gms | German Medical Science

Objectives: Hypoxia is known to beneficially influence chondrogenic differentiation of human bone marrow-derived stromal cells (BMSCs). While the impact of hypoxia on the synthesis of cartilaginous extracellular matrix (ECM) synthesis is well acknowledged, so far very little is known on the effect of hypoxia on ECM degradation. Hence, this study aimed to investigate the differential effects of normoxic and hypoxic conditions on the expression and activity of ECM-modulating enzymes such as matrix metalloproteinases (MMPs) and the resulting impact on ECM development during chondrogenic differentiation of BMSCs.

Methods: BMSCs were cultivated in pellet culture (2x10⁵ cells per pellet) in chondrogenic medium supplemented with TGF-β1 (10 ng/mL) under normoxic (21% O₂) or hypoxic conditions (2% O₂) for 21 days. Cartilaginous ECM development was assessed using histology, immunohistochemistry, and common biochemical quantitative assays for proteoglycans and collagens. The effects of hypoxia on various ECM-modulating enzymes (MMP1, MMP3, MMP13, ADAMTS4, ADAMTS5, TIMP1, TIMP3) were analyzed on mRNA and protein expression level. Additionally, enzyme activity was monitored using a FRET-based general MMP activity assay and by immunohistochemical staining and subsequent quantification (software ImageJ) for specific degradation products (e.g., neoepitope DIPEN after aggrecan cleavage by MMPs) at different time points during chondrogenic differentiation.

Results and Conclusion: After 21 days, pellets cultured under hypoxic conditions exhibited improved chondrogenesis with increased proteoglycan and collagen content, as compared to normoxic conditions. After 5, 10, and 21 days of chondrogenic induction, different MMP expression patterns could be observed in pellets after exposure to hypoxic compared to normoxic conditions using qRT-PCR. Expression levels of MMP13 were distinctly lower (p ≤ 0.05) under hypoxic conditions, whereas TIMP3 expression was increased (p ≤ 0.05). Reduced expression of MMP13 was confirmed on protein level using immunohistochemistry and subsequent quantification (normoxia 240 ± 150 relative immunolabeled area (RIA) and hypoxia 37 ± 18 RIA (p ≤ 0.01)). Remarkably, the MMP-generated aggrecan neoepitope DIPEN was distinctly reduced under hypoxic conditions (immunohistochemical staining). Quantification of DIPEN staining showed a reduction from 10.7 ± 4.3 RIA for normoxic conditions to 2.9 ± 1.5 RIA for hypoxic conditions (p ≤ 0.01). This observation was further supported by reduced general MMP activity under hypoxia (normoxia 6.6 ± 1.1 RFU/mg protein and hypoxia 4.3 ± 0.7 RFU/mg protein (p ≤ 0.05)).

Our results indicate a new mechanism of action of hypoxic conditions during chondrogenesis of BMSCs. Hypoxia appears to not only support ECM synthesis and maturation, but also to protect cartilaginous ECM from degradation by downregulation of MMP expression and especially by attenuation of MMP activity contributing to a higher proteoglycan content in the ECM.