Application Of Bone Marrow Mesenchymal Stem Cells In Repairing Osteoporotic Vertebral Body In Rats

Objective:Implanted the BMSC into the vertebrae of osteoporotic rats which were isolated and cultured in vitro.PTH was used intermittently and in small amounts,osteogenic induction of bone marrow mesenchymal in order to explore the feasibility in the treatment of osteoporotic rats.Methods:1.Foty-two rats were randomly devided into blank control group(sham,n=9)and model group(OVX,n=33).Resected bilateralis ovaries toestablish the model of osteoporosis.In the control group,the same amount of adipose tissue around the ovaries was removed.Ten weeks later,one rats in each group was randomly killed,and the L5 vertebrae were taken for decalcification,fixation,paraffin embedding and the tissu were used to take HE staining to observe the trabecular structural.2.The whole bone marrow adherent method was used to islote and culture the BMSC and morphological changes of each passage were observed during cell passage.BMSCs were identified by flow cytometry.The P3 generation BMSCs with good growth condition and over 80%fusion degree were selected.After osteogenic induction,P3 cells were stained with alizarin red after identification.3.The control group rats(sham,n=8)were devided into model group A,osteoporosis group rats(ovx,n=32)were randomy divided into model group B,PTH treatment group C,BMSC treatment group D,and combined treatment group E,The number of tats in each group was n=8.Group A and Group B were fed normally without special treatment.The group C was subcutaneously injected with PTH(1-34)every other day at a does of 40μg/kg/day;The middle incision of the posterior lumbar region was taken to expose the lumbar 3-5 vertebrae in group D and group E,after adjusted cell concentration 5×10~5/ml,implanted 0.5ml BMSCs through the pedicle.On this foundation,PTH(1-34)was subcutaneously injected very other day in group E and normal saline was subcutaneously injected every other day in group D.After 8 weeks,the levels of serum alkaline phosphatase and osteocalcin were detected by ELISA,the L5 vertebral of rats in each group were taken were taken,decalcify,fix and paraffin embedded,tissue sections were stained with HE.The trabecular structure and osteogenesis of bone marrow mesenchymal stem cells were observed by light microscope.Results:1.Ten weeks after ovariectomy,HE staining of L5 vertebral tissue sections in the model group showed that the arrangement of bone trabeculae was disordered and sparse,and some bone trabeculae were broken compared with the model group,indicating that the osteoporosis model has been done.2.BMSCs were isolated and extracted by whole bone marrow adherent method in viro and then cultureed in constant temperature cell incubator for 72 hours,after half volume fluid has been changed,some adherent cells could be seen under light microscope.after that,the solution was changed every two days.After 10 days,the cells were generally passsaged.The P3 cells were detected by flow cytometry,results:BMSC highly expressed CD29 and CD44,but did not express CD45,the results showed that the extraced cells were BMSCs.The alizarin red staining was performed,it could be seen BMSCs differentiated into osteoblasts,produced mineral nodules and the cells were stained red.3.After 8 weeks,ELISA results show that compared with model group,the serum specific alkaline phosphatase activity of PTH treatment group and combined treatment group was significantly difference(P<0.05),while the PTH treatment group and combination treatment group serum ALP activity has little change,Compared with model group,osteocalcin content in PTH treatment group and combined treatment group was significantly increased,(P<0.05),while serum osteocalcin content in PTH treatment group and combined treatment group was not significantly changed(P>0.05).The histological section showed that the bone trabeculae in the treatment group were more compact and orderly,the medullary cavity was smaller and the arrangement of bone trabeculae was more compact than model group.Conclusion:Only by BMSC transplantation as a means of intervention,there is no significant effect on the treatment of osteoporosis,which may be associated with survival rate of the transplanted cells,and small dose,continuous use of PTH can osteogenic induced BMSC,improve the effect of osteogenesis,and PTH and BMSC are used in combination,can significantly improve the rat osteoporotic vertebral body bone microstructure,play a role for the treatment of osteoporosis.