Engineering cartilage tissue using extracellular matrix-containing hydrogels with decoupled mechanical and biochemical cues [electronic resource]
Keywords: Bioengineering.

Cartilage loss due to diseases or traumatic events represent a major cause of pain and disability for adults. Conventional cell-based therapies for cartilage repair use autologous chondrocytes, which are associated with multiple limitations including insufficient donor supply, donor site morbidity, and tendency to lose phenotype during in vitro expansion. Stem cells offer a promising alternative cell source for cartilage repair given their proliferative potential and ability to undergo chondrogenesis. In contrast to using cells alone, biomimetic matrices such as hydrogels can provide structural and mechanical support, which is particularly important for repairing load bearing tissues such as cartilage. To better mimic the native extracellular matrix (ECM) environment, hydrogels containing ECM based molecules provide a three-dimensional (3D) niche for promoting chondrogenesis. However, increasing ECM molecule concentrations often result in simultaneous changes in the matrix stiffness, which makes it difficult to elucidate the interactive niche signaling between biochemical and mechanical cues on modulating stem cell chondrogenesis. Furthermore, how different types and doses of ECM molecules modulate stem cell chondrogenesis in 3D remains unclear. This thesis aims to bridge the gap of knowledge by developing combinatorial ECM-containing hydrogels with largely decoupled biochemical and mechanical cues as 3D niche for modulating cell-based cartilage regeneration in 3D. To mimic the biochemical cues present in cartilage matrix, three types of ECM molecules including chondroitin sulfate (CS), hyaluronic acid (HA), and heparan sulfate (HS) were chemically conjugated into hydrogels with varying stiffness. The potential of such ECM containing hydrogels as 3D niche for cartilage regeneration was subsequently examined using different cell sources, including adipose derived stem cells (ADSCs), mesenchymal stem cells (MSCs) as well as mixed populations of stem cells and chondrocytes. Specifically, the following aims were achieved: 1. Developed and characterized combinatorial hydrogels containing covalently crosslinked ECM molecules with independently tunable biochemical and mechanical cues as 3D cell niche for supporting cell based cartilage regeneration. 2. Examined the effects of varying biochemical and mechanical niche cues in 3D on modulating stem cell chondrogenesis using ADSCs. 3. Determined the effects of varying ECM doses and hydrogel stiffness on catalyzed cartilage formation by mixed populations of ADSCs and chondrocytes in vitro and in vivo using a mouse subcutaneous model. 4. Compared effects of different ECM molecules in supporting MSC based chondrogenesis in 3D hydrogels with tunable stiffness. 5. Evaluated different methods of hydrogel crosslinking on modulating neocartilage formation by MSCs in 3D hydrogels. 6. Engineered cartilage zonal organization using multi-layered ECM-containing hydrogels and various cell types. The platform reported herein may provide a useful tool for elucidating how ECM biochemical cues and matrix stiffness interact together to regulate cell fate, and for rapidly optimizing ECM-containing scaffolds to support stem cell differentiation and cartilage tissue regeneration using various cell sources. The universal crosslinking mechanisms facilitate mixing and spatial patterning different ECM molecules to recreate tissue zonal organization. While this thesis focuses on cartilage repair as a target tissue type, this platform may be adapted for identifying optimal niche cues for stem cell differentiation towards other lineages and repairing other tissue types.