A scoping review of the influence of clinical contaminants on bond strength in direct adhesive restorative procedures.

OBJECTIVE: Clinical contamination during direct adhesive restorative procedures can affect various adhesive interfaces differently and contribute to bulk failure of the restorations. This review aims to summarise the current knowledge on the influence of a variety of clinical contaminants on the bond strength at various adhesive interfaces during adhesive restorative procedures and identify gaps in the literature for future research. DATA AND SOURCES: An electronic database search was performed in PubMed and EMBASE to identify articles that investigated the influence of contaminants on direct restorative bonding procedures. A data-charting form was developed by two researchers to capture the key characteristics of each eligible study. STUDY SELECTION: The initial search yielded 1,428 articles. Fifty-seven articles published between 1 Jan 2007 and 25 Oct 2023 were included in the final review. Thirty-three of the articles examined the influence of saliva contamination, twelve articles examined the influence of blood contamination, and twenty-five articles examined the influence of other contaminants. CONCLUSION: Saliva contamination exerted less influence on the decrease in bond strength when self-etch systems were used, compared to when etch-and-rinse systems were used. Blood contamination adversely affected the bond strength at the interface between resin composite and dentine, and resin composite and resin-modified glass ionomer cement. Treating contaminated surfaces with water spray for 10-30 s followed by air drying could be effective in recovering bond strength following saliva and blood contamination. CLINICAL SIGNIFICANCE: This scoping review provides a valuable overview of the range of potential clinical contaminants that can influence the bond strength between different interfaces in direct adhesive restorative procedures. Additionally, it identifies potential decontamination protocols that can be followed to restore and enhance bond strength.

Demineralized dentin matrix for bone regeneration in dentistry: A critical update.

Over the last few decades, several new materials and techniques have been developed for bone regeneration. Scaffolds based on demineralized dentin matrix (DDM) present an attractive option due to their availability and several animal and human studies have been conducted to ascertain their utility in regenerative dentistry. The aim of this review was to summarize the recent studies conducted on DDM and used for bone grafts. PubMed, Web of Science, and Scopus were used to search for studies published within the last 10 years. The keywords and terms used were: "demineralized dentine matrix", "bone grafting", "bone augmentation" and "guided tissue regeneration" in various combinations. Original studies (in vitro, animal and human) and systematic reviews were included in the literature search. The literature search initially identified 23 studies (16 animal studies and 7 clinical reports. Most studies included in this review indicate that DDM has demonstrated promising results in a variety of dental and regenerative medicine applications. Further studies are required to completely comprehend its characteristics and prospective applications. Future studies should also focus on optimizing the processing protocols for the production of DDM-based scaffolds.

E-cadherin and TAC in GCF accurately discriminate periodontal health and disease.

OBJECTIVE: To investigate the accuracy of gingival crevicular fluid (GCF) E-cadherin and total antioxidant capacity (TAC) to discriminate periodontal health from disease. SUBJECTS AND METHODS: GCF samples were collected from participants with periodontal health (control), gingivitis, and periodontitis (n = 25 each group). The latter group was further subdivided according to stage (S) and grade. Periodontal parameters were recorded then levels of biomarkers were assayed using ELISA and antioxidant status by use of the Total Antioxidant Capacity Assay for E-cadherin and TAC, respectively. RESULTS: All periodontal parameters were significantly higher in periodontally diseased groups than controls. The GCF E-cadherin significantly increased in gingivitis and periodontitis (S2 to S4) cases as compared to controls. Level of this protein in GCF samples from periodontitis S3 was significantly higher than in gingivitis and S2 groups. The GCF-TAC level was significantly higher in controls than in periodontally diseased groups. No significant differences were observed in the levels of these proteins between grade B and C periodontitis. Both molecules could discriminate periodontal health from gingivitis and periodontitis stages and differentiating periodontitis S3 from gingivitis and other periodontitis stages. CONCLUSIONS: Levels of TAC and unbounded E-cadherin in GCF samples exhibited promising diagnostic abilities to differentiate periodontal health and disease.

Ability of gingival crevicular fluid volume, E-cadherin, and total antioxidant capacity levels for predicting outcomes of nonsurgical periodontal therapy for periodontitis patients.

OBJECTIVES: To determine the potential of gingival crevicular fluid (GCF) volume, E-cadherin and total antioxidant capacity (TAC) levels to predict the outcomes of nonsurgical periodontal therapy (NSPT) for periodontitis patients. BACKGROUND: NSPT is the gold-standard treatment for periodontal pockets < 6 mm in depth, however, successful outcomes are not always guaranteed due to several factors. Periodontitis-associated tissue destruction is evidenced by the increased level of soluble E-cadherin and reduced antioxidants in oral fluids which could be used as predictors for success/failure of NSPT. MATERIALS AND METHODS: Patients with periodontitis (n = 24) were included in this clinical trial and full-mouth periodontal charting was recorded for each patient. GCF samples from periodontal pockets with probing pocket depth (PPD) 4-6 mm from the interproximal surfaces of anterior and premolar teeth were obtained. These sites subsequently received NSPT and were clinically re-evaluated after 1 and 3 months. Levels of GCF E-cadherin and TAC levels were assayed using ELISA. RESULTS: All clinical periodontal parameters were significantly improved 3 months after completion of NSPT. These outcomes were associated with a significant decrease in E-cadherin levels and GCF volume, while TAC levels were significantly increased in samples obtained in follow-up appointments. Binary regression model analysis showed that PPD, GCF volume, E-cadherin, and TAC levels could significantly (p < .05) predict the outcomes of NSPT. The cut-off points for PPD, GCF volume, E-cadherin and TAC were 5 mm, 4 × 10-3, 1267.97 pg/mL and 0.09 µmol/g, respectively. CONCLUSION: NSPT improved clinical parameters along with increased antioxidants capacity and epithelial pocket lining integrity. Discrimination of favorable/unfavorable responsiveness of periodontally diseased sites to NSPT could be possible by using GCF volume, PPD, E-cadherin and TAC level assessments.

In Vitro Models Used in the Formation of Root Caries Lesions-A Review of the Literature.

The management of root caries remains a challenge for clinicians due to its unique anatomical location and structure. There is increasing interest in utilising artificial root caries lesions to develop new strategies for remineralisation. An ideal protocol has not yet been agreed upon. The aim of this review is to provide a structured overview of previously reported in vitro root caries models. The literature was screened and mined for information mainly on substrate selection, model systems utilised, and variables used in the models. Human roots (60%) were the most frequently used substrates, followed by bovine roots (40%). Chemical models (69%) were the most frequently utilised model systems, followed by microbiological models (27%), to form root caries lesions. Acetate buffer solution (80%), pH 5.0 or above (40%), and a demineralisation time of five days (25%) were the common variables used in the chemical systems, while mono-species biofilm was most frequently used (73%) in microbiological models and Streptococcus mutans was the most common bacterial strain utilised in these models (80%). This review highlights the variability amongst the experimental approaches, discusses the advantages and limitations of these approaches, and emphasises that standardisation of experimental conditions along with sustained research will benefit root caries research.

Effects of glycation end-products on the dental pulp in patients with type 2 diabetes.

AIM: This ex vivo study aimed to compare protein expression of advanced glycation end-products (AGE) and receptor (RAGE), and the levels of selected genes associated with inflammation and collagen within dental pulp tissue from patients with type 2 (T2D) diabetes and non-T2D. METHODOLOGY: Noncarious extracted permanent molar teeth from patients with well-controlled T2D (n = 19) and non-T2D (controls) (n = 19) were collected and compared. The coronal pulp was examined using immunohistochemistry (IHC) (n = 10 per group) for anti-AGE and anti-RAGE. Quantitative PCR (n = 9 per group) was used to analyse the gene expression levels of NFKB, S100A12 and COLIA1. Data analyses were performed between the groups using GraphPad Prism using Pearson correlation, Shapiro-Wilk and Mann-Whitney U-tests, and multiple regression using SPSS. RESULTS: AGEs were distributed diffusely throughout the pulp extracellular matrix associated with collagen fibres and were present on several cell types. RAGE was expressed at the pulp-dentine interface and was observed on odontoblasts, immune cells, endothelial cells and fibroblasts. Semi-quantitative analysis of IHC samples showed significantly increased expression of AGE (p < .0001) and RAGE (p = .02) in T2D samples compared with controls. The expression of NFKB (p < .0001), S100A12 (p < .0001) and COLIA1 (p = .01) genes were significantly higher in the T2D pulp, and multivariate logistic regression analysis showed that these findings were not affected by age. CONCLUSION: T2D may exert a similar glycation response in the dental pulp to other body sites. This could occur through activation of NF-κB pathways with a concomitant increase in genes associated with inflammation and collagen.

Development, physicochemical characterization and in-vitro biocompatibility study of dromedary camel dentine derived hydroxyapatite for bone repair.

This study aimed to produce hydroxyapatite from the dentine portion of camel teeth using a defatting and deproteinizing procedure and characterize its physicochemical and biocompatibility properties. Biowaste such as waste camel teeth is a valuable source of hydroxyapatite, the main inorganic constituent of human bone and teeth which is frequently used as bone grafts in the biomedical field. Fourier Transform infrared (FTIR), and micro-Raman spectroscopy confirmed the functional groups as-sociated with hydroxyapatite. X-ray diffraction (XRD) studies showed camel dentine-derived hydroxyapatite (CDHA) corresponded with hydroxyapatite spectra. Scanning electron micros-copy (SEM) demonstrated the presence of dentinal tubules measuring from 1.69-2.91 µm. The inorganic phases of CDHA were primarily constituted of calcium and phosphorus, with trace levels of sodium, magnesium, potassium, and strontium, according to energy dispersive X-ray analysis (EDX) and inductively coupled plasma mass spectrometry (ICP-MS). After 28 days of incubation in simulated body fluid (SBF), the pH of the CDHA scaffold elevated to 9.2. in-vitro biocompatibility studies showed that the CDHA enabled Saos-2 cells to proliferate and express the bone marker osteonectin after 14 days of culture. For applications such as bone augmentation and filling bone gaps, CDHA offers a promising material. However, to evaluate the clinical feasibility of the CDHA, further in-vivo studies are required.

Regenerative endodontic procedures: are they more successful in mature permanent teeth with necrotic pulp than in immature teeth?

DATA SOURCES: Five scientific databases were electronically searched: PubMed, EMBASE, OpenGrey, Web of Science and Cochrane Library.  The search was conducted until 17 February 2022 without any restriction on date of publication but was restricted to English language. Relevant studies were also screened for related publications. The aim of this systematic review and meta-analysis was to evaluate the effectiveness of regenerative endodontic procedures (REPs) in mature and immature permanent teeth with necrotic pulp and to evaluate if the success rate was affected by the stage of root development. STUDY SELECTION: Types of studies: all of the included studies were randomised controlled trials (RCTs). TYPES OF PARTICIPANTS: people with necrotic permanent teeth (immature or mature) treated with regenerative endodontic procedures. Types of interventions: regenerative endodontic procedures. Language: RCTs published in English. EXCLUSION CRITERIA: Types of studies: (1) case reports, (2) retrospective cohort trials, (3) prospective cohort trials, (4) animal trials, (5) in-vitro trials, (6) non-randomised trials. POPULATION: primary teeth. Types of interventions: no details about the clinical procedures. Follow-up period: <6 months. DATA EXTRACTION AND SYNTHESIS: The titles and abstracts of the RCTs identified by the search strategies were independently screened by two reviewers. After the initial screening, the full text of the relevant trials were reassessed against the inclusion and exclusion criteria. Discrepancies and disagreements were resolved by consensus after including a third reviewer. RESULTS: Following the initial electronic and manual searches, a total of 3766 articles were initially identified. This was reduced to 2739 articles after duplicates were removed. However, after the initial screening phase, 35 articles were considered potentially relevant and qualified for full-text scrutiny. Out of the 35 articles, only 27 were considered eligible for inclusion. The differences in the success rate and the asymptomatic rate between the mature and immature permanent teeth with necrotic pulp were not statistically significant. However, the differences between the two groups were statistically significant in the rate of positive response to electrical pulp testing. CONCLUSIONS: Based on the results of this systematic review and meta-analysis, the authors concluded that REPs are an effective therapy and can achieve high success rates for both mature and immature necrotic permanent teeth. It was also concluded that the REPs were more successful in regaining vitality responses for mature compared with immature permanent teeth with necrotic pulps.

Periradicular tissue fluid-derived biomarkers for apical periodontitis: An in vitro methodological and in vivo cross-sectional study.

BACKGROUND: Periradicular tissue fluid (PTF) offers a source of diagnostic, prognostic and predictive biomarkers for endodontic disease. AIMS: (1) To optimize basic parameters for PTF paper point sampling in vitro for subsequent in vivo application. (2) To compare proteomes of PTF from teeth with normal apical tissues (NAT) and asymptomatic apical periodontitis (AAP) using high-throughput panels. METHODOLOGY: (1) To assess volume absorbance, paper points (n = 20) of multiple brands, sizes and sampling durations were inserted into PBS/1%BSA at several depths. Wetted lengths (mm) were measured against standard curves to determine volume absorbance (µL). To assess analyte recovery, paper points (n = 6) loaded with 2 µL recombinant IL-1ß (15.6 ng/mL) were eluted into 250 µL: (i) PBS; (ii) PBS/1% BSA; (iii) PBS/0.1% Tween20; (iv) PBS/0.25 M NaCl. These then underwent: (i) vortexing; (ii) vortexing/centrifugation; (iii) centrifugation; (iv) incubation/vortexing/centrifugation. Sandwich-ELISAs determined analyte recovery (%) against positive controls. (2) Using optimized protocols, PTF was retrieved from permanent teeth with NAT or AAP after accessing root canals. Samples, normalized to total fluid volume (TFV), were analysed to determine proteomic profiles (pg/TFV) of NAT and AAP via O-link Target-48 panel. Correlations between AAP and diagnostic accuracy were explored using principal-component analysis (PCA) and area under receive-operating-characteristic curves (AUC [95% CI]), respectively. Statistical comparisons were made using Mann-Whitney U, anova and post hoc Bonferonni tests (α < .01). RESULTS: (1) UnoDent's 'Classic' points facilitated maximum volume absorbance (p < .05), with no significant differences after 60 s (1.6 µL [1.30-1.73]), 1 mm depth and up to 40/0.02 (2.2 µL [1.98-2.20]). For elution, vortexing (89.3%) and PBS/1% BSA (86.9%) yielded the largest IL-1ß recovery (p < .05). (2) 41 (NAT: 13; AAP: 31) PTF samples proceeded to analysis. The panel detected 18 analytes (CCL-2, -3, -4; CSF-1; CXCL-8, -9; HGF; IL-1ß, -6, -17A, -18; MMP-1, -12; OLR-1; OSM; TNFSF-10, -12; VEGF-A) in ≥75% of AAP samples at statistically higher concentrations (p < .01). CXCL-8, IL-1ß, OLR-1, OSM and TNFSF-12 were strongly correlated to AAP. 'Excellent' diagnostic performance was observed for TNFSF-12 (AUC: 0.94 [95% CI: 0.86-1.00]) and the PCA-derived cluster (AUC: 0.96 [95% CI: 0.89-1.00]). CONCLUSIONS: Optimized PTF sampling parameters were identified in this study. When applied clinically, high-throughput proteomic analyses revealed complex interconnected networks of potential biomarkers. TNFSF-12 discriminated periradicular disease from health the greatest; however, clustering analytes further improved diagnostic accuracy. Additional independent investigations are required to validate these findings.

Characterisation of miRNA Expression in Dental Pulp Cells during Epigenetically-Driven Reparative Processes.

Within regenerative endodontics, exciting opportunities exist for the development of next-generation targeted biomaterials that harness epigenetic machinery, including microRNAs (miRNAs), histone acetylation, and DNA methylation, which are used to control pulpitis and to stimulate repair. Although histone deacetylase inhibitors (HDACi) and DNA methyltransferase inhibitors (DNMTi) induce mineralisation in dental pulp cell (DPC) populations, their interaction with miRNAs during DPC mineralisation is not known. Here, small RNA sequencing and bioinformatic analysis were used to establish a miRNA expression profile for mineralising DPCs in culture. Additionally, the effects of a HDACi, suberoylanilide hydroxamic acid (SAHA), and a DNMTi, 5-aza-2'-deoxycytidine (5-AZA-CdR), on miRNA expression, as well as DPC mineralisation and proliferation, were analysed. Both inhibitors increased mineralisation. However, they reduced cell growth. Epigenetically-enhanced mineralisation was accompanied by widespread changes in miRNA expression. Bioinformatic analysis identified many differentially expressed mature miRNAs that were suggested to have roles in mineralisation and stem cell differentiation, including regulation of the Wnt and MAPK pathways. Selected candidate miRNAs were demonstrated by qRT-PCR to be differentially regulated at various time points in mineralising DPC cultures treated with SAHA or 5-AZA-CdR. These data validated the RNA sequencing analysis and highlighted an increased and dynamic interaction between miRNA and epigenetic modifiers during the DPC reparative processes.

The relationship between sphingosine-1-phosphate receptor 2 and epidermal growth factor in migration and invasion of oral squamous cell carcinoma.

Sphingosine-1-phosphate (S1P) is a lipid mediator and its binding to the S1P receptor 2 (S1PR2) is reported to regulate cytoskeletal organization. Epidermal growth factor (EGF) has been shown to induce migration and invasion in tumour cells. Since binding of S1P to S1PR2 and EGF to the EGF receptors exhibit some overlapping functionality, this study aimed to determine whether S1PR2 was involved in EGF-induced migration and invasion of oral squamous cell carcinoma (OSCC) lines and to identify any potential crosstalk between the two pathways. Migration was investigated using the scratch wound assay while invasion was studied using the transwell invasion and multicellular tumour spheroid (MCTS) assays. Activity of Rac1, a RhoGTPase, was measured using G-LISA (small GTPase activation assays) while S1P production was indirectly measured via the expression of sphingosine kinase (Sphk). S1PR2 inhibition with 10 µM JTE013 reduced EGF-induced migration, invasion and Rac1 activity, however, stimulation of S1PR2 with 10 µM CYM5478 did not enhance the effect of EGF on migration, invasion or Rac1 activity. The data demonstrated a crosstalk between EGF/EGFR and S1P/S1PR2 pathways at the metabolic level. S1PR2 was not involved in EGF production, but EGF promoted S1P production through the upregulation of Sphk1. In conclusion, OSCC lines could not migrate and invade without S1PR2 regulation, even with EGF stimulation. EGF also activated S1PR2 by stimulating S1P production via Sphk1. The potential for S1PR2 to control cellular motility may lead to promising treatments for OSCC patients and potentially prevent or reduce metastasis.

Potential Beneficial Effects of Hydroxyapatite Nanoparticles on Caries Lesions In Vitro-A Review of the Literature.

Dental caries is one of the most common human diseases which can occur in both primary and permanent dentitions throughout the life of an individual. Hydroxyapatite is the major inorganic component of human teeth, consequently, nanosized hydroxyapatite (nHAP) has recently attracted researchers' attention due to its unique properties and potential for caries management. This article provides a contemporary review of the potential beneficial effects of nHAP on caries lesions demonstrated in in vitro studies. Data showed that nHAP has potential to promote mineralization in initial caries, by being incorporated into the porous tooth structure, which resulted from the caries process, and subsequently increased mineral content and hardness. Notably, it is the particle size of nHAP which plays an important role in the mineralization process. Antimicrobial effects of nHAP can also be achieved by metal substitution in nHAP. Dual action property (mineralizing and antimicrobial) and enhanced chemical stability and bioactivity of nHAP can potentially be obtained using metal-substituted fluorhydroxyapatite nanoparticles. This provides a promising synergistic strategy which should be explored in further clinical research to enable the development of dental therapeutics for use in the treatment and management of caries.

Characterization, Quantification, and Visualization of Neutrophil Extracellular Traps.

Following the discovery of neutrophil extracellular traps (NETs) in 2004 by Brinkmann and colleagues, there has been extensive research into the role of NETs in a number of inflammatory diseases, including periodontitis. This chapter describes the current methods for the isolation of peripheral blood neutrophils as well as of oral neutrophils for subsequent NET experiments, including approaches to quantify and visualize NET production, the ability of NETs to entrap and kill bacteria, and the removal of NETs by nuclease-containing plasma.

Enhancing the student learning experience: Co-teaching biochemistry and clinical sciences within the dental curriculum.

Basic knowledge of biochemistry underpins oral and dental care. Undergraduate dental students do not always engage well with basic science teaching due to not appreciating its clinical relevance. Co-teaching provides one approach to overcome students' disengagement and involves two lecturers, with complementary expertise, presenting the curriculum together. This study investigated student experiences and engagement using co-teaching to integrate biochemistry with clinical sciences in the students' second-year dental curriculum. Two successive second year dental student cohorts were co-taught. Content was delivered by a biochemist and an oral biologist, either online (during the 2020 COVID lockdown) or in-person (2021). Each cohort was surveyed at the end of the teaching module using an online questionnaire containing both interval scale and free-text questions. Responses were received from 39 (42%) and 64 (85%) of students in 2020 and 2021, respectively. Students from both cohorts preferred the co-teaching approach with a mean of 8.74 on a 10-point interval scale. In 2020 and 2021, 77% and 76% of participants, respectively, preferred a combined biochemistry and clinical dentistry delivery, either in-person (37%), via Zoom (19%) or via video recording (14%). Thematic analysis of responses revealed students experienced enhanced engagement when co-taught and they attributed this to integration of the curriculum making the content more relevant and stimulating. Students preferred co-teaching to individual subjects being taught by a single teacher. Co-teaching established the relevance of theoretical biochemistry to clinical dental sciences and enhanced the students' learning experience.

Gingival tissue samples from periodontitis patients demonstrate epithelial-mesenchymal transition phenotype.

OBJECTIVE: To determine the expression of key epithelial-mesenchymal transition (EMT) markers in gingival tissue samples collected from patients with periodontitis. BACKGROUND: Epithelial-mesenchymal transition is a process responsible for shifting epithelial-phenotype to mesenchymal-phenotype leading to loss of epithelial-barrier function. Thus, EMT could be involved as a pathogenic mechanism in periodontitis as both conditions share common promoters and signalling pathways. MATERIALS AND METHODS: Gingival tissue samples were collected from patients with periodontitis (case) and healthy periodontium (control). Periodontal parameters including bleeding on probing, probing pocket depth (PPD), and clinical attachment loss were recorded. Paraffinized tissue samples were processed and immunohistochemically stained to determine the expression of key EMT markers which included E-cadherin, ß-catenin, Snail1 and vimentin. RESULTS: The majority of cases (n = 65, 72.2%) were diagnosed with periodontitis stage 3 or 4, grade b or c vs 25 (27.8%) subjects with intact healthy periodontium. Discontinuity of epithelium was detected in up to 80.9% of periodontitis cases associated with reduced number of epithelial layers as compared to controls. Immunohistochemical expression of epithelial markers (E-cadherin and ß-catenin) was significantly downregulated in periodontitis patients as compared with controls. Periodontitis cases exhibited significant upregulation of Snail1 expression. Furthermore, cytoplasmic vimentin (66.2%) and nuclear ß-catenin (27.7%) were solely expressed in periodontally diseased tissues compared with control. Epithelial markers, E-cadherin and ß-catenin, were significantly negatively correlated with increasing PPD, while vimentin showed positive correlation with this parameter. CONCLUSION: There were marked downregulation of epithelial molecules and upregulation of mesenchymal markers in gingival tissues derived from periodontitis patients, suggesting expression of the EMT phenotype in the pathological epithelial lining of periodontal pockets.

Extraction of Hydroxyapatite from Camel Bone for Bone Tissue Engineering Application.

Waste tissues such as mammalian bone are a valuable source from which to extract hydroxyapatite. Camel bone-based hydroxyapatite (CBHA) was extracted from the femur of camel bones using a defatting and deproteinization procedure. The extracted CBHA was mechanically, chemically, physically, morphologically and structurally characterized. Fourier-Transform Infra-Red (FTIR) spectra, Micro-Raman, and X-ray diffraction analysis confirmed successful extraction of hydroxyapatite. The mechanical properties of the CBHA scaffold were measured using a Universal Instron compression tester. Scanning electron microscopy showed the presence of a characteristic interconnected porous architecture with pore diameter ranging from 50-600 µm and micro-computer tomography (Micro-CT) analysis identified a mean porosity of 73.93. Thermogravimetric analysis showed that the CBHA was stable up to 1000 °C and lost only 1.435% of its weight. Inductively coupled plasma-mass spectrometry (ICP-MS) and Energy-dispersive-X-ray (EDX) analysis demonstrated the presence of significant amounts of calcium and phosphorus and trace ions of sodium, magnesium, zinc, lead and strontium. Following 21 days of incubation in simulated body fluid (SBF), the pH fluctuated between 10-10.45 and a gradual increase in weight loss was observed. In conclusion, the extracted CBHA is a promising material for future use in bone tissue regeneration applications.

Exploring the neurogenic differentiation of human dental pulp stem cells.

Human dental pulp stem cells (hDPSCs) have increasingly gained interest as a potential therapy for nerve regeneration in medicine and dentistry, however their neurogenic potential remains a matter of debate. This study aimed to characterize hDPSC neuronal differentiation in comparison with the human SH-SY5Y neuronal stem cell differentiation model. Both hDPSCs and SH-SY5Y could be differentiated to generate typical neuronal-like cells following sequential treatment with all-trans retinoic acid (ATRA) and brain-derived neurotrophic factor (BDNF), as evidenced by significant expression of neuronal proteins ßIII-tubulin (TUBB3) and neurofilament medium (NF-M). Both cell types also expressed multiple neural gene markers including growth-associated protein 43 (GAP43), enolase 2/neuron-specific enolase (ENO2/NSE), synapsin I (SYN1), nestin (NES), and peripherin (PRPH), and exhibited measurable voltage-activated Na+ and K+ currents. In hDPSCs, upregulation of acetylcholinesterase (ACHE), choline O-acetyltransferase (CHAT), sodium channel alpha subunit 9 (SCN9A), POU class 4 homeobox 1 (POU4F1/BRN3A) along with a downregulation of motor neuron and pancreas homeobox 1 (MNX1) indicated that differentiation was more guided toward a cholinergic sensory neuronal lineage. Furthermore, the Extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor U0126 significantly impaired hDPSC neuronal differentiation and was associated with reduction of the ERK1/2 phosphorylation. In conclusion, this study demonstrates that extracellular signal-regulated kinase/Mitogen-activated protein kinase (ERK/MAPK) is necessary for sensory cholinergic neuronal differentiation of hDPSCs. hDPSC-derived cholinergic sensory neuronal-like cells represent a novel model and potential source for neuronal regeneration therapies.

Stage-Dependent Regulation of Dental Pulp Stem Cell Odontogenic Differentiation by Transforming Growth Factor-ß1.

Transforming growth factor-ß1 (TGF-ß1) is an important multifunctional cytokine with dual effects on stem cell differentiation. However, the role of TGF-ß1 on odontogenic differentiation of dental pulp stem cells (DPSCs) remains to be entirely elucidated. In the present study, we initially investigated the effect of TGF-ß1 at a range of concentrations (0.1-5 ng/mL) on the proliferation, cell cycle, and apoptosis of DPSCs. Subsequently, to determine the effect of TGF-ß1 on odontogenic differentiation, alkaline phosphatase (ALP) activity and Alizarin Red S (ARS) staining assays at different concentrations and time points were performed. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis were used to determine the levels of odonto-/osteo-genic differentiation-related gene and protein expression, respectively. For in vivo studies, newly formed tissue was assessed by Masson's trichrome and von Kossa staining. Data indicated that TGF-ß1 inhibited DPSCs proliferation in a concentration-and time-dependent manner (p < 0.05) and induced cell cycle arrest but did not affect apoptosis. ALP activity was enhanced, while ARS reduced gradually with increasing TGF-ß1 concentrations, accompanied by increased expression of early marker genes of odonto-/osteo-genic differentiation and decreased expression of late-stage mineralization marker genes (p < 0.05). ALP expression was elevated in the TGF-ß1-treatment group until 14 days, and the intensity of ARS staining was attenuated at days 14 and 21 (p < 0.05). Compared with the control group, abundant collagen but no mineralized tissues were observed in the TGF-ß1-treatment group in vivo. Overall, these findings indicate that TGF-ß1 promotes odontogenic differentiation of DPSCs at early-stage while inhibiting later-stage mineralization processes.

A 3D Printed Device for In Vitro Generation of Stratified Epithelia at the Air-Liquid Interface.

Air-liquid interface (ALI) cultures are used to produce stratified epithelial tissues in vitro, notably for the production of oral mucosal equivalents. Currently, there are few purpose-built devices, which aim to enhance the ease and reproducibility of generating such tissue. Most ALI cultures utilize stainless steel grids or cell culture inserts to elevate the matrix or scaffold to the surface of the culture media. In this study, a novel buoyant epithelial culture device (BECD) was designed to both contain a fibroblast-seeded collagen hydrogel and float in culture media, thereby automatically maintaining the ALI without further user intervention. BECDs aim to mitigate several issues associated with ALI culture; reducing the chance of media flooding the epithelial layer from physical disturbance, reducing technique sensitivity for less-experienced users, and improving the reproducibility of the epithelia generated. H400 oral squamous cell carcinoma cells cultured in BECDs for 7, 14, and 21 days showed continuous increase in epithelial tissue thickness with expected localization of epithelial differentiation markers: cytokeratin 5, involucrin, and E-cadherin. Fused filament fabrication three-dimensional printing with polypropylene used in BECD production allows for rapid turnover and design iteration, presenting a versatile, adaptable, and useful tool for application in in vitro cell culture.