Roles of Lipids in the Permeability Barriers of Skin and Oral Mucosa.

PubMed searches reveal much literature regarding lipids in barrier function of skin and less literature on lipids in barrier function of the oral mucosa. In terrestrial mammals, birds, and reptiles, the skin's permeability barrier is provided by ceramides, fatty acids, and cholesterol in the outermost layers of the epidermis, the stratum corneum. This layer consists of about 10-20 layers of cornified cells embedded in a lipid matrix. It effectively prevents loss of water and electrolytes from the underlying tissue, and it limits the penetration of potentially harmful substances from the environment. In the oral cavity, the regions of the gingiva and hard palate are covered by keratinized epithelia that much resemble the epidermis. The oral stratum corneum contains a lipid mixture similar to that in the epidermal stratum corneum but in lower amounts and is accordingly more permeable. The superficial regions of the nonkeratinized oral epithelia also provide a permeability barrier. These epithelial regions do contain ceramides, cholesterol, and free fatty acids, which may underlie barrier function. The oral epithelial permeability barriers primarily protect the underlying tissue by preventing the penetration of potentially toxic substances, including microbial products. Transdermal drug delivery, buccal absorption, and lipid-related disease are discussed.

Protein Analysis of Sapienic Acid-Treated Porphyromonas gingivalis Suggests Differential Regulation of Multiple Metabolic Pathways.

UNLABELLED: Lipids endogenous to skin and mucosal surfaces exhibit potent antimicrobial activity against Porphyromonas gingivalis, an important colonizer of the oral cavity implicated in periodontitis. Our previous work demonstrated the antimicrobial activity of the fatty acid sapienic acid (C(16:1Δ6)) against P. gingivalis and found that sapienic acid treatment alters both protein and lipid composition from those in controls. In this study, we further examined whole-cell protein differences between sapienic acid-treated bacteria and untreated controls, and we utilized open-source functional association and annotation programs to explore potential mechanisms for the antimicrobial activity of sapienic acid. Our analyses indicated that sapienic acid treatment induces a unique stress response in P. gingivalis resulting in differential expression of proteins involved in a variety of metabolic pathways. This network of differentially regulated proteins was enriched in protein-protein interactions (P = 2.98 × 10(-8)), including six KEGG pathways (P value ranges, 2.30 × 10(-5) to 0.05) and four Gene Ontology (GO) molecular functions (P value ranges, 0.02 to 0.04), with multiple suggestive enriched relationships in KEGG pathways and GO molecular functions. Upregulated metabolic pathways suggest increases in energy production, lipid metabolism, iron acquisition and processing, and respiration. Combined with a suggested preferential metabolism of serine, which is necessary for fatty acid biosynthesis, these data support our previous findings that the site of sapienic acid antimicrobial activity is likely at the bacterial membrane. IMPORTANCE: P. gingivalis is an important opportunistic pathogen implicated in periodontitis. Affecting nearly 50% of the population, periodontitis is treatable, but the resulting damage is irreversible and eventually progresses to tooth loss. There is a great need for natural products that can be used to treat and/or prevent the overgrowth of periodontal pathogens and increase oral health. Sapienic acid is endogenous to the oral cavity and is a potent antimicrobial agent, suggesting a potential therapeutic or prophylactic use for this fatty acid. This study examines the effects of sapienic acid treatment on P. gingivalis and highlights the membrane as the likely site of antimicrobial activity.

The Effect of Delivery System and Light Activation on Tooth Whitening Efficacy and Hydrogen Peroxide Penetration.

PURPOSE: To evaluate the whitening efficacy of a new two-layer technology in-office system compared to a conventional gel-type system and determine hydrogen peroxide penetration (HPP) into the pulp cavity. MATERIALS AND METHODS: Extracted molars (n = 60) were assigned to group NC: glycerol gel; group QPRO: 20% HP varnish (Zoom Quick Pro, Philips Oral Healthcare); group ZOOM_NL: 25% HP gel (Zoom Chairside Whitening); and group ZOOM_WL: 25% HP gel (Zoom Chairside Whitening) with light-activation. HPP levels were estimated with leucocrystal-violet and horseradish-peroxidase. Instrumental color measurements were performed at baseline (T0 ), 1-day post first whitening (T1 ), 1-day post second whitening (T2 ), 1-day post third whitening (T3 ), and 1-month post whitening (T4 ). One-way analysis of variance followed by post hoc Tukey's HSD test was performed to detect difference in ΔE* and HP penetration levels (α = 0.05). RESULTS: ΔE* of NC was lower than other groups, whereas ΔE* of ZOOM_WL was greater than the other three groups, at T3 and T4 . Mean HPP level obtained from ZOOM_WL (1.568 ± 0.753 µg/mL) was significantly greater than those obtained from the other groups, whereas the mean HPP level observed in NC group (-0.131 ± 0.003 µg/mL) was significantly lower than the other groups. CONCLUSIONS: Tooth whitening efficacy and HPP levels vary based on whitening systems used. CLINICAL SIGNIFICANCE: The two-layer technology in-office varnish system may be an alternative whitening option to reduce chair time in the office. (J Esthet Restor Dent 28:313-320, 2016).

Time Course of Potassium Nitrate Penetration into the Pulp Cavity and the Effect of Penetration Levels on Tooth Whitening Efficacy.

OBJECTIVES: To establish time-course of potassium nitrate (PN) penetration into the pulp cavity, and determine whether PN pretreatment would affect whitening efficacy. MATERIALS AND METHODS: Extracted teeth (n = 100) were randomized into five groups of 20 specimens each. Relief ACP (Philips Oral Healthcare, Los Angeles, CA, USA) was applied for 0, 5, 15, 30, and 60 minutes for groups 15, respectively. A nitrate/nitrite assay kit was used for colorimetric detection of nitrate. Whitening was performed using a Zoom White Speed system (Philips Oral Healthcare) for 60 minutes. Tooth color was measured with a spectrophotometer at baseline (T0 ), 1-day post PN application (T1 ), 1-day post-whitening (T2 ), and 1-month post-whitening (T3 ). Kruskal-Wallis test was used to assess group differences in PN penetration and tooth color change. RESULTS: PN penetration differed among all groups except 2 and 3. There were no differences among groups for any baseline color parameters (p > 0.30). At T2 there was no change relative to baseline for individual components L*, a*, and b*. At T3 and T4 there was significant change relative to baseline for ΔL*, Δb*, and ΔE*, for all groups. CONCLUSIONS: PN penetration is time dependent and pretreatment with PN does not affect whitening efficacy. CLINICAL SIGNIFICANCE: Postassium nitrate penetration into the pulp cavity occurred as early as 5 minutes after application, and pretreatment with potassium nitrate containing desensitizers did not adversely affect tooth whitening efficacy. (J Esthet Restor Dent 28:S14-S22, 2016).

Review of the Mechanism of Tooth Whitening.

PURPOSE: This review integrated the current literature on diffusion of whitening agents, their interactions with stain molecules, and changes to the surface, with the aim of establishing a better understanding of the mechanism underlying tooth whitening. MATERIALS AND METHODS: An electronic PubMed database search, with combinations of the following terms was performed: Tooth Bleaching, Tooth Bleaching Agent, Hydrogen Peroxide, Pharmacokinetics, Tooth Permeability, Oxidation-Reduction, Tooth Demineralization, and Color. RESULTS: Tooth whitening is a dynamic process that involves diffusion of the whitening material to interact with stain molecules and also involves micromorphologic alterations on the surface and changes within the tooth that affect its optical properties. The interaction seems not to be limited to stain molecules, but rather an affinity-based interaction process that also accompanies effects on sound enamel and dentin structures. CONCLUSIONS: This review underlines that supervision by dental health professionals as recommended by the American Dental Association (ADA) Council on Scientific Affairs is critical to achieving a successful and safe whitening outcome. CLINICAL SIGNIFICANCE: The mechanism that underlies tooth whitening with the use of peroxide-based materials is a complex phenomenon encompassing diffusion, interaction, and surfaces changes within the tooth. Therefore, supervision by dental health professionals as recommended by the ADA Council on Scientific Affairs is imperative to achieve a successful and safe whitening outcome.

Effect of menthol on the penetration of tobacco carcinogens and nicotine across porcine oral mucosa ex vivo.

INTRODUCTION: Menthol is a flavored tobacco additive claimed to mask the bitter taste and reduce the harshness of cigarette smoke. (Azzi, C., Zhang, J., Purdon, C. H., Chapman, J. M., Nitcheva, D., Hebert, J. R., et al., 2006, Permeation and reservoir function of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo[a]pyrene (B[a]P) across porcine esophageal tissue in the presence of ethanol and menthol. Carcinogenesis, 27, 137-145). have shown that menthol increased the flux of tobacco carcinogens (TC) across porcine esophagus. As oral mucosa is exposed to both smoke and smokeless tobacco in tobacco users, the objective of this study was to determine whether menthol influenced the penetration of the TC nitrosonornicotine (NNN) across porcine buccal (BM) and floor of mouth (FM) mucosa. METHODS: Porcine BM and FM were collected at slaughter, mounted in perfusion chambers (n = 7/group), and exposed to tritiated NNN ((3)H-NNN; Amersham, activity 1 muCi/ml) and tritiated nicotine ((3)H-nicotine; Sigma) in 3% nicotine/phosphate-buffered saline (0.01 M, pH 7.4) containing 0.01% unlabeled NNN (National Cancer Institute Chemical Carcinogen Repository) +/- 0.08% menthol for 0.5, 1, 2, or 12 hr. K(p) values (cm/min) were determined and statistically analyzed (analysis of variance, Tukey's, p < .05). RESULTS: FM and BM permeability to both (3)H-NNN and (3)H-nicotine was significantly increased (p < .05) with addition of menthol over that of nicotine alone regardless of exposure times. Even short 30-min menthol exposure significantly increased the flux of both compounds, and this was maintained throughout the experiment. DISCUSSION: Menthol enhances penetration of NNN and nicotine through FM and BM in vitro, even after short exposure. This may reflect loading of a superficial epithelial reservoir (Squier, C. A., Kremer, M. J., Bruskin, A., Rose, A., & Haley, J. D., 1999. Oral mucosal permeability and stability of transforming growth factor beta-3 in vitro. Pharmaceutical Research, 16, 1557-1563.), thus delivering menthol and enhancing flux for several hours. Practical implications are for a potentially increased oral exposure to carcinogens among users of menthol-flavored cigarettes and chewing tobacco.

Organotypic human oral tissue models for toxicological studies.

Three-dimensional models of the human oral epithelia have been developed to test the irritation of oral-care products and to provide systems to study the pathology of the oral cavity. The in vitro tissue models, cultured using normal oral epithelial cells and serum free medium, adopt a buccal or gingival phenotype. The buccal tissue (designated ORL-200) is 8-12 cell layers thick and non-cornified; the gingival tissue (designated GIN-100) is 9-13 layers thick and cornified at the apical surface. The tissues express cytokeratins 13 and 14 similar to their corresponding native oral tissues. The MTT viability assay was used to assess inter-lot and intra-lot reproducibility. The MTT average intra-lot coefficient of variation (CV) was less than 10% for both tissues and the time required to reduce tissue viability by 50% (ET-50) following application of 1% Triton-X 100 averaged 1.02+/-0.33 h (n=26) and 7.97+/-0.80 h (n=14) for the buccal and gingival tissues, respectively. The utility of the buccal tissue for irritation studies was examined by testing prototype dentifrice formulations and commercially available products including mouthwashes, toothpastes, and oral cleansers. Use of the MTT ET-50 assay and cytokine release clearly differentiated between the formulations and the oral care products. In conclusion, the oral tissue models represent highly reproducible, non-animal means to screen the irritation potential of newly developed oral care products and should be useful to study the innate immunity, biology, and pathology of the oral mucosa.

Differential cytotoxicity of long-chain bases for human oral gingival epithelial keratinocytes, oral fibroblasts, and dendritic cells.

Long-chain bases, found in the oral cavity, have potent antimicrobial activity against oral pathogens. In an article associated with this dataset, Poulson and colleagues determined the cytotoxicities of long-chain bases (sphingosine, dihydrosphingosine, and phytosphingosine) for human oral gingival epithelial (GE) keratinocytes, oral gingival fibroblasts (GF), dendritic cells (DC), and squamous cell carcinoma (SCC) cell lines [1]. Poulson and colleagues found that GE keratinocytes were more resistant to long-chain bases as compared to GF, DC, and SCC cell lines [1]. In this study, we assess the susceptibility of DC to lower concentrations of long chain bases. 0.2-10.0 µM long-chain bases and GML were not cytotoxic to DC; 40.0-80.0 µM long-chain bases, but not GML, were cytotoxic for DC; and 80.0 µM long-chain bases were cytotoxic to DC and induced cellular damage and death in less than 20 mins. Overall, the LD50 of long-chain bases for GE keratinocytes, GF, and DC were considerably higher than their minimal inhibitory concentrations for oral pathogens, a finding important to pursuing their future potential in treating periodontal and oral infections.

Differential cytotoxicity of long-chain bases for human oral gingival epithelial keratinocytes, oral fibroblasts, and dendritic cells.

Long-chain bases are present in the oral cavity. Previously we determined that sphingosine, dihydrosphingosine, and phytosphingosine have potent antimicrobial activity against oral pathogens. Here, we determined the cytotoxicities of long-chain bases for oral cells, an important step in considering their potential as antimicrobial agents for oral infections. This information would clearly help in establishing prophylactic or therapeutic doses. To assess this, human oral gingival epithelial (GE) keratinocytes, oral gingival fibroblasts (GF), and dendritic cells (DC) were exposed to 10.0-640.0 µM long-chain bases and glycerol monolaurate (GML). The effects of long-chain bases on cell metabolism (conversion of resazurin to resorufin), membrane permeability (uptake of propidium iodide or SYTOX-Green), release of cellular contents (LDH), and cell morphology (confocal microscopy) were all determined. GE keratinocytes were more resistant to long-chain bases as compared to GF and DC, which were more susceptible. For DC, 0.2-10.0 µM long-chain bases and GML were not cytotoxic; 40.0-80.0 µM long-chain bases, but not GML, were cytotoxic; and 80.0 µM long-chain bases induced cellular damage and death in less than 20 min. The LD50 of long-chain bases for GE keratinocytes, GF, and DC were considerably higher than their minimal inhibitory concentrations for oral pathogens, a finding important to pursuing their future potential in treating periodontal and oral infections.

Oral mucosal lipids are antibacterial against Porphyromonas gingivalis, induce ultrastructural damage, and alter bacterial lipid and protein compositions.

Oral mucosal and salivary lipids exhibit potent antimicrobial activity for a variety of Gram-positive and Gram-negative bacteria; however, little is known about their spectrum of antimicrobial activity or mechanisms of action against oral bacteria. In this study, we examine the activity of two fatty acids and three sphingoid bases against Porphyromonas gingivalis, an important colonizer of the oral cavity implicated in periodontitis. Minimal inhibitory concentrations, minimal bactericidal concentrations, and kill kinetics revealed variable, but potent, activity of oral mucosal and salivary lipids against P. gingivalis, indicating that lipid structure may be an important determinant in lipid mechanisms of activity against bacteria, although specific components of bacterial membranes are also likely important. Electron micrographs showed ultrastructural damage induced by sapienic acid and phytosphingosine and confirmed disruption of the bacterial plasma membrane. This information, coupled with the association of treatment lipids with P. gingivalis lipids revealed via thin layer chromatography, suggests that the plasma membrane is a likely target of lipid antibacterial activity. Utilizing a combination of two-dimensional in-gel electrophoresis and Western blot followed by mass spectroscopy and N-terminus degradation sequencing we also show that treatment with sapienic acid induces upregulation of a set of proteins comprising a unique P. gingivalis stress response, including proteins important in fatty acid biosynthesis, metabolism and energy production, protein processing, cell adhesion and virulence. Prophylactic or therapeutic lipid treatments may be beneficial for intervention of infection by supplementing the natural immune function of endogenous lipids on mucosal surfaces.

Presence of wax esters and squalene in human saliva.

OBJECTIVE: The purpose of this study was to determine the presence and relative composition of neutral lipids in human saliva. DESIGN: Whole unstimulated saliva was collected from 12 subjects ranging from 21 to 29 years old. Samples were lyophilized, and lipids were extracted using chloroform-methanol. Lipids were analysed by thin-layer chromatography. RESULTS: Human saliva contains cholesterol, fatty acids, triglycerides, wax esters, cholesterol esters and squalene. The mean total neutral lipid content was 12.1±6.3 µg/ml. CONCLUSIONS: These lipids in human saliva closely resemble the lipids found on the skin surface. These salivary lipids are most likely produced by the sebaceous follicles in the oral mucosa and sebaceous glands associated with major salivary glands.

Structures of the ceramides from porcine palatal stratum corneum.

Ceramides are the major type of lipid found in stratum corneum from the skin, gingiva and hard palate. The present study examined the ceramides of the stratum corneum from the hard palate. Six fractions of ceramides were isolated by preparative thin-layer chromatography. The least polar fraction contained an unusual acyl ceramide (EOS) consisting of long omega-hydroxy acids amide-linked to sphingosine with mostly saturated fatty acids ester-linked to the omega-hydroxyl group. The second and third fractions contained normal fatty acids amide-linked to sphingosine (NS) and phytosphingosine (NP), respectively. In each of these ceramides, the fatty acids consisted of a mixture of saturated and monoenoic species. The three most polar fractions all contain amide-linked alpha-hydroxy acids. The fourth fraction contained long alpha-hydroxy acids amide-linked to sphingosine (ASl), while the fifth fraction contained short alpha-hydroxy acids amide-linked to sphingosine (ASs). The most polar ceramide contained alpha-hydroxy acids amide-linked to phytosphingosine (AP). EOS, NS and NP differed from their epidermal counterparts in terms of the compositions of the normal fatty acids. ASl, ASs and AP from palatal stratum corneum were essentially identical to their epidermal counterparts. The differences between palatal and epidermal EOS, NS and NP contribute to the differences in permeability of palate compared to skin.