Injuries to connective tissues such as ligaments and tendons are common, and rather than healing, repair typically results in fibrosis, or the formation of mechanically inferior and disorganized scar tissue. This fibrotic repair response is due in part to inflammation, during which the injury site is invaded by a number of cell types, including macrophages, neighboring fibroblasts, and homed stem cells or progenitor cells. Activation of macrophages is believed to be modulated by communications with fibroblasts and stem cells, prompting either a pro-fibrotic or a pro-regenerative response. Beyond changes to the cellular microenvironment, fibrosis also results in changes to the organization and mechanical properties of the matrix microenvironment. For healthy fibrous connective tissues, the matrix is comprised of aligned collagen fibers, while scar tissue is disorganized and exhibits weaker mechanical properties than healthy tissue. To date, the nature of the cell-cell and cell-matrix interactions and their relevance in tissue healing or repair remain understudied.

To better understand the cellular and matrix-based cues that direct scar formation versus tissue regeneration, and using anterior cruciate ligament (ACL) injuries as a model, Aim 1 of this thesis tests the hypothesis that in vitro models of cellular communications between fibroblasts, macrophages, and mesenchymal stem cells (MSC) can be used to determine the effects of cellular interactions on macrophage activation and fibrosis. In Aim 2, the contribution of matrix-based cues (alignment and mechanical properties) to the inflammatory and fibrotic response, as well as their modulation of cellular interactions, were examined. Findings from these two aims reveal that 1) communications between native tissue fibroblasts and macrophages drive inflammation and fibrosis, while stem cells modulate the repair process through a combination of trophic signaling and immunomodulatory roles, and 2) matrix alignment and mechanical properties exert combined regulation on cell response during inflammation. From a clinical application perspective, stem cells delivered in conjunction with an engineered matrix that provides the critical cues for driving stem cell immunomodulation and trophic signaling will be essential for promoting tissue regeneration and minimizing fibrosis. In particular, an aligned matrix with an elastic modulus similar to that of developing connective tissue may serve to further minimize inflammation and scar formation, and activate stem cell-guided regeneration of mechanically functional connective tissue.