Effect of Low-Level Diode Laser and Red Light-Emitting Diode on Survival and Osteogenic/Odontogenic Differentiation of Human Dental Pulp Stem Cells.

Background: This investigation set out to compare the impacts of low-level diode laser (LLDL) and red light-emitting diode (LED) on the survival of human dental pulp stem cells (hDPSCs) and osteogenic/odontogenic differentiation. Methods and materials: In this ex vivo experimental study, the experimental groups underwent the irradiation of LLDL (4 J/cm2 energy density) and red LED in the osteogenic medium. Survival of hDPSCs was assessed after 24 and 48 h (n = 9) using the methyl thiazolyl tetrazolium (MTT) assay. The assessment of osteogenic/odontogenic differentiation was conducted using alizarin red staining (ARS; three repetitions). The investigation of osteogenic and odontogenic gene expression was performed at two time points, specifically 24 and 48 h (n = 12). This analysis was performed utilizing real-time reverse-transcription polymerase chain reaction (RT-PCR). The groups were compared at each time point using SPSS version 24. To analyze the data, the Mann-Whitney U test, analysis of variance, Tukey's test, and t-test were utilized. Results: The MTT assay showed that LLDL significantly decreased the survival of hDPSCs after 48 h, compared with other groups (p < 0.05). The qualitative results of ARS revealed that LLDL and red LED increased the osteogenic differentiation of hDPSCs. LLDL and red LED both upregulated the expression of osteogenic/odontogenic genes, including bone sialoprotein (BSP), alkaline phosphatase (ALP), dentin matrix protein 1 (DMP1), and dentin sialophosphoprotein (DSPP), in hDPSCs. The LLDL group exhibited a higher level of gene upregulation (p < 0.0001). Conclusions: The cell survival of hDPSCs was reduced, despite an increase in osteogenic/odontogenic activity. Clinical relevance: Introduction of noninvasive methods in regenerative endodontic treatments.

Interference with the retinoic acid signalling pathway inhibits the initiation of teeth and caudal primary scales in the small-spotted catshark Scyliorhinus canicula.

The retinoic acid (RA) pathway was shown to be important for tooth development in mammals, and suspected to play a key role in tooth evolution in teleosts. The general modalities of development of tooth and "tooth-like" structures (collectively named odontodes) seem to be conserved among all jawed vertebrates, both with regard to histogenesis and genetic regulation. We investigated the putative function of RA signalling in tooth and scale initiation in a cartilaginous fish, the small-spotted catshark Scyliorhinus canicula. To address this issue, we identified the expression pattern of genes from the RA pathway during both tooth and scale development and performed functional experiments by exposing small-spotted catshark embryos to exogenous RA or an inhibitor of RA synthesis. Our results showed that inhibiting RA synthesis affects tooth but not caudal primary scale development while exposure to exogenous RA inhibited both. We also showed that the reduced number of teeth observed with RA exposure is probably due to a specific inhibition of tooth bud initiation while the observed effects of the RA synthesis inhibitor is related to a general delay in embryonic development that interacts with tooth development. This study provides data complementary to previous studies of bony vertebrates and support an involvement of the RA signalling pathway toolkit in odontode initiation in all jawed vertebrates. However, the modalities of RA signalling may vary depending on the target location along the body, and depending on the species lineage.

Acetylsalicylic-acid (ASA) regulation of osteo/odontogenic differentiation and proliferation of human dental pulp stem cells (DPSCs) in vitro.

OBJECTIVE: The study aimed to investigate acetylsalicylic acid (ASA) effects on osteo/odontogenic differentiation and proliferation of dental pulp stem cells (DPSCs) in vitro and the potential involvement of adenosine monophosphate-activated protein kinase (AMPK) pathway in these processes. DESIGN: DPSCs were isolated from third molars pulp tissues of five patients and grown in osteogenic medium alone or supplemented with ASA. Expression of DPSCs markers was tested by flow-cytometry. Cytotoxicity of ASA at concentrations of 10, 50 and 100 µg/ml was tested by MTT and NR assays. Osteo/odontogenic differentiation was analyzed via alizarin red staining and ALP activity. Quantitative PCR (qPCR) was used for osteo/odontogenic markers' (DSPP, BMP2, BMP4, BSP, OCN and RUNX2) and c-Myc expression analysis. AMPK inhibition of ASA-induced osteo/odontogenesis was tested by qPCR of selected markers (DSPP, OCN and RUNX2). RESULTS: Cytotoxicity assays showed that only the highest ASA dose decreased cell viability (89.1 %). The smallest concentration of ASA applied on DPSCs resulted in a remarkable enhancement of osteo/odontogenic differentiation, as judged by increased mineralized nodules' formation, ALP activity and gene expression of analyzed markers (increase between 2 and 30 folds), compared to untreated cells. ASA also increased DPSCs proliferation. Interestingly, AMPK inhibition per se upregulated DSPP, OCN and RUNX2; the gene upregulation was higher when ASA treatment was also included. c-Myc expression level decreased in cultures treated with ASA, indicating undergoing differentiation processes. CONCLUSIONS: Low concentrations of ASA (corresponding to the standard use in cardiovascular patients), were shown to stimulate osteo/odontogenic differentiation of dental pulp stem cells.

Proinflammatory State in the Odontogenesis of Fetuses Exposed to Different Types of Fatty Acids during Pregnancy.

OBJECTIVES: The aim of the present study was to analyze the possible changes caused by the maternal ingestion of different types of fatty acids during pregnancy in the proinflammatory state in the odontogenesis of the fetuses. SUBJECT AND METHODS: Twenty-four jaws (n = 6 per group) of Wistar rats were collected on the 20th day of intrauterine life. Mothers were separated on the first day of pregnancy into 4 groups according to diet, as described below: control group (C) - diet with soy oil as a source of fat; saturated fatty acid group (S) - diet with lard in saturated fatty acids; trans-fatty acid group (T) - diet with vegetable fat, rich in trans-saturated fatty acids; and polyunsaturated fatty acid (PUFA) group - diet with fish oil, rich in PUFAs. RESULTS: Microscopic analysis showed no alterations in tissue development of the teeth between the groups with different lipid diets (T, S, and PUFA) when compared to the control group (C); immunohistochemical analysis for the expression of JAK2, STAT3, P-STAT3, SOCS3, and IL-6 showed no statistically significant difference (p > 0.05) compared to the control group. However, there were changes (p < 0.05) between the T group and the PUFA group in the expression of JAK2. CONCLUSION: Thus, lipid consumption in the maternal diet remains a topic to be explored in embryonic development, despite not causing morphological changes to the tooth germ of rats.

Odontogenesis by Endocytosis of Peptide Embedding Bioactive Glass Composite.

Limited therapeutic options are available for treating deep caries. Those materials with potential of a dual effect to remineralize hard tissue and regenerate defective dentin tissues could be used as a new strategy for deep caries treatment. However, the application of the single component remains a challenge mainly because they lack calcium and phosphorus, are easily degraded, and are difficult to retain in the intricate body fluid environment. Considering the abundant source of calcium and phosphorus as well as the delivery performance of mesoporous bioactive glass (MBG), an amelogenin-derived peptide (QP5), which has a significant role in hard tissue remineralization, was loaded to fabricate a novel composite. After the synthesis of highly ordered MBG using a sol-gel method, the QP5 peptide was loaded increasingly by its extensive porous structure and enhanced electrostatic absorption. When used in an acidic environment, the MBG/QP5 composite presented pH-responsiveness, releasing therapeutic ions and functional peptides in a sequential cascade, and eventually adjusted the pH to a neutral state. The composite was internalized by dental pulp cells through a clathrin-mediated pathway and influenced by cell membrane lipid raft regulation. It could be also transported through the macro-pinocytotic pathway. Compared to the single treatment of peptide QP5 in 48 h, the composite facilitated a higher level of retention of the intracellular peptides. The composite further promoted migration and odontogenesis of dental pulp cells, including the improved activity of alkaline phosphatase, increased formation of mineralized nodules, and upregulated expression of mineralization-related genes compared to using MBG or QP5 alone. The composite further induced the dentin-like layer in a rat pulp capping model. The results suggested that this intelligent material with pH-responsiveness provides a promising alternative treatment method for biomimetic restoration of deep caries.

Photobiomodulation therapy does not depend on the differentiation of dental pulp cells to enhance functional activity associated with angiogenesis and mineralization.

The purpose of this study is to analyze the influence of InGaAlP diode laser (660 nm) with or without an odontogenic medium (OM) in the functional activity of OD-21 cells. Undifferentiated OD-21 pulp cells were cultivated with or without OM and divided into four groups (n = 5): nonirradiated control (C -), nonirradiated + OM (C +), irradiated (L -), and irradiated + OM (L +). Laser application was performed in two sessions of a 24-h interval with an irradiance of 11.3 mW/cm2, energy density of 1 J/cm2, and total cumulative energy/well of 4.6 J. Cell proliferation, VEGF-164 expression, mineralization, and expression of Alp, Runx2, and Dmp1 genes, as well as immunolocalization of RUNX2 and MEPE proteins, were evaluated. Data were analyzed by statistical tests (α = 0.05). All studied groups showed a similar increase in cell proliferation with or without OM. After 7 and 10 days, a significatively higher concentration of VEGF-164 in L - group when compared to C - group was observed. A significant increase in mineralized nodules in the L + was noted when compared to C + in the same conditions. Photobiomodulation upregulated significantly Runx2 and Dmp1 expression after 10 days in L - and after 7 days in L + , with downregulation of Dmp1 after 10 days in L + group. Immunolocalization of RUNX2 and MEPE was expressive after 7 days of culture in the cytoplasm adjacent to the nucleus with a decrease after 10 days, regardless of the presence of OM. Photobiomodulation enhances metabolism associated with angiogenesis, gene expression, and mineralization regardless of the odontogenic medium in OD-21 cells.

Investigation of in vitro odonto/osteogenic capacity of cannabidiol on human dental pulp cell.

INTRODUCTION: Vital pulp treatment (VPT) maintains tooth vitality with certain dental materials by protecting pulp from noxious stimulation and promoting repair through enhancing cell proliferation/differentiation, migration, and inducing odontogenesis. As a non-psychotropic cannabis constituent, cannabidiol (CBD) possesses the properties of analgesic, anti-inflammation, and osteogenesis. Therefore, we hypothesize that CBD may induce the odonto/osteogenesis of human dental pulp cells (HDPCs), a critical feature using as effective pulp capping agent for VPT. MATERIALS AND METHODS: In this in vitro study, the cytotoxicity of CBD on HDPCs was determined by MTT assay. Scratch assay was performed to analyze HDPC migration. The biomineralization was examined by collagen synthesis and calcium nodule formation and related odonto/osteogenic and angiogenic genes. Cannabinoid receptor (CB) specificity was evaluated by Western blotting and Von Kossa staining using specific antagonists AM251 for cannabinoid receptor 1 (CB1) and AM 630 targeted at cannabinoid receptor 2 (CB2). In addition, the underlying molecular mechanism of CBD-induced biomineralization were investigated by examining CB-dependent MAPK signaling pathways. RESULTS: CBD demonstrated bi-phasic effects on HDPC viability in tested concentrations. We found CBD significantly promoted cell migration, enhanced collagen synthesis and mineralized deposits in HDPCs when treated by 1 µM CBD supplemented in the differentiation media. RT-PCR revealed CBD increased the expression of angiogenic and odontogenic genes, such as DSPP, DMP-1, OPN, ALP, Runx2, VEGFR1 and ICAM-1. These effects were via MAPK activation in a manner mainly mediated by CB2. CONCLUSION: The results from this study suggested that CBD can induce odonto/osteogenesis from HDPCs and has the potential to develop new therapeutics in VPT in dentistry.

A novel bio-active adhesive monomer induces odontoblast differentiation: a comparative study.

AIM: To evaluate the in vitro effect of the novel adhesive monomer CMET, a calcium salt of 4-methacryloxyethyl trimellitate (4-MET), on the proliferation, mineralization and differentiation of odontoblast-like cells, comparing with 4-MET, calcium hydroxide (CH) and mineral trioxide aggregate (MTA). METHODOLOGY: Rat odontoblast-like MDPC-23 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% foetal bovine serum. The powder of four tested materials (CMET, 4-MET, CH and MTA) was first dissolved in distilled water (dH2O) and then was diluted by DMEM to yield final concentrations. Solvent (dH2O) was used as a control. Cell viability was assessed using CCK-8 assay. Real-time RT-PCR was used to quantify the mRNA expression of odontogenic markers, cytokines and integrins. Mineralization inducing capacity was evaluated by alkaline phosphatase (ALPase) activity and alizarin red S staining. Statistical analyses were performed using one-way anova and post hoc Tukey's HSD test, with the significance level at 1%. RESULTS: Cell viability was significantly greater in the CMET- (83 to 828 mmol L-1), CH- and MTA-treated (low concentrations) groups than that in the control group (P < 0.01). Higher concentrations of each material decreased the viable cells to different extents (P < 0.01). CMET treatment augmented the expression of several integrin subunits and exhibited the highest mRNA expression levels of odontogenic markers among all groups (P < 0.01). CH and MTA treatment caused significantly greater upregulation of pro-inflammatory cytokines expression than the other groups (P < 0.01). The calcific deposition of MDPC-23 cells was dose-dependently accelerated by the addition of CMET (P < 0.01); the enhancement of mineralization was also found in the fresh prepared CH and MTA treatments. Besides, CMET showed consistency in mineralization induction after 8 weeks storage. Exposure to SB202190, a specific p38 mitogen-activated protein kinases inhibitor, significantly decreased the ALPase activity as well as the mineral deposition which was enhanced by CMET treatment (P < 0.01). CONCLUSIONS: The novel bio-active monomer had the lowest cytotoxicity among all groups and it induced the proliferation, mineralization and differentiation of odontoblast-like cells under appropriate concentrations. This adhesive monomer possesses excellent biocompatibility and hence exhibits great potential in dentine regeneration.

Yunnan Baiyao Conditioned Medium Promotes the Odonto/Osteogenic Capacity of Stem Cells from Apical Papilla via Nuclear Factor Kappa B Signaling Pathway.

Yunnan Baiyao is a traditional Chinese herbal remedy that has long been used for its characteristics of wound healing, bone regeneration, and anti-inflammation. However, the effects of Yunnan Baiyao on the odonto/osteogenic differentiation of stem cells from apical papilla (SCAPs) and the potential mechanisms remain unclear. The aim of this study was to investigate the odonto/osteogenic differentiation effects of Yunnan Baiyao on SCAPs and the underlying mechanisms involved. SCAPs were isolated and cocultured with Yunnan Baiyao conditioned media. The proliferation ability was determined by cell counting kit 8 and flow cytometry. The differentiation capacity and the involvement of NF-κB pathway were investigated by alkaline phosphatase assay, alizarin red staining, immunofluorescence assay, real-time RT-PCR, and western blot analyses. Yunnan Baiyao conditioned medium at the concentration of 50 µg/mL upregulated alkaline phosphatase activity, induced more mineralized nodules, and increased the expression of odonto/osteogenic genes/proteins (e.g., OCN/OCN, OPN/OPN, OSX/OSX, RUNX2/RUNX2, ALP/ALP, COL-I/COL-I, DMP1, DSP/DSPP) of SCAPs. In addition, the expression of cytoplasmic phos-IκBα, phos-P65, and nuclear P65 was significantly increased in Yunnan Baiyao conditioned medium treated SCAPs in a time-dependent manner. Conversely, the differentiation of Yunnan Baiyao conditioned medium treated SCAPs was obviously inhibited when these stem cells were cocultured with the specific NF-κB inhibitor BMS345541. Yunnan Baiyao can promote the odonto/osteogenic differentiation of SCAPs via the NF-κB signaling pathway.

Low-level laser therapy affects dentinogenesis and angiogenesis of in vitro 3D cultures of dentin-pulp complex.

To investigate the effects of gallium-aluminum-arsenide (GaAlAs) diode laser low-level laser therapy (LLLT) on angiogenesis and dentinogenesis of the dentin-pulp complex in a human tooth slice-based in vitro model. Forty tooth slices were prepared from 31 human third molars. Slices were cultured at 37 °C, 5% CO2, and 95% humidity and randomly assigned to one of the following groups: group I: no laser treatment, group II: 660-nm diode laser; energy density = 1 J/cm2, group III: 660-nm diode laser; energy density = 3 J/cm2, group IV: 810-nm diode laser; energy density = 1 J/cm2 and group V: 810-nm diode laser; energy density = 3 J/cm2. LLLT was applied on the third and fifth days of culture. After 7 days, tissues were retrieved for real-time RT-PCR analysis to investigate the expression of VEGF, VEGFR2, DSPP, DMP-1, and BSP in respect to controls. Lower energy density (1 J/cm2) with the 660 nm wavelength showed a statistically significant up-regulation of both angiogenic (VEGF: 15.3-folds and VEGFR2: 3.8-folds) and odontogenic genes (DSPP: 6.1-folds, DMP-1: 3-fold, and BSP: 6.7-folds). While the higher energy density (3 J/cm2) with the 810 nm wavelength resulted in statistically significant up-regulation of odontogenic genes (DSPP: 2.5-folds, DMP-1: 17.7-folds, and BSP: 7.1-folds), however, the angiogenic genes had variable results where VEGF was up-regulated while VEGFR2 was down-regulated. Low-level laser therapy could be a useful tool to promote angiogenesis and dentinogenesis of the dentin-pulp complex when parameters are optimized.

Effect of grape seed extract (GSE) on functional activity and mineralization of OD-21 and MDPC-23 cell lines.

Recent studies on functional tissue regeneration have focused on substances that favor cell proliferation and differentiation, including the bioactive phenolic compounds present in grape seed extract (GSE). The aim of this investigation was to evaluate the stimulatory potential of GSE in the functional activity of undifferentiated pulp cells and odontoblast-like cells. OD-21 and MDPC-23 cell lines were cultivated in odontogenic medium until subconfluence, seeded in 24-well culture plates in a concentration of 2x104/well and divided into: 1) OD-21 without GSE; 2) OD-21+10 µg/mL of GSE; 3) MDPC-23 without GSE; 4) MDPC-23+10 µg/mL of GSE. Cell proliferation, in situ detection of alkaline phosphatase (ALP) and total protein content were assessed after 3, 7 and 10 days, and mineralization was evaluated after 14 days. The data were analyzed by ANOVA statistical tests set at a 5% level of significance. Results revealed that cell proliferation increased after 10 days, and protein content, after 7 days of culture in MDPC-23 cells. In situ ALP staining intensity was higher in undifferentiated pulp cells and odontoblast-like cells after 7 and 10 days, respectively. A discrete increase in MDPC-23 mineralization after GSE treatment was observed despite OD-21 cells presenting a decrease in mineralized nodule deposits. Data suggest that GSE favors functional activity of differentiated cells more broadly than undifferentiated cells (OD-21). More studies with different concentrations of GSE must be conducted to confirm its benefits to cells regarding dentin regeneration.

Effect of grape seed extract (GSE) on functional activity and mineralization of OD-21 and MDPC-23 cell lines

Abstract Recent studies on functional tissue regeneration have focused on substances that favor cell proliferation and differentiation, including the bioactive phenolic compounds present in grape seed extract (GSE). The aim of this investigation was to evaluate the stimulatory potential of GSE in the functional activity of undifferentiated pulp cells and odontoblast-like cells. OD-21 and MDPC-23 cell lines were cultivated in odontogenic medium until subconfluence, seeded in 24-well culture plates in a concentration of 2x104/well and divided into: 1) OD-21 without GSE; 2) OD-21+10 µg/mL of GSE; 3) MDPC-23 without GSE; 4) MDPC-23+10 µg/mL of GSE. Cell proliferation, in situ detection of alkaline phosphatase (ALP) and total protein content were assessed after 3, 7 and 10 days, and mineralization was evaluated after 14 days. The data were analyzed by ANOVA statistical tests set at a 5% level of significance. Results revealed that cell proliferation increased after 10 days, and protein content, after 7 days of culture in MDPC-23 cells. In situ ALP staining intensity was higher in undifferentiated pulp cells and odontoblast-like cells after 7 and 10 days, respectively. A discrete increase in MDPC-23 mineralization after GSE treatment was observed despite OD-21 cells presenting a decrease in mineralized nodule deposits. Data suggest that GSE favors functional activity of differentiated cells more broadly than undifferentiated cells (OD-21). More studies with different concentrations of GSE must be conducted to confirm its benefits to cells regarding dentin regeneration.

EphrinB2 signaling enhances osteogenic/odontogenic differentiation of human dental pulp stem cells.

OBJECTIVE: To investigate the role of the EphrinB2 signaling pathway in the osteogenesis/odontogenesis of human dental pulp stem cells (DPSCs). DESIGN: The endogenous expression levels of EphrinB2 and its cognate receptors EphB2 and EphB4 in DPSCs were analyzed by qRT-PCR and Western blotting after 7, 14 and 21 days of osteogenic/odontogenic induction culture. Additionally, the phosphorylation of EphrinB2, EphB4 and ERK1/2 proteins at early time-points following osteogenic induction, were also investigated by Western blots. Subsequently, we investigated whether supplementation of recombinant EphrinB2-Fc within the induction milieu can enhance the osteogenic/odontogenic differentiation of DPSCs. RESULTS: Endogenous gene and protein expression levels of EphrinB2, EphB2 and EphB4 were upregulated in induced versus non-induced DPSCs, over 21 days of osteogenic/odontogenic induction. Western blots showed increase in phosphorylated EphrinB2, EphB4 and ERK1/2 proteins at early time-points following osteogenic induction. Preliminary investigation of a concentration range (0, 0.5, 1 and 2 µg/ml) of recombinant EphrinB2-Fc within osteogenic induction media, showed that 0.5 µg/ml was optimal for enhancing the osteogenic/odontogenic differentiation of DPSCs over a culture duration of 14 days. Subsequently, more comprehensive qRT-PCR analysis with 0.5 µg/ml EphrinB2-Fc revealed significant upregulation of several key osteogenic marker genes in treated versus untreated DPSCs after 21 days of osteogenic/odontogenic induction. By 7 days of osteogenic induction, DPSCs treated with 0.5 µg/ml EphrinB2-Fc exhibited significantly more calcium mineralization (Alizarin red S staining) and alkaline phosphatase activity than the untreated control. CONCLUSIONS: EphrinB2 signaling plays a key role in the osteogenic/odontogenic differentiation of DPSCs.

Crown heights in the permanent teeth of 47,XYY males.

OBJECTIVE: The results in human sex chromosome aneuploidies had shown that the extra Y chromosome increases permanent and deciduous tooth crown sizes in the mesiodistal and labiolingual directions. The hypothesis of the study was that the additional Y chromosome increases the permanent tooth crown growth in a vertical dimension. We also aimed to observe possible sex difference in the permanent tooth crown height. MATERIAL AND METHODS: Data on 15 47,XYY males or males with an extra Y chromosome, nine male relatives (five brothers and four fathers) and 45 male and 48 female population controls had been gathered previously for Professor Lassi Alvesalo's KVANTTI Research Project. The measurements from panoramic radiographs were performed of all the applicable teeth, except the third molars on both sides of the jaws with a sliding digital calliper. RESULTS: All the mean tooth crown heights in the 47,XYY males were larger than in the male population controls and the differences were statistically significant in six teeth in the maxilla and 10 teeth in the mandible. Apart from few teeth, the crown heights in the 47,XYY males were larger than in their male relatives, but the difference between these groups was significant only in one tooth. The differences between sexes were statistically significant in eight teeth in the maxilla. CONCLUSIONS: Based on previous investigations and this work, it is evident that the impact of the extra Y chromosome during tooth crown development is holistic, increasing permanent tooth sizes in three dimensions in a balanced manner.

Odontogenic differentiation and biomineralization potential of dental pulp stem cells inside Mg-based bioceramic scaffolds under low-level laser treatment.

This study aimed to investigate the potential of low-level laser irradiation (LLLI) to promote odontogenic differentiation and biomineralization by dental pulp stem cells (DPSCs) seeded inside bioceramic scaffolds. Mg-based, Zn-doped bioceramic scaffolds, synthesized by the sol-gel technique, were spotted with DPSCs and exposed to LLLI at 660 nm with maximum output power of 140 mw at fluencies (a) 2 and 4 J/cm2 to evaluate cell viability/proliferation by the MTT assay and (b) 4 J/cm2 to evaluate cell differentiation, using real-time PCR (expression of odontogenic markers) and a p-nitrophenylphosphate (pNPP)-based assay for alkaline phosphatase (ALP) activity measurement. Scanning electron microscopy (SEM) and X-ray diffraction (XRD) analysis were used for structural/chemical characterization of the regenerated tissues. Exposure of the DPSCs/scaffold complexes to the proposed LLLI scheme was associated with statistically significant increase of odontogenesis-related markers (bone morphogenetic protein 2 (BMP-2): 22.4-fold, dentin sialophosphoprotein (DSPP): 28.4-fold, Osterix: 18.5-fold, and Runt-related transcription factor 2 (Runx2): 3.4-fold). ALP activity was significantly increased at 3 and 7 days inside the irradiated compared to that in the non-irradiated SC/DPSC complexes, but gradually decreased until 14 days. Newly formed Ca-P tissue was formed on the SC/DPSC complexes after 28 days of culture that attained the characteristics of bioapatite. Overall, LLLI treatment proved to be beneficial for odontogenic differentiation and biomineralization of DPSCs inside the bioceramic scaffolds, making this therapeutic modality promising for targeted dentin engineering.

Comparative Gene Expression Analysis of the Coronal Pulp and Apical Pulp Complex in Human Immature Teeth.

INTRODUCTION: This study determined the gene expression profiles of the human coronal pulp (CP) and apical pulp complex (APC) with the aim of explaining differences in their functions. METHODS: Total RNA was isolated from the CP and APC, and gene expression was analyzed using complementary DNA microarray technology. Gene ontology analysis was used to classify the biological function. Quantitative reverse-transcription polymerase chain reaction and immunohistochemical staining were performed to verify microarray data. RESULTS: In the microarray analyses, expression increases of at least 2-fold were present in 125 genes in the APC and 139 genes in the CP out of a total of 33,297 genes. Gene ontology class processes found more genes related to immune responses, cell growth and maintenance, and cell adhesion in the APC, whereas transport and neurogenesis genes predominated in the CP. Quantitative reverse-transcription polymerase chain reaction and immunohistochemical staining confirmed the microarray results, with DMP1, CALB1, and GABRB1 strongly expressed in the CP, whereas SMOC2, SHH, BARX1, CX3CR1, SPP1, COL XII, and LAMC2 were strongly expressed in the APC. CONCLUSIONS: The expression levels of genes related to dentin mineralization, neurogenesis, and neurotransmission are higher in the CP in human immature teeth, whereas those of immune-related and tooth development-related genes are higher in the APC.

Odontogenic Differentiation of Human Dental Pulp Stem Cells on Hydrogel Scaffolds Derived from Decellularized Bone Extracellular Matrix and Collagen Type I.

OBJECTIVES: The aim of this study was to evaluate the level of odontogenic differentiation of dental pulp stem cells (DPSCs) on hydrogel scaffolds derived from bone extracellular matrix (bECM) in comparison to those seeded on collagen I (Col-I), one of the main components of dental pulp ECM. METHODS: DPSCs isolated from human third molars were characterized for surface marker expression and odontogenic potential prior to seeding into bECM or Col-I hydrogel scaffolds. The cells were then seeded onto bECM and Col-I hydrogel scaffolds and cultured under basal conditions or with odontogenic and growth factor (GF) supplements. DPSCs cultivated on tissue culture polystyrene (TCPS) with and without supplements were used as controls. Gene expression of dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP-1) and matrix extracellular phosphoglycoprotein (MEPE) was evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and mineral deposition was observed by Von Kossa staining. RESULTS: When DPSCs were cultured on bECM hydrogels, the mRNA expression levels of DSPP, DMP-1 and MEPE genes were significantly upregulated with respect to those cultured on Col-I scaffolds or TCPS in the absence of extra odontogenic inducers. In addition, more mineral deposition was observed on bECM hydrogel scaffolds as demonstrated by Von Kossa staining. Moreover, DSPP, DMP-1 and MEPE mRNA expressions of DPSCs cultured on bECM hydrogels were further upregulated by the addition of GFs or osteo/odontogenic medium compared to Col-I treated cells in the same culture conditions. SIGNIFICANCE: These results demonstrate the potential of the bECM hydrogel scaffolds to stimulate odontogenic differentiation of DPSCs.

Full-length amelogenin influences the differentiation of human dental pulp stem cells.

BACKGROUND: Amelogenin is an extracellular matrix protein well known for its role in the organization and mineralization of enamel. Clinically, it is used for periodontal regeneration and, due to its finding also in predentin and intercellular spaces of dental pulp cells, it has recently been suggested for pulp capping procedures. The aim of this study was to analyse in vitro the effect of the recombinant human full-length amelogenin on the growth and differentiation of human dental pulp stem cells (hDPSCs). METHODS: Human DPSCs were treated with a supplement of amelogenin at a concentration of 10 ng/ml, 100 ng/ml and 1000 ng/ml. The groups were compared to the unstimulated control in terms of cell morphology and proliferation, mineralization and gene expression for ALP (alkaline phosphatase), DMP1 (dentin matrix protein-1) and DSPP (dentin sialophosphoprotein). RESULTS: Amelogenin affects hDPSCs differently than PDL (periodontal ligament) cells and other cell lines. The proliferation rate at two weeks is significantly reduced in presence of the highest concentration of amelogenin as compared to the unstimulated control. hDPSCs treated with low concentrations present a downregulation of DMP1 and DSPP, which is significant for DSPP (p = 0.011), but not for DMP1 (p = 0.395). CONCLUSIONS: These finding suggest that the role of full-length amelogenin is not restricted to participation in tooth structure. It influences the differentiation of hDPSC according to various concentrations and this might impair the clinical results of pulp capping.

Long-Term Fluoride Release from Dental Resins Affects STRO-1+ Cell Behavior.

Fluoride-releasing restorative dental materials can be beneficial to remineralize dentin and help prevent secondary caries. However, the effects of fluoride release from dental materials on the activity of dental pulp stem cells are not known. Here we investigate whether different fluoride release kinetics from dental resins supplemented with modified hydrotalcite (RK-F10) or fluoride-glass filler (RK-FG10) could influence the behavior of a human dental pulp stem cell subpopulation (STRO-1(+) cells) known for its ability to differentiate toward an odontoblast-like phenotype. The 2 resins, characterized by similar physicochemical properties and fluoride content, exhibited different long-term fluoride release kinetics. Our data demonstrate that long-term exposure of STRO-1(+) cells to a continuous release of a low amount of fluoride by RK-F10 increases their migratory response to transforming growth factor ß1 (TGF-ß1) and stromal cell-derived factor 1 (SDF-1), both important promoters of pulp stem cell recruitment. Moreover, the expression patterns of dentin sialoprotein (dspp), dentin matrix protein 1 (dmp1), osteocalcin (ocn), and matrix extracellular phosphoglycoprotein (mepe) indicate a complete odontoblast-like cell differentiation only when STRO-1(+) cells were cultured on RK-F10. On the contrary, RK-FG10, characterized by an initial fluoride release burst and reduced lifetime of the delivery, did not elicit any significant effect on both STRO-1(+) cell migration and differentiation. Taken together, our results highlight the importance of taking into account fluoride release kinetics in addition to fluoride concentration when designing new fluoride-restorative materials.

Genipin, a cross-linking agent, promotes odontogenic differentiation of human dental pulp cells.

INTRODUCTION: The aim of this study was to investigate the effects of genipin, a natural collagen cross-linking agent, on odontogenic differentiation of human dental pulp cells (hDPCs) because the mechanical properties of collagen allow it to serve as a scaffold for engineering of pulp-dentin complex. Furthermore, the role of extracellular signal-regulated kinase (ERK) was investigated as a mediator of the differentiation. METHODS: The odontogenic differentiation was analyzed by alkaline phosphatase activity, real time-polymerase chain reaction, Western blotting, and alizarin red S staining. The morphologic features of hDPCs cultured in genipin-treated collagen were evaluated by scanning electron microscopy. For the assessment of mechanical properties of collagen treated with genipin, the surface roughness and compressive strength were measured. RESULTS: Alkaline phosphatase activity, the expression of odontogenic markers, and mineralized nodule formation increased in the genipin-treated group. Genipin also activated ERK, and treatment with ERK inhibitor blocked the expression of the markers. The cells cultured in genipin-treated collagen spread across the substrate and attached in close proximity to one another. The proliferation and differentiation of hDPCs cultured in genipin-treated collagen were facilitated. The mechanical properties of collagen, such as surface roughness and compressive strength, were increased by treatment with genipin. CONCLUSIONS: Our results show that genipin promotes odontogenic differentiation of hDPCs via the ERK signaling pathway. Furthermore, the enhanced mechanical properties of the collagen scaffold induced by genipin may play important roles in cell fate. Consequently, the application of genipin might be a new strategy for dentin-pulp complex regeneration.