| The bone nonunion,and bone defects.etc.caused by trauma,tumor and osteoporosis have always been the problems to be solved for the orthopedic surgeons.The rapid development of bone tissue engineering is expected to be helpful for overcoming this problem in the future.The bone construction of bone tissue engineering includes seed cells,culture environment and scaffold material.Bone marrow mesenchymal stem cells（BMSCs）,a kind of stem cells derived from mesoderm,exist in the bone marrow and have obvious adherent ability.They have great proliferation and differentiation potential in vitro,and can differentiate into bone,muscle,fat and other cells.They play a key role in the regulation of bone metabolism.The cells that increase and maintain bone mass are mainly the osteoblasts that BMSCs differentiated,and the osteoblasts are important factor for maintaining human skeletal health.BMSCs are relatively easy to obtain,while it has no immunogenicity after in vitro amplification,and they can easily transfecte the outer gene and be passed on for a long time.In addition,the use of BMSCs for autologous transplantation experiments and treatment is almost unrestrained by ethics.The differentiation of BMSCs in vitro can be accurately controlled by multiple factors,and it is considered as the preferred stem cells for transplant experiments and treatments.The incidence of osteoporosis in postmenopausal women was significantly increased,and the rapid decline of estrogen levels in postmenopausal women was the main reason.Hormone replacement therapy（HRT）is the most classic treatment of the disease for 17β-estradiol can promote the osteogenic differentiation of the rat bone marrow mesenchymal stem cells（r BMSCs）,and it has the most significant effect at the concentration of 10-9 mol/L in vitro.With the development of Chinese medicine,the medicine with the ability of "Bushen Zhuanggu" has obvious curative effect in the clinical treatment of osteoporosis,bone nonunion and other diseases.Icariin can increase the activity of alkaline phosphates（ALP）,increase the number of calcified nodules and CFU-FALP,and promote the expression of b FGF,IGF-1,OSX,Runx-2 m RNA.Icariin can regulate the differentiation of BMSCs in two directions,that is,promote BMSCs osteogenic differentiation and inhibit the lipid differentiation of r BMSCs.However,long-term use of estrogen has significant side effects,and the application of epimedium and other traditional Chinese medicine have relatively slow effects.In this study,the effect of 17β-estradiol combined with icariin on osteoblast differentiation of rat bone marrow mesenchymal stem cells was observed which aimed at investigating whether they had synergistic effect and whether they could improve the differentiation efficiency of bone marrow mesenchymal stem cells into osteoblasts with less dose.This study will provide a theoretical basis for clinical medication.The BMSCs required in this study were extracted from the long bone marrow of SD rats of 3 weeks old and cultured by whole bone marrow adherence method.The initially isolated rat bone marrow suspension cells were dense under the microscope.After 12 h,the cells began to adhere to the wall,part of cells stretch,and the cell volume increased.After 72 h,some of the cells protruded with blunt and smooth protrusions,the cells were spindle-shaped,rhombic or polygonal with large and full nucleus,and they have a similar appearance to fibroblasts.After 14 days,the cells fused into pieces,the boundaries became vague,their bodies increased,and their shape became short spindle,and irregular shape,also part of them are focal shape.After 21 days,there were scattered mineralized nodules in the induced cells with a center of poor transmittance.Studies have shown that: BMSCs can express the surface antigen of CD29,CD50,CD90,other mesenchymal cell lines and endothelial cell lines,while they can’t express the surface antigen of CD14,CD45 and other hematopoietic cell lines.In this study,BMSCs surface antigen was identified by flow cytometry,and the results showed that the positive expression rate of CD45 was 0.5%,the positive expression rate of CD29 was 99.6%,and the positive expression rate of CD90 was 99.7%.The results were consistent with the characteristics of rat bone marrow mesenchymal stem cells.The 4th generation BMSCs were taken and digested,and the cell density was adjusted to 1 × 104 / ml.Then the cells were inoculated into 25 ml culture flasks.The cells were divided into control group（group C）,17β-estradiol group（group A）,icariin group（group B）and combined induction group（group A + B）.The 4th generation BMSCs received oriented induction after incubation for 24 h.Group C was treated with basal inducer(dexamethasone 1 × 10-8mol / L,β-glycerophosphate 1 × 10-2mol / L,ascorbic acid 5 × 10-2g / L)17β-estradiol 1 × 10-9mol / L,group B was treated with basal osteogenic inducer and icariin 1 × 10-4mol / L,and group A + B group was treated with basic osteogenic inducer,17β-estradiol 1 × 10-9mol / L and icariin 1 × 10-4mol / L.The induction lasted for 21 days,half of the liquid was replaced every other day,and the cells were passaged for 7 days.BMSCs were inoculated into culture flasks at the concentration of 1 x 104 cells / ml,osteoinductive induction was performed according to the description above.The ALP activity was measured on the 7th day of osteogenic induction,the COL-1 relative concentration was measured on the 14 th day of osteogenic induction,the BGP concentration was measured on the 21 th day of osteogenic induction,and the alizarin red staining was given on the 21 th day of osteogenic induction.The results showed that ALP,COL-1 and BGP relative concentration group of the combined group were higher than single factor induction group,and the ALP,COL-1 and BGP relative concentration of single factor induction group was higher than the control group.The number of calcium nodules in the combined group was higher than that in the single factor induction group,and the number of calcium nodules in the single factor induction group was higher than that in the control group,the difference was statistically significant.（P < 0.05）.This experiment showed that:Firstly,17β-estradiol and icariin could alone induce BMSCs to differentiate into osteoid cells in vitro.Secondly,the combined effect of the two is better than either of them. |
PDF Full Text Request