Experimental Study On Promoting Bone Regeneration Of Rabbit Mandibular Distraction Osteogenesis With The Composite Of Pluronic F-127 Hydrogel And HBMP-2 Gene Modifying Bone Mesenchymal Stem Cells

ObjectiveDistraction Osteogenesis(DO)is an endogenous bone tissue engineering technique in which new bone is regenerated by the mechanism of callus healing.Numerous basic experimental and clinical researches have been carried out since its application in craniofacial field.With the proceeding development of basic study of this technique,DO shows its extensive application prospect in maxillofacial plastic surgery,bone reconstruction following tumor resection and implant restoration in alveolar bone,and has been one of the research hotspots in the field of oromaxillofacial surgery.Although its significan advantages over conventional surgical procedures,DO has been widely applied in the correction of craniofacial anomalies and extremity defects.However,the prolonged distraction and consolidation periods do considerably limit its wider clinical application.Therefore,how to increase the rate of bone regeneration,to shorten consolidation period,to reduce the complication and to enhance regeneration quality have become research hotspots in this field at present.The gene of human bone morphogenetic protein 2(hBMP-2)was transfected into bone mesenchymal stem cells(BMSCs)to construct expression-stable hBMP-2 modified BMSCs.Then the gene modifying cells were seeded in Pluronic F-127 hydrogel,forming cell/gel composite which was then injected to the distraction gap,aiming to achieve the stable and continuous expression of hBMP2 and accelerate bone regeneration with the combination of genetic engineering and tissue engineering techniques.Bone regeneration and remodeling in the distraction gap was observed and detected on the histologic,proteinic and genetic levels,respectively.This study was designed to establish foundation for the extensive application of DO in the treatment of various maxillofacial deformities and defects.Methods1.BMSCs' in-vitro cultivation and directional osteogetic differentiation:Bone marrow was extracted from adult healthy New Zealand's white rabbit by puncturing bilaterial tibia,and then cells were collected by density gradient centrifugation.After 8 to 10 days' culture,primary cells could be digested for passage cultured at 1:3 and every passage cells could be cultured for 3 to 4 days.Cells of passage ? were harvested for osteoanagenesis inducing for 14 to 21 days using osteoblast differentiation medium containing dexamethasone,?-sodium glycerophosphate,ascorbic acid.Mineralization nodules,ALP and BGP in the BMSCs after induction was dectected by Alizarin Red staining,ELISA and IHC methods respectively.2.Liposome-mediated transfection of hBMP-2 gene into BMSCs:The pcDNA3.1 plasmids carrying liposome-mediated hBMP-2 gene were transfected into BMSCs.GFP was observed under fluorescence microscope.The proliferation and growth of tranfected cells was investigated by MTT method.The instantaneous expression of hBMP-2 mRNA was detected by RT-PCR.G418 was used for 2 weeks to screen the stable transfected cells and then the cells were gathered for further use.Immunohistochemistry and Western Blot was used to dectect the expression of BMP-2 in the cells that had been screened and cultured for 4 weeks.3.Cultivation of hBMP-2-BMSCs/Pluronic F-127 hydrogel composite:Pluronic F-127 was prepared in ice bath with its characteristics of which it is water-solutionable under low temperature but solid under room temperature.After sterilization,Pluronic F-127 hydrogel was seeded with hBMP2-BMSCs(2.5×107cells/ml).The cytotoxicity of Pluronic F-127 hydrogel was investigated by MTT method.4.Model establishment of rabbit mandibular distraction osteogenesis:A total of 6 adult healthy New Zealand's white rabbits were used in this study.Distractors were custom-made according to the mandibular anatomy of rabbit.Conventional osteotomy was conducted between right mandibular second and third molar.After a 5-day's latency,distracers were activated at a rate of 0.5 mm/time for twice per day for 7 consecutive days.X-ray images were taken on 2 and 6 weeks consolidations,respectively,to observe bone regeneration and remolding.5.Experimental study on promoting bone regeneration of rabbit mandibular distraction osteogenesis with the composite of Pluronic F-127 hydrogel and hBMP-2 gene modifying BMSCs:Forty-eight New Zealand's white rabbits were ramdomized into four groups(A,B,C and D)with twelve in each.In group A,200?l of hBMP-2-BMSCs/Pluronic F-127 composite was injected into the distraction gap at the first day of consolidation period,while in groups B,C and D the same amount of hBMP2-BMSCs suspension,BMSCs suspension and saline was injected in the distraction gap,respectively.The operated mandibles were harvested on 2-and 6-W consolidations,respectively.The regenerated bone was evaluated by the means of general pathology,radiographics,scanning electron microscopy(SEM),HE staining,immunohistochemistry staining.Results1.Results of the in-vitro cultivation of BMSCs and osteogenic differentiation:8 to 10 days after cultivation,primary BMSCs were overgrowthing for passage cultured.After subculturing,in addition to the preservation of typical morphological feature and growth state of BMSCs,it could be osteogenicly induced successfully.Under microscope,spindle-shaped cells and vortex arrangement were observed.After induction,positive dying of calcium nodules was observed.There was significant difference(p<0.05)in the contents of ALP between uninduced group and induced one in which the content reached highest on 2-week consolidation.In addition,positive staining of BGP was observed by immunohistochemistry.2.Results of the transfection of hBMP-2 gene into BMSCs mediated by liposome:The transfection efficiency of plasmid-carrying hBMP-2 gene was approximately 30%.The fluorescence intensity was rather poor on post-transfection 24h but became stronger after 48h.The result of MTT revealed that the proliferation capacity of transfected BMSCs did not change significantly.And the gene expression of hBMP-2 could be detected.Stable transfected cells were obtained and its stable expression of BMP-2 was dectected by immunohistochemistry and Western Blot after 4 weeks'cultured.3.Results of the cultivation of hBMP-2-BMSCs/Pluronic F-127 composite:Pluronic F-127 was solutive at 4? but solid at 37?.There was no significant change in the proliferation activity of BMSCs in the composite,which was invested by MTT method(p>0.05).4.Results of model establishment of rabbit mandibular distraction osteogenesis:All the experimental animals have survived the whole process without any severe complication.Moreover,distraction proceeded on smoothly.During the whole process,the retention of bone fragment was stable without breakage of distractors or loosening of screws.X-ray images showed that there was significant translucency change of regenerated bone at various consolidays,which corresponded to the clinical principle of distraction osteogenesis.The rabbit model of mandibular distraction osteogenesis was constructed successfully.5.Results of experimental study on promoting bone regeneration of rabbit mandibular distraction osteogenesis with the compound of Pluronic F-127 hydrogel and hBMP-2 gene modifying BMSCs:The results of general pathology,X-ray,SEM and HE staining showed that the quality of regenerated bone of the groups ranked as follows:group A>group B>group C=group D.The gray value of the results of IHC showed that the quality of regenerated bone of group A was obviously higher than that of group B(p<0.05),and group B was higher than group C and D(p<0.05),but there was no significant difference between group C and D(p>0.05).Conclusions1.Rabbit bone mesenchymal stem cells(BMSCs)could be cultivated,amplified and purified by the method of density gradient centrifugation which was convenient and practical.The BMSCs could be directionally induced to osteoblasts.2.Extragenous hBMP-2 gene could be successfully transfected into rabbit BMSCs and got rather stable in-vivo gene and BMP-2 protein expression by the mean of liposome-mediated plasmid transfection.3.The scaffold material of pluronic F-127 had the characteristics of excellent cell compatibility and temperature-sensitivity which was solutionable under low temperature but solid under room temperature.4.Rabbit could be an optimal model animal for large-sample experimental study for its docile disposition,easy raising,favourable operation tolerance,simple management and excellent postoperative healing process.5.The transplantation of the composite of human BMP-2 gene modifying BMSCs and injectable scaffold material Pluronic F-127 could effectively promote bone regeneration in rabbit mandibular distraction,which would provide theoretical and technical foundation for shortening the consolidation period.