Whole cell mechanics of contractile fibroblasts: relations between effective cellular and extracellular matrix moduli.

While much is known about the subcellular structures responsible for the mechanical functioning of a contractile fibroblast, debate exists about how these components combine to endow a cell with its form and mechanical function. We present an analysis of mechanical characterization experiments performed on bio-artificial tissue constructs, which we believe serve as a more realistic testing environment than two-dimensional cell culture. These model tissues capture many features of real tissues with the advantage that they can be engineered to model different physiological and pathological characteristics. We study here a model tissue consisting of reconstituted type I collagen and varying concentrations of activated contractile fibroblasts that is relevant to modelling different stages of wound healing. We applied this system to assess how cell and extracellular matrix (ECM) mechanics vary with cell concentration. Short-term and long-term moduli of the ECM were estimated through analytical and numerical analysis of two-phase elastic solids containing cell-shaped voids. The relative properties of cells were then deduced from the results of numerical analyses of two-phase elastic solids containing mechanically isotropic cells of varying modulus. With increasing cell concentration, the short-term and long-term tangent moduli of the reconstituted collagen ECM increased sharply from a baseline value, while those of the cells decreased monotonically.

Caveolar endocytosis is critical for BK virus infection of human renal proximal tubular epithelial cells.

In recent years, BK virus (BKV) nephritis after renal transplantation has become a severe problem. The exact mechanisms of BKV cell entry and subsequent intracellular trafficking remain unknown. Since human renal proximal tubular epithelial cells (HRPTEC) represent a main natural target of BKV nephritis, analysis of BKV infection of HRPTEC is necessary to obtain additional insights into BKV biology and to develop novel strategies for the treatment of BKV nephritis. We coincubated HRPTEC with BKV and the cholesterol-depleting agents methyl beta cyclodextrin (MBCD) and nystatin (Nys), drugs inhibiting caveolar endocytosis. The percentage of infected cells (detected by immunofluorescence) and the cellular levels of BKV large T antigen expression (detected by Western blot analysis) were significantly decreased in both MBCD- and Nys-treated HPRTEC compared to the level in HRPTEC incubated with BKV alone. HRPTEC infection by BKV was also tested after small interfering RNA (siRNA)-dependent depletion of either the caveolar structural protein caveolin-1 (Cav-1) or clathrin, the major structural protein of clathrin-coated pits. BKV infection was inhibited in HRPTEC transfected with Cav-1 siRNA but not in HRPTEC transfected with clathrin siRNA. The colocalization of labeled BKV particles with either Cav-1 or clathrin was investigated by using fluorescent microscopy and image cross-correlation spectroscopy. The rate of colocalization of BKV with Cav-1 peaked at 4 h after incubation. Colocalization with clathrin was insignificant at all time points. These results suggest that BKV entered into HRPTEC via caveolae, not clathrin-coated pits, and that BKV is maximally associated with caveolae at 4 h after infection, prior to relocation to a different intracellular compartment.

Cellular and matrix contributions to tissue construct stiffness increase with cellular concentration.

The mechanics of bio-artificial tissue constructs result from active and passive contributions of cells and extracellular matrix (ECM). We delineated these for a fibroblast-populated matrix (FPM) consisting of chick embryo fibroblast cells in a type I collagen ECM through mechanical testing, mechanical modeling, and selective biochemical elimination of tissue components. From a series of relaxation tests, we found that contributions to overall tissue mechanics from both cells and ECM increase exponentially with the cell concentration. The force responses in these relaxation tests exhibited a logarithmic decay over the 3600 second test duration. The amplitudes of these responses were nearly linear with the amplitude of the applied stretch. The active component of cellular forces rose dramatically for FPMs containing higher cell concentrations.

Reconstitution of the Frank-Starling mechanism in engineered heart tissues.

According to the Frank-Starling mechanism, as the heart is stretched, it increases its contraction force. Reconstitution of the Frank-Starling mechanism is an important milestone for producing functional heart tissue constructs. Spontaneously contracting engineered heart tissues (EHTs) were reconstituted by growing dissociated chicken embryo cardiomyocytes in collagen matrices. Twitch and baseline tensions were recorded at precisely controlled levels of tissue strain. The EHTs showed a steep increase in twitch tension from 0.47 +/- 0.02 to 0.91 +/- 0.02 mN/mm2 as they were stretched at a constant rate (2.67% per min) from 86% to 100% of the length at which maximum twitch force was exerted. In response to a sudden stretch (3.3%), the twitch tension increased gradually (approximately 60 s) in a Gd3+-sensitive manner, suggesting the presence of stretch-activated Ca2+ channels. A large difference in baseline tension between lengthening (loading) and shortening (unloading) was also recorded. Disruption of nonsarcomeric actin filaments by cytochalasin D and latrunculin B decreased this difference. A simple mechanical model interprets these results in terms of mechanical connections between myocytes and nonmuscle cells. The experimental results strongly suggest that regulation of twitch tension in EHTs is similar to that of natural myocardium.

The relationship between cell and tissue strain in three-dimensional bio-artificial tissues.

Continuum constitutive laws are needed to ensure that bio-artificial tissue constructs replicate the mechanical response of the tissues they replace, and to understand how the constituents of these constructs contribute to their overall mechanical response. One model designed to achieve both of these aims is the Zahalak model, which was modified by Marquez and co-workers to incorporate inhomogeneous strain fields within very thin tissues. When applied to reinterpret previous measurements, the modified Zahalak model predicted higher values of the continuum stiffness of fibroblasts than earlier estimates. In this work, we further modify the Zahalak model to account for inhomogeneous strain fields in constructs whose cell orientations have a significant out-of-plane component. When applied to reinterpret results from the literature, the new model shows that estimates of continuum cell stiffness might need to be revised upward. As in this article's companion, we updated the average cell strain by defining a correction factor ("strain factor"), based upon the elastic response. Three different cell orientation distributions were studied. We derived an approximate scaling model for the strain factor, and validated it against exact and self-consistent (mean-field) solutions from the literature for dilute cell concentrations, and Monte Carlo simulations involving three-dimensional finite element analyses for high cell concentrations.

Thin bio-artificial tissues in plane stress: the relationship between cell and tissue strain, and an improved constitutive model.

Constitutive models are needed to relate the active and passive mechanical properties of cells to the overall mechanical response of bio-artificial tissues. The Zahalak model attempts to explicitly describe this link for a class of bio-artificial tissues. A fundamental assumption made by Zahalak is that cells stretch in perfect registry with a tissue. We show this assumption to be valid only for special cases, and we correct the Zahalak model accordingly. We focus on short-term and very long-term behavior, and therefore consider tissue constituents that are linear in their loading response (although not necessarily linear in unloading). In such cases, the average strain in a cell is related to the macroscopic tissue strain by a scalar we call the "strain factor". We incorporate a model predicting the strain factor into the Zahalak model, and then reinterpret experiments reported by Zahalak and co-workers to determine the in situ stiffness of cells in a tissue construct. We find that, without the modification in this article, the Zahalak model can underpredict cell stiffness by an order of magnitude.