Ression patterns. However, our data indicate that redifferentiation was promoted, hypertrophic
Ression patterns. However, our data indicate that redifferentiation was promoted, hypertrophic and OA-associated degradative genes were suppressed and HIF expression patterns were indistinguishable in both healthy and OA chondrocytes in hypoxic conditions. Healthy and OA chondrocytes both responded with higher COL2A1 and ACAN and lower COL1A1 expression in hypoxic conditions. Hypoxic culture has previously been found to increase COL2A1 and ACAN in nondiseased chondrocytes [8,9] and independently in chondrocytes from joint arthroplasties [24]. In a directcomparison, we found no differences in expression of these genes between healthy and diseased cells, regardless of oxygen tension. Additionally, though COL1A1 was higher in OA chondrocytes than in healthy chondrocytes at 20 oxygen, lower COL1A1 and less collagen I matrix staining was associated with all cells redifferentiated in hypoxia. Collagen prolyl hydroxylases require molecular oxygen, and the decreases in hydroxyproline content at 2 oxygen indicate that this level of hypoxia impairs the activity of these enzymes. It is possible that, when oxygen levels are low enough that these enzymes are impaired in chondrocytes, prolyl hydroxylation and thus secretion of collagen type II is preferentially maintained over other collagens. Consistent with this theory, the COL2A1: COL1A1 ratio was consistently elevated by hypoxia in our studies. We also found that hypoxia increased proteoglycan accumulation by both healthy and OA cells. Excess oxygen may be more detrimental to cells from OA joints,Markway et al. Arthritis Research Therapy 2013, 15:R92 http://arthritis-research.com/content/15/4/RPage 9 ofFigure 4 Oxygen-dependent expression of matrix metalloproteinases in healthy and osteoarthritic chondrocytes. (A) through (D) The relative gene expression levels of MMP1, MMP3, MMP13 and MMP2 in pellet cultures following 2 wk of redifferentiation in either 20 oxygen (gray bars) or 2 oxygen (white bars) were analyzed by real-time quantitative polymerase chain reaction. (E) Gelatin zymography of Vercirnon supplier supernatants pooled from days 9 to 14 of pellet cultures indicates levels of secreted pro-MMP2 and active MMP2 in healthy and osteoarthritic (OA) cells at 20 and 2 oxygen. (F) and (G) The relative gene expression levels of TIMP2 and MMP14 in pellet cultures following 2 wk of redifferentiation in either 20 oxygen (gray bars) or 2 oxygen (white bars) were analyzed by real-time quantitative polymerase chain reaction. All expression values are the mean mRNA levels normalized to 18S ribosomal RNA for chondrocyte pellets from n = 5 donors. Error bars represent 1 SD. Statistical significance was determined by performing independent t-tests (between disease conditions) and paired t-tests (between oxygen levels). *P < 0.05.however, because OA cell pellets had significantly less sGAG/DNA than those of healthy cells maintained at high oxygen. Whether this is due to a greater oxygen sensitivity that results in impaired synthesis, more degradation,disturbed matrix assembly or some combination of these processes remains to be determined. The percentage of sGAGs retained within the matrix was also significantly reduced PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27385778 in OA chondrocytes at 20 oxygen. This may beMarkway et al. Arthritis Research Therapy 2013, 15:R92 http://arthritis-research.com/content/15/4/RPage 10 ofFigure 5 Oxygen-dependent expression of aggrecanases in healthy and osteoarthritic chondrocytes. The relative gene expression levels of ADAMTS4.
