| Objective:1. To obtain rabbit periosteal-derived osteoblasts (POBs) in vitro, and to observe their fundamental growth traits and biological functions. To discuss if there are differences between POBs and ones preserved in a low temperature, then to set up a " bank of osteoblasts" for the following study.2. To constructe two replication-incompetent adenovirus vectors carrying hBMP-2(human bone morphogenetic protein-2) gene and GPF(green fluorescent protein) gene respectively by an in vitro ligation and to identify and amplify the vector.3. To study the effects of Ad-hBMP-2 transfection on biological characteristic of rabbit periosteal-derived osteoblasts and to examine the expression of target gene in POBs. To determine the adenovirus infection rate by using Ad-GFP as a contral gene vector.4. To study the ectopic osteogenic potential of hBMP-2 gene transfer mediated by adenoviral vetor in vivo and periosteal-derived osteoblasts transfected by Ad-hBMP-2 ex vivo.5. To construct tissue-engineered bone with hBMP-2 gene modified POBs and hBMP-2 gene activated TCP and to study the osteogenic potential of these two complex material.6. Distraction osteogenesis (DO) as an ingenerate bone tissue engineering technique has been used in the treatment of craniofacial deficiencies, but it requires a longconsolidation period and has a low but real failure rate. In this study, two techniques of local hBMP-2 gene transfer mediated by adenovirus were used in mandibular DO of a rabbit model to study the effect of gene therapy on new bone formation in DO. Methods:1. The osteoblasts obtained from the tibia periosteum of neonatal New Zealand white rabbit were cultured and then subcultured in vitro in Dulbecco's Modified Eagle's Medium(DMEM) including 20% fetal bovine serum(FBS). The biological characteristics of cultured cells were examined by morphological observation, staining of Alkaline Phosphatase and Staining of Red alizarin. The anabiotic cells were examined in the same way.2. Human bone morphogenetic protein 2 (hBMP-2) gene was ligated into pShuttle 2 vector and then the fragment containing hBMP-2 gene was excised by Pl-Sce I and I-Ceu I endonuclease. After the fragment was combined with adenovirus vector, it was transferred into HEK 293 cells to package adenovirus particles. PCR was used to identify the constructed Ad-hBMP-2 vector. Ad-GFP was constructed using the same method.3. After the adenoviral vectors encoding hBMP-2 gene were transfected into periosteal-derived osteoblasts, the biological characteristics of POBs were examined by cell morphological survey, cell growth curve , ALP staining and Von Kossa staining, and the expression of hBMP-2 in these cells was determined by immunohistochemical staining and Western blot analysis. After the Ad-GFP were transfected into POBs, the adenovirus infection rate was determined under fluorescent microscopy, and the green fluorescent protein was examined in the subcultured POBs.4. Sixteen mature New Zealand rabbits were divided randomly into 4 groups and were injected to the bilateral calf muscle with 4 suspension respectively, which were (1) POBs transfected by Ad-BMP-2 (0.2ml, 107cells, MOI=100), (2)Ad-BMP-2(0.2ml, 109pfu), (3) POBs without transfection(0.2ml, 107cells) and (4) Ad-GFP(0.2ml, 109pfu). The animal were sacrificed at 2 weeks and 4 weeks post injection and the specimens were examined by X-ray, histology and immunohistochemical staining. Group 4 was examined under fluorescent microscopy.5. Bilateral mandibular defects were performed in 28 mature New Zealand rabbits, which were divided randomly into 4 experiment groups(6 animals every group) and a control group(4 animals). The bone defects were implanted with (1)hBMP-2 gene modified tissue-engineered bone, (2)hBMP-2 gene activated TCP, (3)tissue-engineered bone and (4)TCP in the experiment groups, the control group were left without any implantation . All the animals were sacrificed at 4 weeks and 12 weeks post operation and specimens were obtained. X-ray, histology, immunohistochemical staining, new bone fomation and biomechanical test were examined and One-way ANOVA and multiple comparison were used to determine if there were significant difference between different groups.6. Forty eight New Zealand white rabbits which underwent mandibular DO were randomly devided into 4 groups. The dissected bone stumps were fixed in situ for 7 days after operation(latency period), then the distractors were activated at a speed of 0.5 millimeter every 12 hours for 7 days before they were fixedCconsolidation period). On the first day in the consolidation period, the bilateral distraction gaps were injected respectively with (1)107POBs transfected by Ad-BMP-2 with MOI=100 in 0.2ml solution, (2)109pfu Ad-BMP-2 in 0.2ml solution, (3)107POBs without transfection in 0.2ml solution and(4)without any injection. Sixteen animals were killed randomly at the 7th day, the 14th and the 28th day of consolidation period separately. Specimens were observed, and X-ray, histomorphology and immunohistochemical staining were performed. One-way ANOVA and multiple comparison were used to determine if there were statistical difference between the groups at the different consolidation period.Results :1.The cultured cells in vitro were osteoblast-like cells, which were mainly spindle-shaped, polygonal-shaped or irregular-shaped. With histochemical staining, more than 80% cells showed the positive stain of ALP, and the cells could form many minerlization nodes if cultured in conditioned DMEM (including 10%FBS,10mmol/L β-glycerophosphate, 50mg/L Vitamin C) for 40 days. The live ratio of anabiotic cells from the liquid nitrogen was more than 95%, and their major characteristics weresimilar to the subcultured cells.2. Both PCR and restication analysis showed that the recombined vectors pShuttle2-BMP-2 and Adeno-X-BMP-2 contains the target gene. The packaged adenvirus was also identified by PCR with specific primers for BMP-2.3. After transfected by adenoviral vectors, the periosteal-derived osteoblasts showed no significant change in shape, the cell growth curve showed that the growth of infected POBs was not restrained, the ALP staining was stronger and the Von Kossa staining were positive. The expression of hBMP-2 was confirmed in protein levels by western blot at the third day, 2 weeks and 4weeks post the transfection. The green fluorescent protein was detected at 12 hours after the transfection and the average transfection rate was 93%. The green fluorescent protein was also detected in the fifth subcultured cells.4. Radiodense bone image were observed in group 1 and 2 only at 4 weeks post injection, the other animal did not show any abnormity in X-ray image at any time. Two weeks after the injection, with histological examination, numerous chondrocytes and osteoblasts were observed in the fibrous interstitium in group 1, a pile of chondrocytes and osteoblasts with little fibre were observed in group 2, only a few osteoblasts were observed in group 3 and no osteoblasts or chondrocytes were observed in group 4. Immunolabelling for BMP-2 was aggregated in osteoblasts and matrix around in groupl, in osteoblasts and some of chondrocytes and matrix in group2. Four weeks after the injection, mature bone trabecular and marrow cavity were observed in group land 2, chondrocytes and osteoblasts presented in the new bone, only a little immature bone was observed in group3 and no bone formation in group 4. Immunolabelling for BMP-2 presented in osterocytes in the new bone and matrix, there are many positive staining in the marrow of group2, but more weak in the marrow of groupl. The green fluorescent protein was detected in the myocyte of group 4 at both time, but it was weaker at 4 weeks post injection than that at 2 weeks.5. The borderline between implantation and bone defect was still clearly in group 4 at 4 weeks post operation and it was indistinct in other groups at any time. New bone formation was observed in all groups at any time except in group 4 at 4 weeks postoperation. Quantitative analysis demonstrated that a significant difference (P=0.0000) in bone formation among the experimental groups, and multiple comparison showed that there was no difference between group 1 and 2 at both time, but significant difference was observed between them and group3, and group3 had more bome formation than group 4 at any time. Biomechanical testing revealed no statistically significant difference in the maximal compressive strength of new bone formed in group 1 and 2, and their strength was more than that in group 3 and 4 at 4 weeks post operation. Although there was no statistically significant difference among group 1, 2, and 3 at 12 weeks, but the group1 and 2 were similar to the normal mandible while significant difference between group3 and normal mandible.6. All the mandibles of DO animals were prolonged successfully. During the consolidation period, the bone density and the quantity of new bone formation in the distraction gaps of group 1, 2 and 3 were more than group 4, and there were no difference between group 1 and group 2. At the 7th day, no difference were found among group 1, 2 and 3. At the 14th day, the new bone formation of group 1 was more than group 3, and at the 28th day, the bone density and the new bone formation of groupl were significantly more than group 3. Conclusion:1. As one of the seeded cells of bone tissue engineering, POBs have many advantages and could be used in many ways. It was practicable to establish a " Bank of Osteoblasts ", and the anabiotic cells could be used for more experimental studies.2. The Ad-BMP-2 vector was successfully constructed , and the in vitro ligation method was more efficient than traditional homologous recombination.3. After infected efficiently by Ad-BMP-2, the periosteal-derived osteoblasts showed stronger capability of osteogenesis. The in vitro studie demonstrated the potentiality of BMP-2 gene therapy in the future clinical applications.4. The hBMP-2 gene transfer mediated by adenovirus in vivo and ex vivo could induce ectopic bone formation in active animals, and the target gene could be expressed persistently for at least 4 weeks.5. Tissue-engineered bone with hBMP-2 gene modified POBs and hBMP-2 geneactivated TCP had better osteogenic potential than tissue-engineered bone, Ex vivo adenovirus-mediated hBMP-2 gene transfer to allogenous POBs and in vivo gene transfer could enhance bone formation in the repair of mandibular segmental defects and they may become ideal bone substitute for bone reconstruction in the future.6. The model of bilateral mandibular DO in rabbits was successfully established, which can be easily repeated. In the experiment, it showed that transplantation of allogenous POBs had beneficial effect on bone formation in mandibular DO, and transplantation of Ad-hBMP-2 transfected POBs had better osteogenetic potential than POBs, there was no difference observed between the two different local hBMP-2 gene delivery.
|
PDF Full Text Request