Anti-Inflammatory and Protective Effects of Juncus effusus L. Water Extract on Oral Keratinocytes.

Periodontitis is a chronic inflammatory disease caused by periodontopathogenic bacteria that form biofilms in periodontal pockets. The gingival epithelium acts as the first physical barrier in fighting attacks by periodontopathogenic pathogens, such as the primary etiological agent Porphyromonas gingivalis, and various exogenous chemicals, as well as regulates the local innate immune responses. Therefore, the development of novel oral care products to inhibit inflammatory reactions caused by bacterial infection and protect the gingival epithelium is necessary. Juncus effusus L. has generally been used as an indigenous medicine, such as a diuretic, an antipyretic, and an analgesic, in ancient practice. In this study, we examined the effects of a water extract from J. effusus L. on the inhibition of the inflammatory reaction elicited by bacterial infection and protection of the oral epithelium by chemical irritation. Pretreatment of oral epithelial cells with the water extract from J. effusus L. significantly reduced P. gingivalis or its lipopolysaccharide- (LPS-) mediated production of chemokines (interleukin-8 and C-C-chemokine ligand20) in a concentration-dependent manner with comparable to or greater effects than epigallocatechin gallate and protected oral epithelial cells from injury by chemical irritants, cetylpyridinium chloride, and benzethonium chloride. Moreover, the water extract from J. effusus L. in the presence of antimicrobial agents or antifibrinolytics already used as ingredients in mouthwash could significantly reduce the production of chemokines from P. gingivalis LPS-stimulated oral epithelial cells in a concentration-dependent manner. These findings suggest that the water extract from J. effusus L. is potentially useful for oral care to prevent oral infections, such as periodontal infections, and maintain oral epithelial function.

Development and Evaluation of Thermosensitive Hydrogels with Binary Mixture of Scutellariae baicalensis radix Extract and Chitosan for Periodontal Diseases Treatment.

Scutellaria baicalensis root displays anti-inflammatory and antibacterial properties due to the presence of flavonoids, particularly baicalin, baicalein, and wogonin. Our work aimed at developing thermosensitive hydrogels containing a binary mixture of S. baicalensis radix lyophilized extract and chitosan as a novel approach for periodontal diseases treatment. Two types of chitosan were employed in preliminary studies on binary mixtures with S. baicalensis radix lyophilized extract standardized for baicalin, baicalein, and wogonin. Thermosensitive hydrogels were prepared of poloxamer 407, alginate sodium, and cellulose derivatives and evaluated in terms of rheological and mucoadhesive behavior. The presence of chitosan altered the release profile of active compounds but did not affect their in vitro permeation behavior in PAMPA assay. The synergistic effects of S. baicalensis radix lyophilized extract and chitosan toward ferrous ion-chelating activity, inhibition of hyaluronidase, and pathogen growth were observed. The thermosensitive gelling system showed shear-thinning properties, gelation temperature between 25 and 27 °C, and favorable mucoadhesiveness in contact with porcine buccal mucosa, which was enhanced in the presence of binary mixture of S. baicalensis radix extract and chitosan. The release tests showed that baicalin and baicalein were liberated in a prolonged manner with a fast onset from hydrogel formulations.

Investigation of the Oral Retention of Tea Catechins in Humans: An Exploratory Interventional Study.

Green tea catechin ingestion or gargling exhibit anti-viral activity against upper respiratory infection. We hypothesized that retention in the oral cavity could improve the anti-viral effects of catechins. The present study investigated the oral retention of catechins in humans and the effect of catechin beverage viscosity on oral retention. Two intervention studies with different test beverages, beverage-C (40 mL, containing 73.4 mg of catechins) and beverage-XT (40 mL, beverage-C containing 100 mg xanthan gum) were conducted in 20 healthy volunteers (mean age 38.7 years). Catechin concentrations were measured in buccal mucosa samples collected at 10 min, 40 min, and 60 min after ingesting test beverages, and the catechin variability of the tissue after intake was compared between test beverages. As a result, the mean (SEM) concentrations of EGCG were 99.9 (27.2), 58.2 (16.6), and 22.3 (5.7) ng/mg-mucosa at 10, 40, and 60 min, respectively, after ingestion of beverage-XT. Similarly, the catechin concentrations were 86.1 (20.3), 32.2 (5.3), and 27.8 (5.9) ng/mg-mucosa after ingestion of beverage-C. The total retention volume over 60 min tended to be slightly higher after ingestion of beverage-XT, though the difference was not statistically significant. Additional studies are needed to confirm the effect of xanthan gum on improving oral retention of catechins.

Effects of a tart cherry (Prunus cerasus L.) phenolic extract on Porphyromonas gingivalis and its ability to impair the oral epithelial barrier.

Periodontal diseases, including gingivitis and periodontitis, are a global oral health problem. Porphyromonas gingivalis, a key pathogen involved in the onset of periodontitis, is able to colonize the subgingival epithelium and invade the underlying connective tissue due to the contribution of cysteine proteases known as gingipains. In this study, we investigated the effects of a phenolic extract prepared from tart cherry (Prunus cerasus L.) juice on the growth, adherence, and protease activity of P. gingivalis. We also assessed the protective effect of the tart cherry extract on the disruption of the oral epithelial barrier induced by P. gingivalis. The tart cherry extract that contains procyanidins and quercetin and its derivatives (rutinoside, glucoside) as the most important phenolic compounds attenuated P. gingivalis growth, reduced adherence to an experimental basement membrane matrix model, and decreased the protease activities of P. gingivalis. The tart cherry extract also exerted a protective effect on the integrity of the oral epithelial barrier in an in vitro model infected with P. gingivalis. More specifically, the extract prevented a decrease in transepithelial electrical resistance as well as the destruction of tight junction proteins (zonula occludens-1 and occludin). These results suggest that the tart cherry phenolic extract may be a promising natural product for the treatment of periodontitis through its ability to attenuate the virulence properties of P. gingivalis and curtail the ability of this pathogen to impair the oral epithelial barrier.

In search of preventive strategies: novel high-CBD Cannabis sativa extracts modulate ACE2 expression in COVID-19 gateway tissues.

With the current COVID-19 pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there is an urgent need for new therapies and prevention strategies that can help curtail disease spread and reduce mortality. The inhibition of viral entry and thus spread is a plausible therapeutic avenue. SARS-CoV-2 uses receptor-mediated entry into a human host via the angiotensin-converting enzyme 2 (ACE2), which is expressed in lung tissue as well as the oral and nasal mucosa, kidney, testes and gastrointestinal tract. The modulation of ACE2 levels in these gateway tissues may be an effective strategy for decreasing disease susceptibility. Cannabis sativa, especially those high in the anti-inflammatory cannabinoid cannabidiol (CBD), has been found to alter gene expression and inflammation and harbour anti-cancer and anti-inflammatory properties. However, its effects on ACE2 expression remain unknown. Working under a Health Canada research license, we developed over 800 new C. sativa cultivars and hypothesized that high-CBD C. sativa extracts may be used to down-regulate ACE2 expression in target COVID-19 tissues. Using artificial 3D human models of oral, airway and intestinal tissues, we identified 13 high-CBD C. sativa extracts that decrease ACE2 protein levels. Some C. sativa extracts down-regulate serine protease TMPRSS2, another critical protein required for SARS-CoV-2 entry into host cells. While our most effective extracts require further large-scale validation, our study is important for future analyses of the effects of medical cannabis on COVID-19. The extracts of our most successful novel high-CBD C. sativa lines, pending further investigation, may become a useful and safe addition to the prevention/treatment of COVID-19 as an adjunct therapy.

Photobiomodulation modulates inflammation and oral microbiome: a pilot study.

INTRODUCTION: Oral mucositis (OM) is a severe side effect in patients undergoing anticancer therapies, which negatively impacts on their quality of life often leading to either the interruption of the therapy. Photobiomodulation (PBM) is emerging as an effective strategy allowing a faster wound healing. OBJECTIVES: This pilot study aims at verifying whether PBM modulates the inflammatory response in patients and its effect on the oral microbiome composition. MATERIALS AND METHODS: Buccal swabs were collected from four patients affected by OM, both on ulcerated and clinically healthy areas, before and on the last day of PBM therapy, as well as on the first day after treatment discontinuation. The concentration of 38 cytokines and the composition of oral microbiome were measured. RESULTS: Most of the pro-inflammatory cytokines were reduced, whereas anti-inflammatory cytokines resulted up-regulated by PBM. In addition, PBM influenced the composition of oral microbiome, by decreasing the amount of pathogenic species and promoting the growth of commensal bacteria. These changes were even more evident when separately analysing patients who clinically responded to PBM and the only patient who did not respond. CONCLUSIONS: PBM reduces inflammatory burden in patients affected by OM and positively influences the composition of the oral microbiome.

Allergen Stability and Immunological Reactivity during Co-dissolution and Incubation of House Dust Mite and Japanese Cedar Pollen SLIT-Tablets.

Japanese allergic subjects are commonly sensitized to both house dust mite (HDM) and Japanese cedar pollen (JCP) and combined treatment with sublingual immunotherapy (SLIT) tablets is desirable. However, mixing extracts of two non-homologous allergens may compromise allergen stability and affect the clinical outcome. Therefore, we investigated the stability of major allergens and total allergenic reactivity of HDM and JCP SLIT-tablets following dissolution in human saliva or artificial gastric juice. Two fast-dissolving freeze-dried SLIT-tablets were completely dissolved and incubated at 37 °C. Major allergen concentrations and total allergenic reactivity were measured. After mixing and co-incubation of HDM and JCP SLIT tablets in human saliva for 10 min at 37°C, there were no statistically significant changes in major allergen concentrations. In addition, no loss of allergenic reactivity of the mixed two SLIT-tablet solutions was seen. In contrast, complete loss of allergenic reactivity and detectable major allergen concentrations occurred when the two SLIT-tablets were dissolved and incubated in artificial gastric juice. These results demonstrate that HDM or JCP major allergens and the total allergenic reactivity of both SLIT-tablets measured here remain intact after dissolution and co-incubation in human saliva, supporting the possibility of a dual HDM and JCP SLIT-tablet administration regimen if clinically indicated. The complete loss of allergenic reactivity after incubation in artificial gastric juice can furthermore be taken to indicate that the immunological activity of the allergen extracts contained in the two SLIT-tablets is likely to be lost or severely compromised upon swallowing.

Oral interactions between a green tea flavanol extract and red wine anthocyanin extract using a new cell-based model: insights on the effect of different oral epithelia.

Phenolic compounds (PC) are linked to astringency sensation. Astringency studies typically use simple models, with pure PC and/or proteins, far from what is likely to occur in the oral cavity. Different oral models have been developed here, comprising different oral epithelia (buccal mucosa (TR146) and tongue (HSC-3)) and other main oral constituents (human saliva and mucosal pellicle). These models, were used to study the interaction with two PC extracts, one rich in flavanols (a green tea extract) and one rich in anthocyanins (a red wine extract). It was observed that within a family of PC, the PC seem to have a similar binding to both TR146 and HSC-3 cell lines. When the oral constituents occur altogether, flavanols showed a higher interaction, driven by the salivary proteins. Conversely, anthocyanins showed a lower interaction when the oral constituents occur altogether, having a higher interaction only with oral cells. Epigallocatechin gallate, epicatechin gallate, epigallocatechin-3-O(3-O-methyl) gallate were the flavanols with the highest interaction. For the studied anthocyanins (delphinidin-3-glucoside, peonidin-3-glucoside, petunidin-3-glucoside and malvidin-3-glucoside), there was not a marked difference on their interaction ability. Overall, the results support that the different oral constituents can have a different function at different phases of food (PC) intake. These differences can be related to the perception of different astringency sub-qualities.

Adjuvanting Allergen Extracts for Sublingual Immunotherapy: Calcitriol Downregulates CXCL8 Production in Primary Sublingual Epithelial Cells.

Application of allergens onto the sublingual epithelium is used to desensitize allergic individuals, a treatment known as sublingual immunotherapy. However, the response of sublingual epithelial cells to house dust mite allergen and potential tolerance-promoting adjuvants such as Toll-like receptor (TLR) ligands and calcitriol has not been investigated. In order to study this, primary sublingual epithelial cells were isolated from dogs and cultured in vitro. After 24-h incubation with a Dermatophagoides farinae extract, a Dermatophagoides pteronyssinus extract, TLR2 ligands (FSL-1, heat-killed Listeria monocytogenes, Pam3CSK4), a TLR3 ligand (poly I:C), a TLR4 ligand [lipopolysaccharide (LPS)], and calcitriol (1,25-dihydroxyvitamin D3), viability of the cells was analyzed using an MTT test, and their secretion of interleukin 6 (IL-6), IL-10, CXCL8, and transforming growth factor ß1 (TGF-ß1) was measured by enzyme-linked immunosorbent assay. Additionally, to evaluate its potential effect as an adjuvant, sublingual epithelial cells were incubated with calcitriol in combination with a D. farinae extract followed by measurement of CXCL8 secretion. Furthermore, the effect of D. farinae and calcitriol on the transcriptome was assessed by RNA sequencing. The viability of the sublingual epithelial cells was significantly decreased by poly I:C, but not by the other stimuli. CXCL8 secretion was significantly increased by D. farinae extract and all TLR ligands apart from LPS. Calcitriol significantly decreased CXCL8 secretion, and coadministration with D. farinae extract reduced CXCL8 concentrations to levels seen in unstimulated sublingual epithelial cells. Although detectable, TGF-ß1 secretion could not be modulated by any of the stimuli. Interleukin 6 and IL-10 could not be detected at the protein or at the mRNA level. It can be concluded that a D. farinae extract and TLR ligands augment the secretion of the proinflammatory chemokine CXCL8, which might interfere with sublingual desensitization. On the other hand, CXCL8 secretion was reduced by coapplication of calcitriol and a D. farinae extract. Calcitriol therefore seems to be a suitable candidate to be used as adjuvant during sublingual immunotherapy.

Development and evaluation of mucoadhesive buccal dosage forms of lidocaine hydrochloride by ex-vivo permeation studies.

Intraoral lidocaine formulations are applied in children and adults for pain relief. The potential risks associated with orally administered lidocaine due to accidental ingestions were highlighted in a warning letter by the US Food and Drug Administration (FDA). This increases the urgency for a need of a child-appropriate dosage forms. For risk minimization, a novel buccal composite dosage form was developed consisting of a lidocaine containing minitablet centered on top of a bilayered mucoadhesive buccal film, so called composite. The preparation included direct tableting of minitablets as well as film-casting technique. Within a comparability study, the permeation of this composite was classified against marketed lidocaine gel, a single-layer film, and a minitablet. These ex-vivo permeation studies under physiologically related conditions in combination with LC-MS/MS quantification enabled the evaluation of permeation in clinically relevant short-term application. The composite showed comparable permeation to marketed gel (104.26 ± 30.15 µg/cm2 vs 128.17 ± 12.49 µg/cm2 cumulative amount of drug) and a higher permeation compared to film (25.84 ± 6.01 µg/cm2). Therefore, a controlled drug application can be assumed by the composite, whereby the risk of inadvertent swallowing as well as uncontrolled absorbed amount of drug substance may be substantially minimized.

Autophagy and Hsp70 activation alleviate oral epithelial cell death induced by food-derived hypertonicity.

Stable intracellular and intercellular osmolarity is vital for all physiological processes. Although it is the first organ that receives food, the osmolarity around the mouth epithelium has never been systematically investigated. We found that oral epithelial cells are a population of ignored cells routinely exposed to hypertonic environments mainly composed of saline, glucose, etc. in vivo after chewing food. By using cultured oral epithelial cells as an in vitro model, we found that the hypotonic environments caused by both high NaCl and high glucose induced cell death in a dose- and time-dependent manner. Transcriptomics revealed similar expression profiles after high NaCl and high glucose stimulation. Most of the common differentially expressed genes were enriched in "mitophagy" and "autophagy" according to KEGG pathway enrichment analysis. Hypertonic stimulation for 1 to 6 h resulted in autophagosome formation. The activation of autophagy protected cells from high osmolarity-induced cell death. The activation of Hsp70 by the pharmacological activator handelin significantly improved the cell survival rate after hypertonic stimulation. The protective role of Hsp70 activation was partially dependent on autophagy activation, indicating a crosstalk between Hsp70 and autophagy in hypertonic stress response. The extract of the handelin-containing herb Chrysanthemum indicum significantly protected oral epithelial cells from hypertonic-induced death, providing an inexpensive way to protect against hypertonic-induced oral epithelial damage. In conclusion, the present study emphasized the importance of changes in osmolarity in oral health for the first time. The identification of novel compounds or herbal plant extracts that can activate autophagy or HSPs may contribute to oral health and the food industry.

System, Method and Software for Calculation of a Cannabis Drug Efficiency Index for the Reduction of Inflammation.

There are many varieties of Cannabis sativa that differ from each other by composition of cannabinoids, terpenes and other molecules. The medicinal properties of these cultivars are often very different, with some being more efficient than others. This report describes the development of a method and software for the analysis of the efficiency of various cannabis extracts to detect the anti-inflammatory properties of the various cannabis extracts. The method uses high-throughput gene expression profiling data but can potentially use other omics data as well. According to the signaling pathway topology, the gene expression profiles are convoluted into the signaling pathway activities using a signaling pathway impact analysis (SPIA) method. The method was tested by inducing inflammation in human 3D epithelial tissues, including intestine, oral and skin, and then exposing these tissues to various extracts and then performing transcriptome analysis. The analysis showed a different efficiency of the various extracts in restoring the transcriptome changes to the pre-inflammation state, thus allowing to calculate a different cannabis drug efficiency index (CDEI).

Tart cherry (Prunus cerasus L.) fractions inhibit biofilm formation and adherence properties of oral pathogens and enhance oral epithelial barrier function.

Dental caries, candidiasis, and periodontal disease are the most common oral infections affecting a wide range of the population worldwide. The present study investigated the effects of two tart cherry (Prunus cerasus L.) fractions on important oral pathogens, including Candida albicans, Streptococcus mutans, and Fusobacterium nucleatum, as well as on the barrier function of oral epithelial cells. Procyanidins and quercetin and its derivatives were the most important constituents found in the tart cherry fractions. Although the fractions showed poor antimicrobial activity, they inhibited biofilm formation by the three oral pathogens in a dose-dependent manner. The tart cherry fractions also attenuated the adherence of C. albicans and S. mutans to a hydroxylapatite surface as well as the adherence of F. nucleatum to oral epithelial cells. Treating oral epithelial cells with the tart cherry fractions significantly enhanced the barrier function as determined by monitoring the transepithelial electrical resistance. In conclusion, this study showed that the tart cherry fractions and their bioactive constituents could be promising antiplaque compounds by targeting biofilm formation and adherence properties of oral pathogens. Furthermore, its property of increasing the epithelial barrier function may protect against microbial invasion of the underlying connective tissue.

Is rat a good model for assessment of particulate-based taste-masked formulations?

Recently there has been an increased interest to develop specialised dosage forms that are better suited to specific patient populations, such as paediatrics and geriatrics. In these patient populations the acceptability of the oral dosage form can be paramount to the products success. However, many Active Pharmaceutical Ingredients (APIs) are known to cause an aversive taste response. One way to increase the acceptability and to enhance the palatability of the formulation is to design coated taste-masked particulate-based dosage forms. The masking of poorly tasting drugs with physical barriers such as polymer coatings can be utilised to prevent the release of drug within the oral cavity, thus preventing a taste response. However, currently, there are few assessment tools and models available to test the efficiency of these particulate-based taste-masked formulations. The rat brief access taste aversion model has been shown to be useful in assessment of taste for liquid dosage forms. However, the applicability of the rat model for particulate-based taste masked formulations is yet to be assessed. It is not understood whether dissolution, solubility and thus exposure of the drug to taste receptors would be the same in rat and human. Therefore, rat saliva must be compared to human saliva to determine the likelihood that drug release would be similar within the oral cavity for both species. In this study rat saliva was characterised for parameters known to be important for drug dissolution, such as pH, buffer capacity, surface tension, and viscosity. Subsequently dissolution of model bitter tasting compounds, sildenafil citrate and efavirenz, in rat saliva was compared to dissolution in human saliva. For all parameters characterised and for the dissolution of both drugs in rat saliva, a substantial difference was observed when compared to human saliva. This discrepancy in saliva parameters and dissolution of model drugs suggests that preclinical taste evaluation of particulate-based taste-masked formulations suggests rat is not a good model for predicting taste of solid dosage forms or undissolved drug where dissolution is required. Alternative preclinical in vivo models in other species, or improved biorelevant in vitro models should be considered instead.

Mucoadhesive thin films for the simultaneous delivery of microbicide and anti-inflammatory drugs in the treatment of periodontal diseases.

There is an unmet clinical need for new products to address the high percentage of the populous who present with periodontal diseases. Drug dose retention at the point of application would facilitate sustained release and more efficacious treatments. The aim of this study was to evaluate mucoadhesive polymeric thin films for simultaneous in situ delivery chlorhexidine and anti-inflammatory and analgesic drugs. Mucoadhesive thin films were prepared using a polymer mixture containing chlorhexidine (25 mg) ± diclofenac sodium (10 and 50 mg), and lidocaine hydrochloride (10 mg) or betamethasone dipropionate (10 and 50 mg). The films were assessed for in vitro drug release and localised tissue delivery, followed by determination of modulated prostaglandin E2 (PGE2) levels in ex vivo tissue and cytotoxicity using a HaCaT keratinocyte cell line. Antibacterial activity of the chlorhexidine/diclofenac film was determined against planktonic and biofilm bacteria associated with periodontal disease and dental plaque. Chlorhexidine release was consistently low (up to 10% of initial loading) from all films, whereas the release of diclofenac, betamethasone and lidocaine exceeded 50% within 30 min. The 50 mg betamethasone film released up to 4-fold more than the 10 mg film. Statistically significant reduction of PGE2 was observed in ex vivo porcine gingival tissue for films containing chlorhexidine with or without diclofenac, and betamethasone. No cytotoxicity was observed for any film, apart from 50 mg betamethasone at 24 h. Films loaded with chlorhexidine and diclofenac were inhibitory against relevant test bacteria. Between 3 and 6 log10 reductions in bacterial cell recovery was observed after biofilm exposure to the chlorhexidine films irrespective of the presence of the anti-inflammatory or anaesthetic. This work demonstrated that thin film formulations have the potential to simultaneously counter key causative factors in periodontal diseases, namely associated bacteria biofilm and chronic local inflammation.

Recent developments of nanoparticle-delivered dosage forms for buccal delivery.

The improvement of mucoadhesive buccal formulations has attracted profound interest in recent years. Drug permeability and controlled drug release via this route of administration are still a concern for effective therapy, although various strategies have been proposed. Advanced developments in nanotechnology have been investigated with promising clinical applications. The incorporation of nanoparticles in dosage forms for buccal delivery not only ensures efficient delivery but also reduces side effects to biological systems. Many approaches have been suggested to load and deliver nanoparticles containing drugs to the buccal mucosa both in topical and systemic administrations. This paper describes recent advanced concepts to include nanoparticles in dosage forms with improving drug delivery or targeting. The classification and discussion of these technologies are aimed at providing an overview of current strategies for applying controlled drug delivery. All remarkable findings will encourage innovative nanoparticle-delivered dosage forms for buccal delivery in clinical applications.

Micronucleus Cytome Assays in Human Lymphocytes and Buccal Cells.

The micronucleus (MN) assay, applied in different surrogate tissues, is one of the best validated cytogenetic techniques for evaluating chromosomal damage in humans. The cytokinesis-block micronucleus cytome assay in peripheral blood lymphocytes (L-CBMNcyt) is the most frequently used method in biomonitoring human populations to evaluate DNA damage caused by exposure to genotoxic agents, micronutrient deficiency or excess and genetic instability. Furthermore, recent scientific evidence suggests an association between an increased MN frequency in lymphocytes and risk of cancer and other age-related degenerative diseases. The micronucleus cytome assay applied in buccal exfoliated cells (BMNCyt), provides a complementary method for measuring DNA damage and cytotoxic effects in an easily accessible tissue not requiring ex vivo/in vitro culture. The protocol for L-CBMNcyt described here, refers to the use of ex vivo whole blood method, involving 72 h of culture with the block of cytokinesis starting at 44 h. BMNCyt protocol reports the established method for sample collection, processing, slide preparation and scoring.

Novel Human Full-Thickness Three-Dimensional Nonkeratinized Mucous Membrane Model for Pharmacological Studies in Wound Healing.

INTRODUCTION: Efforts are increasingly aiming to develop in vitro models that can provide effective alternatives to in vivo experiments. The main aim of this study was the establishment of an in vitro model of the nonkeratinized mucous membrane that can be used as a standardized tool to evaluate biological and therapeutic effects of pharmaceuticals for mucosal wound healing. METHODS: We established a full-thickness in vitro model of the nonkeratinized mucous membrane. While histological examination was performed to assess morphological characteristics, we utilized gene expression profiling using microarray and qRT-PCR analyses to identify molecular effects of treatment with a dexpanthenol-containing ointment after laser wounding. RESULTS: Performing histological and immunofluorescence analyses we proved that our model mimics the two distinctive layers of the mucous membrane - the stratified squamous epithelium and the lamina propria. We used this model to investigate molecular effects of a dexpanthenol-containing ointment that is commonly used for the wound treatment of mucous membranes. For that purpose, our model exhibits a unique feature in that dexpanthenol and proliferation-enhancing additives that may interfere with our studies are not required for the maintenance of the model culture. After setting standardized lesions with a nonsequential fractional ultrapulsed CO2 laser, topical treatment with the dexpanthenol-containing ointment enhanced wound closure in the model compared to placebo and untreated controls. Furthermore, microarray analysis revealed that the treatment of the laser-wounded model with the dexpanthenol-containing ointment evoked an upregulated expression of various genes related to accelerated wound healing. CONCLUSION: Overall, we verified that this novel mucous membrane model can be utilized in future to monitor ex vivo effects of various topical therapies on mucosa morphology, physiology, and gene expression. Our findings confirm the potential of the model as an in vitro tool for the replacement of pharmacological in vivo studies regarding mucosal wound healing.

Large-Scale and Rapid Preparation of Nanofibrous Meshes and Their Application for Drug-Loaded Multilayer Mucoadhesive Patch Fabrication for Mouth Ulcer Treatment.

Electrospinning provides a simple and convenient method to fabricate nanofibrous meshes. However, the nanofiber productivity is often limited to the laboratory scale, which cannot satisfy the requirements of practical application. In this study, we developed a novel needleless electrospinning spinneret based on a double-ring slit to fabricate drug-loaded nanofibrous meshes. In contrast to the conventional single-needle electrospinning spinneret, our needless spinneret can significantly improve nanofiber productivity due to the simultaneous formation of multiple jets during electrospinning. Curcumin-loaded poly(l-lactic acid) (PLLA) nanofiber meshes with various concentrations and on the large scale were manufactured by employing our developed needleless spinneret-based electrospinning device. We systematically investigated the drug release behaviors, antioxidant properties, anti-inflammatory attributes, and cytotoxicity of the curcumin-loaded PLLA nanofibrous meshes. Furthermore, a bilayer nanofibrous composite mesh was successfully generated by electrospinning curcumin-loaded PLLA solution and diclofenac sodium loaded poly(ethylene oxide) solution in a predetermined time sequence, which revealed potent antibacterial properties. Subsequently, novel mucoadhesive patches were assembled by combining the bilayer composite nanofibrous meshes with (hydroxypropyl)methyl cellulose based mucoadhesive film. The multilayered mucoadhesive patch has excellent adhesion properties on the porcine buccal mucosa. Overall, our double-ring slit spinneret can provide a novel method to rapidly produce large-scale drug-loaded nanofibrous meshes to fabricate mucoadhesive patches. The multiple-layered mucoadhesive patches enable the incorporation of multiple drugs with different targets of action, such as analgesic, anti-inflammatory, and antimicrobial compounds, for mouth ulcer or other oral disease treatments.

Distinct blood and milk 18-carbon fatty acid proportions and buccal bacterial populations in dairy cows differing in reticulorumen pH response to dietary supplementation of rapidly fermentable carbohydrates.

Nine Holstein dairy cows were fed diets with increasing proportions of rapidly fermentable carbohydrates (RFCH) to investigate the effect on reticular pH, milk fat content (MFC), 18-carbon fatty acid proportions in blood plasma and milk, and bacterial community in buccal swab samples. Inter-animal variation was expected in terms of reticular pH response upon higher RFCH proportions, which would be reflected in the occurrence or not of milk fat depression (MFD). Moreover, this variation in occurrence of MFD was hypothesized to be related to differences in blood and milk fatty acid proportions and in the bacterial community in buccal samples. Cows were fed a total mixed ration throughout the experiment, which consisted of 4 periods: adaptation (d 0-4) and low (d 5-18), increasing (d 19-24), and high RFCH (d 25-28). During the increasing RFCH period, the standard concentrate (211 g of starch/kg of dry matter) was gradually and partly replaced by a concentrate high in RFCH (486 g of starch/kg of dry matter). The reticular pH was measured using a bolus and the time below pH 6.00 was calculated on a daily basis. On d 13, 14, 25, 27, and 28, plasma and milk samples were collected and analyzed for 18-carbon fatty acid proportions, and buccal swabs were collected for bacterial community analysis based on 16S rRNA gene amplicon sequencing. Inter-animal variation was observed in terms of reticular pH, which allowed us to divide the cows into 2 groups: tolerant (time below pH 6.00 ≤ 0.1 h/d) and susceptible cows (time below pH 6.00 ≥ 1.26 h/d). The lower reticular pH of susceptible cows was accompanied by lower MFC. Both groups already differed in reticular pH and MFC during the low-RFCH period. Furthermore, higher RFCH amounts did not decrease the reticular pH in either of the 2 groups. Nevertheless, MFD was observed in both groups during the high-RFCH period compared with the low-RFCH period. Lower MFC in animals with lower reticular pH or during the high-RFCH period was associated with a shift in 18-carbon fatty acids toward trans-10 at the expense of trans-11 intermediates, which was observed in plasma as well as in milk samples. Moreover, lower MFC was accompanied by shifts in the relative abundance of specific bacteria in buccal samples. Genera Dialister, Sharpea, Carnobacterium, Acidaminococcus, and uncultured genera belonging to the Betaproteobacteria were more abundant in situations with greater trans-10 proportions.