Primary Human Oral Keratinocytes Research Articles

Cytotoxic, genotoxic, and toxicogenomic effects of heated tobacco products and cigarette smoke in human primary keratinocytes.

INTRODUCTIONHeated tobacco products (HTPs) appear to be less harmful to health than conventional cigarettes (CCs). However, limited analytical data are available to support this claim. This study aimed to compare the cytotoxic, genotoxic, and toxicogenomic effects of HTPs and CCs in carcinogenesis via multistep gene mutations in the oral mucosal cells.METHODSCigarette smoke extract (CSE) was obtained from HTPs and CCs. Primary human oral keratinocytes (HOKs) were treated with 5% and 20% CSE from HTPs and CCs. Cell survival rate assays were performed after 6, 12, and 24 h. After 6 h, DNA double-strand breaks (DSBs) were evaluated using anti-γH2AX antibodies with immunohistochemistry. mRNAs expressions of mediator of DNA damage checkpoint 1 (MDC1) and ataxia telangiectasia and Rad3-related protein (ATR), were analyzed. Expressions of miR-22 and miR-185 were analyzed because miR-22 targets MDC1 and miR-185, ATR.RESULTSThe HOKs had equivalent survival rates after exposure to the same concentrations of CSE from CCs and HTPs. HTPs increased foci formation of γH2AX in HOKs, as did CCs (without CSE vs 20% HTP, p<0.05; without CSE vs 20% CC, p<0.05). Expressions of MDC1 and ATR decreased in cells exposed to CSE from CCs and HTPs (MDC1: without CSE vs 20% HTP, p<0.05; without CSE vs 20% CC, p<0.05; ATR: without CSE vs 20% HTP, p<0.05; without CSE vs 20% CC, p<0.05). Expressions of miR-22 and miR-185 were not significantly increased when exposed to CSE from CCs or HTPs.CONCLUSIONSHTPs and CCs had similar cytotoxic effects. HTPs are genotoxic, can cause DSBs, and have toxicogenomic damage because they inhibit the MDC1 and ATR-CHK1 DNA repair pathways in the oral mucosa. The miRNA-mRNA axis was not related to these inhibitions.

Granulocyte macrophage-colony stimulating factor and keratinocyte growth factor control of early stages of differentiation of oral epithelium.

Oral epithelial differentiation is known to be directed by underlying fibroblasts, but the responsible factor(s) have not been identified. We aimed here to identify fibroblast‐derived factors responsible for oral epithelial differentiation. Primary normal human oral keratinocytes and fibroblasts were isolated from healthy volunteers after informed consent (n = 5) and 3D‐organotypic (3D‐OT) cultures were constructed. Various growth factors were added at a range of 0.1‐100 ng/ml. 3D‐OTs were harvested after ten days and assessed histologically, by immunohistochemistry and the TUNEL method. Epithelium developed in 3D‐OT without fibroblasts showed an undifferentiated phenotype. Addition of granulocyte macrophage‐colony stimulating factor (GM‐CSF) induced expression of cytokeratin 13 in suprabasal cell layers. Admixture of GM‐CSF and keratinocyte growth factor (KGF) induced, in addition, polarization of epidermal growth factor (EGF) receptor and β1‐integrin to basal cell layer and collagen IV deposition. Terminal differentiation with polarization of TUNEL‐positive cells to superficial layers occurred only in the presence of fibroblasts in collagen gels either in direct contact or at distance from normal oral keratinocytes. Taken together, these results show that major aspects of oral epithelial differentiation are regulated by the synergic combination of GM‐CSF and KGF. However, the terminal stage seems to be controlled by other yet unidentified fibroblast‐derived diffusible factor(s).

Epithelial cells release IL-36α in extracellular vesicles following mechanical damage

The epithelium is an integral part of barrier tissues, and plays a critical role in the initiation of the innate immune responses. The pro-inflammatory cytokine IL-36α has been previously reported to be strongly expressed during oral mucosal wound healing, but regulation of IL-36α expression and secretion in the oral mucosa are not well known. The objective of this study was to determine the types of stimuli that lead to expression and secretion of IL-36α in epithelial cells. Maxillary tissues from C57BL/6J mice during wound healing were utilized to identify endogenous expression of IL-36α, β, and γ in oral epithelial tissue. Immortalized HaCaT cells and primary normal human oral keratinocytes were subjected to Escherichia coli derived lipopolysaccharide (LPS), Poly(I:C), heat killed Candida albicans (HKCa), and mechanical damage. IL-36α and IL-1β levels in supernatant were assessed by sandwich ELISA, and expression of pro-inflammatory cytokines and IL-36 family genes were assessed by quantitative real-time PCR in HaCaT cells. Migration ability of keratinocytes was assessed with or without functional IL-36 signaling. IL-36α but not IL-36β or γ levels in the oral epithelium were elevated during wound healing. Treatment of epithelial cells with LPS, Poly(I:C), HKCa and mechanical damage revealed little to no soluble IL-36α in the media supernatant. However, sonication of the supernatant to disrupt the membranes of extracellular vesicles revealed a dose-dependent increase in IL-36α for each of the tested conditions. IL-1 superfamily genes were upregulated following mechanical damage in keratinocytes. Abrogation of IL-36 signaling led to severe inhibition of migration. Our data show for the first time that IL-36α is released primarily in extracellular vesicles by oral keratinocytes. Additionally, we show that IL-36α – but not IL-36β or γ – is upregulated in keratinocytes following mechanical damage, and that IL-36 signaling is important for keratinocyte migration.

Noninvasive Optical Assessment of Implanted Tissue-Engineered Construct Success In Situ.

Quantitative diffuse reflectance spectroscopy (DRS) was developed for label-free, noninvasive, and real-time assessment of implanted tissue-engineered devices manufactured from primary human oral keratinocytes (six batches in two 5-patient cohorts). Constructs were implanted in a murine model for 1 and 3 weeks. DRS evaluated construct success in situ using optical absorption (hemoglobin concentration and oxygenation, attributed to revascularization) and optical scattering (attributed to cellular density and layer thickness). Destructive pre- and postimplantation histology distinguished experimental control from stressed constructs, whereas noninvasive preimplantation measures of keratinocyte glucose consumption and residual glucose in spent culture media did not. In constructs implanted for 1 week, DRS distinguished control due to stressed and compromised from healthy constructs. In constructs implanted for 3 weeks, DRS identified constructs with higher postimplantation success. These results suggest that quantitative DRS is a promising, clinically compatible technology for rapid, noninvasive, and localized tissue assessment to characterize tissue-engineered construct success in vivo. Impact statement Despite the recent advance in tissue engineering and regenerative medicine, there is still a lack of nondestructive tools to longitudinally monitor the implanted tissue-engineered devices. In this study, we demonstrate the potential of quantitative diffuse reflectance spectroscopy as a clinically viable technique for noninvasive, label-free, and rapid characterization of graft success in situ.

Behavior of Primary Human Oral Keratinocytes Grown on Invisalign® SmartTrack® Material

Orthodontic clear aligner treatment is gaining tremendous popularity. The world market leader is Align Technology® and its product Invisalign®. Although numerous patients are treated with Invisalign® aligners, only little is known about the cellular effects of aligner material on oral epithelial cells. In the present study, we aimed to investigate the effects of SmartTrack® clear aligner material on directly cultured primary human oral keratinocytes (HOKs). Cell morphology and behavior were investigated by scanning electron microscopy and bright field microscopy. Aligner effects on viability were detected by cell-counting-kit (CCK)-8 and live/dead staining. Gene expression of several inflammatory and barrier proteins was assessed by qPCR. Cells cultured on tissue culture plastic served as control. Cell proliferation/viability was significantly lower in cells cultured on aligner material (p &lt; 0.05) in comparison to control. Live/dead staining did not reveal an increase in the number of dead cells on aligner surfaces. After two and seven days of incubation, interleukin (IL)-6 expression decreased, and IL-8 expression increased in HOKs cultured on aligner surfaces. The expression of intercellular adhesion molecule 1 (ICAM-1) significantly decreased after seven days. Gene expression of epithelial barrier markers showed that integrin (ITG)-α6 significantly decreased after two and seven days. A significant decrease in ITG-β4 and E-cadherin expression levels compared to control could only be seen after seven days. We did not find any cytotoxic effect, but alterations in the cell’s barrier functions and inflammatory reaction were obvious. Clinical studies are required to give further insights into clinical reactions on the underlying aligner material of this quickly expanding orthodontic appliance.

Semaphorin 4A restricts tumor progression by inhibiting angiogenesis of oral squamous cell carcinoma cells

ObjectiveTo investigate the effects of Semaphorin 4A (Sema4A) on the angiogenesis, migration and invasion of oral squamous cell carcinoma (OSCC) cells. MethodsSema4A expression in OSCC patients was detected by Immunohistochemistry, and its relationship with clinicopathological features and prognosis of patients was analyzed. The mRNA and protein expression of Sema4A in primary human oral keratinocytes (HOKs) and OSCC cells (SCC-25, HSC-3, CAL-27) were determined by Western blotting and qRT-PCR. After HOKs, HSC-3 cells and SCC-25 cells transfected with Control/Sema4A CRISPR activation plasmid, the migration and invasion abilities were detected by Wound healing and Transwell invasion. Tube formation assay was also performed on endothelial cells and the contents of VEGF and bFGF were quantified using qRT-PCR and ELISA. ResultsCytoplasmic Sema4A expression was related to T classification, clinical stage and nodal metastasis of OSCC patients. Patients with low cytoplasmic Sema4A expression showed the higher microvessel density (MVD) and the poorer prognosis in OSCC. Compared with HOK, OSCC cells (SCC-25, HSC-3, CAL-27) declined apparently in Sema4A expression, which was much more significant in metastatic HSC-3 and SCC-25 cells. After HOKs, HSC-3 cells and SCC-25 cells transfected with Sema4A over-expression plasmid, the invasion and migration abilities were decreased. Besides, overexpression of Sema4A could significantly inhibit the tube formation of HUVEC induced by OSCC cells with reductions of angiogenic factors (VEGF and bFGF). ConclusionOver-expression of Sema4A could restrict tumor progression through inhibiting the angiogenesis, invasion and migration of OSCC cells.

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.

Optical Metric Assessed Engineered Tissues Over a Range of Viability States.

Many conventional methods to assess engineered tissue morphology and viability are destructive techniques with limited utility for tissue constructs intended for implantation in patients. Sterile label-free optical molecular imaging methods analyzed tissue endogenous fluorophores without staining, noninvasively and quantitatively assessing engineered tissue, in lieu of destructive assessment methods. The objective of this study is to further investigate label-free optical metrics and their correlation with destructive methods. Tissue-engineered constructs (n = 33 constructs) fabricated with primary human oral keratinocytes (n = 10 patients) under control, thermal stress, and rapamycin treatment manufacturing conditions exhibited a range of tissue viability states, as evaluated by quantitative histology scoring, WST-1 assay, Ki-67 immunostaining imaging, and label-free optical molecular imaging methods. Both histology sections of fixed tissues and cross-sectioned label-free optical images of living tissues provided quantitative spatially selective information on local tissue morphology, but optical methods noninvasively characterized both local tissue morphology and cellular viability at the same living tissue site. Furthermore, optical metrics noninvasively assessed living tissue viability with a statistical significance consistent with the destructive tissue assays WST-1 and histology. Over the range of cell viability states created experimentally, optical metrics noninvasively and quantitatively characterized living tissue viability and correlated with the destructive WST-1 tissue assay. By providing, under sterile conditions, noninvasive metrics that were comparable with conventional destructive tissue assays, label-free optical molecular imaging has the potential to monitor and assess engineered tissue construct viability before surgical implantation.

Emission and Cytotoxicity of Surgical Smoke: Cholesta-3,5-Diene Released from Pyrolysis of Prostate Tissue

Respiratory and lung irritants can be a by-product of the surgical pyrolysis of human tissues. Seven prostate tissues were collected during the transurethral resection of a prostate (TURP). Tissue samples, pyrolyzed in a pyrolysis sampling system, were collected and analyzed for the characterization of aerosols in the surgical smoke. In the pyrolyzed particulate matter (PM) from the TURP, Cholestra-3,5-diene was identified as the most dominant component along with 9-methylanthracene, hentriacontane, and dotriacontane based on the mass fragment structure determined using gas chromatography-mass spectrometry (GC-MS). As a molecular marker, Cholesta-3,5-diene can be associated with a cytotoxic in primary human oral keratinocytes (HOK). In this research, the presence of Cholestra-3,5-diene is reported for the first time as a by-product of surgical pyrolysis.

P‐cadherin Overexpression Enhances Survival Signaling in Dysplastic Oral Epithelia via the Epidermal Growth Factor Receptor

P‐cadherin is a calcium‐dependent transmembrane protein that mediates cell‐cell adhesion within oral epithelial tissue. Previous histological analyses have suggested that P‐cadherin protein levels increase in oral dysplasia and subsequently decrease in oral squamous cell carcinoma (OSCC). The epidermal growth factor receptor (EGFR) gene has also been shown to undergo amplification in the dysplastic stage of oral tumor development. In comparative studies, we found that levels of P‐cadherin were increased two‐fold in dysplastic oral keratinocytes (DOK) compared to primary human oral keratinocytes (HOK) obtained from normal oral mucosa. We hypothesized that in dysplastic oral epithelia, upregulated P‐cadherin cooperates with amplified EGFR to enhance survival signaling and facilitate tumor progression. To further explore the interaction of P‐cadherin and EGFR, DOK cells were retrovirally infected to generate stably‐selected control and P‐cadherin‐overexpressing cell lines. We have previously demonstrated that the DOK cell line exhibits comparatively high levels of EGFR expression. We found that P‐cadherin overexpression increased basal, as well as EGF‐stimulated EGFR phosphorylation levels. Furthermore, this was accompanied by increased phosphorylation of a downstream effector of EGFR, signal transducer and activator of transcription (STAT3). This increased phosphorylation was not dependent on an increase in total STAT3 protein level. STAT3 phosphorylation suggested activation of a pro‐survival mechanism, a finding that was further supported by P‐cadherin‐dependent increases in the anti‐apoptotic protein Bcl‐xL. When exposed to chemotherapeutic agents such as paclitaxel and carboplatin, P‐cadherin overexpression increased cell survival compared to control cells. P‐cadherin‐overexpressing cells treated with paclitaxel also exhibited decreased apoptosis. Further analysis confirmed that the difference in cell death was not due to altered proliferation of the two cell lines. We also explored the possibility that EGFR was activated by P‐cadherin‐mediated adherens junction formation. In contrast to ligand‐dependent signaling, we observed that calcium‐dependent cadherin engagement suppressed the phosphorylation of both EGFR and STAT3. These studies suggest that the upregulation of P‐cadherin, together with increased EGFR expression, promotes tumor progression in dysplastic oral epithelia through the activation of prosurvival signaling pathways.Support or Funding InformationMidwestern University Glendale College of Osteopathic Medicine, College of Health Sciences, College of Veterinary MedicineThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Viral miRNAs Alter Host Cell miRNA Profiles and Modulate Innate Immune Responses.

Prevalence of the members of herpesvirus family in oral inflammatory diseases is increasingly acknowledged suggesting their likely role as an etiological factor. However, the underlying mechanisms remain obscure. In our recent miRNA profiling of healthy and diseased human tooth pulps, elevated expression of human herpesvirus encoded viral microRNAs (v-miRs) were identified. Based on the fold induction and significance values, we selected three v-miRs namely miR-K12-3-3p [Kaposi sarcoma-associated virus (KSHV)], miR-H1 [herpes simplex virus 1 (HSV1)], and miR-UL-70-3p [human cytomegalovirus (HCMV)] to further examine their impact on host cellular functions. We examined their impact on cellular miRNA profiles of primary human oral keratinocytes (HOK). Our results show differential expression of several host miRNAs in v-miR-transfected HOK. High levels of v-miRs were detected in exosomes derived from v-miR transfected HOK as well as the KSHV-infected cell lines. We show that HOK-derived exosomes release their contents into macrophages (Mφ) and alter expression of endogenous miRNAs. Concurrent expression analysis of precursor (pre)-miRNA and mature miRNA suggest transcriptional or posttranscriptional impact of v-miRs on the cellular miRNAs. Employing bioinformatics, we predicted several pathways targeted by deregulated cellular miRNAs that include cytoskeletal organization, endocytosis, and cellular signaling. We validated three novel targets of miR-K12-3-3p and miR-H1 that are involved in endocytic and intracellular trafficking pathways. To evaluate the functional consequence of this regulation, we performed phagocytic uptake of labeled bacteria and noticed significant attenuation in miR-H1 and miR-K12-3-3p but not miR-UL70-3p transfected primary human Mφ. Multiple cytokine analysis of E. coli challenged Mφ revealed marked reduction of secreted cytokine levels with important roles in innate and adaptive immune responses suggesting a role of v-miRs in immune subversion. Our findings reveal that oral disease associated v-miRs can dysregulate functions of key host cells that shape oral mucosal immunity thus exacerbating disease severity and progression.

Comparison of two decellularized dermal equivalents

Immunologically inert allogeneic acellular dermal scaffolds provide a matrix with molecular architecture close to native tissues, which synthetic scaffolds cannot. Not all nature-derived scaffolds possess the same biological and physical properties. The different properties of scaffolds supporting cellular growth used for manufacturing tissue engineered grafts could lead to different implantation results. The scaffold properties should be carefully considered in order to meet the expected outcomes of tissue engineered grafts. In this report, we evaluated the cellular growth on AlloDerm® and Allopatch, 2 acellular scaffolds derived from human cadaver skin, using a fabricated 3D organotypic culture with primary human oral keratinocytes to produce an ex vivo produced oral mucosa equivalent (EVPOME). A well stratified epithelium could be constructed on both scaffolds. AlloDerm® and Allopatch EVPOMEs were also implanted into severe combined immunodeficiency mice to compare the ingrowth of blood vessels into the dermal component of the two EVPOMEs. Blood vessel counts were 3.3 times higher (p=.01) within Allopatch EVPOMEs than within AlloDerm® EVPOMEs. An oral and skin keratinocyte co-culture, separated by a physical barrier to create a cell-free zone, was used to evaluate cell migration on AlloDerm® and Allopatch. Slower cell migration was observed on Allopatch than on AlloDerm®.

Rebamipide suppresses 5-fluorouracil-induced cell death via the activation of Akt/mTOR pathway and regulates the expression of Bcl-2 family proteins

Oral mucositis is a common adverse effect of chemotherapy that limits the required dose of chemotherapeutic agents. Numerous attempts to mitigate chemotherapy-induced oral mucositis have failed to identify an appropriate treatment. Recently, it has been indicated that rebamipide prevents chemoradiotherapy-induced oral mucositis in patients. However, the details of the underlying mechanism involved in the cytoprotective effect of rebamipide remain obscure. In the present study, we investigated the mechanism behind rebamipide cytoprotective effect in the oral mucosa using primary normal human oral keratinocytes (NHOK cells). We found that rebamipide prevented 5-fluorouracil (5-FU)-induced cell death in NHOK cells. In addition, rebamipide increased the levels of phosphorylated Akt and mTOR, enhanced the Bcl-2 and Bcl-xL expressions, and suppressed the expression of Bax and Bim. This is in contrast to 5-FU-induced suppression of Akt and mTOR activation, Bcl-2 and Bcl-xL expressions, and the enhanced expression of Bax and Bim. These findings suggest that rebamipide can potentially be used for the protection of oral mucosa from chemotherapy-induced mucositis. This is the first study that elucidates the specific molecular pathway for the cytoprotective effect of rebamipide.

Histamine H4 receptor signalling in tongue cancer and its potential role in oral carcinogenesis- a short report.

Recent reports indicate that histamine and its novel, high-affinity histamine H4 receptor (H4R) play a role in carcinogenesis, and thus H4R signalling has become a focus of increasing interest in the pathogenesis of many cancers. The roles of H4R in oral epithelial dysplasia (OED) and oral tongue squamous cell carcinoma (OTSCC) are unknown. The purpose of this study was to assess H4R expression in OTSCC patients and in OTSCC-derived cell lines. Biopsies taken from OED, OTSCC and healthy oral mucosa were studied by immunostaining. Primary human oral keratinocytes (HOKs) and two OTSCC-derived cell lines (HSC-3 and SCC-25) were used for the in vitro studies. Quantitative real-time PCR was used to measure oncogene expression in the stimulated HOKs. We found that H4R-immunoreactivity was significantly reduced in the OED and OTSCC samples, especially in the samples with higher histopathological grades and noticeably increased mast cell counts. The presence of H4R in HSC-3 cells had clearly waned, in contrast to the HOKs. Gene expression data indicated that histamine-relevant inflammatory and environmental elements may participate in the regulation of oncogenes. Our results suggest an association between H4R and oral carcinogenesis. Furthermore, our findings raise a potential implication of histamine-mediated factors in the regulation of oncogenes, possibly via mast cells, as crucial components of the tumor microenvironment. The identification of new elements that govern oral cancer development is highly relevant for the development of novel therapeutic approaches in OTSCC.

Rinsing with Saline Promotes Human Gingival Fibroblast Wound Healing In Vitro.

Rinsing the mouth with sodium chloride (NaCl) solution is believed to promote healthy gum and improve oral ulcer healing. Scientific evidence to support this assumption is, however, lacking. This study aims to investigate the effect and clarify underlying mechanisms of short-term rinsing with NaCl on human gingival fibroblast (hGFs) wound healing. Isolated primary hGFs and human normal oral keratinocytes (hNOKs) were rinsed with 0–7.2% NaCl for 2 min, 3 times a day. Scratch-tests, trans-well migration assays and MTT activity were performed. mRNA expression was assessed of type-I collagen, fibronectin and FAK. Changes in FAK and F-actin were detected by immunofluorescence. KCl, NaH2PO4, KH2PO4 were used to clarify the molecules involved. Rinsing with 0.9–1.8% NaCl significantly promoted hGFs cell migration but not proliferation. However, it had no effect on hNOKs. Rinsing with 1.8% NaCl significantly up-regulated the expression of type-I collagen and fibronectin. FAK and F-actin, molecules responsible for cytoskeleton re-organization and cell migration, were also up-regulated. Cl- seemed to be essential since rinsing with KCl resulted in a similar effect as noted with NaCl. In conclusion, short-term rinsing with NaCl promoted hGFs migration, and increased the expression of extracellular matrix as well as cytoskeletal proteins. These data strongly support the long held belief in the benefits of using NaCl mouth-rinse.

Nano-TiO2 penetration of oral mucosa: in vitro analysis using 3D organotypic human buccal mucosa models.

BackgroundOral cavity is a doorway for a variety of products containing titanium dioxide (TiO2) nanoparticles (NPs) (nano‐TiO2) such as food additives, oral healthcare products and dental materials. Their potential to penetrate and affect normal human oral mucosa is not yet determined.ObjectivesTo evaluate the ability of nano‐TiO2 to penetrate the in vitro reconstructed normal human buccal mucosa (RNHBM).Methods RNHBM was generated from primary normal human oral keratinocytes and fibroblasts isolated from buccal oral mucosa of healthy patients (n = 6). The reconstructed tissues were exposed after 10 days to clinically relevant concentrations of spherical or spindle rutile nano‐TiO2 in suspension for short (20 min) and longer time (24 h). Ultrahigh‐resolution imaging (URI) microscopy (CytoViva™, Auburn, AL, USA) was used to assess the depth of penetration into reconstructed tissues.ResultsUltrahigh‐resolution imaging microscopy demonstrated the presence of nano‐TiO2 mostly in the epithelium of RNHBM at both 20 min and 24‐h exposure, and this was shape and doze dependent at 24 h of exposure. The depth of penetration diminished in time at higher concentrations. The exposed epithelium showed increased desquamation but preserved thickness.ConclusionNano‐TiO2 is able to penetrate RNHBM and to activate its barrier function in a doze‐ and time‐dependent manner.

Berberine-induced anticancer activities in FaDu head and neck squamous cell carcinoma cells.

In the present study, we investigated berberine‑induced apoptosis and the signaling pathways underlying its activity in FaDu head and neck squamous cell carcinoma cells. Berberine did not affect the viability of primary human normal oral keratinocytes. In contrast, the cytotoxicity of berberine was significantly increased in FaDu cells stimulated with berberine for 24 h. Furthermore, berberine increased nuclear condensation and apoptosis rates in FaDu cells than those in untreated control cells. Berberine also induced the upregulation of apoptotic ligands, such as FasL and TNF-related apoptosis-inducing ligand, and triggered the activation of caspase-8, -7 and -3, and poly(ADP ribose) polymerase, characteristic of death receptor-dependent extrinsic apoptosis. Moreover, berberine activated the mitochondria‑dependent apoptotic signaling pathway by upregulating pro-apoptotic factors, such as Bax, Bad, Apaf-1, and the active form of caspase-9, and downregulating anti-apoptotic factors, such as Bcl-2 and Bcl-xL. In addition, berberine increased the expression of the tumor suppressor p53 in FaDu cells. The pan-caspase inhibitor Z-VAD-fmk suppressed the activation of caspase-3 and prevented cytotoxicity in FaDu cells treated with berberine. Interestingly, berberine suppressed cell migration through downregulation of vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-2, and MMP-9. Moreover, the phosphorylation of extracellular signal-regulated kinase (ERK1/2) and p38, components of the mitogen-activated protein kinase pathway that are associated with the expression of MMP and VEGF, was suppressed in FaDu cells treated with berberine for 24 h. Therefore, these data suggested that berberine exerted anticancer effects in FaDu cells through induction of apoptosis and suppression of migration. Berberine may have potential applications as a chemotherapeutic agent for the management of head and neck squamous carcinoma.

Akt2 and p-Akt overexpression in oral cancer cells is due to a reduced rate of protein degradation.

To quantitatively measure the increased expression of Akt2 and its phosphorylated form (p-Akt) in oral cancer cell lines and investigate the post-translational mechanism for Akt2 and p-Akt overexpression. Three oral cancer cell lines and three cell lines of primary human oral keratinocytes (HOKs) were cultured and the degrees of Akt2 and p-Akt expression was evaluated by immunoblot analysis and flow cytometry. Each cell line was incubated with cycloheximide, an inhibitor of new protein synthesis, for various times to quantitatively determine the remaining expression levels of Akt2 and p-Akt by flow cytometry. The localization of Akt2 and p-Akt was assessed by immunofluorescence. The levels of Akt2 and p-Akt proteins were significantly higher in cancer cell lines than those in HOKs (P < 0.05). When the new protein synthesis was blocked by cycloheximide treatment, the degradation rate of Akt2 and p-Akt in oral cancer cells was significantly lower than that in HOKs (P < 0.05). Both Akt2 and p-Akt were more intensely stained in the cytoplasm of cancer cells, whereas HOKs expressed Akt2 and p-Akt only minimally. Both Akt2 and p-Akt were overexpressed in oral cancer cells, which may be partly explained by a reduced rate of protein degradation in order to maintain high cytosolic levels.

Proteomics Characterization of Primary Human Oral Epithelial Cells Using a Novel Culture Technique for Use in Tissue Regeneration.

Oral mucosa keratinocytes are widely used in regenerative medicine. The unique cultured cell population "Epithelial-derived Pop-Up Keratinocytes (ePUKs)" was previously reported as undifferentiated cells. Gravity Assisted Cell Sorting (GACS) was used to isolate a small-sized population of undifferentiated cells enriched ePUKs. LC/MS/MS analysis was performed to define the cellular profile of ePUKs of primary human oral mucosa keratinocytes. Small sized ePUKs which showed increased expression of Dickkopf WNT signaling pathway inhibitor 1 (DKK1), serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 1 (SERPINE1), follistatin and tenascin-C were verified by Western blots. These proteins are involved in the regulation of cellular movement, hair follicle development and the maintenance of its stem cell niche. The fabrication of a tissue-engineered oral mucosa, ex vivo produced oral mucosa equivalent (EVPOME), using ePUKs showed increased abundance of these verified proteins. These findings indicate that the specific phenotype of ePUKs and their ability to influence wound healing promotion are implicated by highly expressed cellular movement regulatory proteins. Therefore, ePUKs may be a useful cell source for use in regenerative medicine.

Hypoxia Induces an Undifferentiated Phenotype of Oral Keratinocytes in vitro

The aim of this study was to determine the effects of hypoxia on the proliferating potential and phenotype of primary human oral keratinocytes cultured at ambient oxygen tension (20%) or at different levels of hypoxia (2 and 0.5% O₂). The effects of oxygen tensions on cellular metabolic activity, cell proliferation, clonogenicity and proliferation heterogeneity were measured. Cell cycle profiles were analyzed by a fluorescent-activated cell sorter, and p21^WAF1/CIP1 expression in the G₀/G₁ phase was also concomitantly quantitated. The expression levels of cell cycle regulatory proteins were examined by immunoblotting, and the cellular senescence was assessed by senescence-associated β-galactosidase staining. Basal and suprabasal keratinocyte phenotypes were determined by the expression levels of 14-3-3σ, p75^NTR and α6 integrin. Despite having a lower metabolism, the proliferation rate and clonogenic potential were remarkably enhanced in hypoxic cells. The significantly higher percentage of cells in the G₀/G₁ phase under hypoxia and the expression patterns of cell cycle regulatory proteins in hypoxic cells were indicative of a state of cell cycle arrest in hypoxia. Furthermore, a decrease in the expression of p21^WAF1/CIP1 and p16^INK4A and fewer β-galactosidase-positive cells suggested a quiescent phenotype rather than a senescent one in hypoxic cells. Compared with normoxic cells, the differential expression patterns of keratinocyte phenotypic markers suggest that hypoxic cells that generate minimal reactive oxygen species, suppress the mammalian target of rapamycin activity and express hypoxia-inducible factor-1α favor a basal cell phenotype. Thus, regardless of the predisposition to the state of cell cycle arrest, hypoxic conditions can maintain oral keratinocytes in vitro in an undifferentiated and quiescent state.