Comparison of differentiation markers between normal and two squamous cell carcinoma cell lines in culture.

This study examines differences between cultures of normal human oral epithelial cells and two squamous cell carcinoma cell lines (SCC15 and SCC25) in the expression of structural proteins, adhesion molecules, plasma membrane lipid composition, and intercellular junctions. Based on immunocytochemistry, most normal cell cultures appeared to express more E-cadherin, integrin beta-1, cytokeratin (CK) 14, CK19, and involucrin than SCC cultures. By Western blot analysis, normal cultures expressing high levels of E-cadherin also expressed high levels of involucrin and low levels of CK19. Both SCC cultures demonstrated lower expression of E-cadherin and involucrin, whereas only SCC15 cells showed high levels of CK19. Expression of beta-catenin, an E-cadherin associated protein with potential oncogene function, did not vary among normal and SCC cells. Proportions of saturated fatty acids quantified by thin layer chromatography were higher in the normal cell cultures, than in both SCC cell lines. No morphological differences were evident by transmission electron microscopy (TEM) between normal and SCC cell-cell intercellular junctions. Although no quantitation was attempted, observation suggested that normal cells form more intercellular junctions (TEM observation) and larger intercellular bridges (SEM observation) compared to both SCC cell lines. Of the factors examined, main variations between cultures of normal oral epithelium and the two SCC cell lines examined include the expression of structural and adhesion proteins, lipid composition, and intercellular junctions. The extent of the differences varies according to the stage of terminal differentiation demonstrated by the normal cell cultures.

Subcellular localization of beta-catenin in malignant cell lines and squamous cell carcinomas of the oral cavity.

BACKGROUND: Beta-catenin, an E-cadherin-associated protein involved in cell-cell adhesion and signaling, has been hypothesized to translocate to the nucleus and activate transcription in several human cancers, including oral squamous cell carcinomas (OSCC). METHODS: In the present study, we analyzed the subcellular localization of beta-catenin in cultures of human oral normal and malignant (cell lines SCC15 and SCC25) keratinocytes and in 24 frozen samples of oral squamous cell carcinomas by a double-staining technique for nucleic acids and beta-catenin. Growth potential, as assessed by cell count at different time periods, was established for normal, SCC15 and SCC25 cell lines; oral squamous cell carcinomas were classified according to the histopathological and malignancy indexes. RESULTS: Beta-catenin localized at the plasma membrane in the normal and SCC15 cells, not in the SCC25 cells, where it localized mostly in the perinuclear and nuclear areas. In the growth assays, SCC25 cell lines proliferated faster than in normal and SCC15 cells over a period of 6 days (cell numbers were significantly different, P < 0.0001). Carcinoma sections showed a combination of membranous, cytoplasmic and, in few invading epithelial islands of two tumors, nuclear localization of beta-catenin. CONCLUSIONS: In oral squamous cell carcinomas, nuclear beta-catenin staining was observed only within invading islands of two carcinomas deep in the underlying connective tissue. On the basis of this study, we conclude that intranuclear beta-catenin does not appear to be a common finding in oral squamous cell carcinomas and that a clear association between intranuclear beta-catenin and histopathological and malignancy indexes in vivo could not be established.

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.

Permeability barrier properties of oral keratinocyte cultures: a model of intact human oral mucosa.

OBJECTIVES: The aim of this study was to establish whether an in vitro model of human oral mucosa had similar permeability characteristics to normal oral mucosa. Such a model would have considerable value as an alternative to the use of mucosal biopsies in studies of transmucosal drug delivery. MATERIALS AND METHODS: Keratinocytes obtained from buccal mucosa, hard palate and abdominal skin were seeded onto inert collagen membranes (Cellagen Discs) or dead de-epidermised dermis (DDED) and grown either as submerged or air-liquid interface cultures. Subsequently the ultrastructural characteristics, permeability to water and barrier lipid content of the epithelial cultures were assessed and compared with samples of intact mucosa and skin. RESULTS: All the cultures stratified into multilayered epithelia and displayed features of differentiation including tonofilaments, desmosomes and membrane coating granules. The permeability characteristics and barrier lipid content of the oral mucosal cultures resembled those of intact mucosa. By contrast, epidermal keratinocytes failed to produce a permeability barrier comparable with that of skin and had low levels of barrier associated lipids. CONCLUSIONS: Cultures of human oral mucosal keratinocytes obtained from healthy adults develop similar permeability properties and barrier lipid composition to their site of origin. This model system may be useful for the evaluation of local and systemic oral mucosal drug delivery.

Short-term exposure to alcohol increases the permeability of human oral mucosa.

OBJECTIVES: The aim of this study was to evaluate the effect of short-term exposure to ethanol on the permeability barrier properties of human oral mucosa. MATERIALS AND METHODS: Permeability constants (Kp x 10(-4) cm min(-1)) to tritiated water were determined, for untreated human ventral tongue, and following treatment with phosphate-buffered saline (PBS), 5, 15 or 40% ethanol using an in vitro perfusion chamber system. Some samples were also exposed to fluorescent-labelled albumin and examined by fluorescence microscopy. Permeability barrier lipid composition was assessed in treated and untreated mucosa by heat separation, solvent extraction and thin layer chromatography. RESULTS: Fifteen per cent ethanol significantly increased mucosal permeability (5.8 +/- 0.44; P < 0.05) compared with untreated, PBS and 5% ethanol treated mucosa (4.69 +/- 0.26, 4.48 +/- 0.3 and 4.13 +/- 0.27, respectively). Albumin was restricted to the epithelial surface in control tissue, but extended further through the epithelium and, in some cases, into the connective tissue after treatment with ethanol. Biochemical analysis revealed no significant difference in the epithelial lipid composition of treated and untreated mucosa. CONCLUSIONS: These results suggest that short-term exposure to ethanol may act as a permeability enhancer, possibly by causing molecular rearrangement of the permeability barrier, not as a result of lipid extraction.

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.

Penetration of N-nitrosonornicotine (NNN) across oral mucosa in the presence of ethanol and nicotine.

The effects of ethanol concentrations of 5, 15, 20, 25, 27, 30 and 50% on the penetration of the tobacco-specific carcinogen, nitrosonornicotine (NNN), across porcine oral mucosa were examined using an in vitro perfusion system. Concentrations of ethanol of 25% and above significantly increased the permeability of oral mucosa to NNN, although this increase ceased with 50% ethanol, possibly due to a fixative effect. Nicotine is a consistent component of smoked and smokeless tobacco; the presence of 0.2% nicotine significantly increased the permeability of oral mucosa to NNN and 2% nicotine caused a further increase. Combined use of nicotine and ethanol significantly increased the penetration of NNN across oral mucosa over that of ethanol alone until the concentration of ethanol reached 50%. The results of this study suggest that the synergy between tobacco and alcohol in the etiology of oral cancer may be explained, at least in part, by the local permeabilizing effects of alcohol on the penetration of tobacco-specific (and other) carcinogens across oral mucosa.

Oral mucosal permeability and stability of transforming growth factor beta-3 in vitro.

PURPOSE: To investigate the permeability and localization of topically applied 125I-TGF-beta3 in porcine floor-of-mouth mucosa as a function of concentration and exposure. METHODS: The 125I-TGF-beta3 diluted in three different vehicles was applied to the tissue samples mounted in perfusion cells maintained at 37 degrees C. Flux and Kp values were calculated from the perfusate collected over a 24 hour period. The quantity of 125I-TGF-beta3 present in the tissue was determined by horizontal sectioning and subsequent counting. The stability of 125I-TGF-beta3 in saliva and in the tissue was analyzed by SDS polyacrylamide gradient gel electrophoresis. RESULTS: 125I-TGF-beta3 was relatively stable in saliva and in the epithelium; approximately 50% of the total counts in the deeper epithelium were resident in the 25kDa TGF-beta3 homodimer. A steady-state flux was reached approximately 6 hours post application and Kp value was 4.0+/-0.6 x 10(-6) (mean +/- sem). Penetration of 125I-TGF-beta3 to the basal cell layer was concentration dependent but reached nanomolar concentrations even after extensive surface rinsing, representing over one-thousand fold the IC50 for epithelial cell cycle arrest. CONCLUSIONS: The data suggest that topical application of TGF-beta3 to the oral mucosa in an appropriate vehicle can provide effective therapeutic delivery to the tissue.

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.

Irritant contact stomatitis: a review of the condition.

Several different types of interactions are possible between a chemical, a mixture of chemicals, and associated extrinsic factors (i.e., mechanical irritation) in the oral mucosa. These interactions can be broadly classified as irritative or allergenic in nature. In each case, the pathology usually includes mucosal inflammation. The information compiled and reviewed in this article suggests that, given the broad definition of surface lesions/mucosal abnormalities, there may be a continuum of irritation that can be termed "irritant contact stomatitis." This may be due to the fact that the mouth is lined with highly vascular mucosa that turns over rapidly compared to the skin, and may or may not be covered by keratin. Some regions in the mouth are uniquely sensitive to irritants because they can penetrate through the tissue easily. Key factors involved in the potential development of irritation are: inherent irritation potential of the agent, amount of exposure (concentration, duration, and frequency), ability to penetrate the tissue, and inherent reactivity of the subject as well as other extrinsic factors. Irritation leading to oral mucosal alterations is a common occurrence caused by a wide variety of exposures and insults to the oral cavity. Various irritants such as foods, chemicals, friction, thermal/mechanical injury, metals, spices, and oral care products have been documented to cause irritant reactions in susceptible individuals, particularly if used under exaggerated exposure conditions. It is important to note that most irritation in the oral cavity tends to reverse quickly when the causative agent is removed. Oral irritation is a commonly occurring phenomenon. Thus, it is important that the clinician be aware of the clinical manifestations and etiology of the condition.

Lipids of hamster cheek pouch epithelium.

The hamster cheek pouch is a much used but incompletely understood experimental model. In particular, the cheek pouch epithelial lipids, which are important for permeability barrier function as well as other aspects of epithelial biology, have not been completely characterized. In the present study, the complete lipid class composition has been determined by thin-layer chromatography in conjunction with photodensitometry. The major lipid classes were phospholipids, free sterols, and ceramides. Minor amounts of monohexosylceramides, sterol esters, fatty acids, and triglycerides were also present. Significant amounts of covalently bound omega-hydroxyceramide was also detected. Transmission electron micrographs reveal extensive, largely paired, lipid bilayers in the intercellular spaces of the stratum corneum.

Long-term outcome of extensive skull reconstruction using demineralized perforated bone in Siamese twins joined at the skull vertex.

The successful use of cortical demineralized perforated bone in the treatment of extensive skeletal defects in children is exemplified by this case involving Siamese twins joined at the skull vertex. Four years following extensive skull reconstruction using demineralized perforated bone, an examination revealed successful calvarial reconstruction in one twin. The other twin required additional implants of demineralized perforated bone to fill in defects. However, a histologic examination taken following this additional procedure revealed that these implants neither caused tissue reaction over a 4-year period, nor showed signs of resorption. Bony remodeling and new bone formation were in progress. Compared with other bone substitutes, demineralized perforated bone has proven to be effective in the treatment of large skull defects in children.

Pioneers in oral biology: the migrations of Gottlieb, Kronfeld, Orban, Weinmann, and Sicher from Vienna to America.

Following the annexation of Austria by Hitler's Germany in 1938, officials at the eminent University of Vienna Medical School purged faculty ranks of Jews. Among those forced out were several distinguished physician dentists, several of whom emigrated to the United States. The assimilation of foreign-trained dentists raised questions at national meetings of the AADS and the National Association of Dental Examiners. Already existing ties between dental schools in Chicago and the University of Vienna, including the 1928 appointment of Rudolf Kronfeld to the faculty at Loyola, led to the relocation of Balint Orban, Harry Sicher, and Joseph Peter Weinmann in that city. Bernhard Gottlieb, who had been director of the Dental Institute in Vienna, transplanted less easily, but eventually found a niche at the Baylor College of Dentistry in Dallas. The careers of the Vienna dentist-scientists strengthened the scientific foundations of clinical dentistry in the United States, contributed to the development of a stronger research establishment, and enlarged the scope of oral biology.

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.

Comparative permeability of human vaginal and buccal mucosa to water.

There is currently a resurgence of interest in the oral mucosa as a route for drug delivery. The relative scarcity of human oral mucosa for in vitro permeability studies, and the fact that vaginal mucosa is histologically similar and more abundant than the former, caused us to compare these 2 tissues with respect to their barrier properties to water. Specimens of fresh, clinically-healthy human vaginal and buccal mucosa from non-smokers were taken from excised tissue obtained during vaginal hysterectomies and various oral surgical procedures. Biopsies from each specimen were mounted in flow-through diffusion cells and their permeability to tritiated water determined using a continuous flow-through perfusion system. Specimens were examined histologically before and after permeability experiments and similarities between vaginal and buccal tissues verified. No statistically significant differences between mean steady state flux values (10-16 h) for vaginal and buccal mucosa, respectively, were found. Human vaginal mucosa is therefore as permeable as buccal mucosa to water, and these results warrant further investigation with other compounds to establish whether vaginal mucosa may be a useful model for buccal mucosa for drug permeability studies.

Regional variation in content, composition and organization of porcine epithelial barrier lipids revealed by thin-layer chromatography and transmission electron microscopy.

Epidermis and oral epithelia provide permeability barriers that limit penetration of potentially harmful agents. Barrier function is determined by lipids in the superficial epithelial layers and varies regionally by more than 10-fold. The purpose of this study was to determine whether differences in lipid content, composition or organization could account for this variation in barrier function. Stratum corneum from skin, gingiva and palate and superficial layers from buccal regions and the floor of the mouth were isolated, and lipids were extracted and analysed by thin-layer chromatography. Tissue from each region was examined by electron microscopy. There was an inverse correlation between permeability and ceramide content and a direct correlation with triglyceride content. Electron microscopy revealed that the intercellular space in epidermal stratum corneum contained multiple lipid lamellae displaying an alternating broad-narrow-broad spacing. In palatal and gingival stratum corneum, uniformly spaced lamellae were present at the periphery of dilations of the intercellular space, but the interiors of the dilations contained disorganized lamellae and electron-dense material. In the non-keratinized barriers, there was a single, broad lamella at the cell periphery and occasional short stacks of lamellae traversing the intercellular space. These intercellular lamellae may be derived from a population of membrane-coating granules that contain internal lamellae. The results suggest that ceramides may be important barrier components, even in non-keratinizing epithelia where they are very minor components. Regional differences in the physical organization of barrier lipids may also contribute to differences in barrier function.

Cranioplasty in the growing canine skull using demineralized perforated bone.

This study was designed to test the hypothesis that demineralized perforated bone matrix implant from canine skull and tibia induces new bone formation within the calvarial defect comparable with the bone induced by autogenous graft. We also were interested in determining whether demineralized perforated bone matrix implants from membranous bone have greater osseoinductive capacity in the calvarial area than demineralized perforated bone matrix implants from endochondral bone. Forty 12-week-old purebred beagles were used. Group I consisted of animals with unrepaired surgically created calvarial defects healed by secondary intention (n = 10). Group II consisted of animals with surgically created calvarial defects in which the bone was removed and replaced with an autograft (n = 10). Group III consisted of animals with surgically created calvarial defects in which the bony defect was closed with a demineralized perforated bone matrix implant obtained from beagle calvaria (n = 10). Group IV consisted of animals with surgically created calvarial defects in which the bony defect was closed with a demineralized perforated bone matrix implant obtained from beagle tibia (n = 10). The two control groups (I and II) allowed us to isolate the inductive capacity of demineralized perforated bone matrix implants and compare it with the healing of the bone defects left unrepaired or repaired with calvarial autografts. Animals were sacrificed after 8 and 12 weeks. In the present study we were able to verify that demineralized perforated bone matrix implants are well accepted in the calvarial defects with little tissue reaction and remarkably little osteoclastic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Periosteum in regeneration of palatal defects.

The influence of the periosteum on the regeneration of palatal bone was investigated in this study. Eighty, 8-week-old, purebred beagle dogs were assigned randomly to four groups: (1) unoperated dogs as a group control; dogs in which the mid-third of the palate was surgically removed and (2) left unrepaired (unrepaired controls); (3) repaired with mucosal flaps; (4) repaired with mucoperiosteal flaps. Five animals from each group were killed at 4, 6, 8, and 12 weeks after surgery and coronal sections examined under light microscopy. Among the animals with complete soft tissue healing, 8 of 12 dogs from Group 2, 11 of 20 from Group 3, and 10 of 19 from Group 4 showed complete bone regeneration. No significant differences were found overall in bone thickness and bone density measurements between Group 3 and Group 4. Histologically, a well-differentiated periosteum was present on the maxilla at 4 weeks, even in animals in which the periosteum had not been preserved in the original flap. These results suggest that maintaining the periosteum at surgical closure does not influence bone regeneration in beagles up to 12 weeks of age. We suggest that osteoprogenitor cells, migrating from the undisturbed local periosteum adjacent to the defect, were responsible for the new bone growth in our study.

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.

Free sphingosines in oral epithelium.

Free sphinogosines, intermediates in the biosynthesis of ceramides and inhibitors of protein kinase C, have been found to be present in significant concentrations in epidermis and oral epithelia of the pig (Sus scrofa). Concentrations of sphingosines were higher in the outer portion of epidermis (4.4 mg/g), palatal epithelium (3.9 mg/g) and buccal epithelium (0.7 mg/g) compared to the inner portion of the epithelia (1.0, 0.9 and 0.4 mg/g, respectively). Free sphingosines may provide antimicrobial activity at the epithelial surfaces, and the sphingosine gradient may modulate epithelial differentiation.