The present results found that
The present results found that under osteogenic induction, hydrostatic pressure promoted the transketolase of marker genes for early osteogenic differentiation in BMSCs through the activation of RhoA/ROCK. It was reported that the activated RhoA/ROCK proteins enhanced myosin motor activity through an increase in the level of myosin light chain (MLC) phosphorylation and the inhibition of MLC phosphatase (Amano et al., 1996), which ultimately promoted cytoskeletal assembly. Our results were consistent with the observations of McBeath et al. who had reported that the activation of RhoA/ROCK proteins promoted cytoskeletal assembly, resulting in a well-spread and flattened cell morphology and a differentiation toward the osteoblast lineage (McBeath et al., 2004). In contrast, as it was suggested in earlier studies that, hydrostatic pressure could also simulate the compressive forces borne by the cartilage in the joint cavity well, which could provide a rather favorable biomechanical environment for tissue-engineered cartilage (Carter and Wong, 1988) and promote the differentiation of BMSCs toward the chondroblast lineage (Angele et al., 2003; Miyanishi et al., 2006a, 2006b) even in the absence of chondrogenic differentiation-inducing factors, such as transforming growth factor beta 3 (TGF-β3) (Miyanishi et al., 2006a, 2006b). Studies have also shown that the damage to the cytoskeletal structure (M. Zhang et al., 2006; Z. Zhang et al., 2006) and the inhibition of ROCK activity induced rounded cell morphologies and promoted the expression of chondrogenic marker genes in the BMSCs (Woods and Beier, 2006; Woods et al., 2005). These findings were consistent with our results, which showed that biomechanical stimulation promoted the chondrogenic differentiation process of BMSCs when the BMSC pellets cultured in chondrogenic induction media (at high densities and in suspension culture) were subjected to constant hydrostatic pressure for two or four weeks. Therefore, it could be speculated that the mechanical stimulation may be transduced through an inhibited RhoA/ROCK pathway. This would further decrease the level of MLC phosphorylation, inhibit cytoskeletal assembly, induce rounded cell morphologies and promote the chondrogenic differentiation of BMSCs. However, other studies have shown that Rac1 could inhibit the activity of the downstream protein myosin light-chain kinase (MLCK) through the activation of p21-activated kinase (PAK). This reduced the level of MLC phosphorylation and eventually affected cytoskeletal assembly (Sanders et al., 1999). The activation of Rac1 has also been shown to promote the expression of N-cadherin and ultimately enhance the transcription of Sox-5, Sox-6, Sox-9, Col-II and Aggrecan mRNAs (Woods et al., 2007). These findings suggested that in the present study, hydrostatic pressure may have induced N-cadherin activation, promoted the formation of intercellular connections between the BMSCs, and stimulated chondrogenic differentiation through the activation of Rac1. Our results have well established that RhoA and Rac1 have the potential of regulating stem cell fate via intrinsic mechanisms. To our knowledge, this is the first study showing that hydrostatic pressure could induce RhoA activation and further enhance the early osteogenic differentiation of BMSCs. However, hydrostatic pressure could also up-regulate Rac1 and down-regulate RhoA activities to further enhance the chondrogenic differentiation of the BMSCs. These findings suggest that further studies will be required to determine the complex initial biological mechanisms of bone or cartilage formation and regeneration in response to mechanical stimuli.
Conclusions This study provides new insight to the regulation of the proliferation and the osteogenic/chondrogenic differentiation of BMSCs by hydrostatic pressure. RhoA and Rac1 play important regulatory roles in the pressure-promoted proliferation and differentiation and in F-actin stress fiber assembly and JNK1/2 activation in the BMSCs (Fig. 6). This study showed that the application of hydrostatic pressure can be adopted as an effective stimulation method for inducing the biological activity of BMSCs and that RhoA and Rac1 can be used as effective modulating molecules for controlling the mechanobiological response of BMSCs.