| Partâ… The isolation,cultivation and amplification of adipose derived stem cells(ADSCs)Objective:To investigate the experimental methods of the isolation, culture and amplification of adipose-derived stem cells and the biological characteristics of ADSCs.Methods:The adipose tissue was obtained from SD rats groin, digested byâ… collagenase,and then some cell sediments were obtained after centrifugation,they were cultured and expanded in DMEM with 10% newborn calf serum. The cell morphology and biological characteristics were observed through inverted microscope, Giemmsa staining, cell counting, MTT, flow cytometry method.Results:The cells adhere the wall of flsk and grow fast. The surface markers of the 6th passage cell showed that 95% of the cells expressed CD29,96% of the cells expressed CD44.Conclusion:The isolated and cultured cells from SD rat adipose tissue are of characteristics of stem cell and can be used as seed cells for bone tissue engineeringPart II The osteogenic differentiation of SD rat adipose-derived stem cellsObjective:To investigate the conditions and characteristics when adipose-derived stem cells from SD rats were induced into osteogenic differentiation, To provide a theoretical basis for the application of adipose-derived stem cells in vivoMethods:Used high-sugar DEME supplemented with 10%newborn calf serum, dexamethasone, ascorbic acid, (3-glycerol phosphate, vitamin D as osteogenic inducer, induced the fourth generation SD rat adipose-derived stem cells into osteogenic differentiation, then,observed the cell morphological features through the inverted microscope, Giemmsa staining; detected the expression of intracellular alkaline phosphatase by modified Gomori calcium-cobalt method and alkaline phosphatase detection kit; observed whether there is calcium nodule formation by alizarin red S staining, tetracycline immunofluorescence test;detected the expression of osteocalcin mRNA by immunohistochemical staining and RT-PCR; and detected the expression of collagen type I protein by WeSter-nblotting. Results:After the osteogenic differentiation of SD rat adipose-derived stem cells, they show a short spindle or polygonal shape,the cell volume increases; The alkaline phosphatase expressed positively; there is a calcium nodule formation; the detection of osteocalcin mRNA by RT-PCR showed that the purpose of bands were found in the 280bp segment; I collagen protein expressed; by Western-blot, with the extension of time, type I collagen protein expressed strong graduallyConclusion:SD rat adipose-derived stem cells exhibited the capacity of differentiation into osteoblasts under the differentiated conditions, and have a strong osteogenic differentiation characteristicsPart III The preparation of composite scaffolds of xenogeneic cancellous bone/calcium alginate/PLGA/rat BMP-2Objective:To prepare a biodegradable composite scaffold that can release BMP-2 sustainablely.Methods:To Prepare microspheres of PLGA/rat BMP-2 by emulsification,then, analysis the particle size distribution of PLGA/rat BMP-2 microsphere with a particle size analyzer, wrapped PLGA/rat BMP-2 microspheres through the calcium alginate gel in heterogeneous porous trabecular bone, observed the structure of the composite scaffold through electron microscopy, detect the BMP-2 release of composite scaffold material in vitro through ELISA test, evaluate the biocompatibility of scaffold materials through culturing ADSCs with the composite scaffold.Results:The microspheres of PLGA/rat BMP-2 were round, its sizes were distributed between 120-450nm.The holes of the xenogeneic cancellous bone were interconnected, filled with calcium alginate and PLGA/rat BMP-2 microspheres.The scaffold release BMP-2 stablely in vitro, at the eighth weeks, about 83%of the BMP-2 was released,.The ADSCs grew good when cultured with the liquid extracted from composite scaffolds,it shows the composite scaffolds are no significant cytotoxicity.Conclusion:The xenogeneic cancellous bone/calcium alginate/PLGA-rat/BMP-2 composite scaffold material has a good slow-release function, biocompatibility, and can be as a scaffold for bone tissue engineeringPart IV The osteogenesis of the composite scaffold materials of xenogeneic cancellous bone/calcium alginate/PLGA/rat BMP-2 with rat adipose-derived stem cells in vivoObjective:To study the osteogenesis of SD rat adipose-derived stem cells combined with xenogenic cancellous bone/calcium alginate/PLGA/rat BMP-2 scaffold materials in vivo, to provide a theoretical basis for the clinical application of SD rat adipose-derived stem cells combined with the heterologous cancellous bone-calcium alginate-PLGA-rat BMP-2 scaffold materials for bone tissue engineeringMethods:Take 30 nude mice, divide them into experimental group and control group, for experimental group,implanted the scaffold of xenograft cancellous bone/calcium alginate/PLGA/rat BMP-2 with ADSCs in the back muscle pouches of experimental group, for control group, implanted the scaffold of xenogeneic cancellous bone-calcium alginate-PLGA with ADSCs on the right side of the back muscle pouches, on the left side of the back muscle pouches,implanted the scaffold of xenograft cancellous bone/calcium alginate/PLGA. Take out the scaffolds periodically,observed the situation of osteogensis of scaffolds by HE staining, scanning electron microscopy, x-ray,alkaline phosphatase detection methods in vivo; and observed the osteoid formation by 5-Bromodeoxyuridine immunohistochemical staining; detected the release of BMP-2 from composite scaffold material by ELISA.Results:Adipose derived stem cell adhesioned to pore wall of xenogeneic cancellous bone/calcium alginate/PLGA/rat BMP-2 scaffold scaffold. HE staining and x-ray photographs indicated that there were new bone formation in experimental group; In control group,there were also new bone formation on scaffold of xenogeneic cancellous bone-calcium alginate-PLGA with ADSCs, but the new bone formation was strong in the experimental group;In control group, there were no obvious bone formation on scaffold of xenogeneic cancellous bone-calcium alginate-PLGA, and the scaffold materials degradated gradually; The experimental group could be detected in BMP sustained release; alkaline phosphatase expression in the experimental group showed stronger than the control group; By the 5-Bromodeoxyuridine immunohistochemical staining, the experimental group and the control group of xenogeneic cancellous bone/calcium alginate/PLGA with ADSCs showed positive expression,it confirmed that the osteoid derived from ADSCs.Conclusion:The xenogeneic cancellous bone/calcium alginate/PLGA/rat BMP-2 is an ideal scaffold material and can be used for bone tissue engineering; ADSCs has the ability of osteogenical differentiation, it can be used as the seed cell for bone tissue engineering... |
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