Oral Mucosal Model Research Articles

Development of an oral mucosal irritation test using a three-dimensional human buccal oral mucosal model

The oral mucosa can become irritated by oral care products and lip cosmetics. Therefore, it is important to determine the irritation potential of their ingredients and products during safety evaluations. We developed a method for oral mucosal irritation test using EpiOral, which is a three-dimensional cultured model. Exposure of sodium lauryl sulphate (SLS) to EpiOral showed a dose-dependent decrease in cell viability. Under 120 min exposure conditions, SLS irritation was detected when 60% cell viability was set as a criterion. Evaluation of the irritancy of SLS and four other raw materials used in oral products at three laboratories under the above conditions confirmed good transferability of the test. Focused on the similarity of the oral and eye mucous, 32 chemicals categorised by the UN-GHS eye-irritation classification were evaluated to ensure the reliability of our criteria at these laboratories. The concordance rate between the UN-GHS classification and our test results was 100% for irritants and 60% for non-irritants. The good intra-laboratory reproducibility of our test was confirmed from the evaluation results of negative and positive controls, and the good inter-laboratory reproducibility was confirmed from the results of 32 chemicals. These findings showed that oral mucosal irritation can be evaluated using EpiOral.

Keratin 75 Is a Component of the LINC Complex and Has an Essential Role in Mediating the SOX2 Rapid Healing Response during Wound Repair

Keratin 75 Is a Component of the LINC Complex and Has an Essential Role in Mediating the SOX2 Rapid Healing Response during Wound Repair

Regulation of gingival keratinocyte monocyte chemoattractant protein-1-induced protein (MCPIP)-1 and mucosa-associated lymphoid tissue lymphoma translocation protein (MALT)-1 expressions by periodontal bacteria, lipopolysaccharide, and interleukin-1β.

The aim of this study was to evaluate oral bacteria- and interleukin (IL)-1β-induced protein and mRNA expression profiles of monocyte chemoattractant protein-1-induced protein (MCPIP)-1 and mucosa-associated lymphoid tissue lymphoma translocation protein (MALT)-1 in human gingival keratinocyte monolayers and organotypic oral mucosal models. Human gingival keratinocyte (HMK) monolayers were incubated with Porphyromonas gingivalis, Fusobacterium nucleatum, P. gingivalis lipopolysaccharide (LPS) and IL-1β. The protein levels of MCPIP-1 and MALT-1 were examined by immunoblots and mRNA levels by qPCR. MCPIP-1 and MALT-1 protein expression levels were also analyzed immunohistochemically using an organotypic oral mucosal model. One-way analysis of variance followed by Tukey correction was used in statistical analyses. In keratinocyte monolayers, MCPIP-1 protein expression was suppressed by F. nucleatum and MALT-1 protein expression was suppressed by F. nucleatum, P. gingivalis LPS and IL-1β. P. gingivalis seemed to degrade MCPIP-1 and MALT-1 at all tested time points and degradation was inhibited when P. gingivalis was heat-killed. MCPIP-1 mRNA levels were increased by P. gingivalis, F. nucleatum, and IL-1β, however, no changes were observed in MALT-1 mRNA levels. Gingival keratinocyte MCPIP-1 and MALT-1 mRNA and protein expression responses are regulated by infection and inflammatory mediators. These findings suggest that periodontitis-associated bacteria-induced modifications in MCPIP-1 and MALT-1 responses can be a part of periodontal disease pathogenesis.

The Senescence Effect of Zoledronate on Three-Dimensional Oral Mucosa Model

Zoledronate (ZOL) is an antiresorptive bisphosphonate used to prevent bone loss in skeletal-related disorders, especially in patients with advanced cancer metastatic to bone. However, this medication led to an oral lesion known as medication-related osteonecrosis of the jaw (MRONJ) which also presents clinically as unhealing soft tissue in the jaw. Keratinocytes are thought to be a significant onset factor for MRONJ and recent findings have shown evidence of senescence in keratinocytes. However, little is known about the effect of senescence-associated inflammation that may cause age-associated tissue degeneration, which relates mainly to the expression of extracellular modulators including cytokines known as senescence-associated secretory phenotype (SASP). The aim of this experiment was to investigate the effect of ZOL treatment and senescence-associated secretory phenotype (SASP) if any, on a three-dimensional oral mucosa model (OMM) in which a novel modification was made to reflect the existence of basement membrane (BM) and lamina propria accurately. The ZOL dosage for the model was optimized by exposing the immortalized human oral keratinocyte line (OKF 6/TERT-2) and normal human oral fibroblasts (NHOF) to ZOL at increasing dosages and then histoarchitecture of OMM was examined. Analysis of the model's histology showed significant epithelial thinning upon ZOL treatment and breakage of the BM accompanied by downward proliferation towards the lamina propria. A significant release of SASP molecules (MMP-3 and IL-8) was also detected upon treatment. Therefore, this study suggests that ZOL may impair healing at multiple types of tissues, originally from keratinocytes, by the deleterious effects of the senescence-associated inflammatory response.

Deletion of Non-histidine Domains of Histidine Kinase CHK1 Diminishes the Infectivity of Candida albicans in an Oral Mucosal Model.

ObjectivesThe histidine kinase (HK) CHK1 and other protein kinases in Candida albicans are key players in the development of hyphae. This study is designed to determine the functional roles of the S_Tkc domain (protein kinase) and the GAF domain of C. albicans CHK1 in hyphal formation and mucosal invasion.MethodsThe domain mutants CHK25 (ΔS_TkcCHK1/Δchk1) and CHK26 (ΔS_TkcΔgafCHK1/Δchk1) were first constructed by the his1-URA3-his1 method and confirmed by sequencing and Southern blots. A mouse tongue infection model was used to evaluate the hyphal invasion and fungal loads in each domain mutant, full-gene deletion mutant CHK21 (chk1Δ/chk1Δ), re-constituted strain CHK23 (chk1Δ/CHK1), and wild type (WT) from day 1 to day 5. The degree of invasion and damage to the oral mucosa of mice in each strain-infected group was evaluated in vivo and compared with germ tube rate and hyphal formation in vitro.ResultWhen compared with severe mucosal damage and massive hyphal formation in WT- or CHK23-infected mouse tongues, the deletion of S_Tkc domain (CHK25) caused mild mucosal damage, and fungal invasion was eliminated as we observed in full-gene mutant CHK21. However, the deletion of S_Tkc and GAF (CHK26) partially restored the hyphal invasion and mucosal tissue damage that were exhibited in WT and CHK23. Regardless of the in vivo results, the decreased hyphal formation and germ tube in vitro were less apparent and quite similar between CHK25 and CHK26, especially at the late stage of the log phase where CHK26 was closer to WT and CHK23. However, growth defect and hyphal impairment of both domain mutants were similar to CHK21 in the early stages.ConclusionOur data suggest that both protein kinase (S_Tkc) and GAF domains in C. albicans CHK1 are required for hyphal invasiveness in mucosal tissue. The appropriate initiation of cell growth and hyphal formation at the lag phase is likely mediated by these two functional domains of CHK1 to maintain in vivo infectivity of C. albicans.

3D engineered human gingiva fabricated with electrospun collagen scaffolds provides a platform for in vitro analysis of gingival seal to abutment materials.

In order to advance models of human oral mucosa towards routine use, these models must faithfully mimic the native tissue structure while also being scalable and cost efficient. The goal of this study was to develop a low-cost, keratinized human gingival model with high fidelity to human attached gingiva and demonstrate its utility for studying the implant-tissue interface. Primary human gingival fibroblasts (HGF) and keratinocytes (HGK) were isolated from clinically healthy gingival biopsies. Four matrices, electrospun collagen (ES), decellularized dermis (DD), type I collagen gels (Gel) and released type I collagen gels (Gel-R)) were tested to engineer lamina propria and gingiva. HGF viability was similar in all matrices except for Gel-R, which was significantly decreased. Cell penetration was largely limited to the top layers of all matrices. Histomorphometrically, engineered human gingiva was found to have similar appearance to the native normal human gingiva except absence of rete pegs. Immunohistochemical staining for cell phenotype, differentiation and extracellular matrix composition and organization within 3D engineered gingiva made with electrospun collagen was mostly in agreement with normal gingival tissue staining. Additionally, five types of dental material posts (5-mm diameter x 3-mm height) with different surface characteristics were used [machined titanium, SLA (sandblasted-acid etched) titanium, TiN-coated (titanium nitride-coated) titanium, ceramic, and PEEK (Polyetheretherketone) to investigate peri-implant soft tissue attachment studied by histology and SEM. Engineered epithelial and stromal tissue migration to the implant-gingival tissue interface was observed in machined, SLA, ceramic, and PEEK groups, while TiN was lacking attachment. Taken together, the results suggest that electrospun collagen scaffolds provide a scalable, reproducible and cost-effective lamina propria and 3D engineered gingiva that can be used to explore biomaterial-soft tissue interface.

Cytological Screening Model of Normal Oral Mucosa Exposed to Carcinogens: A Pilot Study

Introduction: Oral cytopathology is able to detect incipient cellular alterations, but it is not routinely applied to this purpose. We aimed to establish a model to screen individuals with no oral lesion exposed to smoking/alcohol, by means of the nuclear area, cell proliferation rate, and analysis of genetic damage. Methods: In this cross-sectional pilot study, 90 patients were allocated into 3 groups: oral cancer group (patients with oral squamous cell carcinoma), tobacco/alcohol group (patients without oral lesions and exposed to these risk factors), and control group (individuals with no lesion and not exposed to tobacco and alcohol). The cytological smears performed in these individuals were stained with Papanicolaou, a silver-staining and a Feulgen reaction. The nuclei of cells were measured, and AgNORs/nucleus and micronuclei (MN) were quantified. The cutoff values were stipulated evaluating the healthy mucosa (control group) and the cancerization field mucosa (oral cancer group). Results: Cutoff values for the screening of individuals exposed to carcinogens were ≥8% of nuclei larger than 100 μm², ≥3.38 AgNOR/nucleus, and ≥3 MN per 1,000 cells. Conclusions: Nuclear area measurement and AgNORs/nucleus and MN quantification identified the incipient phase of oral carcinogenesis. A screening model for individuals without oral lesion exposed to smoking/alcohol was proposed.

Molecular mechanisms of aroma persistence: From noncovalent interactions between aroma compounds and the oral mucosa to metabolization of aroma compounds by saliva and oral cells

The present study aims to reveal the molecular mechanisms underlying aroma persistence, as it plays a major role in food appreciation and quality. A multidisciplinary approach including ex vivo experiments using a novel model of oral mucosa and saliva as well as in vivo dynamic instrumental and sensory experiments was applied. Ex vivo results showed a reduction in aroma release between 7 and 86% in the presence of the thin layer of salivary proteins covering the oral mucosa (mucosal pellicle). This reduction was explained by hydrophobic interactions involving the mucosal pellicle and by the ability of oral cells and saliva to metabolize specific aroma compounds. The in vivo evaluation of exhaled air and perception confirmed the ex vivo findings. In conclusion, this work reveals the need to consider physiological reactions occurring during food oral processing to better understand aroma persistence and open new avenues of research.

Generation of a novel model of bioengineered human oral mucosa with increased vascularization potential.

ObjectiveThe aim of this study was to generate novel models of bioartificial human oral mucosa with increased vascularization potential for future use as an advanced therapies medicinal product, by using different vascular and mesenchymal stem cell sources.BackgroundOral mucosa substitutes could contribute to the clinical treatment of complex diseases affecting the oral cavity. Although several models of artificial oral mucosa have been described, biointegration is a major issue that could be favored by the generation of novel substitutes with increased vascularization potential once grafted in vivo.MethodsThree types of mesenchymal stem cells (MSCs) were obtained from adipose tissue, bone marrow, and dental pulp, and their in vitro potential was evaluated by inducing differentiation to the endothelial lineage using conditioning media. Then, 3D models of human artificial oral mucosa were generated using biocompatible fibrin‐agarose biomaterials combined with human oral mucosa fibroblasts and each type of MSC before and after induction to the endothelial lineage, using human umbilical vein endothelial cells (HUVEC) as controls. The vascularization potential of each oral mucosa substitute was assessed in vitro and in vivo in nude mice.Results In vitro induction of MSCs kept in culture was able to increase the expression of VEGF, CD31, and vWF endothelial markers, especially in bone marrow and dental pulp‐MSCs, and numerous proteins with a role in vasculogenesis become overexpressed. Then, in vivo grafting resulted in a significant increase in blood vessels formation at the interface area between the graft and the host tissues, with significantly positive expression of VEGF, CD31, vWF, and CD34 as compared to negative controls, especially when pre‐differentiated MSCs derived from bone marrow and dental pulp were used. In addition, a significantly higher number of cells committed to the endothelial lineage expressing the same endothelial markers were found within the bioartificial tissue.ConclusionOur results suggest that the use of pre‐differentiated MSCs could contribute to a rapid generation of a vascular network that may favor in vivo biointegration of bioengineered human oral mucosa substitutes.

Regulation of hBD-2, hBD-3, hCAP18/LL37, and Proinflammatory Cytokine Secretion by Human Milk Oligosaccharides in an Organotypic Oral Mucosal Model

Human milk oligosaccharides (HMOs), the third largest solid fraction in human milk, can modulate inflammation through Toll-like receptor signaling, but little is known about their immunomodulatory potential in the oral cavity. In this study, we determined whether the HMOs 2′-fucosyllactose (2′-FL) and 3-fucosyllactose (3-FL) regulate human-beta defensin (hBD)-2 and -3, cathelicidin (hCAP18/LL-37), and cytokine responses in human gingival cells using a three-dimensional oral mucosal culture model. The model was incubated with 0.1% or 1% 2′-FL and 3-FL, alone and in combination, for 5 or 24 h, and hBD-2, hBD-3, and hCAP18/LL-37 were analyzed by immunohistochemistry. The expression profiles of interleukin (IL)-1, IL-1RA, IL-8, and monocyte chemoattractant protein (MCP)-1 were determined by LUMINEX immunoassay. The combination of 1% 2′-FL and 1% 3-FL, and 1% 3-FL alone, for 24 h upregulated hBD-2 protein expression significantly (p < 0.001 and p = 0.016, respectively). No changes in the other antimicrobial peptides or proinflammatory cytokines were observed. Thus, 3-FL, alone and in combination with 2′-FL, stimulates oral mucosal secretion of hBD-2, without effecting a proinflammatory response when studied in an oral mucosal culture model.

Anti-aging effect of an oral disintegrating collagen film: a prospective, single-arm study.

As the average life expectancy increases, skin aging and wrinkles due to photoaging have gained attention. Collagen is closely involved in the process of skin aging. Among the potential methods of drug delivery to the skin, oral disintegrating films show promise for their ability to bypass the loss of active components that is typical of drug absorption via oral administration. This study was conducted to investigate the effect of an oral disintegrating collagen film on skin aging. We performed a prospective, single-arm study in a cohort of 22 women to assess the anti-aging effect of a novel oral disintegrating film containing collagen applied daily over a 12-week period. We measured the clinical indicators of skin integrity and performed immunofluorescence and high-performance liquid chromatography analyses of an exvivo oral mucosa model to compare the absorption rates of collagen films and conventional oral tablets via the mucosa. We found that the oral disintegrating collagen film reduced skin wrinkle depth and number and significantly increased skin elasticity and density. The novel mode of delivery of collagen via oral disintegrating films has a clinically potential anti-aging efficacy and is safe and convenient for daily use.

Directed Transport of CRP Across In Vitro Models of the Blood-Saliva Barrier Strengthens the Feasibility of Salivary CRP as Biomarker for Neonatal Sepsis.

C-reactive protein (CRP) is a commonly used serum biomarker for detecting sepsis in neonates. After the onset of sepsis, serial measurements are necessary to monitor disease progression; therefore, a non-invasive detection method is beneficial for neonatal well-being. While some studies have shown a correlation between serum and salivary CRP levels in septic neonates, the causal link behind this correlation remains unclear. To investigate this relationship, CRP was examined in serum and saliva samples from 18 septic neonates and compared with saliva samples from 22 healthy neonates. While the measured blood and saliva concentrations of the septic neonates varied individually, a correlation of CRP levels between serum and saliva samples was observed over time. To clarify the presence of active transport of CRP across the blood–salivary barrier (BSB), transport studies were performed with CRP using in vitro models of oral mucosa and submandibular salivary gland epithelium. The results showed enhanced transport toward saliva in both models, supporting the clinical relevance for salivary CRP as a biomarker. Furthermore, CRP regulated the expression of the receptor for advanced glycation end products (RAGE) and the addition of soluble RAGE during the transport studies indicated a RAGE-dependent transport process for CRP from blood to saliva.

Investigation of dental elastomers as oral mucosa simulant materials.

ObjectiveTo measure mechanical properties of dental soft liners in tensional stress to identify their suitability as human oral mucosa simulant materials.MethodsEleven different dental elastomers were subjected to tensile testing to obtain their tensile strength and elastic moduli (n = 15/group) according to the ISO‐527 method. Fractured surfaces of one specimen per sample group were examined under the light microscope and scanning electron microscope (SEM). Energy‐dispersive X‐ray spectroscopy (EDS) was performed for the elemental analysis or chemical characterization of each sample group. The obtained data were quantitatively and qualitatively analysed. They were also statistically analysed using SPSS version 25.ResultsThe tensile strength of dental elastomers ranged from 0.43 MPa (±0.09) to 7.41 MPa (±1.11) and had statistically significant differences between tested groups (p = 0.001). Vertex soft heat‐cure soft liner, GC impression silicones and Silagum soft liners were found to have tensile strengths close to that of the oral mucosa reported by previous studies. SEM analysis revealed that the elastomers with higher filler contents showed rough fractured surface with plucking of particles after tensile fracture.ConclusionThis is the first study assessing the suitability of dental elastomers as human oral mucosa simulant materials which can be used for in vitro, mathematical modeling and finite element analysis (FEA) to study masticatory force distribution in oral mucosa. Out of 11 studied, six (Vertex Soft, GC heavy and Light body, Molloplast B, Algin X Ultra and Exaclear) dental elastomers showed similar mechanical properties to the Theil embalmed gingival tissues. Vertex Soft, GC Light body, and Molloplast B may be used for the majority of oral mucosal model when considering tensile strength as the primary factor for mechanical stimulation.

In vitro three-dimensional organotypic culture models of the oral mucosa.

Three-dimensional, organotypic models of the oral mucosa have been developed to study a wide variety of phenomena occurring in the oral cavity. Although a number of models have been developed in academic research labs, only a few models have been commercialized. Models from academic groups offer a broader range of phenotypes while the commercial models are more focused on the oral and gingival mucosa. The commercialized models are manufactured under highly controlled conditions and meet the requirements of quality standards, which leads to high levels of reproducibility. These in vitro models have been used to evaluate the irritancy of oral care products such as toothpastes, mouthwashes, and mucoadhesives. The effects of cigarette smoke on oral cavity tissues have been studied and compared to those of e-cigarettes. Oral tissue models have facilitated investigation of the mechanisms of oral mucositis and oral candidiasis and have been used to examine transbuccal drug delivery rates and the absorption of nanoparticles. Infection studies have investigated the effects of HIV-1 along with the effects of commensal and pathogenic bacteria. More recently, a differentiated oral tissue model has been shown to express the ACE2 receptor, which is known to be important for the receptor-mediated entry of the SARS-CoV-2 coronavirus into human cells and tissues. Hence, oral mucosal models may find application in determining whether viral infection of the oral mucosa is possible and whether such infection has implications vis-a-vis the current COVID-19 pandemic. As is apparent, these models are used in a broad variety of applications and often offer advantages versus animal models in terms of reproducibility, avoiding species extrapolation, and the ethical concerns related to human and animal experimentation. The goals of this paper are to review commercially available models of the human buccal and gingival mucosa and highlight their use to gain a better understanding of a broad range of phenomena affecting tissues in the oral cavity.

Molecular effects of photon irradiation and subsequent aftercare treatment with dexpanthenol-containing ointment or liquid in 3D models of human skin and non-keratinized oral mucosa.

This study aimed to investigate the molecular effects of radiation and subsequent aftercare treatment with dexpanthenol-containing ointment and liquid on established full-thickness 3D skin models depicting acute radiodermatitis and mucositis. To mimic radiomucositis and radiodermatitis, non-keratinized mucous membrane and normal human skin models were irradiated with 5 Gray. Afterwards, models were treated topically every second day with dexpanthenol-containing ointment or liquid in comparison with placebo and untreated controls. On day 7 after irradiation, histological examination showed impairments in irradiated models. In contrast, models treated with dexpanthenol-containing ointment or liquid showed a completely restored epidermal part. While gene expression profiling revealed an induction of genes related to a pro-inflammatory milieu, oxidative stress and an impaired epidermal differentiation after irradiation of the models, aftercare treatment with dexpanthenol-containing ointment or liquid revealed anti-oxidative and anti-inflammatory effects and had a positive effect on epidermal differentiation and structures important for physical and antimicrobial barrier function. Our findings confirm the potential of our established models as in vitro tools for the replacement of pharmacological in vivo studies regarding radiation-induced skin injuries and give indications of the positive effects of dexpanthenol-containing externals after radiation treatments as part of supportive tumor treatment.

Incoming Flow Lamination Increases the Efficiency of Aerosolized Medication Delivery to 3D Oral Mucosal Tissue Models

Background: Inhalable medication devices on the market deliver aerosolized drugs in a turbulent flow, which in complex interaction with oropharyngeal geometry causes the major portion of the drug to deposit locally, while creating significant obstacles for reaching the lower lungs. The unintended deposition of aerosolized medications in the oropharynx is known to have negative effects on oral health. The emergence of numerous new aerosolized medications on the market is very likely to add significantly to the list of side effects. We hypothesized that lamination of the outflow by sequentially subdividing the aerosol flow within the spacer into smaller sub-flows using internal septi of different lengths will improve the delivery of the aerosol to distant targets while reducing its deposition in anterior aspects of the airways, especially in oropharyngeal cavity.

Estudio de las características histológicas y anatómicas de la mucosa oral humana. Un paso hacia la fabricación de mucosa oral artificial hecha a medida

Several pathologies may affect the histological structure and physiology of the human oral mucosa, being necessary to use oral mucosa grafts. In this context, the scarce number of donors has increased the need to develop artificial substitutes of human oral mucosa, most of them, without attending to the histological differences found in the different anatomical areas of the oral cavity. The objective of the present bibliographic review is to carry out a histological description and a morphological classification of the human oral mucosa and to determine the translational potentiality of the new artificial models of oral mucosa.

Inkjet Printed Melatonin on Poly(vinyl alcohol) Oral Films: Uptake in an Ex Vivo Oral Mucosal Pellicle Model

There is a growing interest in the use of melatonin in preventing radiation-induced mucositis with potential beneficial effects including prevention of mucosal damage, the emergence of ulcers and the loss of proliferative progenitor stem cells caused by radiation. Local drug delivery to the oral cavity is influenced by the oral mucosal pellicle which serves as a protective barrier between the oral epithelial surface and the external environment. In this study, we explore two key areas. Firstly, the use of thermal inkjet printing to improve the precision of drug deposition on polymeric oral films. Secondly, investigate melatonin release and penetration across an ex vivo oral mucosal pellicle model into the epithelial cancer cell line (TR146 cells). Our findings show that the deposited melatonin crystallized on the surface of the Polyvinyl Alcohol (PVA) film as the jetted droplets dried, consistent with previous work on in-jetted pharmaceutical solutions. The solid state of the deposited melatonin was further confirmed in attenuated total reflection Fourier transform infrared studies as crystalline. In addition, no new bonds detected which indicates the absence of new chemical bonds and interactions between melatonin and PVA. Investigating melatonin release and penetration across ex vivo oral mucosal pellicle model into the epithelial cancer cell line (TR146 cells) found that the amount of melatonin released was lower in the presence of saliva, likely due to the saliva initiating the formation of the mucosal pellicle through mucin–mucin interactions.

Primary Extracellular Matrix Enables Long-Term Cultivation of Human Tumor Oral Mucosa Models.

3D tumor models clearly outperform 2D cell cultures in recapitulating tissue architecture and drug response. However, their potential in understanding treatment efficacy and resistance development should be better exploited if also long-term effects of treatment could be assessed in vitro. The main disadvantages of the matrices commonly used for in vitro culture are their limited cultivation time and the low comparability with patient-specific matrix properties. Extended cultivation periods are feasible when primary human cells produce the extracellular matrix in situ. Herein, we adapted the hyalograft-3D approach from reconstructed human skin to normal and tumor oral mucosa models and compared the results to bovine collagen-based models. The hyalograft models showed similar morphology and cell proliferation after 7 weeks compared to collagen-based models after 2 weeks of cultivation. Tumor thickness and VEGF expression increased in hyalograft-based tumor models, whereas expression of laminin-332, tenascin C, and hypoxia-inducible factor 1α was lower than in collagen-based models. Taken together, the in situ produced extracellular matrix better confined tumor invasion in the first part of the cultivation period, with continuous tumor proliferation and increasing invasion later on. This proof-of-concept study showed the successful transfer of the hyalograft approach to tumor oral mucosa models and lays the foundation for the assessment of long-term drug treatment effects. Moreover, the use of an animal-derived extracellular matrix is avoided.

Interactions between Candida albicans and Enterococcus faecalis in an Organotypic Oral Epithelial Model.

Candida albicans as an opportunistic pathogen exploits the host immune system and causes a variety of life-threatening infections. The polymorphic nature of this fungus gives it tremendous advantage to breach mucosal barriers and cause oral and disseminated infections. Similar to C. albicans, Enterococcus faecalis is a major opportunistic pathogen, which is of critical concern in immunocompromised patients. There is increasing evidence that E. faecalis co-exists with C. albicans in the human body in disease samples. While the interactive profiles between these two organisms have been studied on abiotic substrates and mouse models, studies on their interactions on human oral mucosal surfaces are non-existent. Here, for the first time, we comprehensively characterized the interactive profiles between laboratory and clinical isolates of C. albicans (SC5314 and BF1) and E. faecalis (OG1RF and P52S) on an organotypic oral mucosal model. Our results demonstrated that the dual species biofilms resulted in profound surface erosion and significantly increased microbial invasion into mucosal compartments, compared to either species alone. Notably, several genes of C. albicans involved in tissue adhesion, hyphal formation, fungal invasion, and biofilm formation were significantly upregulated in the presence of E. faecalis. By contrast, E. faecalis genes involved in quorum sensing, biofilm formation, virulence, and mammalian cell invasion were downregulated. This study highlights the synergistic cross-kingdom interactions between E. faecalis and C. albicans in mucosal tissue invasion.