OSX-Modified Bmmscs Enhance Bone Formation During Distraction Osteogenesis In Rabbits

Distraction osteogenesis (DO) is a well-established technique, originally developed in orthopedic surgery for correction of limb length discrepancies and later also used to treat hereditary malformations in the craniomaxillofacial region. The application of Distraction osteogenesis (DO) to oral and maxillofacial surgery provides a new method dealing with difficult bone defects and deformity. and its effectiveness in clinical practice not only breaks through the traditional surgery theory, but also solve many of the clinical problems traditionally hard to solve.The size, shape and structure of the new bone during distraction osteogenesis are close to the original bone,and bone grafting is unnecessary which avoids many complications.Besides, the soft tissue (such as muscle, skin, nerves, blood vessels) around the distracted region is expanded during the bone distraction without biological changes.however, the long treatment period and the potential of fibrous union or nonunion under some circumstances remain major limitations that hamper further clinical application of DO. Therefore, how to promote the formation and mineralization of the new bone and shorten the treatment period during distraction osteogenesis is becoming the focus of attention of many scholars.Many attempts have been made to improve the technique to accelerate osteogenesis in the distraction gap, including the application of precursor stem cells, demineralized bone matrix, and other inorganic salts, low-intensity ultrasound, direct current or electromagnetic stimulation and hcyperbaric oxygen therapy. The above methods may have certain effect on osteogenesis, but none of these means have been put into a wide range of clinical application.Various growth factors and cytokines have been investigated for their capacity to promote bone regeneration in vivo. Among these, recombinant human bone morphogenetic proteins (rhBMPs) induce robust ectopic bone formation and healing of orthotopic bone defects in several different animal models. Formulations based on bone morphogenetic protein-2 (BMP-2) and BMP-7 have been approved by the FDA for the treatment of severe orthopedic conditions such as spinal fusion and skeletal nonunion. Other growth factors and cytokines, such as transforming growth factor-β1, basic fibroblast growth factor and insulin-like growth factor-1 also have a certain role in promoting new bone formation. However, the doses of recombinant protein required to accelerate healing in humans are significantly higher than the levels expressed during normal bone repair, likely due to suboptimal delivery vehicles and rapid in vivo protein degradation. These supraphysiologicconcentrations are cost-prohibitive to widespread clinical usage and may be problematic if the non-selective targeting of neighboring non-osseous tissues leads to ectopic bone formation, In view of these complications, gene therapy based on over-expression of growth factor became a new strategy strategy for promoting osteogenesisGene therapy with BMP-2, BMP-7, and bBGF as target genes has been used in experimental animals and successfully promoted the new bone formation during distraction osteogenesis.Despite promising results, the clinical feasibility of these growth factor-based gene therapy approaches may be hampered by complex release kinetics and unregulated,ectopic bone formation caused by paracrine signaling to neighboring non-osseous tissues. In addition, the new bone formation during distraction osteogenesis is a very complex process,in which multi-factors and multi-cellular factors participate, while the high expression of several growth factors at the same time is difficult.Therefore, we assume to find a new method of gene therapy based on transcription factor regulation which is the hub and target of a large number of growth factors, cytokines, and power transmission to resolve the disadvantages of gene therapy based on bone growth factors.Many transcription factors are expressed during bone development and fracture healing including Runx2, Smads, Dlx-3,Dlx-5, MSX-2, AP-1, and Osterix. Among these,Runx2 and Osterix have been extensively characterized for their role in regulating the commitment of multipotent MSCs toward the osteoblastic lineage. Runx2 is an essential transcriptional regulator of chondrocyte hypertrophy, osteoblast differentiation, and bone formation. Runx2 can induce new bone formation in multipotential mesenchymal cells, this process might occur more efficiently and lead to bone matrix of better quality in vivo if Runx2 levels are high during the early differentiation stage and low during the osteoblast maturation stage. Osterix is a zinc-finger-containing transcription factor that acts downstream of Runx2 to induce the differentiation of osteoprogenitors into mature osteoblasts,which avoid the problem that Runx2 inhibits osteoblast differentiation of cells in the late stage.In addition, Osx gene therapy for distraction osteogenesis has not been reported to date.Therefore, in this study we select OSX gene for the target gene. Firstly the mandibular distraction osteogenesis model in rabbits was established and recombinant plasmid pEGFP-OSX was constructed in vitro. Then pEGFP-OSX was transiently transfected into rabbit bone marrow MSCs and OSX modified autologuous MSCs were thereafter introduced into rabbit mandibular distraction gap. The effect of OSX gene therapy on callus formation and mineralization were evaluated through radioghaphic,histological and histomorphometric examinations.The results of this study will provide valuable informations for OSX gene therapy to accelerate callus formation and shorten treatment period during distraction osteogenesis,and lay the foundation for eventual clinical application.【Methods】1.Creation of the mandibular distraction osteogenesis model in rabbits. Skeletally mature male New Zealand White rabbits were used in this study. The body weights ranged from 3.0 to 3.5 kg at the beginning of the experiment, without restriction on male and female. Inernal distractors were used which were composed of screw rod for traction, sliding rod, fixed arm, rotating handle as well as rotation joints linking to handle. The distraction distance is 0.4mm after each rotating circle. Anesthesia for all experimental procedures was achieved by intravenous injection of 3% pentobarbital (1 ml/kg), and a 2-cm incision was made along the inferior border of the right mandible. The periosteum and the masseter muscle were incised and carefully elevated. Under constant saline irrigation, the osteotomy was performed on the buccal (outer side) through the anterior part of the mandible (just anterior to the first molar) using a diamond disk. A custom-made distractor was placed and fixed to the mandible with 4 self-tapping screws After 6 days of latency, unilateral mandibular distraction was activated at a rate of 0.4 mm/12 hrs for 10 days, to produce a distraction gap of approximately 8 mm. After distraction was completed, the distractor was left in situ. Half of animals randomly chosen from each group were sacrificed with sodium pentothal injections at the end of week 2 and 6 throughout the consolidation stage respectively. The mandibular samples were harvested for Radiologic and histological examination2.Construction of recombinant plasmid pEGFP-OSX:The target gene was obtained through PCR amplification, purification, digestion, recovery and purification.Enhanced green fluorescent protein vector was Linearized after enzymy digestion, recovery and purification. Then the target gene was connected to the vector, and the new plasmid was identified by PCR amplification and DNA sequencing after competent cell preparation and transformation.3.Autologous bonemarrow stem cells (BMMSCs) were removed from the left tibia and isolated by density gradient centrifugation,and cells of passage 3 were used for the gene transfection in vitro and transplantation in vivo.4.Liposome-mediated in vitro transfection of plasmid pEGFP-OSX to bone marrowMSCs and gene therapy:The third generation of bone MSCs corresponding to each rabbit were obtained and after digestion and counting,passaged to 6-well plates. When MSCs were grown to 80% confluence, they were transfected with pEGFP-OSX using Lipofecta-mineTM2000. Expression of EGFP was observed under fluorescent microscopy to determine the transfection efficiency. Immediately after distraction, autologous bone marrow mesenchymal stem cells (MSCs) transfected with pEGFP-OSX, MSCs, and physiological saline were injected into the distraction gaps of the mandibles in groups A, B, and C,respectively.5. Osteogenic effect of detection:Half of animals from each group were euthanized at 2 and 6 weeks after completion of distraction. The distracted mandibles were removed and processed for radiographic, histological and immunohistochemical examinations. The density of distraction callus was then measured to represent its projectional bone mineral density (BMD).Sixteen serial sections of each specimen were made, ten for the HE staining and were observed in the light microscope t for morphometry analysis; six for the immunohistochemical detection of bone sialoprotein (BSP) expression.Image analysis software was used to analyze bone histomorphometry. Ten discrete sections were measured for each specimens, and the mean measurement results of the specimens were the parameter values.The bone volumes in the newly formed cortical bone area and in the cancellous bone area of the distraction zones were measured.6. Statistical analysis:All data are expressed as means±standard errors of the mean of 3 or more independent experiments.Statistical differences among groups were evaluated by one-way analysis of variance (ANOVA) and post hoc multiple comparison tests (Ducan's multiple range test) were performed when significance was obtained. p<0.05 was considered statistically significant.【Results】1. The animals tolerated the surgical procedures and distraction well.Diet,feces and urine were normal,and mandibular deviation occurred at the end of the distraction. longer mandibles were found in distracted specimens. Radiological and histological study confirmed osteogenesis in gaps in animal models,and the mandibles were extended in accordance with the intended target.2. Through gene recombination technology, the recombinant plasmid pEGFP-OSX was successfully constructed and confirmed the correctness by sequencing analysis3. Bone marrow MSCs were successfully isolated with density gradient centrifugation method, Liposome-mediated transient transfection of pEGFP-OSX to bone marrow MSCs were observed under fluorescence microscope, with 40-45% transfection efficiency at 48 hours.4. Animals in group A showed greater bone formation and earlier mineralization in the distracted callus when compared with that in group B,and similarly increased callus formation was found in group B than group C under radiographic, histological and immunohistochemical examinations.Results of statistical analysis on bone mineral density and histomorphometry showed that group A was significantly better than the group B(P<0.05),group B was significantly better than the group C (P<0.05) in osteogenesis.5. BSP in the sections was detected using immunohistochemical staining using a BSP monoclonal antibody followed by counterstaining with hematoxylin. BSP expression is localized to osteoblasts and osteocytes within newly formed bone at 2 weeks after the end of distraction,and Strong BSP expression of was observed in group A, moderate in group B, and weak in group C.[Conclusion]1. Internal distractors were well designed to meet the requirements of unilateral extension of mandibles in rabbits.The osteogenesis in delayed phase of distraction was similar with fracture healing process, but the osteogenesis in distraction and retention period showed characteristic histological changes2. In this study, stable and highly repetitive rabbit models of unilateral mandibular distraction osteogenesis were successfully established which were suitable for high-volume animal experiments and would provide a good experimental platform for cell and gene therapy.3. MSCs obtained by density gradient centrifugation in vitro has a strong value-added activity, and may be the ideal seed cells for cell therapy and gene therapy. Observed under fluorescence microscope, liposome-mediated transient transfection can make pEGFP-OSX gene expression in MSCs successfully.4.pEGFP-OSX-modified MSCs can more effectively promote new bone formation and mineralization during mandibular distraction osteogenesis compared with MSCs alone under radiographic, histological and immunohistochemical examinations,suggesting that OSX gene therapy is an effective method to promote the mandibular distraction osteogenesis5.Immunohistochemical detection showed increased expression of BSP in cells and bone matrix of distraction gaps in group A,which suggest that the imported OSX gene were effectively expressed and OSX gene play an important role in promoting new bone formation and mineralization.6.Application of ex vivo gene therapy of OSX in autologous MSCs can effectively promote the callus formation and shorten the treatment cycle in distraction osteogenesis. The approach developed in this study may provide an alternative new method to facilitate clinical distraction osteogenesis treatment, especially for the patients whose osteogenic potentials are compromised.