gms | German Medical Science

Objectives: Osteoarthritis (OA) is a degenerative condition affecting a wide range of the adult population. Autologous Chondrocyte Implantation (ACI) has been used to treat focal OA defects, although there are negative long-term outcomes due to poor graft integration and the presence of inflammatory factors, e.g. interleukin-1beta (IL-1β) [1]. Bone marrow derived Mesenchymal Stem Cells (MSCs) are an alternative cell type that can be used due to their chondrogenic capacity. In vivo, cartilage and bone marrow reside under a low oxygen tension, between 2-7% oxygen (physioxia), with recent studies describing enhanced MSC chondrogenesis under this condition [2]. However, MSC chondrogenesis has been shown to be inhibited in the presence of IL-1β [3]. The present investigation sought to understand the effect of physioxia on MSC chondrogenesis and hypothesized that physioxia alleviates IL-1β inhibited chondrogenesis.

Methods: Human MSCs (Male donors; aged 18-60 years, n=9) were split equally for expansion under either 2% (physioxia) or 20% (hyperoxia) oxygen. Chondrogenic pellets (2x 10⁵ MSCs/pellet) were formed by centrifugation in 96-well plates and cultured in the presence of 10ng/ml TGF-β₁ and in combination with 0.1 and 0.5ng/ml IL-1β under their expansion conditions. Pellets removed on day 7, 14 and 21 were measured for their wet weight, pellet GAG. Pellets on day 21 were also evaluated histologically (DMMB (GAG), collagen II and X and MMP-13) and collagen II content. Statistical analysis was performed on quantitative data using a Two-way ANOVA with Tukey post-hoc test, significant differences are stated when p<0.05.

Results and conclusion: Under hyperoxia, a significant inhibition in MSC chondrogenesis was observed in IL-1β treated samples in a dose-dependant manner with respect to pellet wet weight and GAG content (p<0.05). Physioxia alone significantly increased wet weight, GAG and Collagen II content (p<0.05) compared to hyperoxia. Upon administration of IL-1β, a donor dependant response for physioxia was observed, whereby 80% of donors positively responded to low oxygen and 20% were unresponsive. Analysis of responsive donors demonstrated a significant increase in wet weight and GAG content in the presence of 0.1 and 0.5 ng/ml IL-1β under physioxia (p < 0.05). Furthermore, a reduction in the expression of hypertrophy markers (collagen X and MMP-13) was observed under physioxia but only in the presence of 0.1ng/ml IL-1β. Interestingly, physioxia restored pellet wet weight, GAG and collagen II content for 0.1 ng/ml IL-1β treated MSC chondrogenesis to that shown for hyperoxia alone (p>0.05). The underlying molecular mechanisms controlling the physioxic response are to be investigated. In summary, physioxia alone enhances MSC chondrogenesis and alleviates IL-1β inhibited differentiation.