Purpose: Most adult tissues contain stem and progenitor cells known to contribute to tissue maintenance and repair. While tissue-resident mesenchymal progenitor cells (MPCs) have been identified in multiple niches in the adult knee joint, articular cartilage shows poor healing capacity following injury. Previously, it has been demonstrated that the constitutive deletion of the p21 gene, a cell cycle regulator, promotes cartilage regeneration. However, the underlying mechanisms of this regenerative phenotype remain largely unknown, such as signaling pathways and cell types involved. We hypothesized that activation of the cell cycle solely in endogenous adult MPCs would enhance cartilage regeneration in vivo post-injury.
Methods: To test this, we targeted the p21 downstream effector E2F1 and overexpressed it in quiescent MPCs. This was accomplished by developing a conditionally overexpressing E2F1 mouse that was bred to a Hic1CreERT2: Rosa26LSL-TdTomato MPC reporter mouse to generate the Hic1CreERT2; Rosa26LSL-TdTomato; E2F1EGFP offspring. TdTomato reporter gene expression and E2F1 expression was induced in MPCs following administration of tamoxifen at 8 weeks of age. Subsequently, all mice were subjected to full-thickness cartilage defects in the trochlear groove of their distal femur. Knee joints were harvested at 1-, 2-, 4- and 8-weeks post-injury. Histology (2D structure/composition), high-resolution 3D X-ray microscopy (XRM imaging), immunofluorescence (lineage tracking), quantitative proteomics and biomechanical testing outcome measures were employed to assay for cartilage regeneration.
Results: Overexpression of E2F1 in Hic1+ve MPCs resulted in enhanced cartilage regeneration compared to controls. The defect site in control mice lacked proteoglycan staining, surface regularity, and was filled with fibrocartilage-like tissue, while articular cartilage-like tissue was observed in the E2F1 overexpressor by 4-weeks post-injury. Such differences were reflected in the histological score with significant differences between the groups by 4- and 8-weeks post-injury, which was corroborated by the high-resolution 3D imaging of the femurs, indicating an enhanced articular cartilage repair within E2F1 mice. Proteomic analysis demonstrated that extracellular matrix production and secretion were dysregulated between Hic1+ve MPCs derived from the synovium and bone marrow in E2F1 mice, with increased production of matrix proteins observed in synovium-derived MPCs. E2F1mice demonstrated altered stiffness distribution within the repaired tissue area, with lower stiffness values compared to uninjured controls (Figure 1). Interestingly, while Hic1-lineage cells (TdTomato+ve) were seen within the injury site in both strains, by 4-weeks after injury, few to no cells were present. Furthermore, when the articular cartilage layer was nearly regenerated, new chondrocytes at the injury site were found to be TdTomato-ve.
Conclusions: These findings suggest that activation of cell cycle pathways in MPCs promotes cartilage regeneration. Furthermore, while activated MPCs migrated to the injury site, few to no cells were seen within the newly formed fibro or hyaline-like cartilage tissues in control vs. E2F1, respectively. Therefore, while these MPCs are necessary for articular cartilage regeneration, they are not responsible for new tissue formation. Hence, future studies are required to elucidate the role of E2F1 overexpression and the downstream effects on bone and cartilage tissues after injury.
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