| Oral and maxillofacial surgeons are frequently faced with defects in maxilla and mandible due to inflammation, injury and malignant tumor. It is very important to restore lost function by repairing the bone defects owing to oral malignant tumors. At present, there are many restorative methods which have been limited in clinical use because of much disadvantage. Tissue engineering is an exciting and developing field and is applied toward the generation of biological substitutes aimed at the creation , preservation, or restoration of lost organ function. It is significant to restore bone defect employing the tissue engineering technique clinically.Tissue engineering consists of three elements: cells, growth and differentiation factors, and extracellular matrix scaffolds. Osteoblast origins from bone marrow stromal cell which can been induced into an osteoblastic phenotype. There are problems such as aging and weak function in primary cultured osteoblasts , so it is important to modulate cell growth and differentiation by using growth factors. Recently, recombinant human bone morphogenetic protein-2 has been used extensively in vitro and in vivo for safety and efficacy studies. Scaffold material plays a critical role in bone tissue engineering because it is important for cells to adhere,proliferate, differentiate on the scaffold surface. There are few reports on complex of different biodegradable material concerning bone restoration in oral and maxillofacial area. At present, although there are many studies about carries for growth factors, no research has been reported on the carry system for growth factor with biodegradable material and the systemic assess for release process and activity of growth factor. This study is to fabricate compound new biodegradable scaffold which is bioactive and contains sustained growth factor release system. Moreover, a culture complex including primary cells and new scaffold are created in vitro and in vivo. This study aims to observe bone formation by tissue engineering method and evaluate the possibility of bone restoration in predetermined shape employing the method of tissue engineering. It is very important to repair bone defects in clinical use.This study consists of four sections.Section 1The study on primary culture and biological characteristicsof osteoblastIn order to establish a new model and method of osteoblast culture in vitro, the stromal cells of bone marrow was harvested from the ilium of rabbit and normal human. The primary cell culture include two methods: one is to harvest mononuclear stromal cells by centrifuge separation ; the other is to culture with whole bone marrow. The cells were cultured in the presence of 50g/ml Vitamine C. 10mmol/L -Glycerolphosphate, 10-8mol/L dexamethasone. The cells were examined by phase-contrast microscope, light microscope, scanning electron microscope, ALP stain, calvaria stain, cell cycle and DNA analysis and surface antigenic moleculars detection by flow cytometry technique.The result showed that bone marrow stromal cells adhere to floor after 24 to 48 hours and could differeniate into osteoblast. The stromal cellsappeard in all kinds of shapes and prolifered in colony units. The cells fused into monolayer after one week and there was a calcified point in the center of colone after one month. ALP stain and calvaria stain were positive. Scanning electron microscope showed that cells appeared in various shape and had a few cell prominency. The cell cycle and DNA analysis showed that the percentage of Go+Gi and G2+S of primary osteoblast was 88.4%, 11.6% respectively; the percentage of Go+Gi and G2+S of 3T3 cell line was 79. 7%, 20. 3 % respectively. The CD molecular detection of osteoblast showed that the positive cells were CD29(59.00%),CD44(71.52%),CD80(2.27%),CD38(5.86%), CD34(22. 08%), and CD45 (71.45%) respectively.It indicated that bone marrow stromal cell was an appropriate source for osteoblast in bone tissue engineering. Bone marrow stromal cell could be induced to differentiate...
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