Computer-Controlled Biaxial Bioreactor for Investigating Cell-Mediated Homeostasis in Tissue Equivalents

Abstract

Soft biological tissues consist of cells and extracellular matrix (ECM), a network of diverse proteins, glycoproteins, and glycosaminoglycans that surround the cells. The cells actively sense the surrounding ECM and regulate its mechanical state. Cell-seeded collagen or fibrin gels, so-called tissue equivalents, are simple but powerful model systems to study this phenomenon. Nevertheless, few quantitative studies document the stresses that cells establish and maintain in such gels; moreover, most prior data were collected via uniaxial experiments whereas soft tissues are mainly subject to multiaxial loading in vivo. To begin to close this gap between existing experimental data and in vivo conditions, we describe here a computer-controlled bioreactor that enables accurate measurements of the evolution of mechanical tension and deformation of tissue equivalents under well-controlled biaxial loads. This device allows diverse studies, including how cells establish a homeostatic state of biaxial stress and if they maintain it in response to mechanical perturbations. It similarly allows, for example, studies of the impact of cell and matrix density, exogenous growth factors and cytokines, and different types of loading conditions (uniaxial, strip-biaxial, and biaxial) on these processes. As illustrative results, we show that NIH/3T3 fibroblasts establish a homeostatic mechanical state that depends on cell density and collagen concentration. Following perturbations from this homeostatic state, the cells were able to recover biaxial loading similar to homeostatic. Depending on the precise loads, however, they were not always able to fully maintain that state.

Mechanical homeostasis in tissue equivalents: a review

“…This effect was the more pronounced the higher the initial prestretch. Given that prestretch stiffens the collagen matrix, these observations may seem at first glance a contradiction to the ones of Delvoye et al (1991), Zhao (2014), and Eichinger et al (2020). Yet, both may be brought into agreement by noting that cells have been found to respond to changes in stiffness in a biphasic manner (Chan and Odde 2008), that is, there may exist a certain regime where cellular tension increases with the stiffness of the mechanical environment and another regime where the opposite is true.…”

“…Circular, cell-seeded free-floating collagen gels compact strongly over multiple days due to cell contraction (Simon et al 2012. If similar gels are constrained in a uni-or biaxial setting at the boundaries and are therefore not able to deform the gel in the direction of loading, a two-stage response is generally observed: first, cells rapidly build up a certain level of tension (phase I), which is subsequently maintained (phase II) for a prolonged period (Brown et al 1998;Jenkins et al 1999;Brown et al 2002;Sethi et al 2002;Campbell et al 2003;Marenzana et al 2006;Karamichos et al 2007;Dahlmann-Noor et al 2007;Ezra et al 2010;Courderot-masuyer 2017;Eichinger et al 2020). The level of homeostatic tension appears to increase with both collagen concentration and cell density.…”

“…Especially In uniaxially constrained tissue equivalents, the measured tensile force evolves in two characteristic phases: a steep increase (phase I) followed by a plateau (phase II). Here, we show experimental data from Eichinger et al (2020). Each curve is an average of three identical experiments.…”

“…Influence of cell density and matrix composition on tension When using both calf skin fibroblasts (Delvoye et al 1991) and NIH 3T3 fibroblasts (Eichinger et al 2020), tension in the homeostatic state of uniaxial tissue equivalents increases linearly with collagen concentration. Notably, Eichinger et al (2020) demonstrated that higher collagen concentrations increase the steady state tension but not the rate at which tension is built up initially. Moreover, tissue tension increases with cell density, either linearly (Delvoye et al 1991;Eichinger et al 2020) or nonlinearly (Legant et al 2009;Jin et al 2015) depending on the experimental conditions.…”

“…The inability of uniaxially constrained tissue equivalents to reproduce bi-or triaxial stress or strain states, which prevail in vivo, motivated the development of biaxial testing devices in the 2000s (Fig. 2d) (Knezevic et al 2002;Thomopoulos et al 2005Thomopoulos et al , 2007Humphrey et al 2008;Sander et al 2009;Hu et al 2009Hu et al , 2013Lee et al 2018;Eichinger et al 2020). In many cases, however, biaxial settings have been used mainly for fairly general studies of fiber alignment and mechanical properties.…”