| Objective: Masquelet technique is a promising method for bone defect repair.Its core concept is to fill the bone defects with bone cement spacers to induce the biofilm formation in stage I,and to repair the bone defect by filling bone graft in the induced membrane in stage II.In the clinical practice of Masquelet’s technique,the application standard of antibiotics in PMMA has not been unified.In most literatures,it is suggested that broad-spectrum antibiotics with high thermal stability can be added into PMMA,such as vancomycin,gentamicin,tobramycin,etc.However,there is still a great controversy over the dosage.Vancomycin,a classical drug for the treatment of bone infection,is most widely used in combination with bone cement,so it has become one of the preferred drugs in antibiotic bone cement spacers.Therefore,the aim of this study is to investigate the effects of different concentrations of vancomycin in bone cement spacers on the activity of membrane induction in stage I and bone defect repair in stage II of the Masquelet’s technique,to provide theoretical guidance for the clinical application of this technology.Methods: Bone cement spacers with gradient vancomycin concentration were prepared according to vancomycin content of 0 g,1 g,2 g,4 g,6 g,8 g and 10 g in each package of bone cement(40 g)and were divided into seven groups(A-G).(1)Three samples were taken from each group and placed in simulated body fluids for drug release.The concentration of vancomycin was detected by UV spectrophotometer,and the differences of drug release among groups were analyzed.(2)The activities of induced membrane in phase I of Masquelet technique(induced membrane formation)were analyzed.Eighty-four New Zealand white rabbits were randomly divided into seven groups(A-G corresponding to groups of the vancomycin contents in bone cement spacers)to make models of mid-radial defect.vancomycin bone cement spacers were implanted into the bone defects and fixed with intramedullary Kirschner wires.Blood routine and biochemical tests were performed on rabbits in each group to evaluate the toxicity of local vancomycin application at 1,2 and 4 weeks after operation.Besides,X-ray examination was performed to evaluate the reliability of bone defect and spacer fixation.The induced membrane,liver and kidney were collected for detection at 2,4 and 6 weeks after operation.The proliferation characteristics(Ki67,STRO-1,MAC387),angiogenesis(CD31,v WF)and osteogenesis(COL-I,BMP2)of the induced membrane were observed by histological examination,and the cell damages of liver and kidney organs were also observed.The relative expression levels of angiogenic factors(VEGF,TGF-β,v WF),osteogenic factors(COL-I,ALP,RUNX2,BMP2)and stem cell activity-related factors(OCT-4,STRO-1)in the induced membrane were detected by RT-q PCR,and the differences among the groups were compared in the three time periods,respectively.(3)Bone defect repair in the phase II of Masquelet technique(bone transplant within the induced membrane)was evaluated.Thirty-two New Zealand white rabbits were randomly divided into eight groups(experimental groups A-G corresponding to groups of the gradient vancomycin bone cement spacers,and a blank control group H for bone defect).In the experimental groups(A-G),vancomycin bone cement spacers were removed 4 weeks after implantation,and allogeneic bone was implanted into induced membrane.In the blank control group(H),bone defect model was made simultaneously with bone transplantation in the experimental groups,The eight groups were all fixed with intramedullary Kirschner wires to observe the bone healing.X-ray examination was performed at 1,2,6 and 8 weeks postoperatively to show the repair status of bone defects,and Lane-Sandhu radiological scoring was performed.Micro-CT examination,histological examination and Lane-Sandhu histological scoring were performed to evaluate the repair of bone defects 8 weeks after operation,and the repair of bone defects among the 8 groups were compared.Results:(1)0-10 g vancomycin could be mixed with 40 g PMMA bone cement to form spacers,and its release in vitro is both time-and dose-dependent.It showed a burst release in the first 24 hours,and then slowed down and stabilized with time.The concentrations of antibiotics released from vancomycin bone cement spacers in the high-dose groups(8 g and 10 g)were significantly higher than those released in the low-dose groups(1-6 g),and a steady increase in local antibiotic concentrations could be maintained within 4 weeks.(2)The model of Kirschner wire intramedullary fixation in the Masquelet technique of the rabbit radial defect was simple and stable,and there was no Kirschner wire loosening or fixation failure during the two-stage postoperative follow-ups.(3)The blood test results of phase I experiments,that is inducing membrane formation,suggested that local application of vancomycin bone cement spacers(0-10 g)did not lead to obvious abnormalities in blood routine of rabbits.Blood biochemical tests showed that ALT and BUN in the low-dose groups(1-4 g)were not significantly abnormal,while those in the high-dose groups(6-10 g)were higher than the reference values at 1 and 2 weeks after operation,but they showed a trend of gradual decrease.BUN returned to normal 4 weeks after operation.There were no cell damages indicated in histological examinations in the liver and kidney tissues which were collected at 2,4and 6 weeks.(4)In phase I induced membrane formation experiments,histological examinations of the induced membrane collected at 2,4 and6 weeks showed that the induced membrane had obvious proliferative activity,angiogenic and osteogenic ability,especially in the low-dose vancomycin groups(0-4 g).The osteogenic and angiogenic activities in the high-dose vancomycin groups(6-10 g)were relatively poor.Further RT-q PCR results showed that the relative expression levels of angiogenic factors,osteogenic factors and stem cell activity-related factors in vancomycin low-dose groups(0-4 g)were significantly higher than those in high-dose groups(6-10 g)(p < 0.05),and the relative expression of some active factors in 1g and 2 g groups was significantly higher than that in 0 g group(p < 0.05).in addition,most of the bioactive factors in each group reached the peak at 2-4 weeks and gradually decreased after 6weeks.(5)The results of the phase II(i.e.bone defect repair)experiment showed that the the induced intra-membranous bone transplant groups(A-G)achieved bone defect repair,while the blank control group(H)of bone defect did not form effective bone connection.Gross observation,imaging and histological examination showed that the effects of bone defect repair in vancomycin low-dose groups(0-4 g)were significantly better than those in high-dose groups(6-10 g).Compared with the pure bone cement group(0 g),although the external calluses formed in the low-dose vancomycin groups(1-4 g)were smaller and there were still depressions in the bone defects,the cortical bone was thicker and the cancellous bone was more abundant after bone defect repair.H&E staining,Masson’s trichromatic staining and immunofluorescence OCN detection all showed that newborn bone tissues in the low-dose vancomycin groups(0-4 g)were richer and more mature than those in the high-dose vancomycin groups(6-10 g).Radiographic scores at 8 weeks postoperatively showed that the low-dose vancomycin groups(1 g and 2g)had significantly higher scores than the high-dose vancomycin groups(8 g and 10 g)(p < 0.05).The quantitative analysis results of micro-CT showed that TB.N in the experimental group(A-G)was significantly higher than that in the blank control group(H).TB.N in the low-dose groups(0-4 g)was also significantly higher than that in the high-dose groups(6-10 g),and in the low-dose groups,1 g was significantly better than the 4 g group(p < 0.05).Tb.Th in B,C,D,F and G groups were significantly higher than that in blank control group(H),in some low-dose groups significantly better than in high-dose groups(1 g&2 g vs6 g,2 g&4 g vs 8 g&10 g),and in 1-4 g groups significantly better than in0 g group(p < 0.05).BV/TV in the experimental groups(A-G)was also significantly better than that in the blank control group(H),and in some low-dose groups significantly better than in high-dose groups(0 g vs 6g&10 g,1-4 g vs 6-10 g)(p < 0.05).The results of histological scoring showed that the scores of low-dose vancomycin groups(1 g and 2 g)were significantly higher than those of high-dose vancomycin groups(8 g and 10 g)(p < 0.05).Conclusion:(1)The release of antibiotics in vancomycin bone cement spacers is time-and dose-dependent,with the release time lasting for more than 4 weeks.Local application of bone cement spacers loaded with different concentrations of vancomycin did not lead to significant cytotoxicity,nor did it produce significant liver and kidney damage.(2)The induced membranes produced by bone cement spacers loaded with different concentrations of vancomycin showed proliferative activity,angiogenesis and osteogenesis in rabbit radial defects,which were more obvious in low-dose vancomycin groups(0-4 g)than those in high-dose groups(6-10 g).Low doses of vancomycin(1-4 g)did not affect the activity of the induced membranes,while high doses of vancomycin(6-10 g)did inhibit the activity of the induced membrane.(3)Different concentrations of vancomycin bone cement spacers had different effects on the bone defect repair of Masquelet’s technique in rabbits,the osteogenic abilities were not affected in low dose groups(1-4 g),among which 1 g and 2 g of vancomycin(40g of bone cement)were more conducive to bone defect repair,however,in the high dose groups(6-10g),the bone defects repair of the induced membranes could be inhibited. |
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