| NVBG, as an effective method for the repair of bone defects, is still widely used up to now. Conventional view of incorporation of non-vascularized bone graft to the recipient is called creeping substitution. The grafts will finally be totally replaced by the host and all bone-forming cells originate from the recipients. According to this theory, it's impossible to perform NVBG with implants at the same time. Following our previous series studies, this research pays much attention to the turnover of the grafted bone, formation of the new bone, reconstruction of vascularization, and bone-implant osseointegration, using dogs and transgenic mice as NVBG animal model. The purpose of this study is 1. providing a whole picture to the healing process of NVBG with and without dental implants; 2. demonstrating the effect of VEGF and TGF-β1 in bone healing and bone-implant osseointegration; 3. analyzing the fate of the grafts and the origin of the new bone.Part 1: In this part, we developed a NVBG experiment model in dogs, and then studied the cell viability of the grafted bone, new bone formation and angiogenesis, and discussed the rule of healing and roles of healing promoting factors.Methods: A 2.5cm-long-defect was made in one side of 12 native dogs'mandible, and repaired with the same sized iliac bone taken from the pars iliaca of themselves. A rigid fixation was performed with reconstruction plate and steel-wires (Ex vivo time of the graft was less than 15 minutes). Animals were sacrificed at 1, 2, 4 months after surgery respectively and the grafts were carefully harvested and examed by clinic, X-ray, inferior alveolar artery arteriography, histology, capillary vesscle count, bone histomorphometry, VEGF and TGF-β1 immunohistochemistry and TGF-β1 in situ hybridization.Results: One dog suffered from infection and its grafted bone was totally necrosis. We supplemented a dog to continue the study. Others remained healthy during the healing period. X-ray image showed that 1 month after surgery there was a little absorption in the grafted bone and between the connected areas. Bone density of grafted bone was increased in 2 months after surgery, but still lower than that of the normal mandible. In 4 months, there was no distance between the grafted bone and the host, and the density of grafted area was even higher than the host. Arteriography showed the stop of inferior alveolar artery in the operation side.Histologically, 1 month after surgery, parts of the superficial transplanted bone were absorbed and new bone formation was observed around the capillary vesscles. Large amounts of mesenchymal cells and chondrocytes were aggregated. Grafted bone continued to be absorbed and newly formed trabecular grew together and became mineralized in 2 months after surgery. In 4 months, newly formed bone incorporated to the grafted bone and it's difficult to distinguish the host and the grafts.The analysis of the grafted bone lacuna vacancy rate showed that nearly 30 percent of the grafted bone cells survived 1 month after transplantation and the data decrease to 20 percent another month later. The data of bone mineralization deposition rate showed that the marginal parts of the grafted bone was higher than the middle in 1-2 months after surgery, but showed no difference 4 months later. The results of bone histomorphometry showed that trabecular related parameters (TV/BV,Tb.Th and Tb.Sp),osteoid related parameters (OS/BS,O.Th) and bone absorption related parameters(N.Oc/BS,ES/BS)had statistic difference between the marginal position and middle position of the grafted bone in 1-2 months after surgery. For trabecular and osteoid related parameters, marginal parts are higher than middle parts, while it's just opposite in the parameters of bone absorption. Four months later, the statistic difference between the positions of the grafted bone disappeared. In 1, 2 or 4 months after surgery, the marginal parts showed much higher level than the middle parts in all the parameters. From the results of capillary vesscle counts, it can be found that the marginal position, no matter the proximal or the distal, acquired much more blood supply than the middle part. In 1-2 months after surgery, the number of capillary vesscles was much larger in marginal parts, even more than the normal mandible area. Four months later, the data became to decrease to normal level. From above data, it could be presumed that angiogenesis have positive correlation with the new bone formation and negative correlation with bone resorption.Immunohistochemistry staining showed that VEGF localized in the vascular endothelial cells. It showed a strong positive expression in ECs 1-2 months after surgery, while a weak positive expression 4 months later. Immunohistochemistry and in situ hybridization of TGF-β1 revealed that newly formed chondrocytes, osteoblasts and osteoclasts showed a stong positive expression 1 month after surgery and a weak positive expression 2 months after surgery. Four months later only newly formed osteocytes and matrix showed positive expression.The results indicate that: 1. Parts of the NVBG grafted bone survived after transplantation. 2. New bone formation began 1 month after surgery and newly formed trabecular was gradually mineralized and became matured 4 months after surgery. 3. New bone formed arround the capillary vesscles and new bone formation have positive correlation with the angiogenesis. 4. VEGF and TGF-β1 played an important role in osteogenesis and angiogenesis.PartⅡ: Based on the previous studies, in this part we put dental implants, attached with rhVEGF165 and rhTGF-β1, into the transplanted iliac bone and observed the interface between the bone and titanium implants.Methods: Twenty-four dogs were divided into 2 groups and the NVBG was performed the same as before. In group A, two implants were put into the marginal part and the middle part of the transplanted iliac bone. In group B, the implants were attached with rhVEGF165 and rhTGF-β1 first and then were implanted into the grafted bone in the same position as group A. An implant was put into the other side of the mandible as control. Animals were sacrificed at 1, 2, 4 months after surgery respectively and the grafts were carefully harvested and measured by clinic, X-ray, histology, M-CT and bone-implant bonding force test.Results: One dog suffered from infection and the grafted bone with implants was exposed in the mouth. We supplemented a dog to continue the study. X-ray image showed that 1 month after surgery there was an obvious gap between implants and the surrounding bone and 4 months after surgery, no lucency was found in this area. BIC was no more than 5 percent 1 month after surgery and 20 percent 2 months after surgery in experiment group, while was 10 percent and 26 percent respectively in the control. Four months after surgery, 60 percent BIC acquired in experiment group and 70 percent in the control. The test of bone-implant bonding force showed that it's nearly 220N in the position of marginal implants 4 months after surgery. Bone histomorphometry made by M-CT showed that the growth rate of new trabecular formation arounding implants in marginal position were higher than those in middle position. From the analysis of the same position in different time, trabecular around implants showed a gradually bone formation process. In this process control group showed much higher speed than group A or B. In group A, bone tissue mineral density and connectivity density around implant were much higher than that in group B. Connectivity density in marginal position was higher than that in middle position. For the application of the rhVEGF165 and rhTGF-β1, the effectiveness of bone and BIC formation was greater in group A than in group B.The results indicate that: In early healing period of the NVBG with implants, the interface of bone and implant was full of connective tissues, and effective osseointegration was formed 4 months after surgery. VEGF and TGF-β1 can promote osteogenesis and bone implant osseointegration. Part III: In this part, tibia grafts taken from GFP transgenic mice was put into its isogenous'tibia defect and the fate of the grafted bone and the origin of the new bone were analyzed by the GFP fluorescence expression.Methods: 1.Cell culture. GFP-C57BL/6 osteoblasts were cultured and its'propagation ability and GFP fluorescence expression were observed in vitro. 2. Animal study. A 5mm-long-tibia with intact periosteum and parts of muscles was made in 15 GFP-C57BL/6 mice and was put into the same defect of 15 C57BL/6 mice. Animals were sacrificed at 3 days, 1, 2, 3 and 4weeks after surgery respectively and the grafts were harvested and observed by clinic, X-ray, histology and fluorescence microscopy.Results: Two mice were suffered from infection, so we supplemented 2 GFP mice to continue the study. X-ray image showed that in 3 -7d after surgery, the lucency gap between the grafted bone and host bone was obvious. In 2-3 weeks after surgery, porosis around the grafted bone was formed and 4 weeks later, the connection of bone and host formed. Histologically, 3 days: There were lots of blood clots and inflammatory cells in the grafted areas. The transplanted bone was partly absorbed. 1 week: Blood clots were replaced by vascularized granulation tissues. The union between the grafted bone and recipient bone was full of mesenchymal cells undergoing chondrocytic differentiation. Extensive bone absorption was observed at the periphery of the bone graft indicated by the numbers of osteoclasts. 2 weeks: The grafted areas could be divided into five layers according to different tissues: grafted muscles, grafted periosteum, newly formed cartilage, newly formed trabeculae and grafted bone. 3 weeks: The remained grafted bone began to integrate with newly formed mineralized trabecular. 4 weeks: Newly formed bone incorporated to the grafted bone and it's difficult to distinguish them. Bright green light was found in transplanted muscles all the time under fluorescent microscopy. The osteoblasts and newly formed trabeculae and parts of the grafted bone also showed GFP expression.The results indicated that: 1. GFP-C57BL/6 osteoblasts showed normal propagation ability and a stable GFP fluorescence expression was found in the continuous passaged cells in vitro. 2. Parts of non-vascularized grafted bone can keep viability and had the potential of osteogenesis. The grafted periosteum played an important role in new bone formation.
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