Effects Of Aspirin On Differentiation Of Bone Marrow Mesenchymal Stem Cells Into Osteoblasts In Vitro

Osteoporosis is a disease characterized by decreasing of bone mass and degeneration of micro-structure, leading to increase of bone brittleness and easily to become fractured. Osteoporosis is a disease associated with age, the larger scale of old population, the more morbidity of this disease. Osteoporosis will apparently affect the life quality and healthy of old people. China has a large population of old people, with the fast increasing of aging, and with high morbidity of osteoporosis. Despite of the existing of large variety of anti-osteoporosis drugs, all of them have some disadvantages, such as side reaction, little treatment effects, high costs and so on. Recent epidemiology and related researches indicated that low dose of long-term oral taking of aspirin can significantly help elevating bone mass of old people, indicating that aspirin may have some potential effects on anti- Osteoporosis. Aspirin is a frequently used medicine for old people, not only has little side effect, cheap, but can also cure variety of other diseases. It will be clinically significant to investigate the effects of aspirin on osteoporosis, which may offering the theory basis for the clinical therapy of Osteoporosis. Objective:To investigate the effect of aspirin on differentiation of bone marrow mesenchymal stem cells (BMSCs) into osteoblast in vitro, and offer theory basis to further study on the aspirin treatment of osteoporosis.Methods:The tibias and femurs of 4 weeks old SD rat were dissencted on germfree conditions. Bone marrow was extracted and made into single cell suspension. Bone marrow mesenchymal stem cells were cultured through density gradient centrifugation and whole marrow adherence method. Stable generation of bone marrow mesenchymal stem cells (passage 3-6 ) were obtained and applies in the experiment. The cells were induced to osteogenic differentiate in osteogenesis induction medium, without or with different aspirin concentration (0.5, 1, 2, 5,10mmol/L). The experimental groups were divided into low, medium, high aspirin concentration group, in addition to the control group. The morphologic changes of cultured cells were evaluated. The BMSCs proliferation capacity was assayed by CCK-8 method, and the cell growth curve was generated. The osteogenic differentiation markers of the BMSCs were analyzed, which including alkaline phosphatase (ALP) activity and secreted osteocalcin (OC), with enzyme-labeled immunosorbent assay (ELISA) method. The osteogenesis effects of different groups were evaluated by mineralized bone modulus and ALP staining, and the difference was compared through semi-quantitative analysis.Results:We combined density gradient centrifugation and whole marrow adherence method s to generate bone marrow mesenchymal stem cells from the bone marrow mono-nuclear cells. Purified, stable bone marrow mesenchymal stem cells were obtained and applied in the experiment. Primary passage cells are roundish, short spindle, polygonal or irregular shape. And the cell colonys were in different shape and size. Passaged cells have larger size and growth proceed rapidly, presenting spiral shape mostly. The passaged cells are characterized with spindle, trigonal, polygonal shape, and the cell nucleus are oval or circular. From the cell growth curve, we concluded that aspirin could not stimulate the proliferation of bone marrow mesenchymal stem cells, while high concentration of aspirin (>5 mmol/L) had the suppressive effect on BMSCs. Low and medium concentration of aspirin (0.5, 1, 2 mmol/L) stimulated the differentiation of BMSCs into osteogenic cell linages. In different phases, alkaline phosphatase activity and osteocalcin in low and medium concentration of aspirin groups were higher than that of the control group. Alizarin red staining for calcium nodus in aspirin groups was stronger than that in control group in 14 days. Alkaline phosphatase staining showed that cell's osteogenic activity in aspirin groups were significantly higher than control groups.Conclusions:Low and medium concentration of aspirin stimulated the osteogenic differentiation of BMSCs, indicating that aspirin had the ability to enhance the bone metabolism activity. It was suggested that aspirin has the potential of treating osteoporosis through stimulating the osteogenic differentiation of BMSCs, and it may become a novel drug for anti-osteoporosis.