Title: Optimization of Culture Conditions for Cartilage Tissue Engineering Using Synovium-Derived Stem Cells
Description: Osteoarthritis (OA) is the most common joint disease and the leading cause of disability among Americans. OA afflicts 20 million Americans and costs $128 billion in direct medical and work-related losses each year. Nearly 1/3 of OA patients in the United States are over 65 years of age and given the aging population of the "baby boomer" generation, the prevalence of this disease is predicted to increase dramatically in the coming decades. The disease is characterized by the degeneration of cartilage and progressive loss of normal structure and function. However, the harsh loading environment and the avascular nature of mature cartilage lead to a poor intrinsic healing capacity after injury. As a result, cell-based therapies, including tissue engineering strategies for growing clinically relevant grafts, are being intensively researched. An autologous cell source would be ideal for growing clinically relevant engineered cartilage; however, using cells from an osteoarthritic or injured tissue to grow engineered cartilage with mechanical and biochemical properties similar to healthy native tissue poses several challenges, including lack of healthy donor tissues and donor site morbidity.

As a result, the clinical potential of mesenchymal stem cells (MSCs) has driven forward efforts toward their optimization for tissue engineering applications. Of these MSCs, synovium-derived stem cells (SDSCs) are being intensively researched due to their proximity to the defect site and high chondrogenic potential. To address the need for cell-based therapies, functional tissue engineering aims to restore cartilage function by culturing grafts in vitro that recapitulate the mechanical, biochemical, and structural framework of the tissue in order to have an increased chance of integration and survival upon in vivo implantation. While previous work in the lab has explored the utility of physiologically relevant stimuli for creating tissue grafts with chondrocytes, it has not yet been investigated for SDSCs. Therefore, in order to determine the potential of SDSCs as a tissue engineering strategy for growing clinically relevant cartilage grafts, this dissertation had four primary aims: (1) to initially produce tissue growth utilizing synovium-derived stem cells, (2) to utilize additional chemical, physical, and physico-chemical factors to further optimize growth of tissue engineered cartilage using SDSCs, (3) to characterize the response of SDSCs to the factors applied, and (4) to utilize the optimized culture techniques to translate the findings to clinically-relevant human cells.

Our initial studies investigated the potential of using physiologically relevant growth factors during both 2D expansion and 3D culture conditions, from which a baseline culture protocol was established. We then sought to explore additional strategies to further optimize tissue growth. Motivated by the discrepancy in osmolarities between native and in vitro culture conditions, we first assessed the influence of adjusting the osmolarity of the baseline culture media. We found that culturing constructs under a more physiologic osmolarity (400 mOsM) was beneficial for tissue growth. Based on these findings implicating osmolarity as a key influencer of growth potential, we sought to determine and potentially manipulate some of the pathways involved in the osmotic response in an effort to further optimize and characterize our tissue-engineered cartilage constructs. Our results supported the role of the TRPV4 ion channel in our SDSC-seeded constructs as a key mechano-osmosensing mechanism. Through the culturing techniques evaluated, we were able to achieve native mechanical and biochemical measures of juvenile bovine cartilage using SDSCs.

As has been shown in the literature, observed results in other species (bovine or canine) may not always correlate to findings using human cell sources, thereby prompting the emphasis for more relevant pre-clinical models. Therefore, our final studies sought to translate our treatment strategies to clinically relevant human cells from normal (non-diseased) and diseased (OA) SDSCs and chondrocytes in order to determine their utility. We were able to create a complete set of micropellet data for both SDSCs and chondrocytes to allow for comparisons. Overall, our micropellet results indicate that tissue condition (non-diseased vs OA) is the primary determinant of matrix synthesis. The research described in this dissertation has demonstrated the utility of SDSCs for strategies aimed at cartilage regeneration. We present the first studies to grow SDSC-seeded constructs to native properties of juvenile bovine chondrocytes. Therefore, utilization of the culture techniques presented here and other optimization strategies may hold key insights to developing a tissue using autologous/allogeneic SDSCs that can fully recreate native cartilage.

In addition, the findings support the clinical potential of human SDSCs as a cell source for cartilage repair strategies.