Unique osteogenic profile of bone marrow stem cells stimulated in perfusion bioreactor is Rho-ROCK-mediated contractility dependent

by Selleckchem | Sep 14 2023

The fate determination of bone marrow mesenchymal stem/stromal cells (BMSC) is tightly regulated by mechanical cues, including fluid shear stress. Knowledge of mechanobiology in 2D culture has allowed researchers in bone tissue engineering to develop 3D dynamic culture systems with the potential for clinical translation in which the fate and growth of BMSC are mechanically controlled. However, due to the complexity of 3D dynamic cell culture compared to the 2D counterpart, the mechanisms of cell regulation in the dynamic environment remain relatively undescribed. In the present study, we analyzed the cytoskeletal modulation and osteogenic profiles of BMSC under fluid stimuli in a 3D culture condition using a perfusion bioreactor. BMSC subjected to fluid shear stress (mean 1.56 mPa) showed increased actomyosin contractility, accompanied by the upregulation of mechanoreceptors, focal adhesions, and Rho GTPase-mediated signaling molecules. Osteogenic gene expression profiling revealed that fluid shear stress promoted the expression of osteogenic markers differently from chemically induced osteogenesis. Osteogenic marker mRNA expression, type 1 collagen formation, ALP activity, and mineralization were promoted in the dynamic condition, even in the absence of chemical supplementation. The inhibition of cell contractility under flow by Rhosin chloride, Y27632, MLCK inhibitor peptide-18, or Blebbistatin revealed that actomyosin contractility was required for maintaining the proliferative status and mechanically induced osteogenic differentiation in the dynamic culture. The study highlights the cytoskeletal response and unique osteogenic profile of BMSC in this type of dynamic cell culture, stepping toward the clinical translation of mechanically stimulated BMCS for bone regeneration.

Comments:

The present study investigated the effects of fluid shear stress on bone marrow mesenchymal stem/stromal cells (BMSC) in a 3D culture condition using a perfusion bioreactor. The researchers aimed to understand the cytoskeletal modulation and osteogenic profiles of BMSC under fluid stimuli, as well as the role of actomyosin contractility in the dynamic culture system.

The results demonstrated that BMSC subjected to fluid shear stress exhibited increased actomyosin contractility. This was accompanied by the upregulation of mechanoreceptors, focal adhesions, and Rho GTPase-mediated signaling molecules. These findings suggest that mechanical cues, specifically fluid shear stress, play a crucial role in regulating the cytoskeletal structure and signaling pathways involved in cell mechanotransduction.

Furthermore, the study explored the osteogenic gene expression profiling of BMSC under fluid shear stress. It was observed that fluid shear stress promoted the expression of osteogenic markers in a manner distinct from chemically induced osteogenesis. Osteogenic marker mRNA expression, type 1 collagen formation, alkaline phosphatase (ALP) activity, and mineralization were enhanced in the dynamic culture condition, even in the absence of chemical supplementation. These results indicate that the mechanical stimulation provided by fluid shear stress can induce osteogenic differentiation and enhance bone-forming properties of BMSC.

To understand the role of actomyosin contractility in the dynamic culture system, the researchers inhibited cell contractility using various compounds. The inhibition of actomyosin contractility impaired the proliferative status and mechanically induced osteogenic differentiation of BMSC under fluid shear stress. This suggests that actomyosin contractility is necessary for maintaining the proliferative state of BMSC and mediating the mechanically induced osteogenic response in the 3D dynamic culture.

Overall, this study provides valuable insights into the cytoskeletal response and unique osteogenic profile of BMSC in a 3D dynamic cell culture system. These findings contribute to the understanding of how mechanical cues can regulate the fate and growth of BMSC, which has implications for bone tissue engineering and the clinical translation of mechanically stimulated BMSC for bone regeneration.