Shear stresses modulate chondrogenic and osteogenic genes expression in human bone marrow derived stem cells

Abstract

Background: Mechanical stimuli in the microenvironment may provide cues for differentiation, maintenance of phenotypes, and extracellular matrix homeostasis. This study investigated bone marrow-derived stem cells (BMSCs) response to mechanical stresses in terms of differentiation and the extracellular matrix/matrix metalloproteinase genes expression. Materials & Methods: Human BMSCs were exposed to shear stresses (20 rpm or 50 rpm) for 2, 5, or 10 days with or without osteogenic induction medium. The expression of chondrogenic (type II collagen and aggrecan) and osteogenic (type I collagen, alkaline phosphatase, osteopontin, osteocalcin) phenotypic genes and matrix metalloproteinase genes (aggrecanse, MMP-2, and MMP-9) were analyzed by reverse transcription polymerase chain reaction. In vitro differentiation of BMSCs after shear stress treatment were analyzed by von Kossa staining on monolayer cultures and safarin-O staining on pelleted cells. Results: Shear stress at 20 rpm significantly upregulatedexpression of type II collagen on the 5th day, type I collagen on the 2nd, 5th, and 10th day, osteopontin on the 2nd and 5th day, and osteocalcin on the 5th day as compared to shear stress at 50 rpm. Combination of osteogenic induction medium and shear stress at 20 rpm decreased the mRNA expression levels of chondrogenic genes (type II collagen on all time points and aggrecan on the 5th and 10th day) as compared with shear stress alone. It also decreased the expression of osteogenic genes (type I collagen, alkaline phosphatase, and osteopontin) on the 10th day as compared to osteogenic induction medium. Concomitantly, shear stress upregulated aggrecanase on the 10th day, MMP-2 on the 10th day, and MMP-9 on the 5th and 10th day as compared with osteogenic induction medium. Shear stress increased mineralization of the extracellular matrix as shown by von Kossa staining on the 2nd, 5th, and 10th day. Chondrogenic differentiation by cell pellet analysis found similar amount of proteoglycans but less organized extracellular matrix in shear stress-treated BMSCs as compared to control cells. Discussion: Although shear stress could modulate chondrogenic gene expression as well as osteogenic genes expression in BMSCs, the combination of shear stress inhibited the expression of osteogenic phenotypic genes induced by osteogenic induction medium. Parallel studies revealed that shear stress concomitantly modulate the expression of extracellular matrix genes and the matrix metalloproteinase genes. In vitro differentiation analysis found that shear stress may enhance osteogenesis but interfere with chondrogenesis. Our results may provide data for the design or development of in vitro bioreactor systems to control lineage differentiation of stem cells for tissue engineering. More studies on the mechanotransduction pathways are needed in the future.