Detrimental Effects of Collagen Crosslinking on Articular Cartilage Lubrication

Introduction:: Ex vivo studies suggest OA-associated cartilage softening may contribute to elevated shear and friction forces at the articular surface and predispose chondrocytes to acute cell death.^1-2 Thus, there has been great interest in using stiffening mechanisms, such as collagen crosslinking (CXL), to slow OA progression.^3-4 In classical stationary contact area (SCA) tests, OA-like cartilage slid at 1mm/s indeed experiences elevated friction coefficients (µ_k=0.25, vs. 0.15 in healthy tissue).⁵ However, similarly elevated µ_k values have been seen after CXL under identical testing conditions.⁴ Thus, the effects of cartilage stiffness on lubrication remain uncertain. Furthermore, the clinical relevance of SCA-based findings is unclear given that cartilage operates at higher average sliding speeds (30-80mm/s) and markedly lower friction (µ_k< 0.005) in vivo.⁶ Recent work leveraging the convergent stationary contact area (cSCA) reveals that sliding speeds >30mm/s facilitate interstitial fluid recovery and biofidelic µ_k (≤0.02 in saline and ≤0.005 in synovial fluid) via tribological rehydration. Critically, cSCA tribomechanical outcomes appear largely insensitive to of tissue stiffness in both healthy^6-7 and OA-like cartilage.⁸ Here, we examined whether CXL-mediated stiffening alters cartilage lubrication under biofidelic cSCA sliding.


Materials and Methods:: Osteochondral explants (Ø19mm) were extracted from mature bovine femoral condyles. A Ø4mm probe was used to indent each specimen in creep-relaxation (at 50µm/s to a 150mN load), and mechanical properties were determined by fitting the time-dependent normal force and indentation depth to Hertz biphasic theory.^8-9 Tribological testing comprised 30min static compression (7N) and 15min compression+sliding (80mm/s). Effects of CXL were examined in (i) healthy or (ii) OA-like cartilage (approximating ICRS grade 2; generated via chondroitinase-ABC (chABC) digestion).⁸ Explants were crosslinked with 10mM genipin (GP) in PBS for 24hr. Differences between groups were assessed via two-way ANOVA with Tukey’s post-hoc (significance at p< 0.05).


Results, Conclusions, and Discussions:: Results and Discussion: GP treatment of naïve cartilage increased E_y- from 0.53±0.20 to 0.87±0.27 (p< 0.001). There were no significant differences in tribological rehydration-mediated strain recovery after GP (p=0.30). Whereas naïve cartilage experienced near-physiological friction in PBS (µ_k=0.009±0.002), significantly elevated friction was observed after GP (µ_k=0.14±0.03; p< 0.0005). Next, we examined if synergy between tribological rehydration and synovial fluid (SF) lubrication could rescue the lubricity of GP-treated cartilage. Naïve cartilage sustained physiological (µ_k≤0.005) in SF-lubricated cSCA contacts, while GP-treatment led to elevated friction coefficients (µ_k=0.038±0.018; p< 0.005). Finally, we investigated the effects of GP treatment on the lubricity of OA-like cartilage generated via chABC-digestion. Digestion significantly decreased E_y- (p< 0.0005; Fig. 1A) but had no effect on strain recovery or friction coefficients (Fig. 1B-D). Subsequent GP treatment increased E_y- to 0.91±0.20MPa (p< 0.0005); Fig. 1A). These GP-treated tissues experienced significantly elevated friction coefficients of 0.13±0.06 and 0.032±0.017 in PBS- and SF-lubricated contacts, respectively (p< 0.005, Fig. 1C-D).



Conclusions: Here, we introduce the first biofidelic assessment of cartilage lubrication after genipin treatment, which has been proposed for “reinforcing” native and engineered cartilage.³ Despite persistent tribological rehydration, friction coefficients after GP exceeded those in paired naïve tests by 20- and 10-fold in PBS and SF, respectively. Exposure to such elevated friction coefficients should be extremely detrimental to chondrocytes, as multiple explant studies report marked cell death after just 30-60min of sliding at µ_k>0.05.^1,6 The high friction coefficients observed after GP treatment suggest collagen crosslinking is ill-suited for cartilage preservation. More importantly, this work demonstrates that biofidelic lubrication testing must be used to assess whether proposed OA treatments are suitable for the synovial joint microenvironment.




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References (Optional): : ¹Bonnevie (2018) J Biomech. ²Bartell (2015), J Biomech. ³Santos (2022) OA&C Open. ⁴Oungoulian ⁵Bonnevie (2018) JOR. (2014) J Biomech ⁶Farnham (2021) CMBE. ⁷Kupratis (2022) Acta Biomater. ⁸Kupratis (2023) ORS. ⁹Moore (2016) J Tribol.