Contribution of Interstitial Fluid Flow to Fibroblast Alignment and Differentiation

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Fibrosis is often seen in tissues following vessel hyperpermeability, interstitial fluid flow, and edema; examples include secondary lymphedema in skin and pulmonary edema. To determine whether the biophysical changes that interstitial flow induces can influence fibroblast behavior, we developed a tissue culture model of interstitial flow that can be chronically applied to 3‐D cultures. We use it to demonstrate that even very low interstitial fluid flows (of 1–10 microns/sec) can induce human dermal and lung fibroblasts embedded in type I collagen to align perpendicular to the direction of flow and align the collagen extracellular matrix fibers. We saw that attachment to collagen is necessary for alignment, since fibroblasts in fibrin gels do not align, and those grown in fibrin‐collagen mixtures align only weakly. Furthermore, fibroblast alignment corresponds to increased expression of alpha‐smooth muscle actin, signaling that the cells are undergoing differentiation to a fibrotic myofibroblast phenotype. Collagen fiber alignment is demonstrated using confocal reflectance microscopy and FFT analysis. Our results indicate that interstitial flow, which is increased in vivo during wound healing and many pulmonary disorders such as edema, may contribute to interstitial fibrosis. The results also suggest that interstitial flow can be used to develop in vitro models of interstitial fibrosis for studies of directed matrix remodeling and cell orientation.