Collagen cross-linking inhibitor BAPN causes structural changes in the collagen content and blood vessel formation. a, b, c, d demonstrated reconstructed overview of the fish ray containing bones, cartilage, blood vessels and blastema. Amputation plane by the control group 3dpa (a’, a’’, a’’’) and 7dpa (c’, c’’, c’’’) versus BAPN-treated group at 3dpa (b’, b’’, b’’’) and 7dpa (d’, d’’, d’’’) is shown. a–a’’ display perfused, normal blood vessel with well-differentiated endothelial cells (EC), red blood cells (RBC), solitary pigment cells (PC), bones, cartilage (Ca) and compact, dense ECM containing collagen cross-linked fibers (CF). CF are a natural compartment of the cartilage (Ca) during the early regenerative phase (a’); they are also a major component of ECM and located along blood vessel making long sleeves (a’’, arrows) or enclosing the fibroblasts (Fb) and tissue macrophages (MΦ). Higher magnification revealed classical collagen cross-linked fibers (a’’’). In the BAPN-treated fish, at the 3dpa, the intraray regions are enlarged containing dilated blood vessels with impacted RBCs and extravasal RBCs in front of the latter, i.e. bleeding (b, b’). The EC appear seldom (b’’, b’’’, arrows), cover only partially the capillaries and the RBCs are in a direct contact with the amorphous ECM (b’’’). Classical cross-linked CFs are not detectible; instead, Collagen fibrils (Cfl) appear as amorphous material (b’’, b’’’). c/c’ display amputation plane by the control group at 7dpa. Perfused and normal blood vessel is covered by well-differentiated ECs. ECM is compact and dense containing PC, Fb and collagen cross-linked fibers. Collagen is present within the cartilage (Ca) and along blood vessel forming long sleeves (c’’, c’’’, arrows). d/d’ display amputation plane by the BAPN-treated group at 7dpa in which balloon-like enlarged blood vessels (BV) containing densely packed RBCs are documented. Those angioma-like structures are perfused and contains partially ECs (d’’, green arrows). MΦ and Fb are embedded in ECM with loose and amorphous appearance due to the absent classical cross-linked CF; instead of it Cfl are widely presented (d’’, d’’’), left side - zebrafish trunk, right side - zebrafish tail, images acquired by electron microscope.
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