c-Src activation as a potential marker of chemical-induced skin irritation using tissue-engineered skin equivalents.

Skin irritancy to topically applied chemicals is a significant problem that affects millions of people worldwide. New or modified chemical entities must be tested for potential skin irritancy by industry as part of the safety and toxicity profiling process. Many of these tests have now moved to a non-animal-based format to reduce experiments on animals. However, these tests for irritancy potential often rely on monolayer cultures of keratinocytes that are not representative of the skin architecture or tissue-engineered human skin equivalents (HSE) using complex multi-gene expression panels that are often cumbersome and not amenable for high throughput. Here, we show that human skin equivalents increase abundance of several phosphorylated kinases (c-Src, c-Jun, p53, GSK3α/ß) in response to irritant chemical stimulation by phosphokinase array analysis. Specific phosphorylation of c-SrcY419 was confirmed by immunoblotting and was plasma membrane-associated in basal/spinous cells by phospho-specific immunohistochemistry. Moreover, c-SrcY419 phosphorylation in response to the irritants lactic acid and capsaicin was inhibited by the c-Src inhibitors KB-SRC and betaine trimethylglycine. These data provide the first evidence for c-Src specific activation in response to chemical irritants and point to the development of new modes of rapid testing by immunodetection for first-pass screening of potential irritants.

Immune mechanisms in oral lichen planus.

Oral lichen planus (OLP) is a T cell-mediated inflammatory disease of the oral mucosa that has been extensively researched over many years but as yet the mechanisms of pathogenesis are still not fully understood. Whilst the specific aetiological factors driving OLP remain ambiguous, evidence points to the development of a chronic, dysregulated immune response to OLP-mediating antigens presented by innate immune cells and oral keratinocytes leading to increased cytokine, chemokine and adhesion molecule expression. These molecules recruit T cells and mast cells to the diseased site and orchestrate a complex interplay between cells that culminates in keratinocyte cell death, mucosal basement membrane destruction and long-term chronicity of the disease. The main lymphocytes involved are thought to be CD8+ cytotoxic and CD4+ Th1 polarised T cells although recent evidence indicates the involvement of other Th subsets such as Th9, Th17 and Tregs, suggesting that a more complex immune cell relationship exists during the disease process. This review provides an overview of the immune mechanisms at play in OLP pathogenesis with particular emphasis on the role of the different Th subsets and how these recent discoveries may guide research towards identifying potential therapeutic targets.

Computational Modelling for Electrical Impedance Spectroscopy-Based Diagnosis of Oral Potential Malignant Disorders (OPMD).

A multiscale modelling approach has been applied to the simulation of the electrical properties of oral tissue, for the purpose of informing an electrical impedance-based method of oral potential malignant disorder (OPMD) diagnosis. Finite element models of individual cell types, with geometry informed by histological analysis of human oral tissue (normal, hyperplastic and dysplastic), were generated and simulated to obtain electrical parameters. These were then used in a histology-informed tissue scale model, including the electrode geometry of the ZedScan tetrapolar impedance-measurement device. The simulations offer insight into the feasibility of distinguishing moderate dysplasia from severe dysplasia or healthy tissue. For some oral sites, simulated spectra agreed with real measurements previously collected using ZedScan. However, similarities between simulated spectra for dysplastic, keratinised and non-dysplastic but hyperkeratinised tissue suggest that significant keratinisation could cause some OPMD tissues to exhibit larger than expected impedance values. This could lead to misidentification of OPMD spectra as healthy. Sources of uncertainty within the models were identified and potential remedies proposed.

Corticosteroid delivery using oral mucosa equivalents for the treatment of inflammatory mucosal diseases.

Oral lichen planus (OLP) is an immune-mediated disease of the oral mucosa with idiopathic aetiology. It is frequently treated with topical corticosteroids (applied as gels, mouthwashes, or sprays); however, the mucosal exposure times of topical corticosteroids are short because of removal by the constant flow of saliva and mechanical forces. In this study we used cell monolayers, as well as oral mucosal equivalents (OMEs) containing activated T-cells, to examine corticosteroid potency and delivery of clobetasol-17-propionate from a novel electrospun mucoadhesive patch. The OMEs displayed tight junctions, desmosomes, hemidesmosomes, and an efficient permeability barrier. Following application of corticosteroids to cells cultured as monolayers, the degree of cytotoxicity measured correlated to the level of potency recognized for each corticosteroid; by contrast, OMEs were largely unaffected by corticosteroid treatment. Permeation of clobetasol-17-propionate into and through the OMEs was time- and dose-dependent, regardless of whether this corticosteroid was delivered in liquid form or from a mucoadhesive patch, and both liquid- and patch-delivered clobetasol-17-propionate significantly reduced the secretion of interleukin-2 by activated T-cells. This study confirms that OMEs are more suitable models than cell monolayers for evaluating toxicity and drug delivery. After topical exposure, clobetasol-17-propionate accumulated in OMEs at a higher level than betamethasone-17-valerate and hydrocortisone-17-valerate, and exerted its immunosuppressive actions following application via the patch delivery system, highlighting the efficacy of this mode of drug delivery to treat OLP.

Candidalysin Is Required for Neutrophil Recruitment and Virulence During Systemic Candida albicans Infection.

BACKGROUND: Candidalysin is a cytolytic peptide toxin secreted by Candida albicans hyphae and has significantly advanced our understanding of fungal pathogenesis. Candidalysin is critical for mucosal C albicans infections and is known to activate epithelial cells to induce downstream innate immune responses that are associated with protection or immunopathology during oral or vaginal infections. Furthermore, candidalysin activates the NLRP3 inflammasome and causes cytolysis in mononuclear phagocytes. However, the role of candidalysin in driving systemic infections is unknown. METHODS: In this study, using candidalysin-producing and candidalysin-deficient C albicans strains, we show that candidalysin activates mitogen-activated protein kinase (MAPK) signaling and chemokine secretion in endothelial cells in vitro. RESULTS: Candidalysin induces immune activation and neutrophil recruitment in vivo, and it promotes mortality in zebrafish and murine models of systemic fungal infection. CONCLUSIONS: The data demonstrate a key role for candidalysin in neutrophil recruitment and fungal virulence during disseminated systemic C albicans infections.

The IL-1/IL-1R axis induces greater fibroblast-derived chemokine release in human papillomavirus-negative compared to positive oropharyngeal cancer.

Human papillomavirus (HPV) is now recognised as a major aetiological agent in the pathogenesis of oropharyngeal carcinoma (OPC). HPV-positive tumours are associated with better outcomes compared to HPV-negative tumours, possibly due to differences in their aetiology and/or the tumour microenvironment. Increased numbers of tumour-associated leukocytes have been observed in many cancers including OPC, with variable influence on prognosis depending on the leukocyte subpopulation investigated. Whether HPV status influences leukocyte recruitment to OPC remains unknown. This in-vitro study examined differences in the chemoattractant capacity of HPV-positive and HPV-negative OPC cell lines. Gene and protein expression analysis demonstrated that whilst both monocultures of HPV-positive and HPV-negative cell lines, along with normal tonsillar fibroblasts (NTF), expressed low chemokine levels, NTF cultured with conditioned medium from HPV-negative OPC cells expressed significantly higher levels of all chemokines tested compared to NTF incubated with the medium from HPV-positive OPC cell lines. HPV-negative OPC lines expressed IL-1ß mRNA whereas HPV-positive cells did not, and NTF constitutively expressed IL-1R1. Pre-treatment with the IL-R antagonist, anakinra or siRNA to IL-1R1 significantly reduced chemokine secretion from NTF stimulated with conditioned medium from HPV-negative tumour cells or recombinant IL-1ß (p < 0.05). These data suggest that secretion of chemokines is driven by the interaction between HPV-negative OPC cells and stromal fibroblasts through an IL-1/IL-1R-mediated mechanism that is less prominent within the HPV-positive tumour microenvironment. These observations may explain differences in leukocyte sub-populations recruited to HPV-positive versus negative OPC and indicate that HPV status is a key determinant in controlling the inflammatory tumour microenvironment.

Mucoadhesive Electrospun Patch Delivery of Lidocaine to the Oral Mucosa and Investigation of Spatial Distribution in a Tissue Using MALDI-Mass Spectrometry Imaging.

Many oral mucosal conditions cause considerable and prolonged pain that to date has been difficult to alleviate via topical delivery, and the use of injection causes many patients dental anxiety and needle-prick pain. Therefore, developing a noninjectable drug delivery system as an alternative administration procedure may vastly improve the health and wellbeing of these patients. Recent advances in the development of mucoadhesive electrospun patches for the direct delivery of therapeutics to the oral mucosa offer a potential solution, but as yet, the release of local anesthetics from this system and their uptake by oral tissue have not been demonstrated. Here, we demonstrate the fabrication of lidocaine-loaded electrospun fiber patches, drug release, and subsequent uptake and permeation through the porcine buccal mucosa. Lidocaine HCl and lidocaine base were incorporated into the electrospun patches to evaluate the difference in drug permeation for the two drug compositions. Lidocaine released from the lidocaine HCl-containing electrospun patches was significantly quicker than from the lidocaine base patches, with double the amount of drug released from the lidocaine HCl patches in the first 15 min (0.16 ± 0.04 mg) compared to that from the lidocaine base patches (0.07 ± 0.01 mg). The permeation of lidocaine from the lidocaine HCl electrospun patches through ex vivo porcine buccal mucosa was also detected in 15 min, whereas permeation of lidocaine from the lidocaine base patch was not detected. Matrix-assisted laser desorption ionization-mass spectrometry imaging was used to investigate localization of lidocaine within the oral tissue. Lidocaine in the solution as well as from the mucoadhesive patch penetrated into the buccal mucosal tissue in a time-dependent manner and was detectable in the lamina propria after only 15 min. Moreover, the lidocaine released from lidocaine HCl electrospun patches retained biological activity, inhibiting veratridine-mediated opening of voltage-gated sodium channels in SH-SY5Y neuroblastoma cells. These data suggest that a mucoadhesive electrospun patch may be used as a vehicle for rapid uptake and sustained anesthetic drug delivery to treat or prevent oral pain.

HPV-negative, but not HPV-positive, oropharyngeal carcinomas induce fibroblasts to support tumour invasion through micro-environmental release of HGF and IL-6.

Human papillomavirus (HPV) infection is causally related to a subset of oropharyngeal carcinomas (OPC) and is linked to a more favourable prognosis compared to HPV-negative OPC. The mechanisms underlying this effect on prognosis are not fully understood, but interactions with the tumour microenvironment may be pivotal. Here, we investigated the role of the tumour microenvironment in HPV-positive compared to HPV-negative cancer using 2D and 3D modelling of OPC interactions with stromal fibroblasts. HPV-negative, but not HPV-positive, OPC-derived cell lines induced a rapid fibroblast secretory response that supported 2D cancer cell migration and invasion in vitro. Array profiling of this HPV-negative induced fibroblast secretome identified hepatocyte growth factor (HGF) as the principal secreted factor that promoted cancer cell migration. The interaction between HPV-negative cell lines and fibroblasts in 2D was prevented using c-Met (HGF receptor) inhibitors, which further restricted both HPV-negative and positive cell invasion in 3D co-culture models. Furthermore, we discovered a synergistic relationship between HGF and IL-6 in the support of migration that relates JAK activation to HGF responsiveness in HPV-negative lines. In summary, our data show significant differences in the interactions between HPV-positive and HPV-negative OPC cells and stromal fibroblasts. In addition, we, provide in vitro evidence to support the clinical application of c-MET inhibitors in the control of early HPV-negative OPC.

Expression and enzyme activity of cytochrome P450 enzymes CYP3A4 and CYP3A5 in human skin and tissue-engineered skin equivalents.

CYP3A4 and CYP4A5 share specificity for a wide range of xenobiotics with the CYP3 subfamily collectively involved in the biotransformation of approximately 30% of all drugs. CYP3A4/5 mRNA transcripts have been reported in the skin, yet knowledge of their protein expression and function is lacking. In this study, we observed gene and protein expression of CYP3A4/5 in both human skin and tissue-engineered skin equivalents (TESEs), and enzyme activity was detected using the model substrate benzyl-O-methyl-cyanocoumarin. Mass spectrometric analysis of TESE lysates following testosterone application revealed a time-dependent increase in metabolite production, confirming the functional expression of these enzymes in skin.

Cooling of the oral mucosa to prevent adverse effects of chemotherapeutic agents: An in vitro study.

BACKGROUND: The cytotoxic effect of chemotherapeutic agents to the oral mucosa, as a side effect of cancer treatment, is a major problem. Cooling the oral mucosa using ice chips in conjunction with chemotherapy is known to reduce the severity of oral mucositis. However, although the use of ice chips is of clinical value, this method of cooling has inherent problems including discomfort for the patient, non-uniformity and fluctuations in cooling temperature throughout the oral cavity. Furthermore, despite being used clinically, it is not known what reduction in temperature is required to prevent oral mucositis. The aim of this study was therefore to determine in vitro if the cytotoxic effect of 5-fluorouracil (5-FU) on the oral mucosa could be reduced by lowering the temperature during chemotherapeutic treatment. METHODS: Tissue-engineered oral mucosal (TEOM) models were incubated at 20, 25, 30 or 35°C for 30 minutes followed by exposure to a clinically relevant concentration of 5-FU (162 µg/mL) for 2 hours and compared with untreated models (35°C). Cell viability and inflammatory cytokine production (IL-6 and TNF-α) were measured using PrestoBlue® and ELISA, respectively. RESULTS: TEOM models incubated at 20°C showed an increased cell viability and had a reduced IL-6 and TNF-α production compared to models treated with 5-FU incubated at 35°C. CONCLUSION: This study demonstrates a reduced cytotoxic effect to the TEOM by reducing the temperature of the tissue during chemotherapy treatment and suggests that decreasing the temperature to 20°C could have clinical advantages.

Methyl-donor depletion of head and neck cancer cells in vitro establishes a less aggressive tumour cell phenotype.

PURPOSE: DNA methylation plays a fundamental role in the epigenetic control of carcinogenesis and is, in part, influenced by the availability of methyl donors obtained from the diet. In this study, we developed an in-vitro model to investigate whether methyl donor depletion affects the phenotype and gene expression in head and neck squamous cell carcinoma (HNSCC) cells. METHODS: HNSCC cell lines (UD-SCC2 and UPCI-SCC72) were cultured in medium deficient in methionine, folate, and choline or methyl donor complete medium. Cell doubling-time, proliferation, migration, and apoptosis were analysed. The effects of methyl donor depletion on enzymes controlling DNA methylation and the pro-apoptotic factors death-associated protein kinase-1 (DAPK1) and p53 upregulated modulator of apoptosis (PUMA) were examined by quantitative-PCR or immunoblotting. RESULTS: HNSCC cells cultured in methyl donor deplete conditions showed significantly increased cell doubling times, reduced cell proliferation, impaired cell migration, and a dose-dependent increase in apoptosis when compared to cells cultured in complete medium. Methyl donor depletion significantly increased the gene expression of DNMT3a and TET-1, an effect that was reversed upon methyl donor repletion in UD-SCC2 cells. In addition, expression of DAPK1 and PUMA was increased in UD-SCC2 cells cultured in methyl donor deplete compared to complete medium, possibly explaining the observed increase in apoptosis in these cells. CONCLUSION: Taken together, these data show that depleting HNSCC cells of methyl donors reduces the growth and mobility of HNSCC cells, while increasing rates of apoptosis, suggesting that a methyl donor depleted diet may significantly affect the growth of established HNSCC.

Neutrophil-mediated tumour angiogenesis: subversion of immune responses to promote tumour growth.

Neutrophils are rapidly responding, phagocytes that are an essential part of the host innate immune response to invading micro-organisms. Along with other leucocytes they also play a key role in directing repair at sites of tissue damage. Neutrophils accomplish many of their biological functions by releasing enzymes, anti-microbial agents and cytokines when stimulated to degranulate. There is now increasing evidence to show that tumours are able to recruit neutrophils by secreting a number of tumour cell or stromal-derived chemoattractants. Once within the tumour microenvironment neutrophils, like macrophages, are polarised into a pro-tumour phenotype that can foster tumour growth by secreting factors that directly influence tumour cell proliferation, drive immunosuppression and promote tumour angiogenesis. In this review we discuss the likely mechanisms by which neutrophils are recruited into the tumour and then elaborate on how these cells may induce tumour vascularisation by the secretion of powerful pro-angiogenic factors.

Differential virulence of Candida glabrata glycosylation mutants.

The fungus Candida glabrata is an important and increasingly common pathogen of humans, particularly in immunocompromised hosts. Despite this, little is known about the attributes that allow this organism to cause disease or its interaction with the host immune system. However, in common with other fungi, the cell wall of C. glabrata is the initial point of contact between the host and pathogen, and as such, it is likely to play an important role in mediating interactions and hence virulence. Here, we show both through genetic complementation and polysaccharide structural analyses that C. glabrata ANP1, MNN2, and MNN11 encode functional orthologues of the respective Saccharomyces cerevisiae mannosyltransferases. Furthermore, we show that deletion of the C. glabrata Anp1, Mnn2, and Mnn11 mannosyltransferases directly affects the structure of the fungal N-linked mannan, in line with their predicted functions, and this has implications for cell wall integrity and consequently virulence. C. glabrata anp1 and mnn2 mutants showed increased virulence, compared with wild-type (and mnn11) cells. This is in contrast to Candida albicans where inactivation of genes involved in mannan biosynthesis has usually been linked to an attenuation of virulence. In the long term, a better understanding of the attributes that allow C. glabrata to cause disease will provide insights that can be adopted for the development of novel therapeutic and diagnostic approaches.

Polymersome-mediated intracellular delivery of antibiotics to treat Porphyromonas gingivalis-infected oral epithelial cells.

The gram-negative anaerobe Porphyromonas gingivalis colonizes the gingival crevice and is etiologically associated with periodontal disease that can lead to alveolar bone damage and resorption, promoting tooth loss. Although susceptible to antibiotics, P. gingivalis can evade antibiotic killing by residing within gingival keratinocytes. This provides a reservoir of organisms that may recolonize the gingival crevice once antibiotic therapy is complete. Polymersomes are nanosized amphiphilic block copolymer vesicles that can encapsulate drugs. Cells internalize polymersomes by endocytosis into early endosomes, where they are disassembled by the low pH, causing intracellular release of their drug load. In this study, polymersomes were used as vehicles to deliver antibiotics in an attempt to kill intracellular P. gingivalis within monolayers of keratinocytes and organotypic oral mucosal models. Polymersome-encapsulated metronidazole or doxycycline, free metronidazole, or doxycycline, or polymersomes alone as controls, were used, and the number of surviving intracellular P. gingivalis was quantified after host cell lysis. Polymersome-encapsulated metronidazole or doxycycline significantly (P<0.05) reduced the number of intracellular P. gingivalis in both monolayer and organotypic cultures compared to free antibiotic or polymersome alone controls. Polymersomes are effective delivery vehicles for antibiotics that do not normally gain entry to host cells. This approach could be used to treat recurrent periodontitis or other diseases caused by intracellular-dwelling organisms.

Polymersome-mediated delivery of combination anticancer therapy to head and neck cancer cells: 2D and 3D in vitro evaluation.

Polymersomes have the potential to encapsulate and deliver chemotherapeutic drugs into tumor cells, reducing off-target toxicity that often compromises anticancer treatment. Here, we assess the ability of the pH-sensitive poly 2-(methacryloyloxy)ethyl phosphorylcholine (PMPC)- poly 2-(diisopropylamino)ethyl methacrylate (PDPA) polymersomes to encapsulate chemotherapeutic agents for effective combinational anticancer therapy. Polymersome uptake and ability to deliver encapsulated drugs into healthy normal oral cells and oral head and neck squamous cell carcinoma (HNSCC) cells was measured in two and three-dimensional culture systems. PMPC-PDPA polymersomes were more rapidly internalized by HNSCC cells compared to normal oral cells. Polymersome cellular uptake was found to be mediated by class B scavenger receptors. We also observed that these receptors are more highly expressed by cancer cells compared to normal oral cells, enabling polymersome-mediated targeting. Doxorubicin and paclitaxel were encapsulated into pH-sensitive PMPC-PDPA polymersomes with high efficiencies either in isolation or as a dual-load for both singular and combinational delivery. In monolayer culture, only a short exposure to drug-loaded polymersomes was required to elicit a strong cytotoxic effect. When delivered to three-dimensional tumor models, PMPC-PDPA polymersomes were able to penetrate deep into the center of the spheroid resulting in extensive cell damage when loaded with both singular and dual-loaded chemotherapeutics. PMPC-PDPA polymersomes offer a novel system for the effective delivery of chemotherapeutics for the treatment of HNSCC. Moreover, the preferential internalization of PMPC polymersomes by exploiting elevated scavenger receptor expression on cancer cells opens up the opportunity to target polymersomes to tumors.

A cellular automaton model examining the effects of oxygen, hydrogen ions and lactate on early tumour growth.

Some tumours are known to exhibit an extracellular pH that is more acidic than the intracellular, creating a 'reversed pH gradient' across the cell membrane and this has been shown to affect their invasive and metastatic potential. Tumour hypoxia also plays an important role in tumour development and has been directly linked to both tumour morphology and aggressiveness. In this paper, we present a hybrid mathematical model of intracellular pH regulation that examines the effect of oxygen and pH on tumour growth and morphology. In particular, we investigate the impact of pH regulatory mechanisms on the cellular pH gradient and tumour morphology. Analysis of the model shows that: low activity of the Na+/H+ exchanger or a high rate of anaerobic glycolysis can give rise to a "fingering" tumour morphology; and a high activity of the lactate/H+ symporter can result in a reversed transmembrane pH gradient across a large portion of the tumour mass. Also, the reversed pH gradient is spatially heterogeneous within the tumour, with a normal pH gradient observed within an intermediate growth layer within the spheroid. We also include a fractal dimension analysis of the simulated tumour contours, in which we compare the fractal dimensions of the simulated tumour surfaces with those found experimentally via photomicrographs.

Combined mathematical modelling and experimentation to predict polymersome uptake by oral cancer cells.

This study is motivated by understanding and controlling the key physical properties underlying internalisation of nano drug delivery. We consider the internalisation of specific nanometre size delivery vehicles, comprised of self-assembling amphiphilic block copolymers, called polymersomes that have the potential to specifically deliver anticancer therapeutics to tumour cells. The possible benefits of targeted polymersome drug delivery include reduced off-target toxic effects in healthy tissue and increased drug uptake by diseased tissue. Through a combination of in vitro experimentation and mathematical modelling, we develop a validated model of nanoparticle uptake by cells via the clathrin-mediated endocytotic pathway, incorporating receptor binding, clustering and recycling. The model predicts how the characteristics of receptor targeting, and the size and concentration of polymersomes alter uptake by tumour cells. The number of receptors per cell was identified as being the dominant mechanism accounting for the difference between cell types in polymersome uptake rate. FROM THE CLINICAL EDITOR: This article reports on a validated model developed through a combination of in vitro experimentation and mathematical modeling of nanoparticle uptake by cells via the clathrin-mediated endocytotic pathway. The model incorporates receptor binding, clustering, and recycling and predicts how the characteristics of receptor targeting, the size and concentration alter polymersome uptake by cancer cells.

Electrospinning polymersomes into bead-on-string polyethylene oxide fibres for the delivery of biopharmaceuticals to mucosal epithelia.

Fibrous mucoadhesive polymer membranes prepared using electrospinning demonstrate many advantages for mucosal drug delivery compared to other formulations. Previous electrospun membrane formulations have been developed mainly for the delivery of small molecule drugs. There remains great potential to further develop the technology for the delivery of vesicular vectors that allow administration of advanced therapeutic agents. However, there are no previous reports demonstrating the release of intact drug delivery vesicles from electrospun materials. Here, we describe incorporation and release of protein-loaded polymersomes from polyethylene oxide (PEO)-based electrospun membranes. Polymersomes comprising a copolymer of glycerol monomethacrylate (GMA) and hydroxypropyl methacrylate (HPMA) were prepared using polymerization-induced self-assembly and incorporated within PEO membranes using bead-on-string electrospinning at approximately 40 % w/w by polymer mass. Super-resolution fluorescence imaging showed that the vesicles remained intact and retained their encapsulated protein load within the fibre beads. Transmission electron microscopy and dynamic light scattering demonstrated that polymersomes retained their morphology following release from the polymer fibres. F(ab) antibody fragments were encapsulated within polymersomes and then electrospun into membranes. 78 ± 13 % of the F(ab) remained encapsulated within polymersomes during electrospinning and retained functionality when released from electrospun membranes, demonstrating that the formulation is suitable for the delivery of biologics. Membranes were non-irritant to the oral epithelium and fluorescence microscopy detected accumulation of polymersomes within the epithelia following application. This innovative drug delivery approach represents a novel and potentially highly useful method for the administration of large molecular mass therapeutic molecules to diseased mucosal sites.

A tissue-engineered model of T-cell mediated oral mucosal inflammatory disease.

T-cell-mediated oral mucocutaneous inflammatory conditions including oral lichen planus (OLP) are common but development of new treatments aimed at relieving symptoms and controlling OLP progression are hampered by the lack of experimental models. Here, we developed a tissue-engineered oral mucosal equivalent (OME) containing polarised T-cells to replicate OLP pathogenesis. Peripheral blood CD4+ and CD8+ T-cells were isolated, activated and polarised into Th1 and cytotoxic T-cells (Tc). OME were constructed by culturing oral keratinocytes on an oral fibroblast-populated hydrogel to produce a stratified squamous epithelium. OME stimulated with IFN-γ and TNF-α or medium from Th1 cells caused increased secretion of inflammatory cytokines/chemokines. A model of T-cell-mediated inflammatory disease was developed by combining OME on top of a Th1/Tc-containing hydrogel, followed by epithelial stimulation with IFN-γ/TNF-α. T-cell recruitment towards the epithelium was associated with increased secretion of T-cell chemoattractants CCL5, CXCL9 and CXCL10. Histological assessment showed tissue damage associated with cleaved-caspase-3 and altered laminin-5 expression. Treatment with inhibitors directed against JAK, KCa3.1 channels or clobetasol in solution and/or via a mucoadhesive patch prevented cytokine/chemokine release and tissue damage. This disease model has potential to probe for mechanisms of pathogenesis or as a test platform for novel therapeutics or treatment modalities.

An advanced in silico model of the oral mucosa reveals the impact of extracellular spaces on chemical permeation.

Accurately predicting the permeation of chemicals through human epithelial tissues is crucial for pharmaceutical therapeutic design and toxicology. Current mathematical models of multi-layered stratified squamous epithelium such as those in the oral cavity use simplistic 'bricks and mortar' geometries that do not fully account for the complex cellular architecture that may affect chemical permeation in these tissues. Here we aimed to develop a new, advanced mechanistic mathematical model of the human epithelium that more accurately represents chemical tissue permeation. Using measurements of cell size and tortuosity from micrograph images of both human oral (buccal) and tissue-engineered buccal mucosa along with mechanistic mathematical modelling, we show that the convoluted geometry of the extracellular spaces within the epithelium significantly impacts chemical permeation. We next developed an advanced histologically and physiologically-relevant in silico model of buccal mucosal chemical permeation using partial differential equations, fitted to chemical permeation from in vitro assay data derived from tissue-engineered buccal mucosal models and chemicals with known physiochemical properties. Our novel in silico model can predict epithelial permeation kinetics for chemicals with different physicochemical properties in the absence or presence of permeability enhancers. This in vitro - in silico approach constitutes a step-change in the modelling of chemical tissue permeation and has the potential to expedite pharmaceutical innovation by improved and more rapid screening of chemical entities whilst reducing the need for in vivo animal experiments.