The extra domain A of fibronectin facilitates osteoclastogenesis in radicular cysts through vascular endothelial growth factor

Abstract

Aim To analyse the effects of the alternatively spliced fibronectin (FN) gene and its isoforms on osteoclastogenesis in radicular cysts. Methodology Specimens of radicular cysts were collected surgically from 22 patients whose radiolucent periapical areas were measured on digital panoramic radiographs before surgery. The associations between the radiolucent areas and FN isoforms, vascular endothelial growth factor (VEGF) expression or micro‐vessel density, as well as the relationships amongst them, were analysed by immunohistochemical staining using the antibodies IST‐9, BC‐1, P1F11, VEGF and CD34. Fibroblasts isolated from those specimens were used to induce Trap + MNCs, and the effects of induction were assessed by blocking FN containing extra domain A (EDA + FN), COX‐2 or VEGF in vitro. The effects of EDA exon knockout using CRISPR/Cas system were also assessed. Quantitative PCR was used to analyse relative expression of FN isoforms and osteoclastogenic genes. Data were analysed using linear regression, Spearman's rank correlation analysis, chi‐square test and Student's t‐test; P < 0.05 was considered significant. Results Micro‐vessel density and EDA + FN staining were positively associated with the size of radiolucent periapical areas (mm2; P < 0.05), consistent with a positive association between Trap + MNCs and VEGF expression in fibroblasts (P < 0.05). Blocking the interaction between EDA + FN and fibroblasts inhibited Trap + MNC formation. In addition, EDA exon knockout decreased VEGF expression and inhibited Trap + MNC formation to the extent of blocking VEGF by bevacizumab, but osteoclastogenic induction was restored by recombinant VEGF. Using retrospective clinicopathological data, VEGF staining was shown to be positively associated with EDA + FN staining, micro‐vessel density and the size of radiolucent areas (P < 0.05). Conclusion In fibrous capsules of radicular cysts, the alternatively spliced isoform EDA + FN generated by fibroblasts stimulated VEGF expression via an autocrine effect and then facilitated osteoclastogenesis. Both blockage of VEGF and EDA exon knockout could be used to inhibit bone destruction.

Heterogeneity of fibroblasts from radicular cyst influenced osteoclastogenesis and bone destruction

“…In Peking University School and Hospital of Stomatology, in 2018–2019, 19 patients whose radiolucent lesions surrounded the apex with necrotic pulp or filled root were selected and surgically removed, in the Department of Oral and Maxillofacial Surgery; all the lesions were diagnosed as radicular cysts, by the Department of Pathology. The period between radiographic examination and acute periapical periodontitis was no more than one year (Yang et al, 2019). As described previously (Yang et al, 2019; Zizzi et al, 2013), within at least four months before surgical excision, patients had not taken antibiotics or medication for inflammation.…”

“…The period between radiographic examination and acute periapical periodontitis was no more than one year (Yang et al, 2019). As described previously (Yang et al, 2019; Zizzi et al, 2013), within at least four months before surgical excision, patients had not taken antibiotics or medication for inflammation. Every patient has written the consent to use specimens for research, and it was approved by Peking University School of Stomatology Institutional Review Board (Beijing, China; permit number: PKUSSIRB201838112).…”

“…The osteoclast precursor, Raw264.7 cells, was cultured in Dulbecco's modified Eagle's medium (DMEM) (GIBCO), containing 100 g/ml streptomycin, 100 U/ml penicillin, 2 mM L‐glutamine, and 10% fetal bovine serum (FBS). Cells were maintained at 37°C, in the 95% humidity, and atmosphere of 5% CO 2 (Liu & Wang, 2019; Wang et al, 2019; Yang et al, 2019).…”

“…In our previous studies, the fibroblasts that were in contact with monocytes promoted osteoclastogenesis, via unregulated IL‐6 and VEGF‐A (Wang & Li, 2013); the interaction between epithelia and fibroblasts increased the ratio of RANKL/OPG, and hence, osteoclastogenesis was facilitated (Wang, Jiang, Sima, & Li, 2015). However, in radicular cysts, the effects of fibronectin isoforms (EDB + FN, CS1‐FN, and especially EDA + FN) on the fibroblast itself influenced the expression of osteoclastogenesis‐related genes, such as COX‐2, IL‐6, IL‐17, M‐CSF, IL‐1α, TNF‐α, RANKL, OPG, or VEGF‐A (Liu & Wang, 2019; Wang, Wang, Chen, & Zhang, 2019), which led to osteoclastogenesis and bone destruction (Yang, Jiang, Wang, & Li, 2019). Odontogenic cysts are developed from inflammation or odontogenic epithelia rests, the jaw bone was destructed and formed cavities, and, microscopically, cavities are surrounded by lining epithelia and outer fibrous capsules; therefore, it is similar to other pathological conditions (Mizoguchi et al, 2018; Suda et al, 2016; Xie et al, 2018), the inflammatory cells or cytokines, abnormal ECM, epithelia or endothelia in cysts perhaps also facilitating the differentiation of fibroblast subsets to diversify their function in osteoclastogenic induction.…”

“…Apical inflammation stimulates surrounding periodontal ligament, in which the fibroblasts proliferate and form the fibrous capsules circumventing the lining epithelia and cavity of cysts, together with accumulated collagen (Garcia et al, 2007). As the apical lesion enlarging in the jawbone, mesenchymal stroma cells (MSCs) could be recruited in the expanded capsules, participating angiogenesis, ECM synthesis, osteogenesis, and osteoclastogenesis (Yang et al, 2019).…”