Orai1 expression pattern in tooth and craniofacial ectodermal tissues and potential functions during ameloblast differentiation.

BACKGROUND: Orai1 is a plasma membrane protein that forms the pore of the calcium release activated calcium channel. Humans with mutated Orai1 present with hereditary combined immunodeficiency, congenital myopathy and anhidrotic ectodermal dysplasia. Consistent with the ectodermal dysplasia phenotype, enamel formation and mineralization is also abnormal in Orai1 deficient patients. The expression pattern and potential functions of Orai1 in enamel formation remains unclear. To contribute toward understanding the role of Orai1 in amelogenesis we characterized ORAI1 protein developmental pattern in comparison with other ectodermal organs. We also examined the effects of Orai1 down-regulation in ameloblast cell proliferation and differentiation. RESULTS: Our data show strong expression of ORAI1 protein during the ameloblast secretory stage, which weans at the end of the maturation stage. In salivary glands, ORAI1 is expressed mainly in acini cells. ORAI1 expression is also found in hair follicle and oral epithelium. Knockdown of Orai1 expression decreases cell proliferation and results in RNA expression levels changes of key ameloblast genes regulating enamel thickness and mineralization. CONCLUSIONS: This study provides insights in the anhidrotic ectodermal dysplasia phenotype due to Orai1 mutation and highlights the importance of calcium signaling in controlling ameloblast differentiation and maturation during tooth development.

The tick tock of odontogenesis.

Although a big deal of dental research is being focused to the understanding of early stages of tooth development, a huge gap exist on our knowledge on how the dental hard tissues are formed and how this process is controlled daily in order to produce very complex and diverse tooth shapes adapted for specific functions. Emerging evidence suggests that clock genes, a family of genes that controls circadian functions within our bodies, regulate also dental mineralized tissues formation. Enamel formation, for example, is subjected to rhythmical molecular signals that occur on short (24h) periods and control the secretion and maturation of the enamel matrix. Accordingly, gene expression and ameloblast functions are also tightly modulated in regular daily intervals. This review summarizes the current knowledge on the circadian controls of dental mineralized tissues development with a special emphasis on amelogenesis.

Equity-diversity-inclusion (EDI)-related strategies used by dental schools during the admission/selection process: a narrative review.

INTRODUCTION: Decades of evidence have demonstrated a lack of workforce diversity and sustaining disparities in academic dentistry and professional practice. Underrepresented minority students may face challenges and implicit bias during the dental schools' admission/selection process. This review collected papers from different countries to summarize the Equity-Diversity-Inclusion (EDI)-related strategies that dental schools worldwide have used in their admissions process to increase diversity. METHODS: A comprehensive search using MEDLINE (via PubMed), ERIC, Cochrane Reviews, Cochrane Trials, American Psychological Association Psyc Info (EBSCO) and Scopus was done between January and March-2023. All types of articles-designs were included, except comments and editorials, and all articles selected were in English. Two independent investigators screened the articles. Extracted data were general characteristics, study objectives, and EDI-related strategies. RESULTS: Sixteen publications were used to construct this manuscript. The year with the greatest number of publications was 2022. Type of studies were case studies/critical reviews (50%), cross-sectional (including survey and secondary data analysis) (n = 5, 31.25%), qualitative methods of analysis (n = 2, 12.5%), and retrospective/secondary data collection (n = 1, 6.25%). The strategies described in the articles were related to (1) considering the intersectionality of diversity, (2) using noncognitive indicators during the school admissions process to construct a holistic selection process, (3) diversifying, professionalizing, and providing training to admissions persons who had leadership roles with the support from the dental school and the university, and (4) allocating financial investments and analyzing current policies and procedures regarding EDI. CONCLUSIONS: This review aggregated interesting findings, such as: some schools are considering the intersectionality of diversity as a way to include underrepresented minorities and to diversify the students-body. The recent growth in publications on EDI during dental admission/selection process might indicate a positive movement in this field.

YouTube Videos on Nutrition and Dental Caries: Content Analysis.

BACKGROUND: Dental caries is the most common health condition worldwide, and nutrition and dental caries have a strong interconnected relationship. Foods and eating behaviors can be both harmful (eg, sugar) and healthful (eg, meal spacing) for dental caries. YouTube is a popular source for the public to access information. To date, there is no information available on the nutrition and dental caries content of easily accessible YouTube videos. OBJECTIVE: This study aimed to analyze the content of YouTube videos on nutrition and dental caries. METHODS: In total, 6 YouTube searches were conducted using keywords related to nutrition and dental caries. The first 20 videos were selected from each search. Video content was scored (17 possible points; higher scores were associated with more topics covered) by 2 individuals based on the inclusion of information regarding various foods and eating behaviors that impact dental caries risk. For each video, information on video characteristics (ie, view count, length, number of likes, number of dislikes, and video age) was captured. Videos were divided into 2 groups by view rate (views/day); differences in scores and types of nutrition messages between groups were determined using nonparametric statistics. RESULTS: In total, 42 videos were included. Most videos were posted by or featured oral health professionals (24/42, 57%). The mean score was 4.9 (SD 3.4) out of 17 points. Videos with >30 views/day (high view rate; 20/42, 48% videos) had a trend toward a lower score (mean 4.0, SD 3.7) than videos with ≤30 views/day (low view rate; 22/42, 52%; mean 5.8, SD 3.0; P=.06), but this result was not statistically significant. Sugar was the most consistently mentioned topic in the videos (31/42, 74%). No other topics were mentioned in more than 50% of videos. Low-view rate videos were more likely to mention messaging on acidic foods and beverages (P=.04), water (P=.09), and frequency of sugar intake (P=.047) than high-view rate videos. CONCLUSIONS: Overall, the analyzed videos had low scores for nutritional and dental caries content. This study provides insights into the messaging available on nutrition and dental caries for the public and guidance on how to make improvements in this area.

Colorimetric sensing assay based on aptamer-gold nanoparticles for rapid detection of salivary melatonin to monitor circadian rhythm sleep disorders.

Salivary melatonin is a clinically used biomarker for diagnosing circadian rhythm sleep disorders. Current melatonin detection assays are complex, expensive, and in many cases do not adequately measure low levels of salivary melatonin. Precisely measuring melatonin levels at multiple time points is crucial for determining dim light melatonin onset to evaluate its circadian fluctuation as well as the extent of circadian disruption and consequently adapt treatment regimens. Moreover, melatonin low levels in saliva challenges the reliability of routine clinical testing. This paper presents the development of a novel, highly sensitive, yet cost-effective, colorimetric assay for the rapid detection of salivary melatonin utilizing aptamer-AuNPs. Among several types of the aptamer tested, the 36-mer MLT-A-2 aptamer-AuNP probe showed the highest sensitivity with a melatonin limit of detection of 0.0011 nM along with a limit of quantification of 0.0021 nM in saliva. Moreover, our assay showed preferential interaction with melatonin when tested in presence of other structurally similar counter-targets. Taken together, this study provides new parameters for a melatonin assay that meets adequate levels of sensitivity and selectivity. The developed colorimetric assay could be adapted in a point-of-care system for profiling salivary melatonin levels at multiple time points during 24 h, crucial for accurately diagnosing and monitoring circadian rhythm sleep disorders and beyond.

Structural properties and binding mechanism of DNA aptamers sensing saliva melatonin for diagnosis and monitoring of circadian clock and sleep disorders.

Circadian desynchrony with the external light-dark cycle influences the rhythmic secretion of melatonin which is among the first signs of circadian rhythm sleep disorders. An accurate dim light melatonin onset (established indicator of circadian rhythm sleep disorders) measurement requires lengthy assays, and antibody affinities alterations, especially in patients with circadian rhythm disorders whose melatonin salivary levels vary significantly, making antibodies detection mostly inadequate. In contrast, aptamers with their numerous advantages (e.g., target selectivity, structural flexibility in tuning binding affinities, small size, etc.) can become preferable biorecognition molecules for salivary melatonin detection with high sensitivity and specificity. This study thoroughly characterizes the structural property and binding mechanism of a single-stranded DNA aptamer full sequence (MLT-C-1) and its truncated versions (MLT-A-2, MLT-A-4) to decipher its optimal characteristics for saliva melatonin detection. We use circular dichroism spectroscopy to determine aptamers' conformational changes under different ionic strengths and showed that aptamers display a hairpin loop structure where few base pairs in the stem play a significant role in melatonin binding and formation of aptamer stabilized structure. Through microscale thermophoresis, aptamers demonstrated a high binding affinity in saliva samples (MLT-C-1F Kd = 12.5 ± 1.7 nM; MLT-A-4F Kd = 11.2 ± 1.6 nM; MLT-A-2F Kd = 2.4 ± 2.8 nM; limit-of-detection achieved in pM, highest sensitivity attained for MLT-A-2F aptamer with the lowest detection limit of 1.35 pM). Our data suggest that aptamers are promising as biorecognition molecules and provide the baseline parameters for the development of an aptamer-based point-of-care diagnostic system for melatonin detection and accurate profiling of its fluctuations in saliva.

On-demand chlorine dioxide solution enhances odontoblast differentiation through desulfation of cell surface heparan sulfate proteoglycan and subsequent activation of canonical Wnt signaling.

Heparan sulfate proteoglycans (HSPGs) surround the surface of odontoblasts, and their modification affects their affinity for Wnt ligands. This study proposes applying Matching Transformation System® (MA-T), a novel chlorinated oxidant, to enhance dentinogenesis. MA-T treatment in odontoblasts decreased sulfation of HSPG and upregulated the expression of dentin sialophosphoprotein (Dspp) and Dentin Matrix Protein 1 (Dmp1) via activation of canonical Wnt signaling in vitro. Ex vivo application of MA-T also enhanced dentin matrix formation in developing tooth explants. Reanalysis of a public single-cell RNA-seq dataset revealed significant Wnt activity in the odontoblast population, with enrichment for Wnt10a and Wnt6. Silencing assays showed that Wnt10a and Wnt6 were redundant in inducing Dspp and Dmp1 mRNA expression. These Wnt ligands' expression was upregulated by MA-T treatment, and TCF/LEF binding sites are present in their promoters. Furthermore, the Wnt inhibitors Notum and Dkk1 were enriched in odontoblasts, and their expression was also upregulated by MA-T treatment, together suggesting autonomous maintenance of Wnt signaling in odontoblasts. This study provides evidence that MA-T activates dentinogenesis by modifying HSPG and through subsequent activation of Wnt signaling.

Novel trends, challenges and new perspectives for enamel repair and regeneration to treat dental defects.

Dental enamel is the hardest tissue in the human body, providing external protection for the tooth against masticatory forces, temperature changes and chemical stimuli. Once enamel is damaged/altered by genetic defects, dental caries, trauma, and/or dental wear, it cannot repair itself due to the loss of enamel producing cells following the tooth eruption. The current restorative dental materials are unable to replicate physico-mechanical, esthetic features and crystal structures of the native enamel. Thus, development of alternative approaches to repair and regenerate enamel defects is much needed but remains challenging due to the structural and functional complexities involved. This review paper summarizes the clinical aspects to be taken into consideration for the development of optimal therapeutic approaches to tackle dental enamel defects. It also provides a comprehensive overview of the emerging acellular and cellular approaches proposed for enamel remineralization and regeneration. Acellular approaches aim to artificially synthesize or re-mineralize enamel, whereas cell-based strategies aim to mimic the natural process of enamel development given that epithelial cells can be stimulated to produce enamel postnatally during the adult life. The key issues and current challenges are also discussed here, along with new perspectives for future research to advance the field of regenerative dentistry.

Self-Crosslinkable Oxidized Alginate-Carboxymethyl Chitosan Hydrogels as an Injectable Cell Carrier for In Vitro Dental Enamel Regeneration.

Injectable hydrogels, as carriers, offer great potential to incorporate cells or growth factors for dental tissue regeneration. Notably, the development of injectable hydrogels with appropriate structures and properties has been a challenging task, leaving much to be desired in terms of cytocompatibility, antibacterial and self-healing properties, as well as the ability to support dental stem cell functions. This paper presents our study on the development of a novel self-cross-linkable hydrogel composed of oxidized alginate and carboxymethyl chitosan and its characterization as a cell carrier for dental enamel regeneration in vitro. Oxidized alginate was synthesized with 60% theoretical oxidation degree using periodate oxidation and characterized by Fourier Transform Infrared spectroscopy, proton nuclear magnetic resonance spectroscopy, and Ultraviolet-visible absorption spectroscopy. Then, hydrogels were prepared at three varying weight ratios of oxidized alginate to carboxymethyl chitosan (4:1, 3:1, and 2:1) through Schiff base reactions, which was confirmed by Fourier Transform Infrared spectroscopy. The hydrogels were characterized in terms of gelation time, swelling ratio, structure, injectability, self-healing, antibacterial properties, and in vitro characterization for enamel regeneration. The results demonstrated that, among the three hydrogels examined, the one with the highest ratio of oxidized alginate (i.e., 4:1) had the fastest gelation time and the lowest swelling ability, and that all hydrogels were formed with highly porous structures and were able to be injected through a 20-gauge needle without clogging. The injected hydrogels could be rapidly reformed with the self-healing property. The hydrogels also showed antibacterial properties against two cariogenic bacteria: Streptococcus mutans and Streptococcus sobrinus. For in vitro enamel regeneration, a dental epithelial cell line, HAT-7, was examined, demonstrating a high cell viability in the hydrogels during injection. Furthermore, HAT-7 cells encapsulated in the hydrogels showed alkaline phosphatase production and mineral deposition, as well as maintaining their round morphology, after 14 days of in vitro culture. Taken together, this study has provided evidence that the oxidized alginate-carboxymethyl chitosan hydrogels could be used as an injectable cell carrier for dental enamel tissue engineering applications.

Risk Indicators and Treatment Needs of Children 2-5 Years of Age Receiving Dental Treatment under General Anesthesia in Saskatchewan.

BACKGROUND: When compared to national averages in Canada, Saskatchewan has one of the highest rates of dental treatment under general anesthesia (GA) and average costs per child. Thus, the purpose of this cross-sectional study is to explore the risk indicators and treatment needs of children receiving dental treatment under GA in Saskatchewan. METHODS: In this cross-sectional study, we recruited caregivers of children between 24 and 71 months of age in Saskatoon, Canada. Caregivers completed a 40-item questionnaire, which was supplemented with clinical data and then subject to statistical analysis (independent t-tests and one-way ANOVA). RESULTS: A total of 90 caregiver/child dyads were enrolled with the mean age for children being 49.5 ± 12.3 months. The mean age of a child's first dental visit was 34.7 ± 15.3 months with only 37.9% of children having a dental home. The mean deft index was 11.7 ± 3.4, with an average of 10.9 ± 3.5 teeth receiving treatment. Additionally, location of primary residence (p = 0.03), family income (p = 0.04), family size (p = 0.01), parental education (p = 0.03), dental home (p = 0.04), and body mass index (p = 0.04) had a statistically significant association with a higher mean deft. CONCLUSIONS: Our cross-sectional study confirms that children who require dental treatment under GA have a high burden of disease. While individual risk indicators such as diet and oral hygiene play a role in the progression of early childhood caries (ECC), we also demonstrate that children who do not have access to early preventive visits or a dental home are at a higher risk. In addition to improving motivation for oral hygiene at home and nutritional education, improving access to oral health care should be addressed in strategies to reduce ECC.

Gemini surfactant-based nanoparticles T-box1 gene delivery as a novel approach to promote epithelial stem cells differentiation and dental enamel formation.

Enamel is the highest mineralized tissue in the body protecting teeth from external stimuli, infections, and injuries. Enamel lacks the ability to self-repair due to the absence of enamel-producing cells in the erupted teeth. Here, we reported a novel approach to promote enamel-like tissue formation via the delivery of a key ameloblast inducer, T-box1 gene, into a rat dental epithelial stem cell line, HAT-7, using non-viral gene delivery systems based on cationic lipids. We comparatively assessed the lipoplexes prepared from glycyl-lysine-modified gemini surfactants and commercially available 1,2-dioleoyl-3-trimethylammonium-propane lipids at three nitrogen-to phosphate (N/P) ratios of 2.5, 5 and 10. Our findings revealed that physico-chemical characteristics and biological activities of the gemini surfactant-based lipoplexes with a N/P ratio of 5 provide the most optimal outcomes among those examined. HAT-7 cells were transfected with T-box1 gene using the optimal formulation then cultured in conventional 2D cell culture systems. Ameloblast differentiation, mineralization, bio-enamel interface and structure were assessed at different time points over 28 days. Our results showed that our gemini transfection system provides superior gene expression compared to the benchmark agent, while keeping low cytotoxicity levels. T-box1-transfected HAT-7 cells strongly expressed markers of secretory and maturation stages of the ameloblasts, deposited minerals, and produced enamel-like crystals when compared to control cells. Taken together, our gemini surfactant-based T-box1 gene delivery system is effective to accelerate and guide ameloblastic differentiation of dental epithelial stem cells and promote enamel-like tissue formation. This study would represent a significant advance towards the tissue engineering and regeneration of dental enamel.

The Clinical, Microbiological, and Immunological Effects of Probiotic Supplementation on Prevention and Treatment of Periodontal Diseases: A Systematic Review and Meta-Analysis.

(1) Background: Periodontal diseases are a global health concern. They are multi-stage, progressive inflammatory diseases triggered by the inflammation of the gums in response to periodontopathogens and may lead to the destruction of tooth-supporting structures, tooth loss, and systemic health problems. This systematic review and meta-analysis evaluated the effects of probiotic supplementation on the prevention and treatment of periodontal disease based on the assessment of clinical, microbiological, and immunological outcomes. (2) Methods: This study was registered under PROSPERO (CRD42021249120). Six databases were searched: PubMed, MEDLINE, EMBASE, CINAHL, Web of Science, and Dentistry and Oral Science Source. The meta-analysis assessed the effects of probiotic supplementation on the prevention and treatment of periodontal diseases and reported them using Hedge's g standardized mean difference (SMD). (3) Results: Of the 1883 articles initially identified, 64 randomized clinical trials were included in this study. The results of this meta-analysis indicated statistically significant improvements after probiotic supplementation in the majority of the clinical outcomes in periodontal disease patients, including the plaque index (SMD = 0.557, 95% CI: 0.228, 0.885), gingival index, SMD = 0.920, 95% CI: 0.426, 1.414), probing pocket depth (SMD = 0.578, 95% CI: 0.365, 0.790), clinical attachment level (SMD = 0.413, 95% CI: 0.262, 0.563), bleeding on probing (SMD = 0.841, 95% CI: 0.479, 1.20), gingival crevicular fluid volume (SMD = 0.568, 95% CI: 0.235, 0.902), reduction in the subgingival periodontopathogen count of P. gingivalis (SMD = 0.402, 95% CI: 0.120, 0.685), F. nucleatum (SMD = 0.392, 95% CI: 0.127, 0.658), and T. forsythia (SMD = 0.341, 95% CI: 0.050, 0.633), and immunological markers MMP-8 (SMD = 0.819, 95% CI: 0.417, 1.221) and IL-6 (SMD = 0.361, 95% CI: 0.079, 0.644). (4) Conclusions: The results of this study suggest that probiotic supplementation improves clinical parameters, and reduces the periodontopathogen load and pro-inflammatory markers in periodontal disease patients. However, we were unable to assess the preventive role of probiotic supplementation due to the paucity of studies. Further clinical studies are needed to determine the efficacy of probiotic supplementation in the prevention of periodontal diseases.

Bioprinting of alginate-carboxymethyl chitosan scaffolds for enamel tissue engineeringin vitro.

Tissue engineering offers a great potential in regenerative dentistry and to this end, three dimensional (3D) bioprinting has been emerging nowadays to enable the incorporation of living cells into the biomaterials (such a mixture is referred as a bioink in the literature) to create scaffolds. However, the bioinks available for scaffold bioprinting are limited, particularly for dental tissue engineering, due to the complicated, yet compromised, printability, mechanical and biological properties simultaneously imposed on the bioinks. This paper presents our study on the development of a novel bioink from carboxymethyl chitosan (CMC) and alginate (Alg) for bioprinting scaffolds for enamel tissue regeneration. CMC was used due to its antibacterial ability and superior cell interaction properties, while Alg was added to enhance the printability and mechanical properties as well as to regulate the degradation rate. The bioinks with three mixture ratios of Alg and CMC (2-4, 3-3 and 4-2) were prepared, and then printed into the calcium chloride crosslinker solution (100 mM) to form a 3D structure of scaffolds. The printed scaffolds were characterized in terms of structural, swelling, degradation, and mechanical properties, followed by theirin vitrocharacterization for enamel tissue regeneration. The results showed that the bioinks with higher concentrations of Alg were more viscous and needed higher pressure for printing; while the printed scaffolds were highly porous and showed a high degree of printability and structural integrity. The hydrogels with higher CMC ratios had higher swelling ratios, faster degradation rates, and lower compressive modulus. Dental epithelial cell line, HAT-7, could maintain high viability in the printed constructs after 1, 7 and 14 d of culture. HAT-7 cells were also able to maintain their morphology and secrete alkaline phosphatase after 14 d of culture in the 3D printed scaffolds, suggesting the capacity of these cells for mineral deposition and enamel-like tissue formation. Among all combinations Alg4%-CMC2% and in a less degree 2%Alg-4%CMC showed the higher potential to promote ameloblast differentiation, Ca and P deposition and matrix mineralizationin vitro. Taken together, Alg-CMC has been illustrated to be suitable to print scaffolds with dental epithelial cells for enamel tissue regeneration.

Development of a Nutrition Questionnaire for Dental Caries Risk Factors.

BACKGROUND: Few existing tools quickly identify dietary behaviours related to dental caries. The objectives of this study were to (i) create a patient-generated questionnaire identifying these dietary behaviours, (ii) capture information on these dietary behaviours in two specific populations via questionnaire pilot testing and (iii) determine questionnaire test-retest reliability. METHODS: After development, the questionnaire was reviewed by an expert panel. Cognitive interviewing was conducted, followed by pilot testing in a general university campus population (n = 80) and a university dental clinic (n = 10). Retesting was done with the general campus group (n = 53). RESULTS: Most participants reported never receiving dietary advice from professionals regarding caries. Sugary foods were most often consumed as snacks in the evening or afternoon, then breakfast. In total, 41.3% of campus participants consumed high risk items at least a few times per week or more often. Weekly or more frequent consumption of "other" sugary drinks (e.g., iced tea) was common. In total, 77.6% of questionnaire items had a kappa value representing moderate agreement or greater. CONCLUSIONS: Dietary behaviours related to caries were common in this pilot study. Given the high prevalence of caries and low occurrence of prior dietary advice for the same, increased preventive efforts may be warranted.

Systems Biology Approaches and Precision Oral Health: A Circadian Clock Perspective.

A vast majority of the pathophysiological and metabolic processes in humans are temporally controlled by a master circadian clock located centrally in the hypothalamic suprachiasmatic nucleus of the brain, as well as by specialized peripheral oscillators located in other body tissues. This circadian clock system generates a rhythmical diurnal transcriptional-translational cycle in clock genes and protein expression and activities regulating numerous downstream target genes. Clock genes as key regulators of physiological function and dysfunction of the circadian clock have been linked to various diseases and multiple morbidities. Emerging omics technologies permits largescale multi-dimensional investigations of the molecular landscape of a given disease and the comprehensive characterization of its underlying cellular components (e.g., proteins, genes, lipids, metabolites), their mechanism of actions, functional networks and regulatory systems. Ultimately, they can be used to better understand disease and interpatient heterogeneity, individual profile, identify personalized targetable key molecules and pathways, discover novel biomarkers and genetic alterations, which collectively can allow for a better patient stratification into clinically relevant subgroups to improve disease prediction and prevention, early diagnostic, clinical outcomes, therapeutic benefits, patient's quality of life and survival. The use of "omics" technologies has allowed for recent breakthroughs in several scientific domains, including in the field of circadian clock biology. Although studies have explored the role of clock genes using circadiOmics (which integrates circadian omics, such as genomics, transcriptomics, proteomics and metabolomics) in human disease, no such studies have investigated the implications of circadian disruption in oral, head and neck pathologies using multi-omics approaches and linking the omics data to patient-specific circadian profiles. There is a burgeoning body of evidence that circadian clock controls the development and homeostasis of oral and maxillofacial structures, such as salivary glands, teeth and oral epithelium. Hence, in the current era of precision medicine and dentistry and patient-centered health care, it is becoming evident that a multi-omics approach is needed to improve our understanding of the role of circadian clock-controlled key players in the regulation of head and neck pathologies. This review discusses current knowledge on the role of the circadian clock and the contribution of omics-based approaches toward a novel precision health era for diagnosing and treating head and neck pathologies, with an emphasis on oral, head and neck cancer and Sjögren's syndrome.

Multiwalled Carbon Nanotubes for Dental Applications.

Multiwalled carbon nanotubes (MWCNTs) are a particularly promising drug delivery system due to their high surface area allowing high-protein loading, their stability under biological conditions, and their unique interaction with cellular membranes. Studies have shown that covalent attachment of polyethylene glycol (PEG) improves biocompatibility and enhances surface hydrophilicity properties, suggesting that PEGylated MWCNTs are efficient and toxic-safe drug delivery systems. So far, CNTs are used for a broad range of applications in dentistry, especially for dental tissue repair and restorative. Here we present a protocol of protein immobilization onto MWCNTs and describe the procedure for delivering them into the cells after characterization of the nanotubes.

Biological properties of modified bioactive glass on dental pulp cells.

Dental caries is a bacteria-caused condition classified among the most common chronic diseases worldwide. Treatment of dental caries implies the use of materials having regenerative and anti-bacterial properties, and controlling inflammation is critical for successful endodontic regeneration. OBJECTIVES: The aim of this study was to fabricate and characterize a novel composite incorporating sol-gel derived silver-doped bioactive glass (BG) in a chitosan (CS) hydrogel at a 1:1 wt ratio(Ag-BG/CS). METHODS: The effect of Ag-BG/CS on dental pulp cells (DPCs) proliferation was analyzed by CCK-8 assay, whereas the adhesion of DPCs was evaluated by confocal microscopy. The physical morphology of Ag-BG/CS was analyzed by scanning electron microscope. The anti-inflammatory effect of Ag-BG/CS was investigated by quantitative polymerase chain reaction (qPCR). Moreover, the effect of Ag-BG/CS on odontogenic differentiation of DPCs was studied by immunochemical staining, tissue-nonspecific alkaline phosphatase staining, qPCR, and western blot analyses. The antibacterial activity against dental caries key pathogenic bacteria was also evaluated. RESULTS: The results of this study showed that Ag-BG/CS did not affect the proliferation of DPCs, it down-regulated the inflammatory-associated markers (IL-1ß, IL-6, IL-8, TNF-α) of DPCs treated with Escherichia coli lipopolysaccharide (LPS) by inhibiting NF-κB pathway, and enhanced the in vitro odontogenic differentiation potential of DPCs. Furthermore, Ag-BG/CS strongly inhibited Streptococcus mutans and Lactobacillus casei growth. CONCLUSIONS: This novel biomaterial possessed antibacterial and anti-inflammatory activity, also enhanced the odontogenic differentiation potential of LPS-induced inflammatory-reacted dental pulp cells. The material introduced in this study may thus represent a suitable dental pulp-capping material for future clinical applications.

Dental Pulp Stem Cells: Isolation, Characterization, Expansion, and Odontoblast Differentiation for Tissue Engineering.

Tissue engineering is an interdisciplinary area offering a promising approach by the use of stem cells combined with scaffolds and signaling factors for regeneration of damaged or lost tissues. Incorporation of a sufficient number of cells which do not elicit the immunoreaction in the body is a pivotal element for successful tissue formation using this method. Stem cells exhibiting strong capacity to self-renew and differentiate into different cell types are considered as a potent cell source. Among various cell sources, dental pulp stem cells (DPSCs) are widely under investigation due to the fact that they are simply obtainable from extracted third molars or orthodontically extracted teeth and show an excellent potential for clinical application and also their harvesting method is minimally invasive. DPSCs are odontogenic progenitor cells with clonogenic abilities, rapid proliferation rates, and multiple differentiation potentials. Here, we describe protocols that allow 1) the isolation of DPSCs from a single tooth; 2) the characterization of human mesenchymal stem cells markers of DPSCs by flow cytometry; 3) the culture growth of DPSCs in 2D (in cell culture flasks) and 3D (by 3D printing of cell-laden constructs); and 4) the in vivo evaluation of differentiation potential of DPSCs.

Silver-Doped Bioactive Glass/Chitosan Hydrogel with Potential Application in Dental Pulp Repair.

The major indications of a successful inflamed-pulp-capping procedure are the formation of a dense calcified dentin barrier and the preservation of healthy pulp tissue concomitant with elimination of inflammation. Our aim is to evaluate the effects of an injectable silver-doped bioactive glass/chitosan hydrogel (Ag-BG/CS), as a pulp-capping material, and explore the molecular mechanisms of Ag-BG/CS in regards to its bioactive and anti-inflammatory properties. First, the structure and component of the material were analyzed by scanning electron microscopy. Then, the downstream molecular mechanisms and anti-inflammatory effects were characterized by quantitative polymerase chain reaction (qPCR) and Western blot. Finally, a preclinical model of rat pulpitis was used to explore the potential of Ag-BG/CS in controlling pulp inflammation in vivo. The results showed that Ag-BG/CS induced stronger reparative dentin formation and enhanced preservation of vital pulp tissue when compared to the mineral trioxide aggregate (MTA) which is the currently used clinical standard. Ag-BG/CS also significantly increased the phosphorylation of p38 and ERK1/2(p42/44) of dental pulp cells, indicating that Ag-BG/CS enhanced pulpal repair through the mitogen-activated protein kinase (MAPK) pathway. This novel material may represent a superior solution for dental pulp-capping clinical scenarios with specific advantages for cases of early diffuse pulpitis in immature permanent teeth.

Saliva and Gingival Crevicular Fluid (GCF) Collection for Biomarker Screening.

Assaying different biological markers (biomarkers) is commonly used to monitor health status and aid in the diagnosis of diseases. With the recent advances in highly sensitive protein assays, whole saliva (WS) and gingival crevicular fluid (GCF) appear to be fluids that may contain important biomarkers with various applications in dentistry and medicine. Herein, we describe the process of GCF and WS sample collection and preparation for assaying clinically relevant biomarkers in clinical screening trials. Analysis of biomarkers in WS and GCF represents an easy and practical approach for the diagnosis and screening of different pathological conditions particularly in epidemiological surveys.