The Study Of Inductioning The Rabbit Bone Marrow Stromal Stem Cells Differentiate Into Bone Cells In Vitro

objective:Through the way of density gradient centrifugation,separate the bone marrow stromal stem cells BMSCs) from rabbit marrow, to study the rabbit BMSCs culture methods and their growth characteristics in vitro, and induced its differentiation to osteoblast, to explore BMSCs'biological characteristics in vitro and to establishment the osteoblast differentiation system in vitro of a rabbit BMSCs. Observe the process of osteogenesis, in order to providing the basis preparation for future research work.Methods:Anesthetized the rabbits with 2% pentobarbital sodium 30mg/kg dose and killed it.then separation bilateral femur and tibia bone marrow, use the way of density gradient centrifugation combined with adherent culture, purified rabbit BMSCs, preparation of single cell suspension and inoculated with 10% fetal bovine serum L-DMEM culture medium, through the passage amplified, and further removal of non-adherent cells, when cells covered with the bottom of bottle nearly 80%, after adding 0.25% trypsin digestion and subcultured.Taked the 3rd generation logarithmic phase grow well cells and inoculated into pre-coverslip in 24-well plate with 1×107/L cell concentration.the experimental cell is divided into two groups. The experimental group using the osteoblast-induced fluid, directed inducing bone marrow stromal stem cells differentiate into osteoblasts.The control group added equal amount of L-DMEM culture medium containing 10% fetal calf serum continued to culture. Two groups of cells continued to culture (conventional change the liquid every 2 or 3 days) and observe the Osteoblastic differentiation of the cell culture. Main outcome measures:Observation of cell growth and proliferation by the inverted microscope, detection the expression of cell surface markers with flow cytometry and observed the ultrastructure of the cells transmission electron microscopy, I collagen immunohistochemical staining, Von Kossa staining and alkaline phosphatase staining to induce differentiation of osteoblast. And to make statistical analysis.Results:Acquire bone marrow mononuclear cells Rabbit with lymphocyte separation medium by the way of density gradient centrifugation combined with adherent method and inoculate it into L-DMEM culture medium containing 10% fetal bovine serum, After 48h,the adherent cells morphology oval-shaped, polygonal and short spindle-shaped. after 72h, scattered the spindle-shaped adherent cells.after 10-14 days the formation of cloning.when BMSCs adherent sparse, Long bipolar spindle cells toward irregular, cell arrangement disorder and often connected through the protruding between cells. about 3 weeks there a dense layer of adherent cells. At this time the bipolar cells often began arranged in fascicular regular, and some was swirling. Amplified by the passage, the cell further purification, cell morphology was homogeneous, and a long spindle-shaped arrangement of swirling, and the growth rate accelerated, P 1 on behalf of the cell population doubling time is about 37h. P3 cell growth incubation period is 3-4 days, afte 4-6 days into the logarithmic growth phase and after 2 weeks into the plateau. CD34, CD45 negative, CD44-positive by Flow cytometry. Electron microscopy showed BMSCS nucleus was round or irregular type, membrane clear, there are 1-2 nuclear, chromatin coarse, abundant organelles in the cytoplasm, cell surface villi. With Directed osteogenesis inducing medium induced the 3rd generation BMSCs about 7 days and can be observed in a few cells from spindle-shaped into a polygon, with the time pass the polygonal cells gradually increased and gathered into a group, after 14d Von Kossa staining appeared calcium nodules, alkaline phosphatase staining positive, but the control group no calcified nodules and alkaline phosphatase staining were negative. The alkaline phosphatase staining of cells induced clear positive reaction and in cytoplasm showed granular or lumpy gray-black precipitate, alkaline phosphatase active site brownish-black. Von kossa staining showed mineralized nodule of black particles, particle size heterogeneity, suggesting that there are mineralized matrix deposition. Bone induction culture three weeks, immunohistochemical SP method can detect the expression of typeⅠcollagen, in the nodules was around the brown, while the control group was unable to express.Conclusion:The experimental results indicate that bone marrow stromal stem cells cultured in vitro can be obtained large number of relatively high purity of stem cells within a short time, and the cy cle is short, reproducible and can be used as an important source of seed cells for tissue engineering. And explored dexamethasone in bone marrow stromal cells into osteoblasts role in the process, results showed that dexamethasone induced in the experiment group for all cells in the morphological and biological characteristics of both is more characteristics of osteoblasts than the control group, the ALP activity and the formation of calcium nodules have the ability to have stronger than control cells. This confirms the right of BMSC can be transformed into osteoblasts under suitable conditions in vitro, and can detect the expression of specific markers.