Fig. 4

Ligament cells can acquire endothelial-like phenotype and are regulated by VEGFA and PDGF-BB. a, b Representative images of capillary-like structures of human umbilical vein endothelial cells (HUVECs), Human Umbilical Cord Mesenchymal Stem Cells (hUC-MSCs) and Ligament Cells on Matrigel (left) and quantification (right). Scale bar = 100 μm. Values are means ± SD (n = 3). ***P < 0.001, ns not significant by ANOVA. c Changes in the expression level of specified genes in both ligament cells and HUVECs after undergoing endothelial differentiation on Matrigel. Values are means ± SD (n = 3). *P < 0.05, **P < 0.01, ns not significant by Student’s t-test. d Sections of Matrigel spheres loaded or unloaded with ligament cells were stained with H&E. Due to prior ink perfusion through the left ventricle, the deposition of black particles indicated the formation of functional blood vessels. The red arrows indicate the deposition of ink particles (n = 6). Scale bar = 50 μm. e The CD31 immunohistochemistry results on sections of Matrigel spheres reveal black particle deposition on the inner side of the CD31-positive area. The CD31 antibody used in this experiment specifically reacts with humans. The red arrows indicate the deposition of ink particles (n = 6). Scale bar = 50 μm. f Representative images for UEA I (red) and GS-B4 (green) staining of Matrigel spheres loaded or unloaded with OPLL-derived ligament cells (n = 6). Scale bar = 10 μm. g, h Representative images depicting capillary-like structures formed by OPLL-derived ligament cells on Matrigel under various conditions (left): unstimulated (Blank), PBS-treated (Vehicle), and stimulated with VEGFA (100 ng/mL); accompanied by quantitative analysis (right). Scale bar = 100 μm. Values are means  ±  SD (n = 3). *P < 0.05, **P < 0.01, ns not significant by ANOVA. i, j Representative images display capillary-like structures formed by OPLL-derived ligament cells on Matrigel under distinct conditions (left): unstimulated (Blank), ddH₂O (Vehicle), and PDGF-BB (100 ng/mL), including quantification (right). Scale bar = 100 μm. Values are means ± SD (n = 3). *P < 0.05, **P < 0.01, ns not significant by ANOVA. The experiments were performed in three biological replicates