Dentin Sialoprotein Research Articles

Evaluation of reparative dentin formation of ProRoot MTA, Biodentine and BioAggregate using micro-CT and immunohistochemistry

ObjectivesThe purpose of this study was to assess the ability of two new calcium silicate-based pulp-capping materials (Biodentine and BioAggregate) to induce healing in a rat pulp injury model and to compare them with mineral trioxide aggregate (MTA).Materials and MethodsEighteen rats were anesthetized, cavities were prepared and the pulp was capped with either of ProRoot MTA, Biodentine, or BioAggregate. The specimens were scanned using a high-resolution micro-computed tomography (micro-CT) system and were prepared and evaluated histologically and immunohistochemically using dentin sialoprotein (DSP).ResultsOn micro-CT analysis, the ProRoot MTA and Biodentine groups showed significantly thicker hard tissue formation (p < 0.05). On H&E staining, ProRoot MTA showed complete dentin bridge formation with normal pulpal histology. In the Biodentine and BioAggregate groups, a thick, homogeneous hard tissue barrier was observed. The ProRoot MTA specimens showed strong immunopositive reaction for DSP.ConclusionsOur results suggest that calcium silicate-based pulp-capping materials induce favorable effects on reparative processes during vital pulp therapy and that both Biodentine and BioAggregate could be considered as alternatives to ProRoot MTA.

Bioactive glass 45S5-silk fibroin membrane supports proliferation and differentiation of human dental pulp stem cells

To investigate the effect of bioactivity glass 45S5- silk fibroin(BG45S5- SF) membrane on growth, proliferation and differentiation of human dental pulp stem cells(hDPSC), and to provide new ideas and method for the regeneration of pulp-dentine complex. hDPSC seed on pure silk fibroin membrane (protein membrane group) and BG45S5-SF membrane with different concentrations(1 000, 5 000 mg/L, composite membrane group A and B, respectively) were prepared, and the materials were incubated in cell culture fluid for 24 h. No material membrane orifice plate was used as blank control group. Contact angle meter was used to measure surface contact angle of protein membrane and composite membrane group(each group had three repeated holes). Cell proliferation was assessed by cell counting kit- 8 on the 4, 7, 14, and 21 days. The state of adhesion and growth of hDPSC on the materials surface was evaluated by scanning electron microscopy and cytoskeleton staining; and alkaline phosphatase (ALP) activity was measured to evaluate the cell differentiation potential. The expression of odontoblastic differentiation-related genes was measured by real-time PCR. Surface contact angle of the protein membrane group and composite membrane group A and group B were 89.51° ± 0.12°, 70.32° ± 0.07° and 71.31° ± 0.09° respectively. hDPSC adhered well on each materials surface on the 7, 14, 21 days, ALP activity and differentiation genes of composite membrane group A and B rised more significantly than the blank control group and protein membrane group did (P<0.05). Dentin matrix protein1(DMP- 1), dentin sialoprotein(DSP), ALP, osteocalcin(OC) mRNA expression reached peak on the 14 days in group A, and in group B on the 21 days. Bone sialoprotein(BSP) mRNA expression in both group A and B reached peak on the 21 days. BG45S5- SF membrane is able to support the proliferation and showed the potential of odontoblastic differentiation for hDPSC. This finding suggests that BG45S5-SF membrane was a kind of tissue engineering film material with the regeneration potential for pulp-dentine complex.

Combination of Mineral Trioxide Aggregate and Platelet-rich Fibrin Promotes the Odontoblastic Differentiation and Mineralization of Human Dental Pulp Cells via BMP/Smad Signaling Pathway

IntroductionRecent reports have shown that the combined use of platelet-rich fibrin (PRF), an autologous fibrin matrix, and mineral trioxide aggregate (MTA) as root filling material is beneficial for the endodontic management of an open apex. However, the potential of the combination of MTA and PRF as an odontogenic inducer in human dental pulp cells (HDPCs) in vitro has not yet been studied. The purpose of this study was to evaluate the effect of the combination of MTA and PRF on odontoblastic maturation in HDPCs. MethodsHDPCs extracted from third molars were directly cultured with MTA and PRF extract (PRFe). Odontoblastic differentiation of HDPCs was evaluated by measuring the alkaline phosphatase (ALP) activity, and the expression of odontogenesis-related genes was detected using reverse-transcription polymerase chain reaction or Western blot. Mineralization formation was assessed by alizarin red staining. ResultsHDPCs treated with MTA and PRFe significantly up-regulated the expression of dentin sialoprotein and dentin matrix protein-1 and enhanced ALP activity and mineralization compared with those with MTA or PRFe treatment alone. In addition, the combination of MTA and PRFe induced the activation of bone morphogenic proteins (BMP)/Smad, whereas LDN193189, the bone morphogenic protein inhibitor, attenuated dentin sialophosphoprotein and dentin matrix protein-1 expression, ALP activity, and mineralization enhanced by MTA and PRFe treatment. ConclusionsThis study shows that the combination of MTA and PRF has a synergistic effect on the stimulation of odontoblastic differentiation of HDPCs via the modulation of the BMP/Smad signaling pathway.

17β-Estradiol induces odontoblastic differentiation via activation of the c-Src/MAPK pathway in human dental pulp cells.

The present study is aimed at investigating the effects of the exogenous estrogen 17β-estradiol (E2) on odontoblastic differentiation in human dental pulp cells (HDPCs) immotalized with hTERT gene and their molecular mechanism. Proliferation was detected by BrdU assay, and odontoblast differentiation induction was evaluated by the expression of dentin sialophosphoprotein (DSPP), dentin sialoprotein (DSP) and dentin matrix protein1 (DMP1), and alkaline phosphatase (ALP) activity and mineralization. Estrogen receptor-α (ER-α), c-Src, and mitogen-activated protein kinases (MAPKs) were examined and their inhibitors were used to determine the roles on odontogenic induction. E2 significantly promoted the HDPC proliferation, which was mediated by extracellular signal-related kinase 1/2. E2 upregulated DSPP, DSP, and DMP1 as the odontogenic differentiation markers and enhanced ALP activity and mineralization. E2 increased phosphorylation of ER-α and fulvestrant, an ER downregulator, significantly downregulated DSPP, DMP1, and DSP induced by E2. Moreover, E2 treatment activated c-Src and MAPKs upon odontogenic induction, whereas chemical inhibition of c-Src and MAPKs decreased expression of DSPP, DMP1, and DSP and mineralization augmented by E2. Moreover, fulvestrant reduced E2-induced phosphorylation of c-Src and MAPK and inhibition of c-Src by PP2 attenuated activation of MAPKs during E2-induced odontoblastic differentiation. Taken together, these results indicated that E2 stimulates odontoblastic differentiation of HDPCs via coordinated regulation of ER-α, c-Src, and MAPK signaling pathways, which may play a key role in the regeneration of dentin.

Immunoassay analysis of proteins in gingival crevicular fluid samples from resorbing teeth.

To carry out an immunoassay analysis of biomarkers expressed in gingival crevicular fluid (GCF) with the main goal of finding a useful diagnostic pattern to distinguish between resorbing deciduous teeth and nonresorbing controls. A split-mouth design was used in this study with a total of 22 GCF samples collected from 11 patients in the mixed dentition. For each child, one deciduous molar with radiographic evidence of root resorption was used as the test tooth whereas the contralateral first permanent molar with formed roots was used as the control tooth. Samples were processed with immunoassays using a panel of selected biomarkers including interleukin-1 beta (IL-1b), interleukin-1 receptor antagonist (IL-1RA), nuclear factor kappa B ligand (RANKL), osteoprotegerin (OPG), matrix metalloproteinase-9 (MMP-9), and dentin sialoprotein (DSP). There were no statistically significant differences in levels of IL-1b, OPG, and MMP-9 between test and control sites (P > .05). IL-1RA was the only biomarker to show a significant down-regulation (P = .04) in GCF samples collected from resorbing teeth. RANKL data showed a heavily skewed distribution and was deemed unreliable. Only one deciduous GCF sample had detectable levels of DSP; therefore, no further statistical calculation was applicable because of the limited amount of data for this biomarker. This study indicated that IL1-RA is down-regulated in GCF from resorbing primary molars, thus suggesting this cytokine as a potential analyte to be included in a panel that can discriminate between resorbing and nonresorbing teeth.

Enzymatic isolation of viable human odontoblasts.

To improve an enzymatic method previously used for isolation of rat odontoblasts to isolate viable mature human odontoblasts. Collagenase I, collagenase I/hyaluronidase mixture and hyaluronidase were used to extract mature human odontoblasts from the pulp chamber. Detachment of odontoblasts from dentine was determined with field emission scanning electron microscopy (FESEM) and to analyse the significance of differences in tubular diameter, and the t-test was used. MTT-reaction was used to analyse cell viability, and nonparametric Kruskal-Wallis and Mann-Whitney post hoc tests were used to analyse the data. Immunofluorescent staining of dentine sialoprotein (DSP), aquaporin-4 (AQP4) and matrix metalloproteinase-20 (MMP-20) and quantitative PCR (qPCR) of dentine sialophosphoprotein (DSPP) were used to confirm the odontoblastic nature of the cells. MTT-reaction and FESEM demonstrated collagenase I/hyaluronidase resulted in more effective detachment and higher viability than collagenase I alone. Hyaluronidase alone was not able to detach odontoblasts. Immunofluorescence revealed the typical odontoblastic-morphology with one process, and DSP, AQP4 and MMP-20 were detected. Quantitative PCR of DSPP confirmed that the isolated cells expressed this odontoblast-specific gene. The isolation of viable human odontoblasts was successful. The cells demonstrated morphology typical for odontoblasts and expressed characteristic odontoblast-type genes and proteins. This method will enable new approaches, such as apoptosis analysis, for studies using fully differentiated odontoblasts.

Comparative Study of Pulpal Responses to Pulpotomy with ProRoot MTA, RetroMTA, and TheraCal in Dogs' Teeth

IntroductionThis study was conducted to evaluate and compare pulpal responses to ProRoot MTA (Dentsply Tulsa Dental, Tulsa, OK), RetroMTA (Meta Biomed Co, Ltd, Seoul, Korea), and TheraCal (Bisco Inc, Schamburg, IL) in dog partial pulpotomy models. MethodsPartial pulpotomies were performed on 60 beagle teeth. The exposed pulp tissues were randomly capped with either ProRoot MTA (n = 15), RetroMTA (n = 15), TheraCal (n = 15), or interim restorative material as a negative control (n = 15). After 4 weeks, the teeth were extracted and processed for histologic and immunohistochemical examinations using osteocalcin and dentin sialoprotein. Calcific barrier formation, inflammatory reaction, and the odontoblastic layer were evaluated and scored in a blind manner. The areas of newly formed calcific barriers were measured for each group. ResultsIn most of the ProRoot MTA and RetroMTA specimens, continuous calcific barriers were formed, and the pulps contained palisading patterns in the odontoblastic layer that were free of inflammation. However, the TheraCal specimens had lower quality calcific barrier formation, extensive inflammation, and less favorable odontoblastic layer formation. Overall, areas of newly formed calcific barrier were higher in the ProRoot MTA and RetroMTA specimens than in the TheraCal specimens. Also, immunohistochemistry revealed that osteocalcin and dentin sialoprotein were more clearly visible in the ProRoot MTA and RetroMTA specimens than in the TheraCal specimens. ConclusionsRetroMTA could provide an alternative to ProRoot MTA. Both materials produced favorable pulpal responses that were similar in nature, whereas TheraCal produced less favorable pulpal responses.

Effects of In Vitro Osteogenic Induction on In Vivo Tissue Regeneration by Dental Pulp and Periodontal Ligament Stem Cells

IntroductionThe aim of this study was to determine the effects of in vitro odontogenic/cementogenic differentiation on the in vivo tissue regeneration of dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs). MethodsDPSCs and PDLSCs were predifferentiated for 0, 4, or 8 days with an odontogenic/cementogenic medium and then transplanted into subcutaneous pockets in immunocompromised mice. The transplants were harvested 9 weeks after transplantation, and the characteristics of the newly formed tissues in vivo were analyzed by histologic staining; examining alkaline phosphate activity; immunohistochemical staining for osteocalcin, dentin sialoprotein, and type XII collagen; and quantitative real-time polymerase chain reaction to analyze the expression patterns of the following genes: RUNX2, OC, DMP1, DSPP, POSTN, CP23, and Col XII. ResultsIn DPSC transplants, the amount of new tissues was similar in all groups, whereas in predifferentiated transplants the OC and DSPP expression were higher than undifferentiated transplants. Predifferentiated PDLSC transplants generated more hard tissue and expressed higher alkaline phosphatase activity than undifferentiated transplants. In particular, 8-day predifferentiated PDLSC transplants formed tissue closer to the cementum/PDL complex in vivo as confirmed by the higher expression levels of POSTN, CP23, and Col XII. ConclusionsAlthough there was no significant increase in tissue-forming ability among DPSCs after predifferentiation, predifferentiated DPSCs generated hard tissue closer to dentin. Also, predifferentiated PDLSCs appeared to be able to generate higher-quality and greater amounts of tissue for dental regeneration than undifferentiated PDLSCs.

Dentin Sialophosphoprotein–derived Proteins in the Dental Pulp

Porcine dentin sialophosphoprotein (DSPP), the most abundant noncollagenous protein in dentin, is critical for proper mineralization of tooth dentin. DSPP is processed by proteases into 3 major domains: dentin sialoprotein (DSP), dentin glycoprotein (DGP), and dentin phosphoprotein (DPP). There are at least 2 mRNA variants expressed from the Dspp gene: one encodes the full-length DSPP protein (DSP+DGP+DPP); the other encodes only DSP. The shorter transcript is generated through the use of a polyadenylation signal within intron 4, immediately following the DSP coding region (DGP and DPP are encoded by exon 5). We fractionated DSPP-derived proteins from the dental pulp of developing porcine incisors using heparin chromatography. DSP was identified, but little DPP could be detected in any fractions. BMP-1 digestion of DSPP-derived proteins extracted from dental pulp did not generate new DPP bands on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (indicating an absence of intact DSPP), although the results suggested another BMP-1 cleavage site within DSP. We further purified DSPP-derived protein by reversed-phase high-performance liquid chromatography. Its amino acid composition was similar to DSP. Expression of the full-length Dspp mRNA by quantitative real-time polymerase chain reaction analysis was significantly higher in odontoblasts than in pulp, while expression of the DSP-only mRNA was almost equal in odontoblasts and in the body of the pulp. Expression of the full-length Dspp mRNA was also significantly higher than the expression of DSP-only mRNA in odontoblasts. Both the full-length and the DSP-only Dspp mRNA showed only trace expression in the pulp tip. We conclude that use of the 3′ polyadenylation signal in exon 5 predominates in fully differentiated odontoblasts, while both polyadenylation signals are used throughout odontoblast differentiation.

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 β1 (TGF-β1) 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.

Ectopic Hard Tissue Formation by Odonto/Osteogenically In Vitro Differentiated Human Deciduous Teeth Pulp Stem Cells.

There have been many attempts to use the pulp tissue from human deciduous teeth for dentin or bone regeneration. The objective of this study was to determine the effects of odonto/osteogenic in vitro differentiation of deciduous teeth pulp stem cells (DTSCs) on their in vivo hard tissue-forming potential. DTSCs were isolated from extracted deciduous teeth using the outgrowth method. These cells were exposed to odonto/osteogenic stimuli for 4 and 8 days (Day 4 and Day 8 groups, respectively), while cells in the control group were cultured in normal medium. The in vitro differentiated DTSCs and the control DTSCs were transplanted subcutaneously into immunocompromised mice with macroporous biphasic calcium phosphate and sacrificed at 8 weeks post-implantation. The effect of odonto/osteogenic in vitro differentiation was evaluated using alkaline phosphatase (ALP) staining and quantitative reverse transcription polymerase chain reaction (RT-PCR). The in vivo effect was evaluated by qualitative RT-PCR, assessment of ALP activity, histologic analysis, and immunohistochemical staining. The amount of hard tissue was greater in Day 4 group than Day 8 group (p = 0.014). However, Day 8 group generated lamellar bone-like structure, which was immunonegative to anti-human dentin sialoprotein with significantly low expression level of DSPP compared with the control group (p = 0.008). This study demonstrates that odonto/osteogenic in vitro differentiation of DTSCs enhances the formation of bone-like tissue, instead of dentin-like tissue, when transplanted subcutaneously using MBCP as a carrier. The odonto/osteogenic in vitro differentiation of DTSCs may be an effective modification that enhances in vivo bone formation by DTSCs.

Expression of Matrix Metalloproteinase (MMP)-20 and Potential Interaction with Dentin Sialophosphoprotein (DSPP) in Human Major Salivary Glands.

Matrix metalloproteinase-20 (MMP-20) expression is widely regarded as tooth-specific, with expression limited to dental hard tissues. Necessary for sound enamel formation, MMP-20 and MMP-2 proteolytically process dentin sialophosphoprotein (DSPP) into dentin sialoprotein, dentin phosphoprotein, and dentin glycoprotein during tooth formation. In the mid-2000s, three members of the small integrin-binding ligand N-linked glycoproteins (SIBLINGs) were reported to bind specifically with high affinity (nM) to, and activate, three MMPs in vitro: bone sialoprotein with MMP-2; osteopontin with MMP-3; and dentin matrix protein1 with MMP-9. The SIBLING-MMP interaction was confirmed in biological systems such as the ducts of salivary glands, where all five members of the SIBLINGs are expressed. Recently, we documented MMP-20 expression and interaction with DSPP (another member of the SIBLING family) in human oral squamous cell carcinoma. Here we report the expression of MMP-20, and confirm its co-expression and potential interaction with DSPP in human major salivary gland tissues and cell line using immunohistochemistry, immunofluorescence, western blot, quantitative RT-PCR, and proximity ligation assay. This report reinforces our earlier suggestion that the SIBLING-MMP complexes may be involved in the turnover of extracellular proteins damaged by oxidation byproducts in metabolically active duct epithelial systems.

Matrix Metalloproteinase 20–Dentin Sialophosphoprotein Interaction in Oral Cancer

Matrix metalloproteinase 20 (MMP-20), widely regarded as tooth specific, participates with MMP-2 in processing dentin sialophosphoprotein (DSPP) into dentin sialoprotein, dentin phosphoprotein, and dentin glycoprotein. In biochemical system, MMP-2, MMP-3, and MMP-9 bind with high affinity to, and are activated by, specific small integrin-binding ligand N-linked glycoproteins (SIBLINGs): bone sialoprotein, osteopontin, and dentin matrix protein 1, respectively. Subsequent reports documented possible biological relevance of SIBLING-MMP interaction in vivo by showing that SIBLINGs are always coexpressed with their MMP partners. However, the cognate MMPs for 2 other SIBLINGs—DSPP and matrix extracellular phosphogylcoprotein—are yet to be identified. Our goal was to investigate MMP-20 expression and to explore preliminary evidence of its interaction with DSPP in oral squamous cell carcinomas (OSCCs). Immunohistochemistry analysis of sections from 21 cases of archived human OSCC tissues showed immunoreactivity for MMP-20 in 18 (86%) and coexpression with DSPP in all 15 cases (71%) positive for DSPP. Similarly, 28 (93%) of 30 cases of oral epithelial dysplasia were positive for MMP-20. Western blot and quantitative real-time polymerase chain reaction analysis on OSCC cell lines showed upregulation of MMP-20 protein and mRNA, respectively, while immunofluorescence showed coexpression of MMP-20 and DSPP. Colocalization and potential interaction of MMP-20 with dentin sialoprotein was confirmed by coimmunoprecipitation and mass spectrometry analysis of immunoprecipitation product from OSCC cell lysate, and in situ proximity ligation assays. Significantly, results of chromatin immunoprecipation revealed a 9-fold enrichment of DSPP at MMP-20 promoter–proximal elements. Our data provide evidence that MMP-20 has a wider tissue distribution than previously acknowledged. MMP-20–DSPP specific interaction, excluding other MMP-20–SIBLING pairings, identifies MMP-20 as DSPP cognate MMP. Furthermore, the strong DSPP enrichment at the MMP-20 promoter suggests a regulatory role in MMP-20 transcription. These novel findings provide the foundation to explore the mechanisms and significance of DSPP-MMP-20 interaction in oral carcinogenesis.

Nuclear Factor I-C promotes proliferation and differentiation of apical papilla-derived human stem cells in vitro

The transcription factor Nuclear Factor I-C (NFIC) has been implicated in the regulation of tooth root development, where it may be anticipated to impact on the behavior of stem cells from the apical papilla (SCAPs) and root odontoblast activity. We hypothesized that NFIC may provide an important target for promoting dentin/root regeneration. In the present study, the effects of NFIC on the proliferation and differentiation of SCAPs were investigated. Over-expression of NFIC increased cell proliferation, mineralization nodule formation and alkaline phosphatase (ALP) activity in SCAPs. Furthermore, NFIC up-regulated the mRNA levels of odontogenic-related markers, ALP, osteocalcin and collagen type I as well as dentin sialoprotein protein levels. In contrast, knockdown of NFIC by si-RNA inhibited the mineralization capacity of SCAPs and down-regulated the expression of odontogenic-related markers. In conclusion, the results indicated that upregulation of NFIC activity in SCAPs may promote osteo/odontoblastic differentiation of SCAPs.

Four and a Half LIM Domain Protein 2 Enhances Differentiation and Mineralization of Human Dental Pulp Cells

IntroductionFour and a half LIM domain protein 2 (FHL2) is 1 member of the LIM-only protein family, which is a transcriptional cofactor, playing important roles in osteoblast differentiation and bone formation. Our previous studies showed that the FHL2 protein was abundantly expressed in odontoblasts both during tooth development and in mature teeth, indicating that FHL2 might play a role in odontoblast differentiation and dentin formation. The aim of this study was to investigate the effects of FHL2 overexpression on odontoblast differentiation and mineralization of human dental pulp cells (hDPCs), exploring the function of FHL2 on dentin formation. MethodsFHL2 was steadily transfected into hDPCs. Then, alkaline phosphatase activity assay was shown; the formation of mineralized nodules was assessed by alizarin red staining; and the expression of odontoblast differentiation and mineralization-related molecules including alkaline phosphate (ALP), dentin sialoprotein (DSP), bone sialoprotein (BSP), and osteopontin (OPN) at messenger RNA and protein levels were determined by quantitative real-time polymerase chain reaction and Western blot analysis. ResultsIt was found that FHL2 overexpression could increase ALP activity and the formation of mineralized nodules of dental pulp cells. Moreover, FHL2 overexpression could up-regulate the expression of ALP, DSP, BSP, and OPN messenger RNA and protein levels significantly. ConclusionsFHL2 overexpression could enhance the differentiation and mineralization of hDPCs.

Dentin and pulp sense cold stimulus

Dentin hypersensitivity is a common symptom, and recent convergent evidences have reported transient receptor potential (TRP) channels in odontoblasts act as mechanical and thermal molecular sensor, which detect stimulation applied on the exposed dentin surface, to drive multiple odontoblastic cellular functions, such as sensory transduction and/or dentin formation. In the present study, we confirmed expression of TRP melastatin subfamily member-8 (TRPM8) channels in primary cultured cells derived from human dental pulp cells (HPCs) and mouse odontoblast-lineage cells (OLCs) as well as in dentin matrix protein-1 (DMP-1) and dentin sialoprotein (DSP) positive acutely isolated rat odontoblasts from dental pulp tissue slice culture by immunohistochemical analyses. In addition, we detected TRPM8 channel expression on HPCs and OLCs by RT-PCR and Western blotting analyses. These results indicated that both odontoblasts and dental pulp cells express TRPM8 channels in rat, mouse and human, and therefore we hypothesize they may contribute as cold sensor in tooth.

Wntless Regulates Dentin Apposition and Root Elongation in the Mandibular Molar

Wnt signaling plays an essential role in the dental epithelium and mesenchyme during tooth morphogenesis. However, it remains unclear if Wnt ligands, produced from dental mesenchyme, are necessary for odontoblast differentiation and dentin formation. Here, we show that odontoblast-specific disruption of Wntless (Wls), a chaperon protein that regulates Wnt sorting and secretion, leads to severe defects in dentin formation and root elongation. Dentin thickness decreased remarkably and pulp chambers enlarged in the mandibular molars of OC-Cre;WlsCO/CO mice. Although the initial odontoblast differentiation was normal in the mutant crown, odontoblasts became cuboidal and dentin thickness was reduced. In immunohistochemistry, Wnt10a, β-catenin, type I collagen, and dentin sialoprotein were significantly down-regulated in the odontoblasts of mutant crown. In addition, roots were short and root canals were widened. Cell proliferation was reduced in the developing root apex of mutant molars. Furthermore, Wnt10a and Axin2 expression was remarkably decreased in the odontoblasts of mutant roots. Deletion of the Wls gene in odontoblasts appears to reduce canonical Wnt activity, leading to inhibition of odontoblast maturation and root elongation.

Expression and Function of the Actin-severing Protein Adseverin in the Proliferation, Migration, and Differentiation of Dental Pulp Cells

IntroductionAdseverin is an actin-severing and actin-capping protein that is primarily expressed in secretory cells, where it regulates the filamentous actin cytoskeleton during cell differentiation and exocytosis. However, little is known regarding its regulatory role in dental pulp cells (DPCs). This study examined the expression and function of adseverin in the proliferation, migration, and odontoblastic differentiation of DPCs. MethodsDPCs were assayed for morphologic changes, proliferation, migration, alkaline phosphatase activity, and dentin sialoprotein and dentin matrix protein-1 protein levels in vitro after knockdown of adseverin by using small interfering RNA. Tooth germs isolated from Sprague-Dawley rats were processed for immunohistochemistry analysis of adseverin. ResultsAdseverin expression was increased in a time-dependent fashion in the early stage of odontoblastic differentiation. When adseverin expression was suppressed in DPCs, their cellular morphology was altered, and their proliferation, migration, and odontoblastic differentiation were substantially decreased in vitro. Secretory odontoblasts in the tooth germ at day 5 post partum expressed a stronger adseverin signal compared with those at days 1 and 3 post partum. ConclusionsAdseverin may play a crucial role in the proliferation, migration, and odontoblastic differentiation of DPCs via filamentous actin cytoskeleton regulation. However, further investigations are required to clarify the underlying mechanisms.

Human Umbilical Cord Mesenchymal Stem Cells: A New Therapeutic Option for Tooth Regeneration.

Tooth regeneration is considered to be an optimistic approach to replace current treatments for tooth loss. It is important to determine the most suitable seed cells for tooth regeneration. Recently, human umbilical cord mesenchymal stem cells (hUCMSCs) have been regarded as a promising candidate for tissue regeneration. However, it has not been reported whether hUCMSCs can be employed in tooth regeneration. Here, we report that hUCMSCs can be induced into odontoblast-like cells in vitro and in vivo. Induced hUCMSCs expressed dentin-related proteins including dentin sialoprotein (DSP) and dentin matrix protein-1 (DMP-1), and their gene expression levels were similar to those in native pulp tissue cells. Moreover, DSP- and DMP-1-positive calcifications were observed after implantation of hUCMSCs in vivo. These findings reveal that hUCMSCs have an odontogenic differentiation potency to differentiate to odontoblast-like cells with characteristic deposition of dentin-like matrix in vivo. This study clearly demonstrates hUCMSCs as an alternative therapeutic cell source for tooth regeneration.

Controlled release of simvastatin from biodegradable hydrogels promotes odontoblastic differentiation.

The objective of this study was to investigate the odontoblastic differentiation of dental pulp stem cells (DPSC) by biodegradable hydrogels incorporating simvastatin micelles, both in vitro and in vivo. Simvastatin (ST) was incorporated into the micelles of gelatin grafted with L-lactic acid oligomers (LAo) to allow water-solubilization. The simvastatin-LAo-grafted gelatin (LAo-g-gelatin) micelles were mixed with gelatin, followed by chemical crosslinking to form gelatin hydrogels (ST Mi/GH). The ST Mi were released from the gelatin hydrogel granules (GH) through enzymatic degradation. The ST Mi enhanced alkaline phosphatase activity, calcium deposition, and bone morphogenic protein-2 secretion of DPSC. When implanted subcutaneously into mice, the ST Mi/GH treated group exhibited increased dentin sialoprotein and calcium deposition, compared with those treated with GH plus free ST. It is possible to achieve odontoblastic differentiation of DPSC through the controlled release of ST from GH.