Low Oxygen Concentration Enhances Chondrogenesis and Secretome Production in Mesenchymal Stem Cells Cultured in a Three-Dimentional Decellularized Bovine Cartilage Scaffolds

Introduction: Stem cells hold promise for tissue repair due to their secretome, which is influenced by the microenvironment. To improve cartilage regeneration, a three-dimensional (3D) natural cartilage scaffold system has been developed to create a more chondrogenic secretome. As human cartilage is avascular, a hypoxic environment may better mimic in vivo conditions. This study investigates if oxygen levels (normoxic vs. hypoxic) affect MSC chondrogenic potential and secretome composition in this 3D scaffold. Methods: This study used a randomized time series design to investigate how oxygen levels affect the transformation of MSCs into cartilage cells within a 3D cartilage scaffold. A control group (C) was grown in a standard medium. Two experimental groups, P1 and P2, were cultured n a 3D cartilage scaffold under normal (21%) and hypoxic (5%) oxygen concentration. respectively. The hypothesis was that a hypoxic environment would promote superior chondrogenic differentiation. Key markers were evaluated at specific time points using immunohistochemistry and enzyme-linked assay. Results: In the 3D cultures, the cells exposed to low oxygen (hypoxic) generally showed higher expression of markers related to cartilage formation (SOX-9, RUNX-2, Coll-II, and aggrecan) compared to those in normal oxygen levels (normoxic). Additionally, the hypoxic group had consistently higher levels of several growth factors important for tissue development and repair (BMP-2, BMP-7, TGF-β3, IGF-1, and FGF-2) throughout the study. Conclusion: Hypoxic conditions in 3D culture led to increased expression of all analyzed chondrogenic markers in mesenchymal stem cells, suggesting that a low-oxygen environment effectively promotes chondrocyte proliferation and differentiation.