Symptomatic osteoarthritis and other forms of cartilage disease are a burden for millions of adults worldwide. Due to the low vascularity and the intrinsic lack of the ability to regenerate, articular cartilage tissue is a challenge to palliate once damaged, and surgical approaches fall short of creating long-term solutions for cartilage repair. Also, the ability of implanted cartilage or biomaterials to laterally integrate into the diseased cartilage site as well as the role of the subchondral bone region are still not fully understood.

Tissue engineering, which combines materials with cells and bioactive factors to address questions within bioengineering and medicine, has been investigated for the study of osteochondral defects. The most recent studies of osteochondral regeneration have introduced 3D printing techniques, which can increase the degree of control over a printed implant’s cellular and architectural complexity. Among these novel approaches is the bioprinting of continuous composition gradients, which mimic osteochondral physiology and have not been investigated for their mechanical effects nor their biological implications on osteochondral tissue integration. The study of these emergent properties arising from the interactions facilitated by the continuous interface and transition between two phenotypically-distinct, cell-encapsulating bioink phases is the fundamental question of this thesis.

While selected previous works have examined similar questions in vitro with injectable or castable cell culture environments, the adaptation of those promising hydrogels into printable bioinks is still a challenge preventing them from being studied for further questions about more biomimetic architectural complexity and 3D structure – questions that a reliably printable system can address. But despite decades of work in these specific sub-fields of biomaterial 3D printing and bioprinting, standardized methods for how to evaluate the printability and subsequent biological effects of bioinks are still being studied.

The body of work described herein meets the needs of the greater bioprinting and tissue engineering communities by both studying novel bioinks to answer new questions within osteochondral tissue engineering and by documenting the methods, assays, and standards by which the inks were translated from mere hydrogels into a robust set of bioinks that encapsulate living cells. Specifically, we put forward a high-throughput method that evaluates hydrogel polymer systems as candidates for the base material of future bioinks with a given cell type based on viability, proliferation, and immunostaining in 3D culture, using less than 1 million cells and 1 mL per ink to observe and compare biological outcomes. Then, we studied biological additives within a gelatin-based ink that induced bone and cartilage extracellular matrix (ECM) production in encapsulated stem cells, and we also compiled a diverse array of methods to study printability additives, resulting in several novel osteochondral bioinks for extrusion, inkjet, and digital light processing 3D bioprinting. Select bioinks were then used to answer questions about architecture within an osteochondral explant model, and here, we observed that architecture did indeed make a difference in the cells’ ECM deposition and integration into the natural tissue. Altogether, this body of work demonstrates how important 3D factors are when considering tissue-engineered constructs for regenerative medicine, and it also chronicles a vast array of protocols and methods to serve as a reference for broader scientific forums interested in studying those interactions in a 3D environment.