Experimental Study On Transplantation Of BMSCs Transfected With RhPDGF-BB Gene For Promoting Bone Regeneration In Rat Femoral Distraction Osteogenesis

Objective: 1)To establish a reasonable and reproducible distraction osteogenesis model of long bone in rats,and to lay a foundation for further study on the methods and mechanisms of promoting bone regeneration andmineralization in distraction osteogenesis;2)To establish a stable system for obtaining and culturing BMSCs in vitro,and further amplify,purify and identify BMSCs.BMSCs were transfected by lentivirus of rhPDGF BB gene in vitro and the effect on BMSCs was observed;3)The BMSCs transfected by rhPDGF-BB gene were introduced into the femoral distraction space of rats to explore the effect of PDGF factor on the distraction osteogenesis,so as to find a fast and effective treatment method for the treatment of bone defects.Methods: 1)Establishment of unilateral femoral distraction osteogenesis of animal model in rats: male Sprague Dawley(SD)rats were selected as experimental animals,aged<6 months,350-400 g.Using the self-designed lengthening external fixator,10% chloral hydrate was injected into abdominal cavity for anesthesia,and the right middle femur of rats was selected for osteotomy,and then the lengthening external fixator was fixed with 4 titanium screws.After a 7-days of latency period,the femur was slowly distracted at a rate of 0.5mm/day(twice/day),for 14 consecutivedays,with a total lengthening of 7mm.After the distraction period,the distractor was fixed.Three animals were sacrificed at 0,2,4 and 8 weeks after the end of stretching.X-ray examination was performed under anesthesia before animal were sacrificed at different time point.The right femur samples were harvested for general observation.HE staining and saffron green staining were used for histological observation;2)Isolation,culture and identification of rat BMSCs and in vitro lentiviral transfection of rhPDGF BB gene: culture and passage of rat BMSCs.Cell identification was carried out by cell activity detection,ALP staining and alizarin red staining.The third-generation cells with good growth were obtained by density gradient centrifugation for gene transfection and treatment.The recombinant lentivirus vector rh-PDGF-BB carrying both GFP reporter gene and rhPDGF-BB gene was constructed.After amplification and purification,the lentivirus vector rh-PDGF-BB was transfected into BMSCs to observe its effectiveness on BMSCs in vitro;3)The effect of rhPDGF BB transfected bone marrow MSCs on promoting the distraction osteogenesis of femur in rats: 30 SD rats were randomly divided into three groups by using the self-developed extended external fixator.Each group was injected with the same dose of rhPDGF BB modified bone marrow MSCs(experimental group),bone marrow MSCs(experimental control group),PBS(blank control group)and in vivo x-ray examination were performed,which were the day of operation,the day after the end of distraction(21 days after operation),the second weekend of mineralization(35days after operation),the fourth weekend of mineralization(49 days after operation),and the eighth weekend of mineralization(77 days after operation).At the end of the 8th week of mineralization,the animals were sacrificed,and bilateral femurs were harvested,and the muscles on the surface of bone tissue were carefully removed for micro CT,biomechanical and histopathological analysis.Results: 1)In vivo observation of experimental animals,all of them were tolerated to operation and postoperative distraction procedure,and their diet and defecation were normal.Standard X-ray examination and histological observation confirmed that the distraction osteogenesis was successful;2)BMSCs of rats were successfully obtained by density gradient centrifugation.The results of cell activity test showed that the cells grew well.ALP staining and alizarin red staining showed that the cells expressed high level of ALP activity,mineralized extracellular matrix and formed mineralized nodules.The transfected lentiviral rhPDGF BB gene has no significant effect on the growth of BMSCs;3)The BMSCs transfected by rhPDGF-BB gene were applied to the model of femoral distraction osteogenesis.X-ray films showed that 10 lengthened areas in the experimental group achieved bony healing in the eighth week of mineralization,5 in the experimental control group and 4 in the blank control group,respectively.The biomechanical test group was superior to the two control groups,and the torsional strength of the normal femur was the same.Micro CT data showed that in the eighth week of mineralization,the callus in the experimental group was mainly cortical bone,with less cancellous bone,and the marrow cavity was built,forming a structure similar to long tubular bone.In the two control groups,more callus was formed,mainly cancellous bone,with poor reconstruction.The histological section showed that there was cartilage tissue structure in the center of the distraction area of the two control groups at the 8th week of mineralization period,but there was no obvious cartilage tissue in the experimental group,the new bone had been mineralized,the cortical bone was continuous,and the bone marrow cavity began to rebuild,suggesting that the mineralization of the new callus was obvious after the treatment of rhPDGF BB gene transfected BMSCs.The expression of osteocalcin,osteopontin,VEGF and CD31 were higher in the experimental group than that in the control group.Conclusion: 1)The self-designed lengthening external fixator can meet the requirements of unilateral femoral distraction in rats.The process of distraction osteogenesis in latency is similar to that of fracture healing,but the bone regeneration in distraction and mineralization demonstrated characteristic histological changes.In this experiment,a stable animal model of unilateral femoral distraction osteogenesis in rats was successfully established,which has high repeatability and is suitable for conducting animal experiments involved distraction osteogenesis,which provided rationale experimental platform for gene-transfected cell therapy;2)BMSCs were obtained using density gradient centrifugation approach and cultured which demonstrated strong proliferation activity in vitro and indicated that BMSCs is an ideal seed cells for cell therapy and gene therapy.Under fluorescence microscope,rhPDGF BB gene can be successfully expressed in BMSCs by transient transfection of lipid vector;3)X-ray,micro-CT,biomechanics,histology and immunohistochemistry all confirmed that rhPDGF-BB gene transfected BMSCs were better than BMSCs and PBS alone in promoting bone regeneration in rat femoral distraction osteogenesis.rhPDGF-BB gene transfection of BMSCs in vitro for gene therapy can promote bone regeneration and shorten the treatment cycle in distraction osteogenesis,which provides a new strategy for the clinical treatment of distraction osteogenesis.