Mechanoregulation of MSC spheroid immunomodulation.

Mesenchymal stromal cells (MSCs) are widely used in cell-based therapies and tissue regeneration for their potent secretome, which promotes host cell recruitment and modulates inflammation. Compared to monodisperse cells, MSC spheroids exhibit improved viability and increased secretion of immunomodulatory cytokines. While mechanical stimulation of monodisperse cells can increase cytokine production, the influence of mechanical loading on MSC spheroids is unknown. Here, we evaluated the effect of controlled, uniaxial cyclic compression on the secretion of immunomodulatory cytokines by human MSC spheroids and tested the influence of load-induced gene expression on MSC mechanoresponsiveness. We exposed MSC spheroids, entrapped in alginate hydrogels, to three cyclic compressive regimes with varying stress (L) magnitudes (i.e., 5 and 10 kPa) and hold (H) durations (i.e., 30 and 250 s) L5H30, L10H30, and L10H250. We observed changes in cytokine and chemokine expression dependent on the loading regime, where higher stress regimes tended to result in more exaggerated changes. However, only MSC spheroids exposed to L10H30 induced human THP-1 macrophage polarization toward an M2 phenotype compared to static conditions. Static and L10H30 loading facilitated a strong, interlinked F-actin arrangement, while L5H30 and L10H250 disrupted the structure of actin filaments. This was further examined when the actin cytoskeleton was disrupted via Y-27632. We observed downregulation of YAP-related genes, and the levels of secreted inflammatory cytokines were globally decreased. These findings emphasize the essential role of mechanosignaling in mediating the immunomodulatory potential of MSC spheroids.

Hydrogel degradation promotes angiogenic and regenerative potential of cell spheroids for wound healing.

Chronic nonhealing wounds are debilitating and diminish one's quality of life, necessitating the development of improved strategies for effective treatment. Biomaterial- and cell-based therapies offer an alternative treatment compared to conventional wound care for regenerating damaged tissues. Cell-based approaches frequently utilize endothelial cells (ECs) to promote vascularization and mesenchymal stromal cells (MSCs) for their potent secretome that promotes host cell recruitment. Spheroids have improved therapeutic potential over monodisperse cells, while degradable scaffolds can influence cellular processes conducive to long-term tissue regeneration. However, the role of biomaterial degradation on the therapeutic potential of heterotypic EC-MSC spheroids for wound healing is largely unknown. We formed poly(ethylene) glycol (PEG) hydrogels with varying ratios of matrix metalloproteinase (MMP)-degradable and non-degradable crosslinkers to develop three distinct constructs - fully degradable, partially degradable, and non-degradable - and interrogate the influence of degradation rate on engineered cell carriers for wound healing. We found that the vulnerability to degradation was critical for cellular proliferation, while inhibition of degradation impaired spheroid metabolic activity. Higher concentrations of degradable crosslinker promoted robust cell spreading, outgrowth, and secretion of proangiogenic cytokines (i.e., VEGF, HGF) that are critical in wound healing. The degree of degradation dictated the unique secretory profile of spheroids. When applied to a clinically relevant full-thickness ex vivo skin model, degradable spheroid-loaded hydrogels restored stratification of the epidermal layer, confirming the efficacy of scaffolds to promote wound healing. These results highlight the importance of matrix remodeling and its essential role in the therapeutic potential of heterotypic spheroids.

Tuning the Microenvironment to Create Functionally Distinct Mesenchymal Stromal Cell Spheroids.

Mesenchymal stromal cells (MSCs) are under investigation for wound healing and tissue regeneration due to their potent secretome. Compared to monodisperse cells, MSC spheroids exhibit increased cell survival and enhanced secretion of endogenous factors such as vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE2), two key factors in wound repair. We previously upregulated the proangiogenic potential of homotypic MSC spheroids by manipulating microenvironmental culture conditions. However, this approach depends on the responsiveness of host endothelial cells (ECs)-a limitation when attempting to restore large tissue deficits and for patients with chronic wounds in which ECs are dysfunctional and unresponsive. 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. When encapsulated together in engineered protease-degradable hydrogels as a model of cell delivery, VEGFMAX and PGE2,MAX spheroids exhibited robust spreading into the biomaterial and enhanced metabolic activity. The distinct bioactivities of these MSC spheroids demonstrate the highly tunable nature of spheroids and provide a new approach to leverage the therapeutic potential of cell-based therapies.

Multisized Photoannealable Microgels Regulate Cell Spreading, Aggregation, and Macrophage Phenotype through Microporous Void Space.

Microgels are an emerging platform for in vitro models and guiding cell fate due to their inherent porosity and tunability. This work describes a light-based technique for rapidly annealing microgels across a range of diameters. Utilizing 8-arm poly(ethylene) glycol-vinyl sulfone, the number of arms available for crosslinking, functionalization, and annealing is stoichiometrically controlled. Small and large microgels are fabricated to explore how microgel diameter impacts void space and the role of porosity on cell spreading, cell aggregation, and macrophage polarization. Mesenchymal stromal cells spread rapidly in both formulations, yet the smaller microgels permit a higher cell density. When seeded with macrophages, the smaller microgels promote an M1 phenotype, while larger microgels promote an M2 phenotype. As another application, the inherent porosity of annealed microgels is leveraged to induce cell aggregation. Finally, the microgels are implanted to examine how different size microgels influence endogenous cell invasion and macrophage polarization. The use of ultraviolet light allows for microgels to be noninvasively injected into a desired mold or wound defect before annealing, and microgels of different properties combined to create a heterogeneous scaffold. This approach is clinically relevant given its tunability and fast annealing time.

Tissue engineered platforms for studying primary and metastatic neoplasm behavior in bone.

Cancer is the second leading cause of death in the United States, claiming more than 560,000 lives each year. Osteosarcoma (OS) is the most common primary malignant tumor of bone in children and young adults, while bone is a common site of metastasis for tumors initiating from other tissues. The heterogeneity, continual evolution, and complexity of this disease at different stages of tumor progression drives a critical need for physiologically relevant models that capture the dynamic cancer microenvironment and advance chemotherapy techniques. Monolayer cultures have been favored for cell-based research for decades due to their simplicity and scalability. However, the nature of these models makes it impossible to fully describe the biomechanical and biochemical cues present in 3-dimensional (3D) microenvironments, such as ECM stiffness, degradability, surface topography, and adhesivity. Biomaterials have emerged as valuable tools to model the behavior of various cancers by creating highly tunable 3D systems for studying neoplasm behavior, screening chemotherapeutic drugs, and developing novel treatment delivery techniques. This review highlights the recent application of biomaterials toward the development of tumor models, details methods for their tunability, and discusses the clinical and therapeutic applications of these systems.