product with good formation was prepared.This study can provide reference for developing collagen into medical and cosmetic products. |
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