STR/ORT MICE EXHIBIT AN INHERENT ENDOCHONDRAL GROWTH DEFECT AND REDEPLOY TRANSIENT CHONDROCYTE BEHAVIOURS PRIOR TO OSTEOARTHRITIS ONSET

Purpose: Increasing evidence implicates the re-initiation of embryonic processes, responsible for long bone development and growth, in osteoarthritic pathology. We aimed to establish whether an aberrant redeployment of the transient chondrocyte phenotype is observed in the STR/Ort mouse (spontaneous osteoarthritis), and whether this can be attributed to an inherent endochondral growth defect in these mice. Furthermore, we aimed to establish the role of the Wnt inhibitor sclerostin and its downstream target matrix extracellular phosphoglycoprotein (MEPE), a mineralisation inhibitor, in the maintenance of the healthy joint, and in the regulation of these endochondral processes in osteoarthritic joints. Methods: STR/Ort mouse knee joints at advancing osteoarthritis stages and age-matched CBA (control) joints were examined, by: (i) affymetrix mouse gene microarray profiling of articular cartilage (AC) samples, (ii) multiplex PCR analysis of AC and (iii) immunohistochemical labelling of endochondral markers including sclerostin and MEPE. The endochondral growth phenotype of young STR/Ort mice (1-8wks old) was analysed by standard histology, immunohistochemical labelling and by laboratory and synchrotron x-ray computed microtomography. Results: Our microarray studies reveal the ectopic recapitulation of endochondral processes in AC in our STR/Ort mouse model of osteoarthritis evidenced, by increased expression of Col10a1 (2.31 fold), Mmp13 (1.87 fold), Alpl (1.25 fold), Phospho1 (1.13 fold) and Enpp1 (1.59 fold) in a meta-analysis of gene array profiles and increased levels of Ank (P<0.01), Phex (P<0.001), Opn (P<0.05), Dmp1 (P<0.001), Enpp1 (P<0.01) and Ifitm5 (P<0.01) mRNA levels by multiplex-based analysis. Our immunolabelling confirms this expression and reveals collagen type X expression in the AC of STR/Ort mice at all ages suggesting an inherent endochondral growth defect. This was confirmed by growth plate zone analysis in which STR/Ort mice were found to have an increased proliferative zone (PZ) of chondrocytes. Furthermore, immunolabelling revealed collagen type X expression in the hypertrophic zone (HZ) alone of the young CBA mice growth plates; however, in the STR/Ort mouse, collagen type X expression was observed in both the HZ and the PZ of chondrocytes. Immunolabelling for MMP13 revealed a greater expression in STR/Ort mouse growth plates in comparison to age matched CBA mice. Most interestingly, STR/Ort mouse growth plates also show premature closure, evident from 8 weeks of age, as shown by synchrotron radiation-based computer microtomography. At 40 weeks of age growth plate closure in the medial tibia plateau (affected) of the STR/Ort mouse is greater than the lateral (non-affected) compartment, and both compartments in the CBA mouse. This exciting data reveals a direct association between the cartilage-bone transition and osteoarthritis pathology, and suggests that lessons may be learnt from the molecular mechanisms underpinning endochondral development in the pursuit of understanding osteoarthritis development. As such we investigated the role of the MEPE/sclerostin axis in the development of osteoarthritis in the STR/Ort mouse. Our immunolabelling reveals enhanced sclerostin expression at the osteochondral interface, and enhanced MEPE expression predominantly in the AC in the joints of CBA mice and in unaffected regions of STR/Ort mouse joints. At advanced osteoarthritis stages, focal suppression of sclerostin and MEPE expression is observed in regions localised to areas of subchondral bone-thickening and compromised AC integrity. Conclusions: Our data reveals changes in expression patterns in osteoarthritic mouse joints consistent with the aberrant redeployment of endochondral processes in osteoarthritis bone pathology, attributed to an inherent endochondral growth defect. Further investigation into the underpinning mechanisms will identify whether targeting known regulators of long bone development can protect against pathological ossification in osteoarthritis.