| Objectives Preparation of porous Gel-10%SrDCPD repair materials,to explore the internal structure,the physical,chemical properties and preliminary evaluate the biological safety of materials.To build the mandibular defect of rabbit model to implant composite rhBMP2/7 porous Gel-10%SrDCPD and pure Gel-10%SrDCPD materials,preliminary explore the ability of repair rabbit mandibular large area defect of rhBMP2/7 composite porous repair materials and pure Gel-10%SrDCPD material,provide preliminary theoretical basis for clinical application.Methods Experiment 1,CaHPO4·2H2O,SrHPO4 and CaCO3 were used as raw materials to prepare 10%strontium-doped β-TCP powder,and 10%strontium-doped β-TCP powder was mixed with Ca(H2PO4)2·H2O,5wt%Gelatin solution into the mold by glutaraldehyde solution chemical crosslinking and vacuum freeze drying technology,porous Gel-10%SrDCPD scaffold material was prepared.Analysing the structure of material by X-ray diffraction(XRD)and fourier transform infrared spectroscopy(FT-IR),observing the internal structure by scanning electron microscopy(SEM),testing material mechanica by universal material testing machine testing.Experiment 2,1 Extracting leaching solution of 10%Sr-DCPD,Gel-10%SrDCPD.Setting the negative control group was RMPI complete medium,the positive control group was 0.64%phenol solution.After culturing L929 cell 1,3,5,7 days,the number and morphology of cells were observed under inverted microscope,detecting cell toxicity by MTT method.Recording the absorbance(OD)value and calculating the cell proliferation rate(RGR).2 Setting the negative control group was NaCl,the positive control group was deionized water,10%Sr-DCPD,Gel-10%SrDCPD leaching solution was experiment group Ⅰ and group Ⅱ,respectively.Adding diluted rabbit blood,stand for 1 h in constant temperature water-bath water,the supernatant was removed after centrifugation at 1000r/min for 5min,measuring each group OD value and calculating the hemolysis rate.3 Setting to inject Normal saline into the A and C areas of the rabbit’s back skin as control group,inject 10%Sr-DCPD and Gel-10%SrDCPD leaching solution in B,D area from top to bottom.After 24,48,72h,observing the skin redness and swelling in the injection area.Experiment 3,Establishing critical bone defect model of 45 rabbits and randomly divided into the control group,10%Sr-DCPD group,Gel-10%Sr-DCPD group,0.04μg/μLrhBMP2/7Gel-10%SrDCPD group,1μg/μLrhBMP2/7Gel-10%SrDCPD group.Corresponding specimens were taken after 4,8 and 12W,and the growth of new bone in the defect area was evaluated by general observation,CBCT,HE staining and Col-I immunohistochemistry.Results Experiment 1,1 After the XRD detection of Gel-10%SrDCPD,10%Sr-DCPD,comparing with standard card of DCPD.Strontium was successfully introduced into both materials,and after the FT-IR detection,comparing with pure gelatin materials,gelatin in Gel-10%SrDCPD was detected.2 Gel-10%SrDCPD is composed of irregular short rod-shaped crystals,with different number of holes,and the pore size is between 50～300μm,which is better than 10%Sr-DCPD material.3 Under the detection of universal material testing machine,the average pressure of 10%Sr-DCPD,Gel-10%SrDCPD composite material is 18.79±4.63MPa,16.84±3.23MPa,lower than DCPD(28.74±2.93MPa),with statistically significant difference(P<0.001).But there was no significant difference between 10%Sr-DCPD,Gel-10%SrDCPD group(P>0.05),and the addition of gelatin does not affect the compressive strength of the composite material.Experiment 2,1 With the increase of time,except the cell OD value of phenol group gradually decreasing,the other groups showed a growing trend.OD values were significantly different at different time(F=51.592,P<0.01).The OD values were significantly different among different groups(F=75.050>,P<0.01).The Gel-10%SrDCPD group had the highest OD value in 3,5,7 days(P<0.05),suggesting that the Gel-10%SrDCPD group had the strongest proliferation effect on L929 cells.According contrast cytotoxicity grade table,the cell toxicity of two experimental groups in 1,3,5,7 days are at level 0 or 1,the cell toxicity of phenol group level in grade 3 or 4.2 The hemolysis rate of 10%Sr-DCPD,Gel-10%SrDCPD group were 3.33%,4.67%,less than 5%.3 After 24,48,72h,the expe rimental area of 10%Sr-DCPD,Gel-10%SrDCPD and normal saline group were no erythema and swelling,the rating were 0.Experiment 3,postoperative animals had no adverse reactions,and the wound showed no obvious inflammation.The mean optical density value(MOD value)of Col-I immunohistochemical staining showed that there was statistically significant difference in MOD expression between each group at different time points(F=43.604,P<0.01),the change trend of blank group MOD is not obvious,the MOD values of the other four groups all showed a declining trend with the increase of time.The MOD difference between the five groups was statistically significant(F=48.052,P<0.01),Compared with the blank group and the 10%Sr-DCPD group,the MOD values of the other three groups were higher,suggest that the three groups have good osteogenesis.The MOD values of 1μg/μLrhBMP2/7/Gel-10%SrDCPD group was the highest(P<0.05),suggested that the porous composite with adsorption of 1μg/μLrhBMP2/7 had the best osteogenic effect,but the difference was not statistically significant between 0.04μg/μLrhBMP2/7/Gel-10%SrDCPD group and Gel-10%SrDCPD group(P>0.05).Conclusions 1 In the experiment,a porous Gel-10%SrDCPD composite with macropores and micropores was successfully prepared by vacuum freeze-drying method,and the compressive strength of the material was close to that of bone cancellous.2 Gel-10%SrDCPD have no cell toxicity,the hemolysis rate less than 5%and will not cause skin inflammatory reaction,so it is a biological safety repair material.3 rhBMP2/7 cytokines were successfully introduced into the porous materials,animal experiments showed that Gel-10%SrDCPD and the composite rhBMP2/7/Gel-10%SrDCPD porous material all could promote the formation of new bone in vivo,and the effect of 1μg/μL rhBMP2/7/Gel-10%SrDCPD was the best.Figure 26;Table 5;Reference 134... |
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