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Previously, we reported that the fluorapatite (FA)-modified polycaprolactone (PCL) nanofiber could be an odontogenic/osteogenic inductive tissue-engineering scaffold by inducing stem cell differentiation and mineralization. The present study aimed to explore which of the signal pathways affected this differentiation and mineralization process. The Human Signal Transduction PathwayFinder RT2 Profiler PCR Array was used to analyze the involvement of potential signal transduction pathways during human dental pulp stem cell (DPSCs) osteogenic differentiation induced by FA-modified PCL nanofiber scaffolds. Based on the results, perturbation studies of the signaling pathways hedgehog, insulin, and Wnt were performed. Moreover, the autophagy process was studied, as indicated by the expression of the microtubule-associated protein 1 light chain 3A/B-II (LC3-II) and the cell osteogenic phenotypic changes. In a comparison of the cells grown on PCL + FA scaffolds and those on PCL-only scaffolds, the transcript expression of BMP2, BMP4, FOXA2, PTCH1, WNT1, and WNT2 (PCR array-labeled signal proteins of the hedgehog pathway); CEBPB, FASN, and HK2 (PCR array-labeled signal proteins of the insulin pathway); and CCND1, JUN, MYC, TCF7, and WISP1 (PCR array-labeled signal proteins of the Wnt pathway) doubled at day 14 when obvious cell osteogenic differentiation occurred. Phenotypically, in all the perturbation groups at day 14, ALP activity, OPN, and autophagy marker LC3-II expression were coincidently decreased. Consistently, no positive alizarin red staining or von Kossa staining was observed in the specimens from these perturbation groups at day 28. The results showed that when obvious cell differentiation occurred at day 14 on PCL + FA control groups, the inhibition of the hedgehog, insulin, and Wnt pathways significantly decreased DPSC osteogenic differentiation and mineralization. The osteogenic differentiation of DPSCs grown on FA-modified PCL scaffolds appeared to be positively modulated by the hedgehog, insulin, and Wnt signal pathways, which were coordinated with and/or mediated by the cell autophagy process. [ABSTRACT FROM AUTHOR]

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 Me SH 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%). Na OCl [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. [ABSTRACT FROM AUTHOR]

Aim To investigate the effect of simvastatin on lipopolysaccharide ( LPS)-stimulated inflammatory cytokines, cell adhesion molecules and nuclear factor-κB ( NF-κB) transcription factors in human dental pulp cells ( HDPCs). Methodology The effect of LPS and simvastatin on human dental pulp cell ( HDPCs) viability was measured using a 3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyltetrazolium bromide ( MTT) assay. The expression of inflammatory cytokines and cell adhesion molecules was evaluated by reverse-transcription polymerase chain reaction ( RT- PCR), enzyme-linked immunosorbent assay ( ELISA) and Western blot analysis. NF-κB transcription factors were evaluated by Western blot analysis. Statistical analysis was performed with analysis of variance ( anova). Results The viability of cells exposed to different concentrations of E. coli LPS, P. gingivalis LPS and simvastatin was not significantly different compared with that of control cells ( P > 0.05). LPS significantly increased interleukin ( IL)-1β ( P < 0.05) and IL-6 mRNA expression ( P < 0.05) and vascular cell adhesion molecule-1 ( VCAM-1) ( P < 0.05) and intercellular adhesion molecule-1 ( ICAM-1) protein expression ( P < 0.05) in HDPCs. Treatment with simvastatin significantly attenuated LPS-stimulated production of IL-1β, IL-6, VCAM-1 and ICAM-1 ( P < 0.05). Treatment with simvastatin decreased LPS-induced expression of p65 and phosphorylation of IκB and also significantly decreased the phosphorylation of p65 and IκB in the cytoplasm and the level of p65 in the nucleus ( P < 0.05). Conclusions Simvastatin has a suppressing effect on LPS-induced inflammatory cytokine, cell adhesion molecules and NF-κB transcription factors in HDPCs. Therefore, simvastatin might be a useful candidate as a pulp-capping agent in vital pulp therapy. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

As a major intracellular degradation and recycling machinery, autophagy plays an important role in maintaining cellular homeostasis and remodeling during normal development. Our previous study showed that fluorapatite (FA) crystal-coated electrospun polycaprolactone (PCL) was capable of inducing differentiation and mineralization of human dental pulp stem cells. However, how autophagy changes and whether autophagy plays a vital role during these processes is still unknown. In this study, we seeded STEMPRO human adipose-derived stem cells (ASCs) on both PCL+FA and PCL scaffolds to investigate the osteogenic inductive ability of FA crystals and we observed the autophagy changes of these cells. Scanning electron microscopy and fluorescence microscopy images, along with DNA quantitation, showed that both PCL+FA and PCL scaffolds could sustain ASC growth but only the PCL+FA scaffold could sustain cell mineralization. This was confirmed by alkaline phosphatase activity and Alizarin red and Von Kossa staining results. The autophagy RT2 Profiler polymerase chain reaction array analysis showed many autophagy-related genes changes during ASC differentiation. Western blot analysis indicated that several autophagy-related proteins fluctuated during the procedure. Among them, the microtubule-associated protein 1 light chain 3 (LC3)-II protein changes of the ASCs grown on the 2- or 3-dimensional environments at 6 h, 12 h, 1 d, 3 d, 7 d, 14 d, and 21 d reached a peak value at day 7 during osteogenesis. At earlier stages (from day 0 to day 3), the addition of autophagy inhibitors (3-mathyladenine, bafilomycin A1, and NH4Cl) attenuated the expression of osteogenic related markers (osteopontin, alkaline phosphatase activity, Alizarin red, and Von Kossa) compared with the control group. All data indicated that autophagy played an important role in ASC differentiation on the PCL+FA scaffold. Inhibition of autophagy before day 3 strongly inhibited osteogenic differentiation and mineralization of ASCs in the 3-dimensional model. This observation further elucidates the mechanism of autophagy in mesenchymal stem cell osteogenic differentiation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Aim To evaluate both the drug-release profile and the effects on human dental pulp stem cells' (h DPSC) proliferation and viability of novel bi-mix antibiotic-containing scaffolds intended for use as a drug delivery system for root canal disinfection prior to regenerative endodontics. Methodology Polydioxanone ( PDS)-based fibrous scaffolds containing both metronidazole ( MET) and ciprofloxacin ( CIP) at selected ratios were synthesized via electrospinning. Fibre diameter was evaluated based on scanning electron microscopy ( SEM) images. Pure PDS scaffolds and a saturated CIP/MET solution ( i.e. 50 mg of each antibiotic in 1 mL) (hereafter referred to as DAP) served as both negative (nontoxic) and positive (toxic) controls, respectively. High-performance liquid chromatography ( HPLC) was performed to investigate the amount of drug(s) released from the scaffolds. WST-1® proliferation assay was used to evaluate the effect of the scaffolds on cell proliferation. LIVE/DEAD® assay was used to qualitatively assess cell viability. Data obtained from drug release and proliferation assays were statistically analysed at the 5% significance level. Results A burst release of CIP and MET was noted within the first 24 h, followed by a sustained maintenance of the drug(s) concentration for 14 days. A concentration-dependent trend was noticed upon h DPSCs' exposure to all CIP-containing scaffolds, where increasing the CIP concentration resulted in reduced cell proliferation ( P < 0.05) and viability. In groups exposed to pure MET or pure PDS scaffolds, no changes in proliferation were observed. Conclusions Synthesized antibiotic-containing scaffolds had significantly lower effects on h DPSCs proliferation when compared to the saturated CIP/MET solution ( DAP). [ABSTRACT FROM AUTHOR]

Aim To compare the mineralization inductive capacity of Biodentine and Bioaggregate with Mineral trioxide aggregate ( MTA) and to investigate possible signaling pathways of mineralization in human dental pulp cells ( HDPCs). Methodology Viability of HDPCs in response to Biodentine, Bioaggregate, and MTA was measured using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide. To investigate their potential to induce odontoblast differentiation, expression of dentine sialophosphoprotein ( DSPP) and dentine matrix protein1 ( DMP1) m RNA level was evaluated by RT-PCR. For the mineralized nodule assay, Alizarin red staining was performed. To determine the role of MAPK signaling in the odontoblastic differentiation of HDPCs, activated MAPKs were investigated by Western blot and the effect of MAPK inhibitor was examined by Alizarin red S staining. The results were statistically analysed using one-way anova and the Bonferroni test. Results The effects of MTA, Biodentine, and Bioaggregate on cell viability were similar. Biodentine and Bioaggregate enhanced DSPP and DMP1 m RNA expression compared to the control group, but to the same extent as MTA ( P < 0.05). MTA, Biodentine, and Bioaggregate increased the area of calcified nodules compared to the control ( P < 0.01). MTA, Biodentine, and Bioaggregate increased phosphorylation of extracellular signal-regulated kinase ( ERK), p38, and c-Jun N-terminal kinase ( JNK). MAPK inhibitors attenuated mineralized nodule formation, which was increased by MTA, Biodentine, and Bioaggregate, respectively ( P < 0.01). Conclusion Biodentine and Bioaggregate stimulated odontoblastic differentiation and mineralization nodule formation by activating the MAPK pathway as did MTA. This suggests that the new materials could be useful for regenerative endodontic procedures. [ABSTRACT FROM AUTHOR]

Conde MCM, Nedel F, Campos VF, Smith AJ, Nör JE, Demarco FF, Tarquinio SB. Odontoblast RNA stability in different temperature-based protocols for tooth storage. International Endodontic Journal, 45, 266-272, 2012. Abstract 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. [ABSTRACT FROM AUTHOR]

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 · 104 cells per well in 96-well plates. Cell proliferation was evaluated after 24–72 h. 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 microplatereader (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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Caries-induced pulpitis is typically accompanied by an increase in dental pulp microvascular density. However, the mechanisms by which dental pulp cells recognize lipopolysaccharides (LPSs) remain unclear. We hypothesized that Porphyromonas endodontalis and Escherichia coli LPSs induce vascular endothelial growth factor (VEGF) expression in dental pulp stem cells (DPSC) and human dental pulp fibroblasts (HDPF) through mitogen-activated protein kinase (MAPK) signaling. ELISA, semi-quantitative RT-PCR, immunofluorescence, and Western blots were used. Here, we observed that LPSs induced VEGF expression in DPSC and HDPF cells, and both cell types express Toll-like receptor 4 (TLR- 4). Notably, LPS-induced VEGF is associated with phosphorylation of protein kinase C (PKC ζ) and extracellular signal-regulator kinase (ERK1/2) and is dependent upon MAPK activation. Analysis of these data, collectively, unveils a signaling pathway responsible for synthesis of VEGF by pulp cells and suggests a novel therapeutic target for the management of vascular responses in teeth with pulpitis. [ABSTRACT FROM AUTHOR]

Vascular endothelial growth factor (VEGF) signaling is critical for tumor angiogenesis. However, therapies based on inhibition of VEGF receptors (VEGFRs) have shown modest results for patients with cancer. Surprisingly little is known about mechanisms underlying the regulation of VEGFR1 and VEGFR2 expression, the main targets of these drugs. Here, analysis of tissue microarrays revealed an inversely reciprocal pattern of VEGFR regulation in the endothelium of human squamous-cell carcinomas (high VEGFR1, low VEGFR2), as compared with the endothelium of control tissues (low VEGFR1, high VEGFR2). Mechanistic studies demonstrated that VEGF signals through the Akt/ERK pathway to inhibit constitutive ubiquitination and induce rapid VEGFR1 accumulation in endothelial cells. Surprisingly, VEGFR1 is primarily localized in the nucleus of endothelial cells. In contrast, VEGF signals through the JNK/c-Jun pathway to induce endocytosis, nuclear translocation, and downregulation of VEGFR2 via ubiquitination. VEGFR1 signaling is required for endothelial-cell survival, while VEGFR2 regulates capillary tube formation. Notably, the antiangiogenic effect of bevacizumab (anti-VEGF antibody) requires normalization of VEGFR1 and VEGFR2 levels in human squamous-cell carcinomas vascularized with human blood vessels in immunodeficient mice. Collectively, this work demonstrates that VEGF-induced angiogenesis requires inverse regulation of VEGFR1 and VEGFR2 in tumor-associated endothelial cells. [ABSTRACT FROM AUTHOR]

The inflammation observed in the dental pulp of teeth with deep caries lesions is characterized by a significant increase in blood vessel density. It is known that lipoteichoic acid (LTA) from Grampositive cariogenic bacteria induces expression of vascular endothelial growth factor (VEGF) in dental pulp cells. The hypothesis underlying this study was that LTA induces VEGF expression in dental pulp cells through TLR2 and PI3k/Akt signaling. Odontoblast-like cells (MDPC-23) and undifferentiated pulp cells (OD-21) were exposed to LTA from Streptococcus sanguis, and the role of TLR2, PI3K/Akt, and IKK signaling in LTA-induced VEGF expression was evaluated. These studies demonstrated that TLR2 signaling through the PI3K-Akt pathway is necessary for LTA-induced VEGF expression in pulp cells. In contrast, inhibition of IKK signaling did not prevent VEGF up-regulation in response to LTA. Understanding signaling pathways triggered by cariogenic bacteria may reveal novel therapeutic targets for the clinical management of pulpitis. [ABSTRACT FROM AUTHOR]

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°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. Copyright © 2009 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Transformation of small avascular masses of tumor cells into rapidly progressive cancers is triggered by the angiogenic switch, a process that involves vascular endothelial growth factor (VEGF) signaling. We have shown that VEGF enhances the survival and angiogenic potential of endothelial cells by activating the Bcl-2-CXCL8 signaling axis. The purpose of this study was to evaluate the effect of a small-molecule inhibitor of VEGF receptors (PTK/ZK) on the initial stages of head and neck tumor angiogenesis. In vitro, PTK/ ZK blocked head and neck tumor cell (OSCC3 or UM-SCC-17B)-induced Bcl-2 and CXCL8 expression in endothelial cells. Oral administration of PTK/ZK decreased xenograft head and neck tumor microvessel density, and inhibited Bcl-2 and CXCL8 expression in tumor-associated endothelial cells. Analysis of these data demonstrates that PTK/ZK blocks downstream targets of VEGF signaling in endothelial cells, and suggests that PTK/ZK may inhibit the angiogenic switch in head and neck tumors. Abbreviations: HDMEC, human dermal microvascular endothelial cells; VEGF, vascular endothelial growth factor; CXCL8, CXC ligand-8; PTK/ZK, PTK787/ZK222584. [ABSTRACT FROM AUTHOR]

The long-term outcome of replanted avulsed permanent teeth is frequently compromised by lack of revascularization, resulting in pulp necrosis. The purpose of this study was to evaluate the effects of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF-2) on the revascularization of severed human dental pulps. Tooth slices were prepared from non-carious human molars and treated with 0-50 ng/mL rhVEGF165 or rhFGF-2 for 7 days in vitro. Both angiogenic factors enhanced pulp microvessel density compared with untreated controls (p < 0.05). Tooth slices were also treated with 0 or 50 ng/mL rhVEGF165 for one hour prior to implantation into the subcutaneous space of immunodeficient mice. Treatment with rhVEGF165 increased pulp microvessel density in vivo (p < 0.05). These results demonstrate that rhVEGF165 enhanced neovascularization of severed human dental pulps and suggest that topical application of an angiogenic factor prior to replantation might be beneficial for the treatment of avulsed teeth. [ABSTRACT FROM AUTHOR]

The purpose of this study was to compare the cytotoxicity of five one-step dentin-bonding agents on human dental pulp and odontoblast-like cells (MDPC-23). Photopolymerized and unpolymerized samples of these dentin-bonding agents were prepared and incubated with dental pulp or MDPC-23 cells. After 24 or 72 h of incubation, the number of unstained cells with trypan blue was counted. The staining of cells with trypan blue stands for a cytotoxicity. The pulp cell and MDPC-23 cytotoxicity of polymerized sample treatment increased in the order of AQ Bond Plus (AQ)

Antiangiogenic therapies have shown varying results partly because each tumor type secretes a distinct panel of angiogenic factors to sustain its own microvascular network. In addition, recent evidence demonstrated that tumors develop resistance to antiangiogenic therapy by turning on alternate angiogenic pathways when one pathway is therapeutically inhibited. Here, we test the hypothesis that expression of a caspase-based artificial death switch in tumor-associated endothelial cells will disrupt tumor blood vessels and slow down tumor progression irrespective of tumor type. Adenoviral vectors expressing inducible Caspase-9 (iCaspase-9) under transcriptional regulation with the endothelial cell-specific vascular endothelial growth factor receptor-2 (VEGFR2) promoter (Ad-hVEGFR2-iCaspase-9) induced apoptosis of proliferating human dermal microvascular endothelial cells (HDMECs), but not human tumor cells (UM-SCC-17B, head and neck squamous cell carcinoma; HepG2, hepatocellular carcinoma; PC-3, prostate adenocarcinoma; SLK, Kaposi's sarcoma; MCF-7, breast adenocarcinoma). Notably, apoptosis was dependent upon activation of iCaspase-9 with the dimerizer drug AP20187. Local delivery of Ad-hVEGFR2-iCaspase-9 followed by intraperitoneal injection of AP20187 ablated tumor microvessels and inhibited xenografted tumor growth in all tumor models evaluated here. We conclude that a cancer gene therapy strategy based on a transcriptionally targeted viral vector expressing an inducible caspase allows for selective and controlled ablation of microvessels of histopathologically diverse tumor types.Cancer Gene Therapy (2008) 15, 667–675; doi:10.1038/cgt.2008.38; published online 20 June 2008 [ABSTRACT FROM AUTHOR]

Substantial numbers of dendritic cells have been detected in radicular granulomas. To test the hypothesis that local antigen presentation from dendritic cells to T-cells is involved critically in immunological responses within radicular granulomas, we compared characteristics of dendritic cells and macrophages by morphological and biological analyses. Under light microscopy, HLA-DR+ and CD68+ cells showed diverse profiles, including dendritic-shaped cells. Transmission electron microscopy revealed that HLA-DR+ dendritic cells, with long cytoplasmic processes and lacking distinct phagosomes, were concentrated in the lymphocyte-rich area. HLA-DR alpha-chain, CD83, and CD86 mRNAs from HLA-DR+ dendritic cells, and CD28 mRNA from CD28+ T-cells were up-regulated in lymphocyte-rich area. Scanning electron microscopy demonstrated that the density of gold particles on dendritic cells was higher than that on HLA-DR+ macrophages. These results suggest that dendritic cells in radicular granulomas are associated with local defense reactions as stronger antigen-presenting cells, as compared with macrophages. [ABSTRACT FROM AUTHOR]

Expression of Bcl-xL correlates with the clinical outcomes of patients with cancer. While the role of Bcl-2 in angiogenesis is becoming increasingly evident, the function of Bcl-xL in angiogenesis is unclear. Here, we showed that epidermal growth factor (EGF) induces in vitro capillary sprouting and Bcl-xL expression in primary endothelial cells. Bcl-xL-transduced human dermal microvascular endothelial cells (HDMEC-Bcl-xL), but not empty vector control cells, spontaneously organize into capillary-like sprouts. Searching for a mechanism to explain these responses, we observed that Bcl-xL induced expression of the pro-angiogenic chemokines CXC ligand-1 (CXCL1) and CXC ligand-8 (CXCL8), and that blockade of CXC receptor-2 (CXCR2) signaling inhibited spontaneous sprouting of HDMEC-Bcl-xL. Bcl-xL led to Bcl-2 upregulation, but Bcl-2 did not upregulate Bcl-xL, suggesting the existence of a unidirectional crosstalk from Bcl-xL to Bcl-2. EGF and Bcl-xL activate the mitogen-activated protein kinase/ERK pathway resulting in upregulation of vascular endothelial growth factor (VEGF), a known inducer of Bcl-2 in endothelial cells. Inhibition of VEGF receptor signaling in HDMEC-Bcl-xL prevented Bcl-2 upregulation and demonstrated the function of a VEGF-mediated autocrine loop. Bcl-2 downregulation by RNAi blocked CXCL1 and CXCL8 expression downstream of Bcl-xL, and markedly decreased angiogenesis in vivo. We conclude that Bcl-xL functions as a pro-angiogenic signaling molecule controlling Bcl-2 and VEGF expression. These results emphasize a complex interplay between Bcl-2 family members beyond their classical roles in apoptosis.Cell Death and Differentiation (2007) 14, 1657–1666; doi:10.1038/sj.cdd.4402174; published online 15 June 2007 [ABSTRACT FROM AUTHOR]

Tumors of the oral cavity are highly vascularized malignancies. Disruption of neovascular networks was shown to limit the access of nutrients and oxygen to tumor cells and inhibit tumor progression. Here, we evaluated the effect of the activation of an artificial death switch (iCaspase-9) expressed in neovascular endothelial cells on the progression of oral tumors. We used biodegradable scaffolds to co-implant human dermal microvascular endothelial cells stably expressing iCaspase-9 (HDMEC-iCasp9) with oral cancer cells expressing luciferase (OSCC3-luc or UM-SCC-17B-luc) in immunodeficient mice. Alternatively, untransduced HDMEC were co-implanted with oral cancer cells, and a transcriptionaly targeted adenovirus (Ad-VEGFR2-iCasp-9) was injected locally to deliver iCaspase-9 to neovascular endothelial cells. In vivo bioluminescence demonstrated that tumor progression was inhibited, and immunohisto-chemistry showed that microvessel density was decreased, when iCaspase-9 was activated in tumor-associated microvessels. We conclude that activation of iCaspase-9 in neovascular endothelial cells is sufficient to inhibit the progression of xenografted oral tumors. [ABSTRACT FROM AUTHOR]

The purpose of this in vitro study was to evaluate the surface and resin–dentine interface characteristics of permanent tooth dentine cut with diamond or carbide burs and treated with phosphoric acid (PA) or an acidic conditioner. Labial surfaces of permanent incisors were prepared into dentine with high-speed carbide or diamond burs and divided into two halves. Phosphoric acid 36% was applied on one half and non-rinse conditioner (NRC) was applied on the other half. Ten randomly selected scanning electron microscopy (SEM) fields from each specimen ( n = 15) were evaluated. Occlusal surfaces of third molars were divided in two halves for evaluation of the resin–dentine interface. The halves were randomly assigned to one of each conditioner and restored with Prime & Bond NT/Spectrum. Ten specimens were analysed by SEM to evaluate hybrid layer formation and interfacial seal. We observed that surfaces prepared with carbide bur presented less residual smear plugs ( P < 0·05) than surfaces prepared with diamond burs. Surfaces conditioned with NRC, which is a smear layer modifier, presented more residual smear plugs than surfaces conditioned with PA ( P < 0·05). Treatment with PA resulted in more sealed interfaces than specimens treated with NRC. Within the limitations of this study the results showed that carbide burs leave a surface that is more conducive to bonding than diamond burs. [ABSTRACT FROM AUTHOR]

The activation of an inducible caspase (iCaspase-9) mediates apoptosis of neovascular endothelial cells, and overcomes the prosurvival effect of vascular endothelial growth factor or basic fibroblast growth factor. The potential utilization of direct activation of caspases as an antiangiogenic strategy for treatment of angiogenesis-dependent diseases (eg cancer) requires expression of the inducible caspase primarily in the tumor endothelium. The objective of this work was to develop and characterize a transcriptionally targeted adenoviral vector that mediates expression of iCaspase-9 specifically in neovascular endothelial cells. We observed that adenoviral vectors containing the human VEGFR2 promoter induced reporter gene expression primarily in proliferating human dermal microvascular endothelial cells (HDMEC). HDMEC transduced with recombinant adenoviral vectors containing iCaspase-9 under regulation of the VEGFR2 promoter (Ad-hVEGFR2-iCaspase-9) and exposed to a cell-permeable dimerizer drug (AP20187), presented higher caspase-3 activity and apoptosis than controls (P?0.05). Using the SCID Mouse Model of Human Angiogenesis, we observed that local delivery of Ad-hVEGFR2-iCaspase-9 followed by intraperitoneal injection of AP20187 resulted in endothelial cell apoptosis and local ablation of microvessels. We believe that this constitutes the first report of a transcriptionally targeted antiangiogenic adenoviral vector that mediates neovascular disruption upon activation of a caspase-based artificial death switch.Gene Therapy (2005) 12, 320-329. doi:10.1038/sj.gt.3302306 Published online 23 December 2004 [ABSTRACT FROM AUTHOR]

Hernandez EP, Botero TM, Mantellini MG, McDonald NJ, Nör JE.Effect of ProRoot® MTA mixed with chlorhexidine on apoptosis and cell cycle of fibroblasts and macrophagesin vitro.International Endodontic Journal,38, 137–143, 2005.To compare the percentage of apoptotic cells and the cell cycle profile of fibroblasts and macrophages exposed to either ProRoot® mineral trioxide aggregate (MTA) mixed with chlorhexidine (CHX), or exposed to ProRoot® MTA mixed with sterile water.Mouse gingival fibroblasts or mouse macrophages were seeded in six-well plates and allowed to attach overnight. Freshly mixed or set (allowed to dry for 24 h) specimens of tooth-coloured (white) ProRoot® MTA were prepared with 0.12% CHX gluconate (MTA/CHX) or with sterile water (MTA/H2O). The cells were exposed for 24 h to the MTA specimens, which were placed over permeable membrane inserts to avoid direct contact with the cells. Untreated cells served as controls. Propidium iodide staining followed by flow cytometry was used to evaluate the effects of ProRoot® MTA on cell apoptosis and cell cycle. Statistical analyses were performed by one-wayanovafollowed bypost-hoctests with the use of the SigmaStat 2.0 software, and significance was determined atP ≤ 0.05.MTA specimens containing CHX induced apoptosis of macrophages and fibroblasts (P < 0.05). In contrast, no change in the proportion of apoptotic cells was observed when sterile water was used to prepare the specimens (P > 0.05). Cell cycle analysis showed that exposure to MTA/CHX decreased the percentage of fibroblasts and macrophages in S phase (DNA synthesis) as compared with exposure to MTA/H2O (P < 0.05).Thisin vitrostudy demonstrated that the substitution of CHX for sterile water in MTA increases its cytotoxicity. This suggests that the potentially beneficial antimicrobial effect of CHX may be accompanied by an increase in the cytotoxicity of the resulting MTA-based material. [ABSTRACT FROM AUTHOR]

The term apoptosis, also known as programmed cell death (PCD), was coined by developmental biologists a number of years ago to describe a form of cell death characterized by several unique morphological and biochemical features. Genetic studies of the round worm Caeneorhabditis elegans, a simple multicellular organism, first revealed apoptosis to be an integral part of the developmental program. Subsequently, the importance of apoptosis in higher organisms was demonstrated in several eukaryotic systems. In mammals, apoptosis is widespread during embryogenesis and in adult tissues. It is required for normal tissue homeostasis and for clonal selection in the immune system. In both developing and adult organisms, apoptosis plays a central role in reinforcing appropriate cellular patterns and in regulating cell number by eliminating cells that are harmful or no longer needed. It is becoming increasingly clear that disruption in the apoptosis pathway can contribute to the development of a number of developmental, inflammatory, degenerative, and neoplastic diseases. The effector arm of the apoptotic program includes members of the Bcl-2 gene family that function as either death agonists or death antagonists. These proteins participate in an elaborate genetically controlled biochemical pathway that functions to maintain tissue and organ homeostasis and serve as a critical defense mechanism to guard against malignant transformation. Cancer is the result of a series of genetic lesions that include activation of oncogenes and inactivation or loss of tumor suppressor genes. Several groups of investigators have observed that deregulated expression of oncogenes can subvert apoptotic pathways, resulting in prolonged cell survival. In pathological settings such as cancer, members of the Bcl-2 gene family are able to synergize with oncogenes and tumor suppressor genes to transform cells. In this review, we describe the process of apoptosis in mammalian cells and define the role and biochemical pathways through which the Bcl-2 gene family induce and/or protect cells from apoptosis. Last, we will discuss the evidence which suggests that alterations in this pathway may play a central role in tumorigenesis by allowing genetically damaged cells normally destined for elimination to persist, predisposing them to additional mutations and driving them to malignancy. [ABSTRACT FROM AUTHOR]

Previous studies have suggested minor differences between primary and permanent teeth in terms of dentin composition and morphology. Other reports indicated lower bond strengths of resin composites to dentin of primary teeth compared with dentin of permanent teeth; however, no information is available regarding differences in the micromorphology of the resin-dentin interface that may explain these lower bond strengths. Therefore, the purpose of the present study was to compare primary and permanent teeth in terms of the thickness of the hybrid layer developed with two bonding systems. Our hypothesis was that bonding differences previously reported between primary and permanent dentin would be reflected in hybrid layer differences observable in SEM analyses. Twenty human extracted and non-carious teeth were divided into 4 groups: 5 primary and 5 permanent teeth restored with All-Bond 2/Bisfil P system; and 5 primary and 5 permanent teeth restored with Scotchbond Multi-Purpose/Z100. The sample area available on each tooth was divided for the two dentin conditioning times (7 and 15 sec). Measurements of hybrid layer thickness were performed by means of SEM at x13,000. The results of this study indicated that the hybrid layer produced is significantly thicker in primary than in permanent teeth (p = 0.0001), suggesting that primary tooth dentin is more reactive to acid conditioning. No difference was observed in the hybrid layers produced by the two adhesive systems (p = 0.7920). The increased thickness of the hybrid layer in primary teeth (25 to 30%) and the subsequent lack of complete penetration of adhesive resin into previously demineralized dentin may contribute to the lower bond strengths to primary dentin reported in the literature. If a narrower hybrid layer more uniformly infused with resin is the goal of dentin bonding, it is concluded that a differentiated protocol for bonding to primary dentin (with shorter time for dentin conditioning) can be used as a means to reproduce the hybrid layer thickness seen in permanent teeth. [ABSTRACT FROM AUTHOR]

An introduction is presented in which the authors discuss various reports within the issue on topics including precision medicine therapies for head and neck cancer and squamous cell carcinoma, cancer etiology, and cancer immunotherapies.

An introduction is presented in which the editor discusses various reports within the issue on topics including the role of stem cells in craniofacial diseases, research into dental pulp-dentin regeneration, and the use of induced pluripotent stem cells in dental therapeutics.