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.

Potentiality and Inflammatory Marker Expression Are Maintained in Dental Pulp Cell Cultures from Carious Teeth.

OBJECTIVES: This investigation aimed to isolate and culture human dental pulp cells from carious teeth (cHDPCs) and compare their growth characteristics, colony-forming efficiency, mineralization potential and gene expression of Toll-like receptors (TLR)-2, TLR-4, TLR-9, tumour necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, IL-8, IL-17A, 1L-17R, IL-23A, nuclear factor-kappa B (NF-κB), mitogen-activated protein kinase (MAPK1), dentin matrix protein (DMP)-1, dentin sialophospho protein (DSPP), sex determining region Y-box 2 (SOX2) and marker of proliferation Ki-67 (MKi67) with cells isolated from healthy or non-carious teeth (ncHDPCs). METHODS: Pulp tissues were obtained from both healthy and carious teeth (n = 5, each) to generate primary cell lines using the explant culture technique. Cell cultures studies were undertaken by generating growth curves, a colony forming unit and a mineralization assay analysis. The expression of vimentin was assessed using immunocytochemistry (ICC), and the gene expression of above-mentioned genes was determined using quantitative real-time reverse-transcription polymerase chain reaction. RESULTS: ncHDPCs and cHDPCs were successfully isolated and cultured from healthy and inflamed human dental pulp tissue. At passage 4, both HDPC types demonstrated a typical spindle morphology with positive vimentin expression. No statistical difference was observed between ncHDPCs and cHDPCs in their growth characteristics or ability to differentiate into a mineralizing phenotype. ncHDPCs showed a statistically significant higher colony forming efficiency than cHDPCs. The gene expression levels of TLR-2, TLR-4, TLR-9, TNF-α, IL-6, IL-8, IL-17R, IL-23A, NF-κB, MAPK1, DMP1, DSPP and SOX2 were significantly higher in cHDPCs compared with ncHDPC cultures. CONCLUSION: cHDPCs retain their differentiation potential and inflammatory phenotype in vitro. The inflamed tooth pulp contains viable stem/progenitor cell populations which have the potential for expansion, proliferation and differentiation into a mineralizing lineage, similar to cells obtained from healthy pulp tissue. These findings have positive implications for regenerative endodontic procedures.

Type 2 diabetes and the clinically normal pulp: An in vitro study.

AIM: The aim of this study was to investigate the effect of type 2 diabetes (T2D) on clinically normal dental pulp tissue by using special stains and immunohistochemistry (IHC) to determine the morphology of the coronal pulp and distribution of immune markers in non-T2D and T2D groups. METHODOLOGY: Ethics approval for this in vitro pilot study was obtained from the University of Otago Human Ethics Committee (16/069). Twenty extracted permanent molar teeth diagnosed as having clinically normal pulp status were collected. Ten teeth were from participants with well-controlled T2D and ten from participants without diabetes (non-T2D). Each tooth was sectioned transversely at the cemento-enamel junction before the crowns were decalcified and embedded in paraffin. Sections were stained with haematoxylin and eosin, Massons trichrome, and van Gieson stains for histological and morphological evaluation. IHC using anti-CD4, anti-CD68 and anti-CD83 and anti-IL1ß, anti-IL6, anti-IL17, anti-TNF-α, anti-TLR2, anti-TLR4 and anti-FOXP3 identified proteins of interest. Qualitative and semi-quantitative analyses evaluated the morphology of the dental pulp and protein expression. Data analyses were performed with GraphPad Prism, using Student's t-test and multiple regression using SPSS at p < .05. RESULTS: Special stains demonstrated morphological differences in the T2D dental pulp compared with non-T2D. Qualitative analysis indicated that the pulp in the T2D samples was consistently less cellular, less vascular, showed evidence of thickened blood vessel walls, increased pulp calcification and collagen deposition. Semi-quantitative analysis of IHC samples showed the T2D pulp had significantly increased expression of macrophage and dendritic cell markers CD68 (p < .001) and CD83 (p = .04), and there was significantly greater expression of inflammatory cytokines IL1ß (p = .01), IL6 (p < .0001), IL17 (p < .0001) and TNF-α (p = .01). T2D samples showed a significant increase in markers of innate inflammation, TLR2 (p < .001) and TLR4 (p < .001) and decreased expression of regulatory T-cell marker, FOXP3 (p = .01). Multiple regression showed that age-corrected differences were statistically significant. CONCLUSION: Preliminary findings suggest that T2D may exert a similar response in the pulp to complications in other body sites. Hyperglycaemia is associated with changes in the morphology of the clinically normal dental pulp with altered immune cell and cytokine expression.

A critical review of in vitro research methodologies used to study mineralization in human dental pulp cell cultures.

BACKGROUND: The pulp contains a resident population of stem cells which can be stimulated to differentiate in order to repair the tooth by generating a mineralized extracellular matrix. Over recent decades there has been considerable interest in utilizing in vitro cell culture models to study dentinogenesis, with the aim of developing regenerative endodontic procedures, particularly where some vital pulp tissue remains. OBJECTIVES: The purpose of this review is to provide a structured oversight of in vitro research methodologies which have been used to study human pulp mineralization processes. METHOD: The literature was screened in the PubMed database up to March 2021 to identify manuscripts reporting the use of human dental pulp cells to study mineralization. The dataset identified 343 publications initially which were further screened and consequently 166 studies were identified and it was methodologically mined for information on: i) study purpose, ii) source and characterization of cells, iii) mineralizing supplements and concentrations, and iv) assays and markers used to characterize mineralization and differentiation, and the data was used to write this narrative review. RESULTS: Most published studies aimed at characterizing new biological stimulants for mineralization as well as determining the effect of scaffolds and dental (bio)materials. In general, pulp cells were isolated by enzymatic digestion, although the pulp explant technique was also common. For enzymatic digestion, a range of enzymes and concentrations were utilized, although collagenase type I and dispase were the most frequent. Isolated cells were not routinely characterized using either fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) approaches and there was little consistency in terming cultures as dental pulp cells or dental pulp stem cells. A combination of media supplements, at a range of concentrations, of dexamethasone, ascorbic acid and beta-glycerophosphate, were frequently applied as the basis for the experimental conditions. Alizarin Red S (ARS) staining was the method of choice for assessment of mineralization at 21-days. Alkaline phosphatase assay was relatively frequently applied, solely or in combination with ARS staining. Further assessment of differentiation status was performed using transcript or protein markers, with dentine sialophosphoprotein (DSPP), osteocalcin and dentine matrix protein-1 (DMP -1), the most frequent. DISCUSSION: While this review highlights variability among experimental approaches, it does however identify a consensus experimental approach. CONCLUSION: Standardization of experimental conditions and sustained research will significantly benefit endodontic patient outcomes in the future.

Immunohistochemical and immunofluorescence expression profile of lymphatic endothelial cell markers in oral cancer.

Lymphangiogenesis makes an important contribution to the tumour microenvironment (TME), but little is known about this in oral squamous cell carcinoma (OSCC). Archival formalin-fixed paraffin-embedded specimens (28 OSCC, 10 inflamed and 6 normal oral mucosa controls) were processed using immunohistochemistry (IHC) with antibodies against lymphatic markers D2-40 (podoplanin), LYVE-1, VEGFR3 and Prox1. After the endothelial cells had been highlighted by the various markers for lymphatic endothelium, the positive stained cells and vessels were identified and counted in a systematic manner to determine microvessel density. Double-labelling immunofluorescence (DLIF) was used to investigate the specificity of D2-40 and LYVE-1 to lymphatic endothelial cells (LECs) as opposed to blood ECs. There was higher D2-40 and Prox1 lymphatic vessel density (P = .001) in the OSCC group when compared with both control groups. Some malignant keratinocytes expressed lymphatic markers, as did a much smaller number of epithelial cells in the control groups. DLIF showed that no vessels co-expressed D2-40/CD34 or LYVE/CD34. Some D2/40+ LVs were LYVE- . D2-40 was the most specific LEC marker in OSCC tissues. These results establish that the OSCC TME contains significantly more lymphatic vessels expressing D2-40 and Prox1 than the control groups, which may play a role in facilitating lymphatic invasion and metastases.

Curriculum development in final year dentistry to enhance competency and professionalism for contemporary general dental practice.

INTRODUCTION: General dentistry is the most common area of practice, and new dentists must have the competency and skills to safely deliver patient care. In New Zealand (NZ), completion of a 5-year Bachelor of Dental Surgery (BDS) degree enables graduates to register with the Dental Council in NZ. This necessitates that the clinical component of the curriculum in final year dentistry (BDS5) transparently delivers learning opportunities and evaluates competency for independent practice. A review of the BDS5 Clinical Practice course was undertaken in 2015 and a revised curriculum introduced in 2016. CURRICULUM: We present a BDS5 curriculum for a Clinical Practice course that is learner focused with emphasis on comprehensive patient-centred care, competency and professional practice. Learning opportunities and assessment processes are described alongside teacher training. These changes have provided students scaffolding to support clinical and professional development, and accommodate different learning preferences. The outcomes align with the competency requirements of the NZ regulatory body for registration as a general dental practitioner. Since its introduction 3 years ago, ongoing feedback from students and staff has been positive and indicates the curriculum is effective in achieving its objectives. CONCLUSIONS: This curriculum provides a firm foundation for students transitioning to independent clinical practice in the community and supports the professional development of clinical teachers. It may also be translated to other areas of health education to ensure the delivery of quality holistic patient care.

Vascularity and Angiogenic Signaling in the Dentine-Pulp Complex of Immature and Mature Permanent Teeth.

ABSTRACT. OBJECTIVE: This study aimed to examine the protein and gene expression of vascular endothelial growth factor (VEGF) and angiopoietins-1 and 2 in tissue from healthy and inflamed dental pulps. METHODS: Permanent teeth with pulps diagnosed as healthy or reversible pulpitis were used for immunohistochemistry (IHC) and gene expression experiments. For IHC, a whole pulp tissue was excavated from the pulp chamber, and it was formalin-fixed and processed for routine IHC with angiogenic markers anti-VEGF, anti-Ang1, and anti-Ang2. Staining was visualized with diaminobenzidine (DAB), and examined using light microscopy. The distribution of markers in healthy and inflamed pulps was qualitatively and quantitatively analyzed. Real-time quantitative polymerase chain reaction (RT qPCR) was used to ascertain the gene expression levels of ANGPT1, ANGPT2, and TEK in the presence of inflammation. Statistical analysis was performed using the Mann-Whitney test with the statistical significance level set at 0.05. RESULTS: There was increased protein and mRNA expression of VEGF and Ang-1 markers in inflamed pulp samples as compared with that in the healthy pulp tissue. IHC demonstrated intense expression of the VEGF protein on endothelial cells (EC) and some non-ECs, and there was significantly more staining on ECs associated with inflamed tissue (P<0.001). Ang-1 and Ang-2 were significantly expressed on ECs and non-ECs (P<0.05). RT qPCR did not show significant differences in gene expression between healthy and inflamed samples although similar trends were observed to IHC. CONCLUSION: The presence of Ang-1, Ang-2, VEGF, and TEK gene in healthy and mildly inflamed pulp tissue associated with reversible pulpitis indicates that these angiogenic factors may participate in physiological and pathological angiogenesis and healing. The inflammatory process may regulate Ang-1/Ang2/Tie2 signaling; and together with VEGF, these growth factors have an important role in modulating pulp angiogenesis.

Crack Formation Following Root-End Preparation in Roots with the Butterfly Effect.

OBJECTIVE: To investigate apical cracks in roots that exhibit the butterfly effect and that have undergone apical resection and ultrasonic root-end cavity preparation. The effect of the obturation material was also studied. METHODS: Forty extracted single-rooted teeth were decoronated at the cemento-enamel junction. Roots were viewed under a light microscope and coded according to the presence or absence of the butterfly effect. Canals were prepared using ProTaper Next instruments to size X3 and assigned to two obturation groups (gutta-percha and AH Plus, and ProRoot MTA alone). Each contained twenty roots (10 with the butterfly effect and 10 without the butterfly effect). Roots were resected perpendicular to their long axis, 3 mm from the apex, and cavities were cut using ultrasonic retrotips. Resin replicas were used for crack imaging from scanning electron micrographs. Statistical analyses were performed using Stata 13.1 (StataCorp, College Station, TX, USA). RESULTS: Cracks occurred more frequently in teeth with the butterfly effect (80%), with this difference being significant (P=0.001). Most cracks (73%) ran buccolingually. Teeth obturated with MTA developed fewer cracks compared to those obturated with GP and sealer. CONCLUSION: Root-ends with the butterfly effect had a significantly higher number of buccolingual cracks following resection and ultrasonic root-end preparation. This might explain the development of some vertical root fractures, which usually run buccolingually. Canal obturation with MTA may be protective.

Vascularity and VEGF/VEGFR2 Signaling in the Dentine-Pulp Complex of Immature and Mature Permanent Teeth.

OBJECTIVE: To examine the microvessel density (MVD) and spatial distribution of endothelial cells and angiogenic activity in immature and mature permanent teeth using immunohistochemistry. METHODS: Healthy third molars with immature and mature root development were formalin-fixed, decalcified in 10% ethylenediaminetetraacetic acid, and processed for routine immunohistochemistry with endothelial cell markers anti-CD34 and anti-CD146 and angiogenic markers anti-vascular endothelial growth factor (VEGF) and anti-VEGF receptor-2 (VEGFR2). Staining was visualized with diaminobenzidine and examined using light microscopy. The distribution of markers was analyzed qualitatively and quantitatively in the coronal, middle, and apical regions of the dentine-pulp complex. RESULTS: There were spatial differences in protein expression for immature and mature teeth. The pulps of immature teeth were more vascular, had a greater number of CD34+ and CD146+ cells, and a significantly higher MVD in the coronal region than those of mature teeth (P=0.03). The apical papilla contained few blood vessels. VEGF/VEGFR2 activity was significantly greater for immature teeth (P=0.001). VEGF was expressed throughout the pulp-dentine complex, but there was significantly more growth factor coronally (immature P=0.04 and mature P=0.02). VEGFR2 was expressed less than VEGF but was seen on the endothelial cells and single cells unrelated to a vessel lumen. CONCLUSION: The spatial distribution of vascular and angiogenic (VEGF/VEGFR2) markers indicates the potential for altered healing responses in the pulps of immature and mature teeth. Immature teeth have a greater MVD and VEGF/VEGFR2 expression than mature teeth, and the increased expression of these markers in the coronal region of both tooth types is important for pulp healing.

Avulsion and replantation of a primary incisor tooth.

Avulsion of a primary tooth is a serious dental trauma, and the guidelines of the International Association of Dental Traumatology and textbooks in paediatric dentistry do not recommend replantation. Such management can result in severe damage to the supporting structures, and together with avulsion itself is commonly associated with developmental disturbances of the permanent tooth. We report the case of replantation in a 9-month-old child with a successful outcome, in a unique situation where conditions were optimal and careful long-term follow up was possible.

A comparison of phosphor-plate digital images with conventional radiographs for the perceived clarity of fine endodontic files and periapical lesions.

OBJECTIVE: We sought to compare digital images with radiographs for the perceived clarity of small endodontic file tips at 2 different working lengths, as well as for the visualization of periapical bone lesions. STUDY DESIGN: Standardized conventional radiographic and phosphor-plate digital images were taken of 20 extracted permanent mandibular molars with 06 K-files placed in the distal root canal either 2 mm short or flush with the apical foramen. Similar images were obtained from mandibles with teeth that demonstrated large (n = 10) or small (n = 10) periapical lesions. Four evaluators ranked the clarity of the digital image with that of the radiograph. Results were analyzed by using the 2-sided sign test, ordinal logistic regression, and the kappa test. RESULTS: The perceived clarity of an endodontic file tip, at any position, and of a small or large periapical lesion was significantly (P < .01) less on all digital images compared with conventional films. CONCLUSION: Evaluator ratings indicated that the perceived clarity of fine endodontic files and periapical lesions was significantly less with phosphor-plate digital images than with conventional radiographs.