| The anterior cruciate ligament's (ACL) inability to heal post-rupture makes it an attractive target for tissue engineering studies, which support a large clinical need. Development of a fully functional ACL in vitro would provide an efficacious alternative to inadequate current repair options, as well as serving as a tissue model for biomedical research. The objective of this work was to develop a process by which ligament tissue can be generated in vitro, utilizing the sequential application of biochemical and mechanical stimulation. The goal was to monitor the developmental state of adult stem cell seeded constructs, correlating cell response to applied stimulation to further matrix in-growth and tissue development. Initially, bone marrow stromal cell (BMSC) behavior was studied in response to growth factor and medium supplementation, and the combination of biochemical stimulation was identified that supported induction toward fibroblast lineage. In selection of an appropriate biomaterial for three dimensional culture, surface modified silk fibers imparted greater mechanical integrity to tissue engineered constructs and equivalent biocompatibility as compared to cross-linked collagen fibers. BMSC seeded silk matrices responded to the sequential administration of growth factors in static culture with enhanced differentiation and ligament specific extracellular matrix (ECM) production. Sequential stimulation was further explored with the transition of growth factors to dynamic culture to enhance cell specific differentiation and matrix remodeling characteristic of the native ACL. Dynamic culture enhanced BMSC differentiation and the production of ligament specific ECM, providing the framework for in vitro stimulation of ligament development. In summary, an appropriate cell source, biomaterial, and stimulatory regime were combined for in vitro ligament development. The approach and methodology identified in this study offers direction for tissue engineering strategies addressing current clinical needs. |
