Human Dental Pulp Tissue Research Articles

Comparison of ADAMTS Levels in Pulp Tissue Samples of Healthy and Symptomatic Irreversible Pulpitis Teeth

IntroductionThe aim of this study was to compare the levels of different A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS; ie, ADAMTS-1, -4, and -9]) related to the breakdown of the proteoglycans and tissue inhibitor of metalloproteinase 3 (TIMP-3) levels that inhibit ADAMTS in the extracellular matrix of healthy pulp and symptomatic irreversible pulpitis. MethodsPulp tissue samples diagnosed with healthy and symptomatic irreversible pulpitis were collected from 48 participants. Healthy and inflamed pulps were directly transferred to Eppendorf tubes (Labosel, Istanbul, Turkey) and stored at −80°C (Nüve-DF490; Nüve, Ankara, Turkey) until further use. Routine root canal treatment procedures for the teeth were performed, after which the treatment process was completed. The levels of ADAMTS-1, -4, and -9 and TIMP-3 were measured in supernatants of human dental pulp tissue extracts using enzyme-linked immunosorbent assay kits (USCN, Wuhan, China). The independent sample t test and 1-way analysis of variance were used for parametric data, and the Kruskal-Wallis and Mann-Whitney U tests were used for nonparametric data. ResultsThere was a statistically significant difference between ADAMTS-1 and -9 and TIMP-3 levels of the healthy pulp and pulpitis groups (P < .05), but there was no significant difference for ADAMTS-4 levels (P > .05). It was found that the levels of ADAMTS-1 and -9 were higher in the pulpitis group compared with the healthy pulp group, whereas the TIMP-3 level decreased (P < .05). ConclusionsAs a result of this study, it was concluded that ADAMTS-1 and -9 and TIMP-3 might have a role in pulpal inflammation, whereas ADAMTS-4 was not related to pulpal inflammation.

Single-cell characterization of monolayer cultured human dental pulp stem cells with enhanced differentiation capacity

Human dental pulp stem cells (hDPSCs) are easily obtained multipotent cells, however, their potential value in regenerative medicine is hindered by the phenotypic and functional changes after conventional monolayer expansion. Here, we employed single-cell RNA sequencing (scRNA-seq) to comprehensively study the transcriptional difference between the freshly isolated and monolayer cultured DPSCs. The cell cluster analysis based on our scRNA-seq data showed that monolayer culture resulted in a significant cellular composition switch compared to the freshly isolated DPSCs. However, one subpopulation, characterized as MCAM(+)JAG(+)PDGFRA(−), maintained the most transcriptional characteristics compared to their freshly isolated counterparts. Notably, immunofluorescent staining revealed that the MCAM(+)JAG(+)PDGFRA(−) hDPSCs uniquely located in the perivascular region of human dental pulp tissue. Flow-cytometry analysis confirmed that their proportion remained relatively stable (~2%) regardless of physiological senescence or dental caries. Consistent with the annotation of scRNA-seq data, MCAM(+)JAG(+)PDGFRA(−) hDPSCs showed higher proliferation capacity and enhanced in vitro multilineage differentiation potentials (osteogenic, chondrogenic and adipogenic) compared with their counterparts PDGFRA(+) subpopulation. Furthermore, the MCAM(+)JAG(+)PDGFRA(−) hDPSCs showed enhanced bone tissue formation and adipose tissue formation after 4-week subcutaneous implantation in nude mice. Taken together, our study for the first time revealed the cellular composition switch of monolayer cultured hDPSCs compared to the freshly isolated hDPSCs. After in vitro expansion, the MCAM(+)JAG(+)PDGFRA(−) subpopulation resembled the most transcriptional characteristics of fresh hDPSCs which may be beneficial for further tissue regeneration applications.

The induction of inflammation by the cGAS-STING pathway in human dental pulp cells: A laboratory investigation.

To explore the presence of the cGAS-STING inflammatory pathway in human pulp tissue and human dental pulp cells (HDPCs). Pulp tissue was collected from freshly extracted human healthy third molars or third molars with irreversible pulpitis. Quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunoassay (ELISA) were performed to assess IFN-β, TNF and IL-6. Human dental pulp cells prepared from healthy human pulp tissues were transfected with interferon stimulatory DNA (ISD), bacterial genomic DNA, bacterial cyclic dinucleotides c-di-AMP, c-di-GMP or host cyclic dinucleotide cGAMP. SiRNA was used to knock down the endogenous cGAS or STING. G140 and H-151 were used to inhibit cGAS and STING respectively. Amlexanox and BAY 11-7082 were used to inhibit TBK1 and NF-κB respectively. qRT-PCR and ELISA were performed to detect the level of IFN-β, TNF and IL-6. Western blot was performed to evaluate the TBK1, IRF3 and p65 phosphorylation. The Student's t-test and one-way anova were used for statistical analysis. IFN-β, TNF and IL-6 were up-regulated in the inflamed human dental pulp tissue. CGAS and STING mRNA were increased in the inflamed human dental pulp tissue and detected in HDPCs prepared from healthy human pulp tissues. ISD transfection induced TBK1, IRF3 and p65 phosphorylation as well as IFN-β, TNF and IL-6 production. IFN-β, TNF and IL-6 production were also induced by transfection of bacterial and host cyclic dinucleotides or bacteria DNA. ISD or bacteria DNA transfection elevated the intracellular levels of cGAMP. Knock-down of cGAS or STING, as well as using cGAS inhibitor G140 or STING inhibitor H-151 abolished the IFN-β, TNF and IL-6 production induced by ISD transfection. Knock-down of STING or using STING inhibitor H-151 abolished the IFN-β, TNF and IL-6 induction by transfection of bacterial and host cyclic dinucleotides. Both Amlexanox and BAY 11-7082 inhibited IFN-β, TNF and IL-6 production triggered by ISD and cyclic dinucleotides transfection. Human dental pulp cells expressed an intact cGAS-STING signalling axis. The cGAS-STING signalling axis may play an important role in pulp inflammation and immune defence.

Induced Neural Cells from Human Dental Pulp Ameliorate Functional Recovery in a Murine Model of Cerebral Infarction.

Human mesenchymal stem cells are a promising cell source for the treatment of stroke. Their primary mechanism of action occurs via neuroprotective effects by trophic factors, anti-inflammatory effects, and immunomodulation. However, the regeneration of damaged neuronal networks by cell transplantation remains challenging. We hypothesized that cells induced to neural lineages would fit the niche, replace the lesion, and be more effective in improving symptoms compared with stem cells themselves. We investigated the characteristics of induced neural cells from human dental pulp tissue and compared the transplantation effects between these induced neural cells and uninduced dental pulp stem cells. Induced neural cells or dental pulp stem cells were intracerebrally transplanted 5days after cerebral infarction induced by permanent middle cerebral artery occlusion in immunodeficient mice. Effects on functional recovery were also assessed through behavior testing. We used immunohistochemistry and neuron tracing to analyze the differentiation, axonal extension, and connectivity of transplanted cells to the host's neural circuit. Transplantation of induced neural cells from human dental pulp ameliorated functional recovery after cerebral infarction compared with dental pulp stem cells. The induced neural cells comprised both neurons and glia and expressed functional voltage, and they were more related to neurogenesis in terms of transcriptomics. Induced neural cells had a higher viability than did dental pulp stem cells in hypoxic culture. We showed that induced neural cells from dental pulp tissue offer a novel therapeutic approach for recovery after cerebral infarction.

Extreme intrusive force affects the expression of c-Fos and matrix metallopeptidase 9 in human dental pulp tissues: Erratum.

Extreme intrusive force affects the expression of c-Fos and matrix metallopeptidase 9 in human dental pulp tissues: Erratum.

Experimental resin-modified calcium-silicate cement containing N-(2-hydroxyethyl) acrylamide monomer for pulp tissue engineering

AimOur study aimed to measure (1) the flexural strength, (2) shear bond strength to dentin, (3) pH, and (4) calcium (Ca) release of a series of innovative resin-modified calcium-silicate pulp-capping cements (Rm-CSCs). Using an ex-vivo human vital tooth-culture model, we additionally assessed (5) their pulp-healing initiation when brought in direct contact with human dental pulp tissue. MethodologyThree experimental Rm-CSCs, being referred to ‘Exp_HEAA’, ‘Exp_GDM’ and ‘Exp_HEAA/GDM’, contained either 20 wt% N-(2-hydroxyethyl) acrylamide (HEAA), 20 wt% glycerol dimethacrylate (GDM) or 10 wt% HEAA plus 10 wt% GDM, added to a common base composition consisting of 25 wt% urethane dimethacrylate (UDMA), 10 wt% 4-methacryloxyethyl trimellitate anhydride (4-MET), and 5 wt% N,N'-{[(2-acrylamido-2-[(3-acrylamidopropoxy)methyl] propane-1,3-diyl)bis(oxy)]bis-(propane-1,3-diyl)}diacrylamide (FAM-401). As Ca source and radiopacifier, 37 wt% tricalcium silicate powder (TCS) and 3 wt% zirconium oxide (ZrO 2) were respectively added. ResultsAll three experimental Rm-CSCs revealed a significantly higher flexural strength and shear bond strength to dentin (p < 0.05) than the commercial reference Rm-CSC TheraCal LC (Bisco). Exp_HEAA presented with a significantly higher Ca release and pH at 24 h compared with the other Rm-CSCs (p < 0.05). At 1 week, the Ca release and pH of Exp_HEAA and Exp_HEAA/GDM was significantly higher than those of Exp_GDM and TheraCal LC (p < 0.05). Using the ex-vivo human vital tooth culture model, Exp_HEAA revealed pulp-healing initiation capacity as documented by nestin and collagen-I expression. ConclusionsDepending on the formulation, the innovative Rm-CSCs performed favorably for primary properties of relevance regarding pulp capping, this more specifically in terms of flexural strength, bond strength to dentin, as well as alkaline pH and Ca release. However, only Exp_HEAA revealed pulp-healing initiation in direct contact with human dental pulp tissue in the ex-vivo human vital tooth-culture model. This promising outcome for Exp_HEAA should be attributed to the combined use of (1) a novel hydrophilic acrylamide monomer, enabling sufficient polymerization while maintaining adequate hydrophilicity, with (2) the functional monomer 4-MET, possessing chemical bonding potential to dentin, and (3) tricalcium silicate powder to achieve an alkaline pH and to release Ca in a sufficient and controlled way.

LncRNA H19 facilitates the proliferation and differentiation of human dental pulp stem cells via EZH2-dependent LATS1 methylation

Human dental pulp stem cells (hDPSCs) have been recognized as a candidate cell source for tissue engineering. Long non-coding RNAs (lncRNAs) are differentially expressed in inflamed human dental pulp tissues. The present study is aimed at investigating the role of lncRNA H19 in the differentiation potential of hDPSCs. hDPSCs were successfully isolated and cultured, followed by conducting gain and loss-of-function experiments on lncRNA H19 and large tumor suppressor 1 (LATS1) to elucidate their respective biological functions in hDPSCs. lncRNA H19 was able to promote, whereas LATS1 was found to inhibit the differentiation, proliferation, and migration capabilities of hDPSCs. LATS1 was found to activate the Hippo-Yes-associated protein (YAP) signaling pathway by decreasing levels of YAP and Tafazzin (TAZ). The effects of lncRNA H19 on hDPSCs were achieved by repressing LATS1 through enhancer of zeste homolog 2-induced trimethylation of histone 3 at lysine 27. Finally, hDPSCs overexpressing lncRNA H19 and/or LATS1 were transplanted into nude mice. It was shown that lncRNA H19 inhibited LATS1 to promote the production of odontoblasts in vivo. Taken together, lncRNA H19 serves as a contributor to the differentiation potential of hDPSCs via the inhibition of LATS1, therefore highlighting novel therapeutic targets for dental pulp repair.

Bovine-Derived Xenografts Immobilized With Cryopreserved Stem Cells From Human Adipose and Dental Pulp Tissues Promote Bone Regeneration: A Radiographic and Histological Study.

Adipose tissue-derived stem cells (ADSCs) and dental pulp stem cells (DPSCs) have become promising sources for bone tissue engineering. Our study aimed at evaluating bone regeneration potential of cryopreserved ADSCs and DPSCs combined with bovine-derived xenografts with 10% porcine collagen. In vitro studies revealed that although DPSCs had higher proliferative abilities, ADSCs exhibited greater mineral depositions and higher osteogenic-related gene expression, indicating better osteogenic differentiation potential of ADSCs. After applying cryopreserved ADSCs and DPSCs in a critical-sized calvarial defect model, both cryopreserved mesenchymal stem cells significantly improved bone volume density and new bone area at 2, 4, and 8 weeks. Furthermore, the combined treatment with ADSCs and xenografts was more efficient in enhancing bone repair processes compared to combined treatment with DPCSs at all-time points. We also evaluated the sequential early bone healing process both histologically and radiographically, confirming a high agreement between these two methods. Based on these results, we propose grafting of the tissue-engineered construct seeded with cryopreserved ADSCs as a useful strategy in accelerating bone healing processes.

5-aza-2'-deoxycytidine may regulate the inflammatory response of human odontoblast-like cells through the NF-κB pathway.

To explore the role of DNA methylation in the innate immunity of the dental pulp, this study investigated the effect of 5-aza-2'-deoxycytidine (AZA) on lipoteichoic acid (LTA)-induced cytokine production and related intracellular signalling pathways in human odontoblast-like cells (hOBs). hOBs were cultured and differentiated from human dental pulp tissue, and the odontoblastic phenotype of the cells was detected using immunofluorescence, qRT-PCR and Western blotting. hOBs were pretreated with AZA and then stimulated with 10μgmL-1 LTA. The levels of 42 cytokines related to immunity and inflammation were examined using a cytokine antibody array and verified using qRT-PCR and ELISA. The effect of AZA on the LTA-induced NF-κB and MAPK signalling pathways was explored using Western blotting. The cells were treated with the specific NF-κB inhibitor PDTC and MAPK inhibitors (the ERK inhibitor U0126, the p38 inhibitor SB203580, and the JNK inhibitor SP600125) to further confirm the role of the signalling pathways in LTA-treated hOBs. DNA immunoprecipitation-PCR was used to examine the dynamic methylation status of the gene promoters of myeloid differentiation primary response 88 (MyD88) and tumour necrosis factor receptor-associated factor 6 (TRAF6) in the LTA-induced hOBs. Statistical analyses of the differences between two groups were performed using Student's t-test. One-way analysis of variance (anova) or repeated-measures anova with a post hoc Dunnett's test was used to assess the differences between multiple sets of data. P<0.05 was considered to be statistically significant. The odontoblastic markers were significantly higher in hOBs than those in human dental pulp cells (hDPCs) (P<0.05). According to the cytokine antibody array results, hOBs pretreated with AZA had significantly increased production of several inflammatory cytokines (P<0.05), in which the expression levels of IL-6 and IL-8 were the most dramatically increased upon LTA stimulation (P<0.01). Furthermore, AZA resulted in the significant upregulation of p-IKKα/β, p-IκBα, p-p65, p-p38 and p-ERK in LTA-stimulated hOBs (P<0.01). Treatment with the NF-κB pathway inhibitor suppressed both IL-6 and IL-8 expression (P<0.05), whereas inhibitors of the MAPK pathway (SB203580 and SP600125) did not. In LTA-treated hOBs, AZA significantly increased the expression levels of TRAF6 and MyD88 (P<0.05). AZA induced MyD88 promoter hypomethylation but did not affect TRAF6 methylation. AZA regulated the LTA-induced inflammatory response through the NF-κB signal pathway in hOBs. This study highlights the important role of DNA methylation in the immunity defence of odontoblasts during the dental pulp immunity response to caries.

NLRP6-caspase 4 inflammasome activation in response to cariogenic bacterial lipoteichoic acid in human dental pulp inflammation.

To explore the presence and function of NLRP6-caspase 4 inflammasome in human pulp tissue and human dental pulp cells (HDPCs). Pulp tissue was collected from freshly extracted human caries-free third molars and third molars with irreversible pulpitis. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot were performed to assess the expression of NLRP6-caspase 4 inflammasome. HDPCs were prepared from normal human pulp tissues and challenged with Porphyromonas gingivalis LPS. Enzyme-linked immunosorbent assay (ELISA) and qRT-PCR were performed to assess if LPS can upregulate NLRP6 and caspase-4. HDPCs were further challenged with LPS followed with cytosolic Streptococcus mutans lipoteichoic acid (LTA). SiRNA targeting NLRP6 and Casp4 and pharmacology inhibitor Ac-FLTD-CMK and MCC950 were used to assess if Streptococcus mutans LTA can activate the NLRP6 but not the NLRP3 inflammasome. Western blot and ELISA were performed to evaluate inflammasome activation. The Student's t-test and one-way anova were used for statistical analysis. NLRP6-caspase 4 inflammasome was upregulated and activated in inflamed human dental pulp tissue. In HDPCs, Porphyromonas gingivalis LPS upregulated the expression of NLRP6, CASP1 and CASP4 in a type I interferon dependent manner. After LPS priming, cytosolic Streptococcus mutans LTA triggered NLRP6-caspase 4 inflammasome activation. Knockdown of NLRP6 or CASP4 using siRNA or using pharmacology inhibitor Ac-FLTD-CMK but not MCC950 efficiently suppressed inflammasome activation by cytosolic LTA. NLRP6-caspase 4 inflammasome may play an important role in pulp inflammation and immune defence. Inflammatory caspases represent a pharmacological target to restrain pulpal inflammation.

Taxifolin Protects Dental Pulp Stem Cells under Hypoxia and Inflammation Conditions

Background: Dental pulp stem cells (DPSCs) are a unique source for future clinical application in dentistry such as periodontology or endodontics. However, DPSCs are prone to apoptosis under abnormal conditions. Taxifolin is a natural flavonoid and possesses many pharmacological activities including anti-hypoxic and anti-inflammatory. We aimed to elucidate the mechanisms of taxifolin protects DPSC under hypoxia and inflammatory conditions. Methods: DPSCs from human dental pulp tissue was purchased from Lonza (cat. no. PT-5025. Basel, Switzerland)) and identified by DPSC’s biomarkers. DPSC differentiation in vitro following the manufacturers’ instructions. ARS staining and Oil red staining verify the efficiency of differentiation in vitro after 2 weeks. The changes of various genes and proteins were identified by Q-PCR and western-blot, respectively. Cell viability was determined by the CCK-8 method, while apoptosis was determined by Annexin V/PI staining. Results: DPSC differentiation in vitro shows that hypoxia and TNF-α synergistically inhibit the survival and osteogenesis of DPSCs. A final concentration of 10 μM Taxifolin can significantly reduce the apoptosis of DPSCs under inflammation and hypoxia conditions. Taxifolin substantially increases carbonic anhydrase IX (CA9) expression but not HIF1a, and inhibitions of CA9 expression nullify the protective role of taxifolin under hypoxia and inflammatory condition. Conclusion: Taxifolin significantly increased the expression of CA9 when it inhibits DPSC apoptosis and taxifolin synergistically to protect DPSCs against apoptosis with CA9 under hypoxia and inflammatory conditions. Taxifolin can be used as a potential drug for clinical treatment of DPSC-related diseases.

Genotoxicity Induced by Cellular Uptake of Chitosan Nanoparticles in Human Dental Pulp Cells

<i>Objective:</i> Recent <i>in vitro</i> studies have shown that chitosan nanoparticles in several root canal sealers, intracanal medicament, and irrigation solutions could enhance the antimicrobial activity. However, the nanotoxicity of chitosan has not been fully studied. The aim of this study was to evaluate cellular uptake and genotoxicity of various sizes and concentrations of chitosan nanoparticles cultured with human dental pulp cells. <i>Methods:</i> Human dental pulp cells were derived from human dental pulp tissues and cultured for 24 hours with 50 nm and 318 nm FITC-tagged chitosan nanoparticles in concentrations: 0.1 mg/mL, 0.5 mg/mL, and 2 mg/mL as study groups, and 0 mg/mL as a control. The fluorescence intensity of the FITC tagged chitosan nanoparticles was measured using a spectrophotometer to determine the cellular uptake. Genotoxicity was assessed by the Cytokinesis-block micronucleus method and by measuring the fluorescent intensity of the phosphorylated H2AX nuclear foci. Statistical analysis was performed using One-Way ANOVA, post-hoc Tukey, and Chi-square tests. <i>Results:</i> Chitosan nanoparticles were able to internalize the human dental pulp cells and significantly induced micronuclei, nuclear buds, and pH2AX foci at concentrations of 0.5 mg/mL and 2 mg/mL as compared to 0.1 mg/mL (<I>P </i>< 0.01) and control group (<i>P </i>< 0.01). At both concentrations, 0.5 mg/mL and 2 mg/mL, 50 nm chitosan significantly induced higher proportions of micronuclei (<i>P</i>=0.001), nuclear buds (<i>P</i>=0.009), and pH2AX nuclear foci (<i>P</i>=0.00004) as compared to 318 nm chitosan. <i>Conclusion:</i> 50 nm and 318 nm chitosan nanoparticles at concentrations 0.5 mg/mL and 2 mg/mL penetrated human dental pulp cells and induced genotoxicity in dose-dependent and size-associated manners.

Stem Cells Derived from Human Exfoliated Deciduous Teeth (SHED) in Neuronal Disorders: A Review

Most conventional treatments for neurodegenerative diseases fail due to their focus on neuroprotection rather than neurorestoration. Stem cell-based therapies are becoming a potential treatment option for neurodegenerative diseases as they can home in, engraft, differentiate and produce factors for CNS recovery. Stem cells derived from human dental pulp tissue differ from other sources of mesenchymal stem cells due to their embryonic neural crest origin and neurotrophic property. These include both Dental Pulp Stem Cells (DPSCs) from dental pulp tissues of human permanent teeth and Stem cells from Human Exfoliated Deciduous teeth (SHED). SHED offers many advantages over other types of MSCs, such as good proliferative potential, minimal invasive procurement, neuronal differentiation and neurotrophic capacity, and negligible ethical concerns. The therapeutic potential of SHED is attributed to the paracrine action of extracellularly released secreted factors, specifically the secretome, of which exosomes are a key component. SHED and its conditioned media can be effective in neurodegeneration through multiple mechanisms, including cell replacement, paracrine effects, angiogenesis, synaptogenesis, immunomodulation, and apoptosis inhibition, and SHED exosomes offer an ideal refined bed-to-bench formulation in neurodegenerative disorders. However, in spite of these advantages, there are still some limitations of SHED exosome therapy, such as the effectiveness of long-term storage of SHED and their exosomes, the development of a robust GMP-grade manufacturing protocol, optimization of the route of administration, and evaluation of the efficacy and safety in humans. In this review, we have addressed the isolation, collection and properties of SHED along with its therapeutic potential on in vitro and in vivo neuronal disorder models as evident from the published literature.

MicroRNA-223 negatively regulates LPS-induced inflammatory responses by targeting NLRP3 in human dental pulp fibroblasts.

To investigate the effect of miR-223 on 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). Human dental pulp tissue (HDPT) and HDPFs were obtained from impacted third molars. The miR-223 mimics and inhibitor or NLRP3 plasmid were used to upregulate or downregulate miR-223 or NLRP3 in HDPFs, respectively. Computational prediction via TargetScan 5.1 and a luciferase reporter assay was conducted to confirm target association. The mRNA and protein expression of NLRP3, caspase-1, IL-1β and IL-18 was 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 the control groups was determined using one-way analysis of variance; P<0.05 indicated statistical significance. A decrease in miR-223 and an increase in NLRP3 in HDPT occurred during the transformation of reversible pulpitis into irreversible pulpitis compared to that in healthy pulp tissue (P<0.05). The computational prediction and luciferase reporter assay confirmed that NLRP3 was a direct target of miR-223 in HDPFs. The miR-223 inhibitor further promoted ATP plus LPS-induced NLRP3/CASP1 inflammasome pathway activation compared to the ATP plus LPS-induced group (P<0.05). In contrast, the miR-223 mimic significantly inhibited the NLRP3/CASP1 inflammasome pathway activation induced by ATP plus LPS compared to the ATP plus LPS-induced group (P<0.05). MiR-223 served as a negative regulator involved in the control of the production and secretion of proinflammatory cytokines mediated by the NLRP3/CASP1 inflammasome pathway by targeting NLRP3. These data provide insight into the potential regulatory effects of miRNAs on the NLRP3 inflammasome, thus opening up novel potential therapeutic avenues for future endodontic treatment.

Semaphorin3A released from human dental pulp cells inhibits the increase in interleukin-6 and CXC chemokine ligand 10 production induced by tumor necrosis factor-α through suppression of nuclear factor-κB activation.

Human dental pulp cells (HDPCs) play an important role in pulpitis. Semaphorin3A (Sema3A), which is an axon guidance molecule, is a member of the secretory semaphorin family. Recently, Sema3A has been reported to be an osteoprotective factor and to be involved in the immune response. However, the role of Sema3A in dental pulp inflammation remains unknown. The aim of this study was to reveal the existence of Sema3A in human dental pulp tissue and the effect of Sema3A which is released from tumor necrosis factor (TNF)-α-stimulated HDPCs on production of proinflammatory cytokines, such as interleukin (IL)-6 and CXC chemokine ligand 10 (CXCL10), from HDPCs stimulated with TNF-α. Sema3A was detected in inflamed pulp as compared to normal pulp. HDPCs expressed Neuropilin-1(Nrp1) which is Sema3A receptor. TNF-α increased the levels of IL-6 and CXCL10 in HDPCs in time-dependent manner. Sema3A inhibited production of these two cytokines from TNF-α-stimulated HDPCs. TNF-α induced soluble Sema3A production from HDPCs. Moreover, antibody-based neutralization of Sema3A further promoted production of IL-6 and CXCL10 from TNF-α-stimulated HDPCs. Sema3A inhibited nuclear factor (NF)-κB P65 phosphorylation and inhibitor κBα degradation in TNF-α-stimulated HDPCs. These results indicated that Sema3A is induced in human dental pulp, and TNF-α acts on HDPCs to produce Sema3A, which partially inhibits the increase in IL-6 and CXCL10 production induced by TNF-α, and that the inhibition leads to suppression of NF-κB activation. Therefore, it is suggested that Sema3A may regulate inflammation in dental pulp and be novel antiinflammatory target molecule for pulpitis.

Human Dental Pulp Tissue during Orthodontic Tooth Movement: An Immunofluorescence Study.

The orthodontic tooth movement is the last step of several biological processes that take place after the application of external forces. During this process, dental pulp tissue is subjected to structural and protein expression modifications in order to maintain their integrity and functional morphology. The purpose of the present work was to perform an in vivo study, evaluating protein expression modifications in the human dental pulp of patients that have undergone orthodontic tooth movement due to pre-calibrated light force application for 30 days. Dental pulp samples were extracted from molars and premolars of the control group and after 7 and 30 days of treatment; the samples were then processed for immunofluorescence reactions using antibodies against fibronectin, collagen I and vascular endothelial growth factor (VEGF). Our results show that, after 7 days of treatment, all tested proteins change their pattern expression and will reset after 30 days. These data demonstrate that the dental pulp does not involve any irreversible iatrogenic alterations, supporting the efficacy and safety of using pre-calibrated force application to induce orthodontic tooth movement in clinical practice.

MiR-217 Suppresses Osteogenic Differentiation of Human Dental Pulp Stem Cells by Down-Regulating Sirtuin 1

The paper is committed to uncovering the effect of miR-217 on osteogenic differentiation of human dental pulp stem cells (hDPSCs) and its mechanism. hDPSCs were separated from human dental pulp tissues for measurement of stemness. The osteogenic differentiation of hDPSCs was induced in an osteogenic induction medium. The hDPSCs were transfected with miR-217 mimic, miR-217 inhibitor and/or sh-SIRT1 accordingly. The expressions of miR-217 and SIRT1 were detected in hDPSCs after cell transfection and osteogenic differentiation. Calcium nodules were showed by alizarin red staining. Moreover, the expressions of osteogenic differentiation-related genes were also assessed. The binding of miR-217 to SIRT1 was predicted on starBase and further determined by dual-luciferase reporter assay. Down-regulated miR-217 and up-regulated SIRT1 were found during osteogenic differentiation of hDPSCs. The osteogenic differentiation of hDPSCs was suppressed after transfection of miR-217 mimic or sh-SIRT1 while promoted by miR-217 inhibition. Taken together, miR-217 can suppress osteogenic differentiation of hDPSCs by negatively regulating SIRT1.

Clarifying the Tooth-Derived Stem Cells Behavior in a 3D Biomimetic Scaffold for Bone Tissue Engineering Applications.

Massive amounts of cell are needed for creating tissue engineered 3D constructs, which often requires culture on scaffolds under dynamic conditions to facilitate nutrients and oxygen diffusion. Dynamic cultures are expected to improve cell viability and proliferation rate, when compared to static conditions. However, cells from distinct types and/or tissues sources may respond differently to external stimuli and be incompatible with culture under mechanical shear stress. The first aim of this work was to show that dental stem cells are a valuable source for improving bone regeneration potential of artificial grafts. Mesenchymal stem/stromal cells (MSCs) were isolated from human dental follicle (hDFMSC) and pulp tissues (hDPMSC) and shown to express prototypical stem cell markers. The follicle and pulp dental MSCs capacity to differentiate into osteoblast lineage was evaluated after seeding on 3D porous scaffolds of collagen-nanohydroxyapatite/phosphoserine biocomposite cryogel with osteogenic factors in the culture medium. Both tooth-derived MSCs were able to show high ALP activity, express osteogenic gene markers and secrete osteopontin (OPN). Thereafter, designed multicompartment holder adaptable to spinner flasks was used for dynamic culture (50 rpm) of both dental MSCs types within the porous 3D scaffolds. Standard static culture conditions were used as control. Culture under dynamic conditions promoted follicle MSCs proliferation, while improving their spatial distribution within the scaffold. Under dynamic conditions, the biocomposite scaffold promoted MSCs osteogenic differentiation, as suggested by increased alkaline phosphatase (ALP) activity, higher osteogenic gene expression and OPN deposition. In a similar manner, under dynamic conditions, dental pulp MSCs also showed higher ALP activity and proliferation rate, but lower amounts of osteopontin secretion, when compared to static conditions. After implantation, dental follicle MSCs-loaded 3D scaffolds cultured under dynamic conditions showed higher tissue ingrowth and osteogenic differentiation (higher human OPN secretion) than dental pulp cells. Overall, this study explored the use of tooth-derived stem cells as a clinical alternative source for bone tissue engineering, together with an innovative device for dynamic culture of cell-laden 3D scaffolds. Results showed that human MSCs response upon culture on 3D scaffolds, depends on the cells source and the culture regimen. This suggests that both the type of cells and their culture conditions should be carefully adjusted according to the final clinical application.

The effect of photobiomodulation on human dental pulp-derived stem cells: systematic review.

This systematic review assessed if photobiomodulation of human dental pulp tissue improved cell viability, proliferation, and/or differentiation compared with a placebo. This systematic review was conducted in line with PRISMA. PICO question was established; inclusion and exclusion criteria were established before a search had begun. A literature search was conducted through PubMed, Scopus, and Cochrane. Studies were included if published within the last 20years in English language, or where translation was available; laser parameters were mentioned; human dental pulp tissue was studied in vitro. Studies were excluded if non-human dental pulp tissue was studied and where the study was an in vivo study. Out of the total 121 studies found, 109 were excluded. Of the twelve included studies, three full-text articles were not available despite attempts made to contact the respective authors, leaving nine studies. Four of the included studies reported the use of stem cells derived from human deciduous teeth (SHEDs), and five used those from human permanent teeth (DPSCs). Most included studies utilized InGaAlP laser with wavelengths 660nm, and one study with 610nm. Other types of lasers included LED InGaN, and GaAlAs. Out of all included studies, two had a moderate risk of bias, and the rest had a low risk of bias. All studies confirmed positive effects on proliferation. One study also found improved osteogenic differentiation of the stem cells derived from stem cells of deciduous teeth. After assessing SHEDs and DPSCs separately, it is found that photobiomodulation improved cell proliferation in both subgroups. Due to heterogeneity in design protocols and laser parameters, it was not possible to compare the studies together. However, this study indicated that cell viability and proliferation did improve with photobiomodulation.

Functional glucosamine-iron oxide nanocarriers

Abstract