VEGFR1 primes a unique cohort of dental pulp stem cells for vasculogenic differentiation.

Dental pulp stem cells (DPSCs) constitute a unique group of cells endowed with multipotency, self-renewal, and capacity to regenerate the dental pulp tissue. While much has been learned about these cells in recent years, it is still unclear if each DPSC is multipotent or if unique sub-populations of DPSCs are "primed" to undergo specific differentiation paths. The purpose of the present study was to define whether a sub-population of DPSCs was uniquely primed to undergo vasculogenic differentiation. Permanent-tooth DPSCs or stem cells from human exfoliated deciduous teeth (SHED) were flow-sorted for vascular endothelial growth factor receptor 1 (VEGFR1) and exposed to vasculogenic differentiation medium, i.e., Microvascular-Endothelial-Cell-Growth-Medium-2-BulletKit™ supplemented with 50 ng/mL rhVEGF165 in the presence of 0 or 25 µg/mL anti-human VEGF antibody (bevacizumab; Genentech). In addition, sorted SHED (i.e., VEGFR1high or VEGFR1low) were seeded in biodegradable scaffolds and transplanted into the subcutaneous space of immunodeficient mice. Despite proliferating at a similar rate, VEGFR1high generated more in vitro sprouts than VEGFR1low cells (p < 0.05). Blockade of VEGF signaling with bevacizumab inhibited VEGFR1high-derived sprouts, demonstrating specificity of responses. Similarly, VEGFR1high SHED generated more blood vessels when transplanted into murine hosts than VEGFR1low cells (p < 0.05). Collectively, these data demonstrated that DPSCs contain a unique sub-population of cells defined by high VEGFR1 expression that are primed to differentiate into vascular endothelial cells. These data raise the possibility of purifying stem cells with high vasculogenic potential for regeneration of vascularized tissues or for vascular engineering in the treatment of ischemic conditions.

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.

Simvastatin inhibits the expression of inflammatory cytokines and cell adhesion molecules induced by LPS in human dental pulp cells.

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)-1ß (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-1ß, 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.

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.

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.

Dental pulp stem cell responses to novel antibiotic-containing scaffolds for regenerative endodontics.

AIM: To evaluate both the drug-release profile and the effects on human dental pulp stem cells' (hDPSC) 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 hDPSCs' 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 hDPSCs proliferation when compared to the saturated CIP/MET solution (DAP).

Effect of Biodentine and Bioaggregate on odontoblastic differentiation via mitogen-activated protein kinase pathway in human dental pulp cells.

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) mRNA 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 mRNA 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.

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.

Injectable Tissue-Specific Hydrogel System for Pulp-Dentin Regeneration.

The quest for finding a suitable scaffold system that supports cell survival and function and, ultimately, the regeneration of the pulp-dentin complex remains challenging. Herein, we hypothesized that dental pulp stem cells (DPSCs) encapsulated in a collagen-based hydrogel with varying stiffness would regenerate functional dental pulp and dentin when concentrically injected into the tooth slices. Collagen hydrogels with concentrations of 3 mg/mL (Col3) and 10 mg/mL (Col10) were prepared, and their stiffness and microstructure were assessed using a rheometer and scanning electron microscopy, respectively. DPSCs were then encapsulated in the hydrogels, and their viability and differentiation capacity toward endothelial and odontogenic lineages were evaluated using live/dead assay and quantitative real-time polymerase chain reaction. For in vivo experiments, DPSC-encapsulated collagen hydrogels with different stiffness, with or without growth factors, were injected into pulp chambers of dentin tooth slices and implanted subcutaneously in severe combined immunodeficient (SCID) mice. Specifically, vascular endothelial growth factor (VEGF [50 ng/mL]) was loaded into Col3 and bone morphogenetic protein (BMP2 [50 ng/mL]) into Col10. Pulp-dentin regeneration was evaluated by histological and immunofluorescence staining. Data were analyzed using 1-way or 2-way analysis of variance accordingly (α = 0.05). Rheology and microscopy data revealed that Col10 had a stiffness of 8,142 Pa with a more condensed and less porous structure, whereas Col3 had a stiffness of 735 Pa with a loose microstructure. Furthermore, both Col3 and Col10 supported DPSCs' survival. Quantitative polymerase chain reaction showed Col3 promoted significantly higher von Willebrand factor (VWF) and CD31 expression after 7 and 14 d under endothelial differentiation conditions (P < 0.05), whereas Col10 enhanced the expression of dentin sialophosphoprotein (DSPP), alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), and collagen 1 (Col1) after 7, 14, and 21 d of odontogenic differentiation (P < 0.05). Hematoxylin and eosin and immunofluorescence (CD31 and vWF) staining revealed Col10+Col3+DPSCs+GFs enhanced pulp-dentin tissue regeneration. In conclusion, the collagen-based concentric construct modified by growth factors guided the specific lineage differentiation of DPSCs and promoted pulp-dentin tissue regeneration in vivo.

Role of Heparan Sulfate in Vasculogenesis of Dental Pulp Stem Cells.

Dental pulp stem cells (DPSCs) can differentiate into vascular endothelial cells and display sprouting ability. During this process, DPSC responses to the extracellular microenvironment and cell-extracellular matrix interactions are critical in regulating their ultimate cell fate. Heparan sulfate (HS) glycosaminoglycan, a major component of extracellular matrix, plays important roles in various biological cell activities by interacting with growth factors and relative receptors. However, the regulatory function of HS on vasculogenesis of mesenchymal stem cells remains unclear. The objective of this study was to investigate the role of HS in endothelial differentiation and vasculogenesis of DPSCs. Our results show that an HS antagonist suppressed the proliferation and sprouting ability of DPSCs undergoing endothelial differentiation. Furthermore, expression of proangiogenic markers significantly declined with increasing dosages of the HS antagonist; in contrast, expression of stemness marker increased. Silencing of exostosin 1 (EXT1), a crucial glycosyltransferase for HS biosynthesis, in DPSCs using a short hairpin RNA significantly altered their gene expression profile. In addition, EXT1-silenced DPSCs expressed lower levels of endothelial differentiation markers and displayed a reduced vascular formation capacity compared with control DPSCs transduced with scrambled sequences. The sprouting ability of EXT1-silenced DPSCs was rescued by the addition of exogenous HS in vitro. Next, we subcutaneously transplanted biodegradable scaffolds seeded with EXT1-silenced or control DPSCs into immunodeficient mice. Lumen-like structures positive for human CD31 and von Willebrand factor were formed by green fluorescent protein-transduced DPSCs. Numbers of blood-containing vessels were significantly lower in scaffolds loaded with EXT1-silenced DPSCs than specimens implanted with control DPSCs. Collectively, our findings unveil the crucial role of HS on endothelial differentiation and vasculogenesis of DPSCs, opening new perspectives for the application of HS to tissue engineering and dental pulp regeneration.

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.

Tooth slice/scaffold model of dental pulp tissue engineering.

Multipotency is a defining characteristic of post-natal stem cells. The human dental pulp contains a small subpopulation of stem cells that exhibit multipotency, as demonstrated by their ability to differentiate into odontoblasts, neural cells, and vascular endothelial cells. These discoveries highlight the fundamental role of stem cells in the biology of the dental pulp and suggest that these cells are uniquely suited for dental pulp tissue-engineering purposes. The availability of experimental approaches specifically designed for studies of the differentiation potential of dental pulp stem cells has played an important role in these discoveries. The objective of this review is to describe the development and characterization of the Tooth Slice/Scaffold Model of Dental Pulp Tissue Engineering. In addition, we discuss the multipotency of dental pulp stem cells, focusing on the differentiation of these cells into functional odontoblasts and into vascular endothelial cells.

Fabrication of Vascularized DPSC Constructs for Efficient Pulp Regeneration.

Dental pulp regeneration is a promising approach to restore the vitality of necrotic teeth. We have previously reported the fabrication of scaffold-free cell constructs containing only dental pulp stem cells (DPSCs) and their ability to form pulp-like tissue in the pulpless tooth. However, the DPSC construct could not build pulp-like tissue with a full root length because it is difficult to induce blood vessels from a small root canal foramen. Therefore, we hypothesized that vascular structure could be preformed in the DPSC construct by employing endothelial differentiation capability of DPSCs, and vascularized constructs might facilitate dental pulp regeneration in the pulpless tooth. In this study, vascularized DPSC constructs were fabricated by inducing endothelial differentiation, and then we investigated the behavior of differentiated DPSCs, the internal structure of cell constructs, and their pulp regenerative ability in vivo. We observed that DPSCs positive for CD31 and von Willebrand factor were localized at the outer layer of constructs and formed a reticulated lumen structure. The cells constituting the outer layer of the construct expressed endothelial differentiation markers at higher levels than cells in the inner part. These results indicated that DPSCs in the outer layer differentiated into endothelial cells and formed vascular-like structures in the cell construct. Next, a vascularized DPSC construct was transplanted into the human pulpless tooth that was implanted into immunodeficient mice in the subcutaneous space. After 6 wk of implantation, the vascularized construct formed pulp-like tissues with higher density of human CD31-positive blood vessels when compared with specimens implanted with a DPSC construct without prevascularization. These results suggest that the vascular structure formed in the DPSC construct facilitated the blood supply and enhanced pulp regeneration. This study demonstrates that a vascularized DPSC construct is a prospective biomaterial as an implant for novel dental pulp regeneration.

mTOR Inhibition Ablates Cisplatin-Resistant Salivary Gland Cancer Stem Cells.

Patients with advanced salivary gland mucoepidermoid carcinoma (MEC) are treated with surgery and radiotherapy, as current systemic therapies are largely ineffective. As such, current treatment frequently leads to poor long-term survival due to locoregional recurrence or metastases. We have shown that salivary gland cancer stem cells (CSCs) are resistant to platinum-based chemotherapy and drive tumor progression. The purpose of this study was to investigate the effect of therapeutic inhibition of mTOR (mechanistic target of rapamycin) on resistance of CSCs to cisplatin, a prototypic platinum-based chemotherapeutic agent. Viability assays determined the effect of several inhibitors of PI3k/mTOR signaling (e.g., temsirolimus, BKM120, AZD8055, PF4708671) and/or cisplatin on survival of human MEC cells. The impact of mTOR inhibitors and/or cisplatin on MEC stemness was examined with salisphere assays, flow cytometry for ALDH/CD44 (CSC markers for MEC), and Western blots for Bmi-1 expression (marker of stem cell self-renewal). Salivary gland MEC patient-derived xenografts were used to examine the effect of cisplatin and/or temsirolimus on CSCs in vivo. We observed that cisplatin induced mTOR and S6K1 phosphorylation, increased the number and size of MEC salispheres, and induced Bmi-1 expression and the fraction of CSCs in MEC models in vitro. Cisplatin also increased the fraction of CSCs in vivo. In contrast, mTOR inhibition (e.g., temsirolimus) blocked cisplatin-induced Bmi-1 expression and salisphere formation in vitro. Remarkably, temsirolimus slowed down tumor growth and decreased the fraction of CSCs (P < 0.05) even in presence of cisplatin in a short-term in vivo experiment. Collectively, these results demonstrate that therapeutic inhibition of mTOR ablates cytotoxic-resistant CSCs, and they suggest that a combination of an mTOR inhibitor and platinum-based chemotherapy might be beneficial to patients with salivary gland mucoepidermoid carcinoma.

Endothelial-Initiated Crosstalk Regulates Dental Pulp Stem Cell Self-Renewal.

Interactions with the microenvironment modulate the fate of stem cells in perivascular niches in tissues (e.g., bone) and organs (e.g., liver). However, the functional relevance of the molecular crosstalk between endothelial cells and stem cells within the perivascular niche in dental pulps is unclear. Here, we tested the hypothesis that endothelial cell-initiated signaling is necessary to maintain self-renewal of dental pulp stem cells. Confocal microscopy showed that ALDH1high and Bmi-1high stem cells are preferentially localized in close proximity to blood vessels in physiological human dental pulps. Secondary orosphere assays revealed that endothelial cell-derived factors (e.g., interleukin-6 [IL-6]) promote self-renewal of dental pulp stem cells cultured in low-attachment conditions. Mechanistic studies demonstrated that endothelial cell-derived IL-6 activates IL-6R (IL-6 Receptor) and signal transducer and activator of transcription 3 (STAT3) signaling and induces expression of Bmi-1 (master regulator of stem cell self-renewal) in dental pulp stem cells. Transplantation of dental pulp stem cells stably transduced with small hairpin RNA (shRNA)-STAT3 into immunodeficient mice revealed a decrease in the number of blood vessels surrounded by ALDH1high or Bmi-1high cells (perivascular niches) compared to tissues formed upon transplantation of vector control stem cells. And finally, in vitro capillary sprouting assays revealed that inhibition of IL-6 or STAT3 signaling decreases the vasculogenic potential of dental pulp stem cells. Collectively, these data demonstrate that endothelial cell-derived IL-6 enhances the self-renewal of dental pulp stem cells via STAT3 signaling and induction of Bmi-1. These data suggest that a crosstalk between endothelial cells and stem cells within the perivascular niche is required for the maintenance of stem cell pools in dental pulps.

VE-Cadherin and Anastomosis of Blood Vessels Formed by Dental Stem Cells.

It is known that dental pulp stem cells (DPSCs) can be induced to differentiate into vasculogenic endothelial (VE) cells. However, the process that results in sprouting and anastomosis of DPSC-derived vessels remains unclear. Here, we performed studies to understand the mechanisms underpinning the anastomosis of the host vasculature with blood vessels generated by DPSCs (a model for mesenchymal stem cells). VE-cadherin-silenced primary human DPSCs seeded in tooth slice/scaffolds and transplanted into the subcutaneous space of immunodeficient mice generated fewer functional blood vessels (i.e., anastomosed with the host vasculature) than control DPSCs transduced with scrambled sequences. Both VE-cadherin-silenced and mitogen-activated protein kinase kinase 1 (MEK1)-silenced cells showed a decrease in the number of capillary sprouts in vitro. Interestingly, DPSC stably transduced with a VE-cadherin reporter demonstrated that vascular endothelial growth factor (VEGF) induces VE-cadherin expression in sprouting DPSCs undergoing anastomosis, but not in quiescent DPSCs. To begin to understand the mechanisms regulating VE-cadherin, we stably silenced MEK1 and observed that VEGF was no longer able to induce VE-cadherin expression and capillary sprout formation. Notably ERG, a transcriptional factor downstream from MEK/ERK, binds to the promoter region of VE-cadherin (chip assay) and is induced by VEGF in DPSCs. Collectively, these data defined a signaling pathway triggered by VEGF that results in phosphorylation of MEK1/ERK and activation of ERG leading to expression of VE-cadherin, which is required for anastomosis of DPSC-derived blood vessels. In conclusion, these results unveiled a signaling pathway that enables the generation of functional blood vessels upon vasculogenic differentiation of DPSCs.

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.

Unidirectional crosstalk between Bcl-xL and Bcl-2 enhances the angiogenic phenotype of endothelial cells.

Expression of Bcl-x(L) 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-x(L) in angiogenesis is unclear. Here, we showed that epidermal growth factor (EGF) induces in vitro capillary sprouting and Bcl-x(L) expression in primary endothelial cells. Bcl-x(L)-transduced human dermal microvascular endothelial cells (HDMEC-Bcl-x(L)), but not empty vector control cells, spontaneously organize into capillary-like sprouts. Searching for a mechanism to explain these responses, we observed that Bcl-x(L) 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-x(L). Bcl-x(L) led to Bcl-2 upregulation, but Bcl-2 did not upregulate Bcl-x(L), suggesting the existence of a unidirectional crosstalk from Bcl-x(L) to Bcl-2. EGF and Bcl-x(L) 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-x(L) 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-x(L), and markedly decreased angiogenesis in vivo. We conclude that Bcl-x(L) 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.

Antigen-presenting cells in human radicular granulomas.

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.