HYPOXIC CONDITIONING IN 3D MATRIX ENVIRONMENTS POTENTIATES MESENCHYMAL STEM CELL REGENERATIVE SIGNATURES

The limited success of mesenchymal stromal cell (MSC) therapy in cartilage repair necessitates innovative approaches to enhance therapeutic efficacy. Current treatments fail to fully restore damaged cartilage structure and function, resulting in compromised joint mechanics and progressive degeneration. In both primary osteoarthritis and post-traumatic cases, mechanical injury disrupts cartilage homeostasis, leading to progressive tissue degradation. Surgical repair procedures initiated through fibrin clot formation exist, but they typically result in inferior fibrocartilage formation. To address the lack of effective cartilage repair strategies, this study evaluated whether recreating the post-injury microenvironment through 3D fibrin architecture and physiological oxygen tension could influence MSC therapeutic potential for regeneration. Using an immortalized human bone marrow-derived MSC model, cells were cultured in 2D and 3D fibrin environments under normoxic (21% O2) or hypoxic (2% O2) conditions for 72 hours. The 3D fibrin matrices simulate the initial fibrin clot microenvironment that guides natural repair processes. To further elucidate the impact of culture systems on modulating the inflammatory pathophysiology of osteoarthritis, we assessed the influence of pro-inflammatory cytokine stimulation (IFN?/TNFa) within each system. Transcriptome analyses revealed significant molecular and functional differences, with hypoxia-conditioned MSCs showing marked directionality toward a regenerative phenotype in 3D environments. Moreover, cells cultured in 3D hypoxic conditions demonstrated a marked increase in chondrogenic differentiation markers, particularly SOX9, with expression levels six orders of magnitude higher than 2D normoxic culture. This was accompanied by increased expression of cartilage-specific matrix proteins and enhanced cell survival markers. Notably, exposure to pro-inflammatory cytokines further amplified these expression patterns in the 3D hypoxic environment, suggesting robust cellular responses to such conditions. Analyses of metabolic markers highlighted critical adaptations in the 3D hypoxic environment compared to 2D and normoxic conditions, revealing distinct signatures associated with improved cellular resilience, immunomodulation and regenerative capacity. The synergistic effect of 3D architecture and hypoxia created a protective microenvironment that promoted cell survival and chondrogenic function while mitigating effects of IFN?/TNFa. This biomimetic approach offers insight into adapting cell therapy protocols for MSC manufacture in relation to cartilage regeneration. Our findings establish a strategic approach for MSC priming through biomimetic culture conditions that enhance cellular resilience and regenerative capacity. This insight into physiologically-guided cell manufacturing provides a robust foundation to develop more effective cell-based therapies for both traumatic and degenerative osteoarthritis.