title: 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 alpha1, alpha5, and beta1 integrin subunit was performed using flow cytometry. Cell and matrix alignment was studied using light, polarized light, and fluorescent microscopy. Expression of alpha5 and beta1 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.
authors.affiliation: Laboratory for Soft Tissue Research, Sports Medicine and Shoulder Service, The Hospital for Special Surgery, 535 East 70th Street, New York, New York 10021, USA.
authors.forename: D Ross
authors.initials: DR
authors.lastname: Henshaw
authors.forename: Erik
authors.initials: E
authors.lastname: Attia
authors.forename: Madhu
authors.initials: M
authors.lastname: Bhargava
authors.forename: Jo A
authors.initials: JA
authors.lastname: Hannafin
mesh_terms: D000818:Animals; D016118:Anterior Cruciate Ligament / Q000166:cytology* / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism*; D004285:Dogs; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D020100:Hydrogels / Q000737:chemistry / Q000378:metabolism*; D016023:Integrins / Q000378:metabolism*; D040542:Mechanotransduction, Cellular; D013314:Stress, Mechanical; D013718:Tensile Strength; D023822:Tissue Engineering / Q000379:methods
chemical_list: D020100:Hydrogels; D016023:Integrins; D003094:Collagen