CCL2 but not CCR2 is required for articular cartilage regeneration

Abstract

Purpose: Full-thickness cartilage defect (FTCD) injuries in mammals do not demonstrate regenerative capacity. Inflammation within the joint is a characteristic of acute injury and degenerative diseases such as osteoarthritis (OA), however the role of inflammation in articular cartilage regeneration (or lack thereof) remains unclear. Chemokines are cytokines that bind to and activate CC-chemokine receptors, triggering a wide variety of cellular and tissue level responses. The CCL2/CCR2 pair is one of the most potent mediators of monocyte/macrophage migration and infiltration into sites of injury. Recently, conflicting reports on the chondroprotective role of CCL2 and CCR2 in mouse models of OA have been published. Zarebska et al. reported chondropathy scores in CCL2-/- and CCR2-/- mice as being similar to wildtype controls, while Raghu et al. found that CCL2 or CCR2 deficiency protects against mouse OA. Since chemokines are known to be highly promiscuous, it is possible that other receptors/pathways might be playing a role in these single knockout studies. However, to date there are no reports on articular cartilage degeneration and/or regeneration in CCL2-/-CCR2-/- mice. Therefore, to address this gap in the literature and to determine if CCL2 and/or CCR2 play a role in articular cartilage regeneration (instead of solely inhibiting degeneration) we have investigated the ability of CCL2-/-, CCR2-/- and CCL2-/-CCR2-/- mice to regenerate cartilage after FTCD injury. Methods: C57BL/6 (C57), CCL2-/-, CCR2-/- and CCL2-/-CCR2-/- 8 week old mice were used for all experiments. FTCDs (∼0.4mm) were induced in the trochlear groove of femurs. Cartilage regeneration was assessed using a 14-point scoring scale. Immunohistochemistry was used on serial sagittal sections to identify the location of MSCs (Sca1+CD140a+) before and after cartilage injury. Furthermore, immunohistochemistry was used on serial sagittal sections to identify actively proliferating cells (Ki-67+). Data was collected on non-injured joints and 4 weeks and 12 weeks after FTCD injury. Results: Compared to C57 controls, CCR2-/- mice displayed increased cartilage regeneration as early as 4 weeks after FTCD injury (8.0±1.0 vs. 5.38±1.19, respectively) (Fig. 1, Fig. 2A-C, G-I, M-O).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Uninjured joints were examined in all animal strains to determine if increased numbers of MSCs (Sca1+ and/or CD140a+) were present before FTCD injury and no differences in MSCs (localization nor abundance) were observed (Fig. 2D, J, P). MSCs were not visualized within the FTCD area in any mouse strains at any timepoint examined (Fig. 2D-F, J-L, P-R). Cells positive for MSC markers were found to co-express Ki67 in the patella, synovium, subchondral bone, and blood vessels in all animal strains, with no overt differences in triple-positive cell numbers between animal strains at 4 or 12 weeks after injury (Fig. 2E-F, K-L, Q-R). Conclusions: The results of this study suggest that CCL2 is required to activate the regenerative response in mice after FTCD injury. Furthermore, activation of the CCL2-mediated regenerative response is independent of CCR2, as mice deficient in CCR2 demonstrated an inherent healing capacity compared to CCL2-/- or CCL2-/-CCR2-/- mice. The inability of CCL2-/- or CCL2-/-CCR2-/- mice to regenerate cartilage after injury highlights some intrinsic yet unidentified mechanism through which CCL2 contributes to normal cartilage homeostasis. Additional studies are currently underway to elucidate the interplay between the CCL2/CCR2 signaling axis and macrophage recruitment/accumulation in response to cartilage injury.