Tissue engineering aims to recapitulate native tissue function to replace diseased or damaged tissues. Scaffold-free techniques such as the self-assembling process have recently emerged to exploit the natural synthetic ability of cells. Our group, in particular, has used the self-assembling process to engineer various neocartilages including femoral cartilage, the knee meniscus, and the temporomandibular joint disc. For those suffering from degenerative cartilage diseases of the joints, tissue engineered cartilage may prove a viable solution bridging early palliative treatments like microfracture and end-stage irreversible options like total joint replacement. Though we have extensive experience with the self-assembling process, the mechanisms of self-assembly are not well understood. The first global objective of this thesis thus focused on elucidating self-assembly mechanisms, toward developing rational agents to influence the process. The second global objective of this thesis sought to enhance neocartilage tensile properties through the application of novel bioactive stimuli that mimic the osmotic and developmental milieu of articular cartilage. The final objective of this work aimed to engineer—for the first time—articular cartilage with functional native tissue-like tensile stiffness, strength, and anisotropy, through the application of tensile stimulation.

To address these objectives, this thesis 1) elucidated the roles of cadherins and integrins in mediating the self-assembling process, 2) explored the effects of controlling the cellular osmotic environment on functional properties of self-assembled articular chondrocytes, 3) evaluated the use of developmentally critical thyroid hormones to increase neocartilage properties, and 4) investigated the application of tensile mechanical stimulation to enhance anisotropy and tensile properties of neocartilage and explored the in vivo stability of tensile properties.

The results of this work include a proposed mechanism of self-assembly, mediated by cell-cell and cell-matrix adhesion molecules and with a functional cytoskeletal network. Both integrins and cadherins were found to influence the selfassembling process, with integrin-based self-assembly dominating in the presence of surface-bound collagen molecules. Neocartilage generated in the absence of surfacebound collagen was found to exhibit significantly up-regulated collagen production. Finally, it was shown that both an intact myosin-actin network and mediators of contractility (i.e., Rho kinase) are crucial to facilitating robust self-assembly.

This work also demonstrated that recapitulation of the native tissue microenvironment enhanced neocartilage functional properties. Modulation of the osmotic environment via application of a physiologically relevant level of calcium, hyperosmolarity, and a calcium channel agonist were found to beneficially interact, yielding increases in tensile stiffness.

Rationally deriving additional stimuli from the development of growth plate cartilage—specifically, thyroid hormones parathyroid hormone, tri-iodothyronine (T3), and thyroxine (T4)—allowed us to engineer neocartilage exhibiting a tensile stiffness nearly 4-times that of untreated control values. T3, however, is known to elicit hypertrophic responses in growth plate chondrocytes, an undesirable phenotype in neocartilage. Sequential application of T3 and PTH in this work resulted in reduced hypertrophic responses while maintaining the enhanced tensile properties.

Finally, application of a tensile stimulation regimen, in combination with matrix enhancing agents TGF-β1, chondroitinase-ABC, and lysyl oxidase-like protein 2, resulted in scaffold-free neocartilage with tensile properties that, for the first time, are on par with native tissues. Moreover, this work established that tensile stimulation increases expression of matrix remodeling enzymes, the BMP2/SMAD7 signaling pathway, and cell-matrix interactions via integrins. Function of the calcium channel transient receptor potential vanilloid 4 was found to be responsible for mechanotransduction of tensile stimulation. Finally, implantation of treated neocartilages demonstrated maintenance or increases in functional properties.

Collectively, this work elucidated the mechanisms of scaffold-free self-assembly, enabling future work to rationally select agents to beneficially impact this process. Through the application of 1) bioactive stimuli guided by the native osmotic microenvironment and by developmental biology, and 2) a novel tensile stimulation regimen, this thesis achieved tensile properties on par with native articular cartilage. These functional neocartilages can ultimately be used to replace damaged or diseased tissues to restore joint function.