Ligament and tendon injuries remain a persistent clinical challenge, accounting for up to 45% of the 32 million musculoskeletal injuries reported in the U.S. each year. However, current soft tissue repair and reconstruction techniques are limited by insufficient integration with subchondral bone, potentially leading to graft failure and suboptimal functional outcomes. Therefore, there is a pressing clinical need for functional solutions that can enable integrative soft tissue reconstruction via regeneration of the fibrocartilaginous insertion present at the junction between bone and major ligaments and tendons. This fibrocartilaginous enthesis consists of compositionally distinct but structurally continuous tissue regions (non-calcified and calcified fibrocartilage), and it plays a critical role in mediating complex load transfer between soft tissue and bone while minimizing the formation of stress concentrations at the insertion. Given the functional significance of the insertion site and using the anterior cruciate ligament (ACL) as a model tissue, the objective of this thesis is identify and optimize tissue engineering strategies for regeneration of the fibrocartilaginous interface. Thus, the studies detailed in this thesis consist of elucidation of key interface characteristics that can inform interface scaffold design, identification of an optimal cell source, and optimization of chemical and physical stimuli for fibrocartilage formation

To guide biomimetic scaffold design, this thesis began with quantitative mapping of the compositional and structural properties of the native ligament-to-bone interface. As both the aligned collagen matrix structure and distinctive mineral distribution pattern across the insertion were shown to be highly conserved over time, an ideal scaffold for fibrocartilage interface regeneration should therefore consist of aligned fibers and must be able to support the formation of both non-mineralized and mineralized fibrocartilage tissues. Additionally, evaluation of ex vivo behavior of insertion fibrochondrocytes cultured on aligned nanofiber scaffolds indicated that an ideal system for fibrocartilage regeneration should also support cell-mediated deposition of both types I and II collagen as well as proteoglycans. Comparison of potential cell sources for fibrocartilage tissue engineering showed that synovium-derived mesenchymal stem cells (SDSCs) exhibited higher proliferative and fibrochondrogenic differentiation potential compared to bone marrow-derived mesenchymal stem cells. Thus, subsequent studies focused on optimization of culture parameters for SDSC-mediated fibrocartilage formation. Nanofiber scaffolds that provided controlled release of transforming growth factor (TGF)-β3, which is known to play a critical role in development of the insertion as well as in scarless healing, were developed to guide SDSC differentiation. Scaffold-mediated TGF-β3 delivery enhanced cell proliferation and matrix synthesis in a dose-dependent manner, resulting in synthesis of fibrocartilaginous matrix consisting of both type I and II collagen as well as proteoglycans. As mechanical loading is known to also play a critical role in insertion development, a custom bioreactor that mimics the complex loads sustained at the interface was also developed. It was shown that the bioreactor simultaneously generated both tensile and compressive stresses and modulated SDSC matrix synthesis, where deposition of fibrocartilaginous matrix was observed on mechanically loaded scaffolds without any additional chemical co-stimulation. Finally, as a functional scaffold for integrative ACL repair must support the establishment of both non-mineralized and mineralized tissue regions, the combined effects of TGF-β3 and hydroxyapatite (HA) on MSC-mediated formation of mineralized fibrocartilage were also explored. The addition of HA nanoparticles to the scaffold was shown to enhance cell proliferation and matrix synthesis and represents a promising strategy for formation of mineralized fibrocartilage.

Collectively, these observations delineate the importance of bioinspired chemical and physical stimuli in fibrochondrogenic differentiation, and how they can be optimized for stem cell-mediated interface regeneration. These studies yield valuable scaffold design criteria and establish in vitro culture parameters that can be applied to functional integration of soft connective tissue with bone at various critical attachments throughout the musculoskeletal system, including the ligament and tendon-to-bone entheses, as well as for regeneration of other important fibrocartilaginous tissues.