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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.

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.

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.

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 ALDH1 high and Bmi-1 high 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 ALDH1 high or Bmi-1 high 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.

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 RT 2 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.

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., (© 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

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., (© 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

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., (© 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

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)., (© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

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., (© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

In previous studies, fluorapatite (FA) crystal-coated surfaces have been shown to stimulate the differentiation and mineralization of human dental pulp stem cells (DPSCs) in two-dimensional cell culture. However, whether the FA surface can recapitulate these properties in three-dimensional culture is still unknown. This study examined the differences in behavior of human DPSCs cultured on electrospun polycaprolactone (PCL) NanoECM nanofibers with or without the FA crystals. Under near-physiologic conditions, the FA crystals were synthesized on the PCL nanofiber scaffolds. The FA crystals were evenly distributed on the scaffolds. DPSCs were cultured on the PCL+FA or the PCL scaffolds for up to 28 days. Scanning electron microscope images showed that DPSCs attached well to both scaffolds after the initial seeding. However, it appeared that more multicellular aggregates formed on the PCL+FA scaffolds. After 14 days, the cell proliferation on the PCL+FA was slower than that on the PCL-only scaffolds. Interestingly, even without any induction of mineralization, from day 7, the upregulation of several pro-osteogenic molecules (dmp1, dspp, runx2, ocn, spp1, col1a1) was detected in cells seeded on the PCL+FA scaffolds. A significant increase in alkaline phosphatase activity was also seen on FA-coated scaffolds compared with the PCL-only scaffolds at days 14 and 21. At the protein level, osteocalcin expression was induced only in the DPSCs on the PCL+FA surfaces at day 21 and then significantly enhanced at day 28. A similar pattern was observed in those specimens stained with Alizarin red and Von Kossa after 21 and 28 days. These data suggest that the incorporation of FA crystals within the three-dimensional PCL nanofiber scaffolds provided a favorable extracellular matrix microenvironment for the growth, differentiation, and mineralization of human DPSCs. This FA-modified PCL nanofiber scaffold shows promising potential for future bone, dental, and orthopedic regenerative applications., (© International & American Associations for Dental Research.)

The clinical translation of stem-cell-based dental pulp regeneration will require the use of injectable scaffolds. Here, we tested the hypothesis that stem cells from exfoliated deciduous teeth (SHED) can generate a functional dental pulp when injected into full-length root canals. SHED survived and began to express putative markers of odontoblastic differentiation after 7 days when mixed with Puramatrix™ (peptide hydrogel), or after 14 days when mixed with recombinant human Collagen (rhCollagen) type I, and injected into the root canals of human premolars in vitro. Roots of human premolars injected with scaffolds (Puramatrix™ or rhCollagen) containing SHED were implanted subcutaneously into immunodeficient mice (CB-17 SCID). We observed pulp-like tissues with odontoblasts capable of generating new tubular dentin throughout the root canals. Notably, the pulp tissue engineered with SHED injected with either Puramatrix™ or rhCollagen type I presented similar cellularity and vascularization when compared with control human dental pulps. Analysis of these data, collectively, demonstrates that SHED injected into full-length human root canals differentiate into functional odontoblasts, and suggests that such a strategy might facilitate the completion of root formation in necrotic immature permanent teeth.

The discovery that dental pulp stem cells are capable of differentiating into endothelial cells raises the exciting possibility that these cells can be a single source of odontoblasts and vascular networks in dental tissue engineering. The purpose of this study was to begin to define signaling pathways that regulate endothelial differentiation of SHED. Stem cells from exfoliated deciduous teeth (SHED) exposed to endothelial growth medium (EGM-2MV) supplemented with vascular endothelial growth factor (VEGF) differentiated into VEGFR2-positive and CD31-positive endothelial cells in vitro. In vivo, VEGFR1-silenced SHED seeded in tooth slice/ scaffolds and transplanted into immunodeficient mice showed a reduction in human CD31-positive blood vessels as compared with controls (p = 0.02). Exposure of SHED to EGM2-MV supplemented with VEGF induced potent activation of ERK and Akt signaling, while it inhibited phosphorylation of STAT3. Notably, genetic (MEK1 silencing) or chemical (U0126) inhibition of ERK signaling restored constitutive STAT3 phosphorylation and inhibited the differentiation of SHED into endothelial cells. Collectively, analysis of these data unveiled the VEGF/MEK1/ERK signaling pathway as a key regulator of the endothelial differentiation of dental pulp stem cells.

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.

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., (© 2010 International Endodontic Journal.)

Traumatic dental injuries are often irreversible, underscoring the need for therapies that protect dental pulp cells and enhance their regeneration. We hypothesized that generation 5 poly amido amine (PAMAM) dendrimers (G5), functionalized with fluorescein isothiocyanate (FL) and αVβ3-specific, cyclic arginine-glycine-aspartic acid (RGD) peptides, will bind to dental pulp cells (DPCs) and modulate their differentiation. Dental pulp cells and mouse odontoblast-like cells (MDPC-23) (±) treated with G5-FL-RGD were analyzed via Western blot, RT-PCR, and quantitative PCR. Transcription of dental differentiation markers was as follows: Dentin matrix protein (DMP-1), dentin sialoprotein (DSPP), and matrix extracellular phosphoglycoprotein (MEPE) as well as vascular endothelial growth factor (VEGF) all increased via the JNK pathway. Long-term G5-RGD treatment of dental pulp cells resulted in enhanced mineralization as examined via Von Kossa assay, suggesting that PAMAM dendrimers conjugated to cyclic RGD peptides can increase the odontogenic potential of these cells.

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.

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 zeta) 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.

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 Gram-positive 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.

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)

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 rhVEGF(165) 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 rhVEGF(165) for one hour prior to implantation into the subcutaneous space of immunodeficient mice. Treatment with rhVEGF(165) increased pulp microvessel density in vivo (p < 0.05). These results demonstrate that rhVEGF(165) 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.

The application of an adhesive resin near or directly over the pulp was shown to induce pulp inflammation and lack of dentin regeneration. We hypothesize that the absence of dentin bridging is due to adhesive-resin-induced apoptosis of cells responsible for pulp healing and dentin regeneration. Mouse odontoblast-like cells (MDPC-23), undifferentiated pulp cells (OD-21), or macrophages (RAW 264.7) were exposed to SingleBond polymerized for 0-40 seconds. Annexin V and propidium iodide assays demonstrated that SingleBond induced apoptosis of MDPC-23, OD-21, and macrophages. The proportion of apoptotic cells was dependent on the degree of adhesive resin polymerization. Adhesive-resin-induced death of pulp cells was associated with activation of the pro-apoptotic cysteine protease Caspase-3. Interestingly, most cells exposed to adhesive resin that did not undergo apoptosis showed cell-cycle arrest. We conclude that an adhesive resin induces apoptosis and cell-cycle arrest of cells involved in the regeneration of the dentin-pulp complex in vitro.

Vascular endothelial growth factor (VEGF) is a potent inducer of angiogenesis, vascular permeability, and edema. Up-regulation of VEGF expression in the dental pulp may result in increased intra-pulpal pressure, and contribute to pain and irreversible tissue damage. Lipoteichoic acid (LTA) is an amphiphilic molecule from Gram-positive bacteria that has been associated with the pathogenesis of pulpitis. To investigate if LTA regulates expression of VEGF, we exposed mouse odontoblast-like cells (MDPC-23), undifferentiated pulp cells (OD-21), fibroblasts, or macrophages to streptococcal LTA, and evaluated VEGF expression by ELISA and RT-PCR. LTA induced up to a nine-fold increase in VEGF protein expression in macrophages, a 2.4-fold increase in MDPC-23, and a 1.6-fold increase in OD-21 as compared with controls. In contrast, LTA did not induce VEGF expression in fibroblasts. VEGF mRNA expression remained constant upon exposure to LTA, which suggests that VEGF regulation in these cells is primarily post-transcriptional. This work constitutes the first demonstration that lipoteichoic acid is sufficient to induce expression of a pro-angiogenic factor.

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]

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]

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]

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.