Platelet lysate promotes proliferation and angiogenic activity of dental pulp stem cells via store-operated Ca2+ entry

Abstract

Aims

Dental pulp stem cells (DPSCs) had been widely used in nerve tissue engineering. However, the low cell survival and invalid differentiation were urgent problems to be solved for cell-based therapy. Platelet lysate (PL) had attracted attentions for its good biocompatibility, low side effects and abundant growth factors. In this study, we aimed to evaluate the effect and mechanism of PL on the proliferation and angiogenesis of DPSCs.

Materials and methods

In our study, PL was prepared by repeated freeze-thaw methods. The effect of PL on the viability of DPSCs was screened by the Cell Counting Kit-8 (CCK-8) and Live/Dead assays. Then the store-operated Ca²⁺ entry (SOCE) agonist endothelin-1 (ET-1) and inhibitor 2-aminoethoxydiphenyl borate (2-APB) were used to clarify the biological functions of PL on DPSCs. We detected the mitosis of DPSCs by KI67 immunofluorescence staining. Moreover, Ca²⁺ influx in DPSCs was evaluated by fluorescence microscopy and a flow cytometry. The expression of p-SRC, VEGFA and CD31 as well as the number of formed tubules in each group were investigated, so as to further reveal the influence of PL on the DPSCs angiogenic activity.

Results

As for CCK-8 and Live/Dead assays, the results indicated the 0.5% concentration of PL was better than that of 20 ng/ml bFGF and 10% FBS in promoting cell survival within 24 h. KI67 staining showed that 0.5% PL promoted SOCE and cell proliferation. The expression levels of SRC phosphorylation (p-SRC) were increased in the 0.5% PL and ET-1 + 0.5% PL groups, as well as the expressions of endothelial markers CD31 and VEGFA were also increased in the ET-1, 0.5% PL and ET-1 + 0.5% PL groups. However, CD31 and VEGFA were not detected in 2-APB and 2-APB + 0.5% PL groups. After angiogenesis induction, capillary-like structures were observed in ET-1, 0.5% PL and ET-1 + 0.5% PL groups, whereas no vascular structures were observed in 2-APB and 2-APB + 0.5% PL groups.

Conclusion

Our results indicated that PL promoted the proliferation and vessel-formation of DPSCs via the activation of SOCE and the upregulation of p-SRC and VEGFA expressions, respectively. PL might act as a promising cell supplement in DPSCs survival and differentiation, furtherly applied for the repair and regeneration of peripheral nerve injury.