Effect of nicotine on 7,12-dimethylbenz[a]anthracene carcinogenesis in hamster cheek pouch.

We divided 40 male Syrian golden hamsters into four groups of 10 animals each, and we treated both cheek pouches of each hamster three times a week as follows: group 1, 50 microL of sesame oil; group 2, 50 microL of 6% nicotine in sesame oil; group 3, 50 microL of 1% 7,12-dimethylbenz[a]anthracene (DMBA) in sesame oil; and group 4, 50 microL of 1% DMBA in 6% nicotine in sesame oil. Cheek pouches were examined clinically and histologically after 12 weeks of treatment. Hamsters treated with DMBA and nicotine showed significantly (P less than .001) more tumors and a significantly (P less than .05) greater-than-expected proportion of large tumors (greater than or equal to 3-mm diameter) than hamsters treated with DMBA alone. Histologically, there was a greater degree of dysplasia in lesions from the group receiving DMBA plus nicotine than in the DMBA only group. The results suggest that nicotine acts as a cofactor in DMBA tumorigenesis.

Covalently bound lipids in keratinizing epithelia.

Covalently bound lipids have been identified and compared in keratinizing porcine epithelia including epidermis and oral epithelium from palate and gingiva. Stratum corneum was isolated by tryptic digestion, and after extensive extraction of lipids using a series of chloroform-methanol mixtures, the residual tissue was subjected to alkaline hydrolysis to release covalently bound lipids. The lipids so released were analyzed by quantitative thin-layer chromatography. Stratum corneum from each of the three anatomical sites contained omega-hydroxyceramides, omega-hydroxyacids and fatty acids. In epidermal stratum corneum the total covalently bound lipids represented 2.4% of the dry weight of the tissue, but in the oral epithelia this figure was consistently lower: 0.24% in palatal stratum corneum and 0.20% in gingival stratum corneum. Transmission electron microscopy before and after lipid extraction confirms the presence of a lipid envelope in epidermal stratum corneum and demonstrates the absence of this structure in oral stratum corneum.

In vitro reconstitution of stratum corneum lipid lamellae.

In the final stages of differentiation in the epidermis of terrestrial mammals, lipids are extruded into the intercellular spaces. The initially extruded lipid becomes transformed into broad, multilamellar sheets that are found in the intercellular spaces throughout the stratum corneum. These lamellae display an unusual alternating broad-narrow-broad pattern of lucent bands as revealed by transmission electron microscopy (TEM). This arrangement results in two periodicities that can be measured from electron micrographs and are also evident in X-ray diffraction-5 nm (broad) and 13 nm (broad-narrow-broad). The goal of the present study was to reconstitute these lamellae in vitro. Porcine stratum corneum lipids were applied to Millipore filters. The disks were placed in water and heated to 80 degrees C for 1 h. After cooling, the disks were stored over desiccant. At each stage, the disks were prepared for TEM. TEM revealed that the application of the lipid solutions onto the disks resulted to deposition of mostly amorphous material. Heating in water resulted in the formation of many lamellae. The width of the lamellae was uniform and in the range of 5 to 6 nm with no broad-narrow-broad pattern; however, after storage under desiccating conditions, the broad-narrow-broad pattern was reproduced.

The stretching of mouse skin in vivo: effect on epidermal proliferation and thickness.

Small springs were implanted in the backskin of hairless mice to determine the effects of mechanical stretching on epidermal proliferation. The controls consisted of sham operated animals, in which the implanted springs did not exert any tension so as to identify any effect due to surgical trauma, and unoperated animals. There was a significant rise in the mitotic index after one day in both experimental and sham operated animals and a slight thinning of the stretched epidermis. After 2 days the mitotic index and thickness of the epidermis of the stretched skin were greater than that of the sham operated or unoperated control group, and these differences were maintained after 4 days and were significant. At this time the stretched epidermis showed a hyperplastic response with a thickening of all cell layers. It appears that tension due to stretching increases the mitotic activity of the epidermis leading to an increased progenitor cell population and subsequent tissue hyperplasia.

Variations in lipids in different layers of porcine epidermis.

Frozen cryosections, 8 microns thick, were cut parallel to the surface of porcine skin so as to provide separate samples representing various epidermal layers. These samples were dried, extracted with chloroform-methanol mixtures, and the lipids chromatographed on silica gel plates in different solvent systems. After spraying with sulfuric acid and charring, lipids were quantified using a scanning densitometer. It was thus possible to determine lipid concentrations in 12 consecutive epidermal layers, extending 96 microns into the skin. The phospholipids that were characterized all decreased in concentration toward the surface, whereas the neutral lipids and ceramides all increased. Glucosylceramide and acylglucosylceramide reached a peak concentration in the stratum granulosum and then decreased in the surface layers. Cholesterol sulfate reached a maximum concentration in the deeper stratum corneum and then abruptly decreased in the surface layer. These changes in patterns of lipid concentration are consistent with current theories regarding the formation of a water barrier in the stratum corneum that is composed mainly of neutral lipids, and with a possible function of cholesterol sulfate in cellular adhesion in the stratum corneum.

The permeability of skin and oral mucosa to water and horseradish peroxidase as related to the thickness of the permeability barrier.

The permeability of porcine skin and keratinized and nonkeratinized oral mucosa to tritium-labeled water and horseradish peroxidase (HRPO) was determined using perfusion chambers. Small blocks from each tissue were also incubated with HRPO and the extent of penetration visualized microscopically; this enabled measurements to be made of the thickness of the permeability barrier to this water-soluble tracer. Results obtained after inverting the oral mucosa in the chambers or adding metabolic inhibitors indicated that both compounds diffuse across the tissue. The permeability constants derived directly in the study showed that skin was less permeable than oral mucosa and that the floor of the mouth was significantly more permeable than all other regions. When these constants were normalized in terms of a standard permeability barrier thickness and the different tissues compared, the values obtained for skin were again less than those of the oral regions but, of these, the buccal mucosa was significantly higher. The difference in permeability between epidermis and keratinized oral epithelium may be due to differences in the volume density of membrane-coating granules known to exist between the tissues; differences between the oral mucosal regions may reflect differences in the nature of the intercellular barrier material.

Lipid content and water permeability of skin and oral mucosa.

It has been claimed that total lipid content may be the critical factor determining the water permeability of skin. The present study examined this relationship in various oral epithelia and epidermis. Epithelia was heat separated from specimens of porcine skin, gingiva, buccal mucosa, palate, and floor of mouth. Lipids were solvent extracted and separated by thin layer chromatography with appropriate standards. The plates were sprayed with sulfuric acid and charred, and the concentration of lipids was determined by densitometry as mg lipid/gm tissue dry weight. Permeability constants were determined for each tissue by using tritiated water in perfusion chambers. When these values were compared over all regions, total lipid did not appear to be related to the permeability of these tissues. However, in the keratinized regions (epidermis, gingiva, and palate) a lower water permeability was related to a greater content of total lipid, nonpolar lipid, ceramide, and glucosylceramide. In non-keratinized tissues, a lower permeability corresponded to increased amounts of an unidentified glycosylceramide. The role of lipid in the permeability barrier of these tissues was further demonstrated by extracting specimens of skin and oral mucosa with chloroform/methanol and then determining Kp values; in both tissue regions, there was a significant increase in water permeability. Thus, although lipid is a component of the water permeability barrier in both skin and oral mucosa, different lipid components subserve this function in keratinized and non-keratinized tissues.

Comparison of lipids from epidermal and palatal stratum corneum.

Stratum corneum has been isolated by tryptic digestion of porcine epidermis and palatal epithelium, and the lipid concentrations and compositions have been compared by thin-layer chromatography in conjunction with photodensitometry. Palatal stratum corneum contained 47 +/- 6 micrograms lipid/mg tissue or 115 +/- 16 micrograms lipid per cm2 of stratum corneum surface, whereas epidermal stratum corneum contained 105 +/- 17 micrograms lipid/mg tissue or 135 +/- 16 micrograms/cm2. The difference in total lipid content does not account for the tenfold higher permeability constant for the permeation of water through the former tissue compared to the latter; therefore, the difference in permeability must be based on differences in lipid composition. In this regard, palatal stratum corneum includes 12.1% phospholipids, although phospholipids were undetected in epidermal stratum corneum. Differences in the content and location of non-polar liquid-phase lipids may also be of significance for permeability. Other factors that may contribute to the greater permeability of the palatal horny layer relative to epidermal stratum corneum include generally lower proportions of cholesterol, fatty acids, and ceramides, a dramatically lower proportion of the linoleate-containing acylceramide, and a tenfold lower content of covalently bound lipids associated with the corneocyte envelope.

Effects of zinc deficiency on the distribution of membrane-coating granules in rat buccal epithelium.

Nutritional zinc deficiency causes consistent excessive cell proliferation in the epithelium of the buccal mucosa. The number per cell and the intracellular location of membrane-coating granules in this epithelium were investigated in male rats placed at weanling age for a 4-week period on a diet containing 1.2 ppm of Zn and in their pair fed controls. Membrane-coating granules were identified on electron micrographs following their demonstration in thin sections by the use of an alkaline bismuth technique. Counts of membrane-coating granules in the first 4 rows of spinous cells and the last 4 rows of granular cells showed that in the zinc-deficient group (1) the total number of granules per cell was increased; (2) the proportion of granules displaced to the cell periphery was decreased in favor of a higher proportion persisting in the center and (3) there was a marked reduction in number and proportion of granules positioned near the superficial cell membrane. The greater uniformity in the distribution of the granules in the hyperplastic-hypertrophic epithelium of the zinc deficient group suggests weakening of the surface-oriented polarity characteristic of the control tissue.

The permeability of epidermis lacking normal membrane-coating granules: an ultrastructural tracer study of Kyrle-Flegel disease.

Skin lesions from patients with Flegel's disease have been reported to be without membrane-coating granules (Odland bodies). Biopsies of the hyperkeratinized papules of Kyrle-Flegel disease were incubated in vitro with the intercellular tracer, horseradish peroxidase, and the extent of penetration of this substance examined in the light and electron microscopes. The peroxidase was present throughout the corium and extended through the intercellular spaces of the epidermis to a level close to the junction of the granular and keratinized layers; it did not enter the bulk of the thickened stratum corneum of the lesion. Ultrastructural examination revealed the presence of small vesicles in the granular layer, similar in size and shape to membrane-coating granules but lacking a lamellate internal structure; these were occasionally seen fusing with the plasma membrane of the cells. It is suggested that the intercellular permeability barrier to horseradish peroxidase demonstrated in the Kyrle-Flegel lesion may arise from material contributed by these granules to the intercellular space.

Osmium tetroxide and ruthenium tetroxide are complementary reagents for the preparation of epidermal samples for transmission electron microscopy.

Ruthenium tetroxide and osmium tetroxide were compared as post-fixatives in the preparation of human epidermis for transmission electron microscopic examination. Both reagents revealed characteristic lamellar granules within the granular layer and extruded lamellar granule contents in the upper granular layer. The transformation of the granule contents into multilamellar sheets at the interface between the granular and cornified layers and the persistence of these sheets through all levels of the stratum corneum were demonstrated only with ruthenium tetroxide fixation. Therefore, the reactivity of osmium tetroxide with isolated epidermal lipids was examined. The failure of osmium tetroxide to reveal membrane structures in the stratum corneum can be explained by its inability to react with many of the lipid components of these membranes, rather than to selective removal of lipids during tissue processing, as was formerly believed. Ruthenium tetroxide, a stronger oxidizing agent than osmium tetroxide, overcomes this problem but has other severe limitations as a post-fixative.

Biology of oral mucosa and esophagus.

The mucosal lining of the oral cavity and esophagus functions to protect the underlying tissue from mechanical damage and from the entry of microorganisms and toxic materials that may be present in the oropharynx. In different regions, the mucosa shows adaptation to differing mechanical demands: Masticatory mucosa consists of a stratified squamous keratinized epithelium tightly attached to the underlying tissues by a collagenous connective tissue, whereas lining mucosa comprises a nonkeratinized epithelium supported by a more elastic and flexible connective tissue. The epithelium is constantly replaced by cell division in the deeper layers, and turnover is faster in the lining than in the masticatory regions. Chemotherapeutic agents and radiation limit proliferation of the epithelium so that it becomes thin or ulcerated; this will first occur in the lining regions. The principal patterns of epithelial differentiation are represented by keratinization and nonkeratinization. As keratinocytes enter into differentiation, they become larger and begin to flatten and to accumulate cytokeratin filaments. In addition to the keratins, the differentiating keratinocytes synthesize and retain a number of specific proteins, including profilaggrin, involucrin, and other precursors of the thickening of the cell envelope in the most superficial layers. The concept of epithelial homeostasis implies that cell production in the deeper layers will be balanced by loss of cells from the surface. There is a rapid clearance of surface cells, which acts as a protective mechanism by limiting colonization and invasion of microorganisms adherent to the mucosal surface.

Cellular and molecular basis of barrier function in oral epithelium.

The use of the oral mucosa for drug delivery and the erroneous belief that it is a nonkeratinized tissue have given rise to the suggestion that the oral mucosa is a permeable tissue. Such an assumption is not supported by studies which indicate that permeability differs significantly in different oral regions, depending on the pattern of epithelial differentiation. Keratinized regions such as hard palate and gingiva have a permeability which is significantly less than nonkeratinized regions like buccal mucosa and floor of mouth. Nevertheless, all oral regions are more permeable than skin. Associated with these differences in permeability are differences in the type and amount of intercellular lipid; areas of keratinized tissue contain predominantly neutral lipids (ceramides) apparently derived from lamellate membrane-coating granules. In nonkeratinized areas, the lipids consist of as yet uncharacterized glycosyl ceramides that appear to be derived from membrane-coating granules that differ morphologically from those present in nonkeratinized tissue.

Enhancing effect of chitosan on peptide drug delivery across buccal mucosa.

The buccal mucosa represents a potentially important topical route for delivery of peptide or protein drugs with some unique advantages such as the avoidance of hepatic first-pass metabolism and the acidity and protease activity encountered in the gastrointestinal tract. However, the bioavailabilities or relative potencies of intraorally administered peptides are usually quite low, unless permeabilizers are employed. Chitosan, a mucopolysaccharide of marine origin, has been claimed to act both as a bioadhesive and permeabilizer, making it a candidate system for mucosal drug delivery. In this study, the enhancement effect of chitosan in gel form for oral mucosa was investigated with a large bioactive peptide, transforming growth factor-beta (TGF-beta). Chitosan gel was prepared at 2% concentration in dilute lactic acid and TGF-beta was incorporated into the gel. The effect of chitosan as a permeabilizer was determined by measuring the flux of TGF-beta across porcine oral mucosa in an in vitro system. The localization of TGF-beta within the oral mucosa was determined by horizontal sectioning and counting. Chitosan was found to exert a marked permeabilizing effect on buccal mucosa for peptide drug.

Rates of epithelial cell proliferation in the oral mucosa and skin of the tamarin monkey (Saguinus fuscicollis).

Epithelial cell proliferation was determined in skin and various regions of the oral cavity of the tamarin monkey and was expressed by means of two mitotic indices: the number of metaphases per mm of epithelial surface, and the number of metaphases per mm basement membrane. A significant correlation was obtained between the rank ordering of the different regions according to each index. Generally, the non-keratinized tissues of the oral cavity had mitotic rates higher than those of the keratinized oral regions, with the epidermis having the lowest value. The mitotic index also correlated significantly with epithelial thickness, with the thicker regions showing a higher rate of proliferation. These results represent the first comprehensive set of values for a primate and are in general agreement with data obtained from non-primate species; the values support the concept that the oral lining tissue turns over more rapidly than does the masticatory mucosa (Bhaskar, 1980).

Ultrastructural quantitation of connective tissue changes in phenytoin-induced gingival overgrowth in the ferret.

The gingival overgrowth obtained after maintaining ferrets on PHT appeared to be due entirely to the effect of the drug, for inflammation induced by banding had no influence on the action of PHT in eliciting the overgrowth. The significant change observed was an increase in relative volume of interstitial material (ground substance) in response to PHT. Although there was no appreciable alteration in numbers of cells present in the lesion, PHT had a significant effect on the ultrastructure of fibroblasts. These cells showed a decrease in the relative volume of phagosomes, although organelles concerned with synthesis (the rough endoplasmic reticulum and Golgi zones) were not affected. This suggests that the relative increase in ground substance may reflect decreased breakdown of extracellular material within fibroblasts, while synthetic activity is maintained at a constant level. As a consequence, there is an increase in connective tissue volume--an increase which is manifested as an overgrowth.

The permeability of human oral mucosa and skin to water.

Specimens from four regions of oral mucosa (palate, buccal mucosa, lateral border of the tongue, and the floor of the mouth) and of abdominal skin were taken from 58 individuals at autopsy, for determination of permeability constants (Kp) to tritium-labeled water. Comparisons between fresh specimens and those stored at -80 degrees C revealed no significant effect on Kp as a result of freezing; similar results were found with use of specimens from corresponding regions of the pig. Values for Kp were significantly different for all of the tissue regions examined and ranged from 44 +/- 4 x 10(-7) cm/min for skin to 973 +/- 33 x 10(-7) cm/min for the floor of the mouth, which was the most permeable region. Similar differences were evident among corresponding regions of porcine oral mucosa and skin. Moreover, the Kp values obtained for human tissues were not significantly different from those of the pig, except for the floor of the mouth, which was more permeable in human than in pig tissue. The results reveal interesting differences in the permeability of human oral mucosa that might be related to susceptibility to mucosal disease in those conditions where local extrinsic etiological agents are implicated.

Continuous flow mucosal cells for measuring the in-vitro permeability of small tissue samples.

Continuous-flow chambers are described for the measurement of permeability of small tissue samples. The design incorporates a large-capacity donor chamber to permit adequate loading of the applied compound and a low-volume (0.3 mL) receiving chamber that ensures rapid removal of penetrant at relatively low (1.5 mL/h or less) pumping rates. Different sized support disks allow tissue biopsies as small as 4 mm in diameter to be utilized. Comparisons of flux and permeability constants (Kp) for water across oral mucosa indicate that there was no significant difference between values obtained for 10- and 4-mm biopsies. Comparisons of flux and Kp values for porcine oral mucosa and a synthetic membrane between continuous flow and conventional, side-by-side chambers indicated that the latter values were significantly lower, suggesting stasis and inefficient removal of perfusate in the side-by-side design. The Kp values for water obtained in the continuous-flow chambers with pig skin were similar to those published elsewhere for human skin.

Keratinization of the sulcular epithelium--a pointless pursuit?

A considerable amount of effort has been directed at finding methods for modifying the nonkeratinized sulcular epithelium on the assumption that a keratinized surface may offer a better barrier to antigens and bacterial products present in the gingival sulcus. It is argued here that keratinization in itself may not confer greater impermeability, for nonkeratinized epithelia also have been shown to resist the penetration of certain substances. Moreover, few workers have considered the role of junctional epithelium in the initiation of periodontal disease although experimental evidence suggests that this may be a permeable tissue. As formation of a surface with barrier properties seems to be a concomitant of epithelial differentiation while attachment is a property of relatively undifferentiated epithelial cells, attempts to induce junctional epithelium to differentiate could result in a loss of epithelial attachment to the tooth. It is suggested that attempts to keratinize the sulcular region, on theoretical grounds, may be unjustified.

Ultrastructure of regenerating junctional epithelium in the monkey.

It has been established that after gingivectomy the junctional epithelium is reformed from the oral epithelium but there is little information on the regenerative potential of residual junctional epithelium. In this study reformation of the epithelial attachment in the monkey was followed ultrastructurally after surgical removal of all of, or of part of, the original junctional epithelium by internal or external bevel techniques respectively. In both circumstances a new epithelial attachment developed from the adjacent gingival oral epithelium and residual junctional epithelium appeared to persist as small nests of cells adjacent to the cemento-enamel junction. Epithelialization of the gingival wound was rapid, taking place in as little as 5 days after the partial removal of the junctional epithelium by the external bevel technique and by 10 days in wounds in which the junctional epithelium had been completely excised. This difference in the rate of epithelialization seemed to be primarily related to the quantity of coagulum and cell debris present, the greater amount remaining after the internal bevel technique tending to retard epithelial migration and reattachment.