Canine ACL fibroblast integrin expression and cell alignment in response to cyclic tensile strain in three‐dimensional collagen gels

Abstract

Tissue-engineered ligament substitutes have the potential to become an alternative graft source for ligament reconstruction. If this approach is to become viable, one must first understand and define the mechanisms responsible for creation, maintenance, and remodeling of the native anterior cruciate ligament. It is well accepted that mechanical load alters fibroblast phenotypic expression in a variety of cell sources; however, the mechanosensitive pathways responsible for alteration in matrix production, remodeling, and alignment are unknown. We hypothesize that cell surface integrins play a role in this mechanotransduction process, and as such respond to application of cyclic tensile load. Linear 3D collagen gels containing canine ACL fibroblasts were created in Flexercell Tissue-Train Culture Plates. Gels were untethered (control), tethered without external strain (tethered), or tethered and exposed to 2.5% cyclic strain for 2 h per day for 4 days (strain). Quantitation of a1, a5, and b1 integrin subunit was performed using flow cytometry. Cell and matrix alignment was studied using light, polarized light, and fluorescent microscopy. Expression of a5 and b1 integrin subunits was increased significantly in fibroblasts in tethered and strained 3D collagen gels compared with the control, unloaded constructs ( p < 0.05). These integrins are known to function as mechanotransducers in other tissues, implicating a similar role in mechanotransduction in ACL fibroblasts. Histologic analysis of the tethered and strained gels demonstrated a linear arrangement of cells and parallel collagen fibril architecture. In contrast, cell distribution and collagen alignment were disorganized in the control, unloaded gels. The alignment of cells and collagen in the 3D gels parallel to applied strain is similar to the in vivo state. These data add to our understanding of the behavior of ACL fibroblasts in vitro. The ability to manipulate signal transduction pathways may enhance our ability to engineer implantable ACL grafts or to modify ACL healing response. ß

3D collagen cultures under well‐defined dynamic strain: A novel strain device with a porous elastomeric support

“…Because flow and strain are necessarily coupled, we could not differentiate specific responses to flow versus strain, but this alignment was consistent with our previous studies showing that fibroblasts in collagen gels undergoing interstitial flow alone (without matrix compression) align perpendicular to the direction of flow Swartz, 2003, 2006). We note that most of the previous work on fibroblast mechanobiology has been done by applying either tension or confined compression to the cells (Eastwood et al, 1998;Grinnell, 2003;Liu et al, 1999b;Wang et al, 2007), with tensional forces driving alignment of cells parallel to the strain direction (Henshaw et al, 2006;Voge et al, 2008); this is consistent with our findings. This demonstrates the usefulness of this model system for mechanobiology involving 3D dynamic compressive or tensile stresses.…”

Engineering controllable anisotropy in electrospun biodegradable nanofibrous scaffolds for musculoskeletal tissue engineering

“…In 3D, fibroblast-contracted collagen gels develop defined cellular and collagen organization with changing boundary conditions (Girton et al, 2002;Henshaw et al, 2006;Thomopoulos et al, 2005). While useful as a model for neo-tissue formation, these gelatinous scaffolds do not possesses physiologic mechanical properties, and are therefore not suitable for direct implantation.…”

Simulation of the cytoskeletal response of cells on grooved or patterned substrates

“…The response of cells to substrates with ordered textures has received considerable attention [12][13][14][15]. These studies indicate that cells are especially responsive to groove/ridge patterns on the substrate.…”