Cultured Gingival Fibroblasts as a Model of Oral Mucosa Inflammation

Objectives: The objective of this study was to establish parameters of inflammation and cell death in cultured human gingival fibroblasts as an in vitro model of oral mucosa inflammation. Methods: The human gingival fibroblast cell line HGF-1 (ATCC CRL-2014) was cultured in medium containing 10% serum and then serum-starved. In one set of experiments fibroblasts were treated with 0.12% chlorhexidine digluconate for 30 seconds, and then allowed to recover in serum-free medium for various times. In a second set of experiments the fibroblasts were treated with 20 ng/ml TNF-alpha for various times. Untreated cells were controls. After treatment, culture medium was assayed for the pro-inflammatory cytokines IL-8 and IL-1beta by ELISA. Interleukin secretion data was determined as mean concentration in pg/ml +/- SEM. Apoptosis was assayed colorimetrically using the ApoPercentage assay. Apoptotic cells were counted in digitally captured images. Apoptosis data was determined as mean number of apoptotic cells +/- SEM. Statistical analysis was performed using the student's t-test with significance value of p < 0.05. Results: TNF-alpha treatment of human gingival fibroblasts resulted in a significant increase in the secretion of IL-8, and apoptosis, compared to untreated control. IL-8 secretion was not detected in fibroblasts treated with chlorhexidine. IL-1beta secretion was undetected in control, TNF-alpha-treated, and chlorhexidine-treated cells, suggesting either a lack of production of this interleukin, or secretion at a level that is below the detection limit of the ELISA. TNFalpha treatment of human gingival fibroblasts resulted in a significant increase in apoptosis compared to untreated control. Chlorhexidine, in our system, appeared to be very cytotoxic to the fibroblasts when assayed using the apoptosis marker. Conclusions: TNF-alpha-treated, but not chlorhexidine-treated, human gingival fibroblasts are a suitable in vitro model of oral mucosa inflammation. Markers of inflammation in this model include the secretion of IL-8.