Low Oxygen Expansion Improves Subsequent Chondrogenesis of Ovine Bone-Marrow-Derived Mesenchymal Stem Cells in Collagen Type I Hydrogel

Abstract

Background/Objective: A crucial factor when investigating cartilage tissue engineering using mesenchymal stem cells (MSCs) is their application in large-animal models and preclinical trials. However, in vitro studies using cells of these model organisms must proceed. Considering that oxygen tension is an important parameter for stem cell culture, we investigated the effect of low oxygen tension during the expansion of ovine MSCs on colony-forming unit-fibroblast (CFU-F) formation, senescence and subsequent chondrogenesis in pellet culture and a collagen I hydrogel which is in clinical use for matrix-associated autologous chondrocyte transplantation (MACT). Materials and Methods: Ovine MSCs were isolated from bone marrow aspirates and cultured at 5 and 20% O₂ in monolayer. CFU-F formation was detected by Giemsa staining. Senescence was analyzed by detection of senescence-associated β-galactosidase and flow cytometry. Chondrogenic differentiation was carried out in pellet and collagen I hydrogel culture and assessed by gene expression, immunohistochemistry and measurement of sulfated glycosaminoglycans (sGAG). Results: MSCs expanded at 5% O₂ revealed a 2-fold higher CFU-F potential and diminished senescence compared to those expanded at 20% O₂. Most notably, our results show enhanced chondrogenic differentiation in both pellet culture and the MACT-approved collagen I hydrogel. Conclusion: The findings demonstrate that physiologically low oxygen tension during monolayer expansion of ovine MSCs is advantageous in order to improve cartilage tissue engineering in a sheep model. The ovine system is shown to represent an appropriate basis for large-animal studies and preclinical trials on MSC-based cartilage repair.