Bevacizumab or fibronectin gene editing inhibits the osteoclastogenic effects of fibroblasts derived from human radicular cysts

Abstract

Fibronectin (FN) is a main component of extracellular matrix (ECM) in most adult tissues. Under pathological conditions, particularly inflammation, wound healing and tumors, an alternatively spliced exon extra domain A (EDA) is included in the FN protein (EDA + FN), which facilitates cellular proliferation, motility, and aggressiveness in different lesions. In this study we investigated the effects of EDA + FN on bone destruction in human radicular cysts and explored the possibility of editing FN gene or blocking the related paracrine signaling pathway to inhibit the osteoclastogenesis. The specimens of radicular cysts were obtained from 20 patients. We showed that the vessel density was positively associated with both the lesion size (R = 0.49, P = 0.001) and EDA + FN staining (R = 0.26, P = 0.022) in the specimens. We isolated fibroblasts from surgical specimens, and used the CRISPR/Cas system to knockout the EDA exon, or used IST-9 antibody and bevacizumab to block EDA + FN and VEGF, respectively. Compared to control fibroblasts, the fibroblasts from radicular cysts exhibited significantly more Trap + MNCs, the relative expression level of VEGF was positively associated with both the ratio of EDA + FN/total FN (R = 0.271, P = 0.019) and with the number of Trap + MNCs (R = 0.331, P = 0.008). The knockout of the EDA exon significantly decreased VEGF expression in the fibroblasts derived from radicular cysts, leading to significantly decreased osteoclastogenesis; similar results were observed using bevacizumab to block VEGF, but block of EDA + FN with IST-9 antibody had no effect. Furthermore, the inhibitory effects of gene editing on Trap + MNC development were restored by exogenous VEGF. These results suggest that EDA + FN facilitates osteoclastogenesis in the fibrous capsule of radicular cysts, through a mechanism mediated by VEGF via an autocrine effect on the fibroblasts. Bevacizumab inhibits osteoclastogenesis in radicular cysts as effectively as the exclusion of the EDA exon by gene editing.

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 (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.…”

“…Conditioned medium was refreshed at the interval of 1 day for 10 days, and then, cells were stained with tartrate‐resistant acid phosphatase (TRAP) kit (Sigma). Cells containing no less than three nuclei and stained with TRAP (Trap + MNCs) were defined as osteoclast‐like cells (Liu & Wang, 2019; Wang et al, 2019; Yang et al, 2019). The conditioned medium of each CFU was added into four wells, for inducing the Trap + MNCs.…”

“…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.…”