Differentiation Of Pulp Cells Research Articles

Single-cell RNA-seq analysis of rat molars reveals cell identity and driver genes associated with dental mesenchymal cell differentiation

BackgroundThe molecular mechanisms and signaling pathways involved in tooth morphogenesis have been the research focus in the fields of tooth and bone development. However, the cell population in molars at the late bell stage and the mechanisms of hard tissue formation and mineralization remain limited knowledge.ResultsHere, we used the rat mandibular first and second molars as models to perform single-cell RNA sequencing (scRNA-seq) analysis to investigate cell identity and driver genes related to dental mesenchymal cell differentiation during the late bell hard tissue formation stage. We identified seven main cell types and investigated the heterogeneity of mesenchymal cells. Subsequently, we identified novel cell marker genes, including Pclo in dental follicle cells, Wnt10a in pre-odontoblasts, Fst and Igfbp2 in periodontal ligament cells, and validated the expression of Igfbp3 in the apical pulp. The dynamic model revealed three differentiation trajectories within mesenchymal cells, originating from two types of dental follicle cells and apical pulp cells. Apical pulp cell differentiation is associated with the genes Ptn and Satb2, while dental follicle cell differentiation is associated with the genes Tnc, Vim, Slc26a7, and Fgfr1. Cluster-specific regulons were analyzed by pySCENIC. In addition, the odontogenic function of driver gene TNC was verified in the odontoblastic differentiation of human dental pulp stem cells. The expression of osteoclast differentiation factors was found to be increased in macrophages of the mandibular first molar.ConclusionsOur results revealed the cell heterogeneity of molars in the late bell stage and identified driver genes associated with dental mesenchymal cell differentiation. These findings provide potential targets for diagnosing dental hard tissue diseases and tooth regeneration.

Weighted Gene Co-expression Network Analysis (WGCNA) of Wnt Signaling Related to Periodontal Ligament Formation: A Bioinformatics-Based Analysis.

Introduction The Wnt signaling pathway is crucial for tooth development, odontoblast differentiation, and dentin formation. It interacts with epithelial cadherin (E-cadherin) and beta-catenin in tooth development and periodontal ligament (PDL) formation. Dysregulation of Wnt signaling is linked to periodontal diseases, requiring an understanding of therapeutic interventions. Weighted gene co-expression network analysis (WGCNA) can identify co-expressed gene modules. Our study aims to identify hub genes in WGCNA analysis of Wnt signaling-based PDL formation. Methods The study used a microarray datasetGSE201313 from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus to analyze the impact of DMP1 expression on XLH dental pulp cell differentiation and PDL formation. The standardized dataset was used for WGCNA analysis, which generated a co-expression network by calculating pairwise correlations between genes and constructing an adjacency matrix. The topological overlap matrix (TOM) was transformed into a hierarchical clustering tree and then cut into modules or clusters of highly interconnected genes. The module eigengene (ME) was calculated for each module, and the genes within this module were identified as hub genes. Gene ontology (GO) and KEGG pathway enrichment analysis were performed to gain insights into the biological functions of the hub genes. The integrated Differential Expression and Pathway analysis (iDEP) tool (http://bioinformatics.sdstate.edu/idep/; South Dakota State University, Brookings, USA) was used for WGCNA analysis. Results The study used the WGCNA package to analyze 1,000 differentially expressed genes, constructing a gene co-expression network and generating a hierarchical clustering tree and TOM. The analysis reveals a scale-free topology fitting index R2 and mean connectivity for various soft threshold powers, with an R2 value of 5. COL6A1, MMP3, BGN, COL1A2, and FBN2 are hub genes implicated in PDL development. Conclusion The study identified key hub genes, including COL6A1, MMP3, BGN, and FBN2, crucial for PDL formation, tissue remodeling, and cell-matrix interactions, guiding future therapeutic strategies.

Human Dental Pulp Stem Cells Are Subjected to Metabolic Reprogramming and Repressed Proliferation and Migration by the Sympathetic Nervous System via α1B-Adrenergic Receptor

IntroductionHuman dental pulp stem cells (hDPSCs) reside in specialized microenvironments in the dental pulp, termed “niches,” which are composed of diverse cellular components including nerves. Sensory nerves can positively regulate the expansion and differentiation of pulp cells, while the biological effects of the sympathetic nervous system (SNS) on hDPSCs remain elusive. This study is devoted to investigating the effects and underlying mechanisms of the SNS on the proliferation and migration of hDPSCs. MethodsThe distribution of sympathetic nerve fibers in human dental pulp was examined by immunofluorescence staining of tyrosine hydroxylase. The concentration of norepinephrine in healthy and carious human dental pulp tissues was detected using enzyme-linked immunosorbent assay. RNA-sequencing was applied to identify the dominant sympathetic neurotransmitter receptor in hDPSCs. Seahorse metabolic assay, adenosine triphosphate assay, lactate assay, and mitochondrial DNA copy number were performed to determine the level of glycometabolism. Transwell assay, wound healing assay, 5-ethynyl-2'-deoxyuridine staining assay, cell cycle assay, and Cell Counting Kit-8 assay were conducted to analyze the migratory and proliferative capacities of hDPSCs. ResultsSprouting of sympathetic nerve fibers and an increased concentration of norepinephrine were observed in inflammatory pulp tissues. Sympathetic nerve fibers were mainly distributed along blood vessels, and aldehyde dehydrogenase 1–positive hDPSCs resided in close proximity to neurovascular bundles. ADRA1B was identified as the major sympathetic neurotransmitter receptor expressed in hDPSCs, and its expression was enhanced in inflammatory pulp tissues. In addition, the SNS inhibited the proliferation and migration of hDPSCs through metabolic reprogramming via ADRA1B and its crosstalk with serine-threonine kinase and p38 mitogen-activated protein kinase signaling pathways. ConclusionsThis study demonstrates that the SNS can shift the metabolism of hDPSCs from oxidative phosphorylation to anaerobic glycolysis via ADRA1B and its crosstalk with serine-threonine kinase and p38 mitogen-activated protein kinase signaling pathways, thereby inhibiting the proliferative and migratory abilities of hDPSCs. This metabolic shift may facilitate the maintenance of the quiescent state of hDPSCs.

Effects of inflammation in dental pulp cell differentiation and reparative response

The responsiveness of the dentin–pulp complex is possible due to the stimulation of dental pulp cells, which begin to synthesize and secrete dentin matrix. The inflammatory process generated by harmful stimuli should be understood as a natural event of the immune response, resulting in the recruitment of hematopoietic cells, which cross the endothelial barrier and reach the site affected by the injury in order to eliminate the damage and provide an appropriate environment for the restoration of homeostasis. The repair process occurs in the presence of adequate blood supply, absence of infection, and with the participation of pro-inflammatory cytokines, growth factors, extracellular matrix components, and other biologically active molecules. Prostaglandins and leukotrienes are bioactive molecules derived from the metabolism of arachidonic acid, as a result of a variable range of cellular stimuli. The aim of this review is to describe the process of formation and biomineralization of the dentin–pulp complex and how pro-inflammatory events can modify this response, with emphasis on the lipid mediators prostaglandins and leukotrienes derived from arachidonic acid metabolism.

CGRP and Shh Mediate the Dental Pulp Cell Response to Neuron Stimulation

Dental pain is a persistent, detrimental public health issue that requires a better understanding of the mechanisms of tooth pain and inflammation in order to develop more effective treatments. Calcitonin gene-related peptide (CGRP) and dental pulp cells are promising candidates for mediating tooth pain and generating reparative dental tissues, respectively, but their behavior in the context of pulpitis remains elusive. The mouse incisor requires Sonic hedgehog (Shh) secreted from sensory nerves to continuously regenerate. However, it is unknown whether sensory nerves also regulate the comparatively nonregenerative mouse molar through CGRP and Shh. This is an important knowledge gap to fill since mouse incisors differ biologically from human teeth, while mouse and human molars are similar. In this work, we identified that molar pulp cells express CGRP receptor and Gli1, a Hedgehog (Hh) signaling protein found to label a dental stem cell population in the mouse incisor. We also observed in a mouse molar injury model that Hh signaling was activated and Shh expression was upregulated in vivo. We then determined in vitro that Shh and CGRP regulate differentiation of primary mouse molar and incisor pulp cells and a human dental pulp stem cell line. Furthermore, conditioned media from stimulated sensory neurons induced Hh signaling activation and inflammatory gene expression in primary molar pulp cells, which was abolished by inhibition of either Shh or CGRP. Our results suggest that CGRP and Shh signaling may promote an inflammatory response after injury in the molar and that activated sensory nerves secrete CGRP and Shh to regulate molar pulp cell expansion and differentiation into odontoblast-like cells for dentin repair. Thus, CGRP/Shh signaling should be considered for new strategies that seek to manage pain or dentin regeneration in the molar.

Hypoxia‑induced mitophagy regulates proliferation, migration and odontoblastic differentiation of humandental pulp cells through FUN14 domain‑containing 1.

FUN14 domain-containing 1 (FUNDC1) is a receptor that has been previously reported to activate hypoxia-induced mitophagy. However, the potential role of FUNDC1 in the pathophysiology of dental pulp diseases remains unknown. Therefore, present study first collected tissue specimens from patients with pulpitis and from healthy individuals. The results of reverse transcription-quantitative PCR and immunohistochemical staining revealed markedly increased FUNDC1 and hypoxia-inducible factor-1α expression in pulpitis tissue specimens compared with those from healthy individuals. To provide a theoretical basis for the study of the occurrence, development and reparative mechanisms in the dental pulp after tissue injury, the present study then investigated the role of hypoxia-induced mitophagy in the regulation of proliferation, migration and odontoblastic differentiation in human dental pulp cells (HDPCs), in addition, to the possible involvement of FUNDC1. The surface markers and multipotent differentiation capabilities of HDPCs were performed by flow cytometry (surface markers), alizarin red (osteogenic capabilities), alcian blue (chondrogenic capabilities) and oil red O (adipogenic capabilities). Following culture under hypoxia conditions (1% O2) for varying time periods, the proliferation, migration and odontoblastic differentiation of HDPCs were measured using Cell Counting Kit-8, wound healing and Transwell migration assays, alkaline phosphatase staining and activity tests and western blotting (runt-related transcription factor 2, collagen I, osterix and osteopontin), respectively. Immunofluorescence and western blotting were performed to measure the expression levels of hypoxia-inducible factor-1α, pro-fission dynamin-related protein 1, mitochondria-related proteins translocase of inner mitochondrial membrane 23 and translocase of outer mitochondrial membrane 20, in addition to those of autophagy markers (p62, LC3II, Beclin-1 and autophagy-related 5). Transmission electron microscopy was also used to image the autophagosomes and mitochondrial morphology. In addition, to study the functional role of FUNDC1, its expression was silenced by liposome-mediated transfection with small interfering RNA into HDPCs. Compared with those in HDPCs cultured under normoxic conditions (21% O2), the ability of autophagy in HDPCs cultured under hypoxic conditions for 18 h was markedly increased, whilst the proliferation, migration and odontoblastic differentiation were also enhanced. Increased numbers of autophagosomes could also be observed in the hypoxic group. However, FUNDC1 knockdown in HDPCs reversed the aforementioned effects. Overall, data from the present study suggest that hypoxia can promote the proliferation, migration and odontoblastic differentiation of HDPCs, where the underlying mechanism may be associated with the activation of mitophagy downstream of FUNDC1.

Leukotriene B4 loaded in microspheres regulate the expression of genes related to odontoblastic differentiation and biomineralization by dental pulp stem cells

BackgroundLeukotriene B4 (LTB4) is a potent lipid mediator that stimulate the immune response. Because dental pulp inflammation and dentin repair are intrinsically related responses, the aim of this research was to investigate the potential of LTB4 in inducing differentiation of dental pulp stem cells.MethodsMicrospheres (MS) loaded with LTB4 were prepared using an oil emulsion solvent extraction evaporation process and sterility, characterization, efficiency of LTB4 encapsulation and in vitro LTB4 release assay were investigated. Mouse dental pulp stem cells (OD-21) were stimulated with soluble LTB4 or MS loaded with LTB4 (0.01 and 0.1 μM). Cytotoxicity and cell viability was determined by lactate dehydrogenase and methylthiazol tetrazolium assays. Gene expression were measured by quantitative reverse transcription polymerase chain reaction after 3, 6, 24, 48 and 72 h. Mineralized nodule formation was assessed after 28 days of OD-21 cell stimulation with LTB4 in mineralized media or not. Groups were compared using one-way ANOVA test followed by Dunnett’s post-test (α = 0.05).ResultsTreatment with LTB4 or MS loaded with LTB4 (0.01 and 0.1 µm-μM) were not cytotoxic to OD-21 cells. Treatment with LTB4 modulated the expression of the Ibsp (integrin binding sialoprotein) and Runx2 (runt-related transcription factor 2) genes differently depending on the experimental period analyzed. Interestingly LTB4 loaded in microspheres (0.1 μM) allowed long term dental pulp cell differentiation and biomineralization.ConclusionLTB4, soluble or loaded in MS, were not cytotoxic and modulated the expression of the Ibsp and Runx2 genes in cultured OD-21 cells. When LTB4 was incorporated into MS, odontoblast differentiation and mineralization was induced in long term culture.

Histologic evaluation of dentin bridge formation by pachymic acid and biodentine in human tooth culture model

Histologic evaluation of dentin bridge formation by pachymic acid and biodentine in human tooth culture model

Dentin-Pulp Complex Tissue Regeneration via Three-Dimensional Cell Sheet Layering.

The dentin-pulp complex is a unique structure in teeth that contains both hard and soft tissues. Generally, deep caries and trauma cause damage to the dentin-pulp complex, and if left untreated, this damage will progress to irreversible pulpitis. The aim of this study was to fabricate a layered cell sheet composed of rat dental pulp (DP) cells and odontogenic differentiation of pulp (OD) cells and to investigate the ability to regenerate the dentin-pulp complex in a scaffold tooth. We fabricated two single cell sheets composed of DP cells (DP cell sheet) or OD cells (OD cell sheet) and a layered cell sheet made by layering both cells. The characteristics of the fabricated cell sheets were analyzed using light microscopy, scanning electron microscope (SEM), hematoxylin-eosin (HE) staining, and immunohistochemistry (IHC). Furthermore, the cell sheets were transplanted into the subrenal capsule of immunocompromised mice for 8 weeks. After this, the regenerative capacity to form dentin-like tissue was evaluated using micro-computed tomography (micro-CT), HE staining, and IHC. The findings of SEM and IHC confirmed that layered cell sheets fabricated by stacking OD cells and DP cells maintained their cytological characteristics. Micro-CT of layered cell sheet transplants revealed a mineralized capping of the access cavity in the crown area, similar to that of natural dentin. In contrast, the OD cell sheet group demonstrated the formation of irregular fragments of mineralized tissue in the pulp cavity, and the DP cell sheet did not develop any hard tissue. Moreover, bone volume/tissue volume (BV/TV) showed a significant increase in hard tissue formation in the layered cell sheet group compared with that in the single cell sheet group (p < 0.05). HE staining also showed a combination of soft and hard tissue formation in the layered cell sheet group. Furthermore, IHC confirmed that the dentin-like tissue generated from the layered cell sheet expressed characteristic markers of dentin but not bone equivalent to that of a natural tooth. In conclusion, this study demonstrates the feasibility of regenerating dentin-pulp complex using a bioengineered tissue designed to simulate the anatomical structure. Impact statement The dentin-pulp complex can be destroyed by deep caries and trauma, which may cause pulpitis and progress to irreversible pulpitis, apical periodontitis, and even tooth loss. Current treatments cannot maintain pulp health, and teeth can become brittle. We developed a three-dimensional (3D) layered cell sheet using dental pulp cells and odontogenic differentiation of pulp cells for dentin-pulp complex regeneration. Our layered cell sheet enables the regeneration of an organized 3D dentin-pulp-like structure comparable with that of natural teeth. This layered cell sheet technology may contribute to dentin-pulp complex regeneration and provide a novel method for complex tissue engineering.

Biodentine: Characteristics and applications today

Introduction: During the last decades, there was a great interest in the development of bioactive dental material with the ability to interact and induce dental tissues. Biodentine is a calcium silicate-based cement that has beneficial effects on pulp cells and promotes the formation of tertiary dentin. Aim: To analyze the literature on biodentine, as well as its antimicrobial activity, its setting capacity, its clinical applications and its biological properties. Methodology: Using the keywords biodentine, root canal sealing materials, epidemiology, diagnosis, clinical manifestations, antimicrobial resistance, and treatment, the MEDLINE/PubMed and Science Direct databases were searched, with emphasis on the last 5 years. It was evaluated with the PRISMA and AMSTAR-2 guidelines. Results: The antimicrobial activity of Biodentine has been greater against strains such as Streptococcus sanguis, Enterococcus faecalis, Escherichia coli and Candida albicans. Biodentine has benefits such as excellent sealability, biocompatibility, good dimensional stability with the advantage of a setting time of 12 to 13 minutes, less than MTA. Materials used in the vital pulp method must have biocompatibility and bioactivity to promote pulp cell activity. Biodentine induces the differentiation of pulp cells into odontoblast-like cells, formation of mineralized tissue and restorative dentin. Conclusion: The composition and its applications have shown sufficient basis to perform an adequate antimicrobial activity, as well as its setting time improved to 12 minutes. Vital pulp therapy is one of the main uses of this material as it is a widely accepted material for dentin tissues due to its high biological compatibility.

LncRNA MALAT1 Functions as a Competing Endogenous RNA to Regulate BMI1 Expression by Sponging miR-200c/miR-203 in the Control of the Differentiation of Pulp Cells.

Long non-coding RNAs (lncRNAs) and miRNAs (microRNAs) are considered as key regulators of several biological processes, including dental development. In this study, we explored the lncRNAs and miRNAs which are involved in dental development. Real-time PCR was performed to identify the candidate lncRNAs and miRNAs involved in dental development. Bioinformatics analysis and luciferase assay were carried out to establish the regulatory relationships between MALAT1, miR-203 and miR-200c in dental development. Among all candidate lncRNAs, only MALAT1 was highly expressed in differentiated human dental pulp cells (hDPCs), and among all candidate miRNAs which are down-regulated in differentiated hDPCs, miR-203, and miR-200c are most decreased. Furthermore, MALAT1 was up-regulated while miR-203 and miR-200c were down-regulated in differentiated hDPCs in a time-dependent manner. MiR-203 and miR-200c were proved to bind to MALAT1. Moreover, BMI1 was identified as a target gene of miR-203 or miR-200c, and BMI1 was time-dependently decreased in hDPCs cultured with odontogenic medium. On the contrary, dentin sialophosphoprotein (DSPP), dentin matrix protein-1 (DMP-1), osteocalcin (OCN), and alkaline phosphatase (ALP), were time-dependently increased in hDPCs cultured with odontogenic medium. Finally, the overexpression of MALAT1 and the knockdown of miR-203/miR-200c both significantly increased the levels of BMI1, DSPP, DMP-1, OCN, and ALP, while the effect of knockdown of miR-203/miR-200c was much stronger than that of the overexpression of MALAT1. Our results demonstrated that MALAT1 functions as a competing endogenous RNA of miR-203 and miR-200c and accordingly promotes BMI1 expression. Therefore, MALAT1 may serve as a biomarker for dental development.

Odontoblast cell death induces NLRP3 inflammasome-dependent sterile inflammation and regulates dental pulp cell migration, proliferation and differentiation.

To investigate the ability of dead odontoblasts to initiate NLRP3 inflammasome-dependent sterile inflammation and to explore the effect on dental pulp cell (DPCs) migration, proliferation and odontogenic differentiation. Odontoblast-like cells were subjected to freezing-thawing cycles to produce odontoblast necrotic cell lysate (ONCL). DPCs were treated with ONCL to assess proliferation and migration. THP-1 differentiated macrophages stimulated with ONCL and live cell imaging and western blotting were used to assess NLRP3 inflammasome activation. Cytokines were measured with multiplex arrays and ELISA. qPCR, alkaline phosphatase and Alizarin red assays were used to assess odontogenic differentiation of DPCs. Data were analysed using the t-test or anova followed by a Bonferroni post hoc test with the level of significance set at P≤0.05. ONCL induced migration and proliferation of DPCs. Treatment of THP-1 macrophages with ONCL resulted in the release of the inflammatory cytokines IL-1β, IL-6, IL-8, TNFα, IFN-γ, CCL2 and angiogenic growth factors, angiogenin and angiopoietin. This inflammatory response was associated with activation of NFκB, p38MAPK and NLRP3 inflammasome. To confirm that ONCL induced inflammatory response is NLRP3 inflammasome-dependent, treatment with a caspase-1 inhibitor and a specific NLRP3 inhibitor significantly reduced IL-1β release in THP-1 macrophages (P=0.01 and 0.001). Inflammasome activation product, IL-1β, induced odontogenic differentiation of DPCS as evident by the increase in odontogenic genes expression DMP-1, RUNX-2, DSPP and SPP, alkaline phosphatase activity and mineralization. Dead odontoblasts induced NLRP3 inflammasome-dependent sterile inflammation and activated the migration, proliferation and differentiation of DPCs.

Performance of a Biodegradable Composite with Hydroxyapatite as a Scaffold in Pulp Tissue Repair.

Vital pulp therapy is an important endodontic treatment. Strategies using growth factors and biological molecules are effective in developing pulp capping materials based on wound healing by the dentin-pulp complex. Our group developed biodegradable viscoelastic polymer materials for tissue-engineered medical devices. The polymer contents help overcome the poor fracture toughness of hydroxyapatite (HAp)-facilitated osteogenic differentiation of pulp cells. However, the composition of this novel polymer remained unclear. This study evaluated a novel polymer composite, P(CL-co-DLLA) and HAp, as a direct pulp capping carrier for biological molecules. The biocompatibility of the novel polymer composite was evaluated by determining the cytotoxicity and proliferation of human dental stem cells in vitro. The novel polymer composite with BMP-2, which reportedly induced tertiary dentin, was tested as a direct pulp capping material in a rat model. Cytotoxicity and proliferation assays revealed that the biocompatibility of the novel polymer composite was similar to that of the control. The novel polymer composite with BMP-2-induced tertiary dentin, similar to hydraulic calcium-silicate cement, in the direct pulp capping model. The BMP-2 composite upregulated wound healing-related gene expression compared to the novel polymer composite alone. Therefore, we suggest that novel polymer composites could be effective carriers for pulp capping.

Highly Proliferative Immortalized Human Dental Pulp Cells Retain the Odontogenic Phenotype when Combined with a Beta-Tricalcium Phosphate Scaffold and BMP2.

Human dental pulp cells (HDPCs) play a vital role in dentin formation and reparative dentinogenesis, which indicated their potential application in regenerative medicine. However, HDPCs, which can only be obtained from scarce human pulp tissues, also have a limited lifespan in vitro, and stem cells usually lose their original characteristics over a large number of passages. To overcome these challenges, we successfully immortalized human dental pulp cells using the piggyBac system which was employed to efficiently overexpress the SV40 T-Ag, and we then comprehensively described the cell biological behavior. The immortalized human dental pulp cells (iHDPCs) acquired long-term proliferative activity and expressed most HDPC markers. The iHDPCs maintained multiple differentiation potential and could be induced to differentiate into chondrogenic, osteogenic, and adipogenic cells in vitro. We also proved that the iHDPCs gained a stronger ability to migrate than the primary cells, while apoptosis was inhibited. Furthermore, highly proliferative iHDPCs displayed no oncogenicity when subcutaneously implanted into athymic nude mice. Finally, iHDPCs exhibited odontogenic differentiation ability and secreted dentin sialophosphoprotein (DSPP) when combined with a beta-tricalcium phosphate scaffold and bone morphogenetic protein-2 (BMP2) in vivo. Conclusively, the established iHDPCs are a valuable resource for mechanistic study of dental pulp cell differentiation and dental pulp injury repair, as well as for applications in tooth regeneration.

Influence of Adiponectin and Leptin on the Kinetics of Human Pulp Cells

Adipocytokines, such as adiponectin and leptin, are expressed by adipocytes and are known as anti-metabolic syndrome factors. They are also thought to mediate calcification in mineralized tissue. We investigated the effects of adiponectin and leptin on the kinetics of human pulp cells using ELISA and western blot. After gaining informed consent, we obtained human pulp cells from three patients. After cultivation in Dulbecco’s Modified Eagle Medium (DMEM) without serum, cells from the 4th to 6th passages were incubated with various concentrations of adiponectin (1, 10, or 100 ng/mL) in DMEM or leptin (0.1, 1, or 10 ng/mL) in DMEM for 24 h. We confirmed that human pulp cells expressed adiponectin receptor 1 and leptin receptor. Although the proliferation index of these cells as measured by 5-bromo-2'-deoxyuridine (BrdU) incorporation increased in the presence of adiponectin in a dose-dependent manner, pulp cells stimulated with leptin showed no significant changes in BrdU incorporation. Alkaline phosphatase activity showed no significant changes after stimulation with either adipocytokine. Adiponectin induced expression of BMP-2 and osteopontin more strongly than did leptin. In particular, expression of BMP-2 increased in the presence of adiponectin in a dose-dependent manner. In contrast, the expression of dentin sialoprotein increased after stimulation with leptin. The expression of Runx2 was not observed from the cultured pulp cells stimulated with both molecules. These results indicate that adiponectin and leptin contribute to growth and differentiation of human pulp cells and may consequently affect the formation of secondary dentin or reparative dentin in the dentin-pulp complex.

Effects of Papain-Based Gel Used For Caries Removal on Macrophages and Dental Pulp Cells.

Papain-based gel is used for chemical-mechanical caries removal and present antimicrobial and anti-inflammatory activities. However, its effects on dental pulp cells and on macrophages remains largely unknown. Therefore, the aim of this study was to investigate whether the papain-based gel Papacárie Duo® acts as an immunomodulator in lipopolysaccharide (LPS)-activated macrophages and its effects on dental pulp cells . J774.1 macrophage and OD-21 dental pulp cells were stimulated with 0.5% and 5% of Papacárie Duo®, following pre-treatment or not with LPS. After 24 h, a lactate dehydrogenase assay was used to measure cytotoxicity, a tetrazolium-based colorimetric assay (MTT) was used to measure cell viability, and qRT-PCR was used to analyze relative gene expression of Ptgs2, Il10, Tnf, Mmp9, Runx2, Ibsp and Spp1. Papacárie Duo® was cytotoxic and reduced cell viability at 5% but not at 0.5% in both cultures. In macrophages, Papacárie Duo® increased the expression Il10 and LPS-induced Ptgs2, but it did not affect Tnf or Mmp9. In OD-21 cells, Papacárie Duo® inhibited Runx2 and Ibsp expression, but stimulated Spp1 expression. Papain-based gel presented a concentration dependent cytotoxicity, without affecting cell viability, for dental pulp cells and macrophages. Interestingly, the gel presented an inhibitory effect on pulp cell differentiation but modulated the activation of macrophages stimulated with LPS. We speculate that in dental pulp tissue, Papacárie Duo® would impair reparative dentinogenesis but could activate macrophages to perform their role in defense and inflammation.

Interaction of Nel-like molecule 1 with apoptosis related protein 3 with its influence on human dental pulp cells proliferation and differentiation into odontoblasts

Nel-like molecule 1 (Nell-1) is an essential positive regulator of tooth development and odontoblast differentiation. However, its precise mechanism remains undetermined. This study aims to explore the possible receptor or binding protein of Nell-1. Results showed that Nell-1 and Apoptosis related protein 3(APR3) expression levels were high in odontoblasts and inversely correlated. Endogenous Nell-1 co-immunoprecipitated with APR3, and this co-IP was reciprocal. Double immunofluorescence staining revealed that Nell-1 and APR3 colocalized on the nuclear envelope of human dental pulp cells. Nell-1 inhibited the proliferation of these cells co-infected with APR3 through Cyclin D1 downregulation. The interaction of Nell-1 with APR3 stimulated alkaline phosphatase (ALP) activity and promoted the expression and mineralization of DSPP, ALP, OPN, and BSP. The shRNA of APR3 decreased cell differentiation and mineralization. Nell-1 could reciprocally interact with APR3 and stimulate the differentiation and mineralization of human dental pulp cells. Future studies should explore the potential functional connection and the molar mechanism of such interaction.

Pulp Response Capped by Brain Derived Neurotrophic Factor (BDNF)

Background: Four neurotrophins (NTFs) are a family of structurally related proteins with specific effects on the developing nervous system and non-neuronal differentiating cells. The brain-derived neurotrophic factor ( BDNF) has role in differentiation of pulp cells into mineralizing cells,and that was identified as survival factor , development and function of dental tissues.Aim of the study: The study was designed to illustrate pulp response capped by brain-derived neurotrophic factor after mechanical trauma.Materials and Method: Brain derived neurotrophic factor was used as pulp capped material for maxillary anterior teeth of (12 )rat following the mechanical traumatized proceduce.Pulp reaction was studied histologically from the first cutting procedure ,followed by interval periods (48,72,168 hours ) ,Six teeth for each period .Results: Illustrated an enhancement of pulp healing represented by newly apposition of reparative dentine with presences of active angiogenesis followed by globular minerlization that coincides with proceeding interval time.Conclusion: The study suggest that BDNF contributes in pulp healing and can be addressed to a more conservative treatment approach.

Phosphophoryn and Dentin Sialoprotein Effects on Dental Pulp Cell Migration, Proliferation, and Differentiation.

Phosphophoryn (PP) and dentin sialoprotein (DSP) are two of the most abundant dentin matrix non-collagenous proteins, and are derived from dentin sialoprotein-phosphophoryn (DSP-PP) mRNA. Mutations in the DSP-PP gene are linked to dentinogenesis imperfecta II and III. Previously, we reported transient DSP-PP expression in preameloblast cells first, followed by co-expression in preameloblasts and young odontoblasts, and finally sustained expression in odontoblasts. This phenomenon raised the possibility that DSP/PP proteins secreted by preameloblasts might promote dental pulp cell migration toward the dental pulp border and promote dental pulp cell differentiation. To examine the effects of DSP/PP proteins on dental pulp cell development, we investigated:(1) native PP effects on dental pulpcell migration and matrix protein expression; and (2) recombinant DSP/PP protein effects on cell proliferation and differentiation. We found that PP promoted cell migration and the expression of high levels of Col type I and PP in dental pulp cells. The addition of recombinant DSP/PP proteins affected cell proliferation and differentiation in a dental pulp cell line. These findings strongly suggest that DSP/PP may modulate cell migration, cell proliferation and differentiation, thus leading to dentin formation. DSP/PP protein may be useful clinically for pulp tissue regeneration.

Senescence and odontoblastic differentiation of dental pulp cells.

Cellular senescence has been suggested to be involved in physiological changes of cytokine production. Previous studies showed that the concentration of tumor necrosis factor-α (TNF-α) is higher in the blood of aged people compared with that of young people. So far, the precise effects of TNF-α on theodontoblastic differentiation of pulp cells have been controversial. Therefore, we aimed to clarify how this cytokine affected pulp cells during aging. Human dental pulp cells (HDPCs) were cultured until reaching the plateau of their growth, and the cells were isolated at actively (young HDPCs; yHDPCs) or inactively (senescent HDPCs; sHDPCs) proliferating stages. sHDPCs expressed senescence-related molecules while yHDPCs did not. When these HDPCs were cultured in anodontoblast-inductive medium, both young and senescent cells showed mineralization, but mineralization in sHDPCs was lower compared with yHDPCs. However, the administration of TNF-α to this culture medium altered these responses: yHDPCs showed downregulated mineralization, while sHDPCs exhibited significantly increased mineralization. Furthermore, the expression of tumor necrosis factor receptor 1 (TNFR1), a receptor of TNF-α, was significantly upregulated in sHDPCs compared with yHDPCs. Downregulation of TNFR1 expression led to decreased mineralization of TNF-α-treated sHDPCs, whereas restored the reduction in TNF-α-treated yHDPCs. These results suggested that sHDPCs preserved the odontoblastic differentiation capacity and TNF-α promoted odontoblastic differentiation of HDPCs with theprogress of their population doublings through increased expression of TNFR1. Thus, TNF-α might exert a different effect on the odontoblastic differentiation of HDPCs depending on their proliferating activity. In addition, the calcification of pulp chamber with age may be related with increased reactivity of pulp cells to TNF-α.