Abstract
Interstitial fluid flow (IFF) within the extracellular matrix (ECM) produces low magnitude shear stresses on cells. Fluid flow-induced stress (FSS) plays an important role during tissue morphogenesis. To investigate the effect of low FSS generated by IFF on cells, we developed a microfluidic-based cell culture device that can generate multiple low shear stresses. By changing the length and width of the flow-in channels, different continuous low level shear stresses could be generated in individual cell culture chambers. Numerical calculations demonstrate uniform shear stress distributions of the major cell culture area of each chamber. This calculation is further confirmed by the wall shear stress curves. The effects of low FSS on MC3T3-E1 proliferation and differentiation were studied using this device. It was found that FSS ranging from 1.5 to 52.6 µPa promoted MC3T3-E1 proliferation and differentiation, but FSS over 412 µPa inhibited the proliferation and differentiation of MC3T3-E1 cells. FSS ranging from 1.5 to 52.6 µPa also increased the expression of Runx2, a key transcription factor regulating osteoblast differentiation. It is suggested that Runx2 might be an important regulator in low FSS-induced MC3T3-E1 differentiation. This device allows for detailed study of the effect of low FSS on the behaviors of cells; thus, it would be a useful tool for analysis of the effects of IFF-induced shear stresses on cells.
Highlights
Interstitial fluid, flowing within the interstitium of the extracellular matrix (ECM), transports nutrients and signaling molecules between blood vessels, lymphatic capillaries, and ECM
Delaine-Smith et al reported that alkaline phosphatase (ALP) activity, collagen production and calcium deposition were enhanced in human progenitor dermal fibroblasts by flow-induced shear stress (FSS) when these cells were cultured in osteogenic media [17]
Chip Design The microfluidic device developed in this study comprises four cell culture chambers connected with individual flow-in and flowout channels (Fig. 1a, b)
Summary
Interstitial fluid, flowing within the interstitium of the extracellular matrix (ECM), transports nutrients and signaling molecules between blood vessels, lymphatic capillaries, and ECM. Mechanical loading and bending of bones can move the interstitial fluid, which applies shear stresses on osteoblasts and osteocytes. Hayward et al found that interstitial fluid velocity and tissue shear stress are key mechanical stimuli for the differentiation of skeletal tissues. Another in vivo study demonstrated that increased IFF inhibited bone loss in hindlimb-suspended mice [15]. Regarding the signaling pathway regulating FSS-induced osteoblast differentiation, You et al demonstrated that the expression of osteopontin, an important bone matrix protein, was regulated by FSS via intracellular calcium mobilization and activation of mitogen-activated protein kinase (MAPK) in MC3T3-E1 cells [19]. Runx is an essential transcription factor for osteoblast differentiation and bone formation; Runx2-deficient mice completely lack bone formation due to the arrest of osteoblast maturation [21,22]
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