Damage to the fibrocartilage of the knee meniscus leads to nearly 1 million procedures annually in the U.S. and Europe, comprising the single most common source of procedures performed by orthopedic surgeons. Although the knee meniscus is frequently injured due to trauma, age, or disease, this tissue is avascular and thus possesses little healing capacity. Because of this, the knee meniscus is a prime candidate for tissue engineering, and recent advances in the field have brought about a range of new approaches. In particular, scaffold-free and self-organizing methods of tissue synthesis have been gaining attention in recent years. This thesis will investigate the application of novel stimuli, both singularly and in combination, with respect to a recently pioneered scaffold-free technique:​ the selfassembling process.

In line with this, the global objective of this work was to enhance the mechanical properties, biochemical content, and extracellular matrix (ECM) organization of self-assembling knee meniscus fibrocartilage. Towards addressing this objective, this thesis 1) identified a novel stimulus, lysophosphatidic acid (LPA), which increased tensile properties of engineered tissues through a new mechanism, 2) characterized the process of tissue formation in the self-assembling process temporally and under a variety of initial seeding densities, and 3) engineered shape-specific knee meniscus constructs using a combination of lysyl oxidase (LOX) and chondroitinase ABC (C-ABC). Engineered knee meniscus tissue was grown for a variety of culture durations and assayed with multiple techniques, including tensile and compressive testing, histological and immunohistochemical staining, quantitative biochemical assays, high performance liquid chromatography, and scanning electron microscopy.

The results of this work include insights into new mechanisms by which tissue organization may occur, translational improvements in the self-assembling process, fundamental characterization of tissue formation during the self-assembling process, novel findings on the structure-function relationships within the ECM of engineered fibrocartilage, and synergistic and additive effects of biochemical stimuli applied to engineered knee meniscus constructs. When assessing a novel stimulus with which to treat self-assembling tissues, a 1.8-fold increase in tensile properties was achieved, along with beneficial improvements in tissue anisotropy. When a variety of initial seeding densities was investigated, a critical seeding density with superior mechanical and biochemical properties was found, which increased tissue properties by up to 11.8-fold over the historical control while using fewer cells than previously needed. Finally, the application of multiple biophysical agents at a lower seeding density led to a synergistic and additive increases in engineered tissue construct properties, including a 3.8-fold increase in tensile modulus to 3.4 MPa, a 3.2-fold increase in collagen cross-link content to 180 nmol/μg, and a 1.7-fold increase in average collagen fiber diameter to 95 nm. Overall, this thesis underscored the potential for novel biophysical agents to be applied separately and in conjunction to enhance the properties of engineered tissues, and paves the way for future investigations of these agents in scaffold-free and scaffold-based tissue engineering studies.