The fibroblast of radicular cyst facilitate osteoclastogenesis via the autocrine of Fibronectin containing extra domain A

Abstract

Objective To investigate the alternative spliced isoforms of Fibronectin (FN) in the stroma of radicular cysts and analyze the associations between these isoforms and the osteoclastogenic effects of fibroblasts. Methods and materials The specimens of radicular cysts were stained with immunohistochemistry, and the associations between each FN isoform and clinical parameters were assessed. The fibroblasts isolated from cysts or jaw bone were cultured to induce the Trap + MNCs. In the conditioned medium, the Fibronectin containing extra domain A (EDA + FN) was neutralized by antibody IST‐9, and the EDA exon of fibroblasts was knockout by CRISPR/Cas system, for assessing the osteoclastogenic effects. The mRNA level of FN isoforms and the osteoclastogenesis‐related genes were analyzed by quantitive PCR. Results EDA + FN staining was positively associated with the size of the lesions (p < 0.05). In contrast with the controls, the ratio of EDA + FN/total FN in the fibroblasts from radicular cysts was significantly higher (p < 0.05), and positively associate with Trap + MNCs counting, it was consistent with increased expression of COX‐2, IL‐6, IL‐17, and the RANKL/OPG (p < 0.05). The Trap + MNCs counting and osteoclastogenesis‐related genes were decreased by IST‐9 blocking and EDA exon knockout in fibroblasts, but the blockage of the interaction between EDA + FN and pre‐osteoclasts exhibited little effects on Trap + MNCs formation. Conclusion The microenvironment of the fibrous capsule of radicular cysts facilitates the splicing of EDA exon, it endues EDA + FN with autocrine effects on fibroblast itself, and it increases the expression of osteoclastogenesis‐related genes, by which the osteoclastogenesis in radicular cysts could be initiated.

Heterogeneity of fibroblasts from radicular cyst influenced osteoclastogenesis and bone destruction

“…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).…”

“…As described previously(Liu & Wang, 2019), from the surgical specimens of 19 cases of radicular cysts, the fibroblasts were isolated from the fibrous capsules of cysts and routinely cultured in α‐modified Eagle's medium (α‐MEM) (GIBCO). The fibroblasts from the jawbone of 6 patients who were treated with orthognathic surgery were taken as controls (Liu & Wang, 2019; Wang et al, 2019; Yang et al, 2019).…”

“…As described previously(Liu & Wang, 2019), from the surgical specimens of 19 cases of radicular cysts, the fibroblasts were isolated from the fibrous capsules of cysts and routinely cultured in α‐modified Eagle's medium (α‐MEM) (GIBCO). The fibroblasts from the jawbone of 6 patients who were treated with orthognathic surgery were taken as controls (Liu & Wang, 2019; Wang et al, 2019; Yang et al, 2019). As described previously (Wang & Li, 2013), the fibroblasts were seeded into 100 mm dishes at a density of 1,000 cells/dish for 14 days, the aggregations containing no less than 50 cells were defined as a colony formation units (CFUs), and crystal violet was used to stain the cells.…”

“…When fibroblasts had grown to the confluence of 70 ~ 80% (i.e., cells have taken up 70 ~ 80% of the area of a dish) in the 60 mm or 100 mm dishes, serum‐free α‐MEM was used to replace the previous medium, and cells were maintained for seven days. Then, the supernatants were collected and centrifuged for 10 min at 550 g , 40% fresh α‐MEM (GIBCO), and 10% FBS was added into the 50% aliquoted supernatant, to make conditioned medium (Liu & Wang, 2019; Wang et al, 2019; Yang et al, 2019). Only 39 CFUs had survived and grown sufficient fibroblasts for making the conditioned medium.…”

“…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.…”