F-actin Rearrangement And Its Relative Study Of Bone Marrow Stromal Cells(MSCs) Directed By The Rate Of Unidirectional Fluid Shear Stress

There is growing concern for about the effects of fluid shear stress on the regulation of bone marrow stromal cells(MSCs) differentiation. Our previous study has shown that the rate of fluid shear stress regulates the differentiation and cytoskeleton F-actin rearrangement of MSCs. Based on this, we studied the contribution of cytoskeleton F-actin in MSCs differentiation during stimulation by different rate of fluid shear stress. Further we examined the modulation of mechanosensitive, cation-selective channels effects on F-actin reorganization when MSCs were exposed to different rate of fluid shear stress. The main contents and results were as follows:MSCs were from SD rats. The MSCs were treated by Phalloidin and three different rate increase of unidirectional fluid shear stress(linear gradient elution from 0 to 10 dyn/cm2 within 0 min(0-0'), 2 min(0-2') and 20 min(0-20'), respectively.) were loading for 20 min in the flow chamber which designed by our team. After loading, instantaneously observe and quantitative the cytoskeleton F-actin polymerization by fluorescent staining. The results showed that cytoskeleton F-actin in Phalloidin inhibited groups are higher than the static control groups, but are less than the groups without Phalloidin pretreatment under the same loading condition. It also shown that Phalloidin cannot completely inhibit F-actin rearrangement in the loading conditions. In conclusion, different linear increase of fluid shear stress regulated the MSCs cytoskeleton F-actin rearrangement, and the loading way of 0-2' make the cytoskeleton F-actin had a largest polymerization degree. By contrast, the loading way of 0-20' had a smallest effect on the polymerization degree of cytoskeleton F-actin.Base on the results above, the roles of cytoskeleton F-actin in regulating the MSCs differentiation when exposed to different linear increase of fluid shear stress were investigated, the differentiation markers of osteogenic(alkaline phosphatase, ALP) and chondrocyte(glycosaminoglycan, GAG) were determined. Results showed that, three kinds of loading ways applied to MSCs for 20 min and 48 h static cultivation, the groups in Phalloidin inhibition experimental results consistent with the conclusion our laboratory have got, the secretion levels of ALP in 0-2' experimental group was largest, and had a significant difference compared with other groups including the static control groups. However, the secretion levels of GAG in 0-0' experimental group was largest, and had a significant difference compared with other groups including the static control groups. So we confirm that the loading way of 0-2' benefit to differentiation of MSCs to ossification cell line and the loading way of 0-0' promoted differentiation of MSCs to chondrocyte cell line. However, the quantitative results of ALP and GAG of three kind of mechanical loading group in Phalloidin inhibited groups have no significant differences. This indicated that different linear increase of fluid shear stress regulate the recombination of cytoskeleton F-actin and effect the differentiation tendency of MSCs ossification and chondrocyte.The important role of cytoskeleton F-actin in the process of regulating the MSCs differentiation when exposed to different rate increase of unidirectional fluid shear stress were examined. In this part, we also use Phalloidin as cytoskeleton F-actin fixative to pretreat MSCs and three kinds of mechanical loading for 20 min, fresh medium containing Phalloidin or not static cultivation for 48 h, respectively. After that, the levels of ALP and GAG were tested. Results showed that, in the process of static cultivation, no matter Phalloidin containing or not, the results that all quantitative of ALP and GAG in each loading group have no significant differences. The results illustrated that the Phalloidin have no effect on the cell ALP and GAG secretion. This validated the conclusion we get before, different rate increase of unidirectional fluid shear stress by influencing the cytoskeleton F-actin reorganization and regulation the differentiation trend of MSCs ossification and chondrocyte.Previous research has found that mechanosensitive, cation-selective channels and cytoskeleton F-actin rearrangement have a close relationship. But whether it related to the differentiation of MSCs and whether the channels regulated MSCs differentiation by F-actin rearrangement is not yet known. In this context, mechanics sensitive calcium channel blockers(Gadolinium, GdCl3) to pre-treatment of MSCs before the loading, 20 minutes of mechanical loading and 48 hours static training, the expression of differentiation markers of osteogenic and chondrogenic were tested. Comparing the experimental group with blockers pretreatment or not of the expression of differentiation markers. We found that when the MSCs under the stimulation of different rate increase of unidirectional fluid shear stress, cytoskeleton F-actin rearrangement depended on intracellular calcium levels caused by mechanosensitive, cation-selective channels different degree of opening.In conclusion, different rate increase of unidirectional fluid shear stress through mechanosensitive, cation-selective channels regulation cytoskeleton F-actin reorganization futher influence the MSCs differentiation. The research preliminary reveals the mechanism that MSCs differentiation and regulation under fluid shear stress. It could be a reference to the bone/cartilage damage repair.