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Sometimes it is not feasible or convenient to use tissue samples immediately and they are then stored at -85 degrees C after being snap-frozen in liquid N2. Previous studies have shown that, although snap-freezing in liquid N2 does not alter the permeability properties of human buccal and vaginal mucosae towards various markers significantly, some tissue damage does occur. Slight increases in the permeability properties of the frozen/thawed human buccal mucosa towards markers were found, although these were not statistically significant. The damage does, however, not seem to affect the lipid permeability barrier to a meaningful extent. Because porcine buccal mucosa is a popular choice as a substitute for human buccal mucosa in many permeability studies, it was decided to evaluate the effect that snap-freezing, storage and thawing would have on the permeability of this mucosa when compared to human mucosae. Fresh and frozen/thawed buccal mucosa specimens were collected from freshly slaughtered pigs and permeability experiments at 20 degrees C and 1.5 ml/h, over a 24 hour time period, performed using a flow-through diffusion apparatus. The permeants tested were water, arecoline, vasopressin and 17beta-estradiol. Statistically significant differences were obtained between the flux values at steady state for water, arecoline and vasopressin through fresh and frozen/thawed porcine buccal mucosa. The estimated mean steady state flux values for 17beta-estradiol showed no statistically significant differences between the fresh appear to indicate that the lipid barrier of porcine buccal mucosa is not as resistant to snap-freezing, storage and thawing procedures as that of human mucosal tissue.

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Human vaginal mucosa may be used as a model of buccal mucosa for in vitro permeability studies using various chemical compounds. Rectilinear temperature dependence of water flux across vaginal mucosa between 25 degrees C and 41 degrees C has been shown. The objective of this study was to examine the behaviour of the above barrier on fluxes of 17 beta-estradiol at various temperatures. Frozen vaginal mucosa specimens from a single patient were used (snap-frozen in liquid nitrogen and stored at -85 degrees C). The permeability to tritiated 17 beta-estradiol was determined using a continous flow-through perfusion system at temperatures of 20 degrees C, 25 degrees C, 30 degrees C, 37 degrees C and 41 degrees C. Histological examinations were performed before and after permeability experiments. Estimated steady state flux values were used at 20 degrees C, 25 degrees C and 30 degrees C. Estimated and true mean 17 beta-estradiol steady state flux rates (20-24 h) were found to be 415 +/- 27 standard error of the Mean (SEM), 848 +/- 60 SEM, 1355 +/- 77 SEM, 1436 +/- 37 SEM and 1482 +/- 35 SEM cpm.cm-2.min-1, at temperatures of 20 degrees C, 25 degrees C, 30 degrees C, 37 degrees C and 41 degrees C, respectively. A non-linear regression analysis and plot (R2 = 0.9941) displayed a curvilinear flux-temperature relationship. The results from this study showed that, notwithstanding cellular damage, the principal physical permeability barrier governing permeation kinetics was non-linearly temperature-dependent between the temperatures studied, providing further support for the concept that this barrier is lipoidal in nature.

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OBJECTIVES: Permeation of cyclosporin A (CsA) through intact and de-epithelialized human vaginal mucosa in the presence and absence of benzalkonium chloride (BZCl) was tested. MATERIALS AND METHODS: Human vaginal mucosa (snap-frozen in liquid nitrogen, stored at -85 degrees C) had been used for permeability experiments. CsA permeation through thawed frozen intact and de-epithelialized vaginal mucosa was determined using a flow-through diffusion apparatus (20 degrees C, 24 h). Flux rates for CsA across these two mucosae were determined in the presence and absence of 0.01% BZCl. ANOVA and Duncan's multiple range test were used to test for steady-state and an unpaired t-test with Welch's correction was used to test for differences between the mean flux values at each time point (significance level of 5%). A piece of thawed tissue from each patient, before and after de-epithelialization, was placed in formalin and histologically examined. RESULTS: Flux rates of CsA across intact vaginal mucosa tended to increase by 28-46% in the presence of 0.01% BZCl, and CsA across de-epithelialized mucosa by approximately 28%. The latter differences were statistically significantly higher after 10 h. Flux rates across de-epithelialized mucosa were 52-140% higher in the presence of 0.01% BZCl (statistically significantly higher after 12 h). CONCLUSIONS: The permeation of CsA through intact and de-epithelialized human vaginal mucosa can be enhanced by 0.01% BZCl.

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Radiation therapy is an effective way of treating many forms of cancer, however, there are some indications that it may facilitate the development of metastasis. The question arises whether radiation therapy during cancer treatment might result in an alteration of the permeability of the tissues being treated. This alteration in the permeability might lead to metastatic cells escaping from the irradiated tissue, leading to the spread of cancer to other sites in the body. Because of the above implication, we determined the diffusion kinetics of a radioactive marker, 17 beta-oestradiol, through human saphenous vein before and after a single half hour exposure to 60 Gy of 60Co gamma-irradiation. Six clinically healthy saphenous vein specimens (mean patient age +/- standard deviation 57 +/- 13 years; age range 41-77 years) were obtained during cardiac surgery. In vitro flux rates of 17 beta-oestradiol were determined through use of a flow-through diffusion apparatus immediately after irradiation for a period of 24 hours. No statistically significant differences could be demonstrated for the flux rates of 17 beta-oestradiol through the non-irradiated and 60 Gy irradiated saphenous vein tissue. These findings strongly suggest that irradiation at 2 Gy/min and a total dose of 60 Gy would not alter the permeability of the venous wall. We have demonstrated that the in vitro flow-through diffusion method is capable of measuring permeability aspects of endothelial cell layers in saphenous vein biopsies under conditions resembling clinical reality.

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Barrier hand creams, often containing antiseptic agents, may provide a form of protection not only for health care professionals, but also for workers in the chemical and pharmaceutical industries. To evaluate the efficacy of two such barrier creams available on the South African market, the in vitro diffusion of a model compound, benzo[a]pyrene, through human skin at 20 degrees and 37 degrees C was studied. Treated (10 min) and untreated human skin disks (4 mm in diameter) were mounted in flow cells of a continuous flow-through diffusion apparatus. Buffer/tritiated benzo[a]pyrene was collected from the acceptor chambers at 2-hour intervals for a total of 24 hours and counted in a liquid scintillation counter. At 20 degrees C no significant differences could be detected between the flux rates of benzo[a]pyrene across barrier cream treated and untreated skin. However, at 37 degrees C Skinguard significantly increased flux rates of this carcinogen. Skin barrier creams therefore need to be carefully scrutinised with respect to their protective effects because the latter may vary for molecules with different chemical properties.

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Although the definition of burning mouth syndrome (BMS) can vary, the most commonly accepted is that of a burning sensation of normal appearing oral mucosa with no apparent underlying local or systemic contributing factors. The condition can be classified according to the patterns of burning experienced, the severity of the burning, as well as the pattern of onset. The management of these patients is difficult, since they are often seen by numerous clinicians and many unnecessary tests are performed in the hope of finding an underlying physical cause for the burning. No precise information pertaining to the natural history of BMS could be found. This paper consists of a selective review of the literature on BMS as well as a pilot study involving the standardised collection of data on 10 patients (9 women and 1 man) with BMS. These patients will be followed up in the long term in order to gather information pertaining to the natural history of this condition. No detectable local or systemic cause for the burning sensation could be found for any of the 10 subjects. The role of somatisation as a mechanism for burning sensation was investigated and certain proposals have been put forward regarding the management of such patients.

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The scarcity of sizeable specimens of normal oral mucosa for experimental purposes has hampered research on oral epithelium. Because large specimens of viable human vaginal mucosa are readily available and because vaginal and buccal epithelia are microscopically similar, vaginal mucosa has been used successfully to establish a human cyst model in experimental animals. The ultrastructure and distribution of keratin filaments in these epithelia are also similar, as is their permeability to water and a number of chemical substances. Therefore, if vaginal mucosa could be substituted for buccal mucosa it would expedite research on the epithelium of buccal mucosa. To strengthen further the concept that vaginal epithelium could replace buccal epithelium in certain experimental studies, the thickness of these epithelia, their patterns of surface keratinization, the presence or absence of intercellular lipid lamellae and their lipid contents were now compared. Thirty-three specimens of vaginal mucosa from postmenopausal women and 36 of buccal mucosa were investigated. To compare the thickness of the epithelial layers the number of cell layers in sections of 20 vaginal and 20 buccal mucosal specimens were counted in the three thickest and three thinnest regions of each specimen. Surface keratinization was evaluated on sections stained with the Picro-Mallory method. To demonstrate lipid lamellae two vaginal and two buccal mucosa specimens were examined electron microscopically after normal fixation and postfixation in ruthenium tetroxide. Following solvent extraction of 11 vaginal and 14 buccal epithelia, quantitative lipid analyses were performed using thin-layer chromatography. No statistically significant differences were found between the maximum and minimum number of epithelial cell layers. The patterns of surface keratinization and the distribution and appearance of the lipid lamellae in the intercellular spaces were similar. The lipid composition of the two epithelia corresponded, except for the cholesterol esters and glycosylceramides, which were higher in buccal epithelium. Ceramides for vaginal epithelium and triglycerides for buccal epithelium were not determined. Based on structural similarities, a similar lipid composition and earlier findings, it is concluded that vaginal epithelium can be used as a substitute for buccal epithelium in certain in vitro, and possibly for in vivo, studies.

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Areca nut chewing has been implicated in the development of oral cancer and oral submucous fibrosis. Arecoline and arecaidine, which are alkaloids present in the areca nut, are thought to play a major role in the development of adverse effects resulting from this chewing habit. Because these alkaloids appear to be associated with the development of the above diseases, we determined their diffusion kinetics through human vaginal mucosa in the presence and absence of a 1% areca nut extract. Seven clinically healthy vaginal mucosa specimens (mean patient age+/-standard deviation, 52+/-13 years; age range, 38-74 years) were obtained during surgery. In vitro flux values of reduced arecoline and arecaidine (r-arecoline and r-arecaidine) were determined through use of a flow-through diffusion apparatus. Analysis of variance, a Duncan multiple range test, and an unpaired t-test were used to determine steady state kinetics and flux differences over time intervals. The flux values across vaginal mucosa of r-arecoline and r-arecaidine were decreased in the presence of 1% areca nut extract. For r-arecoline, these flux values were significantly lower statistically when compared to those obtained in the absence of areca nut extract. These findings concur with results previously obtained for water, where the astringent action of the tannins present in the areca nut extract was thought to alter the barrier properties of the epithelium, resulting in decreased permeability.

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PURPOSE: To investigate the permeation of cyclosporin A (CsA) through fresh and frozen human corneas in the presence and absence of three penetration enhancers: benzalkonium chloride (BZCl), dimethylsulfoxide (DMSO), and Cremophor-EL. METHODS: Human corneas, unsuitable for transplantation, were either freshly used for permeability experiments or snap-frozen in liquid nitrogen and stored at -85 degrees C. CsA permeation through either fresh or thawed frozen corneal tissue was determined using a flow-through diffusion apparatus (20 degrees C for 24 hours). Flux rates for CsA were determined in the presence and absence of the penetration enhancers 0.01% BZCl, 20% DMSO, and Cremophor-EL (10% and 20%). Analysis of variance and Duncan's multiple-range test were used to test for steady state, and an unpaired Student t test with Welch's correction was used to test for differences between the mean flux values at each time point. A significance level of 5% was used for all of the statistical tests. RESULTS: No statistically significant differences in flux values of CsA could be detected between fresh and frozen corneas. In the presence of Cremophor-EL (10% and 20%) and 0.01% BZCl, statistically significant increases in flux values of CsA before 16 hours and after 16 hours, respectively, could be observed. In the presence of 20% DMSO, no statistically significant increases in flux values could be detected. CONCLUSIONS: The permeation of CsA through human corneas appeared to be enhanced by the presence of BZCl and Cremophor-EL. Additionally, it was shown that the flux rate of CsA across fresh and frozen corneas was not significantly different.

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Because alkaloids from areca nut, arecoline and arecaidine, have been implicated in the development of oral submucous fibrosis, we determined their diffusion kinetics through human buccal and vaginal mucosa. Four clinically healthy vaginal mucosa specimens (mean patient age +/- standard deviation: 47 +/- 15 years; age range: 31-60 years) and 4 buccal mucosa specimens from 2 male patients and 2 female patients (mean patient age +/- standard deviation: 31 +/- 9 years; age range: 17-53 years) were obtained during surgery. In vitro flux rates of reduced arecoline and arecaidine (r-arecoline and r-arecaidine) were determined by use of a flow-through diffusion apparatus. Analysis of variance, a Duncan multiple range test, and an unpaired t-test were used to determine steady state kinetics and flux differences over time intervals. Although statistically significant differences were observed between flux values for both alkaloids and tissues at certain time points, these were not considered to be of biological (clinical) significance. However, the flux rates across both mucosa of r-arecoline were significantly higher statistically than those of rarecaidine. The findings demonstrated the differences in the diffusion kinetics between r-arecoline and r-arecaidine across human buccal and vaginal mucosa, an observation that could be explained in terms of their ionisation characteristics. Additionally, the results obtained further support the hypothesis that human vaginal mucosa can be used as a model for buccal mucosa in studies of permeability to various chemical compounds.

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Continued interest in the various routes by which sumatriptan may be administered prompted us to investigate its passage through buccal mucosa. Because human buccal mucosa is scarce, we proposed using the relatively abundant vaginal mucosa, which has been shown to have comparable diffusion rates for a number of widely varying molecules, as a model of buccal mucosa. In addition, by comparing these two tissues with respect to their permeability to sumatriptan, the human vaginal/buccal mucosa model could be further evaluated. Clinically healthy human vaginal and buccal mucosa specimens were used in the permeability studies. Permeability to sumatriptan was determined using a continuous flow-through diffusion system in the presence and absence of permeation enhancers. No statistically significant differences in permeability could be demonstrated for both mucosae toward sumatriptan. Flux values obtained in the absence and presence of glycodeoxycholate and lauric acid (1:1 molar ratio) to sumatriptan of buccal and vaginal mucosa, respectively, were not significantly different. The results obtained further support the hypothesis of the vaginal/buccal mucosal in vitro permeability model and suggest that this model may be used in conjunction with various absorption enhancers. Further studies on the buccal route of absorption of sumatriptan are thus warranted.

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The in vitro permeability of tritiated water through fresh and frozen human skin was evaluated in the presence and absence of two different barrier creams. Treated (10 min) and untreated fresh and frozen human skin disks (4 mm in diameter) were mounted in flow cells of a continuous flow-through diffusion apparatus. Buffer/tritiated water was collected from the acceptor chambers at 2-hour intervals for a total of 20 h and counted in a liquid scintillation counter. The results indicated that both barrier creams lowered the average flux rates of tritiated water through fresh and frozen skin, but no significant differences could be detected between the two preparations. However, different results may be obtained when compounds with molecular weights much higher than water are used.

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A number of studies have clearly demonstrated that human vaginal mucosa may be used as a model of buccal mucosa for a variety of in vitro permeability studies on drugs and other chemical compounds. Furthermore, at between 25 degrees and 37 degrees C, a linear temperature-dependence of water flux across this mucosa, which was attributed to an increased fluidity of the principal lipoidal permeability barrier, was found to exist. The objective of the present study was to examine the behaviour of the above barrier on water fluxes at normal and elevated temperatures. Clinically healthy human vaginal mucosa specimens were obtained from excess tissue removed during a vaginal hysterectomy from a single patient, snap-frozen in liquid nitrogen and stored for 6 months at -85 degrees C. Seven sections from the mucosa were thawed in phosphate-buffered saline (PBS) and mounted in flow-through diffusion cells (exposed area 0.039 cm2). Their permeability to tritiated water was determined using a continuous flow-through perfusion system at temperatures of 37 degrees, 39 degrees and 41 degrees C. Three permeability experiments were performed at each temperature setting. Specimens were subjected to histological examination before and after permeability experiments. Mean water flux rates at steady state (10-24 h) were found to be 2,356 +/- 71 SEM, 3,020 +/- 38 SEM and 3,659 +/- 116 SEM cpm. cm-2.min-1, at temperatures of 37 degrees, 39 degrees and 41 degrees C, respectively. A linear regression analysis and plot (r2 = 0.99) displayed a slope of 325 +/- 4 SEM cpm.cm-2.min-1/degree C. The results of this study showed that, notwithstanding cellular damage, the principal physical permeability barrier was linearly temperature-dependent between the temperatures studied, providing further support for the concept that this barrier is lipoidal in nature.

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OBJECTIVE: The aim of this study was to compare buccal and vaginal mucosa with respect to their permeability to a potent carcinogen, benzo[a]pyrene. STUDY DESIGN: Six clinically healthy vaginal mucosa specimens (mean patient age +/- standard deviation, 52+/-13.4 years; age range, 37-69 years) and 6 buccal mucosa specimens (from 5 male patients and 1 female patient: mean patient age +/- standard deviation, 32+/-5.2 years; age range, 24-39 years) were obtained during surgery. In vitro flux rates of benzo[a]pyrene across specimens were determined through use of a flow-through diffusion apparatus. Analysis of variance, a Duncan multiple range test, and an unpaired t test were used to determine steady state kinetics and flux differences over time intervals. RESULTS: No statistically significant differences were observed between the overall mean flux values of benzo[a]pyrene across the 2 kinds of mucosa. CONCLUSIONS: The findings further support the hypothesis that human vaginal mucosa can be used as a model for buccal mucosa in studies of permeability to various chemical compounds.

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The permeability to several chemical compounds and the histology of vaginal and buccal mucosa are very similar. Because vaginal mucosa is more abundant, it may be used as a model for the latter. To further develop the vaginal/buccal mucosa model, the objective of the present study was to evaluate the passage of a small polypeptide, vasopressin, across fresh and frozen specimens of these two mucosae. Specimens of fresh buccal and vaginal mucosa were taken from excised tissue obtained following vaginal hysterectomies and various oral surgical procedures. Pieces of buccal and vaginal tissue specimens obtained were used fresh or were snap-frozen and stored at -85 degrees C for periods of up to 10 months. Biopsies from fresh and thawed specimens were mounted in flow-through diffusion cells and their permeability to tritiated vasopressin was determined using a continuous flow-through perfusion system. Specimens were examined histologically before and after freezing as well as before and after permeability experiments and similarities between vaginal and buccal tissues verified. No statistically significant differences between flux values for fresh and frozen vaginal and buccal mucosa, respectively, were found. These results demonstrate that the permeation of vasopressin across fresh and frozen human vaginal and buccal mucosa is for practical purposes similar. These results further support the human vaginal/buccal mucosa model for in vitro permeability studies on therapeutically active compounds.

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In a previous study we demonstrated that human vaginal mucosa was as permeable to water as was buccal mucosa. Water, however, is a very small molecule with a molecular weight of 18 d. To further explore similarities between these two types of mucosa with respect to permeability, it was decided to investigate the passage of two large, hydrophilic molecules across these epithelia. Specimens of fresh, clinically healthy human vaginal and buccal mucosa were taken from excised tissue obtained during vaginal hysterectomies and various oral surgical procedures. Seven biopsy materials from each specimen were mounted in flow-through diffusion cells (exposed area, 0.039 cm2), and their permeability to 4.4- and 12-kd fluorescein-isothiocyanate-labeled dextrans was determined through use of a continuous flow-through perfusion system. Dextran was detected by means of a fluorospectrophotometric method at excitation and emission wave lengths of 498 and 520 nm, respectively. Specimens were examined histologically before and after permeability experiments, and similarities between vaginal and buccal tissues were verified. No statistically significant differences between the flux values of the 4.4-kd dextran across vaginal and buccal mucosa were found. However, for the 12-kd dextran the flux rate across buccal mucosa was significantly higher than the rate across vaginal mucosa. These results demonstrate that human vaginal mucosa is for practical purposes as permeable as buccal mucosa to 4.4-kd hydrophilic molecules. This further supports the hypothesis that vaginal mucosa may be a useful model for studying the passage across buccal mucosa of chemical compounds and therapeutic agents that are less than approximately 4.4 kd in molecular mass. For a 12-kd dextran the flux rate across buccal mucosa is significantly higher than the flux rate across vaginal mucosa, and the model becomes inaccurate.

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Because of the relative scarcity of fresh human oral mucosa specimens for permeability studies, we investigated the use of human vaginal mucosa as a model of the former. In a previous study we demonstrated the comparable diffusion rate of water across human vaginal and buccal mucosa and proposed the use of the former as a suitable model of the latter for in vitro drug permeability studies. To further evaluate the human vaginal/buccal mucosa model, we decided to compare these two tissues with respect to their permeability to 17beta-estradiol. Clinically healthy human vaginal and buccal mucosa specimens were obtained during vaginal hysterectomies and different oral surgical procedures. The permeability of each tissue specimen to 17beta-estradiol was determined through the use of a continuous flow-through diffusion system. Specimens were examined histologically before and after experiments. Mean flux values for 17beta-estradiol across human buccal mucosa tended to be slightly higher than those observed for vaginal tissue, but no statistically significant differences could be demonstrated. The results from this study further support our hypothesis that human vaginal mucosa may be a suitable model of human buccal mucosa for in vitro drug permeability studies.

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The resurgence of interest in the oral mucosa as a route for drug delivery requires a thorough understanding of the permeability of this tissue in health and disease. Previous work has indicated that non-keratinized oral mucosa is more permeable than its keratinized counterpart. It has been suggested that pathological hyperkeratotic mucosa, which was previously non-keratinized, would be more permeable than healthy tissue. Equivocal results obtained from animal studies in which chemical or mechanical irritation was used to induce a hyperplastic and hyperkeratotic epithelium, prompted us to conduct a study on the comparison of the permeability to water of lichen planus lesions and healthy buccal mucosa. Buccal mucosa was obtained from six patients with previously confirmed lichen planus and from six clinically healthy patients. Thawed 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. No statistically significant differences between mean steady state flux values (10-20 h) for lichen planus tissue and healthy buccal mucosa were found. These results warrant further studies with other oral conditions associated with hyperkeratosis to establish whether the nature and course of the condition are determinants for the retention or loss of the epithelium's permeability characteristics.

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The objective of the present study was to develop a single improved technique to culture human vaginal and buccal epithelial cells, whereby the cultured cells can be used in drug permeability studies. Cells were obtained from healthy human vaginal and buccal mucosa following vaginal hysterectomies and various oral surgical procedures. Tissue obtained was washed extensively in phosphate buffered saline (PBS, pH 7.3) containing antibiotics and amphotericin-B. Tissue specimens were cut into small pieces and plated out in 24-well plates. After drying, the full medium was added. Cell growth occurred within 4-6 days from primary explants and confluency was reached within 2-3 weeks. Primary explants yielded epithelial cells with minimal fibroblast contamination. After trypsinization, cells were seeded into collagen-coated wells and onto Transwell membranes. Trypsinized cells grew best on collagen-coated surfaces yielding more than one layer. The average steady state flux for the collagen-coated membranes (ccm's) containing either buccal or vaginal epithelial layers towards water was 6-10 times lower than that found for the cell-free ccm's. Fluxes for cultured cells on ccm's were 3x higher than those obtained for intact buccal and vaginal mucosa. Growth and permeability to water of the vaginal and buccal epithelial cells were comparable, confirming the similarity of these two tissues and the suitability of using the former as a model for the latter in permeability to tritiated water.

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