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Objective To investigate the effect and mechanism of up-regulating calcium-sensing receptor (CaSR) on osteogenic differentiation of complex culture with porous tantalum-bone marrow mesenchymal stem cells (BMSCs). Methods BMSCs were extracted from SD rats and cultured to the third passage, and identified by alizarin red staining and toluidine blue staining after osteogenic and chondrogenic induction. The growth and adhesion of BMSCs on porous tantalum surface were observed by fluorescence microscope. CCK-8 assay was used to detect the proliferation of BMSCs cultured at different concentrations (200, 250, 300, 350, 400 μmol/L) of GdCl3 for 1-5 d, and the optimal concentration was selected. Confocal laser scanning microscopy was used to observe the intracellular calcium distribution after activation of CaSR. The third-generation of BMSCs were divided into control group (with osteogenic induction medium), GdCl3 group (with osteogenic induction medium containing 300 μmol/L GdCl3), porous tantalum group (with domestic porous tantalum material, with osteogenic induction medium) and GdCl3+porous tantalum group (with domestic porous tantalum material, with osteogenic induction medium containing 300 μmol/L GdCl3). At the 7th day of culturing, alkaline phosphatase (ALP) activity was measured in each group. On the 7th, 14th and 21st day of culturing, the protein secretion levels of type I collagen (ColⅠ) and osteopontin (OPN) were detected by ELISA, and the protein expression levels of ColⅠ, OPN, Runt-related transcription factor 2 (Runx2) and Homer 1 were detected by Western blotting. Results Alizarin red and toluidine blue staining showed that the isolated and extracted cells were BMSCs; CCK-8 assay showed that 300 μmol/L GdCl3-treated cells had the highest activity (P<0.05); confocal laser scanning microscopy showed that the intracellular calcium content was significantly increased after activation of CaSR (P<0.05). ALP activity on day 7 and relative expression levels of ColⅠ, OPN, Runx2 and Homer1 protein on days 7, 14 and 21 in GdCl3 group, porous tantalum group and GdCl3+porous tantalum group were higher than those in control group (P<0.05). ALP activity and relative expression levels of ColⅠ, Runx2 and Homer1 protein on day 7 were higher in GdCl3+porous tantalum group than those in GdCl3 group and porous tantalum group; relative expression levels of ColⅠ, OPN, Runx2 and Homer1 protein on day 14 were higher than those in GdCl3 group; relative expression levels of ColⅠ, OPN, Runx2 and Homer1 protein on day 21 were higher than those in porous tantalum group, the secretion levels of ColⅠ on days 7, 14, and 21, and OPN on days 7 and 21 were higher than those in GdCl3 group, porous tantalum group and GdCl3 + porous tantalum group (P<0.01). The secretion levels of ColⅠ on day 21 and OPN on day 14 in GdCl3 group were higher than those in control group (P<0.05). The secretion of OPN protein was higher in GdCl3 group than in control group on the 21st and 14th day of culture (P<0.05). Conclusions After co-culture of porous tantalum with BMSCs, up-regulation of CaSR may promote osteogenic differentiation of BMSCs by activating the expression of Col I, Runx2 and OPN.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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