| Background:With the development of social economy, the incidence of fractures increased gradually. Oral and maxillofacial region is the most common sites for traumatic fracture, because the location of the maxillofacial and its skeletal space within the framework. There are many factors affecting fracture healing, including the fracture type, fracture site, local blood supply, patient age and health status and also the iatrogenic factors. Although most of the fractures can heal after anatomic reduction and fixation, there are still about 5% to 10% of fractures end up with delayed union or nonunion.Maxillofacial bone fracture and bone defects will directly affect the function and appearance of patients with facial appearance, maxillofacial bone is the basis of facial shape, damage of the bones will affect the patient's chewing and pronunciation functions, seriously affecting the quality of life. The main causes of bone defects include:trauma, tumor and postoperative, infection and some bone tissue-related congenital diseases.Many in vitro and in vivo studies have shown that PDGF (platelet-derived growth factor) released by the platelet in the wound area promoted the cell migration, recruitment and cell proliferation of osteoblasts; some in vivo experiments have found that platelet-derived Growth factor-BB can promote fracture healing and cell migration in diabetic animal models by promoting angiogenesis, and can be used in the treatment of osteoporosis. However, the role of PDGF-BB in osteoclast formation has not been clearly defined.Bone healing is a complex, multi-stage process in which many growth factors and cytokines accumulate in the fracture area through diffusion of blood or cell secretion. Different cytokines are involved in different stages and promote fracture healing.At present, the methods of repairing bone defects in clinical and experimental study mainly include bone autograft, allograft, xenograft and artificial material implantation, as well as distraction osteogenesis, prosthetic repair, guided bone regeneration and tissue engineering technology which are commonly used in oral and maxillofacial region. Bone tissue engineering technology is widely used in bone defect research and treatment.Purposes:To study the regulation effect of PDGF-BB on the proliferation, differentiation and mineralization of mesenchymal cells and osteoblasts in vitro, and to investigate the role of PDGF-BB on the formation of osteoclasts in RAW264.7 cell line and BMMs (bone marrow mononuclear macrophages), and the effects of PDGF-BB regulation in osteoblast-osteoclast co-culture conditions, osteoclast-cell interaction and its mechanism;To study the effect of PDGF-BB on osteoclast formation in vivo and to investigate the role of PDGF-BB in the process of rat mandibular fracture healing, and explore its mechanism on the process of fracture healing;To study the effect of PDGF-BB on the sustained-release properties and biocompatibility of the composited porous scaffold, and investigate the effect of PDGF-BB on the healing of cranial defects in rats for providing the experimental and theoretical basis for clinical application of tissue engineering methods in the treatment of bone defects.Materials and Methods:Primary bone marrow mesenchymal cells, osteoblasts, mononuclear cells and RAW264.7 (mouse monocyte-macrophage cell line) were cultured in vitro to study the effects of PDGF-BB on these cells. (1) Study the effect of PDGF-BB on the cell differentiation, cell proliferation and the expression of related genes in bone marrow stromal cells; (2) Investigate the effect of PDGF-BB on the differentiation and proliferation of osteoblasts; (3) Investigate the effect of PDGF-BB on osteoclast formation in vitro; Culturing primary mononuclear cells and RAW264.7 cell line to study the effect of PDGF-BB on osteoblast differentiation, proliferation and osteoblast differentiation of primary osteoblast; (AG-1295), JAK2 inhibitor (AG490), and STAT3 inhibitor (S3i-201) were used in the experiment, and the effects of PDGFR-? on the osteoclast formation and osteoclast precursor cell chemotaxis; (4) Study the effects and the mechanism PDGF-BB osteoblast-osteoclasts co-culture system and the relationship between them;The model of mandibular fracture was established by evaluated by histology and radiation imaging examination to study the effect of PDGF-BB on mandibular fracture healing. (1) Design and make different types of mandibular fracture models by measuring and evaluating the morphology of mandible. The fracture models of mandible were evaluated by histological and radiation imaging examination, and the optimal fracture healing model was selected. (2) The osteoclast formation of PDGF-BB in animal experiment was studied by TRAP staining at 1,2,3 weeks after operation. (2) The effect of PDGF-BB on the osteoclast formation in animal experiment was studied. (3) PDGF-BB was used in the experimental group. The effect of PDGF-BB on fracture healing was studied by histological and radiation imaging analysis;Chitosan-mesoporous silica composite scaffolds loaded with rhPDGF-BB (recombinant human platelet-derived growth factor) were used to repair the skull bone defects in rats. (1) The chitosan and SBA-15 composites were prepared by mixing the chitosan and mesoporous silica. The chitosan and SBA-15 composites and the loaded PDGF-BB proteins were prepared by freeze-drying. All the scaffolds were measured by the method of Scanning Electron Microscope (2) Evaluate the cytotoxicity and biocompatibility of the scaffolds in vitro:PDGF-BB was evaluated by CCK-8 cell activity assay and alizarin red staining after mineralization induction. (3) Construct the model of critical cranial defect in rats, implant different groups of materials in the bone defect, and study the effect of PDGF-BB on the proliferation of osteoblasts. Quantitative x-ray examination, micro-CT and histology were used to detect the new bone formation in the rat skull defect model, and evaluate the ability of the composite scaffold materials in the promotion of bone defect healing.Results:1. PDGF-BB can promote the proliferation of bone marrow stromal cells, and transcription levels of some osteoblast differentiation-related gene were increased. PDGF-BB promoted osteogenic differentiation and mineralization formation of bone marrow stromal cells; PDGF-BB could promote the expression of alkaline phosphatase and osteocalcin in the primary osteoblasts of mice. PDGF-BB could promote the expression of osteocalcin, nuclear transcription factor Runx2, bone morphogenetic protein and other mineralization-related gene transcription level increased in osteoblasts; the transcription level of angiogenesis and cell migration releated gene-metal matrix metalloproteinase-9 was also significantly increased. PI3K inhibitor LY294002 can inhibit or even downregulate the transcription of these genes;2. PDGF-BB can promote the formation of osteoclasts, and this promoting effect can be reduced by JAK2 inhibitor AG490, PDGF-R? inhibitor AG-1295 and STAT3 inhibitor S3I-201; PDGF-BB could promote the osteoclast formation in vitro, and AG490 and AG1295 inhibit this effect. PDGF-BB promoted the phosphorylation of ERK1/2, Akt and STAT3 in RAW264.7 cells. AG490 could inhibit PDGF-BB-induced STAT3 phosphorylation; PDGF-BB up-regulates the expression of NFATcl, DC-STAMP and BCL-2 in osteoclastogenesis. AG-1295, AG490 and S3I-201 can reduce these effects; PDGF-BB enhanced the migration of RAW264.7 cells, promoted osteoclast formation by promoting osteoclast precursor cell chemotaxis, and promoted MMP-9 protein expression; PDGFR-? inhibitor and JAK2 inhibitor inhibited cell migration promoted by PDGF-BB;3. In the osteoblast-osteoclast co-culture system, there is less osteoclasts formation compare with the directly promotion of PDGF-BB on the osteoclast formation. The possible mechanism is that PDGF-BB promotes the secretion of nitric oxide by osteoblasts, thereby inhibiting the formation of osteoclasts. The promoting effects of PDGF-BB on synthesis of nitric oxide by osteoblasts can be reduced by JAK2 inhibitor AG490, PDGF-R? inhibitor AG-1295 and STAT3 inhibitors S3I-201. In addition, PDGF-BB could promote the expression of MCP-1 in osteoblasts, which could also be inhibited by inhibitors of PDGF-R? and JAK2/STAT3;4. Through the measurement and evaluation of the mandibular bone and anatomical structure, we designed the mandibular approach and the merhod for the construction of mandibular fracture model, and measured by histology, radiation imaging examination, and mechanical properties, to select the optimal fracture model for the subsequent experimental study. In the evaluation of the fracture model, we found that the crevice defect group could simulate the biological processes of fracture healing better, including the process of hematoma formation, fibrous callus formation and bone callus formation, and can ensure the uniformity of the experimental study. In addition, we used this model and investigated the normal healing process with the histological evaluation. We found that the fracture model of the mandible can be used for studying the bone formation in the process of fracture healing, and also the osteoclast formation;5. By using our mandibular fracture model, we found that PDGF-BB promotes osteoclast formation in vivo. At the first week of fracture healing, osteoclast formation was not found in the control group, but PDGF-BB group could found the osteoclast formation, indicating that PDGF-BB promoted the formation of osteoclasts; PDGF-R? inhibitor AG-1295, JAK2 inhibitor AG490 significantly inhibited PDGF-BB-induced osteoclast formation. The osteoclast formation in the PDGF-BB group was significantly higher than that in the control group at the second week of the mandibular fracture, and the osteoclast formation was inhibited in the AG-1295 and AG490 groups; exogenous PDGF-BB could promoted the healing of fracture in the histological and radiological examination;6. Chitosan/SBA-15 composite scaffolds have the potential to promote bone regeneration and have good porosity and stress properties. Chitosan/SBA-15 composite scaffolds exhibited good biocompatibility and sustained release of the composite scaffolds in vitro, the proliferation and osteogenic differentiation of rat bone marrow stromal cells were also promoted. In the model of cranial bone defect, the semi-quantitative X-ray and Micro-CT imaging results and the histological examination showed that rhPDGF-BB Chitosan/SBA-15 composite scaffolds could promote new bone formation in the critical bone defect of the rat skull. The new bone formed in the CTS/S20 group was significantly higher than the other groups.Conclusion:1. PDGF-BB could promote the proliferation of mesenchymal stem cells and promote its mineralization, and also promoted the proliferation and differentiation of osteoblasts; PDGF-BB can promote osteoclast formation directly through PDGF-R?-JAK2-STAT3 pathway; PDGF-BB can promote osteoblasts to secrete nitric oxide and other products through the JAK2-STAT3 pathway in osteoblast-osteoclast co-culture system, and indirectly inhibit the formation of osteoclasts. PDGF-BB can also promote osteoclastogenesis which may be related to the expression of MMP-9 and MCP-1 protein by PDGF-BB;2. We designed the mandibular fracture model, which can be used for studying the fracture healing process, and also the osteoclast formation in the process of the fracture healing; PDGF-BB can promote the formation of osteoclasts in vivo, the promoting effect was related to JAK2/STAT3 pathway; PDGF-BB can promote the healing of rat mandibular fracture in rats;3. The rhPDGF-BB-loaded chitosan-mesoporous silica composite scaffold has good biological activity and biocompatibility, and has good ability on promoting the cell adhesion. This scaffold and tissue engineering method has a good application prospect for the treatment of large area bone defect in craniofacial region. |
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