Objective The current study was undertaken to adapt Equilibrium Partitioning of an Ionic Contrast agent via microcomputed tomography (EPIC-µCT) to mouse articular cartilage, which presents a particular challenge because it is thin (~30 µm) and has a small volume (0.2 – 0.4 mm3), meaning there is only approximately 2 – 4 µg of chondroitin sulfate glycosaminoglycan per joint surface cartilage. Design Using 6 µm isotropic voxels and the negatively charged contrast agent ioxaglate (Hexabrix), we optimized contrast agent concentration and incubation time, assessed two methods of tissue preservation (formalin fixation and freezing), examined the effect of ex vivo chondroitinase ABC digestion on x-ray attenuation, assessed accuracy and precision, compared young and skeletally mature cartilage, and determined patterns of degradation in a murine cartilage damage model induced by treadmill running. Results The optimal concentration of the contrast agent was 15%, formalin fixation was preferred to freezing, and 2 hours of incubation was needed to reach contrast agent equilibrium with formalin fixed specimens. There was good agreement with histologic measurements of cartilage thickness, although µCT overestimated thickness by 13% (~5 µm) in 6 week old mice. Enzymatic release of 0.8 µg of choindrotin sulfate (about 40% of the total) increased x-ray attenuation by ~17%. There was a 15% increase in x-ray attenuation in 14 week old mice compared to 6 week old mice (p < 0.001) and this corresponded to ~65% decrease in chondroitin sulfate content at 14 weeks. The older mice also had reductions of 33% in cartilage thickness and 44% in cartilage volume (p < 0.001). Treadmill running induced a 16% decrease in cartilage thickness (p = 0.012) and a 12% increase in x-ray attenuation (p = 0.006) in 14 week old mice. Conclusion This technique enables non-destructive visualization and quantification of murine femoral articular cartilage in three dimensions with anatomic specificity and should prove to be a useful new tool in studying degeneration of cartilage in mouse models.

Objective Articular cartilage defects commonly result from traumatic injury and predispose to degenerative joint diseases. To test the hypothesis that aberrant healing responses and chronic inflammation lead to osteoarthritis, we examined spatiotemporal changes in joint tissues after cartilage injury in murine knees. Since intra-articular injection of hyaluronan (HA) can attenuate injury-induced osteoarthritis in wild-type (WT) mice, we investigated a role for HA in the response to cartilage injury in mice lacking HA synthase 1 (Has1−/−). Design Femoral groove cartilage of WT and Has1−/− mice was debrided to generate a non-bleeding wound. Macroscopic imaging, histology, and gene expression were used to evaluate naïve, sham-operated, and injured joints. Results Acute responses (1–2 weeks) in injured joints from WT mice included synovial hyperplasia with HA deposition and joint-wide increases in expression of genes associated with inflammation, fibrosis, and extracellular matrix (ECM) production. By 4 weeks, some resurfacing of damaged cartilage occurred, and early cell responses were normalized. Cartilage damage in Has1−/− mice also induced early responses; however, at 4 weeks, inflammation and fibrosis genes remained elevated with widespread cartilage degeneration and fibrotic scarring in the synovium and joint capsule. Conclusions We conclude that the ineffective repair of injured cartilage in Has1−/− joints can be at least partly explained by the markedly enhanced expression of particular genes in pathways linked to ECM turnover, IL-17/IL-6 cytokine signaling, and apoptosis. Notably, Has1 ablation does not alter gross HA content in the ECM, suggesting that HAS1 has a unique function in the metabolism of inflammatory HA matrices.

We conclude that progressive articular CA damage in post-injury OA results primarily from biomechanical, cell biologic and mediator changes that promote a fibroblastic phenotype in joint cells. Since ADAMTS5 and TGFβ1 appear to control this process, agents which interfere with their activities may not only enhance endogenous CA repair in vivo, but also improve the properties of tissue-engineered CA for implantation.

The present study is the first study for the efficacy of DEI for cartilage lesions in an animal joint, from very early signs through erosion down to subchondral bone, representing the spectrum of cartilage changes occurring in human osteoarthritis (OA). Here we show that DEI allows the visualization of cartilage lesions in intact canine knee joints with good accuracy. Hence, DEI may be applicable for following joint degeneration in animal models of OA.

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