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Objective: Calcium silicate cements (CSCs) are often used in endodontics despite some limitations related to their physical properties and antibacterial efficacy. This study aimed to develop and demonstrate the viability of a series of CSCs that were produced by sol-gel method and further modified with mesoporous bioactive glass nanoparticles (MBGNs) and collagen, for endodontic therapy., Methods: Calcium silicate (CS) particles and MBGNs were synthesized by the sol-gel method, and their elemental, molecular, and physical microstructure was characterized. Three CSCs were developed by mixing the CS with distilled water (CS+H 2 O), 10 mg/mL collagen solution (CS+colH 2 O), and MBGNs (10 %) (CSmbgn+colH 2 O). The mixing (MT) and setting (ST) times of the CSCs were determined, while the setting reaction was monitored in real-time. Antibacterial efficacy against Enterococcus faecalis (E. faecalis) and regenerative potential on dental pulp stem cells (DPSCs) were also analyzed., Results: The CS+H 2 O displayed a ST comparable to commercial products, while CSmbgn+colH 2 O achieved the longest MT of 68 s and the shortest ST of 8 min. All the experimental CSCs inhibited the growth of E. faecalis. Additionally, compared to the control group, CSCs supported cell proliferation and spreading and mineralized matrix production, regardless of their composition., Significance: Tested CSCs presented potential as candidates for pulp therapy procedures. Future research should investigate the pulp regeneration mechanisms alongside rigorous antibacterial evaluations, preferably with multi-organism biofilms, executed over extended periods., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Objectives: To investigate the cytotoxicity on human dental pulp cells (HDPCs) and Streptococcus mutans (S.mutans) biofilm formation on universal resin composite cements (UCs)., Methods: Three UCs (RelyX Universal, 3 M Oral Care - RXU; Panavia SA Cement Universal, Kuraray Noritake - PSAU; SoloCem, Coltene - SCM) and one 'gold-standard' multi-step cement (Panavia V5, Kuraray Noritake - PV5) were used following two polymerization protocols (light-cured - LC; self-cured - SC). Cytotoxicity (MTT) tests were performed after 1, 3 and 7 days of direct contact. Carboxy-2',7'-dichlorodihydrofluorescein diacetate was used to detect the release of reactive oxygen species (ROS), and interleukin 6 (IL-6) expression was analyzed by IL-6 proquantum high sensitivity immunoassay. S. mutans biofilms were grown on UCs samples in a bioreactor for 24 h, then adherent viable biomass was assessed using MTT assay. For microbiological procedures, half of UCs samples underwent accelerated aging. Data were statistically analyzed (α = 0.05)., Results: The highest cytotoxicity was observed for PSAU SC, RXU SC, and PV5 SC at day 1, then for SC RXU after 3 days, and SC PSAU, LC PV5 and SCM after 1-week (p < 0.05). There was no increase in IL-6 expression after 1 day, while it increased depending on the group at 3 and 7 days. The highest ROS expression after 12 h was recorded for PSAU SC, PV5 SC and PV5 LC. Biofilm formation was as follows: RXU > > PSAU = PV5 > SCM, while light-curing systematically decreased biofilm formation (≈-33 %). Aging leveled out differences between UCs and between polymerization protocols., Significance: The choice of cement brand, rather than category, and polymerization protocol influence cell viability and microbiological behavior. Light-curing is beneficial for reducing the harmful pulpal effect that UCs may possess., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest. Uros Josic acknowledges Academy of Dental Materials Early Investigator Award., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Objectives: To evaluate the effects of dentin biomodification agents (Proanthocyanidin (PAC), Cardol (CD) and Cardol-methacrylate (CDMA) on dentin hydrophilicity by contact angle measurement, viability of dental pulp stem cells (DPSCs) and nanomechanical properties of the hybrid layer (HL)., Methods: CDMA monomer was synthesized from cardol through methacrylic acid esterification. Human extracted third molars were used for all experiments. For nanomechanical tests, specimens were divided in four groups according to the primer solutions (CD, CDMA, PAC and control) were applied before adhesive and composite coating. Nanomechanical properties of the HL were analyzed by nanoindentation test using a Berkovich probe in a nanoindenter. Wettability test was performed on dentin surfaces after 1 min biomodification and measured by contact angle analysis. Cytotoxicity was assessed by a MTT assay with DPSCs after 48 and 72 h. Data were analyzed with Student's t test or Two-way ANOVA and Tukey HSD test (p < 0.05)., Results: CD and CDMA solutions achieved greater hydrophobicity and increased the water-surface contact angles when compared to PAC and control groups (p < 0.05). PAC group showed a greater reduction of elastic modulus in nanoindentation experiments when compared to CD and CDMA groups (p < 0.05) after 4 months of aging. CD inhibited cell proliferation compared to all further materials (p < 0.05), whilst CDMA and PAC indicated no cell cytotoxicity to human DPSCs., Significance: Cardol-methacrylate provided significantly higher hydrophobicity to dentin and demonstrated remarkable potential as collagen crosslinking, attaining the lowest decrease of HL's mechanical properties. Furthermore, such monomer did not affect pulp cytotoxicity, thereby highlighting promising feasibility for clinical applications., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Objective: Drug-loaded non-resorbable polymeric nanoparticles (NPs) are proposed as an adjunctive treatment for pulp regenerative strategies. The present in vitro investigation aimed to evaluate the effectiveness of tideglusib-doped nanoparticles (TDg-NPs) in mitigating the adverse effects of bacterial lipopolysaccharide endotoxin (LPS) on the viability, morphology, migration, differentiation and mineralization potential of human dental pulp stem cells (hDPSCs)., Methods: Cell viability, proliferation, and differentiation were assessed using a MTT assay, cell migration evaluation, cell cytoskeleton staining analysis, Alizarin Red S staining and expression of the odontogenic related genes by a real-time quantitative polymerase chain reaction (RT-qPCR) were also performed. Cells were tested both with and without stimulation with LPS at various time points. One-way ANOVA and Tukey's test were employed for statistical analysis (p < 0.05)., Results: Adequate cell viability was encountered in all groups and at every tested time point (24, 48, 72 and 168 h), without differences among the groups (p > 0.05). The analysis of cell cytoskeleton showed nuclear alteration in cultures with undoped NPs after LPS stimulation. These cells exhibited an in blue diffuse and multifocal appearance. Some nuclei looked fragmented and condensed. hDPSCs after LPS stimulation but in the presence of TDg-NPs exhibited less nuclei changes. LPS induced down-regulation of Alkaline phosphatase, Osteonectin and Collagen1 gene markers, after 21d. LPS half-reduced the cells production of calcium deposits in all groups (p < 0.05), except in the group with TDg-NPs (decrease about 10 %)., Significance: LPS induced lower mineral deposition and cytoskeletal disorganization in hDPSCs. These effects were counteracted by TDg-NPs, enhancing osteogenic differentiation and mineralization., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2024. Published by Elsevier Inc.)

Objectives: The aim of this study was to assess the cytotoxicity of novel polymerization co-initiators and their effect on cytokine release from human dental pulp stem cells (hDPSCs), comparing them with commonly used co-initiators., Methods: Cells were isolated from the dental pulp of healthy human third molars. The new co-initiators, namely HDa1, HD4, HD1, and MHPTm, were evaluated and compared with the compounds dimethylaminoethyl amine benzoate (EDAB) and 2-(dimethylamino)ethyl methacrylate (DMAEMA). These compounds were diluted in dimethylsulfoxide (DMSO) at concentrations ranging from 1 to 8 mM. hDPSCs were seeded onto 96-well plates and incubated for 48 h. Subsequently, the cells were exposed to different concentrations of the co-initiators mentioned for 24 h. After this period, the culture medium was removed, and mitochondrial metabolism was evaluated using the MTT assay, while cytokine release (IL-1β, IL-6, IL-8, IL-10, TNF-α) was analyzed by the MAGPIX assay. Cells without exposure to the tested compounds served as controls. The data were analyzed using one-way ANOVA and Tukey's test., Results: The compounds showed low toxicity, with 8 mM concentration causing the most significant reduction in mitochondrial metabolism. MHPTm was the most toxic co-initiator tested (compound bearing an amine functionality). All compounds up-regulated TNF-α, IL-10, IL-6, and IL-8, with HD4 exhibiting the most pronounced increase in IL-6 and IL-8., Significance: The newly proposed co-initiators demonstrated reduced impact on mitochondrial metabolism, comparable to some traditional co-initiators. Despite their lower toxicity, HD4 increased IL-6 and IL-8 release, suggesting its potential involvement in triggering an inflammatory reaction, particularly in the short term., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Objectives: Evaluate a new light-cured material with better properties for vital pulp therapy., Methods: Light-cured resin materials consisted of polyethylene glycol (600) diacrylate mixed with different ratios of TCP to HA. In addition to the temperature change (n = 5 for each subgroup) were tested, cell viability and Alizarin Red Staining (ARS) assay were also tested in vitro on human dental pulp cells (n = 6 for each subgroup). Lastly, the material was then compared with Biodentine and control groups in the molars of Wistar rats in vivo for histology assessment., Results: The temperature change for the new materials were under 5 degrees Celsius. For the in vitro assessments, there was no significant difference on day 3 and day 7 for cell viability test. ARS assay showed significantly higher mineralized nodule formation when treated without induction medium for Group D and Biodentine on day 10 compared to Group C and control. On the contrary, Biodentine and control groups treated with induction medium showed significant higher mineralization than the new materials. Histology assessments demonstrated higher mineralized content in Group D and Biodentine on week 3 and week 6. The inflammatory cells in the dental pulp complex of the Biodentine group resolved on week 6 while the inflammation resolved in Group D on week 3., Significance: The new material exhibits low heat production, low cytotoxicity, and good calcium ion release capability. Compared to traditional materials, it has shorter setting time and better aesthetic outcomes, making it highly suitable for use in vital pulp therapy., Competing Interests: Declaration of Competing Interest To the best of our knowledge, all authors have no conflict of interest, financial or otherwise., (Copyright © 2024. Published by Elsevier Inc.)

Objectives: The purpose of this study was evaluating the performance of new Bis-EFMA based bulk-fill composites with common methacrylate based composites and commercial dental composites., Methods: The Bis-EFMA monomer was synthesized and the novel Bis-EFMA based bulk-fill composites were prepared. The resin composite samples were co-cultured with human gingival epithelial cells and human dental pulp stem cells to test the biocompatibility. The edge adaptation was observed under a combination of stereoscope and scanning electron microscope. The internal hardness was measured using a Vickers microhardness tester after one-time filling of cavities prepared in extracted teeth. After friction and wear test on the surface of the resin composites, the surface morphology and volume wear of each group were measured by the optical profilometer. The color stability was measured by a colorimeter., Results: Direct contact with human gingival epithelial cells and human dental pulp stem cells did not cause significant changes in their growth density and morphology, indicating good biocompatibility of Bis-EFMA group (p > 0.05). The continuous margin proportion of the Bis-EFMA group was as good as commercial bulk-fill composites (p > 0.05). The sectional microhardness results showed that the Bis-EFMA group had the highest microhardness. After the friction and wear test, the volume wear of the Bis-EFMA group was minimal, indicating its good wear resistance and mechanical strength. Color changes in all resin groups after 28 days of immersion were within the clinically acceptable range., Significance: The addition of Bis-EFMA demonstrated excellent biocompatibility, edge adaptation and color stability comparable to commonly used clinical bulk-fill composites, along with preferable mechanical strength, friction and wear resistance. Bis-EFMA based bulk-fill composites have the potential to be employed as a bulk filling material in commercial dental composite applications., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Objectives: Silicon-releasing biomaterials are widely used in the field of dentistry. However, unlike bone, very little is known about the role of silicon on dental tissue formation and repair. This study investigates the influence of silicic acid on the survival, differentiation and mineralizing ability of human dental pulp stem cells (hDPSCs) in 3D pulp-like environments METHODS: Dense type I collagen hydrogels seeded with hDPSCs were cultured over 4 weeks in the presence of silicic acid at physiological (10 μM) and supraphysiological (100 μM) concentrations. Cell viability and proliferation were studied by Alamar Blue and live/dead staining. The collagen network was investigated using second harmonic generation imaging. Mineral deposition was monitored by histology and scanning electron microscopy. Gene expression of mineralization- and matrix remodeling-associated proteins was studied by qPCR., Results: Presence of silicic acid did not show any significant influence on cell survival, metabolic activity and gene expression of key mineralization-related proteins (ALP, OCN, BSP). However, it induced enhanced cell clustering and delayed expression of matrix remodeling-associated proteins (MMP13, Col I). OPN expression and mineral deposition were inhibited at 100 μM. It could be inferred that silicic acid has no direct cellular effect but rather interacts with the collagen network, leading to a modification of the cell-matrix interface., Significance: Our results offer advanced insights on the possible role of silicic acid, as released by pulp capping calcium silicates biomaterials, in reparative dentine formation. More globally, these results interrogate the possible role of Si in pulp pathophysiology., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Growth differentiation factor 11 (GDF11) is considered to be a potential molecular target for treating pulpitis. However, whether GDF11 regulates osteogenic/odontogenic differentiation of dental pulp stem cells (DPSCs) to mediate pulpitis process remains unclear., Methods: Lipopolysaccharide (LPS) was used to induce inflammation conditions in DPSCs. The levels of GDF11, sirtuin 3 (SIRT3), forkhead box O-3 (FOXO3), osteogenic/odontogenic differentiation-related markers were measured by quantitative real-time PCR (qRT-PCR) and western blot (WB). Immunofluorescence staining was used to measure mitophagy. Mitophagy-related proteins were analyzed by WB, and the levels of inflammation factors were examined using qRT-PCR, ELISA and immunohistochemistry. Alkaline phosphatase activity and alizarin red S intensity were evaluated to assess osteogenic differentiation. Acute pulp (AP) injury rat model was constructed to study the role of oe-GDF11 in vivo., Results: GDF11 was downregulated in LPS-induced DPSCs, and LPS suppressed osteogenic/odontogenic differentiation and mitophagy. GDF11 overexpression promoted osteogenic/odontogenic differentiation in DPSCs through the activation of mitophagy. Furthermore, GDF11 upregulated SIRT3 to enhance FOXO3 expression by inhibiting its acetylation. GDF11 ameliorated LPS-induced inflammation and promoted osteogenic/odontogenic differentiation in DPSCs via enhancing SIRT3/FOXO3-mediated mitophagy. Besides, GDF11 overexpression suppressed inflammation and promoted dentin repair in AP rat models., Conclusion: GDF11 promoted SIRT3/FOXO3-mediated mitophagy to accelerate osteogenic/odontogenic differentiation in DPSCs, providing a novel target for pulpitis treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objectives: This study aimed to assess antimicrobial efficacy, cytotoxicity, and cytokine release (IL-1b, IL-6, IL-10, TNF-α) from human dental pulp stem cells (hDPSCs) of chitosan (CH) and hydroxyapatite (HAp)-modified glass ionomer cements (GIC)., Methods: GICs with varied CH and HAp concentrations (0 %, 0.16 %, 2 %, 5 %, 10 %) were tested against S. mutans for 24 h or 7 days. Antimicrobial activity was measured using an MTT test. Cytotoxicity evaluation followed for optimal concentrations, analyzing mitochondrial activity and apoptosis in hDPSCs. Cytokine release was assessed with MAGPIX. Antimicrobial analysis used Shapiro-Wilk, Kruskal-Wallis, and Dunnett tests. Two-way ANOVA, Tukey, and Dunnett tests were applied for hDP metabolism and cytokine release., Results: CH 2 % and HAp 5 % significantly enhanced GIC antimicrobial activity, especially after seven days. In immediate analysis, all materials showed reduced mitochondrial activity compared to the control. After 24 h, CH demonstrated mitochondrial metabolism similar to the control. All groups exhibited mild cytotoxicity (∼30 % cell death). Only IL-6 was influenced, with reduced release in experimental groups., Significance: CH 2 % and HAp 5 % were most effective for antibacterial effects. GIC-CH 2 % emerged as the most promising formula, displaying significant antibacterial effects with reduced hDPSC toxicity., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Objective: to compare conventional nanohybrid (Ceram.x Spectra) and ormocer-based (Admira fusion) dental composite resins effects on human dental pulp stem cells (hDPSCs) in terms of cytotoxicity, self-renewal, migration and osteogenic differentiation., Methods: hDPSCs were cultured in presence of different dilutions (undiluted, form 1:2 to 1:100) of CeramX (CX) and Admira fusion (AD) eluates and viability assay in standard or osteogenic conditions were performed. Samples and eluates were prepared according to ISO 10993-12. In addition, apoptosis, self-renewal and migration activity evaluations were carried out. Osteogenic differentiation potential was tested by Alkaline Phosphatase Activity, alizarin red staining and gene expression of specific markers (ALP, RUNX2, OCN, OPN and COL1α1). Statistical analysis was performed by means of a One-way analysis of variance (One-way ANOVA) followed by a Tukey's test for multiple comparison; results were presented as mean ± standard error of mean (SEM)., Results: Admira Fusion demonstrated to be highly biocompatible and showed positive effects on hDPSCs proliferation and differentiation; on the contrary, conventional nanohybrid composite showed to be more cytotoxic and without any notable effect on stem cells differentiation. Moreover, the obtained results were further corroborated by a significant upregulation of osteogenic differentiation markers obtained in presence of ormocer-based composite resin eluate. Specifically, in AD 1:50 group expression levels of ALP, Runx2, Col1α1 were double than control (ALP, p = 0.045; Runx2, p = 0.003; Col1α1, p = 0.001) and CX 1:50 (ALP, p = 0.006; RUNX2, p = 0.029; Col1α1, p = 0.005). Moreover, in the same group, OPN and OCN resulted about 5 times more expressed as compared to control (OPN, p = 0.009; OCN, p = 0.0005) and CX 1:50 (OPN, p = 0.012; OCN, p = 0.0006)., Significance: The less cytotoxicity obtained by AD than conventional nanohybrid composite may be attributed to a reduced monomers release in the oral environment, supporting the hypothesis of limited adverse effect and enhanced healing potential, mainly when the material is positioned in close contact with pulp tissue., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Objectives: The biological responses of MTA and Biodentine™ has been assessed on a three-dimensional, tissue-engineered organotypic deciduous pulp analogue., Methods: Human endothelial (HUVEC) and dental mesenchymal stem cells (SHED) at a ratio of 3:1, were incorporated into a collagen I/fibrin hydrogel; succeeding Biodentine™ and MTA cylindrical specimens were placed in direct contact with the pulp analogue 48 h later. Cell viability/proliferation and morphology were evaluated through live/dead staining, MTT assay and Scanning Electron Microscopy (SEM), and expression of angiogenic, odontogenic markers through real time PCR., Results: Viable cells dominated at day 3 after treatment presenting typical morphology, firmly attached within the hydrogel structures, as shown by live/dead staining and SEM images. MTT assay at day 1 presented a significant increase of cell proliferation in Biodentine™ group. Real-time PCR showed significant upregulation of odontogenic markers DSPP, BMP-2 (day 3,6), RUNX2, ALP (day 3) in contact with Biodentine™ compared to MTA and the control, whereas MTA promoted significant upregulation of DSPP, BMP-2, RUNX2, Osterix (day 3) and ALP (day 6) compared to the control. MSX1 presented downregulation in both experimental groups. Expression of angiogenic markers VEGFa and ANGPT-1 at day 3 was significantly upregulated in contact with Biodentine™ and MTA respectively, while the receptors VEGFR1, VEGFR2 and Tie-2, as well as PECAM-1 were downregulated., Significance: Both calcium silicate-based materials are biocompatible and exert positive angiogenic and odontogenic effects, although Biodentine™ during the first days of culture, seems to induce higher cell proliferation and provoke a more profound odontogenic and angiogenic response from SHED., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Objectives: to assess the impact of universal adhesives, cured with single-peak and polywave LEDs, on the metabolic activity and cytokine release of human dental pulp stem cells (hDPSCs). In addition, analyze the degree of conversion (DC) of the adhesives cured with the different LEDs., Methods: Discs (5 mm diameter, 1 mm thick) were prepared using three universal adhesives: Single Bond Universal (SBU, 3 M ESPE), Optibond Universal (OBU, Kerr), and Zipbond Universal (ZBU, SDI). These discs were cured for 40 s using a single-peak (DeepCure, 3 M ESPE) or a polywave light-emmiting diode (LED) curing unit (Valo Grand, Ultradent). After 24 h, the specimens were placed in 24-well culture plates, each containing 1 mL of culture medium for 24 h. hDPSCs (1.8 ×10 4 ) were seeded in 96-well plates and allowed to grow for 24 h. Subsequently, the cells were exposed to the extracts (culture medium containing eluates from the adhesive discs) for an additional 24 h. Cells not exposed to the extracts were used as a control group. The mitochondrial metabolism was assessed using the MTT assay and the cytokine release evaluated through MAGPIX. The degree of conversion of the adhesives was analyzed using FTIR (n = 5). The results were analyzed by ANOVA two-way and Tukey's test., Results: OBU and ZBU eluates caused a statistically significant reduction in mitochondrial metabolism, regardless of the LED used, indicating their cytotoxicity. In contrast, SBU did not significantly affect the MTT results, resembling the control group. A higher release of cytokines IL-1, IL-6, IL-10, and TNF-α were found in association to ZBU. SBU, on the other hand, increased the release of IL-8. OBU did not influenced the cytokine release. SBU presented the higher DC, while OBU and ZBU had similar DC, lower than SBU., Significance: In conclusion, universal adhesives exhibit toxicity towards hDPSCs, but the extent of toxicity varies depending on the adhesive material. ZBU was associated with increased cytokine release, particularly pro-inflammatory mediators, from hDPSCs. The different LEDs did not influenced the cytotoxicity of the evaluated adhesives., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Damage to somatosensory "barrel" cortex reduces the rats' behavioral sensitivity in discrimination of tactile stimuli. Here, we examined how transplantation of stem cells into the lesioned barrel cortex can help in recovery of sensory capacities. We induced mechanical lesions in the right barrel cortex area of male rats. Three days after lesioning, rats received one of three transplantation types: un-differentiated dental pulp stem cells (U-DPSCs) or differentiated dental pulp stem cells (D-DPSCs), or cell medium (vehicle). A fourth group of rats were control without any Surgery. For 4 consecutive weeks, starting one week after transplantation, we evaluated the rats' preference to explore novel textures as a measure of sensory discrimination ability, also measured the expression of glial fibrillary acidic protein (GFAP), Olig 2, nestin, neuronal nuclei (NeuN), brain-derived neurotrophic factor (BDNF) and neuroligin1 by immunohistochemistry and western blotting. Unilateral mechanical lesion decreased the rats' preferential exploration of novel textures compared to the control group across the 4-week behavioral tests. Following stem cell therapy, the rats' performance significantly improved at week 2-4 compared to the vehicle group. Compared to the control group, there was a significant decrease in the expression of nestin, NeuN, Olig 2, BDNF, neuroligin1 and a significant increase in the expression of GFAP in the vehicle group. The expression of the neural markers was significantly higher in DPSCs compared with the vehicle group whereas GFAP level was lower in DPSCs compared to vehicle. We found that DPSCs therapy affected a range of neuronal markers in the barrel cortex post lesion, and improved the rats' recovery for sensory discrimination., (Copyright © 2021. Published by Elsevier Inc.)

Stem cell therapy provides a ray of hope for treating neurodegenerative diseases (ND). Bone marrow mesenchymal stem cells (BM-MSC) were extensively investigated for their role in neuroregeneration. However, drawbacks like painful bone marrow extraction, less proliferation and poor CNS engraftment following systemic injections of BM-MSC prompt us to search for alternate/appropriate source of MSC for treating ND. In this context, dental pulp stem cells (DPSC) could be an alternative to BM-MSC as it possess both mesenchymal and neural characteristic features due to its origin from ectoderm, ease of isolation, higher proliferation index and better neuroprotection. A study on the migration potential of DPSC compared to BM-MSC in a neurodegenerative condition is warranted. Given the neural crest origin, we hypothesize that DPSC possess better migration towards neurodegenerative milieu as compared to BM-MSC. In this prospect, we investigated the migration potential of DPSC in an in vitro neurodegenerative condition. Towards this, transwell, Matrigel and chorioallantoic membrane (CAM) migration assays were carried-out by seeding hippocampal neurons in the lower chamber and treated with 300 μM kainic acid (KA) for 6 h to induce neurodegeneration. Subsequently, the upper chamber of transwell was loaded with DPSC/BM-MSC and their migration potential was assessed following 24 h of incubation. Our results revealed that the migration potential of DPSC/BM-MSC was comparable in non-degenerative condition. However, following injury the migration potential of DPSC towards the degenerating site was significantly higher as compared to BM-MSC. Furthermore, upon exposure of naïve DPSC/BM-MSCs to culture medium derived from neurodegenerative milieu resulted in significant upregulation of homing factors like SDF-1alpha, CXCR-4, VCAM-1, VLA-4, CD44, MMP-2 suggesting that the superior migration potential of DPSC might be due to prompt expression of homing factors in DPSC compared to BM-MSCs., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Diabetes mellitus is one of the most common health threatening disorders. Patients with chronic diabetes are at high risk of contracting oral diseases, including dental pulp damage. In this study, we reviewed how Teneligliptin, a commonly used anti-diabetic agent, protected dental pulp cells from lipopolysaccharide (LPS)-induced cytotoxicity and improved their viability. The dental pulp cells treated with Teneligliptin were resistant to LPS-induced reactive oxygen species (ROS) and its byproduct 4-hydroxynonenal (4-HNE) generation. The Teneligliptin recovered LPS-induced a reduction of cellular glutathione and produced cytokine including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). Mechanistically, we found that Teneligliptin suppressed LPS- that caused an expression of the cell surface receptor toll like receptor 4 (TLR-4) and the activation of JNK kinase and activator protein 1 (AP1) as well as the nuclear factor-κB (NF-κB) signal pathways. Collectively, our study demonstrates that the molecular mechanism Teneligliptin is a protective anti-diabetic agent in dental pulp cells and it has the potential to treat diabetes-associated dental pulp diseases., (Copyright © 2019. Published by Elsevier B.V.)

Current saliva-based stimulation therapies for radiotherapy-induced xerostomia are not fully effective due to the presence of damaged secretory epithelia and nerves in the salivary gland (SG). Hence, three-dimensional bio-engineered organoids are essential to regenerate the damaged SG. Herein, a recently validated three-dimensional (3D) biofabrication system, the magnetic 3D bioprinting (M3DB), is tested to generate innervated secretory epithelial organoids from a neural crest-derived mesenchymal stem cell, the human dental pulp stem cell (hDPSC). Cells are tagged with magnetic nanoparticles (MNP) and spatially arranged with magnet dots to generate 3D spheroids. Next, a SG epithelial differentiation stage was completed with fibroblast growth factor 10 (4-400 ng/ml) to recapitulate SG epithelial morphogenesis and neurogenesis. The SG organoids were then transplanted into ex vivo model to evaluate their epithelial growth and innervation. M3DB-formed spheroids exhibited both high cell viability rate (>90%) and stable ATP intracellular activity compared to MNP-free spheroids. After differentiation, spheroids expressed SG epithelial compartments including secretory epithelial, ductal, myoepithelial, and neuronal. Fabricated organoids also produced salivary α-amylase upon FGF10 stimulation, and intracellular calcium mobilization and trans-epithelial resistance was elicited upon neurostimulation with different neurotransmitters. After transplantation, the SG-like organoids significantly stimulated epithelial and neuronal growth in damaged SG. It is the first time bio-functional innervated SG-like organoids are bioprinted. Thus, this is an important step towards SG regeneration and the treatment of radiotherapy-induced xerostomia., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Objective: Collagen fibrils aid in anchoring resin composite restorations to the dentine substrate. The aim of the study was to investigate effect of non-enzymatic glycation on bond strength and durability of demineralized dentine specimens in a modified two-step etch-and-rinse dentine adhesive., Methods: Dentine surfaces were etched with 37% phosphoric acid, bonded with respective in vitro ethanol and acetone adhesives modified with (m/m, 0, 1%, 2% and 3% ribose), restored with restorative composite-resin, and sectioned into resin-dentine slabs and beams to be stored for 24h or 12 months in artificial saliva. Bond-strength testing was performed with bond failure analysis. Pentosidine assay was performed on demineralized ribose modified dentine specimens with HPLC sensitive fluorescent detection. The structural variations of ribose-modified dentine were analysed using TEM and human dental pulpal cells were used for cell viability. Three-point bending test of ribose-modified dentine beams were performed and depth of penetration of adhesives evaluated with micro-Raman spectroscopy. The MMP-2 and cathepsin K activities in ribose-treated dentine powder were also quantified using ELISA. Bond strength data was expressed using two-way ANOVA followed by Tukey's test. Paired T tests were used to analyse the specimens for pentosidine crosslinks. The modulus of elasticity and dentinal MMP-2 and cathepsin K concentrations was separately analyzed using one-way ANOVA., Results: The incorporation of RB in the experimental two-step etch-and-rinse adhesive at 1% improved the adhesive bond strength without adversely affecting the degree of polymerisation. The newly developed adhesive increases the resistance of dentine collagen to degradation by inhibiting endogenous matrix metalloproteinases and cysteine cathepsins. The application of RB to acid-etched dentine helps maintain the mechanical properties., Significance: The incorporation of 1%RB can be considered as a potential candidate stabilizing resin dentine bond., (Copyright © 2018 The Academy of Dental Materials. Published by Elsevier Inc. All rights reserved.)

Objective: To evaluate the in vitro cytotoxicity of Equia Forte (GC, Tokyo, Japan) and Ionostar Molar (Voco, Cuxhaven, Germany) on human dental pulp stem cells (hDPSCs)., Methods: hDPSCs isolated from third molars were exposed to several dilutions of Equia Forte and Ionostar Molar eluates (1/1, 1/2 and 1/4). These eluates were obtained by storing material samples in respective cell culture medium for 24h (n=40). hDPSCs in basal growth culture medium were the control. Cell viability and cell migration assays were performed using the MTT and wound-healing assays, respectively. Also, induction of apoptosis and changes in cell phenotype were evaluated by flow cytometry. Changes in cell morphology were analysed by immunocytofluorescence staining. To evaluate cell attachment to the different materials, hDPSCs were directly seeded onto the material surfaces and analyzed by scanning electron microscopy (SEM). The chemical composition of the materials was determined by energy dispersive X-ray (EDX) and eluates were analyzed by inductively coupled plasma-mass spectrometry (ICP-MS). Statistical analysis was performed with analysis of variance (ANOVA) and Student's t-test (α<0.05)., Results: Undiluted Equia Forte extracts led to a similar cell proliferation rates than the control group from 72h onwards. There were no significance differences between Equia Forte and Ionostar Molar in terms of cell apoptosis and phenotype. However, in presence of Equia extracts the migration capacity of hDPSCs was higher than in presence of Ionostar Molar (p<0.05). Also, SEM studies showed a higher degree of cell attachment when Equia Forte extracts were used. Finally, EDX analysis pointed to different weight percentages of C, O and Ca ions in glass ionomer cements, while other elements such as La, Al, Si, W, Mo and F were also detected., Significance: In summary, Equia Forte promoted better biological responses in hDPSCs than Ionostar Molar., (Copyright © 2018 The Academy of Dental Materials. Published by Elsevier Inc. All rights reserved.)

Objectives: To evaluate the effect of the eluates from 3 freshly-mixed and setting hydraulic calcium-silicate cements (hCSCs) on human dental pulp cells (HDPCs) and to examine the effect of a newly developed hCSC containing phosphopullulan (PPL) on HDPCs., Methods: Human dental pulp cells, previously characterized as mesenchymal stem cells, were used. To collect the eluates, disks occupying the whole surface of a 12-well plate were prepared using an experimental hCSC containing phosphopullulan (GC), Nex-Cem MTA (GC), Biodentine (Septodont) or a zinc-oxide (ZnO) eugenol cement (material-related negative control). Immediately after preparing the disks (non-set), 3ml of Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) were added. The medium was left in contact with the disks for 24h before being collected. Four different dilutions were prepared (100%, 50%, 25% and 10%) and cell-cytotoxicity, cell-proliferation, cell-migration and odontogenic differentiation were tested. The cell-cytotoxicity and cell-proliferation assays were performed by XTT-colorimetric assay at different time points. The cell-migration ability was tested with the wound-healing assay and the odontogenic differentiation capacity of hCSCs on HDPCs was tested with RT-PCR., Results: Considering all experimental data together, the eluates from 3 freshly-mixed and setting hCSCs appeared not cytotoxic toward HDPCs. Moreover, all three cements stimulated proliferation, migration and odontogenic differentiation of HDPCs., Significance: The use of freshly-mixed and setting hCSCs is an appropriate approach to test the effect of the materials on human dental pulp cells. The experimental material containing PPL is non-cytotoxic and positively stimulates HDPCs., (Copyright © 2018 The Academy of Dental Materials. Published by Elsevier Inc. All rights reserved.)

Objective: Early responses of human pulp to Prime&Bond/phosphoric acid, Clearfil SE Bond, Clearfil S3 Bond and Dycal were investigated ex vivo., Materials and Methods: The three adhesives, Dycal or buffer (DPBS) were applied directly onto the pulp of human teeth slices that were placed in culture for 4 days. Cell viability was monitored by the MTT assay during the culture period. After 4 days, tissue integrity was examined by hematoxylin-eosin staining. Vimentin levels were assessed by Western blotting. TUNEL assay was applied for apoptotic cell detection at specific pulp areas., Results: Profound reduction of cell viability and tissue integrity was observed in adhesive-treated groups, while the impact of Dycal was found to be less harmful. Extended apoptosis was caused mostly by the Clearfil SE and Prime&Bond. All adhesives reduced Vimentin levels., Significance: The study provides evidence that early pulp responses to direct capping with different adhesive systems or calcium hydroxide may vary significantly and underline the need for further studies in relevant ex vivo systems., (Copyright © 2018 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.)

Objective: A systematic characterization of hybrid scaffolds, fabricated based on combinatorial additive manufacturing technique and freeze-drying method, is presented as a new platform for osteoblastic differentiation of dental pulp cells (DPCs)., Methods: The scaffolds were consisted of a collagenous matrix embedded in a 3D-printed beta-tricalcium phosphate (β-TCP) as the mineral phase. The developed construct design was intended to achieve mechanical robustness owing to 3D-printed β-TCP scaffold, and biologically active 3D cell culture matrix pertaining to the Collagen extracellular matrix. The β-TCP precursor formulations were investigated for their flow-ability at various temperatures, which optimized for fabrication of 3D printed scaffolds with interconnected porosity. The hybrid constructs were characterized by 3D laser scanning microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and compressive strength testing., Results: The in vitro characterization of scaffolds revealed that the hybrid β-TCP/Collagen constructs offer superior DPCs proliferation and alkaline phosphatase (ALP) activity compared to the 3D-printed β-TCP scaffold over three weeks. Moreover, it was found that the incorporation of TCP into the Collagen matrix improves the ALP activity., Significance: The presented results converge to suggest the developed 3D-printed β-TCP/Collagen hybrid constructs as a new platform for osteoblastic differentiation of DPCs for craniomaxillofacial bone regeneration., (Copyright © 2017. Published by Elsevier Ltd.)

Objective: Zinc oxide eugenol (ZOE) is widely used as a therapeutic dental restorative material. However, ZOE has poor mechanical properties and high cytotoxicity toward human dental pulp stem cells (hDPSCs) due to the release of Zn ions. In this study, zirconia-incorporated ZOE (ZZrOE) was developed to reduce the cytotoxicity and improve the mechanical properties of ZOE with sustained therapeutic effects on inflamed hDPSCs in terms of inflammatory gene expression levels compared with those of the original material., Methods: After the setting time and mechanical properties of ZZrOE incorporating varying amounts of zirconia (0, 5, 10, and 20wt% in powder) were characterized, the surface morphology and composition of the resulting ZZrOE materials were investigated. The ions and chemicals released into the cell culture medium from ZOE and ZZrOE (3cm 2 /mL) were measured by inductively coupled plasma atomic emission spectroscopy and gas chromatography, respectively. After testing cytotoxicity against hDPSCs using the above extracts, the therapeutic effects on lipopolysaccharide-inflamed hDPSCs in terms of compromising the upregulation of inflammatory response-related mRNA expression were tested using real-time PCR., Results: ZZrOE 20% exhibited increased compressive strength (∼45%), 3-point flexural strength (∼150%) and hardness (∼75%), as well as a similar setting time (∼90%), compared with those of ZOE. After the rough surface of ZZrOE was observed, significantly fewer released Zn ions and eugenol (∼40% of that from ZOE) were detected in ZZrOE 20%. ZZrOE showed less cytotoxicity because of the lower amount of Zn ions released from ZOE while showing sustained inhibition of inflammatory marker (e.g., interleukin 1β, 6 and 8) mRNA levels., Significance: The improved mechanical properties and cytocompatibility, as well as the sustained therapeutic effects on inflamed hDPSCs, were investigated in ZZrOE compared with those of ZOE. Therefore, ZZrOE has the potential to be used as an alternative to ZOE as a dental restorative material., (Copyright © 2017 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.)

Cinnamaldehyde (CA) has various functional properties, such as anti-cancer, anti-microbial, anti-inflammatory, and anti-oxidant activities. This study examined the intracellular signaling mechanisms of CA on the oxidative stress response in human dental pulp cells (hDPCs). The results showed that CA did not have any cell cytotoxicity or cause morphological changes at concentrations up to 50µM. A CA treatment strongly up-regulated the cellular protein level of heme oxygenase-1 (HO-1) and promoted Nrf2 translocation to the nucleus. CA-mediated Nrf2/HO-1 activation reduced the level of reactive oxygen species and protected the hDPCs from H 2 O 2 -induced oxidative stress, which induces apoptosis. Moreover, HO-1 depletion by siRNA attenuated the CA-mediated cell protection against oxidative stress. These results indicate that CA protects hDPCs dysfunction under oxidative stress conditions, and this effect is mediated by Nrf2 activation and the up-regulation of HO-1. Overall, these observations suggest that CA is a potential therapeutic agent for cell protection against oxidative stress., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Activation of the G protein-coupled calcium-sensing receptor (CaSR) has crucial roles in skeletal development and bone turnover. Our recent study has identified a role for activated CaSR in the osteogenic differentiation of human periodontal ligament stem cells. Furthermore, odontoblasts residing inside the tooth pulp chamber play a central role in dentin formation. However, it remains unclear how CaSR activation affects the odontoblastic differentiation of human dental pulp cells (HDPCs). We have investigated the odontoblastic differentiation of HDPCs exposed to elevated levels of extracellular calcium (Ca) and strontium (Sr), and the contribution of CaSR and the L-type voltage-dependent calcium channel (L-VDCC) to this process. Immunochemical staining of rat dental pulp tissue demonstrated that CaSR was expressed at high levels in the odontoblastic layer, moderate levels in the sublayer, and low levels in the central pulp tissue. Although normal HDPCs expressed low levels of CaSR, stimulation with Ca or Sr promoted both CaSR expression and odontoblastic differentiation of HDPCs along with increased expression of odontoblastic makers. These effects were inhibited by treatment with a CaSR antagonist, whereas treatment with an L-VDCC inhibitor had no effect. Additionally, knockdown of CaSR with siRNA suppressed odontoblastic differentiation of Ca- and Sr-treated HDPCs. ERK1/2 phosphorylation was observed in Ca- and Sr-treated HDPCs, whereas CaSR antagonist treatment or CaSR knockdown blocked ERK1/2 phosphorylation. Furthermore, inhibition of ERK1/2 suppressed mineralization of Ca- and Sr-treated HDPCs. These results suggest that elevated concentrations of extracellular Ca and Sr induce odontoblastic differentiation of HDPCs through CaSR activation and the ERK1/2 phosphorylation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Objective: This study investigated the mechanical and in vitro biological properties (in immortalized human dental pulp stem cells (ihDPSCs)) of bioactive glass nanoparticle (BGN)-incorporated glass ionomer cement (GIC) with or without chitosan as a binder., Methods: After the BGNs were synthesized and characterized, three experimental GICs and a control (conventional GIC) that differed in the additive incorporated into a commercial GIC liquid (Hy-bond, Shofu, Japan) were produced: BG5 (5wt% of BGNs), CL0.5 (0.5wt% of chitosan), and BG5+CL0.5 (5wt% of BGNs and 0.5wt% of chitosan). After the net setting time was determined, weight change and bioactivity were analyzed in simulated body fluid (SBF) at 37°C. Mechanical properties (compressive strength, diametral tensile strength, flexural strength and modulus) were measured according to the incubation time (up to 28 days) in SBF. Cytotoxicity (1day) and biomineralization (14 days), assessed by alizarin red staining, were investigated using an extract from GIC and ihDPSCs. Data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test; p<0.05., Results: BGNs were sol-gel synthesized to be approximately 42nm in diameter with a spherical morphology and amorphous structure. After the bioactivity and suspension ability of the BGNs were confirmed, all the experimental GIC groups had setting times of less than 6min and approximately 1% weight loss after 28days of incubation. In addition, BGNs incorporated into GIC (BG5 and BG5+CL0.5) exhibited surface bioactivity. The mechanical properties were increased in the BGN-incorporated GICs compared to those in the control (p<0.05). Without cytotoxicity, the biomineralization capacity was ranked in the order BG5, BG5+CL0.5, control, and CL0.5 (p<0.05)., Significance: BGN-incorporated GIC showed enhanced mechanical properties such as compressive, diametral tensile and flexural strength as well as in vitro biomineralization properties in ihDPSCs without cytotoxicity. Therefore, the developed BGN-incorporated GIC is a promising restorative dental material, although further in vivo investigation is needed before clinical application., (Copyright © 2017 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.)

Objective: The study assessed the cytotoxicity and immunomodulatory/anti-inflammatory effect of extract from zinc oxide-eugenol (ZOE)-based dental materials during setting using immortalized human dental pulp stem cells (IHDPSCs) and mouse bone marrow monocytes (IMBMMs), and identified the responsible extract component., Methods: In accord with the ISO 10993-12, we extracted a mixture of ZOE cement and sealer after a specified time. The extract was analyzed by two types of mass spectrometry (ICP-MS and GC-MS). Cell viability was evaluated with extract and serial concentrations of ZnCl 2 , ZnSO 4 , and eugenol liquid by WST assay. The immunomodulatory/anti-inflammatory effect of a ZOE component was determined by RT-PCR to detect the downregulatory effect of inflammatory mRNA expression after lipopolysaccharide (LPS)-induced inflammation., Results: Zn 2+ and eugenol (2-20ppm) were detected in the ZOE cement and sealer extracts. During the early stage of setting, significant cytotoxicity was observed in IHDPSCs and IMBMMs (p<0.05). The half maximal effective concentration of Zn 2+ was 5-8ppm, whereas that of eugenol could not be detected within 80ppm. After extract treatment, the expression of inflammatory mRNA was significantly lower in inflamed IHDPSCs, but not inflamed IMBMMs, than in the LPS control (p<0.05). However, eugenol, not Zn 2+ , at 5-20ppm downregulated inflammatory mRNA expression in the inflamed IMBMMs with and without the exchange of LPS-pretreated medium., Significance: ZOE was highly cytotoxic, especially during setting, to both cells due to Zn 2+ while the immunomodulatory/anti-inflammatory effect of ZOE was induced by eugenol., (Copyright © 2016 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.)

Objective: The objective was to investigate the potential of graphene (Gp) to induce odontogenic and osteogenic differentiation in dental pulp stem cells (DPSC)., Methods: Gp was produced by chemical vapor deposition. DPSC were seeded on Gp or glass (Gl). Cells were maintained in culture medium for 28 days. Every two days, culture medium from Gp was used to treat cells on Gl and vice versa. Mineralization and differentiation of DPSC on all substrates were evaluated after 14 and 28 days by alizarin red S staining, qPCR, immunofluorescence and FACS. Statistics were performed with two-way ANOVA and multiple comparisons were performed using Tukey's post hoc test at a pre-set significance level of 5%., Results: After 14 and 28 days, Gp induced higher levels of mineralization as compared to Gl. Odontoblastic genes (MSX-1, PAX and DMP) were down-regulated and osteogenic genes and proteins (RUNX2, COL and OCN) were significantly upregulated on Gp comparing to Gl (p<0.05 for all cases). Medium from Gp induced downregulation of odontoblastic genes and increased bone-related gene and protein on Gl., Significance: Graphene induced osteogenic and not odontoblastic differentiation of DPSC without the use of chemical inducers for osteogenesis. Graphene has the potential to be used as a substrate for craniofacial bone tissue engineering research., (Copyright © 2016 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.)

Achieving and maintaining safe and reliable lineage specific differentiation of stem cells is important for clinical translation of tissue engineering strategies. In an effort to circumvent the multitude of problems arising from the usage of growth factors and growth factor delivery systems, we have explored the use of exosomes as biomimetic tools to induce stem cell differentiation. Working on the hypothesis that cell-type specific exosomes can trigger lineage-specific differentiation of stem cells, we have evaluated the potential of exosomes derived from dental pulp cells cultured on under growth and odontogenic differentiation conditions to induce odontogenic differentiation of naïve human dental pulp stem cells (DPSCs) and human bone marrow derived stromal cells (HMSCs) in vitro and in vivo. Results indicate that the exosomes can bind to matrix proteins such as type I collagen and fibronectin enabling them to be tethered to biomaterials. The exosomes are endocytosed by both DPSCs and HMSCs in a dose-dependent and saturable manner via the caveolar endocytic mechanism and trigger the P38 mitogen activated protein kinase (MAPK) pathway. In addition, the exosomes also trigger the increased expression of genes required for odontogenic differentiation. When tested in vivo in a tooth root slice model with DPSCs, the exosomes triggered regeneration of dental pulp-like tissue. However, our results indicate that exosomes isolated under odontogenic conditions are better inducers of stem cell differentiation and tissue regeneration. Overall, our results highlight the potential exosomes as biomimetic tools to induce lineage specific differentiation of stem cells. Our results also show the importance of considering the source and state of exosome donor cells before a choice is made for therapeutic applications., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Objectives: An experimental discoloration-free calcium aluminosilicate cement has been developed with the intention of maximizing the beneficial attributes of tricalcium silicate cements and calcium aluminate cements. The present study examined the effects of this experimental cement (Quick-Set2) on the mineralogenic characteristics of osteogenic lineage-committed human dental pulp stem cells (hDPSCs), by comparing the cellular responses with a commercially available tricalcium silicate cement (white mineral trioxide aggregate (ProRoot(®) MTA); WMTA)., Methods: The osteogenic potential of hDPSCs exposed to the cements was examined using qRT-PCR for osteogenic gene expressions, Western blot for osteogenic-related protein expressions, alkaline phosphatase enzyme activity, Alizarin red S staining, Fourier transform infrared spectroscopy and transmission electron microscopy of extracellular calcium deposits., Results: Results of the six assays indicated that osteogenic differentiation of hDPSCs was significantly enhanced after exposure to the tricalcium silicate cement or the experimental calcium aluminosilicate cement, with the former demonstrating better mineralogenic stimulation capacity., Significance: The better osteogenic stimulating effect of the tricalcium silicate cement on hDPSCs may be due to its relatively higher silicate content, or higher OH(-) and Ca(2+) release. Further investigations with the use of in vivo animal models are required to validate the potential augmenting osteogenic effects of the experimental discoloration-free calcium aluminosilicate cement., (Published by Elsevier Ltd.)

Objective: The aim of this study was to evaluate the cytotoxicity and differentiation potential of a graphene oxide (GO)-based substrate using dental pulp stem cell (DPSC)., Methods: GO was obtained via chemical exfoliation of graphite using the modified Hummer's method and dispersed in water-methanol solution. 250μL of 1.5mg/mL solution were added to a cover slip and allowed to dry (25°C, 24h). GO-based substrate was characterized by Raman spectroscopy, AFM and contact angle. DPSC were seeded on GO and glass (control). Cell attachment and proliferation were evaluated by polymeric F-actin staining, SEM and MTS assay for five days. mRNA expression of MSX-1, PAX-9, RUNX2, COL I, DMP-1 and DSPP were evaluated by qPCR (7 and 14 days). Statistical analyses were performed by either Mann-Whitney, one or two-way Anova followed by and Tukey's post hoc analysis (α=0.05)., Results: Peaks at 1587cm(-1) and 1340cm(-1) (G and D band) and ID/IG of 0.83 were observed for GO with Raman. AFM showed that GO was randomly deposited and created a rougher surface comparing to the control. Cells successfully adhered on both substrates. There was no difference in cell proliferation after 5 days. Cells on GO presented higher expression for all genes tested except MSX-1 and RUNX2 for 7 days., Significance: GO-based substrate allowed DPSC attachment, proliferation and increased the expression of several genes that are upregulated in mineral-producing cells. These findings open opportunities to the use of GO alone or in combination with dental materials to improve their bioactivity and beyond., (Copyright © 2016 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.)

Objective: Acids, such as those used in adhesive dentistry, have been shown to solubilize bioactive molecules from dentin. These dentin matrix components (DMC) may promote cell proliferation and differentiation, and ultimately contribute to dentin regeneration. The objective of this study was to evaluate the potential for varying concentrations of DMC extracted from human dentin by phosphoric acid of a range of pHs to stimulate proliferation and mineralization of two different cultured pulp cell populations., Methods: DMC were solubilized from powdered human dentin (7 days - 4°C) by phosphoric acid of pH 1, 3, and 5 and also, EDTA. Extracts were dialyzed for 7 days against distilled water and lyophilized. Undifferentiated mouse dental pulp cells (OD-21) and cells of the odontoblast-like cell line (MDPC-23) were seeded in six-well plates (1×10(5)) and cultured for 24h in DMEM (Dulbecco's modified Eagle's medium) containing 10% (v/v) FBS (fetal bovine serum). The cells were washed with serum-free medium and then treated with different concentrations of DMC (0.01, 0.1, 1.0 and 10.0μg/ml) daily in serum free medium for 7 days. After 3, 5 (MDPC-23 only), and 7 days of treatment, cell proliferation was measured using 10vol% Alamar blue solution, which was added to each well for 1h. Cell numbers were first measured by cell counting (Trypan blue; n=5) and Alamar blue fluorescence to validate the assay, which was then used for the subsequent assessments of proliferation. Mineralization was assessed by Alizarin Red S assay after 12 days exposure to DMC (n=5). Controls were media-only (DMEM) and dexamethasone (DEX; positive control). Results were analysed by ANOVA/Tukey's (p≤0.05)., Results: There was a linear correlation between cell counts and Alamar blue fluorescence (R(2)>0.96 for both cell types) , verifying the validity of the Alamar blue assay for these cell types. In general, there was a dose-dependent trend for enhanced cell proliferation with higher concentration of DMC for both cell lines, especially at 10.0μg/ml. DEX exposure resulted in significantly higher mineralization, but did not affect cell proliferation. DMC exposure demonstrated significantly greater mineralization than media-only control for 10μg/ml for all extracts, and at lower concentrations for EDTA and pH 5 extracts., Significance: Human dentin matrix components solubilized by acids at pH levels found in commercial dentin adhesives enhanced cell proliferation and mineralization of mouse and rat undifferentiated dental pulp cells when presented in adequate concentration., (Copyright © 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.)

Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovial hyperplasia and chronic inflammation, which lead to the progressive destruction of cartilage and bone in the joints. Numerous studies have reported that administrations of various types of MSCs improve arthritis symptoms in animal models, by paracrine mechanisms. However, the therapeutic effects of the secreted factors alone, without the cell graft, have been uncertain. Here, we show that a single intravenous administration of serum-free conditioned medium (CM) from human deciduous dental pulp stem cells (SHED-CM) into anti-collagen type II antibody-induced arthritis (CAIA), a mouse model of rheumatoid arthritis (RA), markedly improved the arthritis symptoms and joint destruction. The therapeutic efficacy of SHED-CM was associated with an induction of anti-inflammatory M2 macrophages in the CAIA joints and the abrogation of RANKL expression. SHED-CM specifically depleted of an M2 macrophage inducer, the secreted ectodomain of sialic acid-binding Ig-like lectin-9 (ED-Siglec-9), exhibited a reduced ability to induce M2-related gene expression and attenuate CAIA. SHED-CM also inhibited the RANKL-induced osteoclastogenesis in vitro. Collectively, our findings suggest that SHED-CM provides multifaceted therapeutic effects for treating CAIA, including the ED-Siglec-9-dependent induction of M2 macrophage polarization and inhibition of osteoclastogenesis. Thus, SHED-CM may represent a novel anti-inflammatory and reparative therapy for RA., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objectives: In pulpal revascularization, a protective material is placed coronal to the blood clot to prevent recontamination and to facilitate osteogenic differentiation of mesenchymal stem cells to produce new dental tissues. Although mineral trioxide aggregate (MTA) has been the material of choice for clot protection, it is easily displaced into the clot during condensation. The present study evaluated the effects of recently introduced calcium silicate cements (Biodentine and TheraCal LC) on the viability and osteogenic differentiation of human dental pulp stem cells (hDPSCs) by comparing with MTA Angelus., Methods: Cell viability was assessed using XTT assay and flow cytometry. The osteogenic potential of hDPSCs exposed to calcium silicate cements was examined using qRT-PCR for osteogenic gene expressions, alkaline phosphatase enzyme activity, Alizarin red S staining and transmission electron microscopy of extracellular calcium deposits. Parametric statistical methods were employed for analyses of significant difference among groups, with α=0.05., Results: The cytotoxic effects of Biodentine and TheraCal LC on hDPSCs were time- and concentration-dependent. Osteogenic differentiation of hDPSCs was enhanced after exposure to Biodentine that was depleted of its cytotoxic components. This effect was less readily observed in hDPSCs exposed to TheraCal LC, although both cements supported extracellular mineralization better than the positive control (zinc oxide-eugenol-based cement)., Significance: A favorable tissue response is anticipated to occur with the use of Biodentine as a blood clot-protecting material for pulpal revascularization. Further investigations with the use of in vivo animal models are required to validate the potential adverse biological effects of TheraCal LC on hDPSCs., (Copyright © 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.)

Numerous studies have indicated dental pulp stem cells (DPSCs) potency to differentiate into several types of cell lineages. Oligodendrocyte lineage transcription factor 2 (OLIG2) plays an important role in the oligodendrogenic pathway. In this study, a tetracycline (Tet)-inducible system expressing OLIG2 gene was transfected into human DPSCs to direct their differentiation toward oligodendrocyte progenitor cells (OPCs). Following induction, the expression of stage-specific markers was studied by Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR), immunocytochemistry and western blotting. In the following, the cells were transplanted into the mouse model of local sciatic demyelination damage by lysolecithin. Recovery of lysolecithin-induced lesions in sciatic nerve was studied by treadmill exercise, von Frey filament test and hind paw withdrawal in response to a thermal stimulus. Improvement of behavioral symptoms was efficiently observed from the second week to the sixth week post-transplantation. Our findings showed that exogenous expression of the OLIG2 gene by a Tet-regulated system could be used as an efficient way to induce the differentiation of DPSCs into functional oligodendrocytes. Meanwhile, the DPSC-derived OPCs have relevant therapeutic potential in the animal model of sciatic nerve injury and therefore might represent a valuable tool for stem cell-based therapy in inflammatory and degenerative diseases of the peripheral and central nervous systems (CNSs)., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

LIM homeobox 8 (Lhx8) is a highly conserved transcriptional factor with recently illustrated roles in cholinergic and GABAergic differentiation, and is expressed in neural crest derived craniofacial tissues during development. However, Lhx8 functions and signaling pathways are largely elusive. Here we showed that Lhx8 regulates dental mesenchyme differentiation and function via Wnt and TGFβ pathways. Lhx8 expression was restricted to dental mesenchyme from E11.5 to a peak at E14.5, and absent in dental epithelium. By reconstituting dental epithelium and mesenchyme in an E16.5 tooth organ, Lhx8 knockdown accelerated dental mesenchyme differentiation; conversely, Lhx8 overexpression attenuated dentin formation. Lhx8 overexpressed adult human dental pulp stem/progenitor cells in β-tricalcium phosphate cubes attenuated mineralized matrix production in vivo. Gene profiling revealed that postnatal dental pulp stem/progenitor cells upon Lhx8 overexpression modified matrix related gene expression including Dspp, Cola1 and osteocalcin. Lhx8 transcriptionally activated Wnt and TGFβ pathways, and its attenuation upregulated multiple dentinogenesis genes. Together, Lhx8 regulates dentin development and regeneration by fine-turning Wnt and TGFβ signaling., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Objectives: The photoinitiator diphenyl-(2,4,6-trimethylbenzoyl)phosphine oxide (TPO) is more reactive than a camphorquinone/amine (CQ) system, and TPO-based adhesives obtained a higher degree of conversion (DC) with fewer leached monomers. The hypothesis tested here is that a TPO-based adhesive is less toxic than a CQ-based adhesive., Methods: A CQ-based adhesive (SBU-CQ) (Scotchbond Universal, 3M ESPE) and its experimental counterpart with TPO (SBU-TPO) were tested for cytotoxicity in human pulp-derived cells (tHPC). Oxidative stress was analyzed by the generation of reactive oxygen species (ROS) and by the expression of antioxidant enzymes. A dentin barrier test (DBT) was used to evaluate cell viability in simulated clinical circumstances., Results: Unpolymerized SBU-TPO was significantly more toxic than SBU-CQ after a 24h exposure, and TPO alone (EC50=0.06mM) was more cytotoxic than CQ (EC50=0.88mM), EDMAB (EC50=0.68mM) or CQ/EDMAB (EC50=0.50mM). Cultures preincubated with BSO (l-buthionine sulfoximine), an inhibitor of glutathione synthesis, indicated a minor role of glutathione in cytotoxic responses toward the adhesives. Although the generation of ROS was not detected, a differential expression of enzymatic antioxidants revealed that cells exposed to unpolymerized SBU-TPO or SBU-CQ are subject to oxidative stress. Polymerized SBU-TPO was more cytotoxic than SBU-CQ under specific experimental conditions only, but no cytotoxicity was detected in a DBT with a 200μm dentin barrier., Significance: Not only DC and monomer-release determine the biocompatibility of adhesives, but also the cytotoxicity of the (photo-)initiator should be taken into account. Addition of TPO rendered a universal adhesive more toxic compared to CQ; however, this effect could be annulled by a thin dentin barrier., (Copyright © 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.)

Objectives: Aim of this study was to investigate whether Dental Pulp Stem Cells-DPSCs responses to pulp injury caused by resinous monomers is be mediated through activation of Wnt/β-catenin signaling., Methods: DPSCs cultures were established from third molars of healthy donors and characterized for stem cell markers with flow cytometry. Cells were exposed to TEGDMA (T: 0.5-2mM) with or without presence of the Wnt-1 ligand (W:25-100ng/ml) or the GSK3β inhibitor Lithium (L:1-10mM), used both as activators of Wnt/β-catenin signaling. Cell viability was evaluated by MTT assay, cell cycle profiles by flow cytometry and expression of key molecules of Wnt/β-catenin signaling by Real-time PCR and Western Blot., Results: DPSC exposure to TEGDMA caused a concentration-dependent cytotoxicity, accompanied by G1 arrest at lower and G2/M arrest at higher concentrations or after prolonged exposure. Lithium caused a dual effect, by stimulating/inhibiting cell proliferation at lower/higher concentrations respectively and causing a G2/M arrest in a concentration-dependent manner. Wnt signaling could be activated in DPSCs after Lithium or Wnt-1 treatment, as shown by accumulation of β-catenin, its translocation into the nucleus and enhanced expression of key pathway players, like LEF1 and Cyclin D1. Importantly, exposure to TEGDMA caused a more pronounced activation of the pathway, whereas cumulative effects were observed after T/L or T/W co-treatment, indicating a very strong activation of Wnt signaling after treatment of already "activated" (by Lithium or Wnt-1) cells with TEGDMA., Significance: These findings highlight the important role of Wnt canonical signaling in pulp repair responses to common injuries., (Copyright © 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.)

Critical morphological requirements for pulp regeneration are tissues replete with vascularisation, neuron formation, and dentin deposition. Autophagy was recently shown to be related to angiogenesis, neural differentiation, and osteogenesis. The present study aimed to investigate the involvement of autophagy in stromal cell-derived factor-1α (SDF-1α)-mediated dental pulp stem cell (DPSC) migration and pulp regeneration, and identify its presence during pulp revascularisation of pulpectomised dog teeth with complete apical closure. In vitro studies showed that SDF-1α enhanced DPSCs migration and optimised focal adhesion formation and stress fibre assembly, which were accompanied by autophagy. Moreover, autophagy inhibitors significantly suppressed, whereas autophagy activator substantially augmented SDF-1α-stimulated DPSCs migration. Furthermore, after ectopic transplantation of tooth fragment/silk fibroin scaffold with DPSCs into nude mice, pulp-like tissues with vascularity, well-organised fibrous matrix formation, and new dentin deposition along the dentinal wall were generated in SDF-1α-loaded samples accompanied by autophagy. More importantly, in a pulp revascularisation model in situ, SDF-1α-loaded silk fibroin scaffolds improved the de novo ingrowth of pulp-like tissues in pulpectomised mature dog teeth, which correlated with the punctuated LC3 and Atg5 expressions, indicating autophagy. Our findings provide novel insights into the pulp regeneration mechanism, and SDF-1α shows promise for future clinical application in pulp revascularisation., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Objective: To determine the in vitro cytotoxicity of dental composites containing bioactive glass fillers., Methods: Dental composites (50:50 Bis-GMA/TEGDMA resin: 72.5wt% filler, 67.5%Sr-glass and 5% OX50) containing different concentrations (0, 5, 10 and 15wt%) of two sol-gel bioactive glasses, BAG65 (65mole% SiO2, 31mole% CaO, 4mole% P2O5) and BAG61 (3mole% F added) were evaluated for cytotoxicity using Alamar Blue assay. First, composite extracts were obtained from 7 day incubations of composites in cell culture medium at 37°C. Undifferentiated pulp cells (OD-21) were exposed to dilutions of the original extracts for 3, 5, and 7 days. Then freshly cured composite disks were incubated with OD-21 cells (n=5) for 2 days. Subsequently, fresh composite disks were incubated in culture medium at 37°C for 7 days, and then the extracted disks were incubated with OD-21 cells for 2 days. Finally, fresh composites disks were light cured for 3, 5, and 20s and incubated with OD-21 cells (n=5) for 1, 3, 5, and 7 days. To verify that the three different curing modes produced different levels of degree of conversion (DC), the DC of each composite was determined by FTIR. Groups (n=5) were compared with ANOVA/Tukey's (α≤0.05)., Results: Extracts from all composites significantly reduced cell viability until a dilution of 1:8 or lower, where the extract became equal to the control. All freshly-cured composites showed significantly reduced cell viability at two days. However, no reduction in cell viability was observed for any composite that had been previously soaked in media before exposure to the cells. Composites with reduced DC (3s vs. 20s cure), as verified by FTIR, showed significantly reduced cell viability., Significance: The results show that the composites, independent of composition, had equivalent potency in terms of reducing the viability of the cells in culture. Soaking the composites for 7 days before exposing them to the cells suggested that the "toxic" components had been extracted and the materials were no longer cytotoxic. The results demonstrate that the cytotoxicity of composites with and without BAG must predominantly be attributed to the release of residual monomers, and not to the presence of the BAG., (Copyright © 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.)

Tooth development involves sequential interactions between dental epithelial and mesenchymal cells. Our previous studies demonstrated that preameloblast-conditioned medium (PA-CM) induces the odontogenic differentiation of human dental pulp cells (hDPCs), and the novel protein Cpne7 in PA-CM was suggested as a candidate signaling molecule. In the present study, we investigated biological function and mechanisms of Cpne7 in regulation of odontoblast differentiation. Cpne7 was expressed in preameloblasts and secreted extracellularly during ameloblast differentiation. After secretion, Cpne7 protein was translocated to differentiating odontoblasts. In odontoblasts, Cpne7 promoted odontoblastic markers and the expression of Dspp in vitro. Cpne7 also induced odontoblast differentiation and promoted dentin/pulp-like tissue formation in hDPCs in vivo. Moreover, Cpne7 induced differentiation into odontoblasts of non-dental mesenchymal stem cells in vitro, and promoted formation of dentin-like tissues including the structure of dentinal tubules in vivo. Mechanistically, Cpne7 interacted with Nucleolin and modulated odontoblast differentiation via the control of Dspp expression. These results suggest Cpne7 is a diffusible signaling molecule that is secreted by preameloblasts, and regulates the differentiation of mesenchymal cells of dental or non-dental origin into odontoblasts., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Objectives: Bioactive calcium phosphate cement (CPC) has been used widely to repair bone defects because of its excellent biocompatibility and bioactivity. However, the poor handling properties, low initial mechanical strength, and long setting time of CPC limit its application in vital pulp therapy (VPT). The aim of this study was to synthesize biphasic calcium phosphate/sulfate cements and evaluate the feasibility of applying these cements in VPT., Methods: The physical, chemical, and mechanical properties of CPC were improved by mixing the cement with various amounts of α-calcium sulfate hemihydrate (CSH). The hydration products and crystalline phases of the materials were characterized using scanning electron microscopy and X-ray diffraction analysis. In addition, the physical properties, such as the setting time, compressive strength, viscosity, and pH were determined. Water-soluble tetrazolium salt-1 and lactase dehydrogenase were used to evaluate cell viability and cytotoxicity., Results: The developed CPC (CPC/CSH cement), which contains 50wt% CSH cement, exhibited no obvious temperature increase or pH change during setting when it was used as a paste. The initial setting time of the CPC/CSH biphasic cement was substantially shorter than that of CPC, and the initial mechanical strength was 23.7±5.6MPa. The CPC/CSH cement exhibited higher viscosity than CPC and, thus, featured acceptable handling properties. X-ray diffraction analysis revealed that the relative peak intensity for hydroxyapatite increased, and the intensity for calcium sulfate dehydrate decreased as the amount of CPC was increased. The cell viability and cytotoxicity test results indicated that the CPC/CSH cement did not harm dental pulp cells., Significance: The developed CPC/CSH biphasic cement exhibits substantial potential for application in VPT., (Copyright © 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.)

Objectives: Aim of this study was to investigate the effects of dental pulp stem cells' (DPSCs) secretome, expressed through their culture conditioned medium (CM), on biological endpoints related to pulp repair and on TEGDMA-induced cytotoxicity., Methods: DPSCs cultures were established and characterized for stem cell markers with flow cytometry. CM was collected from DPSCs under serum deprivation conditions (SDC) and normal serum conditions (NSC) at various time-points. CM effects on DPSCs viability, migration and mineralization potential were evaluated by MTT assay, transwell insert and in vitro scratch assay and Alizarin Red staining/quantification respectively. TEGDMA (0.25-2.0mM) cytotoxicity regarding the same biological endpoints was tested in the presence/absence of CM. TGF-β1 and FGF-2 secretion in CM was measured by ELISA., Results: CM collected under SDC (4d) was able to increase cell viability by 20-25% and to reduce TEGDMA cytotoxicity by 20% (p<0.05). CM positive effects were not obvious when collected under NSC. Transwell assay showed significant increase (26%, p<0.05) of DPSCs' migration after CM exposure, whereas both migration assays could not support a migration rate improvement in TEGDMA-treated cultures exposed to CM compared to TEGDMA alone. CM significantly (p<0.01) increased DPSCs mineralization potential and completely counteracted TEGDMA cytotoxicity on this process. ELISA analysis showed a time-dependent increase of TGF-β1 and a TEGDMA concentration-dependent increase of both TGF-β1 and FGF-2 in CM., Significance: These findings suggest that DPSCs secretome increases their viability, migration and mineralization potential and counteracts TEGDMA-induced cytotoxicy, revealing a novel mechanism of DPSCs autocrine signaling on pulp repair processes., (Copyright © 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.)

Human adult dental pulp stem cells (DPSCs) are self-renewing stem cells that originate from the neural crest during development and remain within the dental pulp niche through adulthood. Due to their multi-lineage differentiation potential and their relative ease of access they represent an exciting alternative for autologous stem cell-based therapies in neurodegenerative diseases. In animal models, DPSCs transplanted into the brain differentiate into functional neurons or astrocytes in response to local environmental cues that appear to influence the fate of the surviving cells. Here we tested the hypothesis that DPSCs might be able to respond to factors present in the retina enabling the regenerative potential of these cells. We evaluated the response of DPSCs to conditioned media from organotypic explants from control and chemically damaged rat retinas. To evaluate cell differentiation, we analyzed the expression of glial fibrillary acidic protein (GFAP), early neuronal and retinal markers (polysialic acid-neural cell adhesion molecule (PSA-NCAM); Pax6; Ascl1; NeuroD1) and the late photoreceptor marker rhodopsin, by immunofluorescence and reverse transcription polymerase chain reaction (RT-PCR). Exposure of DPSC cultures to conditioned media from control retinas induced a 39% reduction on the number of DPSCs that expressed GFAP; the expression of Pax6, Ascl1, PSA-NCAM or NeuroD1 was undetectable or did not change significantly. Expression of rhodopsin was not detectable in control or after exposure of the cultures with retinal conditioned media. By contrast, 44% of DPSCs exposed to conditioned media from damaged retinas were immunopositive to this protein. This response could not be reproduced when conditioned media from Müller-enriched primary cultures was used. Finally, quantitative RT-PCR was performed to compare the relative expression of glial cell-derived neurotrophic factor (GDNF), nerve growth factor (NGF), ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) in DPSC co-cultured with retinal organotypic explants, where BDNF mRNA expression was significantly upregulated in retinal-exposed cultures. Our data demonstrate that DPSC cultures respond to cues from the rat retina and differentiate to express retinal neuronal markers., (Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.)

Recently, human dental pulp stem cells (DPSCs) isolated from inflamed dental pulp tissue have been demonstrated to retain some of their pluripotency and regenerative potential. However, the effects of periodontal inflammation due to periodontitis and its progression on the properties of DPSCs within periodontally compromised teeth remain unknown. In this study, DPSCs were isolated from discarded human teeth that were extracted due to aggressive periodontitis (AgP) and divided into three experimental groups (Groups A, B and C) based on the degree of inflammation-induced bone resorption approaching the apex of the tooth root before tooth extraction. DPSCs derived from impacted or non-functional third molars of matched patients were used as a control. Mesenchymal stem cell (MSC)-like characteristics, including colony-forming ability, proliferation, cell cycle, cell surface antigens, multi-lineage differentiation capability and in vivo tissue regeneration potential, were all evaluated in a patient-matched comparison. It was found that STRO-1- and CD146-positive DPSCs can be isolated from human teeth, even in very severe cases of AgP. Periodontal inflammation and its progression had an obvious impact on the characteristics of DPSCs isolated from periodontally affected teeth. Although all the isolated DPSCs in Groups A, B and C showed decreased colony-forming ability and proliferation rate (P < 0.05), the decreases were not consistent with the degree of periodontitis. Furthermore, the cells did not necessarily show significantly diminished in vitro multi-differentiation potential. Only DPSCs from Group A and the Control group formed dentin-like matrix in vivo when cell-seeded biomaterials were transplanted directly into an ectopic transplantation model. However, when cell-seeded scaffolds were placed in the root fragments of human teeth, all the cells formed significant dentin- and pulp-like tissues. The ability of DPSCs to generate dental tissues decreased when the cells were isolated from periodontally compromised teeth (P < 0.05). Again, increased periodontal destruction was not necessarily followed by a decrease in the amount of dentin- and pulp-like tissue formed. These findings provide preliminary evidence that periodontally compromised teeth might contain putative stem cells with certain MSC properties, as long as the vitality of the pulp has not been totally damaged. Whether these cells can serve as a source of autologous multipotent MSCs for clinical regenerative therapies warrants further investigation with larger sample sizes and various types of periodontitis., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Mesenchymal stem cells (MSCs) isolated from human postnatal dental pulp and periodontal ligament (PDL) tissues can give rise to multilineage differentiation in vitro and generate related dental tissues in vivo. However, the cell properties of human dental pulp stem cells (DPSCs) and PDL stem cells (PDLSCs) after in vivo implantation remain largely unidentified. In this study, cells were re-isolated from in vivo-generated dental pulp-like and PDL-like tissues (termed re-DPCs and re-PDLCs, respectively) as a result of ectopic transplantation of human DPSC and PDLSC sheets. The cell characteristics in terms of colony-forming ability, cell surface antigens and multi-differentiation potentials were all evaluated before and after implantation. It was found that re-DPCs and re-PDLCs were of human and mesenchymal origin and positive for MSC markers such as STRO-1, CD146, CD29, CD90 and CD105; and, to some extent, re-DPCs could maintain their colony forming abilities. Moreover, both cell types were able to form mineral deposits and differentiate into adipocytes and chondrocytes; however, quantitative analysis and related gene expression determination showed that the osteo-/chondro-differentiation capabilities of re-DPCs and re-PDLCs were significantly reduced compared to those of DPSCs and PDLSCs, respectively (P < 0.05); re-PDLCs showed a greater reduction potential than re-DPCs. We conclude that DPSCs and PDLSCs may maintain their MSC characteristics after in vivo implantation and, compared to PDLSCs, DPSCs appear much more stable under in vivo conditions. These findings provide additional cellular and molecular evidence that supports expanding the use of dental tissue-derived stem cells in cell therapy and tissue engineering., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The age-associated decline in the regenerative abilities of mesenchymal stem cells (MSCs) may be due to age-related changes in reduction in number, intrinsic properties of MSCs and extrinsic factors of the extracellular environment (the stem cell niche). The effect of age on the efficacy of MSC transplantation on regeneration, however, has not been clearly demonstrated due to variable methods of isolation of MSCs and variations in stem cell populations. In this study, dental pulp stem cell (DPSC) subsets were isolated from young and aged dog teeth based on their migratory response to granulocyte-colony stimulating factor (G-CSF) (MDPSCs). In order to study the age-associated changes, their biological properties and stability were compared and the regenerative potential was examined in a pulpectomized tooth model in aged dogs. MDPSCs from aged dogs were efficiently enriched in stem cells, expressing trophic factors with high proliferation, migration and anti-apoptotic effects as in MDPSCs from young dogs. However, pulp regeneration was retarded 120 days after autologous transplantation of aged MDPSCs. We further demonstrated that isolated periodontal ligament stem cells (PDLSCs) from aged dogs, representative of migrating stem cells from outside of the tooth compartment to regenerate pulp tissue, had lower proliferation, migration and anti-apoptotic abilities. These results therefore provide a better understanding of the mechanisms involved in the age-dependent decline in pulp regeneration, which are attributed to a decrease in the regenerative potential of resident stem cells., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Dentin regeneration remains a great challenge in clinic. Dental pulp stem cells (DPSCs) actively contribute to dentinogenesis, which is orchestrated by a spectrum of signaling factors. However, the exact mechanism underlying the reparative dentin regeneration process is largely unknown and the application of DPSCs in the repair of dentin defect is thus limited. Here, using a rat reparative dentin regeneration model, we observed that DPSCs underwent a proliferation phase followed by a differentiation phase after dental injury. A transient elevation of nephroblastoma overexpressed (NOV, or CCN3) expression correlated with this progressive dental tissue restoration process. Further studies revealed that over-expression of CCN3 promoted human DPSCs proliferation via activation of Notch. Moreover, using cocultured cells (DPSCs/CCN3 and DPSCs) in vitro and the cocultured cells-poly (lactic-co-glycolic acid) (PLGA) scaffold complex in vivo, we demonstrated that CCN3 was capable of promoting mineralization in a non-cell autonomous manner through promoting secretion of BMP2. CCN3 can promote dentinogenesis by coordinating proliferation and odontoblastic differentiation of DPSCs via modulating Notch and BMP2 signaling pathways and CCN3 is a promising therapeutic target in dentin tissue engineering., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Human dental pulp stem cells (DPSCs) contain subsets of progenitor/stem cells with high angiogenic, neurogenic and regenerative potential useful for cell therapy. It is essential to develop a safe and efficacious method to isolate the clinical-grade DPSCs subsets from a small amount of pulp tissue without using conventional flow cytometry. Thus, a method for isolation of DPSCs subsets based on their migratory response to optimized concentration of 100 ng/ml of granulocyte-colony stimulating factor (G-CSF) was determined in this study. The DPSCs mobilized by G-CSF (MDPSCs) were enriched for CD105, C-X-C chemokine receptor type 4 (CXCR-4) and G-CSF receptor (G-CSFR) positive cells, demonstrating stem cell properties including high proliferation rate and stability. The absence of abnormalities/aberrations in karyotype and lack of tumor formation after transplantation in an immunodeficient mouse were demonstrated. The conditioned medium of MDPSCs exhibited anti-apoptotic activity, enhanced migration and immunomodulatory properties. Furthermore, transplantation of MDPSCs accelerated vasculogenesis in an ischemic hindlimb model and augmented regenerated pulp tissue in an ectopic tooth root model compared to that of colony-derived DPSCs, indicating higher regenerative potential of MDPSCs. In conclusion, this isolation method for DPSCs subsets is safe and efficacious, having utility for potential clinical applications to autologous cell transplantation., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Mesenchymal stem cells (MSCs) have been used for cell therapy in various experimental disease models. However, the regenerative potential of MSCs from different tissue sources and the influence of the tissue niche have not been investigated. In this study, we compared the regenerative potential of dental pulp, bone marrow and adipose tissue-derived CD31(-) side population (SP) cells isolated from an individual porcine source. Pulp CD31(-) SP cells expressed the highest levels of angiogenic/neurotrophic factors and had the highest migration activity. Conditioned medium from pulp CD31(-) SP cells produced potent anti-apoptotic activity and neurite outgrowth, compared to those from bone marrow and adipose CD31(-) SP cells. Transplantation of pulp CD31(-) SP cells in a mouse hindlimb ischemia model produced higher blood flow and capillary density than transplantation of bone marrow and adipose CD31(-) SP cells. Motor function recovery and infarct size reduction were greater with pulp CD31(-) SP cells. Pulp CD31(-) SP cells induced maximal angiogenesis, neurogenesis and pulp regeneration in ectopic transplantation models compared to other tissue sources. These results demonstrate that pulp stem cells have higher angiogenic, neurogenic and regenerative potential and may therefore be superior to bone marrow and adipose stem cells for cell therapy., (Copyright © 2012 Elsevier Ltd. All rights reserved.)