| Objective:To study the effect of bone marrow mesenchymal stem cells(BMSCs) labeled by 5-bromodeoxyuridine (BrdU) and determine the the optimal concentration and the best mark time mark in vitro. Observed the growth,proliferation and osteogenic differentiation of BMSCs through cultured the BMSCs labeled BrdU and Fibrin Sealant (FS) in vitro. To evaluate the feasibility of Fibrin Sealant as a cell carrier then provided experimental evidence in which tissue engineering of BMSCs/FS labeld BrdU Used vivo.Method:1.Isolation and culturing of bone marrow mesenchymal stem cells in vitro and to detect the best concentration and best time of BrdU:Choosing 2 months old New Zealand rabbits and removing Bilateral femur under sterile, Use of whole bone marrow culture, the phenotypes(CD34,CD44 or CD90) of BMSCs were identified by flow cytometry, 1×104cells/ml were incubated in sixteen 6-well plate at the third passage when High purity confirmed BMSCs(Each culture plate placed in advance climbing film cell line), A total of four groups (A,B,C,D),Each of four plates for a group:Containing BrdU were added to a final concentration of 5umol/L,1 Oumol/L,15umol/L,20umol/L of L-DMEM complete culture medium dark, After the 24h,48h,72h,96h the same time period in culture, Take each group of cells within a culture plate climbing films Bank Immunohistochemical staining, comparing the rate of BrdU-positive cells labeled with Different concentrations and different times through Basing on cell labeling efficiency to determe the optimal concentration and the best time of BrdU labeled.2.Implanted the BMSCs labed BrdU to osteoblast culture in vitro after being spreaded Fibrin Sealant on the bottom of culture plate:Adopting the best concentration of BrdU labeled bone marrow stromal stem cell to mark, the experiment is divided into three groups:group A(the control group):adding P3 generation of unlabeled BMSCs into osteogenic culture medium; group B(BrdU group):adding P3 generation of labeled BMSCs into osteogenic culture medium; group C(the experimental group):Putting the FS to thin plates at the end of tile then inoculation BMSCs labeled BrdU with Osteogenic culture medium Keep in Dark Place. In accordance with the purposes behind the detection of three groups of cells plant into 24,96-well culture plate, each 6 holes of culture plate were randomly selected as the test specimen number. In culture for 2,4,6,8,10,12h adherent cells when the line rate detection,after 1d,2d,4d,7d,14d,21d of Osteogenesis, it is separate to observe under light microscope, to test Growth kinetics (MTT), Trypan blue staining, GENMED cell alkaline phosphatase (ALP) activity staining and GENMED cell von Cusa (Von-Kossa) Improved staining, at last we will test the expression of ALP and calcium with quantificational method.3.The BMSCs labeled BrdU were directly put into Fibrin Sealant to mix culture:Let the cells collected, digested and centrifuged after Selecting the P3 generation of BrdU labeled BMSCs, About 1×10(?) BMSCs mixed into fibrinogen tube, then mixed with thrombin of Tube with an equal amount of liquid so that the BMSCs/FS Complex of the formation by BrdU labeled,plant In the culture plate bottom and curture with L-DMEM complete culture medium,it is the experimental group. The control group is not marked with the P3 generation of BMSCs implanted BrdU culture plate by adding L-DMEM complete culture medium. At 1,3,7,14,21d, the experimental group were observed BMSCs grown and proliferation in FS use Light Microscopic. Simultaneously FDA-PI two-color fluorescence detection rates of living cells to judge the livability of BMSCs in FS. Alkaline phosphatase (ALP) activity is the last indicator that to test the differentiate into osteoblasts of Two groups BMSCs. It is to study the proliferation and differentiation of BMSCs when BrdU and FS exist simultaneously, FS in vitro to determine whether the BrdU labeled BMSCs presence of proliferation and degradation of FS outside the state.These experimental data with SPSS17.0 statistical software for statistical analysis, P<0.05 was statistically significant.Result:1.Isolation and culturing of bone marrow mesenchymal stem cells in vitro and to detect the best concentration and best time of BrdU:P3 generation of BMSCs by flow cytometry showed that Bone marrow stromal cell purity was above 90%, BrdU positive mark on the nucleus and showing brown nuclei were labeled. The concentration of the marker, four different rates of detection of the concentration at the same time, lOumol/L,15umol/L,20umol/L concentration of labeled rate than 5umol/L concentration of labeling efficiency, with significant differences (P<0.05), 10umol/L,15umol/L,20umol/L concentration of labeling rate compared with that no significant difference (P>0.05). In terms of marking time, at this four groups, the same group 48h,72h,96h and 24h labeling efficiency labeling rate, compared with a significant difference (P<0.05), it is no statistical significance that comparisons with groups 48h,72h,96h at the same group(p>0.05). So that in 10umol/L concentration and the concentration of labeled 48h is the best mark and the best markers.2.Implanted the BMSCs labed BrdU to osteoblast culture in vitro after being spreaded Fibrin Sealant on the bottom of culture plate:After the induction of the bone cell morphology from spindle into a cubic, and cell volume were increased, it begin to find small number of nodules of calcium In the week after osteogenic, as growing and big as time. The same time C (experimental group) compare with A (control group) and B (BrdU group), cell adhesion was significantly higher (P<0.05). GENMED alkaline phosphatase (ALP) activity of cells staining and GENMED von Cusa (Von-Kossa) Improved staining, we find that three groups of cells increased with incubation time, positive cells gradually increased and calcium nodules. From the Quantitative detection of alkaline phosphatase and calcium, At the same time, C (experimental group) compared with the A (control group) and B (BrdU group) that the two groups showed no significant difference, P> 0.05, no statistically significant.3.The BMSCs labeled BrdU were directly put into Fibrin Sealant to mix culture:Bone marrow stromal cells labeled by BrdU was well survive and proliferate in fibrin glue, some cells were short spindle shape after 3 days and the edge of fibrin glue begin to degrade after 6 days, some cells loss to culture plates; Cultured for 14 days, cells grew well, Most of fibrin glue degradation, shedding of cells increased, the growth of adherent cells with normal morphology;fibrin glue completes degradation After three weeks. BMSCs over 95% living cells after the FDA-PI two-color fluorescence assay results so BMSCs in fibrin glue can grow well. Through testing the two groups Cell culture medium alkaline phosphatase (ALP),it is No significant difference compared with activityThe experimental group and the control group(p>0.05), we find that have no great affect when BMSCs that wrapped after the fibrin glue about osteogenic differentiation of BMSCs.Conclusion:1. BrdU labeled is simple, fast, safe and more sensitive. it can reflect cell proliferation and tracing the ideal index to monitor the transplanted cells.2.10 umol/L marker concentration and the labeled time at 48h are the best indicators.3. The cells adhered to the well after Planted BMSCs cultured on the surface of fibrin sealant,it shows that fibrin sealant has good biocompatibility, for no apparent BMSCs of exclusion.4. Fibrin sealant has good degradation under BMSCs, which is an ideal for tissue engineering and transplantation of cell carrier scaffolds.5. The fibrin sealant constructed engineering organization after The BrdU labeled BMSCs have no significant adverse effecting that growth,proliferation and osteogenic differentiation of biological characteristics so that provides a theoretical basis for further in vivo experiments.
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