Characterization of cellular and matrix alterations in the early pathogenesis of osteochondritis dissecans in pigs using second harmonic generation and two-photon excitation fluorescence microscopy.

Osteochondritis dissecans is a joint disease that is observed in several species. The disease can develop as a cause of ischemic chondronecrosis in the epiphyseal growth cartilage. Some lesions of chondronecrosis undergo spontaneous resolution, but it is not possible to predict whether a lesion will resolve or progress and require intervention. Proliferation of cells into clusters occurs at the lesion margin, but it is unclear if the clusters have a repair function. The aims of the current study were to examine clusters and potential matrix changes in response to ischemic chondronecrosis in the distal femur of 10 pigs aged 70-180 days using advanced microscopy based on two-photon excitation fluorescence and second harmonic generation. These microscopy techniques can perform 3D imaging of cells and collagen without staining. The results indicated a lower collagen density in the chondronecrotic areas compared to the normal growth cartilage, and fissures and breaks in the matrix integrity were demonstrated that potentially can propagate and cause osteochondritis dissecans. A higher number of cells in clusters was correlated with reduction in collagen density in the lesions. Some of the cells in the clusters had a morphology similar to progenitor cells, suggesting a potential repair role of the clusters. The study has shed further light on the secondary responses after initial lesion formation, which information can be of potential use to create models that can predict lesion progression and that may hence avoid unnecessary interventions in the future.