UC Davis

Tissue engineering strategies to regenerate damaged tissues and restore tissue function integrate cells, materials as scaffolds, and biochemical factors to synergistically regulate cell behavior. Mesenchymal stromal cells (MSCs) have great therapeutic potential and are often leveraged in cell-based therapies due to their ability to self-renew in vivo, capacity to differentiate towards various cell lineages, and their bioactive secretome. However, a major challenge to the translation of MSC-based therapies is ensuring MSC viability and functionality after transplantation to the injury site. This limitation is overcome by aggregating MSCs into spheroids, which are also advantageous for their increased cell viability, proangiogenic, anti-inflammatory, and tissue regenerative potential. Biomaterials are also readily used as delivery vehicles to direct the therapeutic function of MSCs. Moreover, dynamic mechanical loads on MSCs have been reported to instruct cell behavior and differentiation. We therefore sought to enhance the therapeutic potential of MSC spheroids by fine-tuning the microenvironmental culture conditions during MSC spheroid formation, the biomaterial carrier, and the external physical stimuli for tissue engineering applications.The spheroid microenvironment can be independently tuned to engineer MSC spheroids with distinct therapeutic capabilities. Previously, we developed an MSC spheroid that enhanced wound healing by simultaneously upregulating the secretion of anti-inflammatory and proangiogenic cytokines. However, because upregulated secretion of two factors was linked, the secretory potential of MSCs was not fully exploited compared to enhancing the secretion levels of these individual factors. To address this challenge, we used a Design of Experiments (DOE) approach to engineer functionally distinct MSC spheroids that maximize VEGF production (VEGFMAX) or PGE2 production (PGE2,MAX) while incorporating ECs that could serve as the basic building blocks for vessel formation. VEGFMAX produced 22.7-fold more VEGF with enhanced endothelial cell migration compared to PGE2,MAX, while PGE2,MAX produced 16.7-fold more PGE2 with accelerated keratinocyte migration compared to VEGFMAX. We demonstrated the unique bioactive functionalities (i.e., promoting angiogenesis or epithelialization) of these individual spheroids and, for the first time, the capability of leveraging them in a modular fashion. Furthermore, by incorporating ECs as building blocks for the formation of new vasculature, this approach overcomes the challenges of depending on the recruitment and responsiveness of host ECs. This study offers the unique opportunity to independently tune MSC spheroids and leverage them modularly to maximize specific growth factor secretions for regenerative therapies. Biomaterial carrier properties, specifically hydrogel degradability, directs the angiogenic and regenerative potential of co-culture EC-MSC spheroids for wound healing. Degradation plays a critical role in extracellular matrix (ECM) remodeling, facilitating the migration of host cells (e.g., ECs, fibroblasts) to the wound bed. Despite the established importance of degradation, the influence of matrix degradability on EC-MSC spheroid behavior remains largely unknown. To address this gap in knowledge, we engineered hydrogels with different rates of degradation by varying the ratio of matrix metalloproteinase (MMP)-degradable (i.e., GPQ-A) and non-degradable (i.e., PEG-DT) crosslinkers in a 4-arm poly(ethylene) glycol (PEG) maleimide (PEG-4MAL) hydrogel. Matrix degradability significantly influenced cell proliferation and metabolic activity. Furthermore, more degradable scaffolds promoted robust cell spreading, outgrowth, and secretion of proangiogenic cytokines (i.e., VEGF, HGF) by EC-MSC spheroids. Treatment of burn injuries developed in an ex vivo human skin model with EC-MSC spheroid-loaded degradable hydrogels increased expression of cytokeratin, reduced epidermal detachment, and restored epidermal stratification compared to non-degradable counterparts. This work is the first to establish the effects of hydrogel degradation rate on EC-MSC spheroid potential and emphasizes the need to carefully consider the degradative properties of biomaterial cell carriers. Compressive mechanical load, a physiological force experienced by most cells has been established to promote chondrogenesis. However, there is limited understanding of its role on the immunomodulatory potential of MSCs. Furthermore, majority of the current approaches that investigate the effects of compression utilize systems that produce loads using a fluid medium, which do not recapitulate the physical compressive forces experienced in vivo. On the other hand, systems that do capture this physical aspect with platens focus on studying monodisperse MSCs, leaving the effects of compressive load on MSC spheroids unknown. We addressed these limitations by using a bioreactor that generated controlled, uniaxial cyclic compression to interrogate the influence of compressive load on MSC spheroid behavior. Load magnitude (i.e., 5 or 10 kPa) and hold duration (i.e., 30 or 250 s) influenced proinflammatory (i.e., IL-1β, IL-6, IL-8, IL-17A), anti-inflammatory (i.e., IL-10), and inflammatory cell recruiting (i.e., GM-CSF, ICAM-1, CCL2) cytokines. Larger compressive loads and shorter durations upregulated proinflammatory cytokine production, larger compressive loads and longer durations downregulated anti-inflammatory cytokine production, and smaller compressive loads and shorter durations enhanced proangiogenic cytokine secretion compared to static conditions. While under compressive load, MSCs regulate their immunomodulatory behavior via mechanosensing. These data emphasize the importance of cyclic compressive load on the immunomodulatory potential of MSC spheroids. Overall, the results of this thesis establish that the spheroid microenvironment can be tuned to engineer functionally distinct spheroids, hydrogel degradability promotes angiogenic and pro-regenerative EC-MSC capabilities, and cyclic compressive loading directs the immunomodulatory potential of MSC spheroids. These findings demonstrate that microenvironmental and biophysical properties potentiate the therapeutic potential of MSC spheroids for tissue engineering applications.