Experimental Study On The Effects Of The Membrane Combined With Adv-BMP-2 Regional Gene Therapy For Senile Bone Defects

ObjectiveThe purpose of this study was to evaluate weather the guided boneregeneration technique combined with Adv-BMP-2 regional gene therapycould promote the bone regenerating function of senile bone defects, andto explore a new way to treat senile bone defects.1.To obtain, culture in vitro, and identify the rat's osteoblasts; to amplify,purify and determine the concentration of Adv-BMP-2; and to investigatethe effects of Adv-BMP-2 transfection on biological characteristic ofosteoblasts.2.To study the affection of TNF-αon the growth of osteoblasts and itsregulating function on the expression of BMP receptors.3.To establish the animal model of alveolar bone defects withosteoporosis.4.To carry on the BMP-2 gene transfer ex vivo, and to study the effect ofAdv-BMP-2 on repairing alveolar bone defects of osteoporosis rat.5.To compare effectiveness of variant deepfreeze periosteum and e-PTFEmembranes with red bone marrow on bone regeneration in surgicallycreated bone defects.6.To carry on the guided bone regeneration technique combined withAdv-BMP-2 regional gene therapy on the aged animal models (in vivo),and to compare the effect with other types of bone graft materials.Methods1. Osteoblast's isolation, culture in vitro and identification. The primary osteoblast-like cells was picked from the Wistar neonate rat, and cultured in vitro. The osteoblasts were determined through phase-contrast microscope's observation; tetrazolium salts chromatometry detection, alkali phosphatase activity measurement and dyeing with alizarin Bordeaux. Adenovirus vectors containing BMP-2 gene were reproduced and purified by cesium chloride density gradient ultracentrifugation. Biological activity identification was carried out using infectious dose. After the adenoviral vectors encoding BMP-2 gene were transfected into osteoblast, the biological characteristics of the osteoblast were examined.2.The influence of TNF-αwith different concentration on the proliferation and differentiation of the osteoblasts was evaluated through MTT and PNTT methods. On the premise of that the cell quantity was equal, RT-PCR technique was used to examine the regulation function of TNF-αwith different concentration to the expressing of osteoblast BMP receptors.3.Establishment of animal model. A total of 40 three months old female Wistar rats were randomly divided into four groups: control group (group A), ovariectomy group (group B), bone defects group (group C), and ovariectomy with bone defects group (group D). 8thW, 12thW after surgery, the weight change of rats was observed, ALP, Ca, P content in blood serum and womb weight coefficient were examined; jaw bone density and the bone mineral content were determined through DEXA technique.4.Twenty female Wistar rats were randomly divided into two groups. Experimental group (group A): Osteoblast transfected by Adv-BMP-2 and Collagen Sponge group (group A/C); Control group (group B): Ovariectomy with bone defects group, (group OB). The animals were killed respectively at the 8th weeks and 12th week; morphological observation, radiographic examination, histological determination were performed.5.Healthy Wistar rats were killed and periosteum of the parietal bone was got; and variant deepfreeze periosteum (VDP) was made of the periosteum. Twenty -four adult Wistar rats were randomly divided into three groups. Bone defects approximately 0.5 cm×0.5 cm was produced on the mandible ramus. Group A: control group (SO); group B: e-PTFE membrane/ red bone marrow; group C: variant deepfreeze periosteum / red bone marrow (VDP/ RBM) group. The animals were killed respectively at the 4th and 8th week; the specimens were obtained and examined by X-ray, histology.6. Forty female aged Wistar rats, 22 months, (weight 330±32g), SPF (no specific pathogen animal) were randomly divided into five groups: group A was control group; group B was VDP / Adv-BMP-2/CS group; group C was VDP /perioglas group; group D was VDP / HPPA group; group E was VDP /Adv- GFP / CS group. Full thickness bone defects approximately 0.5 cm×0.5 cm was produced in the mandible rumus, group BCD were respectively implanted with different materials. As a blank control group, group A was not implant with any material. The animals were sacrificed respectively at the 8th week and the 12th week and the specimens were examined by X-ray, histology and immunohistochemical staining. Group E was examined with fluorescent microscopy.Results1.The osteoblasts obtained from the Wistar neonate rat were cultured. The biological characteristics of cultured cells were examined through morphological observation, staining of Alkaline Phosphatase and Staining of Red alizarin. That cultured cells had typical mature osteoblasts characteristics. Detecting by MTT method we found that the transfected osteoblasts proliferated with higher speed than the control group. Through detecting the alkali phosphatase activity, we found that the transfected osteoblasts generated more alkali phosphatase than the control group.2.The results showed that when the concentration of TNF-αwas greater than 50 ng / ml, the proliferation of osteoblasts were significantly depressed. When the concentration of TNF-αwas higher than 5 ng / ml,the osteoblast differentiation was significantly depressed. All kinds ofconcentration of TNF-αcould inhibit BMP-IA and BMPR-II geneexpression, at the mean time could promote the BMP-IB gene expression.In a word,TNF-αplays an inhibitory role to the BMPR complex.3.All signals showed that twelve weeks after the surgery, the alveolarbone defect animal model of osteoporosis rat had been successfullyestablished.4.We found that Adv-BMP-2 could promote the repairing process of theosteoporosis rat alveolar bone defects.5.Eight weeks after surgery, we compared the effects of variantdeepfreeze periosteum and the e-PTFE with the control group, the resultsshowed that variant deepfreeze periosteum and the e-PTFE may bothguide the bone defects to form the bone tissue.6.Different types of biological material influenced the response of frontalzone organization at early time. The guided bone regeneration techniquecombined with Adv-BMP-2 regional gene therapy guided the new boneformation earlier than other methods.6.1.Radiographic observations: Eight weeks after surgery, demarcationline in the bone defect area of the control group was clear, small amountsof high-density image were shown at the edge of the defect area. Cottonor fog- like high-densisy image was shown in the bone defect regions ofgroup B, C and D, with clear boundaries between material and bone.Differences between each group were not obvious. 12 weeks after surgery:the control group still had approximately 2mm bone defect. Bone densityof repair area of group B was the highest, which was similar withperiphery bone tissue and the border was blurred. Bone defects of group C,group D were also completely repaired, and the demarcation line was alsoambiguous, but the density of defect area was slightly lower than that ofthe periphery bone tissue.6.2.Histological observation: 8 weeks after surgery, osteoblasts and small amounts of new bones were detected in the peripheral areas of defected bone in group A, the central area of bone defect showed massive fiber formation. Group group B, C, and D all had some new bone formed in different degrees. In group B, newly-formed bone increased significantly surrounding the transplanted bone area; the osteoblast proliferation was active; the new bone form from the circumference to the center; calcium deposition increased. In group C, the white transparent particles of Perioglas material were almost completely dissolved, leaving holes behind. Surrounding the holes, basal bone formed. This could be the microscopic mechanism of its bone induction function. Thin layer of connective tissue could be found around small amounts of materials, and obvious bone metrocytes, the bone cells, the osteoid tissue could be detected in the particle gap. In group D, all materials were surrounded by large amounts of osteoid, but the calcification degree of the osteoid and cell appearance in the matrix were different. The material had connected with the bone tissue into an entirety; the new bone tissue surrounded the material; the newborn bone's reconstruction was obvious. 12 weeks after surgery in group A, the osteoblast and ossein were still limited in the two sides, the central was filled with the fibrous connective tissue, just small amouts of blood vessels were seen and the bone formation had not been detected .In group B, the defect area was almost filled up with new bone tissue, left just a little fibrous tissue, and the trabecular bone could be seen in peripheral area. In group C, bony bridge connected the centre and the fringe of the defected area; holes left by Perioglas degradation were completely substituted by new bone. The osteoid tissue and the bone tissue in bone defect increased. In group D, the material of implanted HPPA was almost covered by the bone tissue, and there were large amount of new bone formation and a high degree of calcification, with unclear boundaries.6.3.Statistical analysis result: 8 weeks after surgery, the effect of group B was better than group C(P<0.05) and better than group D ( P<0.05); there was no significant difference between group C and group D (P>0/05); 12weeks after surgery, the new bone formation of group B, C, D weresignificantly better than group A(P<0.01).6.4.Osteoblasts were traced with Green fluorescent protein in vivo of rat:In group E, two weeks after transplantation, frozen section and continualparaffin section were observed under the fluorescence microscope,net-framed material could be seen with green fluorescent light, andosteoblats could be detected in the transplanted area at fourth week aftertransplantation, the same scene were found.Conclusion1.As one of the seeded cells of bone tissue engineering, osteoblast havemany advantages and could be used in many ways. After infectedefficiently by Adv-BMP-2, osteoblasts showed stronger capability ofosteogenesis. The study demonstrated the potentiality of BMP-2 genetherapy in the future clinical applications.2.TNF-αplays an inhibitory role to the BMPR complex.3.The alveolar bone defect animal model of osteoporosis rat wassuccessfully established.4.The BMP-2 gene transfer mediated by adenovirus ex vivo could inducebone formation of osteoporosis rat alveolar bone defect.5.Variant deepfreeze periosteum and the e-PTFE may both guide the bonedefect to form the bone tissue. This study demonstrates favorableregenerative outcomes by the use of two different types of membranesthat could be used as alternatives for guided tissue regeneration (GTR).6.The guided bone regeneration technique combined with Adv-BMP-2regional gene therapy (in vivo) in aged rats guided the new boneformation earlier than other materials. It was an ideal bone substitutematerials, especially for the senile bone defects.