Heterogeneity of fibroblasts from radicular cyst influenced osteoclastogenesis and bone destruction.

Aim To analyze the heterogeneity of fibroblasts isolated from the fibrous capsules of radicular cysts and explore the effects of fibroblast subsets on bone destruction. Methodology Radicular cysts were divided into groups according to varying perilesional sclerosis identified by radiograph. Colony-forming units (CFUs) were isolated from the fibrous capsules of cysts, by which Trap + MNCs were induced, and the expression of osteoclastogenesis-related genes was compared among groups by real-time PCR. The variances in gene profiles of CFUs were identified by principal component analysis, and then, CFUs were divided into subsets using cluster analysis. The induction of Trap + MNCs and related gene expression was compared among subsets, and osteoclastogenic induction was blocked by IST-9 or bevacizumab. The fibroblast subsets in cysts were investigated by retrospective immunostaining with IST-9, VEGF-A, and CD34. A fibroblast subset that underwent gene editing by CRISPR/Cas was injected into the site of bone defects in animal models, and the in vivo effects on osteoclastogenesis were investigated. Results The fibroblast CFUs isolated from radicular cysts with perilesional unsclerotized cysts induced more Trap + MNCs than those with perilesional sclerotic cysts (p < .05). Most fibroblast CFUs from unsclerotized cysts belonged to Cluster 2, which induced more Trap + MNCs (p < .05) and highly expressed genes facilitating osteoclastogenesis; these results were different from those of Cluster 1 (p < .05), in which most CFUs were isolated from perilesional sclerotic cysts or controls (p < .05). The high expression of EDA + FN and VEGF-A was investigated in both the fibroblasts of Cluster 2 and the fibrous capsules of unsclerotized cysts (p < .05), and the number of Trap + MNCs induced by Cluster 2 was decreased by treatment with IST-9 and bevacizumab (p < .05). Consistently, EDA exon exclusion significantly decreased the osteoclastogenic induction of fibroblasts from Cluster 2 in vivo (p < .05). Conclusion The fibrous capsules of radicular cysts contain heterogeneous fibroblasts that can form subsets exhibiting different effects on osteoclastogenesis. The subset, which depending on the autocrine effects of EDA + FN on VEGF-A, mainly contributes to the osteoclastogenesis and bone destruction of radicular cysts. The regulation of the proportion of subsets is a possible strategy for artificially interfering with osteoclastogenesis.