Design,preparation And Biological Characteristic Study Of Ternary Biomaterial Ano-hydroxyapatite/L-RADA16-RGD

ObjectiveA novel ternary biomaterial composed of n HA and L-RADA16-RGDpolypeptide was prepared in the present research;then by material testing and experiment in vivo and in vitro,its structural composition was analyzed and evaluated,mechanical properties were tested,and bio-safety,biocompatibility and bioactivity in vivo and in vitro were observed.Method(1)powdered n HA were ground and was sintered at 1500°C for 5h.After sintering,n HAwas crushed and mixed with chemical bubbling materials by injection molding to prepare porousn HA.Mechanical splines,square slices andwaferswere prepared by different molds;besides,porousn HA was made into a porous bone filling material by chemical bubbling method.The characterization and composition of n HA,L-RADA16-RGDpolypeptide and n HA/L-RADA16-RGD were observed by canning electron microscopy(SEM),transmission electron microscope(TEM).(2)DMEM/F12 containing 10% FBS was used as the extraction medium.In this experiment,the extract solution of porousn HA and n HA/L-RADA16-RGDmaterialwere prepared and the DMEM/F12 as a blank control.The 1st day,4thday and 7th day after co-culture were selected as observation points,cell proliferation was tested by CCK-8,and cell cycle was tested by flow cytometry to evaluate the biocompatibility of this composite with cells as well as its cytotoxicity to cells.(3)MC3T3-E1 and n HA/L-RADA16-RGD wafers were co-cultured in a 6-well plate,and MC3T3-E1 was cultured through osteogenic induction.Calcium nodule formation was observed by alizarin red staining,the expression of osteogenic-related proteins including collagen I(COL-I),osteopontin(OPN),osteocalcin(OCN)was tested by western blot(WB),and the bioactivity of this biological material was evaluated.(4)A bone defect model was built for the lateral femoral condyle of SD rat,into which the porous bone filling materials made from n HA/L-RADA16-RGD and porousn HA respectively were implanted,and observations were performed on the 30 th day and 90 th day after the implantation of the filling materials.The growth of the bones around the implanted materialswas observed by micro computed temography(Micro-CT)and histological staining;then,the biochemical indexes of animal liver and kidney functions,as well as hepatic and renal tissues,were detected in order to comprehensively evaluate the bio-safety,biocompatibility and bioactivity of n HA/L-RADA16-RGD.Results(1)SEM showed that porousn HA and L-RADA16-RGD were well combined with each other,the observation of L-RADA16-RGD polypeptide showed that this polypeptide composed by nano-fiber,and become three-dimensional scaffold.observation of n HA/L-RADA16-RGDshows that L-RADA16-RGDpolypeptideshapethethree-dimensional scaffold in the porous of the n HA materials.(2)The proliferation of cells co-cultured with DMEM/F12,n HA extract or n HA/L-RADA16-RGD extract was tested by CCK-8.It is found that the number of MC3T3-E1 cells increased with the increasing incubation time for each group.The cells in the experimental group exhibited a similar proliferation capacity compared to other groups.There was no significant difference between the experimental group and other groups at each observation time point(P>0.05).In addition,cell cycle detection by flow cytometry suggested the cell cycles in the n HA/L-RADA16-RGD group,n HA group and blank control group at different observation points There was a similarity in cell cycle between the groups on the 1st and 7th days.But the cell cycle test on the 4th day showed that there were obviously more cells in the n HA/L-RADA16-RGD group than in other group at the S and G2 M phase,showing a statistically significant difference(P<0.05).(3)After 21-day osteogenic induction culture,alizarin red staining showed that as could be seen by the naked eye,the color in the n HA/L-RADA16-RGD group was darker than the n HA group and control group;as could be seen under the microscope,there were more calcium nodules generated in the n HA/L-RADA16-RGD group than the n HA group and control group.After the dye on the composite surface was washed away,a quantitative test was conducted,suggesting that the absorbance of n HA/L-RADA16-RGD,n HA and the control was 0.37 ± 0.04?0.22 ± 0.01?0.19 ± 0.03 respectively,showing a statistically significant difference(P<0.05).WB-based test on the expression level of the osteogenesis-related proteins indicated that the expression level of osteogenic proteins was higher in the n HA/L-RADA16-RGD group than in the n HA group and control group at all the observation points,and that the difference was statistically significant(P<0.05).(4)In the analysis of the bone defect model,the results of the analysis by Micro-CT suggested that the trabecular bone volume(BV),relative trabecular bone volume(BV/TV),trabecular bone number(Tb.N)and trabecular bone thickness around the implants in the n HA/L-RADA16-RGD group at observation points were higher than in n HA group,and trabecular bone separation(Tb.Sp)was lower in n HA/L-RADA16-RGD group,suggesting statistical differences between the groups(P<0.05).Hard tissue slice staining suggested that bones grew well in the pores of the implants in both groups,and bones were combined closely with the implant surface,but bones grew better in the pores of the implant in the n HA/L-RADA16-RGD group.ConclusionNon-degradable ternary composite n HA/L-RADA16-RGD was successfully prepared.n HA/L-RADA16-RGD showed excellent bio-safety,biocompatibility and bioactivity in the experiments in vivo and vitro.This reveals its further research values and prospects.