Abstract
INTRODUCTION
The generation of cartilage from progenitor cells for the purpose of cartilage repair is often hampered by unwanted ossification of the generated tissue due to endochondral ossification. Our in vitro data show that celecoxib is able to suppress the hypertrophic differentiation phase of endochondral ossification in differentiating human bone marrow stem cells via inhibition of prostaglandin signalling. Continuing on our earlier studies our goal is to further improve the engineering of hyaline cartilage for the treatment of cartilage defects, by determining if celecoxib released from poly(D,L-lactic acid)microspheres is able to prevent unwanted ossification in an in vivo model for the subperiosteal cartilage generation.
METHODS
A 2% (m/v) low melting agarose was injected between the bone and periosteum at the upper medial side of the tibia of both legs of New Zealand white rabbits (DEC 2012–151). The agarose was left unloaded or (n=8) or loaded (n=7) with celecoxib-loaded PGLA microspheres (poly(D,L-lactic acid) microspheres were loaded with 20% (w/w) Celecoxib (Pfizer)). Fourteen days post-injection, rabbits were euthanised. The developed subperiosteal cartilage tissue was analysed for weight, GAG and DNA content. In addition, RT-qPCR and (immuno)histochemistry were performed for key markers of different phases of endochondral ossification.
RESULTS
The Functional release of celecoxib from poly(D,L-lactic acid) microspheres was confirmed in vitro by decreased prostaglandin E2 levels in cell culture. The subperiosteal cartilage tissue from the celecoxib group was significantly higher in weight and DNA content as compared to the control condition. GAG content was not significantly different between groups. No significant differences in chondrogenic marker expression (COL2A1, SOX9, ACAN and PTHrP) were detected, but levels of hypertrophic markers COL10A1, RUNX2 and ALPL were significantly decreased. COL1A1 expression was not significantly different between groups.
DISCUSSION
In summary, subperiosteal generation of cartilage was successful when an agarose bio-gel was injected subperiosteally. Supplementation of the agarose gel with celecoxib-loaded microspheres favourably changed the weight of the generated cartilage tissue, combined with significantly lower expression levels of indicators of chondrocyte hypertrophy, while leaving chondrogenic differentiation capacity unaltered. These data hold the promise that local supplementation of celecoxib during in vivo cartilage regeneration protects the tissue from adverse hypertrophic differentiation.
