A tissue-engineered ligament is a promising alternative to autograft for anterior cruciate ligament (ACL) reconstruction, but strategies must be developed to form oriented tissues with high tensile strength. To accomplish this, we propose to seed ligament progenitor cells onto oriented fiber scaffolds and to use cyclic mechanical stretch on the cells to stimulate deposition of a ligament-like extracellular matrix (ECM). Here, electrospinning of a poly(esterurethane urea) (PEUUR) onto a rotating target will be used to create an oriented fiber mesh that will guide cell alignment and suppress cell reorientation in response to stretch. Further, our PEUUR is elastomeric, biocompatible, and degradable, making it a suitable substrate for both in vitro cyclic mechanical stretch and implantation in vivo.

For this study electrospun meshes will be formed with mean fiber diameters of 0.8 mm and orientation marked by an angular standard deviation of 30°, as our previous studies have shown that these characteristics ensure that ligament progenitor cells assume a spindle-shaped morphology and an orientation parallel to fiber alignment. Next, ligament progenitor cells – derived from bone marrow explants – will be cultured on our electrospun substrates and exposed intermittently to uniaxial cyclic stretch in a bioreactor that we have developed (0.5 Hz, 8% strain, 60 min daily). These experimental conditions have been shown to induce expression of the ECM proteins collagen types I and III, decorin, and tenascin-C. After performing cyclic stretch, cells will be analyzed for the expression of ECM proteins and for cell morphology (e.g., projected area, aspect ratio, and orientation relative to the axis of stretch). Control groups will include cells grown on fiber meshes but not exposed to stretch, and cells grown on smooth polydimethylsiloxane elastomer films. This study will serve as an initial condition for a series of systematic studies to probe the effect of mechanical stretch regimens on the formation of ligament-like tissues, and will provide materials for establishing a subcutaneous implantation model for characterizing integration and vascularization.