Mesenchymal stem cells (MSC) regulate their behavior by sensing mechano-environmental factors.
Accumulated evidence indicates that appropriate mechanical force including fluid shear stress enhances the osteogenic property of MSC on 3D polymeric scaffolds even without the presence of osteogenic cocktail (i.e., dexamethasone and beta-glycerophosphate). However, despite a common understanding of how cytoskeleton transmits mechanical stimuli, a detailed role of cytoskeleton in fluid flow-induced osteogenesis is not fully understood. The aim of the present study was therefore to assess cytoskeletal modulation under fluid force and then explore causal relationship with altered cell growth and flow-induced osteogenic differentiation by using a perfusion bioreactor.
MSC from Lewis rat bone marrow (rBMSC) were seeded on 3D microporous scaffolds made of Poly(L-lactide-co-trimethylene carbonate) and subjected to laminar flow excreting shear stress at 1 mPa on an average for 14 days in a perfusion bioreactor. Cytoskeletal modulation was assessed by, but not limited to, RT-qPCR array, cell morphological analysis, enzymic activity of Rho-associated protein kinase (ROCK), and level of phosphorylation of myosin light chain. Transcriptional profile of osteogenesis-related markers in rBMSC under fluid stimuli was compared with that induced by the osteogenic cocktails and a static control without osteo-inducement. To evaluate the role of cytoskeletal modulation in flow-induced osteogenesis, pharmacologic inhibition of cytoskeletal modulators, namely, Rho GTPases, ROCK, myosin light chain kinase and non-muscle myosin II ATPases, was performed to induce cell relaxation during perfusion culture. With the inhibitors, cell growth and osteogenic differentiation were further evaluated.
Under the fluid flow, rBMSC significantly altered the expression pattern of mRNA related to cell morphogenesis and focal adhesion including Pkt2, Prkca, Rock1 and Rock2. This was accompanied with cell morphological alternation characterized by cell contraction and actin stabilizayion, and the enhanced phosphorylation of myosin light chain was observed. In such a condition, rBMSC upregulated a number of osteogenic markers including Runx2, Sp7, Col1a1, Bmp2, ALPL and Spp1. Interestingly, the mRNA expression pattern of osteogenic markers differed from dexamethasone-induced osteogenesis. The inhibition of cytoskeletal modulation cascade from Rho activation to actomyosin contraction at different levels suppressed the flow-induced upregulation of the osteogenic markers. Despite the fact that cell proliferation was suppressed by fluid flow, the inhibition aggravated cell growth even further while the inhibitors did not show a notable suppressive effect on proliferation in the static control.
The present study using a perfusion bioreactor demonstrated that rBMSC responded to a low level of fluid stimuli by cytoskeletal moduration, which was associated with altered cell growth and osteogenic differentiation on 3D polymeric scaffolds. The inhibition of cell contraction revealed that cytoskeletal modulation under fluid stimuli was required for maintaining the proliferative state while it directed rBMSC fate towards an osteogenic lineage.