Hypoxia of neonatal meniscus: maturation towards a fibro-chondrocytes phenotype through the modulation of vascular markers

Introduction: Meniscus is a complex tissue made of a vascular outer zone and an avascular inner zone with chondrocytes existing under physiological conditions of sustained hypoxia. Using a hypoxic chamber to provide controlled hypoxia, this study was performed to determine whether persistent hypoxia enhances the differentiation of menisci tissue harvested from neonatal pigs. Materials and Methods: Meniscal bodies were in vitro cultured under hypoxia or normoxia conditions until 14 days. Samples were analysed at 0 (T0), 7 (T1), 10 (T2) and 14 (T3) days by immunofluorescence and RT-PCR [Type II Collagen (COL2A1), SRY-Box Transcription Factor 9 (SOX-9), Type I Collagen (COL1A1), Hypoxia Inducible Factor 1 Subunit Alpha (HIF-1α) and the vascular markers, the Vascular Endothelial Growth Factor A (VEGF) and Platelet And Endothelial Cell Adhesion Molecule 1 (CD31)]. Results: A maturation shifting was observed by immunofluorescence and RT-PCR analysis in hypoxic treatment characterized by 1) up-regulation of COL2A1 at T2 (P <0.05); 2) reduction in SOX9 expression (not significant); 3) greater increase of VEGF at T3 (P <0.001); 4) down-regulation of CD31 with a peak growth at T2 and a significantly decrease at T3 (P<0.05). Conclusion: This study shows that hypoxia can be considered as a booster to induce the differentiation of neonatal fibroblast-like meniscal cells into a mature fibro-cartilaginous phenotype by acting on angiogenesis markers: hypoxia induces VEGF, which is a chondrocyte survival factor during development, and it is also required for postnatal differentiation of chondrocytes. These results open considerably opportunities in the field of meniscus tissue engineering.