Epithelial necrotic cells provoke exacerbated inflammatory response in gingival fibroblasts

Abstract

Abstract Objectives: to study how cellular lysates could affect the expression of inflammatory cytokines of periodontal-related cells. Materials and Methods: cell suspensions were submitted to different treatments: lysate 1 – no treatment; lysate 2 – sonication; and lysate 3 – freeze/thawing. All lysates were centrifuged, and the supernatants were used for experimentation. Cell viability assays, RT-qPCR of IL1, IL6 and IL8, IL6 immunoassay, and immunofluorescence of NF-kB were applied to verify the inflammatory crosstalk potential of damaged cells over healthy plated cells. Further, titanium discs and collagen membranes were treated with the cell lysates and checked for IL8 RT-qPCR expression. Results: lysates obtained upon sonication or freeze/thawing of oral squamous cell carcinoma cell lines provoked a robust increase in the expression of IL1, IL6, and IL8 by gingival fibroblasts, which was confirmed by immunoassays for IL6. Lysates obtained from the gingival fibroblasts failed to increase the expression of inflammatory cytokines in oral squamous cell carcinoma cells. Epithelial cell lysates caused the activation of the NF-kB signalling cascade in gingival fibroblasts as indicated by the phosphorylation and nuclear translocation of p65, indicating inflammation. Finally, epithelial lysates could adhere to the titanium and collagen membrane surfaces and increase IL8 expression by gingival fibroblasts. 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, prostheses maladaptation, implant drilling, and teeth preparation.