Osteoarthritis (OA) is the leading cause of disability in the US and one in two people are expected to develop knee OA by age 85. OA is disease of the entire joint, affecting not only the cartilage, but also the bone and synovium. The avascularity, low cellularity, and slow proliferation of chondrocytes all limit the natural regenerative capacity of cartilage in addition to the chronic inflammation prevalent in the joint space.

Cell therapies, such as autologous chondrocyte implantation (ACI), offer promising options for treating persistent cartilage lesions, but the inability to expand chondrocytes to sufficient numbers without adversely affecting their phenotype remains a significant problem. While synthetic microcarrier culture can improve the scalability of chondrocyte expansion over conventional monolayer methods by providing a high surface area-tovolume ratio, de-differentiation remains a problem for long-term expansion. Therefore, decellularized cartilage microcarriers (DC-µCs) that retain structural and biochemical cues of the native extracellular matrix (ECM) may provide an improved means to culture and deliver chondrocytes for ACI therapies. While ACI is promising, it is not indicated for cartilage damage associated with OA or other inflammatory diseases of the joint and is better for defects resulting from trauma. This lack of efficacy of cellular therapies is likely due to the inflammatory environment cells are exposed to upon implantation since multiple inflammatory mediators have been shown to play a pivotal role in the initiation and perpetuation of OA. Anti-inflammatory therapies with single molecular inhibitors do not effectively modulate the complex inflammatory environment presented in OA. Thus, novel therapies capable of modulating multiple signaling pathways and cell types are needed.

Mesenchymal stem cells (MSCs) are an adult multipotent stem cell population that can regulate multiple immune cells involved in innate and adaptive immunity, largely through paracrine mechanisms. Additionally, human amniotic membrane (AM) has emerged as a potential therapy for OA as it provides an abundant source of multiple immunoregulatory proteins, promotes stem cells proliferation, promotes pro-healing macrophage phenotype, and modulates cell secretion in vivo.

Therefore, the objective of this proposal was to engineer an improved cartilage repair strategy by combining cells and ECM materials to address problems with both cartilage repair and OA-associated inflammation. In Chapter 3, we developed decellularized cartilage microcarriers that retain endogenous extracellular matrix proteins to both expand and deliver chondrocytes while retaining their phenotype. In Chapter 4, robust characterization of the influence of culture format, donor variability, and media composition demonstrated that aggregated MSCS have enhanced sensitivity to changes in the local microenvironment, which can be tailored to enhance immunomodulatory paracrine activity. Moreover, MSC single cells and spheroids reduced inflammation in activated synoviocyte cultures in a dose-dependent manner and reduced OA progression when delivered to a rodent model of OA. Finally, in Chapter 5, we investigated the interaction between MSCs and human amniotic membrane and the influence of cell-cell and cell-ECM therein on the modulation of inflammation, both in vitro and in vivo. Overall, this work broadens current understanding of cartilage tissue engineering and immunomodulation, providing valuable information that can be used to develop strategies to improve efficacy of osteoarthritis treatments.