Preparation, Characterization And Biological Comparison Of Nano-hydroxyapatite And Calcined Bone/type I Collagen Composite Scaffolds

Bone defect is a serious health problem facing the society at present.Commonly used methods for bone defects are autologous bone,allogeneic bone and synthetic bone repairing,but these methods are the corresponding defects.At present,commonly used bone tissue engineering materials are natural polymer materials and natural inorganic materials,as well as their composite materials.The main components of bone are collagen(CoL)and hydroxyapatite(HA).According to the particle size,HA was divided into nano-hydroxyapatite powder and micron-sized bioceramics.Synthetic nano-hydroxyapatite(nHA)was confirmed to have good clinical Efficacy.Calcined bone(TBC)is the animal bone by removing collagen though high temperature calcination,with HA as its main component of the inorganic material.TBC has a high similar crystal structure and pore structure and to natural bone,which is considered to be the most suitable inorganic material to prepare bone replacement materials.Objective:In this study,nHA?TBC and CoL were combined to prepare nHA/CoL and TBC/CoL composite scaffolds respectively.The physical and chemical properties of the two composite scaffolds were evaluated as well as the effects of the two inorganic materials on CoL.To compare the promoting ability of osteoblastic ability of nHA and TBC,the osteogenesis-related gene expression in MC3T3-E1 cells on different types of composite scaffolds were evaluated.Methods:The collagen was dissolved by acid-dissolving method and the collagen scaffold was prepared by freezedrying.The scanning electron microscopy(SEM)was used to observe the morphology of the scaffolds.Besides the porosity,pore size and tensile modulus of the stent were also being examined.The orthogonal experiment was designed to select the optimum collagen concentration and the preparation conditions.nHA/CoL and TBC/CoL scaffolds were prepared by mixing with nHA and TBC with the optimum collagen concentration,and the physical and chemical properties of the scaffolds were analyzed.The surface characterization,element composition and distribution of the inorganic materials and composite scaffolds,including the chemical structure of the scaffolds and the crystal orientation of the inorganic salt crystals in the collagen,were mainly analyzed.Meanwhile,the residual of glutaraldehyde and hydrophilicity of scaffolds were also measured.In order to evaluate the biological properties of the scaffolds,the proliferation,apoptosis and cycle of the cells grown on the materials were tested.Moreover,the morphology of the cells on the scaffold material was observed by SEM,which was to evaluate the cell compatibility of the scaffold.Lastly,to compare the promoting osteoblast differentiation ability of the osteogenesis-related cells,the expression of osteogenic genes in MC3T3-E1 cells cultured in scaffolds was detected by Real-Time PCR.Results:The optimum concentration of collagen scaffold was 3%by orthogonal design.The optimum cross-linking conditions were 0.2%of glutaraldehyde,25? of temperature and 120 min of cross-linking.The porosity of three scaffolds was 100?300?m and the porosity were all above 70%which were consistent with demand of the bone tissue engineering.The elastic modulus of nHA/CoL scaffolds was 7.87±0.23MPa which was higher than that of TBC/CoL scaffolds and CoL scaffolds(P<0.01).The results of FIRT and XRD show that CoL was mineralized in composite scaffolds.In addition to the HA phase,there are beta-tricalcium phosphate(?-TCP)crystals in TBC.The HPLC results showed that the residue of glutaraldehyde in the material extract was lower than the detection limit,which was lower than the minimum requirement of pharmacopoeia.The contact angle test results show that the contact angle of the composite scaffold is larger than that of the CoL group,in which the TBC/CoL group has the largest contact angle and the worst hydrophobicity.The cytotoxicity test showed that the toxicity of the material is grade 1,in line with the national standard in the medical device toxicity requirements.The cell proliferation rate on CoL scaffolds was higher than that on composite scaffolds on day 1 and day 3,and the proliferation rate of the three groups was similar on the 5th day and the 7th day.However,on the 9th day,the rate of the cell proliferation on the composite scaffolds was greater than that on the CoL scaffolds.The poptosis test showed that the apoptotic rate of TBC/CoL scaffold was the highest on day 1,while the apoptotic rate was the same on day 3 between nHA/CoL and TBC/CoL scaffolds,but higher than that of CoL group.The results of cell cycle test further confirmed the proliferation results.The SEM results of the morphology of the cells on the scaffolds showed groups was similar on the 5th day and the 7th day.However,on the 9th day,the rate of the cell proliferation on the composite scaffolds was greater than that on the CoL scaffolds.The poptosis test showed that the apoptotic rate of TBC/CoL scaffold was the highest on day 1,while the apoptotic rate was the same on day 3 between nHA/CoL and TBC/CoL scaffolds,but higher than that of CoL group.The results of cell cycle test further confirmed the proliferation results.The SEM results of the morphology of the cells on the scaffolds showed that the CoL material was more favorable for the early adhesion of the cells,but on the 14th and 21st day,the cells on the composite scaffolds were more robust judged from the secretion of metabolites.What's more,the results of the SEM also showed that the cells had a degradation effect on the materials in contact with them.The results of Real-Time PCR showed that the composite scaffolds could promote the expression of osteogenesis-related genes in MC3T3-E1 cells under osteogenic induction factor.Conclusion:The difference of surface structure and composition of nHA and TBC leads to the difference of mechanical properties and surface roughness of collagen scaffolds,which in turn affect the adhesion and proliferation of cells in scaffolds and the expression of osteogenesis related genes.The final results showed that nHA and TBC could promote the expression of osteogenic genes in MC3T3-E1 cells under the stimulation of osteogenic induction factor,and the effect of nHA on the late osteogenesis and mineralization was stronger than that of TBC.Ability to promote osteogenesis and the osteogenesis mechanism of the two kinds of composite scaffold in vivo remains to be further studied.