Experimental Study Of BMSCs Combined With PTH In The Treatment Of Osteoporosis In Rats

Chapter 1 Rat bone marrow mesenchymal stem cells culture,identification,osteogenic differentiation and USPIO marking Objective:To detect the cell viability and biological characteristics of BMSCs from rats,which is isolated,cultured,amplificated,identified and labeled with USPIO in vitro.Methods:1.The femurs of rats were isolated under aseptic conditions,and BMSCs were cultured and expanded by full adherence method.2.Identify the surface antigen of the cultured cells by flow cytometry and clarify the purity of the target cells.3.The osteogenic differentiation of the expanded BMSCs was performed,and the alkaline phosphatase and alizarin red staining were used to identify the osteogenic differentiation ability.4.The amplified BMSCs were subjected to PLL-transfected USPIO labeling,and Prussian blue staining confirmed that USPIO was successfully transfected into BMSCs cells.The growth curve of labeled cells was drawn by MTT method,and the effect of USPIO on the proliferation ability of BMSCs was determined.The biological activity of labeled cells was detected by trypan blue exclusion staining.Results:1.After 24 hours,the extracted cells were not uniform in shape,mostly spherical,partially adherent;after 48 hours of culture,most of the cells were adherent,and the cell morphology was spindle-shaped or fusiform.2.The results of antigenic phenotype detection by flow cytometry showed that the expression rate of CD29(99.86%)and CD90(99.73%)was extremely high,and CD34(6.48%)and CD45(0.94%)were hardly expressed,which proved that culture and expansion were carried out.The increased cells are BMSCs with extremely high purity.3.Under the light microscope of BMSCs stained with alkaline phosphatase,there were gray-black nodules between the cell clusters;after staining with alizarin red,large red staining and red mineralized nodules were observed.4.Light yellow fine iron particles were observed in BMSCs cells labeled with USPIO;most of the cytoplasm was blue after Prussian blue staining.The proliferation rate of the labeled cells was slow 2 days before the MTT assay,the logarithmic phase of proliferation was entered on the 3rd day,and the proliferation plateau was observed on the 4th to 5th days.The labeling cell trypan blue rejection rate was as high as 97%.Conclusion:1.Full-adherent culture method can successfully culture and expand ideal seed cells.2.After induction,BMSCs can produce calcium nodules,which prove that they have osteogenic differentiation ability.3.USPIO can effectively label BMSCs,and has no effect on its proliferative ability and biological characteristics,and can effectively perform in vitro tracing.Chaper 2 Construction and identification of rat osteoporosis modelObjective:To construct a rat osteoporosis model by excising the bilateral ovaries of female ratsand to determine whether the osteoporosis model was successful.Methods:1.Excision of bilateral ovaries in female rats.2.Detection of estrogen levels in ovariectomized rats by rat estrogen ELISA kit and determination of femoral neck related bone parameters by Micro-CT: bone volume fraction(BV/TV),trabecular thickness(Tb.Th),Indexes such as trabecular bone number(Tb.N)and trabecular bone separation(Tb.Sp).Results:The level of estrogen in ovariectomized rats was significantly decreased.Micro-CT measured femoral neck bone index and found that Tb.Th,Tb.N,BV/TV decreased,and Tb.Sp increased.Conclusion:The rat osteoporosis model can be successfully constructed by excising the bilateral ovaries of female rats.Chapter 3 Experimental study of BMSCs combined with PTH in the treatment of osteoporosis in rats Objective:USPIO-labeled BMSCs were implanted into the femoral neck of osteoporotic rats by in-line MRI in vitro tracing,which proved that the target cells were successfullyimplanted into rats.Finally,the femoral neck bone index was measured by Micro-CT,and the therapeutic effect of BMSCs and PTH on osteoporosis rats was observed.Methods:1.A total of 50 female rats were randomly divided into 5 groups.After 12 weeks,rats in groups A and B were not treated with intervention.Group A was the normal control group,group B was the osteoporosis blank control group;(PTH intervention group)Rat PTH 1-34 was injected subcutaneously at the dose of 60 ?g/kg once every other day for12 weeks.In group D(BMSCs intervention group),the concentration of 0.5 ml of femoral neck was 5×108 cells/ml.The USPIO-BMSCs;group E(BMSCs combined with PTH intervention group)applied PTH factors simultaneously on the basis of group D,and the injection concentration,dose and frequency were consistent with group C.2.All rats were sacrificed 12 weeks after intervention.Micro-CT was used to measure the femoral neck of five groups of rats: bone volume fraction(BV/TV),trabecular thickness(Tb.Th),trabecular bone The number of bone(Tb.N)and trabecular bone separation(Tb.Sp)were observed to observe the osteogenesis.Results:1.MRI tracer found that USPIO-labeled BMSCs were successfully implanted into the femoral neck of osteoporosis rats.2.Compared with the treatment group alone,BV/TV,Tb.Th,Tb.N were significantly increased,while Tb.Sp was significantly decreased,the difference was statistically significant(P < 0.05),but still not normal.Group level.Conclusion:The effect of BMSCs combined with PTH in the treatment of local osteoporosis is superior to the effect of treatment alone.