Human Oral Tissues Research Articles

Troglitazone, a Selective Ligand for PPARγ, Induces Cell-cycle Arrest in Human Oral SCC Cells.

We attempted to clarify the role of Peroxisome proliferator-activated receptor γ (PPARγ) and its ligand, troglitazone (TRO) on oral squamous cell carcinoma (SCC). The expression of PPARγ gene was examined in 47 human oral SCC tissues and two human oral SCC cell lines, CA9-22 and HSC-4. The effects of TRO on the growth and cell-cycle progression of human oral SCC cells were examined. PPARγ mRNA was detected in 20 of 47 oral SCC tissues and two human oral SCC cells. TRO significantly suppressed the growth of the cells, but did not induce apoptosis. CA9-22 cells treated with TRO showed an increased fraction in the G1 phase and decreased fractions in the S and G2-M phases. TRO did not induce apoptosis in oral SCC cells, but did inhibit the growth of the cells by arresting the cell cycle at G1 phase.

Effects of electronic cigarette liquid on monolayer and 3D tissue-engineered models of human gingival mucosa.

Background There is limited data available on potential biological effects of E-cigarettes on human oral tissues. The aim of this study was to evaluate the effects of E-cigarette liquid on the proliferation of normal and cancerous monolayer and 3D models of human oral mucosa and oral wound healing after short-term and medium-term exposure. Methods Normal human oral fibroblasts (NOF), immortalized OKF6-TERET-2 human oral keratinocytes, and cancerous TR146 keratinocyte monolayer cultures and 3D tissue engineered oral mucosal models were exposed to different concentrations (0.1%, 1%, 5% and 10%) of E-cigarette liquid (12 mg/ml nicotine) for 1 hour daily for three days and for 7 days. Tissue viability was monitored using the PrestoBlue assay. Wounds were also produced in the middle surface of the monolayer systems vertically using a disposable cell scraper. The alterations in the cell morphology and wound healing were visualized using light microscopy and histological examination.Results Statistical analysis showed medium-term exposure of TR146 keratinocytes to 5% and 10% E-liquid concentrations significantly increased the viability of the cancer cells compared to the negative control. Short-term exposure of NOFs to 10% E-liquid significantly reduced the cell viability, whereas medium-term exposure to all E-liquid concentrations significantly reduced the NOF cells’ viability. OKF6 cells exhibited significantly lower viability following short-term and mediumterm exposure to all E-cigarette concentrations compared to the negative control. 3D oral mucosal model containing normal oral fibroblasts and keratinocytes showed significant reduction in tissue viability after exposure to 10% E-liquid, whereas medium-term exposure resulted in significantly lower viability in 5% and 10% concentration groups compared to the negative control. There was a statistically significant difference in wound healing times of both NOF and OKF6 cells after exposure to 1%, 5% and 10% E-cigarette liquid.Conclusion Medium-term exposure to high concentrations of the E-cigarette liquid had cytotoxic effects on normal human oral fibroblasts and OKF6 keratinocytes, but a stimulatory cumulative effect on the growth of cancerous TR146 keratinocyte cells as assessed by the PrestoBlue assay and histological evaluation of 3D oral mucosal models. In addition, E-liquid exposure prolonged the wound healing of NOF and OKF6 oral mucosa cells.

The effects of cannabis use on oral health.

Cannabis, also known as marijuana, is one of the most commonly used substances for medical and recreational purposes globally. With the trend of global legalization of medical use of cannabis and even the recreational use, the prevalence of recreational use of cannabis has increased markedly over the past few years. Correspondingly, the potential health concerns related to cannabis consumption have also increased. Therefore, it is necessary for oral healthcare providers to understand the effects of cannabis use on oral health. This review briefly summarizes the components of cannabis, biologic activities on tissues, and mechanisms of action in human cells and tissues. Oral tissue expression of cannabinoid receptors and the potential association of cannabis to oral diseases are also examined. The goals of this review are to (1) elaborate the basic biology and physiology of cannabis in human oral tissues, and (2) provide a better understanding the effects of its use and abuse on oral health. Due to insufficient information, more well-designed studies should be conducted. It is urgent to include cannabis usage into dental patient health records.

PD-l1 conjugated all-trans retinoic acid nanoparticles for targeted treatment of oral dysplasia and oral squamous cell carcinoma

Objectives The present study conjugated all-trans retinoic acid (ATRA) with poly(lactic acid-co-glycolic acid)-block-poly(ethylene glycol) (PLGA-PEG) nanocarriers, and combined with anti–programmed death-ligand 1 (PD-L1) antibody to achieve targeted therapy for oral squamous cell carcinoma (OSCC) and oral dysplasia. Study Design The molecular mechanism of ATRA and PD-L1 was studied by using Cell Counting Kit-8 (CCK8), flow cytometry, Western blot, and immunohistochemistry. The effect of ATRA on OSCC and oral dysplasia cell proliferation inhibition, apoptosis induction, and PD-L1 downregulation was significantly enhanced after signal transducer and activator of transcription 3 (STAT3) signaling blockade. PD-L1 was highly expressed in patients with oral leukoplakia and OSCC compared with normal controls. By the measurement of dynamic light scattering, the average particle size and zeta potential of ATRA–PLGA–PEG and ATRA–PLGA-PEG–PD-L1 was 97 nm and –3.6 mV, 105 nm and –5.7 mV, respectively. The release of ATRA in nanoparticles reached 55% within 36 hours. The maximum encapsulation efficiency and loading capacity of ATRA into the nanoparticles reached 76.29% and 12.18%, respectively. The PEG-PLGA nanocarrier had almost no cytotoxicity, and the nanomedicine significantly inhibited cell proliferation of DOK and CAL27 cells on the in vitro cytotoxicity test. After 1 hour of incubation, the Nile red group showed few fluorescence intensity, whereas the 2 nanoparticle groups were readily detectable under confocal microscopy. Moreover, nanoparticles enhanced anticancer activity and reduced side effects compared with free ATRA in C3 H tumor-bearing mice. Furthermore, ATRA–PLGA-PEG–PD-L1 nanoparticles could target the tumor more effectively compared with the control group, as shown by whole-animal fluorescence imaging. Multicolor immunohistochemistry revealed effective regulation of expression of the related proteins after nanoparticle treatment. Results The mechanism of ATRA and PD-L1 downregulation was closely associated with STAT3 signaling inhibition in both OSCC and oral dysplasia. PD-L1, which was highly expressed in human oral leukoplakia and OSCC tissues, proved to be a specific target site for cancer therapy. The ATRA–PLGA-PEG–PD-L1 nanoparticles had lower toxicity and higher biocompatibility and targeted the tumor more specifically compare with free ATRA, both in vitro and in vivo. Conclusions These findings suggested that ATRA induced antitumor effects via STAT3 signaling inhibition in oral dysplasia and OSCC. The use of ATRA–PLGA-PEG–PD-L1 nanomedicine was an effective and nontoxic targeted therapy for oral dysplasia and OSCC, both in vitro and in vivo.

Red-Emissive Guanylated Polyene-Functionalized Carbon Dots Arm Oral Epithelia against Invasive Fungal Infections.

Oral candidiasis as a highly prevalent and recurrent infection in medically compromised individuals is mainly caused by the opportunistic fungal pathogen Candida albicans. This epithelial infection, if not controlled effectively, can progress to life-threatening systemic conditions and complications. The efficacy of current frontline antifungals is limited due to their poor bioavailability and systemic toxicity. As such, an efficient intervention is essential for controlling disease progression and recurrence. Herein, a theranostic nanoplatform (CD-Gu+-AmB) was developed to track the penetration of antifungals and perturb the invasion of C. albicans at oral epithelial tissues, via decorating the homemade red-emissive carbon dots (CD) with positively charged guanidine groups (Gu+) followed by conjugation with antifungal polyene (amphotericin B, AmB) in a reacting site-controllable manner. The generated CD-Gu+-AmB favorably gathered within the Candida cells and exhibited potent antifungal effects in both planktonic and biofilm forms. It selectively accumulated in the nuclei of human oral keratinocytes and exhibited undetectable toxicity to the host cells. Moreover, we reported for the first time the penetration and exfoliation profiles of CD in a three-dimensional organotypic model of human oral epithelial tissues, demonstrating that the extra- and intracellular accumulation of CD-Gu+-AmB effectively resisted the invasion of C. albicans by forming a "shielding" layer throughout the entire tissue. This study establishes a multifunctional CD-based theranostic nanoplatform functioning as a traceable and topically applied antifungal to arm oral epithelia, thereby shedding light on early intervention of mucosal candidiasis for oral and general health.

A Dual-modality Smartphone Microendoscope for Quantifying the Physiological and Morphological Properties of Epithelial Tissues

We report a nonconcurrent dual-modality fiber-optic microendoscope (named SmartME) that integrates quantitative diffuse reflectance spectroscopy (DRS) and high-resolution fluorescence imaging (FLI) into a smartphone platform. The FLI module has a spatial resolution of ~3.5 µm, which allows the determination of the nuclear-cytoplasmic ratio (N/C) of epithelial tissues. The DRS has a spectral resolution of ~2 nm and can measure the total hemoglobin concentration (THC) and scattering properties of epithelial tissues with mean errors of 4.7% and 6.9%, respectively, which are comparable to the errors achieved with a benchtop spectrometer. Our preliminary in vivo studies from a single healthy human subject demonstrate that the SmartME can noninvasively quantify the tissue parameters of normal human oral mucosa tissues, including labial mucosa tissue, gingival tissue, and tongue dorsum tissue. The THCs of the three oral mucosa tissues are significantly different from each other (p ≤ 0.003). The reduced scattering coefficients of the gingival and labial tissues are significantly different from those of the tongue dorsum tissue (p < 0.001) but are not significantly different from each other. The N/Cs for all three tissue types are similar. The SmartME has great potential to be used as a portable, cost-effective, and globally connected tool to quantify the THC and scattering properties of tissues in vivo.

Fusobacterium nucleatum Facilitates Apoptosis, ROS Generation, and Inflammatory Cytokine Production by Activating AKT/MAPK and NF-κB Signaling Pathways in Human Gingival Fibroblasts.

Fusobacterium nucleatum (F. nucleatum) plays key roles in the initiation and progression of periodontitis. However, the pathogenic effect of F. nucleatum on human oral tissues and cells has not been fully evaluated. In this study, we aimed to analyze the pathogenic effects of F. nucleatum on human gingival fibroblasts (GFs) and clarify the potential mechanisms. RNA-sequencing analysis confirmed that F. nucleatum significantly altered the gene expression of GF as the stimulation time increased. Cell counting and EdU-labeling assays indicated that F. nucleatum inhibited GF proliferation and promoted cell apoptosis in a time- and dose-dependent manner. In addition, cell apoptosis, intracellular reactive oxygen species (ROS) generation, and proinflammatory cytokine production were dramatically elevated after F. nucleatum stimulation. Furthermore, we found that the AKT/MAPK and NF-κB signaling pathways were significantly activated by F. nucleatum infection and that a large number of genes related to cellular proliferation, apoptosis, ROS, and inflammatory cytokine production downstream of AKT/MAPK and NF-κB signaling pathways were significantly altered in F. nucleatum-stimulated GFs. These findings suggest that F. nucleatum inhibits GF proliferation and promotes cell apoptosis, ROS generation, and inflammatory cytokine production partly by activating the AKT/MAPK and NF-κB signaling pathways. Our study opens a new window for understanding the pathogenic effects of periodontal pathogens on the host oral system.

Repression of metastasis-associated protein 2 for inhibiting metastasis of human oral cancer cells by promoting the p-cofilin-1/ LC3-II expression.

The overexpression of metastasis-associated protein 2 (MTA2) contributes to human tumor progression and metastasis in various tumor cells. However, the role of MTA2 in human oral cancer progression remains unknown. MTA2 expression in human oral tumor tissues and cell lines was measured by immunohistochemistry and Western blotting. Cell proliferation and cell cycle were analyzed using MTT assay and flow cytometry. The effects of MTA2 on oral cell migration and invasion were investigated using migration and invasion assays. The expression of MTA2, p-cofilin-1, and MTA2-induced LC3-II levels were measured using Western blotting and an immunofluorescence assay. Based on the human oral cancer tissue array and TCGA database, we found that MTA2 was increased in oral cancer tissues than in non-tumor oral tissues (P<.01). Moreover, MTA2 is significantly associated with tumor grade (P<.01) and the overall survival rate of patients with grade III tumor (P<.05). MTA2 expression in oral cancer cells was markedly higher than that in normal oral cells. Cell proliferation and cell cycle were not significantly changed in the cells inhibited by MTA2. MTA2 knockdown can inhibit cell migration and invasion of human oral cancer cells. Furthermore, we suggest that MTA2 inhibition enhances p-cofilin and LC3-II expression, and the knockdown of LC3-II expression in cells inhibited by MTA2 had the opposite effect. These results indicate that MTA2 may serve as a candidate prognostic biomarker and that targeting autophagy is a potential therapeutic strategy for treating human oral cancer.

Abstract 30: The development of a 3D cell-culture model of drug-induced oral mucositis

Abstract Purpose: Mucositis is a debilitating disease affecting cancer patients. Mucositis is an adverse effect of radiation or chemotherapy. In the past, our lab has developed a model of radiation-caused mucositis using a validated human tissue substitute of oral mucosa and studied the post radiation cellular and molecular stresses and their prevention. In this project, we wished to develop an in-vitro model of chemical-, or drug-induced oral mucositis. For this purpose, we used a three dimensional (3-D) cell culture model of oral mucosa and as the drug to induce mucositis we used everolimus. Everolimus belongs to rapalogs which represent important therapies against cancer but one of its adverse-effects is mucositis. Mucositis caused by everolimus develops early in therapy and progresses quickly to grade 3 making patients to postpone life-saving therapies. Thus, it is very important to understand and prevent the development of everolimus-caused mucositis. Methods: The 3-D cell cultures of primary human oral keratinocytes grown on top of primary human oral fibroblasts were purchased from MatTek Corporation. These 3-D cell cultures (tissues) are similar to human oral mucosal tissue and have been validated as its substitutes. Different 3-D tissues were treated for 24, 40 and 60 hours with 8, 16, 32, and 64 ng/ml of everolimus. Tissues which received no treatment were used as controls. We then evaluated the tissue histology using H and E staining, and gene expression using RNA-sequencing. Results: Tissues which were treated with everolimus show signs of distress based on the concentration of everolimus and the duration of the treatment. Low and medium concentration of everolimus (8ng/ml) and (16 or 32 ng/ml), respectively, show no, or minimal tissue distress after 24 hours of treatment duration. Tissues which received a high concentration of everolimus (64 ng/ml) even at a short duration of treatment, show signs of distress, as evidenced by atypical proliferation at the keratinous epithelium, vacuoles, or shredded keratin layers, as well as, higher apoptosis, and expression of pro-inflammatory cytokine genes. In general, the higher the concentration of everolimus and the longer the duration of the treatment, the more the signs of the stress in tissues. Conclusion: The cell and molecular stress that the 3-D tissue cultures of human oral mucosa experience depends on the drug concentration and the duration of treatment. Acknowledgment We thank Novartis for their support. Citation Format: Maria P. Lambros, QinQin Fei, Jonathan Moreno. The development of a 3D cell-culture model of drug-induced oral mucositis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 30.

Evaluation of a novel oral mucosa in vitro implantation model for analysis of molecular interactions with dental abutment surfaces.

BackgroundAbutment surfaces are being designed to promote gingival soft tissue attachment and integration. This forms a seal around prosthetics and consequently ensures long‐term implant survival. New scalable and reproducible models are necessary to evaluate and quantify the performance of these surfaces.PurposeTo evaluate a novel implantation model by histomorphometric and immunohistochemical characterization of the interactions between human oral gingival tissue and titanium abutments with either novel anodized or conventional machined surface.Materials and MethodsAbutments were inserted into an organotypic reconstructed human gingiva (RHG) model consisting of differentiated gingival epithelium cells on a fibroblast populated lamina propria hydrogel following a tissue punch. Epithelial attachment, down‐growth along the abutment surface, and phenotype were assessed via histomorphology, scanning electron microscopy, and immunohistochemistry 10 days after implantation.ResultsThe down‐growing epithelium transitioned from a gingival margin to a sulcular and junctional epithelium. The sulcus depth and junctional epithelial length were similar to previously reported pre‐clinical and clinical lengths. A collagen IV/laminin 5 basement membrane formed between the epithelium and the underlying connective tissue. The RHG expanded in thickness approximately 2‐fold at the abutment surface. The model allowed the evaluation of protein expression of adhering soft tissue cells for both tested abutments.ConclusionsThe RHG model is the first in vitro 3D model to enable the assessment of not only human epithelial tissue attachment to dental abutments but also the expression of protein markers involved in soft tissue attachment and integration. The two abutments showed no noticeable difference in epithelial attachment.

A novel humanized anti-PD-1 monoclonal antibody potentiates therapy in oral squamous cell carcinoma.

Currently, immune checkpoint inhibitors have been shown to extend the survival of many cancer patients. However, few studies have focused on immune checkpoint inhibition for the treatment of patients with oral squamous cell carcinoma (OSCC). Here, by screening at an early stage, we obtained a strain of anti-PD-1 monoclonal antibody (mAb) that targets programmed cell death-1 (PD-1) does not contain the CH1 and CL fragment. In this study, the role of our novel mAb was tested in the treatment of OSCC in vitro and in vivo. We found that our novel mAb can significantly augment T cell mediated cytokine secretion, target cellular lytic and apoptotic abilities, and inhibit tumor growth and inflammation in vivo. The PD-L1 blockade was accompanied by the inhibition of AKT and ERK1/2, thus suggesting that the PD-L1/PD-1 signaling pathway may play an important immunopreventive role in the tumorigenic properties of OSCC cells by modulating the AKT and ERK1/2 pathways. Additionally, PD-L1 staining was observed both in human OSCC tissues and normal oral mucous tissue adjacent to the tumor, which occurred at different rates. Taken together, these results indicated that our novel anti-PD-1 mAb may be used as a clinical therapy in human OSCC development and progression.

Decreased expression of folate transport proteins in oral cancer

The purpose of this study was to assess the expression of the 3 major folate transporters-folate receptors (FRs), reduced folate carrier (RFC), and proton-coupled folate transporter (PCFT)-in oral squamous cell carcinoma (OSCC). We hypothesized that patterns of expression of folate transporters would be different in OSCC compared with normal oral epithelium. We used immunohistochemistry to examine the expression of FR, RFC, and PCFT in 15 primary specimens collected from patients with OSCC, 2 human cadaveric samples of OSCC, and 12 normal human cadaveric oral tissues from a medical gross anatomy laboratory. Possible correlations between the expression of each folate transporter and patients' clinical data were determined. All 3 folate transporters were highly expressed in normal oral epithelium. In contrast, OSCC samples generally demonstrated low expression of FR, RFC, and PCFT, with wide distribution in the invading cancer cells. There were no differences in folate transporter expression between OSCC samples collected from patients and from human cadavers. The lowest expression of FR and PCFT characterized less-differentiated tumors, and the lowest expression of RFC correlated with higher lymph node involvement. Human oral cancer samples expressed decreased amounts of all 3 major folate transport proteins compared with controls from normal cadaveric oral tissues.

Angiotensin (1-7) inhibits arecoline-induced migration and collagen synthesis in human oral myofibroblasts via inhibiting NLRP3 inflammasome activation.

Arecoline induces oral submucous fibrosis (OSF) via promoting the reactive oxygen species (ROS). Angiotensin (1-7) (Ang-(1-7))protects against fibrosis by counteracting angiotensin II (Ang-II) via the Mas receptor. However, the effects of Ang-(1-7) on OSF remain unknown. NOD-like receptors (NLRs) family pyrin domain containing 3 (NLRP3) inflammasome is identified as the novel mechanism of fibrosis. Whereas the effects of arecoline on NLRP3 inflammasome remain unclear. We aimed to explore the effect of Ang-(1-7) on NLRP3 inflammasome in human oral myofibroblasts. In vivo, activation of NLRP3 inflammasomes with an increase of Ang-II type 1 receptor (AT1R) protein level and ROS production in human oral fibrosis tissues. Ang-(1-7) improved arecoline-induced rats OSF, reduced protein levels of NADPH oxidase 4 (NOX4) and the NLRP3 inflammasome. In vitro, arecoline increased ROS along with upregulation of the angiotensin-converting enzyme (ACE)/Ang-II/AT1R axis and NLRP3 inflammasome/interleukin-1β axis in human oral myofibroblasts, which were reduced by NOX4 inhibitor VAS2870, ROS scavenger N-acetylcysteine, and NOX4 small interfering RNA (siRNA). Furthermore, arecoline induced collagen synthesis or migration via the Smad or RhoA-ROCK pathway respectively, which could be inhibited by NLRP3 siRNA or caspase-1 blocker VX-765. Ang-(1-7) shifted the balance of RAS toward the ACE2/Ang-(1-7)/Mas axis, inhibited arecoline-induced ROS and NLRP3 inflammasome activation, leading to attenuation of migration or collagen synthesis. In summary, Ang-(1-7) attenuates arecoline-induced migration and collagen synthesis via inhibiting NLRP3 inflammasome in human oral myofibroblasts.

Abstract 487: MicroRNA regulation of oral squamous cell carcinoma

Abstract Oral squamous cell carcinoma (OSCC) remains the sixth most common human cancer with high mortality rate worldwide. Understanding the molecular mechanisms is important for development of an effective treatment for the disease. MicroRNA is a short, noncoded nucleotide playing an important role in tumorigenesis and metastasis. This study is to identify novel microRNAs and their targets in order to understand the molecular mechanisms underlying OSCC and metastasis using label-free proteomics and microRNA microarray techniques. Human oral tissue biopsies from three groups of subjects who had benign, localized carcinoma and distal carcinoma were collected from the archived tissue bank at Pathology in Creighton University. Total RNA and proteins were extracted from the core biopsy of the same subject. MicroRNA identification was performed by using Affymetrix GeneChip miRNA arrays and comprehensive data analysis. Protein was analyzed by using label-free LTQ orbit trap mass spectrometry. Forty-four miRNAs were upregulated significantly, including let-7b, miR-720, miR-21, and miR-1274b, while 41 miRNAs were remarkably downregulated, including miR-195, and miR-932, at localized and distal carcinoma compared to benign tissues. Additionally, as compared to benign, 38 miRNAs upregulated at local carcinoma were downregulated significantly at distal carcinoma, while other 17 miRNAs downregulated significantly at localized carcinoma were upregulated gigantically at distal carcinoma. Those miRNAs were further validated by qRT-PCR and FISH. Among those upregulated at both local and distal, miR-720 was further studied, and tryptase alpha-1 as its target was identified. MicroRNA-720 mimics suppressed significantly the expression of tryptase alpha-1 in cultured human OSCC stem cell lines. Results from miR-720 tissue microarray analysis showed that 98.8% of sensitivity and 100% of specificity for OSCC detection were achieved, suggesting that miR-720 may be developed as a novel biomarker for detection of human OSCC. Citation Format: Gary Guishan Xiao, Min Cao. MicroRNA regulation of oral squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 487.

Characterization and subtraction of luminescence background signals in high‐wavenumber Raman spectra of human tissue

AbstractRaman spectroscopy in the high‐wavenumber spectral region (HWR) is particularly suited for fiber‐optic in vivo medical applications. The most‐used fiber‐optic materials have negligible Raman signal in the HWR. This enables the use of simple and cheap single‐fiber‐optic probes that can be fitted in endoscopes and needles. The HWR generally shows less tissue luminescence than the fingerprint region. However, the luminescence can still be stronger than the Raman signal. Hardware‐ and software‐based strategies have been developed to correct for these luminescence signals. Typically, hardware‐based strategies are more complex and expensive than software‐based solutions. Effective software strategies have almost exclusively been developed for the fingerprint region. First‐order polynomial baseline fitting (PBF) is the most common background/luminescence estimation employed for the HWR. The goal of this study was to characterize the luminescence background signals of HW spectra of human oral tissue and compare the performance of two algorithms for correction of these background signals: PBF and multiple regression fitting (MRF). In the MRF method, we introduce here, prior knowledge of the range of Raman signals that can be obtained from the tissues of interest is explicitly used. MRF is more robust than PBF because it does not require an a priori choice of the polynomial order for fitting the background signal. This is important because, as we show, no single polynomial order can optimally characterize all backgrounds that are encountered in HW tissue spectra. We conclude that MRF should be the preferred method for background subtraction in the HWR.

Increased expression of high-mobility group nucleosomal-binding domain 2 protein in various tumor cell lines.

High mobility group nucleosomal-binding domain 2 (HMGN2) is an abundant non-histone nuclear protein of vertebrates and invertebrates. The aim of the present study was to characterize the endogenous expression of HMGN2 in various types of tumor cell. Western blotting was performed to analyze HMGN2 expression in the following tumor cell lines: H1975, HSC-4, MDA-MB-468, MDA-MB-231, SCC-25 and THP-1. Periodontal ligament cells (PDLCs) were included as a noncancerous control. HMGN2 was detected in human oral squamous cell carcinoma tissues by immunohistochemical analysis. The results demonstrated that the expression of HMGN2 was increased in the majority of tumor cell lines, particularly MDA-MB-468 and THP-1 cells, compared with PDLCs. The expression of HMGN2 in oral squamous cell carcinoma tissue was significantly increased compared with the expression in normal tissue. Furthermore, the expression of HMGN2 in metastatic oral squamous cell carcinoma tissues was increased compared with that in its non-metastatic counterpart. These results indicated that HMGN2 may serve an important function in the growth and metastasis of tumor cells.

Oral mucosa tissue gene expression profiling before, during, and after radiation therapy for tonsil squamous cell carcinoma.

BackgroundRadiation-therapy (RT) induces mucositis, a clinically challenging condition with limited prophylactic interventions and no predictive tests. In this pilot study, we applied global gene-expression analysis on serial human oral mucosa tissue and blood cells from patients with tonsil squamous cell cancer (TSCC) to identify genes involved in mucositis pathogenesis.Methods and findingsEight patients with TSCC each provided consecutive buccal biopsies and blood cells before, after 7 days of RT treatment, and 20 days following RT. We monitored clinical mucositis and performed gene-expression analysis on tissue samples. We obtained control tissue from nine healthy individuals. After RT, expression was upregulated in apoptosis inducer and inhibitor genes, EDA2R and MDM2, and in POLH, a DNA-repair polymerase. Expression was downregulated in six members of the histone cluster family, e.g., HIST1H3B. Gene expression related to proliferation and differentiation was altered, including MKI67 (downregulated), which encodes the Ki-67-proliferation marker, and KRT16 (upregulated), which encodes keratin16. These alterations were not associated with the clinical mucositis grade. However, the expression of LY6G6C, which encodes a surface immunoregulatory protein, was upregulated before treatment in three cases of clinical none/mild mucositis, but not in four cases of ulcerative mucositis.ConclusionRT caused molecular changes related to apoptosis, DNA-damage, DNA-repair, and proliferation without a correlation to the severity of clinical mucositis. LY6G6C may be a potential protective biomarker for ulcerative mucositis. Based on these results, our study model of consecutive human biopsies will be useful in designing a prospective clinical validation trial to characterize molecular mucositis and identify predictive biomarkers.

Natural substitutes for formalin: A boon to histopathology!!

Introduction:In routine tissue processing, formalin has been proved as efficient as fixative since inception and hazards associated with it are of major safety and health concern.Aim:The aim of this study is to compare the efficacy of natural fixatives such as jaggery and Khandsari over formalin.Materials and Methods:Ninety normal tissue specimen collected during minor oral surgical procedures were included in this study. Thirty specimen each were fixed in 30% jaggery (Group-A), 30% Khandsari (Group-B) and 10% formalin (Group-C). The slides obtained after tissue processing were analyzed for the quality of fixation. The tissue sections were assessed for cellular outline, cytoplasmic details, nuclear details, staining quality and overall morphology. Each criterion was rated on a scale of 1–4. (one for poor and four for excellent). The study was double-blinded and subjected to the Kruskal–Wallis ANOVA and Chi-square test.Results:The cellular outline is excellent in 90% (Group-C) followed by 36.67% (Group-B) of specimens. With respect to cytoplasmic staining 83.33% (Group-C) of tissues showed excellent results followed by 60% (Group-B), 33.33% (Group-A). Nuclear details are excellent in 86.67% (Group-C) followed by 83.33% (Group-B), 36.67% (Group-A) of specimens. With respect to staining quality 93.33% (Group-C) followed by 50% (Group-B), 26.67% (Group-A). Overall morphology is excellent in 90% (Group-C) followed by 46.67% (Group-B).Conclusion:In the present study, Khandsari was on par with formalin and our effort of using Khandsari and jaggery for tissue fixation in human oral tissues yielded good results.

In vivo imaging of human oral hard and soft tissues by polarization-sensitive optical coherence tomography.

Since optical coherence tomography (OCT) provides three-dimensional high-resolution images of biological tissue, the benefit of polarization contrast in the field of dentistry is highlighted in this study. Polarization-sensitive OCT (PS OCT) with phase-sensitive recording is used for imaging dental and mucosal tissues in the human oral cavity in vivo. An enhanced polarization contrast of oral structures is reached by analyzing the signals of the co- and crosspolarized channels of the swept source PS OCT system quantitatively with respect to reflectivity, retardation, optic axis orientation, and depolarization. The calculation of these polarization parameters enables a high tissue-specific contrast imaging for the detailed physical interpretation of human oral hard and soft tissues. For the proof-of-principle, imaging of composite restorations and mineralization defects at premolars as well as gingival, lingual, and labial oral mucosa was performed in vivo within the anterior oral cavity. The achieved contrast-enhanced results of the investigated human oral tissues by means of polarization-sensitive imaging are evaluated by the comparison with conventional intensity-based OCT.

The characterization of human oral mucosal fibroblasts and their use as feeder cells in cultivated epithelial sheets.

Aim:To characterize human oral mucosa middle interstitial tissue fibroblasts (hOMFs) and their application in the cultivation of epithelial sheets.Methodology:hOMFs were cultured with methylcellulose to form cell clusters. hOMFs amplified in adhesive culture were analyzed by flow cytometry, and were found to differentiate into multiple cell types suitable for the cultivation of human corneal epithelial sheets. hOMFs were expanded from clusters to analyze CD56 and PDGFRα expression.Results:These cells showed similar differentiation patterns as keratocytes, and similar expression patterns as mesenchymal and neural cells. Furthermore, we established human corneal epithelial sheets using hOMFs.Conclusion:hOMFs may be of neural crest origin and possess multipotent differentiation capacity, and are suitable for use as an autologous cell source for corneal regeneration.