Effects of Platelet-Rich Plasma on BMSCs Differentiation Into SC in Vitro
Objective: To explore effect of platelet-rich plasma (PRP) on rabbit BMSCs differentiation into SC in vitro and to detect secretory function of the differentiated cells.
Methods: BMSCs isolated from 5 mL bone marrow of 2-month-old New Zealand white rabbit were cultured using density gradient centrifugation and adherence screening methods. A total of 5 mL femoral vein blood was obtained from rabbits to prepare PRP using modified Appel method. The BMSCs at passage 3 were divided into three groups: the combined induction group, in which the cells were cultured with complete medium containing PRP after beta-mercaptoethanol and retinoic acid inductions; the simple induction group, in which the cells were cultured with L-DMEM complete medium without PRP after beta -mercaptoethanol and retinoic acid induction; the control group, in which the cells were cultured with L-DMEM complete medium. Growth condition of the cells in each group was observed using inverted microscope. cell identification was conducted at 4, 7, 9, and 11 days after culture using immunofluorescence staining method, and NGF content was detected by ELISA method. NGF mRNA expression was assayed by RT-PCR 11 days after culture.
Results: Most cells in the combined induction and the simple induction group were out of BMSCs typical cell morphology 4 days after culture; cells in the combined induction group were out of BMSCs typical cell morphology and changed into cells resembling SC in terms of morphology and contour 9 days after culture. The cells in the control group showed no obvious morphological changes. S-100 protein expression in the cells was evident in the combined induction and the simple induction group at each time point after induced culture; the positive expression rate of cell in each group was increased over time, and significant differences were evident between the combined induction group and the simple induction group 7, 9, and 11 days after culture (P < 0.05). Control group was negative for the expression. There were significant differences when comparing the control group with the combined induction group or the simple induction group in terms of NGF content at each time point (P < 0.01). Significant difference was evident between the combined induction group and the simple induction group 7, 9, and 11 days after culture (P < 0.05), and no significant difference was noted 4 days after culture (P > 0.05). Relative intensity of NGF mRNA expression in the combined induction group was greater than that of the simple induction group 11 days after culture (P < 0.05).
Conclusion: Rabbit BMSCs can differentiate into SC excreting NGF under certain induction condition in vitro. PRP can remarkably promote BMSCs differentiation into SC.
