Abstract
Purpose
Whilst it is known that oxidative stress can cause early degenerative changes observed in experimental osteoarthritis and that a major drawback of current cartilage and intervertebral disc tissue engineering is that human mesenchymal stem cells (MSCs) from osteoarthritis (OA) patients express type X collagen, a marker of late-stage chondrocyte hypertrophy (associated with endochondral ossification), little is known whether the expression of type X collagen in MSCs from OA patients can be related to oxidative stress or inflammatory reactions that occur during this disease.
Method
Human MSCs were obtained from aspirates from the intramedullary canal of donors undergoing total hip replacement for OA. Bone marrow aspirates were processed essentially as previously described. Briefly, non-adherent cells were discarded after 72h of culture and the adherent ones were expanded for 2–3 passages. MSCs from normal donor (control) were obtained from Lonza. Cells were then lysed and protein expression was detected by Western blot using specific antibodies directed against type X collagen, as well as the antioxidant enzymes Mn-superoxide dismutase (MnSOD), catalase (CAT) and glutathione peroxidase-1 (GPx-1) and inflammation related proteins cyclooxygenase-1 (COX-1) and intercellular adhesion molecule-1 (ICAM-1). GAPDH was used as a housekeeping gene and served to normalize the results. Correlations between the expressions of the different proteins were realized using the correlation Z test with StatView (SAS Institute).
Results
Results confirmed that type X collagen was over-expressed in MSCs from OA patients when compared to expression in cells of normal donors. MnSOD, CAT, and COX-1 were also over-expressed. Results showed that the expression of MnSOD strongly correlated to the expression of type X collagen (r=0.79; p=0.03). The expression of CAT weakly correlated to the expression of type X collagen (r=0.67; p=0.10) whereas GPx was not expressed in MSCs from OA patients. Regarding inflammatory reaction, results showed that COX-1 expression strongly correlated to type X collagen expression (r=0.77; p=0.004). ICAM-1 was weakly expressed and no correlation with the expression of type X collagen was observed. Interestingly, COX-1 expression was highly correlated to the expression MnSOD (r=0.92; p=0.0001) and the expression of CAT (r=−0.82; p=0.02).
Conclusion
We showed that the level of anti-oxidant enzymes correlates with type X collagen expression in MSCs from OA patients. This suggests that oxidative stress may lead to the up-regulation of stem cell hypertrophy. Results also suggest that prostaglandin production though COX-1 activity is associated with anti-oxidant enzyme expression (MnSOD) and hypertrophy (type X collagen expression). Further studies are however necessary to better understand whether the increased expression of these proteins is the cause or the effect of type X collagen over-expression in MSCs from OA patients.
