Transient Scleraxis Overexpression Combined with Cyclic Strain Enhances Ligament Cell Differentiation

Efforts to generate tissue-engineered anterior cruciate ligament replacements are limited by a lack of methods to derive mature ligament cells. Viral overexpression of the tendon/ligament marker scleraxis (Scx) can drive cell differentiation; however, the use of viral vectors hampers translation to clinical use. In this study, C3H10T1/2 cells were transiently transfected with expression vectors containing the full-length murine Scx cDNA and cultured in three-dimensional collagen hydrogels under static or cyclic strain for up to 14 days. β-galactosidase (LacZ) transfected cells served as controls. Cell morphology and gene expression for ligament-related genes, in addition to contraction (hydrogel width), mechanical properties, and glycosaminoglycan (GAG) and DNA content of hydrogels, were quantified and compared over time, between Scx and LacZ groups, and between static and cyclically strained constructs. Increased Scx expression was maintained for the entire 14-day study in both static and cyclically strained constructs. In static culture, overexpression of Scx resulted in greater cell elongation and construct contraction compared to LacZ controls. There were no differences in gene expression, DNA, or GAG content between Scx and LacZ constructs cultured under static conditions and no differences in DNA content between Scx and LacZ constructs. When exposed to cyclic strain, Scx-overexpressing cells maintained the elongated phenotype exhibited in static constructs, increased GAG production compared to static culture, and increased expression of the ligament-related genes collagen type I, decorin, and tenascin-C compared to strained LacZ controls. Cyclically strained constructs containing Scx-overexpressing cells had increased maximum load and stiffness compared to LacZ controls. The maintenance of increased Scx expression throughout the 14 day study and subsequent increases in ligament marker gene expression and mechanical properties with cyclic, but not static strain, suggest that transient transfection may be a viable alternative to viral transduction of Scx for ligament engineering studies and support a synergistic effect of Scx and mechanical strain on driving early ligament cell differentiation.