Human Dental Pulp Tissue Research Articles

Establishing Three-Dimensional Explant Culture of Human Dental Pulp Tissue.

Mesenchymal stem cells in the dental tissue indicate a disposition for differentiation into diverse dental lineages and contain enormous potential as the important means for regenerative medicine in dentistry. Among various dental tissues, the dental pulp contains stem cells, progenitor cells and odontoblasts for maintaining dentin homeostasis. The conventional culture of stem cells holds a limit as the living tissue constitutes the three-dimensional (3D) structure. Recent development in the organoid cultures have successfully recapitulated 3D structure and advanced to the assembling of different types. In the current study, the protocol for 3D explant culture of the human dental pulp tissue has been established by adopting the organoid culture. After isolating dental pulp from human tooth, the intact tissue was placed between two layers for Matrigel with addition of the culture medium. The reticular outgrowth of pre-odontoblast layer continued for a month and the random accumulation of dentin was observed near the end. Electron microscopy showed the cellular organization and in situ development of dentin, and immunohistochemistry exhibited the expression of odontoblast and stem cell markers in the outgrowth area. Three-dimensional explant culture of human dental pulp will provide a novel platform for understanding stem cell biology inside the tooth and developing the regenerative medicine.

Effect of four direct pulp capping agents on human dental pulp tissue.

Direct pulp capping (DPC) is a vital pulp therapy, wherein accidental or carious pulp exposures can be capped with various materials to induce reparative dentine formation. One of the major factors that determine the success of direct pulp capping procedures is the material used. Therefore, it is of interest to evaluate and compare the efficacy of a new material, Tristrontium aluminate in comparison with the widely used materials such as ProRoot MTA, Biodentine & TheraCal LC. Hence, 40 premolars scheduled for extraction for orthodontic treatment, were equally divided into 4 groups and iatrogenic DPC was done using one of the study materials. The premolars were then extracted after 90 days & assessed for the formation of Dentine Bridge and pulpal response elicited by the materials using CBCT and histopathological analysis. All the materials tested in the study were successful in inducing dentine bridge formation & maintaining pulp vitality. Thus, the novelty of Tristrontium aluminate for potential applications in future is eminent.

MicroRNA-181b attenuates lipopolysaccharide-induced inflammatory responses in pulpitis via the PLAU/AKT/NF-κB axis

ObjectiveThis study aimed to investigate the role and underlying mechanisms of microRNA (miRNA)-181b in the inflammatory response in pulpitis. MethodsQuantitative reverse-transcription polymerase chain reaction (qRT-PCR), fluorescence in situ hybridization (FISH), and immunofluorescence techniques were used to determine the miRNA-181b and urokinase-type plasminogen activator (PLAU) expression levels in inflamed human dental pulp tissues (HDPTs) and lipopolysaccharide (LPS)-stimulated human dental pulp cells (hDPCs). The targets of miRNA-181b were identified and confirmed using a bioinformatics analysis, RNA sequencing, and dual-luciferase gene reporter assays. The effect of miRNA-181b or PLAU on proinflammatory cytokine expression in hDPCs was examined using qRT-PCR and western blotting. RNA sequencing was conducted to examine the signaling pathways implicated in miRNA-181b-mediated pulpitis. Western blotting and qRT-PCR were used to determine the miRNA-181b /PLAU/AKT/NF-κB signaling axis in pulpitis. A rat pulpitis model was created to observe the histopathological changes in the dental pulp tissue after the topical application of miRNA-181b agomir. ResultsA significant decrease in miRNA-181b and an increase in PLAU were observed in HDPTs compared to the healthy controls, and these two factors showed a negative correlation. MiRNA-181b directly targeted PLAU. The miRNA-181b inhibitor resulted in a significant upregulation of IL-1β, IL-6 and TNF-α, whereas the knockdown of PLAU reversed this proinflammatory effect. Conversely, PLAU overexpression prevented the anti-inflammatory effects of the miRNA-181b mimics. Mechanistically, miRNA-181b inhibited the AKT/NF-κB pathway by targeting PLAU. In vivo application of the miRNA-181b agomir to inflamed pulp tissue alleviated inflammation. ConclusionMiRNA-181b targets PLAU, negatively regulating pro-inflammatory cytokine expression via the AKT/NF-κB signaling pathway.

IWP-2 modulates the immunomodulatory properties of human dental pulp stem cells invitro.

To investigate the effect of IWP-2, Wnt inhibitor, on human dental pulp stem cells (hDPSCs) responses. hDPSCs were isolated from human dental pulp tissues. Cells were treated with 25 μM IWP-2 for 24 h, and subsequently, the gene expression profile was examined using high-throughput RNA sequencing. The mRNA expression was analysed using qPCR. The effect of IWP-2 was investigated in both normal and LPS-induced hDPSCs (inflamed hDPSCs). CD4+ T cells and CD14+ monocyte-derived macrophages were cultured with conditioned media of IWP-2 treated hDPSCs to observe the immunosuppressive property. RNA sequencing indicated that IWP-2 significantly downregulated several KEGG pathways, including cytokine-cytokine receptor interaction, IL-17 signalling pathway, and TNF signalling pathway. In both normal and inflamed conditions, IWP-2 markedly upregulated TGFB1 mRNA expression while the mRNA expression of pro-inflammatory cytokines, TNFA, IL1B, IFNG, and IL6, was inhibited. In the inhibition experiment, the pretreatment with p38, MAPK, or PI3K inhibitors abolished the effects of IWP-2 in LPS-induced inflammation. In terms of immune cells, IWP-2-treated-inflamed hDPSCs conditioned media attenuated T cell proliferation and regulated regulatory T cell differentiation. In addition, the migratory property of macrophage was decreased after being exposed to IWP-2-treated inflamed hDPSCs conditioned media. IWP-2 suppressed inflammatory cytokine expression in both normal and inflamed hDPSCs. Moreover, hDPSCs exerted the immunosuppressive property after IWP-2 treatment. These results suggest the role of Wnt in inflammatory responses and immunomodulation in dental pulp tissues.

Histologic and proteomic profile of two methods to decellularize human dental pulp tissue

ObjectiveThe present study compared the structural and proteomic architecture of extracellular matrices (ECM) of decellularized human dental pulp using two previously described protocols. DesignPulp tissue from 150 molars was extracted and three treatments took place, based on the Matoug-Elwerfelli Group (MG) and the Song Group (SG) protocols and an untreated pulp group (CG), to examine histoarchitecture and the effectiveness of the decellularization process, using histological analysis (n = 12) and scanning electron microscopy (SEM) (n = 3). Protein extraction took place using 100 mg dry weight of pulp, in triplicates for each group, and the shotgun proteome analysis was performed by nanoUPLC-MSE. Proteins were identified using the revised human UNIPROT database attached to the PLGS search engine. ResultsHistological analysis and SEM demonstrated that ECM in MG was more preserved. Proteome analysis showed that the decellularized process in MG maintained approximately 69.56% of proteins identified in untreated pulp tissue while SG maintained 28.26%. ConclusionsECM appears to be suitable as a potential biological scaffold for pulp revascularization and regeneration procedures, especially those processed according to the Matoug-Elwerfelli protocol. This finding can collaborate to enhance clinical solutions for young permanent teeth that have suffered necrosis.

Three-Dimensional Culture of Human Dental Pulp Tissue: A Preliminary Experimental In Vitro Model for Regenerative Endodontic Procedures

Introduction: Dental pulp regeneration is fundamental in dentistry and endodontics; however, few in vitro experimental models are available to study its biological process. This study aimed to develop a three-dimensional (3D) culture model of human dental pulp-like tissue mimicking the possible complexity of human pulp tissue. This new and innovative human-like tissue model could be used for testing drugs and materials, particularly those involved in regenerative endodontics. Methods: Vital pulp tissue samples were obtained from human third molars (n = 4) immediately after extraction and cultured in a 3D fibrin matrix to create a sustainable ex vivo experimental model. The angiogenesis degrees and the nitric oxide levels were evaluated following the culture of pulp-like tissues in the fibrin matrix for 21 days. The expression of Transforming growth factor- beta (TGF-β1), TGF-β2, TGF-β3, and their relevant receptors, tumor necrosis factor (TNF) and vascular endothelial growth factor A (VEGFA) was evaluated using a reverse transcription polymerase chain reaction (RT-PCR) method. Results: Pulp tissue angiogenesis was initiated, and completed on days 7 and 21, and pulp-like tissue cells expressed TGF-β1, TGF-β2, TGF-β3, and their relevant receptors, TNF and VEGFA. Conclusion: This model provided a precise observation of dental pulp angiogenesis at early stages.

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.

Public RNA-seq data-based identification and functional analyses reveal that MXRA5 retains proliferative and migratory abilities of dental pulp stem cells

Dental pulp stem cells (DPSC) usually remain quiescent in the dental pulp tissue; however, once the dental pulp tissue is injured, DPSCs potently proliferate and migrate into the injury microenvironment and contribute to immuno-modulation and tissue repair. However, the key molecules that physiologically support the potent proliferation and migration of DPSCs have not been revealed. In this study, we searched publicly available transcriptome raw data sets, which contain comparable (i.e., equivalently cultured) DPSC and mesenchymal stem cell data. Three data sets were extracted from the Gene Expression Omnibus database and then processed and analyzed. MXRA5 was identified as the predominant DPSC-enriched gene associated with the extracellular matrix. MXRA5 is detected in human dental pulp tissues. Loss of MXRA5 drastically decreases the proliferation and migration of DSPCs, concomitantly with reduced expression of the genes associated with the cell cycle and microtubules. In addition to the known full-length isoform of MXRA5, a novel splice variant of MXRA5 was cloned in DPSCs. Recombinant MXRA5 coded by the novel splice variant potently induced the haptotaxis migration of DPSCs, which was inhibited by microtubule inhibitors. Collectively, MXRA5 is a key extracellular matrix protein in dental pulp tissue for maintaining the proliferation and migration of DPSCs.

MicroRNA-4691-3p inhibits the inflammatory response by targeting STING in human dental pulp cells: A laboratory investigation.

The regulation of human dental pulp inflammation is not fully understood. This study aims to investigate the effect of miR-4691-3p on the cGAS-STING signalling cascade and its downstream cytokines production in human dental pulp cells (HDPCs). Normal dental pulp tissue and pulp tissue with irreversible pulpitis from third molars were collected. HDPCs were isolated from pulp tissue. The expression of STING mRNA and miR-4691-3p was measured by quantitative real-time PCR. Bioinformatic computation via TargetScanHuman 8.0 and a luciferase reporter assay was used to identify the targets of miR-4691-3p. A miR-4691-3p mimic and inhibitor were used to upregulate or downregulate miR-4691-3p expression in HDPCs. HDPCs were transfected with c-di-AMP, c-di-GMP, cGAMP, interferon stimulatory DNA (ISD) and bacterial genomic DNA. Immunoblot was performed to detect the phosphorylation of TBK1, p65 and IRF3. Enzyme-linked immunoassay was performed to detect the cytokines including IFN-β, TNF or IL-6 downstream of cGAS-STING. MiR-4691-3p expression was increased in human dental pulp tissue with irreversible pulpitis. Treatment of HDPCs using recombinant human IFN-β, TNF or IL-6 also upregulated miR-4691-3p. The bioinformatic prediction and luciferase reporter assay confirmed that STING was a direct target of miR-4691-3p. The miR-4691-3p mimic suppressed STING expression, the phosphorylation of TBK1, p65 and IRF3, and the IFN-β, TNF or IL-6 production. In contrast, the miR-4691-3p inhibitor enhanced the STING expression, the phosphorylation of TBK1, p65 and IRF3 and the IFN-β, TNF or IL-6 production. MiR-4691-3p negatively regulates the cGAS-STING pathway by directly targeting STING. This provides insight to utilize miRNA-dependent regulatory effect to treat endodontic disease as well as STING-dependent systemic inflammatory disease.

Semaphorin 4D Induces Vasculogenic Differentiation of Dental Pulp Stem Cells.

This work aimed to evaluate the effect of Semaphorin 4D (SEMA4D) signaling through Plexin B1 on the vasculogenic differentiation of dental pulp stem cells. We assessed the protein expression of SEMA4D and Plexin B1 in dental pulp stem cells (DPSC) from permanent human teeth and stem cells from human exfoliated deciduous (SHED) teeth using Western blots. Their expression in human dental pulp tissues and DPSC-engineered dental pulps was determined using immunofluorescence. We then exposed dental pulp stem cells to recombinant human SEMA4D (rhSEMA4D), evaluated the expression of endothelial cell differentiation markers, and assessed the vasculogenic potential of rhSEMA4D using an in vitro sprouting assay. Lastly, Plexin B1 was silenced to ascertain its role in SEMA4D-mediated vasculogenic differentiation. We found that SEMA4D and Plexin B1 are expressed in DPSC, SHED, and human dental pulp tissues. rhSEMA4D (25-100 ng/mL) induced the expression of endothelial markers, i.e., vascular endothelial growth factor receptor (VEGFR)-2, cluster of differentiation (CD)-31, and tyrosine kinase with immunoglobulin-like and EGF-like domains (Tie)-2, in dental pulp stem cells and promoted capillary-like sprouting in vitro (p < 0.05). Furthermore, Plexin B1 silencing abrogated the vasculogenic differentiation of dental pulp stem cells and significantly inhibited capillary sprouting upon exposure to rhSEMA4D. Collectively, these data provide evidence that SEMA4D induces vasculogenic differentiation of dental pulp stem cells through Plexin B1 signaling.

Efficacy of Chlorine Dioxide as an Endodontic Irrigant for Pulp Dissolution in vitro

Determine the efficacy of 5 % chlorine dioxide as an endodontic irrigant for pulp dissolution. Thirty five samples of human dental pulp were obtained, previously weighed and immersed in three solutions= 5 % ClO 2, 5.25 % NaOCl and saline (control group), for 10 minutes at 32 ºC; they were dried and reweighed. Then the weight loss was compared to the original weight and analyzed statistically. 5.25 % NaOCl and 5 % ClO 2 dissolved the dental pulp samples more effectively than saline (p> 0.001). No statistically significant difference was found between the tissue dissolving proper ties of 5.25 % NaOCl and 5 % ClO2 (p=0.893). 5 % ClO2 is effective in dissolving human dental pulp tissue.

Temporospatial Expression of Neuropeptide Substance P in Dental Pulp Stem Cells During Odontoblastic Differentiation in Vitro and Reparative Dentinogenesis in Vivo

IntroductionSubstance 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. MethodsDental 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. ResultsSP 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. ConclusionsSP contributes to odontoblastic differentiation during reparative dentin formation by binding to the NK1R.

CHARACTERIZATION OF A NOVEL CROSS LINKED PRF UNDER COMPRESSION INTENDED FOR PULP TISSUE ENGINEERING

Knowledge of biological and mechanical properties of native tissues is critical for biomaterial design and synthesis for tissue engineering. So far, selection of biomaterials for dental pulp regeneration has been done randomly or based on experience mainly due to the scarcity of data of biomechanical properties of human dental pulp tissue. This study, for the rst time, characterizes the physical properties of human dental pulp tissue harvested from wisdom teeth, under compression and compared it with PRF and a novel crosslinked PRF (C-PRF) prepared by crosslinking PRF with tannic acid. The results revealed that conventional PRF compressive sterength is very less compared to dental pulp tissue and comparable compressive strength of the novel crosslinked PRF(C-PRF). Taken collectively, crosslinked PRF will better suit for pulp tissue engineering purposes.

RNA-sequencing analysis reveals the potential molecular mechanism of RAD54B in the proliferation of inflamed human dental pulp cells.

To investigate the role of RAD54B in the proliferation of inflamed human dental pulp cells (hDPCs) induced by lipopolysaccharide (LPS). Normal, carious and pulpitic human dental pulp tissues were collected. Total RNA was subjected to RNA-sequencing (seq) and gene expression profiles were studied by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Differentially expressed genes (DEGs) in homologous recombination repair (HRR) were validated with qRT-PCR. The expression of RAD54B and TNF-α in human dental pulp tissues was detected using immunohistochemistry. HDPCs were cultured and RAD54B level in hDPCs was detected after LPS stimulation using western blot. CCK-8 was used to investigate the proliferation of hDPCs transfected with negative control (Nc) small interfering RNA (siRNA), RAD54B siRNA, P53 siRNA or both siRNAs with or without LPS stimulation. Flow cytometry was used to detect the cell cycle distribution, and western blot and immunofluorescence were used to analyse the expression of RAD54B, P53 and P21 under the above treatments. One-way and two-way anova followed by least significant difference posttest were used for statistical analysis. RNA-seq results identified DEGs amongst the three groups. KEGG pathway analysis revealed enrichment of DEGs in the replication and repair pathway. HRR and non-homologous end joining (NHEJ) components were further verified and qRT-PCR results were basically consistent with the sequencing data. RAD54B, an HRR accessory factor highly expressed in carious and pulpitic tissues as compared to that in normal pulps, was chosen as our gene of interest. High RAD54B expression was confirmed in inflamed human dental pulp tissues and LPS-stimulated hDPCs. Upon RAD54B knockdown, P53 and P21 expressions in hDPCs were upregulated whereas the proliferation was significantly downregulated, accompanied by increased G2/M phase arrest. After inhibiting P53 expression in RAD54B-knockdown hDPCs, P21 expression and cell proliferation were reversed. Gene expression profiles of normal, carious and pulpitic human dental pulp tissues were revealed. HRR components were elucidated to function in dental pulp inflammation. Amongst the DEGs in HRR, RAD54B regulated the proliferation of inflamed hDPCs via P53/P21 signalling. This research deepens our understanding of dental pulp inflammation and provides new insight to clarify the underlying mechanisms.

MicroRNA-22 suppresses NLRP3/CASP1 inflammasome pathway-mediated proinflammatory cytokine production by targeting the HIF-1α and NLRP3 in human dental pulp fibroblasts.

To investigate the synergetic regulatory effect of miR-22 on HIF-1α and NLRP3, subsequently regulating the production of the NLRP3/CASP1 inflammasome pathway-mediated proinflammatory cytokines IL-1β and IL-18 in human dental pulp fibroblasts (HDPFs) during the progression of pulpitis. Fluorescence in situ hybridization (FISH) and immunofluorescence (IF) were performed to determine the localization of miR-22-3p, NLRP3 and HIF-1α in human dental pulp tissues (HDPTs). The miR-22 mimics and inhibitor or plasmid of NLRP3 or HIF-1α were used to upregulate or downregulate miR-22 or NLRP3 or HIF-1α in HDPFs, respectively. Computational prediction via TargetScan 5.1 and a luciferase reporter assay were conducted to confirm target association. The mRNA and protein expression of HIF-1α, NLRP3, caspase-1, IL-1β and IL-18 were determined by qRT-PCR and western blotting, respectively. The release of IL-1β and IL-18 was analysed by ELISA. The significance of the differences between the experimental and control groups was determined by one-way analysis of variance, p < .05 indicated statistical significance. A decrease in miR-22 and an increase in HIF-1α and NLRP3 in HDPTs occurred during the transformation of reversible pulpitis into irreversible pulpitis compared with that in the healthy pulp tissues (p < .05). In the normal HDPTs, miR-22-3p was extensively expressed in dental pulp cells. HIF-1α and NLRP3 were mainly expressed in the odontoblasts and vascular endothelial cells. Whereas in the inflamed HDPTs, the odontoblast layers were disrupted. HDPFs were positive for miR-22-3p, HIF-1α and NLRP3. Computational prediction via TargetScan 5.1 and luciferase reporter assays confirmed that both NLRP3 and HIF-1α were direct targets of miR-22 in HDPFs. The miR-22 inhibitor further promoted the activation of NLRP3/CASP1 inflammasome pathway induced by ATP plus LPS and hypoxia (p < .05). In contrast, the miR-22 mimic significantly inhibited the NLRP3/CASP1 inflammasome pathway activation induced by ATP plus LPS and hypoxia (p < .05). MiR-22, as a synergetic negative regulator, is involved in controlling the secretion of proinflammatory cytokines mediated by the NLRP3/CASP1 inflammasome pathway by targeting NLRP3 and HIF-1α. These results provide a novel function and mechanism of miR-22-HIF-1α-NLRP3 signalling in the control of proinflammatory cytokine secretion, thus indicating a potential therapeutic strategy for future endodontic treatment.

Potentiality and Inflammatory Marker Expression Are Maintained in Dental Pulp Cell Cultures from Carious Teeth.

Objectives: This investigation aimed to isolate and culture human dental pulp cells from carious teeth (cHDPCs) and compare their growth characteristics, colony-forming efficiency, mineralization potential and gene expression of Toll-like receptors (TLR)-2, TLR-4, TLR-9, tumour necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-8, IL-17A, 1L-17R, IL-23A, nuclear factor-kappa B (NF-κB), mitogen-activated protein kinase (MAPK1), dentin matrix protein (DMP)-1, dentin sialophospho protein (DSPP), sex determining region Y-box 2 (SOX2) and marker of proliferation Ki-67 (MKi67) with cells isolated from healthy or non-carious teeth (ncHDPCs). Methods: Pulp tissues were obtained from both healthy and carious teeth (n = 5, each) to generate primary cell lines using the explant culture technique. Cell cultures studies were undertaken by generating growth curves, a colony forming unit and a mineralization assay analysis. The expression of vimentin was assessed using immunocytochemistry (ICC), and the gene expression of above-mentioned genes was determined using quantitative real-time reverse-transcription polymerase chain reaction. Results: ncHDPCs and cHDPCs were successfully isolated and cultured from healthy and inflamed human dental pulp tissue. At passage 4, both HDPC types demonstrated a typical spindle morphology with positive vimentin expression. No statistical difference was observed between ncHDPCs and cHDPCs in their growth characteristics or ability to differentiate into a mineralizing phenotype. ncHDPCs showed a statistically significant higher colony forming efficiency than cHDPCs. The gene expression levels of TLR-2, TLR-4, TLR-9, TNF-α, IL-6, IL-8, IL-17R, IL-23A, NF-κB, MAPK1, DMP1, DSPP and SOX2 were significantly higher in cHDPCs compared with ncHDPC cultures. Conclusion: cHDPCs retain their differentiation potential and inflammatory phenotype in vitro. The inflamed tooth pulp contains viable stem/progenitor cell populations which have the potential for expansion, proliferation and differentiation into a mineralizing lineage, similar to cells obtained from healthy pulp tissue. These findings have positive implications for regenerative endodontic procedures.

Alpha- Smooth Muscle Actin Expression in The Human Dental Pulp Tissue of Mobile Teeth

Tooth mobility is a degree of tooth looseness in its bony socket. A slight degree is accepted as a normal physiologic mobility of all types of the teeth in the oral cavity. During the tooth mobility due to different causes several biological processes take place, during these biological processes the dental pulp tissue is subjected to structural and protein expression modifications in order to maintain their integrity and functional morphology. The aim of this study is to evaluate Alpha- smooth muscle actin expression in the human dental pulp tissue of mobile teeth.Method: Acrosssectional study of 66 extracted teeth with their pulp tissue (49 with mobility and 17 as non-mobile control group); the samples were processed, and the immunohistochemicalreaction of the Alpha- smooth muscle actin (α-SMA) marker for each sample were analyzed.Results: Among the mobile teeth group (29) were with mild to moderate mobility, (20) with sever mobility. The Alpha- smooth muscle actin (α-SMA) positive results were obtained in (7) samples of the teeth with sever mobility and (21) samples of teeth with mild to moderate mobility were compared to (9) positive cases of control group. Conclusion: The data demonstrate that the dental pulp tissue does not expresses any irreversible histological alterations with mild to moderate teeth mobility compared to the irreversible changes that obtained in the pulp tissue of the sever mobile group. In conclusion the type of treatment required for the mobile teeth will depend on the responses of the pulp and the supporting periapical tissues to determine the outcome of the involved teeth sequel

Identification of Dental Stem Cells Similar to Skeletal Stem Cells

Stem and progenitor cells play important roles in the development and maintenance of teeth and bone. Surface markers expressed in bone marrow–derived mesenchymal stem cells are also expressed in dental tissue–derived stem cells. Mouse skeletal stem cells (mSSCs, CD45−Ter119−Tie2−CD51+Thy−6C3−CD105−CD200+) and human skeletal stem cells (hSSCs, CD45−CD235a−TIE2−CD31−CD146−PDPN+CD73+CD164+) have been identified in bone and shown to play important roles in skeletal development and regeneration. However, it is unclear whether dental tissues also harbor mSSC or hSSC populations. Here, we employed rainbow tracers and found that clonal expansion occurred in mouse dental tissues similar to that in bone. We sorted the mSSC population from mouse periodontal ligament (mPDL) tissue and mouse dental pulp (mDP) tissue in the lower incisors by fluorescence-activated cell sorting (FACS). In addition, we demonstrated that mPDL-derived skeletal stem cells (mPDL-SSCs) and mDP-derived skeletal stem cells (mDP-SSCs) have similar clonogenic capacity, as well as cementogenic and odontogenic potential, but not adipogenic potential, similar to the characteristics of mSSCs. Moreover, we found that the dental tissue–derived mSSC population plays an important role in repairing clipped incisors. Importantly, we sorted the hSSC population from human periodontal ligament (hPDL) and human dental pulp (hDP) tissue in molars and identified its stem cell characteristics. Finally, hPDL-like and hDP-like structures were generated after transplanting hPDL-SSCs and hDP-SSCs beneath the renal capsules. In conclusion, we demonstrated that mouse and human PDL and DP tissues harbor dental stem cells similar to mSSCs and hSSCs, respectively, providing a precise stem cell population for the exploration of dental diseases.

Leptin in Dental Pulp and Periapical Tissues: A Narrative Review.

Leptin is a non-glycosylated 16 kDa protein synthesized mainly in adipose cells. The main function of leptin is to regulate energy homeostasis and weight control in a central manner. There is increasing evidence that leptin also has systemic effects, acting as a link between innate and acquired immune responses. The expression of leptin and its receptor in human dental pulp and periradicular tissues have already been described, as well as several stimulatory effects of leptin protein expression in dental and periodontal tissues. The aim of this paper was to review and to compile the reported scientific literature on the role and effects of leptin in the dental pulp and periapical tissues. Twelve articles accomplished the inclusion criteria, and a comprehensive narrative review was carried out. Review of the available scientific literature concluded that leptin has the following effects on pulpal and periapical physiology: 1) Stimulates odontogenic differentiation of dental pulp stem cells (DPSCs), 2) Increases the expression of dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1), odontoblastic proteins involved in odontoblastic differentiation and dentin mineralization, 3) Stimulates vascular endothelial growth factor (VEGF) expression in human dental pulp tissue and primary cultured cells of human dental pulp (hDPCs), 4) Stimulates angiogenesis in rat dental pulp cells, and 5) Induces the expression of interleucinas 6 and 8 in human periodontal ligament cells (hPDLCs). There is evidence which suggests that leptin is implicated in the dentin mineralization process and in pulpal and periapical inflammatory and reparative responses.

Extracellular vesicles from mesenchymal stem cells of dental pulp and adipose tissue display distinct transcriptomic characteristics suggestive of potential therapeutic targets.

Objective: Mesenchymal stem cells (MSCs) are isolated from various human tissues and used for therapy, in which beneficial effects are attributed mainly to mesenchymal stem cell-derived extracellular vesicles (MSC-EVs). Whereas MSCs of diverse tissue types share cardinal stem cell features, it is becoming evident that MSCs of each tissue type possess unique properties as well. For designing efficient stem cellbased therapies, it is crucial to understand the unique properties associated with MSCs and MSC-EVs of each tissue type. Such unique properties can be analyzed through transcriptomic approaches using comprehensive gene expression databases and sophisticated analytical tools. Here, we comparatively studied the transcriptomes in MSC-EVs of dental pulp and adipose tissue. Additionally, the transcriptomes of MSC-EVs were compared with the cellular transcriptomes of MSCs for the same tissue types. Methods: MSCs were cultured from human dental pulp and adipose tissue specimens. Conditioned culture media were collected to prepare MSC-EVs, from which RNAs were isolated and subjected to next-generation sequencing for transcriptomic analysis. Gene expression signatures in MSC-EVs of each tissue type were investigated using gene set analysis. Results: MSC-EVs obtained from dental pulp-derived MSCs showed distinct transcriptomic signatures of neurogenesis and neural retina development while MSC-EVs of adipose tissue-derived MSCs showed signatures of mitochondrial activity and skeletal system development. The transcriptomes of MSC-EVs resembled the cellular transcriptomes of MSCs, and the genes associated with neurogenesis were highly expressed in both MSCs and MSC-EVs of dental pulp. Adipose tissue-derived MSCs and MSC-EVs highly expressed genes associated with angiogenesis, hair growth, and dermal matrices. Conclusion: The clear and distinct signatures of neurogenesis and neural retina development in dental pulp-derived MSC-EVs imply neurodegenerative disorders and retinal diseases as putative therapeutic targets. In contrast, the transcripts in adipose tissue-derived MSC-EVs could be useful in rejuvenating the skin and musculoskeletal system. Further insights into MSC-EVs of divergent tissue types may expand the list of potential therapeutic targets.