The Reparative Function of MMP13 in Tertiary Reactionary Dentinogenesis after Tooth Injury.

MMP13 gene expression increases up to 2000-fold in mineralizing dental pulp cells (DPCs), with research previously demonstrating that global MMP13 deletion resulted in critical alterations in the dentine phenotype, affecting dentine-tubule regularity, the odontoblast palisade, and significantly reducing the dentine volume. Global MMP13-KO and wild-type mice of a range of ages had their molar teeth injured to stimulate reactionary tertiary dentinogenesis. The response was measured qualitatively and quantitatively using histology, immunohistochemistry, micro-CT, and qRT-PCR in order to assess changes in the nature and volume of dentine deposited as well as mechanistic links. MMP13 loss affected the reactionary tertiary dentine quality and volume after cuspal injury and reduced Nestin expression in a non-exposure injury model, as well as mechanistic links between MMP13 and the Wnt-responsive gene Axin2. Acute pulpal injury and pulp exposure to oral fluids in mice teeth showed upregulation of the MMP13 in vivo, with an increase in the gene expression of Mmp8, Mmp9, and Mmp13 evident. These results indicate that MMP13 is involved in tertiary reactionary dentine formation after tooth injury in vivo, potentially acting as a key molecule in the dental pulp during dentine-pulp repair processes.

Dynamic Alterations in Acetylation and Modulation of Histone Deacetylase Expression Evident in the Dentine-Pulp Complex during Dentinogenesis.

Epigenetic modulation, including histone modification, alters gene expression and controls cell fate. Histone deacetylases (HDACs) are identified as important regulators of dental pulp cell (DPC) mineralisation processes. Currently, there is a paucity of information regarding the nature of histone modification and HDAC expression in the dentine-pulp complex during dentinogenesis. The aim of this study was to investigate post-translational histone modulation and HDAC expression during DPC mineralisation and the expression of Class I/II HDACs during tooth development and in adult teeth. HDAC expression (isoforms -1 to -6) was analysed in mineralising primary rat DPCs using qRT-PCR and Western blot with mass spectrometry being used to analyse post-translational histone modifications. Maxillary molar teeth from postnatal and adult rats were analysed using immunohistochemical (IHC) staining for HDACs (1-6). HDAC-1, -2, and -4 protein expression increased until days 7 and 11, but decreased at days 14 and 21, while other HDAC expression increased continuously for 21 days. The Class II mineralisation-associated HDAC-4 was strongly expressed in postnatal sample odontoblasts and DPCs, but weakly in adult teeth, while other Class II HDACs (-5, -6) were relatively strongly expressed in postnatal DPCs and adult odontoblasts. Among Class I HDACs, HDAC-1 showed high expression in postnatal teeth, notably in ameloblasts and odontoblasts. HDAC-2 and -3 had extremely low expression in the rat dentine-pulp complex. Significant increases in acetylation were noted during DPC mineralisation processes, while trimethylation H3K9 and H3K27 marks decreased, and the HDAC-inhibitor suberoylanilide hydroxamic acid (SAHA) enhanced H3K27me3. These results highlight a dynamic alteration in histone acetylation during mineralisation and indicate the relevance of Class II HDAC expression in tooth development and regenerative processes.

A WWP2-PTEN-KLF5 signaling axis regulates odontoblast differentiation and dentinogenesis in mice.

WW domain-containing E3 Ubiquitin-protein ligase 2 (WWP2) has been found to positively regulate odontoblastic differentiation by monoubiquitinating the transcription factor Kruppel-like factor 5 (KLF5) in a cell culture system. However, the in vivo role of WWP2 in mouse teeth remains unknown. To explore this, here we generated Wwp2 knockout (Wwp2 KO) mice. We found that molars in Wwp2 KO mice exhibited thinner dentin, widened predentin, and reduced numbers of dentinal tubules. In addition, expression of the odontoblast differentiation markers Dspp and Dmp1 was decreased in the odontoblast layers of Wwp2 KO mice. These findings demonstrate that WWP2 may facilitate odontoblast differentiation and dentinogenesis. Furthermore, we show for the first time that phosphatase and tensin homolog (PTEN), a tumor suppressor, is expressed in dental papilla cells and odontoblasts of mouse molars and acts as a negative regulator of odontoblastic differentiation. Further investigation indicated that PTEN is targeted by WWP2 for degradation during odontoblastic differentiation. We demonstrate PTEN physically interacts with and inhibits the transcriptional activity of KLF5 on Dspp and Dmp1. Finally, we found WWP2 was able to suppress the interaction between PTEN and KLF5, which diminished the inhibition effect of PTEN on KLF5. Taken together, this study confirms the essential role of WWP2 and the WWP2-PTEN-KLF5 signaling axis in odontoblast differentiation and dentinogenesis in vivo.

Identification of DSPP novel variants and phenotype analysis in dentinogenesis dysplasia Shields type II patients.

OBJECTIVES: To investigate the genetic causes and teeth characteristics of dentin dysplasia Shields type II(DD-II) in three Chinese families. MATERIALS AND METHODS: Data from three Chinese families affected with DD-II were collected. Whole-exome sequencing (WES) and whole-genome sequencing (WGS) were conducted to screen for variations, and Sanger sequencing was used to verify mutation sites. The physical and chemical characteristics of the affected teeth including tooth structure, hardness, mineral content, and ultrastructure were investigated. RESULTS: A novel frameshift deletion mutation c.1871_1874del(p.Ser624fs) in DSPP was found in families A and B, while no pathogenic mutation was found in family C. The affected teeth's pulp cavities were obliterated, and the root canals were smaller than normal teeth and irregularly distributed comprising a network. The patients' teeth also had reduced dentin hardness and highly irregular dentinal tubules. The Mg content of the teeth was significantly lower than that of the controls, but the Na content was obviously higher than that of the controls. CONCLUSIONS: A novel frameshift deletion mutation, c.1871_1874del (p.Ser624fs), in the DPP region of the DSPP gene causes DD-II. The DD-II teeth demonstrated compromised mechanical properties and changed ultrastructure, suggesting an impaired function of DPP. Our findings expand the mutational spectrum of the DSPP gene and strengthen the understanding of clinical phenotypes related to the frameshift deletion in the DPP region of the DSPP gene. CLINICAL RELEVANCE: A DSPP mutation can alter the characteristics of the affected teeth, including tooth structure, hardness, mineral content, and ultrastructure.

METTL3 enhances dentinogenesis differentiation of dental pulp stem cells via increasing GDF6 and STC1 mRNA stability.

BACKGROUND: The dentinogenesis differentiation of dental pulp stem cells (DPSCs) is controlled by the spatio-temporal expression of differentiation related genes. RNA N6-methyladenosine (m6A) methylation, one of the most abundant internal epigenetic modification in mRNA, influences various events in RNA processing, stem cell pluripotency and differentiation. Methyltransferase like 3 (METTL3), one of the essential regulators, involves in the process of dentin formation and root development, while mechanism of METTL3-mediated RNA m6A methylation in DPSC dentinogenesis differentiation is still unclear. METHODS: Immunofluorescence staining and MeRIP-seq were performed to establish m6A modification profile in dentinogenesis differentiation. Lentivirus were used to knockdown or overexpression of METTL3. The dentinogenesis differentiation was analyzed by alkaline phosphatase, alizarin red staining and real time RT-PCR. RNA stability assay was determined by actinomycin D. A direct pulp capping model was established with rat molars to reveal the role of METTL3 in tertiary dentin formation. RESULTS: Dynamic characteristics of RNA m6A methylation in dentinogenesis differentiation were demonstrated by MeRIP-seq. Methyltransferases (METTL3 and METTL14) and demethylases (FTO and ALKBH5) were gradually up-regulated during dentinogenesis process. Methyltransferase METTL3 was selected for further study. Knockdown of METTL3 impaired the DPSCs dentinogenesis differentiation, and overexpression of METTL3 promoted the differentiation. METTL3-mediated m6A regulated the mRNA stabiliy of GDF6 and STC1. Furthermore, overexpression of METTL3 promoted tertiary dentin formation in direct pulp capping model. CONCLUSION: The modification of m6A showed dynamic characteristics during DPSCs dentinogenesis differentiation. METTL3-mediated m6A regulated in dentinogenesis differentiation through affecting the mRNA stability of GDF6 and STC1. METTL3 overexpression promoted tertiary dentin formation in vitro, suggesting its promising application in vital pulp therapy (VPT).

The Effect of Calcium-Silicate Cements on Reparative Dentinogenesis Following Direct Pulp Capping on Animal Models.

Dental pulp vitality is a desideratum for preserving the health and functionality of the tooth. In certain clinical situations that lead to pulp exposure, bioactive agents are used in direct pulp-capping procedures to stimulate the dentin-pulp complex and activate reparative dentinogenesis. Hydraulic calcium-silicate cements, derived from Portland cement, can induce the formation of a new dentin bridge at the interface between the biomaterial and the dental pulp. Odontoblasts are molecularly activated, and, if necessary, undifferentiated stem cells in the dental pulp can differentiate into odontoblasts. An extensive review of literature was conducted on MedLine/PubMed database to evaluate the histological outcomes of direct pulp capping with hydraulic calcium-silicate cements performed on animal models. Overall, irrespective of their physico-chemical properties and the molecular mechanisms involved in pulp healing, the effects of cements on tertiary dentin formation and pulp vitality preservation were positive. Histological examinations showed different degrees of dental pulp inflammatory response and complete/incomplete dentin bridge formation during the pulp healing process at different follow-up periods. Calcium silicate materials have the ability to induce reparative dentinogenesis when applied over exposed pulps, with different behaviors, as related to the animal model used, pulpal inflammatory responses, and quality of dentin bridges.

Depletion of SNRNP200 inhibits the osteo-/dentinogenic differentiation and cell proliferation potential of stem cells from the apical papilla.

BACKGROUND: Tissue regeneration mediated by mesenchymal stem cells (MSCs) is deemed a desirable way to repair teeth and craniomaxillofacial tissue defects. Nevertheless, the molecular mechanisms about cell proliferation and committed differentiation of MSCs remain obscure. Previous researches have proved that lysine demethylase 2A (KDM2A) performed significant function in the regulation of MSC proliferation and differentiation. SNRNP200, as a co-binding factor of KDM2A, its potential effect in regulating MSCs' function is still unclear. Therefore, stem cells from the apical papilla (SCAPs) were used to investigate the function of SNRNP200 in this research. METHODS: The alkaline phosphatase (ALP) activity assay, Alizarin Red staining, and osteogenesis-related gene expressions were used to examine osteo-/dentinogenic differentiation potential. Carboxyfluorescein diacetate, succinimidyl ester (CFSE) and cell cycle analysis were applied to detect the cell proliferation. Western blot analysis was used to evaluate the expressions of cell cycle-related proteins. RESULTS: Depletion of SNRNP200 caused an obvious decrease of ALP activity, mineralization formation and the expressions of osteo-/dentinogenic genes including RUNX2, DSPP, DMP1 and BSP. Meanwhile, CFSE and cell cycle assays revealed that knock-down of SNRNP200 inhibited the cell proliferation and blocked cell cycle at the G2/M and S phase in SCAPs. In addition, it was found that depletion of SNRNP200 up-regulated p21 and p53, and down-regulated the CDK1, CyclinB, CyclinE and CDK2. CONCLUSIONS: Depletion of SNRNP200 repressed osteo-/dentinogenic differentiation potentials and restrained cell proliferation through blocking cell cycle progression at the G2/M and S phase, further revealing that SNRNP200 has crucial effects on preserving the proliferation and differentiation potentials of dental tissue-derived MSCs.

Coordinated expression of p300 and HDAC3 upregulates histone acetylation during dentinogenesis.

Cellular differentiation is caused by highly controlled modifications in the gene expression but rarely involves a change in the DNA sequence itself. Histone acetylation is a major epigenetic factor that adds an acetyl group to histone proteins, thus altering their interaction with DNA and nuclear proteins. Illumination of the histone acetylation during dentinogenesis is important for odontoblast differentiation and dentinogenesis. In the current study, we aimed to discover the roles and regulation of acetylation at histone 3 lysine 9 (H3K9ac) and H3K27ac during dentinogenesis. We first found that both of these modifications were enhanced during odontoblast differentiation and dentinogenesis. These modifications are dynamically catalyzed by histone acetyltransferases (HATs) and deacetylases (HDACs), among which HDAC3 was decreased while p300 increased during odontoblast differentiation. Moreover, overexpression of HDAC3 or knockdown p300 inhibited odontoblast differentiation in vitro, and inhibition of HDAC3 and p300 with trichostatin A or C646 regulated odontoblast differentiation. Taken together, the results of our present study suggest that histone acetylation is involved in dentinogenesis and coordinated expression of p300- and HDAC3-regulated odontoblast differentiation through upregulating histone acetylation.

Distal-less homeobox 5 promotes the osteo-/dentinogenic differentiation potential of stem cells from apical papilla by activating histone demethylase KDM4B through a positive feedback mechanism.

Understanding the mechanism of osteo-/dentinogenic differentiation is beneficial for jaw bone and dental tissue regeneration. DLX5 is highly expressed in dental tissue-derived mesenchymal stem cells (MSCs) and is upregulated by lysine-specific demethylase 4B (KDM4B), enabling it to regulate osteo-/dentinogenic differentiation, while the function of DLX5 in osteo-/dentinogenesis has not been thoroughly elucidated to date. Therefore, we investigated DLX5 function using stem cells from apical papilla (SCAPs). SCAPs were obtained from the human wisdom tooth. Alkaline phosphatase (ALP) assay, Alizarin red staining (ARS), quantitative analysis of calcium, osteo-/dentinogenesis-related gene expression and in vivo transplantation were used to determine the osteo-/dentinogenic differentiation potential. Luciferase and ChIP assays were used to investigate the physical relationship between DLX5 and KDM4B. DLX5 and KDM4B were upregulated during osteogenic induction and were induced by BMP4 in SCAPs. Next, we found that DLX5 enhanced ALP activity, mineralization in vitro, and the expression of dentin sialophosphoprotein (DSPP), dentin matrix acidic phosphoprotein 1 (DMP1), osteopontin (OPN), and the key transcription factor osterix (OSX). Moreover, transplant experiments showed that DLX5 promoted osteo-/dentinogenesis in vivo. Interestingly, DLX5 enhanced KDM4B transcription by directly binding with its promoter. In addition, KDM4B upregulated DLX5 in SCAPs. These results indicate that DLX5 and KDM4B are positive effectors of BMP signaling and regulate each other via a positive feedback mechanism. DLX5 enhanced osteo-/dentinogenic differentiation via upregulated KDM4B in SCAPs, suggesting that activation of the DLX5/KDM4B signaling pathway might serve as an intrinsic mechanism that promotes tissue regeneration mediated by dental-derived MSCs.

Immunohistochemistry and gene expression of GLUT1, RUNX2 and MTOR in reparative dentinogenesis.

OBJECTIVES: To determine glucose transporter 1 (GLUT1) and runt-related transcription factor 2 (RUNX2) expression during reparative dentinogenesis after pulpotomy with mineral trioxide aggregate (MTA) capping. SUBJECTS AND METHODS: Eight-week-old male Wistar rats were used. Pulp of the upper left first molar was exposed and capped with MTA. The upper right first molar of the same animal was used as a control. After collecting molars at various time points, GLUT1, RUNX2 and mammalian target of rapamycin (MTOR) were examined by immunohistochemistry. mRNA levels of Slc2a1 (encoding GLUT1), Runx2, Nestin and Mtor were determined by real-time PCR. RESULTS: Pulp exhibited progressive formation of reparative dentine lined with GLUT1- and MTOR-immunoreactive odontoblast-like cells at 5 days after pulpotomy. RUNX2 was detected in nuclei of most pulp tissue cells at day 5 after pulpotomy. Double immunofluorescence staining revealed GLUT1 immunoreactivity on odontoblast-like cells positive for Nestin or RUNX2, 5 days after pulpotomy. Slc2a1, Runx2, Nestin and Mtor mRNA levels were significantly upregulated on days 3-5 after pulpotomy. CONCLUSIONS: After rat molar pulpotomy, dental pulp induced formation of reparative dentine with colocalization of GLUT1 and Nestin or RUNX2. Moreover, mRNA levels of Slc2a1, Runx2, Nestin and Mtor were significantly upregulated in pulpotomized dental pulp.

Irradiation with blue light-emitting diode enhances osteogenic differentiation of stem cells from the apical papilla.

This study aimed to evaluate the effects of low-energy blue LED irradiation on the osteogenic differentiation of stem cells from the apical papilla (SCAPs). SCAPs were derived from human tooth root tips and were irradiated with 0 (control group), 1 J/cm2, 2 J/cm2, 3 J/cm2, or 4 J/cm2 blue light in osteogenic induction medium. Cell proliferation was analyzed using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Osteogenic differentiation activity was evaluated by monitoring alkaline phosphatase (ALP), alizarin red staining, and real-time polymerase chain reaction (RT-PCR). The results of the MTT assay indicated that SCAPs in the LED groups exhibited a lower proliferation rate than those in the control group, and there were statistically differences between the 2 J/cm2, 3 J/cm2, and 4 J/cm2 groups and the control group (P < 0.05). The results of the ALP and alizarin red analyses showed that blue LED promoted osteogenic differentiation of the SCAPs. And 4 J/cm2 blue light upregulates the expression levels of the osteogenic/dentinogenic genes ALP, dentin sialophosphoprotein (DSPP), dentin matrix protein-1 (DMP-1), and osteocalcin (OCN) in SCAPs. Our results confirmed that low-energy blue LED at 1 J/cm2, 2 J/cm2, 3 J/cm2, and 4 J/cm2 could inhibit the proliferation of SCAPs and promotes osteogenic differentiation of SCAPs. Further in vitro studies are required to explore the mechanisms of the effects by low-energy blue LED.

IGFBP5 enhances the dentinogenesis potential of dental pulp stem cells via JNK and ErK signalling pathways.

BACKGROUND: Dental stem cell transplantation has become a new method for tooth tissue regeneration. However, its molecular mechanism of the dentinogenic differentiation is still unclear, limited its application. Our previous studies found that insulin-like growth factor-binding protein 5 (IGFBP5) can promote the osteogenic differentiation of periodontal ligament stem cells and the regeneration of periodontal tissues. This study aims to clarify the effect and mechanism of IGFBP5 on the dentinogenesis of dental pulp stem cells (DPSCs). OBJECTIVE AND METHODS: Lentiviral IGFBP5 shRNA was used to knock-down of IGFBP5. And recombinant human IGFBP5 protein (rhIGFBP5) was used to treat DPSCs. Alkaline phosphatase (ALP) staining, Alizarin red staining, quantitative calcium analysis, real-time RT-PCR and Western Blot were used to detect dentinogenic differentiation markers and related signalling pathways. Transplantation in nude mice was used to detect the dentin regeneration in vivo. RESULTS: Depletion of IGFBP5 inhibited ALP activity and the mineralisation and reduced the expressions of osteo/dentinogenic differentiation markers BSP, DMP-1 and DSPP in DPSCs. 0.05 ng/mL rhIGFBP5 promoted ALP activity, the mineralisation and the expressions of BSP, DMP-1 and DSPP in DPSCs. In addition, 0.05 ng/mL rhIGFBP5 could promote DPSC-mediated dentin-like tissues formation in vivo. Western blot results showed that IGFBP5 activated JNK and Erk signalling pathways in DPSCs. Furthermore, inhibition of JNK pathway by SP600125, the expression of p-JNK and p-Erk was reduced, while inhibition of Erk pathway by PD98059, only p-Erk expression was decreased. CONCLUSIONS: Our results demonstrated that IGFBP5 could promote the dentinogenic differentiation and dentinogenesis potential of DPSCs via JNK and ErK signalling pathways.

Zebrafish sp7 mutants show tooth cycling independent of attachment, eruption and poor differentiation of teeth.

The capacity to fully replace teeth continuously makes zebrafish an attractive model to explore regeneration and tooth development. The requirement of attachment bone for the appearance of replacement teeth has been hypothesized but not yet investigated. The transcription factor sp7 (osterix) is known in mammals to play an important role during odontoblast differentiation and root formation. Here we study tooth replacement in the absence of attachment bone using sp7 zebrafish mutants. We analysed the pattern of tooth replacement at different stages of development and demonstrated that in zebrafish lacking sp7, attachment bone is never present, independent of the stage of tooth development or fish age, yet replacement is not interrupted. Without bone of attachment we observed abnormal orientation of teeth, and abnormal connection of pulp cavities of predecessor and replacement teeth. Mutants lacking sp7 show arrested dentinogenesis, with non-polarization of odontoblasts and only a thin layer of dentin deposited. Osteoclast activity was observed in sp7 mutants; due to the lack of bone of attachment, remodelling was diminished but nevertheless present along the pharyngeal bone. We conclude that tooth replacement is ongoing in the sp7 mutant despite poor differentiation and defective attachment. Without bone of attachment tooth orientation and pulp organization are compromised.

EDTA conditioning of circumpulpal dentine induces dentinogenic events in pulpotomized miniature swine teeth.

AIM: To investigate pulp responses after pulpotomy and EDTA conditioning of pulp chamber dentinal walls with or without the placement of a collagenous scaffold in the experimental model of miniature swine teeth. METHODOLOGY: Forty-two fully developed permanent premolars and molars of healthy miniature swines were used. After preparation of pulp exposures through Class I cavities, the tissue of the pulp chamber was completely removed. The haemorrhage was controlled, and the root pulp was protected using a polyurethane film. The circumpulpal pulp chamber dentine was treated for 3 min with normal saline (group 1), or 17% EDTA solution (groups 2 and 3). The film was removed, and the pulp chamber cavities were left empty (groups 1 and 2), or filled with swine collagenous sponge (group 3). The access cavities were restored with a Teflon disc and glass ionomer. Teeth were evaluated histo-morphologically after 10 weeks. Data were compared using the nonparametric Fisher's exact test. RESULTS: Teeth after treatment of dentine with saline (group 1) were associated with no or only traces of hard tissue formation along the root canal walls. Atubular tertiary dentine deposition in the form of matrix deposition along root canal walls, or dentine bridge formation at the orifice of root canals or complete pulp canal obliteration, was found after treatment of dentine with EDTA in both experiments (groups 2 and 3). Significantly different types of mineralization in the root canals of groups 2 and 3 were seen (P = 0.001). Tissue changes in the pulp cavity, characterized by soft tissue growth and osteodentine or atubular tertiary dentine formation, were only seen after EDTA conditioning of dentine, in 6.2% of the teeth without scaffold and 64.7% of the teeth with scaffold application. Newly deposited mineralized matrix in the pulp chamber was always in continuation with hard tissue deposited in the root canals. CONCLUSIONS: The EDTA conditioning of pulp cavity dentinal walls after pulpotomy induced dentinogenic events in the root pulp. Application of collagenous scaffold in the pulp chamber enhanced soft tissue growth and mineralized tissue formation along the treated circumpulpal dentine.

Activation of αSMA expressing perivascular cells during reactionary dentinogenesis.

AIM: To examine the contribution of perivascular cells expressing αSMA to reactionary dentinogenesis. METHODOLOGY: An inducible, Cre-loxP in vivo fate-mapping approach was used to examine the contribution of the descendants of cells expressing the αSMA-CreERT2 transgene to reactionary dentinogenesis in mice molars. Reactionary dentinogenesis was induced by experimental mild injury to dentine without pulp exposure. The Student's t test was used to determine statistical significance at *P ≤ 0.05. RESULTS: The lineage tracing experiments revealed that mild injury to dentine first led to activation of αSMA-tdTomato+ cells in the entire pulp chamber. The percentage of areas occupied by αSMA-tdTomato+ in injured (7.5 ± 0.7%) teeth were significantly higher than in teeth without injury (2 ± 0.5%). After their activation, αSMA-tdTomato+ cells migrated towards the site of injury, gave rise to pulp cells and a few odontoblasts that became integrated into the existing odontoblast layer expressing Col2.3-GFP and Dspp. CONCLUSION: Mild insult to dentine activated perivascular αSMA-tdTomato+ cells giving rise to pulp cells as well as a few odontoblasts that were integrated into the pre-existing odontoblast layer.

Use of 0.4-Tesla static magnetic field to promote reparative dentine formation of dental pulp stem cells through activation of p38 MAPK signalling pathway.

AIM: To investigate whether static magnetic fields (SMFs) have a positive effect on the migration and dentinogenesis of dental pulp stem cells (DPSCs) to promote reparative dentine formation. METHODOLOGY: In vitro scratch assays and a traumatic pulp exposure model were performed to evaluate the effect of 0.4-Tesla (T) SMF on DPSC migration. The cytoskeletons of the DPSCs were identified by fluorescence immunostaining and compared with those of a sham-exposed group. Dentinogenic evaluation was performed by analysing the expressions of DMP-1 and DSPP marker genes using a quantitative real-time polymerase chain reaction (qRT-PCR) process. Furthermore, the formation of calcified deposits was examined by staining the dentinogenic DPSCs with Alizarin Red S dye. Finally, the role played by the p38 MAPK signalling pathway in the migration and dentinogenesis of DPSCs under 0.4-T SMF was investigated by incorporating p38 inhibitor (SB203580) into the in vitro DPSC experiments. The Student's t-test and the Kruskal-Wallis test followed by Dunn's post hoc test with a significance level of P < 0.05 were used for statistical analysis. RESULTS: The scratch assay results revealed that the application of 0.4-T SMF enhanced DPSCs migration towards the scratch wound (P < 0.05). The cytoskeletons of the SMF-treated DPSCs were found to be aligned perpendicular to the scratch wound. After 20 days of culture, the SMF-treated group had a greater number of out-grown cells than the sham-exposed group (nonmagnetized control). For the SMF-treated group, the DMP-1 (P < 0.05) and DSPP genes (P < 0.05), analysed by qRT-PCR, exhibited a higher expression. The distribution of calcified nodules was also found to be denser in the SMF-treated group when stained with Alizarin Red S dye (P < 0.05). Given the incorporation of p38 inhibitor SB203580 into the DPSCs, cell migration and dentinogenesis were suppressed. No difference was found between the SMF-treated and sham-exposed cells (P > 0.05). CONCLUSION: 0.4-T SMF enhanced DPSC migration and dentinogenesis through the activation of the p38 MAPK-related 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.

Promoting dentinogenesis of DPSCs through inhibiting microRNA-218 by using magnetic nanocarrier delivery.

PURPOSE: The purposes of this study are to explore the roles of microRNA-218 (miR-218) delivered by a newly designed magnetic nanocarrier: GCC-Fe3O4 (GCC-Fe) in dentinogenesis potentials of human dental pulp stem cells (DPSCs). METHODS: Human DPSCs were obtained from impacted wisdom teeth of healthy donors under the permission of National Taiwan University Hospital institutional review board (NTUH IRB). Meanwhile, the transfection efficiency of GCC-Fe was evaluated. After transfecting miR-218 (GFm) and miR-218 inhibitor (GFmi) into DPSCs for 24 h, the dentinogenesis potentials of DPSCs were then evaluated with Alizarin Red S (ARS) staining with or without induction for 1, 4, and 9 days. Possible signaling pathway was further investigated by Western Blotting. RESULTS: We found that the magnetic GCC-Fe3O4 nanocarrier was serum endurable with about 90% transfection efficiency in DPSCs under normal culture condition. Results of ARS staining indicated that miR-218 was negatively regulating dentinogenesis potentials of DPSCs after induction. When the miR-218 inhibitor was delivered, calcium deposits in DPSCs were increased significantly. We also discovered that the effects of miR-218 were further regulated through the MAPK/ERK pathway. CONCLUSION: We identified that miR-218 had a negative regulation role in the dentinogenesis of DPSCs. By inhibiting miR-218, the mineralization potentials of DPSCs were promoted after induction. In addition, we also confirmed that the highly efficient magnetic GCC-Fe3O4 nanocarrier not only was suitable for gene manipulation in biomedical studies, but also ideal for future clinical applications due to its serum endurable property.

WIF1 enhanced dentinogenic differentiation in stem cells from apical papilla.

BACKGROUND: Odontogenic mesenchymal stem cells (MSCs) isolated from tooth tissues are a reliable resource that can be utilized for dental tissue regeneration. Exploration of the mechanisms underlying the regulation of their differentiation may be helpful for investigating potential clinical applications. The stem cell niche plays an important role in maintaining cell functioning. Previous studies found that Wnt inhibitory factor 1 (WIF1) is more highly expressed in apical papilla tissues than in stem cells from apical papilla (SCAPs) using microarray analysis. However, the function of WIF1 in SCAPs remains unclear. In the present study, we investigated the function of WIF1 during dentinogenic differentiation in SCAPs. METHODS: A retrovirus containing HA-WIF1 was used to overexpress WIF1 in SCAPs. Using Western blot analysis, we verified the expression of HA-WIF1. Alkaline phosphatase (ALP) activity assays, Alizarin Red staining and quantitative calcium analysis were performed to investigate the in vitro potential for dentinogenic differentiation in SCAPs. The expression of dentinogenesis-associated genes DSPP, DMP1, Runx2 and OSX were assayed using real-time RT-PCR. Transplantation experiments were used to measure dentinogenesis potential in vivo. RESULTS: The real time RT-PCR results showed that WIF1 was more highly expressed in apical papilla tissues than in SCAPs, and its expression was increased during the process of dentinogenic differentiation. Overexpression of WIF1 enhanced ALP activity and mineralization in vitro, as well as the expression of DSPP, DMP1 and OSX in SCAPs. Moreover, in vivo transplantation experiments revealed that dentinogenesis in SCAPs was enhanced by WIF1 overexpression. CONCLUSION: These results suggest that WIF1 may enhance dentinogenic differentiation potential in dental MSCs via its regulation of OSX and identified potential target genes that could be useful for improving dental tissue regeneration.

Accelerated dentinogenesis by inhibiting the mitochondrial fission factor, dynamin related protein 1.

Undifferentiated odontogenic epithelium and dental papilla cells differentiate into ameloblasts and odontoblasts, respectively, both of which are essential for tooth development. These differentiation processes involve dramatic functional and morphological changes of the cells. For these changes to occur, activation of mitochondrial functions, including ATP production, is extremely important. In addition, these changes are closely related to mitochondrial fission and fusion, known as mitochondrial dynamics. However, few studies have focused on the role of mitochondrial dynamics in tooth development. The purpose of this study was to clarify this role. We used mouse tooth germ organ cultures and a mouse dental papilla cell line with the ability to differentiate into odontoblasts, in combination with knockdown of the mitochondrial fission factor, dynamin related protein (DRP)1. In organ cultures of the mouse first molar, tooth germ developed to the early bell stage. The amount of dentin formed under DRP1 inhibition was significantly larger than that of the control. In experiments using a mouse dental papilla cell line, differentiation into odontoblasts was enhanced by inhibiting DRP1. This was associated with increased mitochondrial elongation and ATP production compared to the control. These results suggest that DRP1 inhibition accelerates dentin formation through mitochondrial elongation and activation. This raises the possibility that DRP1 might be a therapeutic target for developmental disorders of teeth.