SummaryWe investigated the involvement of oral epithelial cells via two cytokines (IL-6 and TNF-a a a a ) and one chemokine (IL-8) in local defences against live yeast (C andida albicans ) and bacteria ( Streptococcus salivarius ) using an engineered human oral mucosa model. We report that the yeast changed from the blastospore to the hyphal form and induced significant tissue disorganization at later contact periods (24 and 48 h) compared to the bacteria. However, this effect did not reduce the viability or total number of epithelial cells. Gene activation analyses revealed that IL-6, IL-8 and TNF-a a a a mRNA levels rose in tissues in contact with live C. albicans or S. salivarius. Gene activation was followed by an upregulation of protein secretion. IL-6 levels were higher after contact with C. albicans than with S. salivarius . IL-8 levels after contact with S. salivarius were higher than with C. albicans . Our study suggests that S. salivarius is more efficient at inducing proinflammatory mediator release than C. albicans . These results provide additional evidence for the contribution of oral epithelial cells to the inflammatory response against fungi and bacteria.

Under physiological conditions, matrix metalloproteinases (MMPs) are involved in the remodeling and turnover of periodontal tissue and their activity is tightly regulated by tissue inhibitors of metalloproteinases (TIMPs). Disturbances in the balance between MMPs and TIMPs may result in excessive tissue destruction. We previously used an engineered human oral mucosa (EHOM) model to demonstrate that Porphyromonas gingivalis, a major etiological agent of periodontitis, infiltrates connective tissue and induces significant loss of attachment of the stratified epithelium from the basement membrane. The aim of the present study was to investigate the effect of P. gingivalis on the expression and production of MMP-2, MMP-9, TIMP-1, and TIMP-2 by oral fibroblasts and epithelial cells. The EHOM model was infected with P. gingivalis ATCC 33277 or its derivative gingipain-null mutant (KDP128) for different periods of time. MMP and TIMP mRNA expression was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) analysis, while protein secretion into the culture medium was assessed by enzyme-linked immunosorbent assays. P. gingivalis significantly up-regulated MMP-2 and MMP-9 mRNA expression by oral epithelial cells. This MMP gene activation was paralleled by TIMP-2 gene activation. However, only MMP-9 mRNA expression was significantly enhanced by the gingipain-null mutant. At 8 and 24 h post-infection, P. gingivalis increased significantly the MMP-9 protein level compared to the uninfected EHOM model. The present study reports the ability of P. gingivalis to regulate MMP and TIMP production by oral cells, a phenomenon that may contribute to tissue destruction.

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The use of dentifrice is part of an oral prophylaxis that aims at keeping bacteria in check within the dental plaque. When introduced into the oral cavity, dentifrice also comes in close contact with the oral epithelium. Our goal was to evaluate the effects of dentifrices on tissue structure and pro-inflammatory mediator release by epithelial cells. For this purpose, tri-dimensional engineered human oral mucosa (EHOM) was produced using normal human palatal fibroblasts and epithelial cells. EHOMs were either treated with Aquafresh(R) or Crest(R) for 1, 4, 8, and 24 h, or untreated, then used for cell viability assessment and structural analyses. Cultured supernatants were used to evaluate cytokine (interleukin (IL)-1beta, IL-8 and tumor necrosis factor (TNF)-alpha) secretion, and metalloproteinase (MMP)-2 and -9 activities. The present in vitro study using engineered oral mucosa confirms that dentifrices (Aquafresh and Crest) contribute to tissue desquamation. The desquamation was substantial at 24 h of contact but was limited to the upper layers of the treated tissues. Cell death in these tissues was not increased, suggesting that the dentifrice had accelerated desquamation of the layers containing differentiated cells. Measurement of cytokines revealed that dentifrices up-regulated IL-1beta while down-regulating IL-8 and TNF-alpha secretion, thus indicating an impaired cascade of inflammatory responses. These dentifrices may also impair normal repair mechanisms as suggested by an up-regulation of gelatinase activities. In conclusion, this study suggested that, via cytokines, dentifrice contributes to the modulation of the inflammatory (pro-inflammatory/anti-inflammatory responses) process.

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