Predicting intention of Norwegian dental health-care workers to use nanomaterials: An application of the augmented theory of planned behavior.

Due to the rapid development of nanotechnology and its integration into dentistry, there is a need for information on the factors influencing the decision of dental health-care workers to use nanomaterials. Based on a national survey among Norwegian dentists and dental hygienists, this study applied the theory of planned behavior (TPB), augmented with past behavior and perceived risk, to predict the intention to use dental nanomaterials in the future and to assess whether an augmented TPB model operates equivalently across professional groups. Structural equation modelling was used to assess whether the hypothesized model fits the data. Of 1792 eligible participants, 851 responded to an electronic survey. Attitudes and perceived behavioral control had the strongest effect on intention, followed by past behavior and subjective norms. Risk perceptions had an indirect effect on intention. Multigroup comparison confirmed invariance of the model across professional groups. This study supports the validity of the augmented TPB model to explain the intention of Norwegian dentists and dental hygienists to use nanomaterials. The strongest influence on intention is given by the attitudes toward nanomaterials and perceived confidence in their use. The findings of the study have implications for management of the use of nanomaterials in dentistry by policy makers.

Knowledge, opinions, and practices related to oral cancer prevention and oral mucosal examination among dentists in Moldova, Belarus and Armenia: a multi-country cross-sectional study.

INTRODUCTION: Moldova, Belarus, and Armenia are post-Soviet countries with a high rate of heavy smokers and a relatively high age-standardized incidence of oral cancer. However, to our knowledge, there is lack of available information on dentists' knowledge on prevention of oral cancer in the countries in question. Accordingly, this study aimed to assess the knowledge, opinions, and practices related to oral cancer prevention and oral mucosal examination among dentists in Moldova, Belarus, and Armenia. METHODS: This was a multi-country, cross-sectional study based on a self-administered questionnaire. A structured questionnaire was distributed to 3534 dentists (797 in Chisinau, Moldova, 1349 in Minsk, Belarus, and 1388 in Yerevan, Armenia). Dentists' knowledge about risk factors for oral cancer development and its clinical picture, current practices and opinions with regard to oral mucosal screening and oral cancer prevention, and their consistency to perform oral mucosal examination were assessed. A knowledge score ranging from 0 to 14 points was generated based on each dentist's answer to the questionnaire. RESULTS: A total of 1316 dentists responded, achieving an overall response rate of 37.2% (34.5% in Moldova; 52.3% in Belarus; 24.2% in Armenia). Most dentists in the three countries correctly identified tobacco (83.8-98.2%) and prior oral cancer lesions (84.0-96.3%) as risk factors for oral cancer. Most dentists correctly identified leukoplakia as a lesion with malignant potential (68.7% in Moldova; 88.5% in Belarus; 69.9% in Armenia), while erythroplakia was identified by much fewer in all three countries. Less than 52% of dentists identified the tongue, rim of tongue, and floor of mouth as the most common sites for oral cancer. The mean knowledge score for all countries combined was 7.5 ± 2.7. The most commonly reported barriers to perform oral mucosal examination were lack of training, knowledge, and experience. CONCLUSIONS: This study highlights the need for improved oral cancer-related education and training on oral mucosal examination for dentists in Moldova, Belarus, and Armenia. Such skills are essential to enhance oral cancer prevention and to improve the prognostic outcome by early detection.

Use of nanomaterials in dentistry: covariates of risk and benefit perceptions among dentists and dental hygienists in Norway.

Objective: Focusing dentists and dental hygienists employed in the Public Dental Health Services (PDHSs) in Norway, this study set out to assess whether socio-demographic factors, familiarity with nanotechnology and social trust are associated with dental health care workers' perceived risks and benefits of use of nanomaterials in dentistry and whether those associations varied according to professional status. It was hypothesized that increased knowledge, trust in stakeholders and familiarity with nanomaterials would decrease the risk and increase benefit perceptions among dental health care workers.Methods: Electronic questionnaires were administered to a census of 1792 dentists and dental hygienists.Results: About 64% and 69% of respondents perceived respectively, risk and benefits associated with use of nanomaterials. Multiple variable logistic regression revealed that dentists were more likely than hygienists to perceive risks (OR = 1.9, 95% CI 1.1-3.3) and benefits (OR = 3.6, 95% CI 2.1-6.2). Having experience with dental nanomaterials (OR = 2.2, 95% CI 1.3-3.7) and feeling safe (OR = 6.6, 95% CI 3.1-14.2) increased perceived benefits. Having moderate or much correct knowledge about nanotechnology (OR = 2.3, 95% CI 1.5-3.5) increased the likelihood of perceived risk.Conclusions: Policy makers should consider the factors that influence dental health care workers' risk and benefit perceptions associated with the use of nanomaterials in dentistry.

Knowledge about nanotechnology and intention to use nanomaterials: A comparative study among dental students in Norway and Romania.

BACKGROUND: The introduction of innovative nanotechnologies in medicine and dentistry may initiate a need for curriculum revision at the universities. The aim of this study was to assess dental students' knowledge and attitudes related to nanotechnology. Covariates of students' intention to use nanomaterials in their future dental practice were evaluated using the theory of planned behaviour (TPB). METHODS: Dental students at Norwegian and Romanian Universities were invited to participate. A self-administered structured questionnaire including socio-demographics and Ajzen's TPB components was used. FINDINGS: A total of 212 out of 732 dental students participated in the survey: 52 Norwegian and 160 Romanian. Most students reported to have little knowledge about nanotechnology (Norwegians = 44.2% vs Romanians = 46.9%, P < .05). More than 90% of the students in both countries reported that they wanted to get more information about nanotechnology. Mean knowledge score was similar for Norwegian and Romanian students (4.4 ± 1.7 vs 4.2 ± 1.4, P > .05). Romanian students had more positive attitude, stronger subjective norms and stronger perceived behavioural control towards nanotechnology compared to their Norwegian counterparts. Intention to use nanomaterials in the total sample was most strongly influenced by attitude towards the use of dental nanomaterials (beta = 0.42, P < .001). CONCLUSION: Dental students in Norway and Romania demonstrated limited knowledge about nanotechnology. Intention to use nanomaterials was primarily influenced by attitudes. A clear desire for more information about the application of nanotechnology in dentistry was expressed by the respondents indicating a need for curriculum modification.

Isolation and characterization of cells derived from human epithelial rests of Malassez.

The epithelial rests of Malassez (ERMs) might represent a valuable source of oral epithelial cells with stem cell properties. The purpose of this study was to isolate and characterize cells derived from human ERM, and compare them with cells derived from matched normal oral mucosa (NOM). Matched tissue specimens of the periodontal ligament of extracted tooth and NOM were collected. Cells were isolated in culture, then characterized by immunohistochemistry and flow cytometry for expression of pancytokeratin, ESA, PDGFRB, CD31 and CD44. 3D organotypic cultures were constructed by growing epithelial cells on top of fibroblast-populated collagen gels. Both ERM and NOM-isolated cells expressed the markers of epithelial lineage (ESA and pancytokeratin), and to some extent PDGFR, an indicator of a more mesenchymal phenotype, but not the endothelial cell marker CD31. Cells with epithelial morphology were isolated from periodontium of cervical, middle and apical parts of the root, but contained a significantly lower percentage of ESA and pancytokeratin-positive cells than when isolating cells from NOM (p < 0.001). ERM cells expressed a significantly higher percentage of the stem cell-related molecule CD44 (cervical 92.93 ± 0.25%, middle 93.8 ± 0.26%, apical 94.36 ± 0.41%) than cells isolated from NOM (27.8 ± 1.47%, p < 0.001). When grown in 3D organotypic cultures and in collagen gels, ERM cells formed a less differentiated epithelium than NOM cells, but expressing pancytokeratin and vimentin. In conclusion, epithelial cells could be isolated from human periodontium and grown in culture; their in vitro characterization indicates that they have a less differentiated phenotype compared with cells derived from normal oral epithelium.

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

BACKGROUND: Oral 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. OBJECTIVES: To 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. RESULTS: Ultrahigh-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. CONCLUSION: Nano-TiO2 is able to penetrate RNHBM and to activate its barrier function in a doze- and time-dependent manner.

Xeno-free generation of human induced pluripotent stem cells from donor-matched fibroblasts isolated from dermal and oral tissues.

BACKGROUND: Induced pluripotent stem cells (iPS) can be generated from various somatic cells and can subsequently be differentiated to multiple cell types of the body. This makes them highly promising for cellular therapy in regenerative medicine. However, to facilitate their clinical use and to ensure safety, iPS culturing protocols must be compliant with good manufacturing practice guidelines and devoid of xenogenic products. Therefore, we aimed to compare the efficiency of using humanized culture conditions, specifically human platelet lysate to fetal bovine serum, for iPS generation from different sources, and to evaluate their stemness. METHODS: iPS were generated via a platelet lysate or fetal bovine serum-based culturing protocol from matched dermal, buccal and gingival human fibroblasts, isolated from healthy donors (n = 2) after informed consent, via episomal plasmid transfection. Pluripotency, genotype and phenotype of iPS, generated by both protocols, were then assessed by various methods. RESULTS: More attempts were generally required to successfully reprogram xeno-free fibroblasts to iPS, as compared to xenogenic cultured fibroblasts. Furthermore, oral fibroblasts generally required more attempts for successful iPS generation as opposed to dermal fibroblasts. Morphologically, all iPS generated from fibroblasts formed tight colonies surrounded by a reflective "whitish" outer rim, typical for iPS. They also expressed pluripotency markers at both gene (SOX2, OCT4, NANOG) and protein level (SOX2, OCT4). Upon stimulation, all iPS showed ability to differentiate into the three primary germ layers via expression of lineage-specific markers for mesoderm (MESP1, OSR1, HOPX), endoderm (GATA4) and ectoderm (PAX6, RAX). Genome analysis revealed several amplifications and deletions within the chromosomes of each iPS type. CONCLUSIONS: The xeno-free protocol had a lower reprogramming efficiency compared to the standard xenogenic protocol. The oral fibroblasts generally proved to be more difficult to reprogram than dermal fibroblasts. Xeno-free dermal, buccal and gingival fibroblasts can successfully generate iPS with a comparable genotype/phenotype to their xenogenic counterparts.

Characterization of immune cell infiltrate in tumor stroma and epithelial compartments in oral squamous cell carcinomas of Sudanese patients.

BACKGROUND: Tumor immune infiltrate has been explored in oral squamous cell carcinoma (OSCC), but studies on simultaneous characterization of multiple immune cell subtypes separately in stromal and intraepithelial tumor compartments are limited. OBJECTIVES: We aimed to investigate the immune cell infiltrate in OSCC by using immunohistochemistry (IHC) for a panel of inflammatory cells in stromal and epithelial tumor compartments for a better characterization of the tumors. METHODS: Thirty-six OSCC lesions and nine normal oral mucosa (NOM) samples from patients attending Khartoum Dental Teaching Hospital, Sudan were investigated for presence of tumor infiltrating lymphocytes, tumor-associated macrophages, tumor-associated neutrophils, and PD-L1 positive cells in the inflammatory infiltrate by single and double IHC. Digital quantitative analysis (Aperio Technologies Inc.) was performed separately for stromal and epithelial compartments. RESULTS: OSCC cases displayed a higher inflammatory infiltrate in the associated stroma, but not in the epithelial compartment when compared to NOM. The immunosuppressive type of inflammatory infiltrate, that is, T regulatory cells (FoxP3+ cells) was identified to be significantly higher in the epithelial compartment of tumors with advanced clinical state. An immunoscore developed by combining intraepithelial FoxP3+ and CD4+ cells was found significantly higher in lesions from elderly patients, localized at toombak dipping-related sites, poorly differentiated OSCCs, or with loco-regional lymph node spreading. CONCLUSIONS: Despite heavy immune cell infiltration in tumor-associated stroma, the majority of OSCCs in this cohort displayed a low intraepithelial immune infiltration. An immunoscore based on combined CD4 and FoxP3 intraepithelial expression may serve as an indicator of advanced tumor progression and should be further investigated for its use as potential prognostic biomarker in OSCC.

Epithelial PD-L1 expression at tumor front predicts overall survival in a cohort of oral squamous cell carcinomas from Sudan.

BACKGROUND: We recently described the tumor immune microenvironment (TIME) in oral squamous cell carcinomas (OSCC) from Sudan by assessing the core of the lesions. However, the invasive tumor front (ITF) is the most active part of OSCC lesions; thus, TIME should also be characterized at the ITF in this patient cohort. OBJECTIVES: We aimed to evaluate patterns of immune cell infiltration at the ITF in a cohort of OSCC patients from Sudan previously investigated at the tumor center and their association with clinicopathological parameters. METHODS: This study was performed on a prospective cohort of 22 OSCC patients attending Khartoum Dental Teaching Hospital with a median follow-up of 48 months. Inflammatory infiltrate densities of CD4-, CD8-, FoxP3-, CD20-, CD66b-, M1 (CD80/CD68)-, M2 (CD163/CD68)-, and PD-L1-positive cells were assessed at the ITF by immunohistochemistry, followed by digital quantitative analysis at the stromal and epithelial compartments separately. Histopathological parameters such as the worst pattern of invasion, differentiation, and tumor budding (TB) were also assessed. Correlations between clinicopathological parameters and survival analysis were investigated using SPSS. RESULTS: All inflammatory cell subsets investigated were found to be higher in the stromal compartment as compared to the epithelial one, except for the PD-L1+ subset. Stromal infiltration with the CD8+ cell subset was associated with low TB. Kaplan-Meier analyses identified higher epithelial and stromal CD4+ cell subsets. The presence of PD-L1 was found to be associated with unfavorable overall survival. Further, Cox's regression analysis using an age- and tumor-stage-adjusted model identified epithelial PD-L1 expression at the ITF as the only independent prognosticator. CONCLUSIONS: Epithelial PD-L1 expression at the ITF was found to be an independent prognostic biomarker for OSCC in a cohort of Sudanese patients.

Ectopic Bone Tissue Engineering in Mice Using Human Gingiva or Bone Marrow-Derived Stromal/Progenitor Cells in Scaffold-Hydrogel Constructs.

Three-dimensional (3D) spheroid culture can promote the osteogenic differentiation and bone regeneration capacity of mesenchymal stromal cells (MSC). Gingiva-derived progenitor cells (GPC) represent a less invasive alternative to bone marrow MSC (BMSC) for clinical applications. The aim of this study was to test the in vivo bone forming potential of human GPC and BMSC cultured as 3D spheroids or dissociated cells (2D). 2D and 3D cells encapsulated in constructs of human platelet lysate hydrogels (HPLG) and 3D-printed poly (L-lactide-co-trimethylene carbonate) scaffolds (HPLG-PLATMC) were implanted subcutaneously in nude mice; cell-free HPLG-PLATMC constructs served as a control. Mineralization was assessed using micro-computed tomography (µCT), histology, scanning electron microscopy (SEM) and in situ hybridization (ISH). After 4-8 weeks, µCT revealed greater mineralization in 3D-BMSC vs. 2D-BMSC and 3D-GPC (p < 0.05), and a similar trend in 2D-GPC vs. 2D-BMSC (p > 0.05). After 8 weeks, greater mineralization was observed in cell-free constructs vs. all 2D- and 3D-cell groups (p < 0.05). Histology and SEM revealed an irregular but similar mineralization pattern in all groups. ISH revealed similar numbers of 2D and 3D BMSC/GPC within and/or surrounding the mineralized areas. In summary, spheroid culture promoted ectopic mineralization in constructs of BMSC, while constructs of dissociated GPC and BMSC performed similarly. The combination of HPLG and PLATMC represents a promising scaffold for bone tissue engineering applications.

Corrigendum: Ectopic Bone Tissue Engineering in Mice Using Human Gingiva or Bone Marrow-Derived Stromal/Progenitor Cells in Scaffold-Hydrogel Constructs.

[This corrects the article DOI: 10.3389/fbioe.2021.783468.].

Analysis of Salivary Mycobiome in a Cohort of Oral Squamous Cell Carcinoma Patients From Sudan Identifies Higher Salivary Carriage of Malassezia as an Independent and Favorable Predictor of Overall Survival.

Background: Microbial dysbiosis and microbiome-induced inflammation have emerged as important factors in oral squamous cell carcinoma (OSCC) tumorigenesis during the last two decades. However, the "rare biosphere" of the oral microbiome, including fungi, has been sparsely investigated. This study aimed to characterize the salivary mycobiome in a prospective Sudanese cohort of OSCC patients and to explore patterns of diversities associated with overall survival (OS). Materials and Methods: Unstimulated saliva samples (n = 72) were collected from patients diagnosed with OSCC (n = 59) and from non-OSCC control volunteers (n = 13). DNA was extracted using a combined enzymatic-mechanical extraction protocol. The salivary mycobiome was assessed using a next-generation sequencing (NGS)-based methodology by amplifying the ITS2 region. The impact of the abundance of different fungal genera on the survival of OSCC patients was analyzed using Kaplan-Meier and Cox regression survival analyses (SPPS). Results: Sixteen genera were identified exclusively in the saliva of OSCC patients. Candida, Malassezia, Saccharomyces, Aspergillus, and Cyberlindnera were the most relatively abundant fungal genera in both groups and showed higher abundance in OSCC patients. Kaplan-Meier survival analysis showed higher salivary carriage of the Candida genus significantly associated with poor OS of OSCC patients (Breslow test: p = 0.043). In contrast, the higher salivary carriage of Malassezia showed a significant association with favorable OS in OSCC patients (Breslow test: p = 0.039). The Cox proportional hazards multiple regression model was applied to adjust the salivary carriage of both Candida and Malassezia according to age (p = 0.029) and identified the genus Malassezia as an independent predictor of OS (hazard ratio = 0.383, 95% CI = 0.16-0.93, p = 0.03). Conclusion: The fungal compositional patterns in saliva from OSCC patients were different from those of individuals without OSCC. The fungal genus Malassezia was identified as a putative prognostic biomarker and therapeutic target for OSCC.

Nanodiamond modified copolymer scaffolds affects tumour progression of early neoplastic oral keratinocytes.

This study aimed to evaluate the tumorigenic potential of functionalising poly(LLA-co-CL) scaffolds. The copolymer scaffolds were functionalised with nanodiamonds (nDP) or with nDP and physisorbed BMP-2 (nDP-PHY) to enhance osteoinductivity. Culturing early neoplastic dysplastic keratinocytes (DOK(Luc)) on nDP modified scaffolds reduced significantly their subsequent sphere formation ability and decreased significantly the cells' proliferation in the supra-basal layers of in vitro 3D oral neoplastic mucosa (3D-OT) when compared to DOK(Luc) previously cultured on nDP-PHY scaffolds. Using an in vivo non-invasive environmentally-induced oral carcinogenesis model, nDP scaffolds were observed to reduce bioluminescence intensity of tumours formed by DOK(Luc) + carcinoma associated fibroblasts (CAF). nDP modification was also found to promote differentiation of DOK(Luc) both in vitro in 3D-OT and in vivo in xenografts formed by DOK(Luc) alone. The nDP-PHY scaffold had the highest number of invasive tumours formed by DOK(Luc) + CAF outside the scaffold area compared to the nDP and control scaffolds. In conclusion, in vitro and in vivo results presented here demonstrate that nDP modified copolymer scaffolds are able to decrease the tumorigenic potential of DOK(Luc), while confirming concerns for the therapeutic use of BMP-2 for reconstruction of bone defects in oral cancer patients due to its tumour promoting capabilities.

MicroRNA alterations and associated aberrant DNA methylation patterns across multiple sample types in oral squamous cell carcinoma.

BACKGROUND: MicroRNA (miRNA) expression is broadly altered in cancer, but few studies have investigated miRNA deregulation in oral squamous cell carcinoma (OSCC). Epigenetic mechanisms are involved in the regulation of >30 miRNA genes in a range of tissues, and we aimed to investigate this further in OSCC. METHODS: TaqMan® qRT-PCR arrays and individual assays were used to profile miRNA expression in a panel of 25 tumors with matched adjacent tissues from patients with OSCC, and 8 control paired oral stroma and epithelium from healthy volunteers. Associated DNA methylation changes of candidate epigenetically deregulated miRNA genes were measured in the same samples using the MassArray® mass spectrometry platform. MiRNA expression and DNA methylation changes were also investigated in FACS sorted CD44(high) oral cancer stem cells from primary tumor samples (CSCs), and in oral rinse and saliva from 15 OSCC patients and 7 healthy volunteers. RESULTS: MiRNA expression patterns were consistent in healthy oral epithelium and stroma, but broadly altered in both tumor and adjacent tissue from OSCC patients. MiR-375 is repressed and miR-127 activated in OSCC, and we confirm previous reports of miR-137 hypermethylation in oral cancer. The miR-200 s/miR-205 were epigenetically activated in tumors vs normal tissues, but repressed in the absence of DNA hypermethylation specifically in CD44(high) oral CSCs. Aberrant miR-375 and miR-200a expression and miR-200c-141 methylation could be detected in and distinguish OSCC patient oral rinse and saliva from healthy volunteers, suggesting a potential clinical application for OSCC specific miRNA signatures in oral fluids. CONCLUSIONS: MiRNA expression and DNA methylation changes are a common event in OSCC, and we suggest miR-375, miR-127, miR-137, the miR-200 family and miR-205 as promising candidates for future investigations. Although overall activated in OSCC, miR-200/miR-205 suppression in oral CSCs indicate that cell specific silencing of these miRNAs may drive tumor expansion and progression.

Dual effects of sodium lauryl sulphate on human oral epithelial structure.

Sodium lauryl sulphate (SLS) is a common detergent known to cause irritation and inflammatory reactions in skin. SLS is also the most commonly used toothpaste detergent and has been related to intraoral adverse effects. However, its specific biological effects on the oral mucosa (OM) have not yet been identified. The objective of this study was to investigate the putative effects of SLS on human oral epithelium using a novel in vitro reconstructed three-dimensional cell culture model. Reconstructed human OM, generated from primary normal human oral keratinocytes and fibroblasts, was exposed to clinically relevant concentrations of SLS (range 0.015-1.5%). The cultured tissues were evaluated by histomorphometry, immunohistochemistry (Ki-67, epithelial (E)-cadherin, alpha6-, beta1-integrins, cleaved caspase-3) and the TUNEL method. Increased epithelial thickness, enhanced proliferation (Ki-67), a more pronounced expression of E-cadherin throughout all epithelial cell layers and single TUNEL-positive cells in the middle spinous cell layers were observed in cultures exposed to low concentrations (0.015%) of SLS. At exposure to higher SLS concentrations (>or=0.15%), epithelial thickness, cell proliferation and E-cadherin expression gradually decreased and in the central areas of exposed regions, cells detached from each other and underwent cell death. In conclusion, clinically relevant concentrations of SLS have dual effects on reconstituted human OM; although occasional cell death within the epithelium was also observed, the increased epithelial thickness, proliferation and E-cadherin expression induced at lower concentrations might be associated with a protective mucosal response, whereas at higher concentrations a more destructive type of reaction predominated.