Bianca de Freitas Machado (Dresden, DE), Franziska Alt (Dresden, DE), Dr. Vera Hintze (Dresden, DE), Dr. Poh Soo Lee (Dresden, DE)
Abstract text (incl. figure legends and references)
Objectives
This study aimed to investigate the combined effects of cell density, medium pH, oxygen tension, and chondroitin sulfate (CSA) supplementation on the chondrogenic differentiation potential of hMSC seeded in a collagen (Col)-based artificial extracellular matrix (aECM) scaffold. Thereby, recapitulating the initial stage of endochondral ossification in vitro and achieve bone constructs with near-physiological properties.
Materials & Methods
Col and Col+CSA scaffolds were prepared and characterized. Different parameters, including pH values of basal media, cell seeding densities, and media supplements, were investigated. Scaffolds were seeded at a density of 1x106 hMSCs/cm3 and maintained for up to 35 days. The changes in mechanical stiffness and scaffold morphology were monitored. The effects of hypoxia and normoxia on cell viability and gene expression were assessed.
Results
As shown in Fig1, gene expression analysis demonstrated significant Sox9 up-regulation, a crucial regulator of chondrocyte commitment, in acidic medium on d30. CSA supplementation showed similar effects to standard growth factors for chondrogenesis, with increased expression of Aggrecan, a major cartilage component, in acidic conditions. Runx2 expression, associated with osteoblast differentiation and chondrocyte hypertrophy, increased after 30 days, indicating the initiation of an osteogenic fate. Furthermore, the DNA assays for cell viability revealed no significant differences between hypoxic and normoxic conditions. Further, increased calcium concentration was observed in DMEM-cultivated samples.
Conclusion
These findings highlighted the potential of subtle fluctuations in extracellular pH and oxygen tension to influence chondrocyte metabolism and marker expression. The potential of these samples to proceed with osteogenesis is now investigated.
Fig1: Normoxia vs. Hypoxia and their interplay with medium pH, CSA to support chondrogenic differentiation of hMSC.