Effects Of L-type Calcium Channels On Rat Bone Mesenchymal Stem Cells And Osteogenic Differentiation In Vitro

Bone mesenchymal stem cells(BMSCs), as seed cells, has become increasingly depth in bone tissue engineering research. So how to abtain a large number of good functional BMSCs, and how to motivate osteogenic differentiation of BMSCs are hotspots at present. There are many facts affecting the growth and osteogenic differentiation of BMSCs in vitro conditions, such as serum, nutrient medium, cell density, hydrogenion concentration(pH), oxygen concentration, temperature, humidity, shearing force, growth factor, etc. L-type calcium channels are voltage-gated calcium channels allowing Ca2+ to enter in to cells, which are involved in cell proliferation and cell functional activity. Research has showed that there are L-type calcium channels on osteoblasts, and these channels are involved in osteoblasts proliferation and functional activity. Osteoblasts derived from BMSCs, then, we hyhothesized that L-type calcium channels may also be involved in the proliferation and osteogenic differentiation of BMSCs.By investigating response of rat bone mesenchymal stem cells (rBMSCs) and osteogenic differentiation of it after blocking L-type calcium channels, we can observe the effects of L-type calcium channels on growth and osteogenic differentiation of BMSCs. We emphasized in the following aspects:(1) Sprague Dawled rats bone mesenchymal stem cells were obtained by using whole marrow attachment method in vitro. The method of osteogenic/adipogenic differentiation of rBMSCs was employed for identification. Result: after 7 days of adipogenic differentiation of third-generation cells, Oil red staining showed that there were obvious red cell lipid droplets in cells. After 21 days of osteogenic differentiation, alizarin red staining showed the apparent extracellular matrix mineralization nodules. The results showed that the cells we abtained were BMSCs which had multilineage differentiation potential.(2) RT-PCR was used to detect the mRNA expression of calcium channels BMSCs. Result: a high expression of mRNA in alC, CCHL2a, alG, CACNA1 was detected by RT-PCR. The result confirmed that there were L-type calcium channels on Sprague Dawled rats marrow mesenchymal stem cells, as well as, we also discovered the T- type calcium channels on the BMSCs.(3) The L-type calcium channel blocker, nifedipin, was use to block the L-type calcium channels. Methyl thiazol tetrazolium(MTT) assay and flow cytometry were applied to measure the cellular proliferation and cellular cycle of rBMSCs. Result: the cells in test group proliferated slower than those in the control group after blocking L-type calcium channels. There was significant difference in two groups after continuouse cultured for 3 days. After 72 hours stimulation, the percentage of phase S(19.22±4.91)% was decreased compared with the control group(45.33±8.64)%, There was significant difference between two groups. The result indicated that L-type calcium channels blocker could inhibit DNA synthesis of cells and then inhibit cell proliferation.(4) The activity of alkaline phosphatase (ALP) and content of calcium nodules was measured to evaluate the ability of osteogenic differentiation of BMSCs. Result: after blocking L-type calcium channels for 1, 3, 5 days, activity of ALP was also decreased significantly compared with the control group, there were significant differences between two groups. After osteogenic differentiation of rBMSCs for 21 days, the calcification rate of test group(4.29±2.24) % was decreased compared with the control group(23.66±5.52) % after stimulation of Nif. So, the result suggest that L-type calcium channels may be involved in osteogenic differentiation of bone mesenchymal stem cells.