A scoping review of root canal revascularization: relevant aspects for clinical success and tissue formation.

The aim of this scoping study was to evaluate the survival rate and nature of tissue formed inside root canals of human immature permanent teeth with necrotic pulps (NIPT) under root canal revascularization (RCR). The search was performed in SciVerse Scopus®, PubMed/MEDLINE, Web of Science®, BIREME and in the grey literature up to November 2015. The keywords were selected using MeSH terms and DECs. Two independent reviewers scrutinized the records obtained considering specific inclusion criteria. The included studies were evaluated in accordance with a modified Arksey and O' Malley's framework. From 375 studies that were evaluated, 75 were included. A total of 367 NIPT were submitted to RCR, from which only 21 needed further endodontic treatment. The weighted mean follow-up time was 17.6 months. The data were derived mainly from case reports (69%) or small case series (15%). NaOCl [0.5-6%] was applied as the disinfecting solution in almost all studies. Triple antibiotic paste was as effective as Ca(OH)2 as on intracanal medicament. De novo tissue was cementum and poorly mineralized bone positive to bone sialoprotein (BSP) but negative to dentine sialoprotein (DSP). Failures were associated mainly with reinfection of the root canal. The majority of included studies reported a significant increase in both root length and width. However, as most of these data came from case reports, they must be interpreted with care, as most were focused on treatment successes (not failures). Therefore, well-designed randomized controlled trials comparing RCR with available apexification treatments are needed to address this gap in the literature.

Stem cell-based pulp tissue engineering: variables enrolled in translation from the bench to the bedside, a systematic review of literature.

Stem cell-based therapy (SC-BT) is emerging as an alternative for endodontic therapies. The interaction between stem cells and scaffolds plays a crucial role in the generation of a 'friendly cell' microenvironment. The aim of this systematic review was to explore techniques applied to regenerate the pulp-dentine complex tissue using SC-BT. An electronic search into the SciVerse Scopus (SS), ISI Web Science (IWS) and Entrez PubMed (EP) using specific keywords was performed. Specific inclusion and exclusion criteria were predetermined. The search yielded papers, out of which full-text papers were included in the final analyses. Data extraction pooled the results in four main topics: (a) influence of the chemical properties of the scaffolds over cell behaviour; (b) influence of the physical characteristics of scaffolds over cell behaviour; (c) strategies applied to improve the stem cell/scaffold interface; and (d) influence of cue microenvironment on stem cell differentiation towards odontoblast-like cells and pulp-like tissue formation. The relationship between the scaffolds, the environment and the growth factors released from dentine are critical for de novo pulp tissue regeneration. The preconditioning of dentine walls with ethylenediaminetetraacetic acid (EDTA) was imperative for successful pulp-dentine complex regeneration. An analyses of the grouped results revealed that pulp regeneration was an attainable goal.

The dental pulp stem cell niche based on aldehyde dehydrogenase 1 expression.

AIM: To detect cells expressing the stem cell marker ALDH1 (aldehyde dehydrogenase1) in the pulp of human permanent teeth and to investigate the expression of ALDH1 in isolated dental pulp cells. METHODOLOGY: Pulp tissue was collected and processed for immunohistochemistry to detect ALDH1-, STRO-1- and CD90-positive cells. In addition, cells were isolated and analysed by flow cytometry for ALDH1 activity and for the cell surface markers CD44, CD73, CD90, STRO-1 and CD45. Cells were also examined for multidifferentiation capacity. Within these cells, an ALDH1(+) cell subpopulation was selected and evaluated for multidifferentiation capacity. RESULTS: The immunohistochemistry analyses showed that ALDH1-, CD90- and STRO-1-positive cells were located mainly in the perivascular areas and nerve fibres of dental pulps. Cells on the fifth passage had high expression for CD44, CD73 and CD90, whereas moderate labelling was observed for STRO-1 and ALDH1 in flow cytometry analysis. On the same passages, cells were able to differentiate into osteogenic, adipogenic and chondrogenic lineages. The ALDH1(+) cell subpopulation also demonstrated multilineage differentiation ability. CONCLUSIONS: Dental pulp stem cells reside in the vicinity of blood vessels and nerve fibres, indicating the possible existence of more than one stem cell niche in dental pulps. Furthermore, ALDH1 was expressed by isolated dental pulp cells, which had mesenchymal stem cell characteristics. Thus, it can be suggested that ALDH1 may be used as a DPSC marker.

Immunoprofiling of oral squamous cell carcinomas reveals high p63 and survivin expression.

BACKGROUND: Cancer is a multifactorial disease composed of cells that show somatic mutations and epigenetic changes. The aim of this study was to investigate the expression of proteins involved in the development and maintenance of epithelia, cell cycle regulation, and apoptosis in human oral squamous cell carcinoma (OSCC) tissue samples. METHODS: A tissue microarray containing 65 primary human OSCC specimens was immunolabeled for bcl-2, survivin, epidermal growth factor receptor (EGFR), p21, p53, p63, and cleaved caspase-3. RESULTS: Samples were scored for percentage of positively stained tumor cells and staining intensity. A total immunostaining score was also calculated, using the product of percentage and intensity scores. All specimens showed high scores, > 75%, for p63 and survivin, and 75.4% of the specimens also presented high EGFR expression. All cases showed p53-positive cells. p21 showed a diffuse staining pattern. The percentage of cells positive for cleaved caspase-3 and bcl-2 was low. CONCLUSIONS: The high frequency of tumor cells expressing p63 and survivin highlights the role of these proteins in the malignant transformation of oral epithelium. Collectively, our results suggest that p63 and survivin may constitute attractive targets for cancer therapy in patients with OSCC.

Odontoblast RNA stability in different temperature-based protocols for tooth storage.

AIM: To evaluate the effect of four tooth storage temperature-based methods on quality of RNA obtained from cells retrieved from human dental pulps and human pre-dentine. METHODOLOGY: RNA was isolated from dental pulp tissue and from cells retrieved by scraping the pre-dentine of freshly extracted human third molars (n = 15) using TRIzol(®) reagent. Teeth were randomly assigned to the following temperature conditions: immediate RNA isolation after tooth extraction, liquid nitrogen (24 h), -80 °C (24 h), 20 °C (24 h) and 4 °C (6 h). RNA integrity was checked by the density of 28S and 18S ribosomal RNA. RT-PCR was used to analyse the expression of odontoblast makers (DSPP, DMP1 and MEPE) and the housekeeping gene GAPDH. RESULTS: All experimental conditions evaluated preserved RNA integrity. The three odontoblastic markers were amplified from the pulp tissue and from the cells associated with pre-dentine. CONCLUSION: The four storage options allowed RNA isolation for RT-PCR analysis. These findings may facilitate the use of clinically derived human dental pulp and odontoblasts for endodontic research.

Comparative analysis of two colorimetric assays in dental pulp cell density.

AIM: To compare and contrast two colorimetric assays used for the measurement of proliferation using two dental pulp cell types: dental pulp stem cells (DPSC) and human dental pulp fibroblasts (HDPF). METHODOLOGY: Dental pulp stem cells or HDPF were seeded at 0.25×10(4) cells per well in 96-well plates. Cell proliferation was evaluated after 24-72h. At the end of the experimental period, the sulforhodamine B (SRB) assay or a water-soluble tetrazolium salt (WST-1) assay was performed. Optical densities were determined in a microplate reader (Genius; TECAN). Data were analysed by Student's t-test (comparison between cell types) and one-way anova followed by Tukey test (time-point intervals). Pearson' correlation tests were performed to compare the two assays for each cell line. RESULTS: Both assays showed that DPSC had higher proliferation rates than HDPF. A positive significant correlation between the two colorimetric assays tested for both cell types DPSC (Pearson's correlation coefficient=0.847; P<0.05) and HDPF (Pearson's correlation coefficient=0.775; P<0.05). CONCLUSION: Both tests demonstrated similar trends of cell proliferation, and thus are both appropriate for the evaluation of DPSC and HDPF. The choice of assay is therefore one of the practical applications. SRB stained plates can be dried and stored so may have utility in laboratories where data may require review or when access to analytical equipment is limited. WST-1 assays have the benefit of both ease and speed and may have utility in laboratories requiring either high throughput or rapid analyses.

SHED differentiate into functional odontoblasts and endothelium.

Studies on mechanisms underlying the differentiation of dental pulp stem cells are critical for the understanding of the biology of odontogenesis and for dental tissue engineering. Here, we tested the hypothesis that stem cells from exfoliated deciduous teeth (SHED) differentiate into functional odontoblasts and endothelial cells. SHED were seeded in tooth slice/scaffolds and implanted subcutaneously into immunodeficient mice. SHED differentiated into functional odontoblasts that generated tubular dentin, as determined by tetracycline staining and confocal microscopy. These cells also differentiated into vascular endothelial cells, as determined by beta-galactosidase staining of LacZ-tagged SHED. In vitro, vascular endothelial growth factor (VEGF) induced SHED to express VEGFR2, CD31, and VE-Cadherin (markers of endothelium) and to organize into capillary-like sprouts. VEGF induced ERK and AKT phosphorylation (indicative of differentiation), while inhibiting phosphorylation of STAT3 (indicative of 'stemness'). Collectively, this work demonstrates that SHED can differentiate into angiogenic endothelial cells and odontoblasts capable of generating tubular dentin.

Dentin-derived BMP-2 and odontoblast differentiation.

It is known that stem cells from exfoliated deciduous teeth (SHED) can be induced to differentiate into odontoblasts. However, the nature of dentin-derived morphogenic signals required for dental pulp stem cell differentiation remains unclear. The hypothesis underlying this work is that dentin-derived Bone Morphogenetic Proteins (BMP) are necessary for the differentiation of SHED into odontoblasts. We observed that SHED express markers of odontoblastic differentiation (DSPP, DMP-1, MEPE) when seeded in human tooth slice/scaffolds and cultured in vitro, or implanted subcutaneously into immunodeficient mice. In contrast, SHED cultured in deproteinized tooth slice/scaffolds, or scaffolds without a tooth slice, do not express these markers. SHED express the BMP receptors BMPR-IA, BMPR-IB, and BMPR-II. Notably, blockade of BMP-2 signaling inhibited the expression of markers of odontoblastic differentiation by SHED cultured in tooth slice/scaffolds. Collectively, this work demonstrates that dentin-derived BMP-2 is required to induce the differentiation of SHED into odontoblasts.

Chlorhexidine Inhibits the Proteolytic Activity of Root and Coronal Carious Dentin in vitro.

The purpose of this study was to evaluate the effect of chlorhexidine on the proteolytic activity of carious coronal and root dentin collected from patients. Sound dentin from freshly extracted human teeth was used as a control. Dentin fragments were mixed with a synthetic substrate for proteolytic enzymes (N-benzoyl-DL-arginine-naphthylamide--BANA) and the suspensions mixed with either 0.12% chlorhexidine digluconate or distilled water. These mixtures were incubated for 18 h at 37 degrees C, color was developed by the addition of 0.1% Fast Garnet and their optical density was recorded spectrophotometrically. BANA hydrolysis measured by the optical density of incubated specimens was detected in all tested groups, but was significantly higher for carious than for sound dentin (p < 0.05). The proteolytic activity was reduced for carious coronal and root dentin by chlorhexidine (p < 0.05; 50 and 30%, respectively). Chlorhexidine also reduced the proteolytic activity in sound root dentin (p < 0.05; 20%). Conversely, changes in the proteolytic activity of sound coronal dentin were not observed in the presence of chlorhexidine. The reduction in proteolytic activity by chlorhexidine was significantly higher in carious coronal dentin than in carious root dentin (p < 0.05). In conclusion, part of the effect of chlorhexidine in controlling caries progression in humans may be due to a decrease in the proteolytic activity of carious coronal and root dentin. Because of the prolonged incubation time in the present study, similar results may be obtained clinically with prolonged dentin exposure to chlorhexidine, e.g. chlorhexidine-containing varnishes.

A model for clinical evaluation of the effect of antimicrobial agents on carious dentin.

PURPOSE: To evaluate, longitudinally, the effect of a chlorhexidine varnish on the proteolytic activity of dentin caries in vivo. MATERIALS AND METHODS: 20 permanent molars and 8 primary molars with carious lesions in dentin were studied in subjects 18-35 yrs old (n=20), and 5-6 yrs old (n=8) respectively. These lesions were clinically evaluated according to texture and color. Carious dentin specimens were obtained by means of biopsies performed with a #4 carbide bur at the initial visit (TO) before application of a 10% chlorhexidine varnish and 2, 4, 8, and 12 wks thereafter. The dentin biopsies were immersed in Sorensen's buffer, vortexed for 30 s, and mixed with a 1.67 mM solution of n-benzoyl-DL-arginine-naphthylamide (BANA), a substrate for proteolytic enzymes. Samples were incubated overnight at 37 degrees C and color was developed with 0.1% fast garnet. The optical density (OD) of reaction mixtures was recorded photometrically. All teeth were grouped for analysis, as Mann-Whitney tests revealed no statistically significant differences between median values for OD for both age groups. ANOVA was used to compare progressive inhibition of proteolytic activity in dentin caries samples over time. RESULTS: The average proteolytic activity at the dentin substrates (OD) at TO and 2, 4, 8 and 12 wks thereafter were 0.794+/-0.089, 0.741+/-0.071, 0.676+/-0.087, 0.600+/-0.094, and 0.508+/-0.108 respectively. The chlorhexidine varnish mediated a significant inhibition of the proteolytic activity present in dentin caries after 12 wks (P<0.0001). At T0, 100% of the carious lesions examined were characterized as soft upon exploration. After 12 wks, 54% (15/28) of the lesions were partially hardened and 46% (13/28) hardened/nonprogressing. The dentin color was yellow/light brown in 100% of the lesions at baseline, and dark brown/black in 86% (24/28) after 12 wks. CLINICAL SIGNIFICANCE: This study demonstrated that chlorhexidine varnishes arrested active caries in vivo and inhibited the proteolytic activity present in these lesions. These findings strengthen the rationale for including chlorhexidine in the overall treatment strategy for patients with high caries activity.

SEM study of a self-etching primer adhesive system used for dentin bonding in primary and permanent teeth.

PURPOSE: The purpose of this study was to evaluate the interfacial micromorphology of direct esthetic restorations bonded to primary or permanent tooth dentin with a self-etching primer adhesive system. METHODS: Superficial dentin at the occlusal surface of 15 primary and 15 permanent molars was exposed with a carbide bur. Prompt-L-Pop was applied in one half of each surface. A control bonding system, Single Bond or Vitremer Primer, was used in the other half Teeth were restored either with a composite resin (Filtek Z250), a compomer (Hytac), or a resin-modified glass ionomer (Vitremer). Twenty-five scanning electron microscope fields from 5 teeth were evaluated blindly by two investigators for each condition. RESULTS: In this study, a significant difference in quality of the interfacial seal was not observed when restorations performed in primary teeth were compared to restorations in permanent teeth. Interfacial gaps were observed in most restorations bonded with Prompt-L-Pop and restored with Filtek Z250 (9/10), Hytac (9/10), or Vitremer (5/10). No interfacial gaps were observed in teeth bonded with Single Bond and restored with Filtek Z250 (0/10) or Hytac (0/10), while all teeth bonded with Vitremer Primer and restored with Vitremer presented gaps (10/10). To understand the reason for the interfacial gaps observed with Prompt-L-Pop, we examined if this system generated a hybrid layer at the dentin/restorative material interface. All surfaces bonded with Single Bond and restored with Filtek Z250 or Hytac presented a visible hybrid layer. In contrast, 0/10 (Z250) and only 3/10 (Hytac) restorations bonded with Prompt-L-Pop showed signs of a hybrid layer. CONCLUSION: The self-etching primer adhesive system Prompt-L-Pop failed to generate sealed interfaces consistently between the dentin of primary and permanent teeth and the composite resin or the compomer evaluated in this study.

Unstimulated salivary flow rates of young children.

OBJECTIVE: Studies on salivary flow rates in human beings have mainly been carried out with adults. The purpose of this study was to determine the unstimulated salivary flow rates of children 4 to 7 years old. In addition, the relative contributions of the variables age, gender, race, height, body weight, dentition status, use of prescription medication, and health status (information obtained from parents) to the unstimulated salivary flow rates of children were also studied. STUDY DESIGN: Data were obtained from children (n = 447) at 2 sites in the United States (site 1, southeast Michigan; site 2, northern Michigan) and at 5 sites in Brazil (site 3, Porto Alegre; site 4, São Paulo; site 5, Belém; and sites 6 and 7, sites in Rio de Janeiro). In northern Michigan (site 2) the participants were cognitively or developmentally disabled, or both. In Rio de Janeiro (site 7), a group of 8- to 12-year-olds served as a control group. Saliva samples were collected for 3 minutes between 9 AM and noon in the spring or summer, and the saliva rate was determined gravimetrically. Data were analyzed by analysis of variance, bivariate analysis, and regression analysis. RESULTS: The secretion rates at the 7 sites were (in milliliters per minute) 0.19 +/- 0.15, 0.23 +/- 0.28, 0.34 +/- 0.23, 0.48 +/- 0.37, 0.25 +/- 0.27, 0.37 +/- 0.28, and 0.61 +/- 0.34, respectively. There were significant differences among sites (P <.0001). The older group (site 7) had flow rates that were significantly higher than the flow rates of any other group. In addition, children from Michigan (sites 1 and 2) had significantly lower rates than most groups of children in Brazil. Girls had lower unstimulated salivary flow rates than boys did at all the sites, but the differences were not statistically significant. Race was shown not to affect the flow rates. The use of any prescription medication by children in the previous 3 months was associated with lower salivary flow rates than were found in children not using prescription medication. Children who were in good health and who had no previous medical conditions had higher flow rates--but not significantly so. Higher flow rates occurred in children with mixed dentition than in children with primary dentition, although again the differences were not statistically significant. Regression analysis revealed weight to be of significance in explaining the variability of the unstimulated salivary flow rates at 2 sites, height at 1 site, the use of prescription medication at 2 sites, and age at 1 site. CONCLUSIONS: The unstimulated salivary flow rates in children in the northern United States are comparable with those reported for Japanese children, whereas the flow rates of children in Brazil are comparable with those reported for North American and European adults. In addition, none of the demographic variables/parameters tested contributed consistently to the variability of the unstimulated salivary flow rates in children at the 7 sites assessed in this study.