| Summary: | Culturing articular chondrocytes under physiological oxygen tension exerts positive effects on their extracellular matrix synthesis. The underlying molecular mechanisms which enhance the chondrocytic phenotype are, however, still insufficiently elucidated. The TGF-β superfamily of growth factors, and the prototypic TGF-β isoforms in particular, are crucial in maintaining matrix homeostasis of these cells. We employed a feedback-controlled table-top bioreactor to investigate the role of TGF-β in microtissues of human chondrocytes over a wider range of physiological oxygen tensions (i.e., physoxia). We compared 1%, 2.5%, and 5% of partial oxygen pressure (pO<sub>2</sub>) to the ‘normoxic’ 20%. We confirmed physoxic conditions through the induction of marker genes (<i>PHD3</i>, <i>VEGF</i>) and oxygen tension-dependent chondrocytic markers (<i>SOX9</i>, <i>COL2A1</i>). We identified 2.5% pO<sub>2</sub> as an oxygen tension optimally improving chondrocytic marker expression (<i>ACAN</i>, <i>COL2A1</i>), while suppressing de-differentiation markers (<i>COL1A1,</i> <i>COL3A1</i>). Expression of TGF-β isoform 2 (<i>TGFB2</i>) was, relatively, most responsive to 2.5% pO<sub>2</sub>, while all three isoforms were induced by physoxia. We found TGF-β receptors <i>ALK1</i> and <i>ALK5</i> to be regulated by oxygen tension on the mRNA and protein level. In addition, expression of type III co-receptors betaglycan and endoglin appeared to be regulated by oxygen tension as well. R-Smad signaling confirmed that physoxia divergently regulated phosphorylation of Smad1/5/8 and Smad2/3. Pharmacological inhibition of canonical ALK5-mediated signaling abrogated physoxia-induced <i>COL2A1</i> and <i>PAI-1</i> expression. Physoxia altered expression of hypertrophy markers and that of matrix metalloproteases and their activity, as well as expression ratios of specific proteins (Sp)/Krüppel-like transcription factor family members SP1 and SP3, proving a molecular concept of ECM marker regulation. Keeping oxygen levels tightly balanced within a physiological range is important for optimal chondrocytic marker expression. Our study provides novel insights into transcriptional regulations in chondrocytes under physoxic <i>in vitro</i> conditions and may contribute to improving future cell-based articular cartilage repair strategies.
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