Fig. 2

LOXL2 and blood vessels were elevated in OPLL and ligament cell characterization. a, b Representative images for IF staining (left) of LOXL2 (red) and quantification (right). Scale bar = 100 μm. Values are means ± SD. n = 3 for the non-OPLL group and n = 5 for the OPLL group, ***P < 0.001 by Student’s t-test. c, d Representative images for IF staining (left) of EMCN (green) and CD31 (red) of non-OPLL or OPLL samples and corresponding quantitative analysis (right). Scale bar = 100 μm. Values are means ± SD. n = 3 for the non-OPLL group and n = 5 for the OPLL group, *P < 0.05 by Student’s t-test. e, f Representative images of LOXL2 protein of primary culture cells derived from non-OPLL or OPLL by western blotting (left) and quantification (right). Values are means ± SD. n = 5, *P < 0.05 by Student’s t-test. g Representative images of trilineage differentiation of ligament cells. The left panels represent Alcian blue staining for chondrogenic differentiation; the middle panels represent ALP staining for osteogenic differentiation, and the right panels represent oil red staining for adipogenic differentiation. The upper parts represent ligament cells in the growth medium (GM), and the lower parts represent ligament cells in the differentiation medium (DM). h Ligament cells derived from OPLL expressed typical MSC surface markers, positive for CD73 (99.9%), CD90 (99.5%), CD105 (99.3%), and negative for CD45 (0%). n = 5. i High-throughput sequencing was performed on ligament cells derived from OPLL and non-OPLL samples (GSE69787). The GO pathway enrichment analysis unveiled significant differences in angiogenesis, bone metabolism, and signaling pathways governing vasculogenesis while identifying co-regulatory network presence. The experiments were performed in three biological replicates