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Mesenchymal stem cells (MSCs) are multipotent, potent pro-osteoarthritis and chondrocyte-inducing factors. To date, however, the clinical potential of MSC-based treatments for cartilage repair has not yet been realized. The majority of previous studies have used MSCs that are expanded in monolayers. In this study, we developed a novel synthetic hydrogel for in vitro chondrogenesis that resembles the macrostructure of articular cartilage. We compared the chondrogenic capacity of human adipose-derived MSCs (AD-MSCs) expanded in monolayers or in the aforementioned hydrogel. The hydrogel-expanded AD-MSCs showed higher chondrogenic potential than the monolayer-expanded AD-MSCs, demonstrated by greater chondrogenic gene expression and matrix accumulation. Moreover, the hydrogel-expanded AD-MSCs exhibited improved cell distribution, alignment, and mineral deposition compared to the monolayer-expanded AD-MSCs. Hydrogel-expanded AD-MSCs also showed a better capacity to differentiate to osteoblasts than monolayer-expanded AD-MSCs did. The upregulation of interleukin (IL)-6, IL-1β, and tumor necrosis factor-α was significantly inhibited by hydrogel expansion. Consequently, cartilage-specific extracellular matrix production, including glycosaminoglycan and collagen type II, was enhanced in hydrogel-expanded AD-MSCs. The hydrogel cultured AD-MSCs also demonstrated a reduction in immunogenic capacities, including cytokine release, indoleamine 2,3-dioxygenase, and major histocompatibility complex class I and II molecule expression, when compared with those of monolayer-cultured AD-MSCs. Collectively, the hydrogel may enhance the chondrogenic
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