Human β-defensin-2 and IL-6 secretions by gingival epithelial cells were maintained over multiple cell passages following exposure to cigarette smoke.

<p>Confluent (80%) gingival epithelial cell cultures were exposed to whole cigarette smoke for 15 min or left untreated, followed by culture for 24 h, after which supernatants were collected and used to determine the levels of HBD2 or IL-6 by ELISA. Data are expressed as the means+SD (n = 5).</p

The effect of whole cigarette smoke on TLR2, human β-defensin-2, and IL-6 expression by gingival epithelial cells was maintained over multiple cell passages.

<p>Confluent (80%) gingival epithelial cell cultures were exposed to whole cigarette smoke for 15 min or left untreated, followed by culture for 3 h, after which the cells were detached and used either to extract total RNA or to subculture for 4 days. This subculture was repeated twice every 3 days for a total culture period of 10 days. Extracted RNAs from the different conditions were analyzed by qRT-PCR. Data are expressed as the means+SD (n = 5); *, p<0.0001.</p

Whole cigarette smoke promoted human β-defensin-2 and -3 secretions through the ERK1/2 MAP kinase and NFκB signaling pathways.

<p>Confluent (80%) gingival epithelial cell cultures were incubated with 10 µM of ERK inhibitor (PD98059), 10 µM of p38 inhibitor (SB202190), 10 µM of JNK inhibitor (SP600125), or 10 µM of NF-κB inhibitor (IKK-2) for 45 min before exposure or not to cigarette smoke for 15 mn. 24 hours later, supernatants were collected and used to measure the β-defensin secretion levels by ELISA kits. Data are expressed the means+SD, (n = 4).</p

Human IL-1β and IL-6 gene and protein expression levels increased after normal human gingival epithelial cells were exposed to whole cigarette smoke.

<p>Confluent (80%) gingival epithelial cell cultures were exposed to whole cigarette smoke for 15 or 30 min or left untreated and then cultured for various time periods prior to gene expression analysis by qRT-PCR (5a and 5c). Data are expressed as the means+SD (n = 5); *, p<0.01. IL-1β and IL-6 levels were quantified by ELISA (5b and 5d) of the cells exposed to cigarette smoke and post-cultured for 24 h. Data are expressed the means+SD, (n = 6). **, p<0.001.</p

Human β-defensin-2 and -3 gene and protein expression levels increased when normal human gingival epithelial cells were exposed to cigarette smoke.

<p>Confluent (80%) gingival epithelial cell cultures were exposed to whole cigarette smoke for 15 or 30 min or left untreated and subsequently cultured for various time periods prior to gene expression analysis by qRT-PCR (3a and 3c). Data are expressed as the means+SD (n = 5); *, p<0.01. The β-defensin secretion levels were quantified by ELISA (3b and 3d) of the supernatant of the cells exposed to cigarette smoke and post-cultured for 24 h. Data are expressed the means+SD, (n = 6). **, p<0.0001.</p

Primer sequences used for the qRT-PCR.

<p>Primer sequences used for the qRT-PCR.</p

Whole cigarette smoke promoted human β-defensin-2 and -3 expression through the ERK1/2 MAP kinase and NFκB signaling pathways.

<p>Confluent (80%) gingival epithelial cell cultures were incubated with 10 µM of ERK inhibitor (PD98059), 10 µM of p38 inhibitor (SB202190), 10 µM of JNK inhibitor (SP600125), or 10 µM of NFκB inhibitor (IKK-2) for 45 min before exposure to cigarette smoke for 15 or 30 min. Six hours later, total RNA was extracted, and HBD gene expression was analyzed by qRT-PCR (n = 6). *, p<0.05; **, p<0.01 when comparing values obtained in the presence and absence of inhibitor. The results are expressed as the means+SD, (n = 5).</p

Effect of ES on fibroblast α-SMA expression.

<p>Dermal fibroblasts were seeded on conductive PPy/HE/PLLA membranes followed by exposure to 50 or 200 mV/mm for various periods. The cells were then detached from the conductive membranes, washed, seeded on coverslips, and cultured up to 70% confluence. The cells were then stained using relevant monoclonal antibodies. Cytoplasmically immunolabelled myofibroblasts are presented.</p

Effect of ES on dermal fibroblast viability.

<p>Cells were seeded on the conductive PPy/HE/PLLA membranes and cultured for 24 h, then exposed to ES of 50 or 200 mV/mm for 2, 4, or 6 h, and subsequently cultured for an additional 24 h. The cells were then detached from each membrane and cell viability was ascertained by trypan blue exclusion assay (<i>n</i> = 5). The ES-exposed and non-exposed cultures were compared, with the difference considered significant at p<0.05.</p