Double Phosphonic Acid Salt Combined With BMSCs Experimental Research For The Treatment Of Vertebral Osteoporosis

Chapter 1 between rat bone marrow mesenchymal stem cells in vitro isolation,culture,identification and markingObjective:Through,amplification,the induced BMSCs were isolated and cultured in vitro,and study the biological characteristics and morphologic features of BMSCs.Using fluorescence labeling BMSCs,by depicting,comparing to compare its growth curve fluorescent effects on BMSCs cell activity.Methods:1.Sterile conditions in rat femur and tibia,use whole bone marrow adherent culture method separation and purification of BMSCs.For cell covered bottle around 90%,to extend the 1:2.2.The wall with flow cytometry instrument to detect cell surface antigen,appraisal is for the purpose of the cell.3.Select growth in good condition of P3 generation of cells,osteogenesis induced 21 days,alizarin red and von Kossa staining method improved,observe its ability to produce calcium nodules.4.Use the CM-Dil labeling BMSCs,ability to observe its mark.Through the comparison of labeled cells count and number,and draw the growth curve,clear CM-Dil proliferation ability of BMSCs.Results:1.The SD rat bone marrow cells attached training after 24 h section stick wall,is not to the size of the spherical;Most of the cell wall,after 48h a fusiform or star;After 6 to 7 d can be represented for the first time.2.The cultivation of more than 99%of the adherent cells expressed CD29,CD44 and CD90,no expression of CD34,CD45.Prove that the cultured cells of BMSCs.3.Alizarin red staining mineralized nodules are visible red,Von Kossa staining improved light visible are dyed red cell mass there is massive black nodules.Ectomesenchymal stem cells have been proved that calcium nodules is induced into osteoblast and produced.4 CM-Dil instant of BMSCs markup rate was 100%,through the observation,counting,comparing the cell morphology and cell number,found no significant difference.Conclusion:1.The whole bone marrow adherent culture method can obtain the ideal BMSCs.2.After BMSCs after induction ability to produce calcium nodules.3 CM-Dil BMSCs can effectively marks,and will not affect its biological properties,is an effective method for in vitro tracer.Chapter2 Osteoporosis model in ratsObjective:Effective vertebral osteoporosis rats model was established to identify whether osteoporosis.Methods:1.The female rats with bilateral oophorectomy.2.By observing the rat vaginal smear,confirm whether ovaries removed successfully.Results:Ovaries removed rats sexual cycle,no longer stay for a week of vaginal smears were between emotional period.Conclusion:1.By removing bilateral ovaries rats osteoporosis model method is feasible.2.Through continuous observation rat vaginal smears can judge castrated rats successfully.Chapter 3 Between bone marrow mesenchymal stem cellscombined double phosphonic acid salt experimental research for the treatment of osteoporosis vertebral bodyObjective:In the early stage of the vertebral osteoporosis model was established based on the given corresponding vertebral transplantation in rats of different concentration of BMSCs and double phosphonic acid salt treatment,by measuring the relevant indicators,judge the curative effect.Methods:1.12 weeks not mated female rats 100,were randomly divided into 5 groups,each group of 20.(1)the control group;(2)castration group;(3)castration + transplantation group;(4)castration + double phosphonic acid salt group;(5)castration + +transplantation double phosphonic acid salt group.2.Adopt the method of removal of bilateral ovaries osteoporosis model was constructed.Castration after 12 weeks,the CM-Dil markers of P3 generation of BMSCs,with 1 ml syringe to 5 × 105/104/ml and 5 ml,5 × 106/ml,5×108/107/ml and 5 ml volume concentration of cell suspension injection respectively(3)and(5)group rats L2-L6 vertebral body,L1 vertebral injection volume of fluorescence suspension,etc.(4)and(5)group rats given Allen phosphonic acid sodium lavage treatment(8.2 mg/kg),once a week,for 12 weeks.All rats were fed on the same conditions.3.After treatment of 4 w and 8 w,12 w executed corresponding set of 6 rats respectively.4.Through the hard grinding mechanism for bone grinding,fluorescence microscope to observe whether stem cell transplant success.5.Through the MicroCT measures related to bone metabolism indicators,to determine whether to have curative effect.Results:1.Stem cell transplantation after 12 w make bone grinding,fluorescence microscope,new trabecular bone can be observed in a red fluorescence.2.The resection of bilateral ovaries rats after 12 w respectively by Micro-CT detection,found that rats compared with(1)(2)the group group rats whose vertebral BMD,Tb.N,Tb.Th,BV/TV is dropped significantly.BMD was reduced by 37.86%(P<0.05);Tb.N was reduced by 77.25%(P<0.05);Tb.Th was reduced by 43.48%(P<0.05);BV/TV was reduced by 66.67%(P<0.05).3.Micro-CT detection,(4)and(2)group,compared the ALE treatment can significantly improve the rat cancellous bone microstructure parameters of the vertebral bodies,BMD increased by 16.19%(P<0.05);Tb.N increased by 49.61%(P<0.05);Tb.Th increased by 57.69%(P<0.05);BV/TV increased by 55.56%(P<0.05).4.Micro-CT detection,(3)group rats L1 and(2)in the rat vertebral bone microstructure parameters.With the increase of concentration of BMSCs transplantation,L2-L6 vertebral body presents the BV/TV,Tb.Th,Tb.N,increasing BMD BS/BV,Tb.Sp gradually decrease trend..5.Micro-CT detection,(5)and(3)group compared to the L2-L6 vertebral bone mineral density are higher,L1 vertebral body bone mineral density and bone mineral density close to(4)group,(5)and(1)group compared with L6 vertebral bone mineral density is close to,but the former is still lower than the latter.Conclusion:1.Fluorescence labeling of stem cell transplant success the vertebral bodies.2.By removing the rat bilateral ovaries to success due to the osteoporosis,with extended time,castrated rats bone osteoporosis.3.Allen phosphonic acid sodium can slow the progress of osteoporosis.4.Stem cells can improve the bone mass,increase bone density,but does not increase within 12 w to normal vertebral levels.With the increase of number of transplanted stem cells,vertebral body bone mineral density is increased,and the transplantation of the experiment,the best concentration.5.Stem cell transplantation joint Allen phosphonic acid sodium improve osteoporosis is better than single use stem cell transplantation and separate the effect of alendronate phosphonic acid sodium,but still can not reach normal bone level.