Effects of Low-Intensity Pulsed Ultrasound on the Proliferation of Human Umbilical Cord Mesenchymal Stem Cells and Their Chondrogenic Differentiation via Bmp2 /Smad Signaling Pathway

BackgroundAlthough Low-intensity pulsed ultrasound (LIPUS) regiment has been applied to the treatment of cartilage injury clinically, the effect of LIPUS stress loading on the proliferation and differentiation of hUCMSCs is still controversial. Yet the specific mechanical mechanism of LIPUS stimulates cartilage differentiation of hUCMSCs has not been clarified.ObjectiveTo investigate the effects of low intensity pulsed ultrasound (LIPUS) on proliferation and chondrogenic differentiation of human umbilical cord mesenchymal stem cells (hUCMSCs) via BMP2 /smad signaling pathway in vitro.MethodThe hUCMSCs at P4 were randomly divided into control group(no LIPUS), L30 group (30mW/cm2), L50 group (50mW/cm2), L80 group (80mW/cm2), each LIPUS group respectively loaded 5, 10, 20 min/d, the cell proliferation activity was detected by CCK-8 assay after continuous irradiation for 5 days. For chondrogenic differentiation, hUCMSCs were divided 4 groups as follows: control group, Noggin group, LIPUS group, Noggin+LIPUS group, LIPUS irradiation was given 50w/cm2, 5min/d. Cartilage formation after 3 weeks was evaluated by Alician blue staining for GAG and COL II immunohistochemical staining. Furthermore, RT-PCR analysis of Sox-9, COL II Aggrecan, BMP2, Smad1, Smad5 and Smad9. Result： (1) The proliferation activity of L50 group(50w/cm2 , 5min/d) was noticeably higher compared with other groups, Alcian blue staining of GAG , COL II fluorescent staining in the LIPUS group were much stronger, quantitative RT-PCR assays showed that the expression of COL II, GAG, Sox-9, smad1, smad5 and smad9 in LIPUS group was significantly increased. Conclusion：The suitable LIPUS irradiation can promote cell proliferation, 50mW/cm2 intensity at 5min/d is most significant. And it can promote chondrogenesis of hUCMSCs via upregulating MP2 /smads signaling pathway.