Studies About Osteogenic Factors(BMP9,OPG) Of Tissue Engineering Artificial Bone

Bone Morphogenetic Proteins (BMPs) were originally found in bone matrix and described to act as bone growth factors. The BMP family of cytokines comprises over 20 kinds of different ligands that belong to the Transforming Growth Factorβ(TGF-β) superfamily. The main biological function of BMPs is inducing osteogenic differentiation, they can induce preosteoblasts such as bone marrow stromal cells, synovial cells and fibroblasts to form bone and cartilage tissue. BMPs play important roles during the development of many tissues and organ systems including heart, lung, kidney, eyes, reproductive tissue, nervous system, placenta and placental connections, vasculogenesis and angiogenesis, fat tissue.Among the 20 BMPs, BMP2,BMP4,BMP6,BMP7 and BMP9 have the ability of inducing osteogenic differentiation . The proteins of rhBMP2 and rhBMP7 have been used in the treatment of clinical Orthopedic diseases such as bone defect, nonunion , delayed union and spinal fusion.BMP9 (also known as GDF-2) has more stronger ability of inducing osteogenic differentiation than that of BMP2 and BMP7. It has been proved by an experiment of C3H10 and C2H12 cells in vitro and nude mouse in vivo. The studys about the osteogenic ability of BMP9 in large animals have not been reported.The results of an experiment in cells and in nude mouses indicate that BMP9 has a synergy effect with BMP2,BMP4,BMP6 and BMP7, and the synergy effect of BMP9 and BMP2 is strongest. All these results support that BMP9 would be a new choice for tissue engineering and it has become necessary to detect the osteogenic ability of BMP9 and synergy effects of BMP9/BMP2 in large animal models, especially in weight-bearing bone defect models. In order to ensure the accuracy of experimental results of the synergy effects between BMPs, avoiding the mutual interference of gene expression and the errors of concentration detection is essential.Osteoprotegerin (OPG), also known as an osteoclastogenesis inhibitory factor, is a Secreted glycoprotein composed of 401 amino acids. OPG can promote new bone formation and inhibit bone resorption by regulating RANKL / RANK / OPG system, so as to achieve the role of promoting bone formation as a bone growth factor. RANKL is a homologue of OPG ligand, mainly expressed in osteoblasts and on the surface of bone marrow stromal cells, RANK mainly expressed on the surface of osteoclasts and its precursor cells. The binding of RANK and RANKL can promote the activation and mature of osteoclasts and accelerate bone resorption. OPG is a decoy receptor for RANKL, OPG and RANK binds with RANKL competitively. OPG can reduce the binding of RANK and RANKL and inhibit the differentiation and maturation of osteoclasts,so the bone absorption is reduced. OPG can also promote the formation of calcified nodules and alkaline phosphatase and induced bone marrow mesenchymal stem cells into osteoblasts, and it can be seen as a bone growth factor.osteoactivin capsule was derived from pure traditional Chinese medicine extraction, it has been used in the treatment of osteoporosis for many years. Its main ingredient is icariin, which can promote the secretion of OPG from osteoblasts. Osteoactivin capsule is effective in treating osteoporosis by regulating bone metabolism, it can induce bone formation and has fewer side effects, these advantages indicate that osteoactivin capsule would be a new choice for the treatment of OPF and bone defect accomplished with osteoporosis. To select a appropriate surgical approach that not only can provide good reduction and fixation but also protect the sites around fracture is very important in the treatment of OPF.The aims of this study: 1, To observe the osteogenic ability of BMP9-mediated artificial bone in weight-bearing bone defect model in large animals( critical bone defect in the middle of goat femur) 2, To construct co-expression adenovirus of BMP2 and BMP9, and give a tool for verification and further study of synergy effects between BMP2 and BMP9 3, To detect the osteogenic ability of osteoprotegerin capsule in OPF model(rabbit radius OPF) and discuss the possibility that OPG acts as a part of artificial bone for tissue engineering. 4,To introduce a new operation style for the treatment of OPFThe first part: Methods: 12 goats of 12-18 months were selected and critical bone defect models were established in the middle of femur. The animals were randomly divided into two groups,the experimental group was given artificial bone composed of Ad-BMP9, BMSCs and gelatin sponge, control group was given Ad-GFP, BMSCs and gelatin sponge. X-ray examination was taken at 1 day and 2, 4, 8, 20 weeks after operation and Gross observation, CT examination, Histological staining, biomechanical test was detected at 20 weeks when the animals were sacrificed. Results: X-ray showed the callus of the experimental group was formed earlier, Gross observation and Histological staining showed the new bone of the experimental group was more mature, better remolding and better biomechanical properties of the new bone of the experimental group were demonstrated by CT and biomechanical test separately. Conclusion: tissue engineering artificial bone mediated by BMP9 can promote the healing of goat femur defect effectually.Another results of this part: BMP9 can promote Adipogenic differentiation in vivo; The morphology and biomechanical properties of new bone of experimental group are worse than the normal bone.The second part: Methods :The genes of BMP2 and BMP9 were amplified from AdEasy vector by PCR and sub-cloned into pASG2 vector. The co-expression shuttle plasmid pASG2-BMP2,9 was confirmed by restriction endonuclease digestion, PCR and gene sequencing, then pASG2– BMP2, 9 was electro-transducted into competent AdEasier cells to acquire recombinant adenovirus plasmid . Following , the recombinant vector was transfected into HEK293 cells and high-titer recombinant adenovirus (AdBMP2,9) was gained after rounds of amplification. The expression and bone induction capacity of AdBMP2, 9 was observed in C3H10 cells. Results :AdBMP2, 9 was constructed successfully and the virus titer was 1010IU/ml after amplification. AdBMP2, 9 can express and induce alkaline phosphat ase activity in C3H10 cells. Conclusion :the recombinant adenovirus co-expressing BMP2 and BMP9 was constructed successfully and provide a useful tool for bone tissue engineering.The third part: Methods: 8-12 month-old female New Zealand's rabbits were ovariectomized for 10 weeks to establish typeⅠosteoporosis model, then radial bone segments for 3mm were resected to establish osteoporotic fracture model. The animals were randomly divided into two groups, experimental group and control group were given 1g/Kg ? d osteoactivin Capsules and starch paste 5ml through intragastric administration separately. Shadowgraph, blood biochemical parameters, HE staining, pcna immunohistochemistry and biomechanical testing were detected at 2,4,8 weeks. Results: The callus and trabecular of the experimental group increased more rapidly than the control group, the mechanical strength of bone callus increased. Conclusion: osteoactivin Capsules can Shortened the time of osteoporotic fracture healing and increase the intensity of bone callus.The fourth part: Methods: Between March 2006 and August 2007, 60 patients with osteoporotic vertebral fractures were treated. Kyphoplasty was performed in 40 patients (test group) and conservative treatment was performed in 20 patients as control (control group). In test group, there were 6 males and 34 females with an average age of 68.7 years (range, 56-78 years). The disease duration was 10-18 months (mean, 12 months). A total of 73 vertebral bodies fractured. In control group, there were 5 males and 15 females with an average age of 70.1 years (range, 57-80 years). The disease duration was 9-16 months (mean, 13 months). A total of 41 vertebral bodies fractured. There was no significant difference in the general data between 2 groups (P > 0.05). Results: All incisions healed by first intention in test group, and no leakage of bone cement occurred. The patients of 2 groups were followed up 36-38 months. The visual analogue scale (VAS) scores, European Vertebral Osteoporosis Study (EVOS) questionnaire scores, anterior and middle vertebral column heights, and Cobb angles of test group at 1-3 days, 12 and 36 months after treatment were significantly improved when compared with those before operation (P < 0.05); but there was no significant difference between before treatment and after treatment in control group (P > 0.05). After 12 and 36 months, the VAS scores, EVOS scores, anterior and middle vertebral column heights, and Cobb angles of test group were better than those of control group (P < 0.05). The incidence of vertebral re-fractures was higher in control group than in test group after 36 months (χ~2=16.347, P=0.015). Conclusion : Kyphoplasty can effectively rel ieve pain and restore the function after the procedure. The risk of vertebral re-fractures after kyphoplasty can be reduced in comparison with conservative treatment.In summary, the osteoinductive capacity of BMP9 was confirmed in bone defect models, the synergy effect between BMP2 and BMP9 was found, OPG and PKP can accelerate the healing of OPF. BMP9,AdBMP2,9 and OPG provide new options for tissue engineering artificial bone. The combination of artificial bone and minimally invasive surgery would give better treatment for diseases such as bone defect, fracture nonunion and OPF.