Oral squamous carcinoma cell lysates provoke exacerbated inflammatory response in gingival fibroblasts

Abstract

Abstract Objectives To study whether damaged epithelial cells and gingival fibroblast could affect the expression of inflammatory cytokines in healthy cells. Materials and methods Cell suspensions were submitted to different treatments to obtain the lysates: no treatment (supernatant control), sonication, and freeze/thawing. All treatments were centrifuged, and the supernatants of the lysates were used for experimentation. Cell viability assays, RT-qPCR of IL1, IL6 and IL8, IL6 immunoassay, and immunofluorescence of NF-kB p65 were applied to verify the inflammatory crosstalk of damaged cells over healthy plated cells. Furthermore, titanium discs and collagen membranes were treated with lysates and checked for IL8 expression by RT-qPCR. Results Lysates obtained upon sonication or freeze/thawing of oral squamous carcinoma cell lines provoked a robust increase in the expression of IL1, IL6, and IL8 by gingival fibroblasts, which was confirmed by IL6 immunoassays. Lysates obtained from the gingival fibroblasts failed to increase the expression of inflammatory cytokines in oral squamous carcinoma cells. Additionally, oral squamous carcinoma cell lysates caused the activation of the NF-kB signalling cascade in gingival fibroblasts as indicated by the phosphorylation and nuclear translocation of p65. Finally, oral squamous carcinoma cell lysates adhered to the titanium and collagen membrane surfaces and increased IL8 expression by gingival fibroblasts growing in these materials. Conclusions Injured oral epithelial cells can release factors that incite gingival fibroblasts to become pro-inflammatory. Clinical relevance Injuries affecting the oral mucosa generate epithelial fragments that may reach the underlying connective tissue and provoke inflammation. These injuries are routinely caused by mastication, sonication for teeth cleaning, teeth preparation, prostheses maladaptation, and implant drilling.