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Heparan sulfate (HS) is a major component of dental pulp tissue. We previously reported that inhibiting HS biosynthesis impedes endothelial differentiation of dental pulp stem cells (DPSCs). However, the underlying mechanisms by which exogenous HS induces DPSC differentiation and pulp tissue regeneration remain unknown. This study explores the impact of exogenous HS on vasculogenesis and dentinogenesis of DPSCs both in vitro and in vivo. Human-derived DPSCs were cultured in endothelial and odontogenic differentiation media and treated with HS. Endothelial differentiation of DPSCs was investigated by real-time polymerase chain reaction and capillary sprouting assay. Odontogenic differentiation was assessed through real-time polymerase chain reaction and detection of mineralized dentin-like deposition. Additionally, the influence of HS on pulp tissue was assessed with a direct pulp capping model, in which HS was delivered to exposed pulp tissue in rats. Gelatin sponges were loaded with either phosphate-buffered saline or 101–102 μg/mL HS and placed onto the pulp tissue. Following a 28-day period, tissues were investigated by histological analysis and micro–computed tomography imaging. HS treatment markedly increased expression levels of key endothelial and odontogenic genes, enhanced the formation of capillary-like structures, and promoted the deposition of mineralized matrices. Treatment of exposed pulp tissue with HS in the in vivo pulp capping study induced formation of capillaries and reparative dentin. Exogenous HS effectively promoted vasculogenesis and dentinogenesis of DPSCs in vitro and induced reparative dentin formation in vivo , highlighting its therapeutic potential for pulp capping treatment. [ABSTRACT FROM AUTHOR]

Prostaglandin E 2 (PGE 2) exerts biological actions through its transport pathway involving intracellular synthesis, extracellular transport, and receptor binding. This study aimed to determine the localization of the components of the PGE 2 -transporting pathway in human dental pulp and explore the relevance of PGE 2 receptors (EP2/EP4) to angiogenesis and dentinogenesis. Protein localization of microsomal PGE 2 (mPGES)synthase, PGE 2 transporters (multidrug resistance-associated protein-4 [MRP4] and prostaglandin transporter [PGT]), and EP2/EP4 was analyzed using double immunofluorescence staining. Tooth slices from human third molars were cultured with or without butaprost (EP2 agonist) or rivenprost (EP4 agonist) for 1 week. Morphometric analysis of endothelial cell filopodia was performed to evaluate angiogenesis, and real-time polymerase chain reaction was performed to evaluate angiogenesis and odontoblast differentiation markers. MRP4 and PGT were colocalized with mPGES and EP2/EP4 in odontoblasts and endothelial cells. Furthermore, MRP4 was colocalized with mPGES and EP4 in human leukocyte antigen-DR-expressing dendritic cells. In the tooth slice culture, EP2/EP4 agonists induced significant increases in the number and length of filopodia and mRNA expression of angiogenesis markers (vascular endothelial growth factor and fibroblast growth factor-2) and odontoblast differentiation markers (dentin sialophosphoprotein and collagen type 1). PGE 2 -producing enzyme (mPGES), transporters (MRP4 and PGT), and PGE 2 -specific receptors (EP2/EP4) were immunolocalized in various cellular components of the human dental pulp. EP2/EP4 agonists promoted endothelial cell filopodia generation and upregulated angiogenesis- and odontoblast differentiation-related genes, suggesting that PGE 2 binding to EP2/EP4 is associated with angiogenic and dentinogenic responses. [ABSTRACT FROM AUTHOR]

The differentiation of dental pulp cells (DPCs) plays an important role in the repair of dental pulp injury. Bone morphogenetic protein 9 (BMP9) is one of the most effective BMPs to induce the differentiation of stem cells. However, the role of BMP9 in promoting the odontogenic differentiation of DPCs and dentinogenesis is worth knowing. Fluorescence in situ hybridization and immunohistochemistry staining were performed to detect the BMP9 expression in human dental pulp. BMP9 was overexpressed in human DPCs (hDPCs), and the mineralization of hDPCs was tested by alkaline phosphatase staining and alizarin red staining. The expression of odontogenic differentiation-related genes was examined by quantitative real-time polymerase chain reaction and western blotting. The subcutaneous transplantation experiment was performed to test the odonto-induction ability of BMP9 in vivo. The rat direct pulp-capping experiment was performed to test the function of BMP9 in promoting dentin formation. BMP9 showed an increased expression in odontoblast layer at both the mRNA and protein levels. BMP9 enhanced the mineralization and induced the expression of odontogenic differentiation-related genes in hDPCs. More mineralized nodules, and increased expression of dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP1) were detected in the beta-tricalcium phosphate scaffold/cells composites of BMP9 group compared with the control group. Meanwhile, there was thicker reparative dentin formation in the BMP9 group in the rat pulp exposure experiment. BMP9 participates in the process of DPC differentiation and promotes DPC mineralization and dentinogenesis. BMP9 might be a potential therapeutic target in the repair of dental pulp injury. [ABSTRACT FROM AUTHOR]

Abstract Introduction Neurotrophic factors play a significant role in the innervation of the pulp-dentin complex during and after organogenesis. There have been numerous bioactive molecules identified in the dentin extracellular matrix; however, the expression of neurotrophic factors in the dentin matrix and their biological activity are largely unknown. The purpose of this study was to characterize the relative expression of neurotrophic factors in human dentin matrix proteins (DMPs) and their effect on neurite outgrowth of trigeminal (TG) neurons. Methods Dentin was powdered in liquid nitrogen from noncarious human third molar teeth. DMPs were solubilized through an EDTA extraction method, dialyzed, and lyophilized until use. The relative expression of nerve growth factor, brain-derived neurotrophic factor, glial cell−line derived neurotrophic factor, neurotrophin 3, and neurotrophin 4/5 was determined by the enzyme-linked immunosorbent assay. Rat TG neurons were cultured and exposed to different concentrations of DMPs (1–105 ng/mL) or vehicle, and a quantitative neurite outgrowth assay was performed. Results Human DMPs contained all of the tested neurotrophic factors, with glial cell−line derived neurotrophic factor and neurotrophin 4/5 found at the highest levels. DMPs were able to promote the neurite outgrowth of rat TG neurons at an optimum concentration of 10–102 ng/mL, whereas the effect was partially inhibited at higher concentrations (>103 ng/mL). Conclusions The human dentin extracellular matrix is a rich reservoir for neurotrophic factors that are key components for neuronal homeostasis, differentiation, and regeneration. These data suggest that neurotrophins in DMPs could play an important role as signaling molecules for the innervation of the pulp-dentin complex during the processes of tooth formation, repair, and regeneration. [ABSTRACT FROM AUTHOR]

Introduction: Prostaglandin E 2 (PGE 2 ) exerts biological actions through its transport pathway involving intracellular synthesis, extracellular transport, and receptor binding. This study aimed to determine the localization of the components of the PGE 2 -transporting pathway in human dental pulp and explore the relevance of PGE 2 receptors (EP2/EP4) to angiogenesis and dentinogenesis., Methods: Protein localization of microsomal PGE 2 (mPGES)synthase, PGE 2 transporters (multidrug resistance-associated protein-4 [MRP4] and prostaglandin transporter [PGT]), and EP2/EP4 was analyzed using double immunofluorescence staining. Tooth slices from human third molars were cultured with or without butaprost (EP2 agonist) or rivenprost (EP4 agonist) for 1 week. Morphometric analysis of endothelial cell filopodia was performed to evaluate angiogenesis, and real-time polymerase chain reaction was performed to evaluate angiogenesis and odontoblast differentiation markers., Results: MRP4 and PGT were colocalized with mPGES and EP2/EP4 in odontoblasts and endothelial cells. Furthermore, MRP4 was colocalized with mPGES and EP4 in human leukocyte antigen-DR-expressing dendritic cells. In the tooth slice culture, EP2/EP4 agonists induced significant increases in the number and length of filopodia and mRNA expression of angiogenesis markers (vascular endothelial growth factor and fibroblast growth factor-2) and odontoblast differentiation markers (dentin sialophosphoprotein and collagen type 1)., Conclusions: PGE 2 -producing enzyme (mPGES), transporters (MRP4 and PGT), and PGE 2 -specific receptors (EP2/EP4) were immunolocalized in various cellular components of the human dental pulp. EP2/EP4 agonists promoted endothelial cell filopodia generation and upregulated angiogenesis- and odontoblast differentiation-related genes, suggesting that PGE 2 binding to EP2/EP4 is associated with angiogenic and dentinogenic responses., (Copyright © 2023 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction Adenosine 5′-triphosphate (ATP) is a potent signaling molecule that regulates diverse biological activities in cells. Its effects on human dental pulp cells (HDPCs) remain unknown. This study aimed to examine the effects of ATP on proliferation and differentiation of HDPCs. Methods Reverse transcription polymerase chain reaction was performed to explore the mRNA expression of P2 receptor subtypes. Cell Counting Kit-8 test and flow cytometry analysis were used to examine the effects of ATP on proliferation and cell cycle of HDPCs. The effects of ATP on differentiation of HDPCs were examined by using alizarin red S staining, energy-dispersive x-ray analysis, Western blot analysis, and real-time polymerase chain reaction. Results The purinoceptors P2X3, P2X4, P2X5, P2X7, and all P2Y receptor subtypes were confirmed to present in HDPCs. ATP enhanced HDPC proliferation at 10 μmol/L concentration. However, it inhibited cell proliferation by arresting the cell cycle in G 0 G 1 phase ( P < .05 versus control) and induced odontoblastic differentiation, ERK/MAPK activation, and dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP) mRNA transcriptions at 800 μmol/L concentration. Suramin, an ATP receptor antagonist, inhibited ERK/MAPK activation and HDPC odontoblastic differentiation ( P < .05 versus control). Conclusions Extracellular ATP activates P2 receptors and downstream signaling events that induce HDPC odontogenic differentiation. Thus, ATP may promote dental pulp tissue healing and repair through P2 signaling. Results provide new insights into the molecular regulation of pulpal wound healing. [ABSTRACT FROM AUTHOR]

Introduction: Substance P (SP) is a neuropeptide released from the nervous fibers in response to injury. In addition to its association with pain and reactions to anxiety and stress, SP exerts various physiological functions by binding to the neurokinin-1 receptor (NK1R). However, the expression and role of SP in reparative dentinogenesis remain elusive. Here, we explored whether SP is involved in odontoblastic differentiation during reparative dentinogenesis., Methods: Dental pulp stem cells (DPSCs) were isolated from healthy human dental pulp tissues and subjected to odontoblastic differentiation. The expression of SP and NK1R during odontoblastic differentiation was investigated in vitro. The effects of SP on odontoblastic differentiation of DPSCs were evaluated using alizarin red staining, alkaline phosphatase staining, and real-time polymerase chain reaction. After direct pulp capping with mineral trioxide aggregate, the expression of SP and NK1R during reparative dentin formation in rats were identified using histological and immunohistochemical staining., Results: SP and NK1R expression increased during the odontoblastic differentiation of DPSCs. SP translocated to the nucleus when DPSCs were exposed to differentiation medium. NK1R was always present in the nuclei of DPSCs and odontoblast-like cells. Additionally, we discovered that 10 -8  M SP marginally enhanced the odontoblastic differentiation of DPSCs, and that these effects could be impaired by the NK1R antagonist. Furthermore, SP and NK1R were expressed in odontoblast-like and dental pulp cells during reparative dentin formation in vivo., Conclusions: SP contributes to odontoblastic differentiation during reparative dentin formation by binding to the NK1R., (Copyright © 2023. Published by Elsevier Inc.)

Introduction Quick-Set (Primus Consulting, Bradenton, FL) is a calcium aluminosilicate cement that is a potential alternative to mineral trioxide aggregate (MTA) with greater acid resistance and faster setting. The purpose of this study was to compare the effects of Quick-Set and MTA on pulpal tissues in response to pulpotomy procedures. Methods The pulp chambers of 42 maxillary teeth in 7 beagle dogs were accessed, and the coronal pulpal tissue was removed. Pulpotomy procedures were performed, placing the experimental materials directly over the radicular pulp tissues. The dogs were sacrificed at 70 days, and the teeth and surrounding tissues were removed and prepared for histologic analysis. The sections of the pulpotomy areas were scored for inflammation, pulp tissue organization, reactionary dentin formation, and quality of dentinogenesis. Results The Quick-Set group exhibited significantly more pulpal inflammation ( P = .002) and significantly less pulp tissue organization ( P = .004). No significant difference was noted for reactionary dentin formation ( P = .526) and quality of dentinogenesis ( P = .436). Conclusions Compared with ProRoot White MTA (Dentsply Tulsa Dental Specialties, Tulsa, OK), Quick-Set exhibited more pulpal inflammation and decreased pulp tissue organization. No significant differences were noted for reactionary dentin formation and quality of dentinogenesis. [ABSTRACT FROM AUTHOR]

Introduction The aim of the present study was to evaluate comparatively the bioactivity potential of a calcium silicate–based material (Biodentine; Septodont, Saint-Maur-des-fosses Cedex, France) after the restoration of deep dentinal cavities of miniature swine teeth with or without the application of a calcium hydroxide–containing pulp protective base (Dycal; Caulk Lab, Milford, DE). Methods Thirty-three permanent teeth (premolars, canines, and incisors) of 3 miniature swine were used. Class V cavities were prepared on the buccal surface of teeth. The cavities were restored with Biodentine in the presence (control group) or absence (experimental group) of a Dycal protective base. The pulpal tissue responses were histologically and histomorphometrically assessed at postoperative periods of 3 and 8 weeks. Three specimens were further evaluated with scanning electron microscopy. The maximum thickness of the postoperatively formed mineralized matrix beneath the cavity floor was measured. Data were statistically analyzed by the Kruskal-Wallis and the Mann-Whitney U tests. Results A bacterial staining reaction along the cavity walls or intense inflammatory infiltration in the pulp was not detected in any of the specimens. A continuous zone of the postoperatively formed mineralized matrix mostly of atubular structure with scattered defects and cellular inclusions and occasionally followed by a thin zone with tubular morphology was detected in all specimens of the control group and 13 of 18 experimental group teeth. In the remaining teeth of the experimental group, a separate zone composed of the osteotypic mineralized matrix and soft tissues was noted between the circumpulpal and the newly formed matrix. Scanning electron microscopy confirmed the fibrous structural morphology of the tertiary dentin. A significantly higher rate of the postoperatively formed mineralized matrix had been formed in the teeth of the experimental group in both periods of 3 and 8 weeks ( P < .01). Conclusions The present investigation indicates that under the present experimental conditions tertiary dentin with occasional intermediate formation of osteodentin is observed after the application of Biodentine in the presence or absence of a Dycal protective base. The thickness of the tertiary dentine zone was significantly higher in the absence of Dycal. [ABSTRACT FROM AUTHOR]

Introduction Beta-2 adrenergic receptor has been found within the osteoblast membrane meditating bone remodeling. Propranolol is a sympatholytic beta antagonist commonly used as long-term medication for the management of many common diseases such as hypertension. This study was performed to verify the presence of this receptor on odontoblasts in rats and, if present, to investigate its possible association with tertiary dentinogenesis. Methods Twenty male Sprague-Dawley rats (9 weeks old) were randomly assigned to 4 groups: CP0.8 group, cavity preparation + propranolol treatment (0.8 mg/day, n = 5); CP4 group, cavity preparation + propranolol treatment (4.0 mg/day, n = 7); CON group, cavity preparation + saline treatment (0.2 mL/day, n = 5); and NT group, no treatment ( n = 3). Cavity preparation was performed on the mesial aspect of the maxillary first molars bilaterally. After 2 weeks, the tertiary dentinogenesis (CP0.8, CP4, and CON) was examined by hematoxylin-eosin staining, and the localization of beta-2 adrenergic receptor (NT) was examined by immunohistochemistry. Results The beta-2 adrenergic receptor immunoreactivity was observed in the odontoblastic layer in normal rat molar dental pulp. The tertiary dentinogenesis beneath the prepared cavity was significantly higher in the rats receiving 2-week systemic administration of propranolol than in those without the propranolol treatment. The higher-dose treatment of propranolol ( P < .001) presented more effective up-regulation of tertiary dentinogenesis than the lower-dose treatment ( P < .01). Conclusions These results indicate that the sympathetic nervous system decreases tertiary dentin formation via beta-2 adrenergic receptors located on rat odontoblasts. It suggests that adrenergic beta antagonist is expected to use in the treatment of inducing tertiary dentin formation to protect dental pulp. [ABSTRACT FROM AUTHOR]

Introduction Leptin, an inflammation-related adipokine, and its receptor (LEPR) are expressed in human dental pulp. Dentin sialophosphoprotein (DSPP) is involved in dentinogenesis and the dental pulp reparative response. The cell type expressing LEPR in dental human pulp and the resultant effect of the binding of leptin to LEPR remain unknown. This study describes the immmunohistochemical localization of LEPR and the effect of leptin on DSPP expression in human dental pulp. Methods Twenty-five dental pulp specimens were obtained from freshly extracted caries-free and restoration-free human third molars. LEPR localization was examined by immunohistochemistry using the antihuman LEPR monoclonal antibody. The effect of leptin on DSPP expression was determined by immunoblot analysis and quantitative real-time polymerase chain reaction. Results Immunoreactivity for LEPR concentrated in the odontoblast layer but was not evident in the central zone of the dental pulp. Leptin dose dependently stimulated DSPP expression. Western blot analysis revealed the presence of a protein with an apparent molecular weight of ∼00 kDa, the estimated molecular weight of DSPP. The expression of DSPP messenger RNA was confirmed by quantitative real-time polymerase chain reaction, and the size of the amplified fragments (298 bp) was confirmed by agarose gel electrophoresis. Conclusions The present study shows that human dental pulp is immunoreactive for LEPR, with the immunoreactivity concentrated in the odontoblast layer, and that leptin stimulates, in a dose-dependent manner, DSPP protein and messenger RNA (mRNA) expression in human dental pulp. These findings further support the functional role of leptin in the dentin mineralization process and/or in dental pulp reparative and immune responses. [ABSTRACT FROM AUTHOR]

Introduction During dentinogenesis, growth factors become entrapped in the dentin matrix that can later be released by demineralization. Their effect on pulpal stem cell migration, proliferation, and differentiation could be beneficial for regenerative endodontic therapies. However, precondition for success, as for conventional root canal treatment, will be sufficient disinfection of the root canal system. Various irrigation solutions and intracanal dressings are available for clinical use. The aim of this study was 2-fold: to identify a demineralizing solution suitable for growth factor release directly from dentin and to evaluate whether commonly used disinfectants for endodontic treatment will compromise this effect. Methods Dentin disks were prepared from extracted human teeth and treated with EDTA or citric acid at different concentrations or pH for different exposure periods. The amount of transforming growth factor-β1 (TGF-β1), fibroblast growth factor 2, and vascular endothelial growth factor were quantified via enzyme-linked immunosorbent assay and visualized by gold labeling. Subsequently, different irrigation solutions (5.25% sodium hypochloride, 0.12% chlorhexidine digluconate) and intracanal dressings (corticoid-antibiotic paste, calcium hydroxide: water-based and oil-based, triple antibiotic paste, chlorhexidine gel) were tested, and the release of TGF-β1 was measured after a subsequent conditioning step with EDTA. Results Conditioning with 10% EDTA at pH 7 rendered the highest amounts of TGF-β1 among all test solutions. Fibroblast growth factor 2 and vascular endothelial growth factor were detected after EDTA conditioning at minute concentrations. Irrigation with chlorhexidine before EDTA conditioning increased TGF-β1 release; sodium hypochloride had the opposite effect. All tested intracanal dressings interfered with TGF-β1 release except water-based calcium hydroxide. Conclusions Growth factors can be released directly from dentin via EDTA conditioning. The use of disinfecting solutions or medicaments can amplify or attenuate this effect. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: Mineral trioxide aggregate (MTA) has been widely used in clinical apexification and apexogenesis. However, the effects of MTA on the stem cells from apical papilla (SCAPs) and the precise mechanism of apexogenesis have not been elucidated in detail. Methods: Multiple colony-derived stem cells were isolated from the apical papillae, and the effects of MTA on the proliferation and differentiation of SCAPs were investigated both in vitro and in vivo. Activation of nuclear factor kappa B (NFκB) pathway in MTA-treated SCAPs was analyzed by immunofluorescence assay and Western blot. Results: MTA at the concentration of 2 mg/mL did not affect the proliferation activity of SCAPs. However, 2 mg/mL MTA-treated SCAPs presented the ultrastructural changes, up-regulated alkaline phosphatase, increased calcium deposition, up-regulated expression of odontoblast markers (dentin sialoprotein and dentin sialophosphoprotein) and odonto/osteoblast markers (runt-related transcription factor 2 and osteocalcin), suggesting that MTA enhanced the odonto/osteoblastic differentiation of SCAPs in vitro. In vivo results confirmed that MTA can promote the regular dentinogenesis of SCAPs. Moreover, MTA-treated SCAPs exhibited the up-regulated cytoplasmic phos-IκBα and phos-P65, enhanced nuclear P65, and increased nuclear translocation of P65. When co-treated with BMS345541 (the specific NFκB inhibitor), MTA-mediated odonto/osteoblastic differentiation was significantly attenuated. Conclusions: MTA at the concentration of 2 mg/mL can improve the odonto/osteogenic capacity of SCAPs via the activation of NFκB pathway. [Copyright &y& Elsevier]

Abstract: Introduction: The chronic nature of diabetes mellitus (DM) raises the risk of oral complication diseases. In general, DM causes oxidative stress to organs. This study aimed to evaluate the cellular change of dental pulp cells against glucose oxidative stress by glucose oxidase with a high glucose state. The purpose of this study was to test the antioxidant character of davallialactone and to reduce the pathogenesis of dental pulp cells against glucose oxidative stress. Methods: The glucose oxidase with a high glucose concentration was tested for hydroxy peroxide (H2O2) production, cellular toxicity, reactive oxygen species (ROS) formation, induction of inflammatory molecules and disturbance of dentin mineralization in human dental pulp cells. The anti-oxidant effect of Davallilactone was investigated to restore dental pulp cells' vitality and dentin mineralization via reduction of H2O2 production, cellular toxicity, ROS formation and inflammatory molecules. Results: The treatment of glucose oxidase with a high glucose concentration increased H2O2 production, cellular toxicity, and inflammatory molecules and disturbed dentin mineralization by reducing pulp cell activity. However, davallialactone reduced H2O2 production, cellular toxicity, ROS formation, inflammatory molecules, and dentin mineralization disturbances even with a long-term glucose oxidative stress state. Conclusions: The results of this study imply that the development of oral complications is related to the irreversible damage of dental pulp cells by DM-induced oxidative stress. Davallialactone, a natural antioxidant, may be useful to treat complicated oral disease, representing an improvement for pulp vital therapy. [Copyright &y& Elsevier]

Abstract: Introduction: Calcium aluminosilicate cements are fast-setting, acid-resistant, bioactive cements that may be used as root-repair materials. This study examined the osteogenic/dentinogenic potential of an experimental calcium aluminosilicate cement (Quick-Set) by using a murine odontoblast-like cell model. Methods: Quick-Set and white ProRoot MTA (WMTA) were mixed with the proprietary gel or deionized water, allowed to set completely in 100% relative humidity, and aged in complete growth medium for 2 weeks until rendered non-cytotoxic. Similarly aged Teflon disks were used as negative control. The MDPC-23 cell line was used for evaluating changes in mRNA expressions of genes associated with osteogenic/dentinogenic differentiation and mineralization (quantitative reverse transcription polymerase chain reaction), alkaline phosphatase enzyme production, and extracellular matrix mineralization (alizarin red S staining). Results: After MDPC-23 cells were incubated with the materials in osteogenic differentiation medium for 1 week, both cements showed up-regulation in ALP and DSPP expression. Fold increases in these 2 genes were not significantly different between Quick-Set and WMTA. Both cements showed no statistically significant up-regulation/down-regulation in RUNX2, OCN, BSP, and DMP1 gene expression compared with Teflon. Alkaline phosphatase activity of cells cultured on Quick-Set and WMTA were not significantly different at 1 week or 2 weeks but were significantly higher (P < .05) than Teflon in both weeks. Both cements showed significantly higher calcium deposition compared with Teflon after 3 weeks of incubation in mineralizing medium (P < .001). Differences between Quick-Set and WMTA were not statistically significant. Conclusions: The experimental calcium aluminosilicate cement exhibits similar osteogenic/dentinogenic properties to WMTA and may be a potential substitute for commercially available tricalcium silicate cements. [Copyright &y& Elsevier]

Abstract: Introduction: The molecular mechanisms behind odontoblast differentiation remain obscure. Lymphoid enhancer-binding factor 1 (Lef1) is a transcription factor that mediates Wnt signaling and has been suggested to regulate dentin sialophosphoprotein (Dspp) expression in vitro. This study aimed to clarify their precise relationship in the process of odontoblast differentiation in vivo. Methods: The detailed spatiotemporal expression patterns of Lef1 and Dspp together with other known and putative odontoblast differentiation markers such as P21 and heat-shock protein 25 (Hsp25) were examined by in situ hybridization and immunohistochemistry on paraffin sections of rat incisors and developing molars at postnatal days 1–100. To observe odontoblast regeneration following tooth injury, a cavity was prepared on the upper first molar of 10-week-old rats and the expressions of Lef1 and Dspp were investigated. Results: Following undifferentiated state expressing none of these examined markers, preodontoblasts begun to express P21, Lef1 and Hsp25 according to their progress of differentiation, although Dspp was undetectable. Immature odontoblasts commenced transcribing Dspp simultaneously with dentin calcification. Lef1, Dspp and Hsp25 were co-expressed in mature odontoblasts. In contrast to continuously growing incisors, Lef1, Dspp and P21 were down-regulated in the resting odontoblasts in molars when primary dentin formation was completed. Remarkably, Lef1 expression also preceded Dspp expression in newly differentiated odontoblast-like cells during the pulpal healing process after tooth injury. Conclusions: Lef1 expression precedes Dspp expression without exception in both primary and reparative dentinogeneses. Our results suggest that Lef1 might play a key role in odontoblast differentiation through regulating Dspp expression. [Copyright &y& Elsevier]

Abstract: Introduction: Dental pulp functions include pulp cell activity involvement in dentin formation. In this study we investigated the age-related changes in dental pulp cells that may influence pulp cell activity for restoring pulp function. Methods: Human dental pulp cells (HDPCs) were serially subcultured until spontaneously arrested. Altered expression of chronic inflammatory molecules and age-related molecules were determined by Western blotting. Odontogenic functions impaired by senescence were assayed by Western blotting, reverse transcriptase polymerase chain reaction, alkaline phosphatase activity, and alizarin red S staining. To understand the mechanism of aging process by stress-induced premature senescence (SIPS), the cells were treated with H2O2. Replicative senescence and SIPS were also compared. Results: Replicative senescence of HDPCs was characterized by senescence-associated β-galactosidase activity and reactive oxygen species formation. These cells exhibited altered expression of chronic inflammatory molecules such as intracellular adhesion molecule-1, vascular cell adhesion molecule-1, peroxisome proliferator activated receptor-gamma, and heme oxygenase-1 and age-related molecules such as p53, p21, phosphorylated-extracellular signal-regulated kinase, and c-myb. SIPS cell results were similar to replicative senescence. Furthermore, HDPCs decreased odontogenic markers such as dentin sialophosphoprotein and dentin matrix-1 and osteogenic markers such as bone morphogenetic protein-2 and -7, runt-related transcription factor-2, osteopontin, alkaline phosphatase activity, and mineralized nodule formation by replicative senescence and SIPS. Conclusions: This study suggests that development of aging-related molecules in pulp cells offers understanding of cellular mechanisms and biological events responsible for tooth preservation and maintenance strategies for healthy teeth across the life span. [Copyright &y& Elsevier]

Abstract: Introduction: The aim of this study was to analyze the contribution of nonresident progenitor/stem cells and hematopoietic cells to reparative dentinogenesis. Methods: Parabiosis was established between C57BL/6-TgN(ACTbEGFP)10sb/J transgenic mice (GFP+) and C57BL/6 wild-type mice (GFP−) to ensure blood cross-circulation between animals. Reparative dentinogenesis was stimulated by pulp exposures and capping on the first maxillary molar in the GFP− mice. Histologic sections of injured molars from GFP− mice were analyzed by epifluorescence microscopy to examine the contributions of GFP+ cells (nonresident progenitor cells and hematopoietic cells originating from GFP+ mice) to reparative dentinogenesis. Results: GFP+ cells were detected in close association with reparative dentin formed at the site of pulp exposure in the maxillary first molars of the GFP− mice. Conclusions: The present study suggests the participation of the nonresident progenitor cells and hematopoietic cells in reparative dentinogenesis. [Copyright &y& Elsevier]

Abstract: Introduction: Human dental apical papilla cells (APCs) have mineralization potential, which plays a key role in the root development of young permanent teeth. Limited literature is available about APC mineralization in the presence of inflammatory cytokines. The purpose of this study was to investigate the effects of tumor necrosis factor-α (TNF-α) on APC mineralization. Methods: APC cultures were established with the enzymatic dissociation method in vitro. The viability of APCs treated with TNF-α was investigated using methyl-thiazol-tetrazolium assays. Cells were then cultured in osteo-/dentinogenic medium with TNF-α, and mineralization was assessed by alizarin red S staining. Bone sialoprotein (BSP) and dentin sialoprotein (DSP) were analyzed using immunocytochemistry. Mineralization genes such as BSP, dentin sialophosphoprotein (DSPP), osteocalcin (OCN), and dentin matrix acidicphosphoprotein-1 (DMP1) were determined with real-time polymerase chain reaction analyses. Results: The viability of cultured cells was higher with TNF-α concentrations of 10 ng/mL and 50 ng/mL than with 5 ng/mL or in the control group. Alizarin red S staining showed that APCs had a higher mineralization activity when the osteo-/dentinogenic culture medium contained 10 ng/mL TNF-α. Immunocytochemical detection showed that the expression of BSP and DSP was positive in APCs after they were induced in osteo-/dentinogenic medium. The expression of mineralization genes differed when treated with 10 ng/mL TNF-α (ie, the expression of DSPP mRNA increased on days 7 and 14, whereas the expression of DSPP mRNA decreased on day 21). Conclusions: TNF-α may promote APC mineralization in short-term cultures and inhibit the mineralization in long-term cultures. [Copyright &y& Elsevier]

Abstract: Introduction: The equilibrium of inhibitors and inducers plays an important role in the mineralization of dentin during dentinogenesis. High-temperature requirement protein A1 (HtrA1) is a novel bone mineralization inhibitor involved in physiological and pathological bone formation. However, the expression of HtrA1 in tooth mineralization is unknown. The purpose of the present study was to investigate the localization and quantity of HtrA1 and its possible related molecules during induced reparative dentin formation. Methods: Rats were randomly sacrificed after direct pulp capping on days 0, 7, 14, and 21. Maxillary segments were obtained and routinely prepared for histological analysis, immunohistochemistry, quantum dots–based double immunofluorescence, and CRi’s Nuance imaging system (CRI, Woburn, MA)–based quantitative determination. Results: The square measure values of reparative dentin significantly increased on day 7 and continued to increase until day 21. HtrA1, matrix Gla protein (MGP), nestin, and bone sialoprotein were positively stained and colocalized in the odontoblasts and/or odontoblast-like cells zone and reparative dentin during induced reparative dentin formation. The expressions of HtrA1 and MGP were significantly enhanced after direct pulp capping on day 7 and did not significantly change between days 7, 14, and day 21. Both expressions of HtrA1 and MGP were positively correlated with the square measure values of reparative dentin; however, no correlation was found between the expressions of HtrA1 and MGP. Conclusions: HtrA1 could be observed and might possibly be involved in the process of reparative dentin formation associated with MGP. [Copyright &y& Elsevier]

Abstract: Introduction: Dental pulp can be exposed to hypoxic conditions in case of trauma or inflammation. Dental pulp cells (DPCs) have mineralization potential, which plays a key role in pulp repair and reparative dentinogenesis process. Little information is available about DPC mineralization in hypoxic condition. The purpose of this study was to assess the influence of hypoxia on DPC mineralization to pave the way for a better understanding of dental pulp regeneration and reparative dentin formation. Methods: Human DPCs were obtained by using tissue explant technique in vitro and cultured in normoxia (20% O2) or hypoxia (5% O2). Cell viability was investigated by methyl-thiazol-tetrazolium assay. Cell mineralization was assessed by von Kossa staining and alizarin red S staining. Important mineral genes such as osteocalcin (OCN), dentin matrix acidic phosphoprotein-1 (DMP-1), bone sialoprotein (BSP), and dentin sialophosphoprotein (DSPP) were determined by real-time polymerase chain reaction. Results: Cell viability of DPCs increased more in hypoxia than in normoxia from day 3 to day 5. Von Kossa staining and alizarin red S staining showed DPCs in hypoxia had higher mineralization activity than in normoxia. Expression of mRNAs for OCN, DMP-1, BSP, and DSPP was greater in hypoxia than in normoxia. Conclusions: These results imply that hypoxia promotes DPC mineralization. [Copyright &y& Elsevier]

Abstract: Introduction: Although the presence of the stromal cell–derived factor (SDF)-1α–CXCR4 axis has been reported in dental pulp tissue, little has been known about the underlying regulation of this axis in dental pulp stem cells (DPSCs). The purpose of this study was to investigate whether inflammation or hypoxia can regulate this axis in cultured human dental pulp cells (DPCs). Methods: Primary cultures of DPCs were stimulated by various concentrations of lipopolysaccharide (LPS) for 48 hours, and the production of SDF-1α or CXCR4 was assessed through the enzyme-linked immunosorbent assay and Western blotting, respectively. Additionally, DPCs were incubated in a hypoxic condition (1% O2) for 24 hours, and the cell proliferation ability was detected by methylthiazol tetrazolum assay. Quantitative reverse-transcription polymerase chain reaction (RT-PCR) was used to observe messenger RNA level changes of hypoxia inducible factor-1α(HIF-α), SDF-1α, and CXCR4. The effects of hypoxia on cell migration ability were further confirmed by transmigration assay. Results: All concentrations of LPS inhibited SDF-1α production except that 1 μg/mL LPS increased the expression of CXCR4. Hypoxia promoted the proliferation of DPCs in a 24-hour culture period. Quantitative RT-PCR showed that messenger RNA levels of HIF-α and CXCR4 increased, whereas SDF-1α decreased in hypoxic DPCs. Transmigration assay indicated that hypoxia increased the migration ability of DPCs. Conclusions: These results suggested that inflammation and hypoxia might play an important role in regulating the SDF-1α-CXCR4 axis, which further recruits DPSCs to participate in reparative dentinogenesis. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: Regenerative endodontic procedures use the differentiation potential of embryonic and adult pulp progenitor cell populations to reconstitute dental structures. Methods: An in-depth search of the literature was accomplished to review biologic knowledge from basic research on tooth morphogenesis and differentiation, root development, dentin-pulp regeneration, pulp revascularization and apexification, experimental and clinical studies on the dentinogenic differentiation potential of progenitor cells in the embryonic dental papilla, dental pulp, and associated mesenchymal tissues of the developing root. Results: Odontogenic potential is determined during early tooth morphogenesis in the odontogenic mesenchyme. Progenitor cells from the odontogenic mesenchyme give rise to primary dentin-forming cells (odontoblasts) in the presence of stage-specific enamel epithelium and/or basement membrane and tertiary dentin-forming cells (odontoblast-like cells) in experimental conditions. The specificity of odontogenic mesenchymal cells to form tertiary dentin might be related to the repertoire of signaling pathways operated by the temporospatial pattern of epithelial-mesenchymal interactions during tooth formation. Dental papilla cells isolated from tooth germs before the onset of odontoblast differentiation have not shown any competence to become odontoblasts in the absence of enamel epithelium. On the other hand, the specificity of progenitor cells in the mesenchymal cell populations of the developing root apex remains to be determined. Conclusions: It seems evident that the dental pulp might be only used as a source of progenitor cells with dentinogenic competence for the regeneration of the dentin-pulp complex. The nature of dental or apical papilla progenitor cells in terms of their specificity for dentin regeneration has to be first characterized. [Copyright &y& Elsevier]

Abstract: Introduction: We have previously shown that the p38 gene is highly expressed in odontoblasts during active primary dentinogenesis, but is drastically down-regulated as cells become quiescent in secondary dentinogenesis. Based on these observations, we hypothesized that p38 expression might be upregulated, and the protein activated by phosphorylation, when odontoblasts are stimulated such as during tertiary reactionary dentinogenesis. Methods: We stimulated immortalized, odontoblast-like MDPC-23 cells, alone or in combination, with heat-inactivated Streptococcus mutans, EDTA-extracted dentine matrix proteins (DMPs), or growth factors, including transforming growth factor (TGF)-β1, tumor necrosis factor-α (TNF-α), and adrenomedullin (ADM). We used ELISA to measure the resulting phosphorylation of the p38 protein, as well as its degree of nuclear translocation. Results: Our results suggest that the p38-MAPKinase pathway is activated during odontoblast stimulation in tertiary dentinogenesis by both p38 phosphorylation and enhanced nuclear translocation. Conclusions: Data indicate that odontoblast behaviour therefore potentially recapitulates that during active primary dentinogenesis. [Copyright &y& Elsevier]

Abstract: Introduction: In recent years, the use of high-throughput transcriptomics and proteomics has expanded rapidly in molecular biology and biomedical science. These methods, including DNA microarray and suppression subtractive hybridization at the mRNA level and 2-dimensional electrophoresis and antibody arrays at the protein level, enable studying the expression levels of thousands of genes and proteins simultaneously and thus allow forming genome-wide expression profiles and evaluation of biologic signaling networks. Methods: This review discusses the most used high-throughput expression analysis methods and their use in pulp biologic research. Results: The use of these methods in pulp biology has been limited but is expanding. The methods have been used to compare pulp and bone marrow stem cells and to study the function of pulp tissue in vivo and in vitro. Conclusions: Even though the adoption of the high-throughput transcriptomic and proteomic techniques in pulp biology has been fairly slow, their use is increasing and will significantly increase the understanding of pulp tissue physiology and pathology. The comprehensive data of the transcriptome and proteome of the pulp tissue and the odontoblasts will facilitate the understanding of their functions during health and disease and provide novel target molecules for diagnosis and treatment. Identification of the genes controlling odontoblast differentiation might lead to development of methods enabling induction of reparative dentin formation under carious lesions. Identification of the genes active during dentinogenesis might lead to recognition of regulatory factors, which would cause secondary dentinogenesis to proceed at the rate of primary dentinogenesis. [Copyright &y& Elsevier]

Abstract: Introduction: Pulp regeneration therapy is important to overcome the limitations of conventional therapy to induce reparative dentinogenesis. In the present study, we examined the effects of controlled release of different dosages of fibroblast growth factor–2 (FGF-2) from gelatin hydrogels to regenerate the dentin-pulp complex. Methods: After the amputation of dental pulp of rat molars, gelatin hydrogels incorporating various dosages of FGF-2 were individually implanted into dentin defects above the sites of the amputated pulps. Histologic changes as well as the expression of dentin matrix protein–1 (DMP-1) and nestin in the dentin defect area above the amputated pulp were analyzed. Results: We found that controlled release of high doses of FGF-2 from gelatin hydrogels induced DMP-1–positive calcified particles in the proliferating pulp, whereas a moderate dose of FGF-2 induced DMP-1–positive dentinal bridge on the surface of the proliferating pulp. These findings indicate that the dosage of released FGF-2 has an influence on the structure of calcified tissue regenerated in dentin defects. In addition, pulp cells near calcified tissues regenerated in dentin defects were nestin-negative, suggesting that the calcified tissues might be osteodentin. Conclusions: Our results showed that the dentin regeneration on amputated pulp, not reparative dentin formation toward amputated pulp, can be regulated by adjusting the dosage of FGF-2 incorporated in biodegradable gelatin hydrogels. [Copyright &y& Elsevier]

Introduction: Oxytocin (OT) is a neurohypophysial hormone that plays a role in lactation and parturition and exerts diverse biological actions via the OT receptor. Recently, several studies have reported that OT stimulates bone formation by osteoblasts in osteoporosis. We focused on OT and hypothesized that OT can stimulate the differentiation of odontoblasts as well as osteoblasts. The aim of this study was to verify whether OT is an essential factor in dentinogenesis; we examined the effects of OT on dentinogenesis using a long-term culture system of rat dental pulp cells., Methods: Using a culture system of rat dental pulp cells with Otr knocked out by CRISPR-Cas9 genome editing, we examined the effects of OT on odontoblastlike cell differentiation as reflected by dentin formation., Results: We confirmed that OT stimulated mineralized nodule formation and the expression of both dentin sialoprotein and bone Gla protein messenger RNAs (mRNAs) in the culture system. Interestingly, the cultured cells treated with OT also exhibited an increase of both Wnt10a and Lef-1 mRNA. The Otr knockout cells showed inhibition of nodule formation and mRNA expression, and these phenomena remained despite OT treatment. These results indicate the following: OT regulates odontoblastlike cell differentiation via the OT receptor, it stimulates dentin formation, and the Wnt canonical pathway is closely related to these effects., Conclusions: The present results suggest that OT can promote odontoblastlike cell differentiation, resulting in increased dentin formation, and that OT could be an important factor for dentinogenesis., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Abstract: Introduction: After a traumatic injury to the upper central incisors of a 7-year-old patient, avulsion of tooth #9 and pulp exposure after crown fracture of tooth #8 were managed. Methods: After immediate replacement of tooth #9 in its socket, teeth were splinted for 3 weeks. No endodontic treatment was performed on tooth #9, but pulp capping was performed with mineral trioxide aggregate (Pro Root MTA; Dentsply Maillefer, Ballaigues, Switzerland) on #8, and both teeth were restored with composite resin. The teeth were monitored every 3 months for 2 years. Results: At 24 months, both teeth responded positively to electrometric tests and roots showed normal development, but an abnormal reduction in the size of the root canal space of tooth#8 was observed. Conclusion: Based on these observations, we discuss odontoblast behavior in the context of the stage of dentinogenesis and the probable disturbance of regulation of the physiologic dentinogenic secretory processes in the pathologic situation. [Copyright &y& Elsevier]