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[Interventional effect of platelet lysate on osteogenic differentiation of BMSCs in rats].

OBJECTIVE
To explore the interventional effect of platelet lysate (PL) on osteogenic differentiation of BMSCs by induction in rats in vitro.


METHODS
Twenty-four clean-grade adult Wistar rats, weighing from 250 g to 300 g, male or female, were included in this study. PL was obtained through three times of centrifugation and repeated freeze-thaw for the blood aspirated from cardiac cavities in 16 Wistar rats. ELISA assay was conducted to detect the concentration of growth factors PDGF, TGF-beta1, IGF-1 and VEGF in PL. The BMSCs harvested by flushing femurs of 8 adult Wistar rats were isolated, cultivated and expanded in vitro. The cells at the 4 passage were performed for osteogenic differentiation by induction in three groups of A (5% PL of final concentration in basic induction medium), B (1% PL of final concentration in basic induction medium), and C (no presence of PL in basic induction medium as a control). The morphological changes of the cells were dynamically observed with inverted phase contrast microscope during the whole period. At different time-points, ALP staining (7 days) and ALP/TP (2, 8, 12 days) of the cells were detected to evaluate ALP activity, and the mineral formation in extracellular matrix was examined with Alizarin red staining which provided quantitative analysis of mineral deposits.


RESULTS
ELISA assay showed that the content of PDGF, TGF-beta1, IGF-1 and VEGF in PL reached (300 +/- 30), (140 +/- 25), (80 +/- 35), (70 +/- 20) pg/mL, respectively. Morphological observation displayed BMSCs in group A or B gradually turned from spindle-shape to square- or polygon-shape as the morphological type of osteoblast-like cells at 7 days. The cells in group A showed slower shape changes but higher proliferation than that in group B or C. Moreover, at the 20 days, the cells in group A still displayed dense growth and produced obviously decreased amount of mineral deposits in ECM when compared with group B or C. At the 7 days, the cells of group A showed smaller amount of granules positive for ALP staining in cytoplasm when compared with groups B and C, and displayed marked reduction in ALP activity assay at the 2, 8, and 10 days compared with that of groups B and C (P < 0.05). At the 20 days, Alizarin red staining showed the number of mineral deposits in groups A, B and C were 7.67 +/- 1.10, 12.87 +/- 0.81 and 15.59 +/- 0.25, respectively, while the area of mineral deposits were (161 778.70 +/- 44 550.80), (337 349.70 +/- 56 083.24), and (415 921.70 +/- 71 725.39) pixels, respectively. The number of mineral deposits and the area of mineral deposits in group A were smaller than those in groups B and C (P < 0.05). But there was no statistically significant difference between groups B and C (P > 0.05).


CONCLUSION
PL is a kind of system carrying various growth factors. Exposure of PL inhibits both ALP activity and mineral formation of BMCs in a dose-dependent way under the osteogenic induction environment.