Summary

Stem cell is an important kind of seed cell in cell therapy and tissue engineering. Studies have shown that mechanical stimulation may play a role in regulating proliferative activity, delaying senescence, and maintaining differentiation potential to stem cell. Effect of extracorporeal shock wave on proliferation, senescence and differentiation ability to vascular endothelial cell and Schwann cells of rat mesenchymal stem cells is unknown.

Abstract

Purpose

The purpose of this study was to research the effect of extracorporeal shock wave on proliferation, senescence and differentiation ability to vascular endothelial cells and Schwann cells of rat mesenchymal stem cells

Methods

The rat bone marrow mesenchymal stem cells (BMSCs) were harvested and identified. The stem cells cultured in vitro were divided into 5 groups as A, B, C, D and E. The cells were treated with radial shock waves (0.4 bar, 3Hz, and 600 shocks) 0 to 4 times, respectively. Every group of cells were passaged every 4 days until the 16th passage. The cell proliferation was analyzed by CCK-8 and flow cytometry after treated by shock wave. After shock wave treated, the cells in each group were stained by β-galactosidase and the activity of β-galactosidase was determined by spectrophotometry; The expresion of CD34, CD45, CD90 and CD105 were measured by flow cytometry; The cells were stained by caspase and quantitatively analyzed; Annexin V / PI was used to quantitatively analyze the ratio of apoptotic / necrotic cells. The 16th passage cells treated with clasical chemical stimulation were induced to vascular endothelial cells and Schwann cells. The Vascular endothelial cells and schwann cells differentiation ability were evaluated by specific antibody fluorescence staining and flow cytometry, respectively.

Results

The radial shock wave (0.4bar, 3Hz, 600 shocks) could promote BMSCs proliferation. Compared with the untreated group, multiple ESW treatment can maintain BMSCs with a high proliferative activity (P <0.05), prevent BMSCs proliferation capacity decline seriously in vitro. β-galactosidase activity increased with the passages, and the activity of β-galactosidase of more than 3 times of ESW treated group was significantly lower than that of untreated group (P <0.05). After cultured in vitro, the positive expresion of CD34 and CD45 was increased, and the positive rate of CD90 and CD105 was decreased. Many times of ESW treated could delay this change, the expresion of CD molecule on cell surface was closer to that of primary. The OD value of caspase fluorescence in ESW treated more than 3 times group was lower. The percentage of cells in ESW treated more than 3 times group that had apoptosis / necrosis was lower than that of the control group without ESW treated (P <0.05). VEGF-R1, VEGF-R2 and CD34-positive vascular endothelial cells were analyzed by immunofluorescence staining. Flow cytometry analysis showed that the positive rate of specific antibody staining in group D and group E was significantly higher than that in ESW untreated group (P <0.05). The S100β, P75 and GFAP positive and P0-negative Schwann cells were observed by immunofluorescence staining. The positive rate of S100β and P75 staining in group D and group E was significantly higher than in ESW untreated group (P <0.05).

Conclusion

Extracorporeal shock wave (0.4bar，3Hz，600 shocks) can promote the proliferation of BMSCs, to inhibit the aging, apoptosis and necrosis of BMSCs in vitro. The differentiation ability to vascular endothelium and Schwann cells of the 16th passage BMSCs become weaker. The shock wave intervention can keep the differentiation ability at a high level.