gms | German Medical Science

Objectives: Osteoarthritis, a major cause of disability world-wide, causes aggravating cartilage erosion, immobility and disabling pain in all affected joints but still evades efficient pharmacological or cellular therapies. Intriguingly, recent studies determined that HS-deficient (HS^-) mice are protected from OA in joint-destabilisation models, pointing at a potential of HS-interference for OA-attenuating therapies. However, the underlying mechanisms remained elusive. One possibility is that a reduced mechano-response of chondrocytes protects HS^--cartilage from load-induced degeneration. Our aim was to clarify the role of HS for the mechano-response of chondrocytes and elucidate pathways relevant for chondro-protection.

Methods: HS^--cartilage was tissue-engineered by agarose culture of Ext1^gt/gt mouse primary chondrocytes hypomorphic in Exostosin1 (Ext1), one major HS-polymerizing enzyme. Quality of engineered cartilage matured for 2 weeks was assessed by histology (SafO, IHC), glycosaminoglycan (GAG) quantification and RT-qPCR. The molecular response to cyclic unconfined compression in a bioreactor was determined by transcriptome profiling, bioinformatic data-processing, RT-qPCR and WB. Statistical significance was tested by Kruskal-Wallis and Mann-Whitney U-test with p<0.05 considered significant.

Results and Conclusion: HS^--chondrocytes expressed Ext1-mRNA at 3-6 % of WT levels throughout culture and matured into HS-deficient engineered cartilage. During differentiation, Col2a1-mRNA rose by 2000-fold and Sox9 by 120-fold confirming successful cartilage maturation in both groups. Remarkably, the GAG/DNA production in HS^--cartilage was increased to 150 % of WT-levels. A higher sensitivity of HS^--chondrocytes to BMP stimulation was evident by Western blotting. Loading similarly activated ERK and P38 signalling. Transcriptome analysis identified the novel mechano-induced genes Inhba, Timp1, Ngf, Fosl1, Dhrs9 and Itga5 in both groups. Bioinformatic overrepresentation analysis of all mechano-regulated genes (>1.5-fold) delivered "negative regulation of apoptotic process" for HS^--cartilage, indicating an anti-apoptotic expression signature upon loading. Strikingly, the apoptosis-regulator Bnip3 was downregulated exclusively in Ext1^gt/gt tissue by loading providing evidence of an altered mechano-response of HS^--cartilage.

Overall, HS-deficiency promoted an anti-apoptotic gene expression signature upon loading that together with elevated GAG-production and higher BMP-sensitivity of chondrocytes may protect HS-deficient cartilage from load-induced damage. Bnip3 as HS-modulated mechano-response gene invites future translational studies on its mechanistic role during OA-progression and -protection in vivo.