Performances Of Bone-induced Porous Materials Compositing Stingray Skin Collagen With Oyster

Successful regeneration of bone defects remains a major challenge in clinical orthopedics,thus it is important significance to prepare a suitable biomimetic scaffold for stimulating bone regeneration.Because of its excellent biological activity,safety and low immunogenicity,marine collagen has more advantages than terrestrial animal collagen,which has attracted extensive attention of biomedical researchers.In recent years,marine collagen has developed from simple extraction to the preparation of tissue engineering scaffolds.However,the simple marine collagen scaffolds have some problems,such as poor mechanical properties,poor plasticity and rapid degradation,which limit their application.Therefore,in order to improve these defects,researchers often design composite scaffolds or introduce crosslinking agents.In 2004,an article in Science reported that the biomineralization process of oyster shell was similar to that of human bone formation,and proved that oyster shell was a bioactive bone injury repair material.Nevertheless,oyster shells have yet to be developed in the field of preparing bone tissue engineering scaffolds.Based on this,two bone induced composite porous scaffolds are prepared around natural biomaterials,marine collagen and oyster shell powder.The specific research contents are as follows:(1)Stingray skin collagen(Col)is prepared by acidase enzymatic method.UV,ATR-FTIR,CD,AFM,SDS-PAGE,DSC,amino acid analysis,cytotoxicity and cell adhesion are tested.The results show that the secondary structure and triple helix structure of stingray skin collagen remain intact;Three peptide chains α1、 α2、 β are distributed at about 130 k Da,119 k Da and200 k Da,respectively.The denaturation temperature of stingray skin collagen is 106.4℃;Rich in glycine and characteristic amino acids,it retains the nutrients of stingray skin collagen,respectively.Stingray skin collagen has no cytotoxicity,can promote the growth of mouse embryonic osteoblast precursor cells(MC3T3-E1),and has good cell adhesion.(2)Using stingray skin collagen and oyster shell powder(OSP)as raw materials,a new bioactive material-stingray skin collagen/oyster bone induced composite scaffold(Col-OSP)is prepared by genipin crosslinking,porogen and freeze-drying method for the first time.ATRFTIR,SEM,compression,swelling,in vitro degradation,cytotoxicity,cell proliferation,cell adhesion,alkaline phosphatase activity,alizarin red staining and RT-PCR are tested.The results show that the prepared Col-OSP composite scaffolds has interconnected three-dimensional porous structure,which could maintain the transportation of nutrients;Compared with the simple Col scaffold,the mechanical properties of Col-OSP composite scaffold are enhanced,and the swelling rate and degradation rate are reduced,which is more in line with the preparation requirements of bone tissue engineering scaffold.Col-OSP composite scaffold is not only nontoxic,but also promotes the proliferation,adhesion and differentiation of MC3T3-E1 cells and stimulates the expression of osteogenic related genes osteocalcin(OCN),type I collagen(COL-I)and RUNX2 of MC3T3-E1 cells.(3)In order to avoid the side effects caused by cross linker,a bioactive hydrogel scaffold is prepared by using simple schiff base reaction.Sodium alginate(OSA)and carboxymethyl chitosan(CMCS)are crosslinked by natural biomaterials as the main porous framework of hydrogels.In order to improve the physical and chemical properties and bioactivities of hydrogels,OSP and Col are introduced into OSA-CMCS precursors to form OSA-CMCS-OSPCol composite hydrogels.ATR-FTIR,gel time analysis,SEM,compression,swelling,in vitro degradation,self healing analysis,cytotoxicity,cell proliferation,alkaline phosphatase activity and alizarin red staining are carried out.The results show that the gel time of OSA-CMCS-OSPCol hydrogel is 114±12 s,which is self-healing and suitable for repairing irregular bone defects.After introducing OSP and Col,the porous structure of hydrogels becomes more dense,which not only improves the compressive strength of hydrogels(the maximum compressive strength is68.6±6.9 k Pa),but also reduces the swelling and degradation properties of hydrogels.In addition,OSA-CMCS-OSP-Col hydrogel is not only non-toxic,but also promotes the proliferation and osteogenic differentiation of MC3T3-E1 cells.In conclusion,based on stingray skin collagen and oyster shell powder,this paper designs two new bone induced composite porous scaffolds,which can promote the proliferation,differentiation and expression of related genes of MC3T3-E1 cells,and has a certain application prospect in the field of bone tissue engineering scaffolds.