| Objective:Construction of rat osteoporotic model by oviposition;isolation,culture,identification,induction of differentiation,and USPIO-labeled rat BMSCs;BMSCs traced in rats by MRI.Finally,Micro-CT was used to determine and observe the osteogenic effect of PTH and BMSCs in the vertebral body of osteoporotic rats.Methods:1.All rats were divided into sham-operated group(SHAM,n=10)and osteoporosis model group(OVX,n=40).Osteoporosis model was established by classical excision of bilateral ovarian ovariectomy in OVX group.In the SHAM group,only the same size of adipose tissue around the ovary was removed.After 12 weeks,serum estrogen levels in two groups of rats were detected by ELISA and lumbar 6 vertebral bone mineral density.2.All adherent methods were used to isolate,culture,and increase BMSCs in vitro.Flow cytometry was used to identify whether the extracted cells were BMSCs.Osteogenic differentiation?alkaline phosphatase staining and alizarin red staining of BMSCs after identification.3.USPIO-labeled BMSCs transfected with PLL,and BMSCs containing iron Prussian blue staining identified and transfected BMSCs proliferation(MTT method),draw its growth curve.Trypan blue dye exclusion was used to test cell viability.4.The SHAM rats were group A.The OVX rats were randomly average divided into groups B,C,D,and E.The rats in groups A and B were not treated.The rats in group C were injected with Rat PTH 1-34,subcutaneously at a dose of 60?g/kg body weight,once every other day,for 12 consecutive days;group D rats exposed lumbar 5?6vertebrae,adjusted cell concentration 5×10~8/ml,transfused through the pedicle 0.5ml USPIO labeled BMSCs;E group large On the basis of subcutaneous injection of PTH,rats were injected with BMSCs at the lumbar spine 5 and 6,and the PTH injection concentration,frequency,and cell concentration were consistent with those of the C and D groups,respectively.MR examinations were performed in groups D and E to prove that BMSCs were successfully implanted in the vertebral body.All rats were sacrificed after 12 weeks and examined by Micro-CT of the lumbar 5 and 6 vertebral bodies to observe the changes of bone microstructure.Results:1.After 12 weeks of oviposition,the serum estrogen level and lumbar 6 vertebral bone mineral density in the OVX group was significantly lower than that in the SHAM group,demonstrating the successful construction of a rat model of osteoporosis.2.After the cells were extracted in vitro,they were of different sizes.After 24 hours,some cells adhered to the culture medium.After 48 hours,the medium was exchanged for half a day.After 5 to 6 days,the cells were generally passaged.Flow cytometry results showed that the cells expressed CD29 and CD90,which were strongly positive;CD34and CD45 were not expressed.Description This study successfully acquired and expanded BMSCs.About 10 days after osteogenic differentiation of BMSCs,alkaline phosphatase staining was performed.Dark blue to gray-black clumpy nodules were seen in the cytoplasm;about 21 days after induction of differentiation,alizarin red staining was performed.Microscopic observation showed that the cells were stained red.3.USPIO-labeled BMSCs were observed to adhere to the wall after the cells were filled with light yellow iron particles;most of the cytoplasm was visible blue after Prussian blue staining.The number of cells in the first 2 days was not changed by the MTT assay,but the cells entered the proliferative active phase from the 3rd day and the4th to 5th days were the cell proliferation phase.Trypan blue dye rejection rate of 97%.4.D?E group rats in the lumbar 5?6 vertebral body found irregular lumps or lumps of low signal.The results of Micro-CT showed that in group C,compared with group B,Tb.N increased by 41.7%(P<0.05),Tb.Th increased by 45.2%(P<0.05),Tb.Sp decreased by 44.5%(P<0.05),BV/TV increased by 39.1%(P<0.05),BMD increased by30.3%(P<0.05),and group D compared with group B,Tb.N increased by 28.1%(P<0.05),Tb.Th increased by 21.5%(P<0.05),Tb.Sp decreased by 31.4%(P<0.05),and BV/TV increased by 16.8%(P<0.05),BMD increased by 19.2%(P<0.05);In group E,compared with group B,Tb.N increased by 48.2%(P<0.05),Tb.Th increased by51.4%(P<0.05),Tb.Sp decreased by 56.0%(P<0.05),and BV/TV increased by47.1%(P<0.05),BMD increased by 42.7%(P<0.05).It was proved that both PTH and BMSCs can increase the bone volume fraction of osteoporotic vertebral and lumbar vertebral bone mineral density,increase the number and thickness of trabecular bone,and reduce the degree of trabecular bone separation,but the combined use of both is better than its single use.Conclusion:The combined use of PTH and BMSCs was superior to the two alone in improving bone volume fraction and bone microarchitecture of osteoporotic vertebral bodies. |
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