Purpose. A major drawback of current cartilage and intervertebral disc (IVD) tissue engineering is that human mesenchymal stem cells (MSCs) from osteoarthritic (OA) patients express high levels of type X collagen. Type X collagen is a marker of late stage chondrocyte hypertrophy, linked with endochondral ossification, which precedes bone formation. However, it has been shown that a novel plasma-polymer, called nitrogen-rich plasma-polymerized ethylene (PPE:N), is able to inhibit type X collagen expression in committed MSCs. The aim of this study was to determine if the decreased expression of type X collagen, induced by the PPE:N surfaces is maintained when MSCs are removed from the surface and transferred to pellet cultures in the presence of serum and growth factor free chondrogenic media. Method. Human MSCs were obtained from aspirates from the intramedullary canal of donors undergoing total hip replacement for OA. Cells were expanded for 2–3 passages and then cultured on polystyrene dishes and on two different PPE:N surfaces: high (H) and low (L) pressure deposition. Cells were transferred for 7 additional days in chondrogenic serum free media (DMEM high glucose supplemented with 2 mM L-glutamine, 20 mM HEPES, 45 mM NaHCO3, 100 U/ml penicillin, 100 ug/ml streptomycin, 1 mg/ml bovine serum albumin, 5 ug/ml insulin, 50 ug/ml ascorbic acid, 5 ng/ml sodium selenite, 5 ug/ml transferrin) in pellet culture or on PS cell culture dishes. RNA was extracted using a standard TRIzol protocol. RT-PCR was realized using Superscript II (RT) and Taq polymerase (PCR) with primers specific for type I and X collagen. GAPDH was used as a housekeeping gene and served to normalize the results. Results. As observed in previous studies, type X collagen mRNA level was suppressed when cultured on both H- and L-PPE:N. HPPE:N was more effective in decreasing type X collagen expression than LPPE:N (55 vs. 78 % of control OA cells). Results also showed that the decreased type X collagen mRNA level was maintained not only when cells were removed from the PPE:N surfaces and transferred to new polystyrene culture dishes in the presence of chondrogenic media, but also when transferred to pellet cultures. Culturing MSCs from OA patients on PPE:N surfaces and in pellet culture had however no effect on the level of type I collagen mRNA. Conclusion. The present study confirmed the potential of PPE:N surfaces in suppressing type X collagen expression in MSCs from OA patients. More importantly, when these cells are transferred to pellet cultures, type X collagen suppression is maintained. These results may lead us one step closer to the production of large amounts of reprogrammed MSCs for tissue engineering applications

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

Currently, there is no animal model in which to evaluate the underlying physiological processes leading to the heterotopic ossification (HO) which forms in most combat-related and blast wounds. We sought to reproduce the ossification that forms under these circumstances in a rat by emulating patterns of injury seen in patients with severe injuries resulting from blasts. We investigated whether exposure to blast overpressure increased the prevalence of HO after transfemoral amputation performed within the zone of injury. We exposed rats to a blast overpressure alone (BOP-CTL), crush injury and femoral fracture followed by amputation through the zone of injury (AMP-CTL) or a combination of these (BOP-AMP). The presence of HO was evaluated using radiographs, micro-CT and histology. HO developed in none of nine BOP-CTL, six of nine AMP-CTL, and in all 20 BOP-AMP rats. Exposure to blast overpressure increased the prevalence of HO.

This model may thus be used to elucidate cellular and molecular pathways of HO, the effect of varying intensities of blast overpressure, and to evaluate new means of prophylaxis and treatment of heterotopic ossification.

Cite this article: Bone Joint J 2015;97-B:572–6