Figure 4.

Canal invasion precedes endothelial cell recruitment and is led by chondrocyte extracellular matrix (ECM) degradation. (A): Canal initiation was led by Tomato^H cells (white arrows) and trailed by blood vessels stained with the endothelial marker endomucin. Bar = 50 μm. (Ba): Canal invasion was preceded by an “empty space” between the canal front (dotted white line) and the surrounding chondrocytes (dotted yellow line), containing only Ter119⁺ erythroid lineage cells (yellow arrow). Bar = 20 μm. (Bb): Within the canal, CD45⁺ hematopoietic cells were either occasionally associated with immature vasculature (yellow arrow) or away from endothelial cells (blue arrow). Double-headed arrow shows gap between canal front and epiphyseal chondrocytes. Bar = 20 μm. (C): Immunofluorescent images show intense staining for chondrocyte ECM degradation around the invading canal. Double-headed arrow marks gap between canal and epiphyseal chondrocytes. Bar = 50 μm. (D): Staining with H&E confirmed a cell-free gap, with residual fiber or matrix components in the observed empty space (black arrows). Bar = 50 μm.