Experimental Study Of NAR/HA/PDA/CMCS Composite Scaffold In The Treatment Of Large Tibial Defects In Rats

Objective1.Objective to prepare NAR/HA/PDA/CMCS composite scaffold and evaluate its compatibility in rats;2.In vitro cell experiment was carried out to explore the ability of NAR/HA/PDA/CMCS composite scaffold to promote the adhesion and proliferation of rat ROB cells,and the expression of related osteogenic factors;3.To construct the rats fracture defect model with suitable scaffold materials,and to review and analyze the failure cases;4.According to the model established in the previous step,the osteogenic effect of NAR/HA/PDA/CMCS composite scaffold was evaluated in rats.Method1.Biocompatibility Evaluation48 SD rats were randomly divided into model group,blank stent group and NAR stent group.Through subcutaneous toxicity test,acute toxicity test and in vivo contact test,the abnormal skin condition after subcutaneous injection of extract,general condition and weight change after intraperitoneal injection of a large amount of extract,blood analysis,blood biochemistry and serum inflammatory factors after partial bone tissue defect and stent filling were analyzed The biocompatibility of NAR/HA/PDA/CMCS composite scaffolds was evaluated by pathological staining of liver and kidney;2.Cell Experiment in VitroThe third generation of ROB cells were seeded on the composite scaffolds with different concentrations of NAR.CCK8 method were used to detect the proliferation of cells on the composite scaffolds with different concentrations of NAR.The expression of osteogenic factors was evaluated by WB,PCR and ELISA.3.Establishment of a Rat Model of Long Tubular Bone Fracture DefectSixteen 10 weeks old male SD rats from SPF were randomly divided into two groups.Four 1 mm Kirschner wires and four rings were used to construct the right tibial fracture model.They were randomly divided into 4 groups:1 week,2 weeks,4 weeks and 8 weeks after operation.The quality of model construction was evaluated according to the above time points,and 55 cases with previous failure were selected for retrospective analysis;4.In Vivo Experiment in RatsForty eight SPF SD rats were randomly divided into 4 groups according to random number table:model group,blank stent group,drug loaded stent group,distraction osteogenesis positive control group(DO group),n=12 After 45 d and 90 d,6 rats in each group were randomly selected for X-ray and micro CT examination.The osteogenesis of the defect area was evaluated from three aspects:bone formation,bone connection and bone shaping.On the 60th day after operation,3 rats in each group were killed by decapitation under random anesthesia.The tibia specimens were stained with he,and the related osteogenic genes VEGF,HIF-1a,Runx-2 and BMP-2 were detected by q-PCR.Results1.Biocompatibility EvaluationAfter subcutaneous injection of the extract,there were no special reactions such as eschar and erythema.After acute high-dose intraperitoneal injection of the extract,there were no death,normal diet,activity and feces.The body weight showed a trend of continuous growth.The body weight growth at each detection time point was statistically significant(P<0.05),and there was no significant difference between the groups(P>0.05).The results of in vivo contact test showed that the blood analysis indexes(white blood cells,red blood cells and platelets)of rats fluctuated in the normal range on 1d,3d,7d and 14d after operation,but ALT and AST were higher than the standard value.Considering that the model group was only operated without any material implantation,and the abnormal value gradually approached the standard range within 14 days,the study speculated that the abnormal liver function of rats might come from the use of anesthetics during operation,the use of antibiotics after operation,the stress reaction of rats after operation,and the obesity of rats themselves,but the ALT and AST of NAR stent group on the 14th day were significantly lower than those of model group and blank stent group(P<0.05);2.Cell Experiment in VitroAfter the third generation of rat rob cells(ROB)were implanted in composite scaffolds,the results showed that:the cells of five groups continued to proliferate within 72 hours after implantation,the cell proliferation of NAR High-Dose Group was better than that of low-dose group and blank scaffold group,close to the positive control group,the expression of osteogenic related factors VEGF,HIF-1a,Runx-2,BMP-2:Positive Control Group>NAR High Dose Group>NAR Medium Dose Group>NAR Low Dose Group>Blank Stent Group,the difference was statistically significant(P<0.05);3.Establishment of Long Tubular Fracture Defect Model in rats and Analysis of Failure CasesThe tibial fracture model of rats established by self-made annular external fixator was stable and effective,and had no significant effect on the activity of rats.The fracture recovered well,and bone healing could occur within 8 weeks.The results are repeatable.The main reasons for the failure of plate fixation were improper selection of filling materials in the defect area,poor fixation materials and operation methods,and ineffective braking after operation.4.Involve Experiment in RatsIn the model group with the osteotomy range of 4 mm in vivo,bone healing could not occur within 90 days after operation,and the new bone tissue in the defect area was less than 50%,which was in line with the concept of critical bone defect.The lane Sandhu imaging score and pathological staining results on 45d and 90d after osteotomy showed that the osteogenic effect of NAR/HA/PDA/CMCS composite scaffold was better than that of HA/PDA/CMCS scaffold and 0.2mm/d distraction osteogenesis(the difference was statistically significant,P<0.05).All the rats in the three groups had bone healing within90 days after operation.At 45d after operation,VEGF,HIF-1a,Runx-2:NAR Group>Blank Group>DO Group>Model Group,the difference was statistically significant(P<0.05),BMP-2:DO Group>NAR Group>Blank Group>Model Group,the difference was statistically significant(P<0.05).90 days after operation:osteogenic gene expression:DO Group>NAR Group>Blank Group>Model Group,the difference was statistically significant(P<0.05).Conclusion1.Biocompatibility EvaluationThe NAR/HA/PDA/CMCS composite scaffold has no toxicity and good biocompatibility,which is suitable for the next research of bone formation in vivo and in vivo;2.In Vitro Cell ExperimentThe osteogenic proliferation ability of drug-loaded stents and the expression of VEGF,HIF-1a,Runx-2 and BMP-2 increased with the increase of NAR dose,and approached the Positive Control Group(TGF-β).The composite scaffold(high concentration)with2×10~6mol/L concentration NAR has good early osteogenic efficiency,and it is suitable for the next in vivo experiment;3.Establishment of the model of long tubular fracture defect in rats and analysis of failure casesThe model of tibial fracture was stable and effective by using the self-made external fixation frame,which had no obvious effect on the activity of the rats.The fracture recovered well.Bone healing could occur within 8 weeks,which could avoid the disadvantages of traditional plate internal fixation model.The results of the study were repeatable;4.In vivo experiment in ratsThe results of imaging scores at 45d and 90d after osteotomy and pathological staining at 60d after osteotomy showed that,the osteogenic effect of the NAR Group was better than that of Blank stent and distraction osteogenesis at 0.2mm/d rate.The expression of bone related genes in NAR Group was better than that of other groups at 45d after osteotomy,and the expression of bone related genes in DO group was better than that of other groups at 90d after osteotomy.