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Cerebral ischemia-reperfusion injury (I/RI) is one of the principal pathogenic factors in the poor prognosis of ischemic stroke, for which current therapeutic options to enhance neurological recovery are notably insufficient. Dental pulp stem cell-derived extracellular vesicles (DPSC-EVs) have promising prospects in stroke treatment and the specific underlying mechanisms have yet to be fully elucidated. The present study observed that DPSC-EVs ameliorated the degree of cerebral edema and infarct volume by reducing the apoptosis of neurons. Furthermore, the miRNA sequencing and functional enrichment analysis identified that miR-877–3p as a key component in DPSC-EVs, contributing to neuroprotection and anti-apoptotic effects. Following target prediction and dual-luciferase assay indicated that miR-877–3p interacted with Bcl-2-associated transcription factor (Bclaf1) to play a function. The miR-877–3p inhibitor or Bclaf1 overexpression reversed the neuroprotective effects of DPSC-EVs. The findings reveal a novel therapeutic pathway where miR-877–3p, transferred via DPSC-EVs, confers neuroprotection against cerebral I/RI, highlighting its potential in promoting neuronal survival and recovery post-ischemia.

Background/purpose: Cell pyroptosis and gingival inflammation have been implicated in periodontitis progression. Our previous study revealed that AR-A014418, a pharmacological inhibitor of glycogen synthase kinase-3β (GSK-3β), can enhance the migratory and osteogenic differentiation abilities of rat dental pulp stem cells (rDPSCs). The present study aimed to explore the effect of AR on the inflammation of rDPSCs. Materials and methods: The primary rDPSCs were isolated and identified by flow cytometry, as well as Oil red O and Alizarin Red S staining. The rDPSCs were cultured and exposed to lipopolysaccharide (LPS) before treating them with different concentrations of AR-A014418. The cell viability was detected using the CCK-8 assay. The generation and secretion of pro-inflammatory cytokines (IL-18, TNF-α, L-1β, and IL-6) were examined by qPCR and ELISA, respectively. To investigate the activation of the NLRP3 inflammasome, the expression levels of pro-caspase 1, cleaved caspase 1, as well as NLRP3 were analyzed by western blotting and immunofluorescence, respectively. Results: In the rDPSCs, LPS prohibited cell viability and enhanced the generation and secretion of pro-inflammatory cytokines. LPS upregulated NLRP3 and cleaved caspase-1 protein levels and promoted ASC speck formation in the rDPSCs. AR-A014418 administration effectively blocked the LPS-induced inflammation of the rDPSCs in a dose-dependent way. Mechanistically, AR-A014418 significantly restrained the up-regulation of NLRP3 and cleaved caspase-1 in LPS-treated rDPSCs. Conclusion: Collectively, our findings suggest that AR-A014418 significantly mitigates LPS-induced inflammation of rDPSCs by blocking the activation of the NLRP3 inflammasome.

Background/purpose: Human dental pulp stem cells (hDPSCs) are an emerging source of mesenchymal stem cells (MSCs) for bone tissue regeneration and engineering. In bone regeneration using transplanted MSCs, the extracellular environment or co-injected drugs can affect their success or failure. In this study, we investigated the effects and signaling mechanisms of lidocaine on osteogenic differentiation of hDPSCs after inducing inflammatory conditions with lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNF-α). Materials and methods: To investigate the effect of lidocaine on the osteogenic differentiation of LPS/TNF-α-treated hDPSCs, alkaline phosphatase (ALP) and Alizarin red S (ARS) staining were conducted. The expression of osteogenesis-related genes was assessed using quantitative real-time polymerase chain reaction and western blotting. The expression of mitogen-activated protein kinases was analyzed to evaluate the effect of lidocaine on osteogenic differentiation of LPS/TNF-α-treated hDPSCs. Results: Various concentrations of lidocaine (0.05, 0.2, and 1 mM) further decreased ALP and ARS staining of LPS/TNF-α-treated hDPSCs. Similarly, the mRNA and protein expression of osteogenesis-related genes was suppressed via lidocaine treatment in LPS/TNF-α-treated hDPSCs. Lidocaine treatment downregulated the protein expression of p-ERK and p-JNK in LPS/TNF-α-treated hDPSCs. Conclusion: Lidocaine intensified the inhibition of osteogenic differentiation on inflammation-induced hDPSCs by inhibiting the ERK and JNK signaling pathways. This in vitro study suggested that lidocaine may have an inhibitory effect on bone regeneration.

Background/purpose: Various studies have used stem cells in the field of bone tissue engineering to repair bone defects. Dental pulp stem cells (DPSCs) have multipotent properties and can be acquired in a noninvasive manner; therefore, they are frequently used in experiments in regenerative medicine. The objective of this study was to investigate the odontogenic/osteogenic differentiation of human DPSCs (hDPSCs) using propofol, a widely used intravenous anesthetic agent. Materials and methods: Alkaline phosphatase (ALP) staining was used to investigate the effects of various concentrations of propofol (5, 20, 50 and 100 μM) on the osteogenic differentiation of hDPSCs. Real-time qPCR and Western blot analysis were used to detect the effect of propofol on the expression of odontogenic/osteogenic genes, such as DMP1, RUNX2, OCN, and BMP2. Odontogenic/osteogenic differentiation of hDPSCs was estimated at days 7 and 14. Results: ALP staining of hDPSCs was significantly decreased by propofol treatment. The mRNA expression of DMP1, RUNX2, OCN, and BMP2 decreased after propofol treatment for 14 days. The protein expression of DMP1 and BMP2 was decreased by propofol at days 7 and 14, and that of RUNX2 was decreased by propofol at day 14 only. Conclusion: Propofol attenuated odontogenic/osteogenic differentiation of hDPSCs in vitro. This result suggests that propofol, which is widely used for dental sedation, may inhibit the odontogenic/osteogenic differentiation of hDPSCs.

Large bone tissue defects present a significant clinical challenge due to the lack of stem cells and an osteogenic microenvironment, leading to fibrotic healing and impaired bone regeneration. Microsphere-based cell-on three-dimensional (3D) culture systems show great promise for constructing osteogenic microtissues. However, the underlying mechanisms require further investigation. In this study, we propose a simple, scalable framework for highly efficient osteogenic microtissue construction, utilizing gelatin methacryloyl (GelMA) microspheres and dental pulp stem cells (DPSCs). The GelMA microspheres provide an extensive, scalable 3D framework for the autonomous adhesion, migration, and proliferation of DPSCs. Within the enormous 3D space created by the microspheres, DPSCs anchor to the microspheres and neighboring cells, inducing intrinsic tensile stress and simulating a mechanical force akin to "rock climbing training". Transcriptomic sequencing results reveal that the 3D spatial and mechanical microenvironment modulates biological processes involved in cell adhesion, extracellular matrix organization, and the positive regulation of cell migration. Further investigations demonstrate that triggering the FAK/YAP pathway mediate mechanical driven differentiation of DPSCs into the osteoblastic lineage in the excellent osteogenic microtissues. Moreover, this simple scalable 3D framework strategy is expected to enable the efficient and large-scale preparation of stem cell-based microtissues., 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 the paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background/purpose: Dental pulp stem cells (DPSCs) contribute to the regeneration of various tissues and have superior proliferation, immune privilege, and anti-inflammation properties to other mesenchymal stem cells. 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside (THSG) not only enhances the aforementioned properties of DPSCs but also promotes self-renewal and reprogramming-like ability. However, whether THSG enhances the aforementioned properties and abilities through direct or indirect interaction mechanisms remains unclear. To address this knowledge gap, we examined the effects of THSG-stimulated DPSC-derived conditioned medium (THSG-CM) on the activity and anti-inflammation properties of cells. Materials and methods: DPSCs were treated with various concentrations of THSG to produce THSG-CM, which was then collected, analyzed, and lyophilized. A cytokine profiling antibody assay was used to compare protein components between THSG-treated and nontreated CM. Human skin fibroblasts (HSFs) and human gingival fibroblasts (HGFs) were used to investigate the effect of THSG-CM on cell proliferation, anti-inflammation, and wound healing abilities; for this investigation, MTS assay, quantitative real-time PCR analysis, and 2-well silicone inserts wound model were conducted. Results: We observed that THSG enhanced the secretion of growth- and immune-associated proteins in THSG-CM and increased the proliferation of HSFs and HGFs. Furthermore, THSG-CM significantly attenuated lipopolysaccharide-stimulated mRNA levels of cytokines in both cells and improved wound healing abilities. Conclusion: We conclude that THSG-CM had more beneficial effects on cell activity and anti-inflammation in the HSFs and HGFs than DPSC-derived CM. DPSC-derived CM can be developed into a cell-free regenerative strategy in the future, and its therapeutic efficacy may be improved by THSG-CM.

The study of regenerative dentistry receives a fast growing interest. The potential ability of the dentin-pulp complex to regenerate is both promising and perplexing. To answer the challenging nature of the dental environment, scientists have developed various combinations of biomaterial scaffolds, stem cells, and incorporation of several growth factors. One of the crucial elements of this tissue engineering plan is the selection and fabrication of scaffolds. However, further findings suggest that cell behavior hugely depends on mechanical signaling. Nanotopography modifies scaffolds to alter cell migration and differentiation. However, to the best of the author's knowledge, there are very few studies addressing the correlation between nanotopography and dentin-pulp complex regeneration. Therefore, this article presents a comprehensive review of these studies and suggests a direction for future developments, particularly in the incorporation of nanotopography design for dentin-pulp complex regeneration.

Science and commerce advance together and the stem cell field is no exception. With the promise of cures for conditions as diverse as cancer, autism, neural degeneration, organ replacement and addiction, long-term preservation of dental stem cells is a growth market. The discovery nearly twenty years ago, of viable, multipotent, stem cells in dental pulp from both baby and adult teeth initiated, and drives, this market.The dental stem cell preservation services, “tooth banks”, focus on the collection of a child’s baby teeth, as they are shed naturally, and storage of the stem cells from within the pulp for therapeutic use in later years should the child require them. This review focuses on the procedures related to these stem cell storage services and may serve as an introduction for many to the practice of “tooth banking”.

This study was conducted to investigate the odontogenic proliferation and differentiation of dental pulp stem cells (DPSCs) after induction by nanoparticle mineral trioxide (NMT). DPSCs were isolated from permanent teeth and placed in tubes containing Dulbecco's modified Eagle's medium, followed by immunocytochemistry analysis. The viability of DPSCs exposed to NMT was measured using MTT assay with trypan blue dye exclusion. Alkaline phosphatase (ALP) activity was evaluated using ALP colorimetric reactions by reacting NMT supernatants with fluorescent-specific ALP substrates. The concentration of osteocalcin was determined using an instant human osteocalcin enzyme-linked immunosorbent assay (ELISA) kit. A human dentin sialophosphoprotein (DSPP) ELISA kit coated with anti-human DSPP antibody was employed to measure DSPP levels. There was a significant difference between ALP activity after exposing the cells to NMT and trioxide mineral aggregate on days 3, 7, and 21. Osteocalcin activity showed a significant difference on days 3, 7, 14, and 21. There was a significant difference in DSPP levels on days 7 and 21. DPSCs exposed to NMT and to trioxide mineral aggregate showed extracellular matrix formation on day 7 and 14, respectively. Furthermore, NMT may effectively increase the proliferation and differentiation of DPSCs as well as their maturation toward odontoblasts.

Mesenchymal stem/stromal cell (MSC) spheroids generated in a three-dimensional (3D) culture system serve as a surrogate model that maintain stem cell characteristics since these mimic the in vivo behavior of cells and tissue more closely. Our study involved a detailed characterization of the spheroids generated in ultra-low attachment flasks. The spheroids were evaluated and compared for their morphology, structural integrity, viability, proliferation, biocomponents, stem cell phenotype and differentiation abilities with monolayer culture derived cells (2D culture). The in-vivo therapeutic efficacy of DPSCs derived from 2D and 3D culture was also assessed by transplanting them in an animal model of the critical-sized calvarial defect. DPSCs formed compact and well-organized multicellular spheroids when cultured in ultra-low attachment condition with superior stemness, differentiation, and regenerative abilities than monolayer cells. They maintained lower proliferative state and showed marked difference in the cellular biocomponents such as lipid, amide and nucleic acid between DPSCs from 2D and 3D cultures. The scaffold-free 3D culture efficiently preserves DPSCs intrinsic properties and functionality by maintaining them in the state close to the native tissues. The scaffold free 3D culture methods allow easy collection of a large number of multicellular spheroids of DPSCs and therefore, this can be adopted as a feasible and efficient method of generating robust spheroids for various in-vitro and in-vivo therapeutic applications., 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 © 2023 Elsevier Ltd. All rights reserved.)

Introduction: The objective of this study was to determine the effectiveness of several antibiotic-loaded hydrogel scaffolds against Enterococcus faecalis, as well as their ability to stimulate proliferation and mineralization of dental pulp stem cells., Methods: Fibrin (Fg) or chitosan-fibrin hydrogels (Ch) were prepared using 12.5 mg/mL fibrinogen and 0.4% (w/v) chitosan. Triple antibiotics, clindamycin-modified triple antibiotic paste, or double antibiotics were loaded in gels (1 mg/mL). Antibacterial effect against E. faecalis biofilm was determined by using colony-forming units (CFUs) and confocal laser scanning microscope (CLSM). Cell viability and morphology were determined by loading cells into different gels at 7 and 14 days using the water-soluble tetrazolium salt-1 cell viability assay and Live & Dead cell analysis. Mineralization was detected by using alkaline phosphatase and alizarin red staining activity., Results: Antibiotic-loaded Fg gel and Ch gel alone without antibiotics resulted in a significant reduction in CFUs compared with the positive control (P < .05). When antibiotics were loaded in Ch gel, there were no CFUs detected in any groups (P < .05). CLSM images showed dense red areas with mostly dead bacteria on the dentin surface in antibiotic-loaded Ch groups, which showed significantly less live bacteria compared with the other groups (P < .05). Triple antibiotic-loaded Fg and Ch gels resulted in a dramatic decrease in the mineralized nodule formation compared with all other gel groups (P < .05). Ch hydrogels resulted in round cell morphology up to 7 days. Ch alone or with double antibiotic paste showed more cell spreading with spindle-shaped morphology at 14 days and higher alkaline phosphatase activity compared with other antibiotic-loaded Ch groups (P > .05)., Conclusions: Double antibiotic-loaded Ch gel appears to enhance the antibacterial properties while maintaining higher cell viability, cell spreading, and mineralization activity, compared with all the other scaffolds investigated., (Published by Elsevier Inc.)

Introduction: Dental pulp is a major composition in the pulp-dentin complex, which serves as protective system against dental trauma/infection. Functional dental pulp regeneration is highly desirable after pulpitis or pulp necrosis. However, endodontic regeneration has remained challenging for decades because of the deconstructive microenvironment and the lack of functional cells within the root canal system. The present study developed a decellularized matrix hydrogel derived from human dental pulp (hDDPM-G), which might serve as a growth-permissive microenvironment for dental pulp regeneration., Methods: Human dental pulps extracted from healthy wisdom teeth were decellularized and digested and then underwent sol-gel transition to form hDDPM-G. The protein compositions were identified by proteomic analysis. Human dental pulp stem cells (hDPSCs) were seeded on hDDPM-G-coated surfaces and evaluated by immunofluorescence staining, transwell migration, and Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) assays. Induced hDPSC differentiation was examined in vitro and characterized by immunostaining, Western blotting, and reverse transcription polymerase chain reaction., Results: Complete decellularization was implemented. Protein contents found in the human decellularized dental pulp matrix were identified to contribute in promoting cell proliferation, migration, and regulation of stem cell differentiation. The hDDPM-G-coated surfaces promoted hDPSC adhesion, migration, and proliferation. Furthermore, hDDPM-G coatings facilitated odontoblastlike, neural-like, and angiogenic differentiation of the seeded hDPSCs after being cultured in induction media for 14 days., Conclusions: This study showed that hDDPM-G effectively contributed in promoting hDPSC proliferation and migration and induced multidirectional differentiation. Considering the injectability and gelation at body temperature, hDDPM-G may hold translational potential for endodontic regeneration., (Copyright © 2020 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

LIM homeobox 8 (LHX8) is expressed during embryonic development of craniofacial tissues, including bone and teeth. In a previous study, the overexpression of LHX8 inhibited osteodifferentiation of human dental pulp stem cells (DPSCs). In this study, a cDNA microarray analysis was performed to reveal the molecular changes which occur in response to LHX8 overexpression in DPSCs and discover possible targets for an osteoinductive agent. There were 345 differentially expressed genes (DEGs) in response to osteoinductive signaling and 53 DEGs in response to LHX8 overexpression and osteoinductive signaling, respectively. Thirty-eight genes were common in both conditions, and among these, genes upregulated in LHX8 DPSCs but downregulated in osteodifferentiated DPSCs were chosen. Five of them had commercial inhibitors available. Among the tested inhibitors, ML323, which target DNA-binding protein inhibitor ID-1, promoted osteodifferentiation of DPSCs. In conclusion, inhibition of ID-1 led to increased osteogenesis of human DPSCs., 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 © 2020 Elsevier Inc. All rights reserved.)

Supernumerary tooth (ST) may arise from uncertain developmental abnormalities or underlying genetic causes, and the extraction at the early age is recommended. Dental pulp stem cells (DPSCs) are the valuable resource for the regeneration of tooth and related craniofacial structures. DPSCs isolated from ST (sDPSCs) have not been fully characterized despite the potential in the applications. The objectives of this study are the efficient isolation of sDPSCs and the analysis of the properties as stem cells. sDPSCs were established by hammer-cracking and separation of the intact pulp from ST. sDPSCs in the culture were examined by light microscope and flow cytometer for the morphology and the surface marker expression. sDPSCs exhibited the cellular morphology of typical mesenchymal stem cells and expressed CD44, CD73, CD90, CD105 and CD166, but not CD14, CD34 or CD45. sDPSCs showed the differentiation potential toward osteogenic, chondrogenic and adipogenic lineages. During osteogenic differentiation, the stimulation by Oncostatin M enhanced the differentiation and significantly increased the expression of genes involved in the hard tissue repair, such as BMP2, BMP4, BMP6 and RUNX2. sDPSCs can be effectively derived from ST and displays the characteristics of mesenchymal stem cells in the maintenance and the differentiation. sDPSCs satisfies the quality as DPSCs thus provide the valuable resource to the regenerative therapy., 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 © 2020 Elsevier Inc. All rights reserved.)

Dental caries and trauma, particularly in childhood, are among the most prevalent teeth problems, which result in the creation of cavities and probably tooth loss. Thus, novel regenerative approaches with high efficiency and less toxicity are required. Stem cell therapy along with the implementation of scaffolds has provided excellent opportunities in the regeneration of teeth structure. Hyaluronic acid (HA) hydrogels have enticed great attention in the field of regenerative medicine. The unique chemical and structural properties of HA and its derivatives have enabled their application in tissue engineering. Several factors such as the location and type of the lesion, teeth age, the type of capping materials determine the success rate of pulp therapy. HA hydrogels have been considered as biocompatible and safe scaffold supports in human dental cell therapies., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Introduction: The nuclear enzyme poly(adenosine phosphate ribose) polymerase 1 (PARP-1) has been implicated in the maintenance and differentiation of several stem cells. The role of PARP-1 in dental pulp stem cell (DPSC) differentiation, especially in the context of its ability to modulate nerve regeneration factors, has not been investigated. Regeneration of neuronal components in pulp tissue is important for the assessment of tooth vitality. Brain-derived neurotrophic factor (BDNF) is known to play an integral signaling factor during nerve regeneration. In this study, we identified the role of PARP-1 in the modulation of BDNF in DPSC differentiation into odontoblastlike cells., Methods: Human DPSCs were prepared from healthy molars and cultured in regular and osteogenic media treated with PARP-1 antagonist and PARP-1 exogeneous protein. Polymerase chain reaction and immunohistochemistry analysis for BDNF and various differentiation markers were performed., Results: Our polymerase chain reaction results showed that differentiated cells show odontoblastlike properties because they express odontogenic markers such as dentin sialophosphoprotein and dentin matrix protein 1. Both PARP-1 inhibitor and protein did not affect odontogenic differentiation and proliferation because the number of the differentiated cells was unaffected, and the expression of dentin sialophosphoprotein and dentin matrix protein 1 was not significantly changed. There is the possibility that PARP-1 treatment induces DPSCs into the unique cell lineage. Some differentiated cells show a very unique morphology with large irregular cytoplasm and an oval nucleus. Moreover, PARP-1 inhibition significantly increased BDNF secretion in DPSC-derived odontoblastlike cells. This observation was also confirmed by immunohistochemistry., Conclusions: Taken together, our results indicate PARP-1 as a negative regulator in BDNF secretion during odontogenic DPSC differentiation, showing its potential application for translational nerve regeneration strategies to improve dental pulp tissue vitality assessments., (Copyright © 2018 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Dental pulp stem cells (DPSCs) derived from the human dental pulp tissue have multiple differentiation capabilities, such as osteo/odontogenic differentiation. Therefore, DPSCs are deemed as ideal stem cell sources for tissue regeneration. As new nanomaterials based on DNA, tetrahedral DNA nanostructures (TDNs) have tremendous potential for biomedical applications. Here, the authors aimed to explore the part played by TDNs in proliferation and osteo/odontogenic differentiation of DPSCs, and attempted to investigate if these cellular responses could be driven by activating the canonical Notch signaling pathway. Upon exposure to TDNs, proliferation and osteo/odontogenic differentiation of DPSCs were dramatically enhanced, accompanied by up regulation of Notch signaling. In general, our study suggested that TDNs can significantly promote proliferation and osteo/odontogenic differentiation of DPSCs, and this remarkable discovery can be applied in tissue engineering and regenerative medicine to develop a significant and novel method for bone and dental tissue regeneration., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Rett syndrome is an X-linked neurodevelopmental disorder associated with psychomotor impairments, autonomic dysfunctions and autism. Patients with Rett syndrome have loss-of-function mutations in MECP2, the gene encoding methyl-CpG-binding protein 2 (MeCP2). Abnormal biogenic amine signaling and mitochondrial function have been found in patients with Rett syndrome; however, few studies have analyzed the association between these factors. This study investigated the functional relationships between mitochondria and the neuronal differentiation of the MeCP2-deficient stem cells from the exfoliated deciduous teeth of a child with Rett syndrome. An enrolled subject in this study was a 5-year-old girl carrying a large deletion that included the methyl-CpG-binding domain, transcriptional repression domain, and nuclear localization signal of MECP2. Using the single-cell isolation technique, we found that the two populations of MeCP2-expressing and MeCP2-deficient stem cells kept their MECP2 expression profiles throughout the stages of cell proliferation and neuronal differentiation in vitro. Neurite outgrowth and branching were attenuated in MeCP2-deficient dopaminergic neurons. MeCP2-deficient cells showed reduced mitochondrial membrane potential, ATP production, restricted mitochondrial distribution in neurites, and lower expression of a central mitochondrial fission factor, dynamin-related protein 1 than MeCP2-expressing cells. These data indicated that MeCP2-deficiency dysregulates the expression of mitochondrial factors required for the maturation of dopaminergic neurons. This study also provides insight into the pathogenic mechanism underlying dysfunction of the intracerebral dopaminergic signaling pathway in Rett syndrome., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The aim of this study was to evaluate the effects of several additives on the setting time and cytotoxicity of accelerated-set mineral trioxide aggregate (MTA) on stem cells of human dental pulp. ProRoot white MTA (WMTA) (Dentsply Tulsa Dental, Johnson City, TN) was mixed with various additives including distilled water, 2.5% disodium hydrogen phosphate (Na2HPO4) (Merck, Darmstadt, Germany), K-Y Jelly (Johnson & Johnson, Markham, ON, Canada), and 5% and 10% calcium chloride (CaCl2) (Merck). The setting times were evaluated using a Vicat apparatus (Alsa Lab, Istanbul, Turkey). Human dental pulp stem cells were isolated and seeded into 48-well plates at 2 × 10(3) cells per well and incubated with MTA samples for 24 hours, 3 days, and 7 days. Cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. MTA mixed with 10% CaCl2 showed the lowest setting time (P < .05). According to the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium results on the 1st, 3rd, and 7th days, a statistically significant difference was found (P < .05) between MTA groups and the control group. MTA mixed with K-Y Jelly in all groups showed the lowest cell viability at all time points (P < .05). The cell viability of MTA mixed with distilled water, 5% CaCl2, 10% CaCl2, and Na2HPO4 increased significantly through time (P < .05). This in vitro study found MTA mixed with 5% and 10% CaCl2 and Na2HPO4 is biocompatible with dental pulp stem cells in terms of cell viability. Further in vitro and in vivo investigations are required to prove the clinical applications of MTA mixed with various additives., (Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: The aim of this study was to determine the effects of in vitro odontogenic/cementogenic differentiation on the in vivo tissue regeneration of dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs)., Methods: DPSCs and PDLSCs were predifferentiated for 0, 4, or 8 days with an odontogenic/cementogenic medium and then transplanted into subcutaneous pockets in immunocompromised mice. The transplants were harvested 9 weeks after transplantation, and the characteristics of the newly formed tissues in vivo were analyzed by histologic staining; examining alkaline phosphate activity; immunohistochemical staining for osteocalcin, dentin sialoprotein, and type XII collagen; and quantitative real-time polymerase chain reaction to analyze the expression patterns of the following genes: RUNX2, OC, DMP1, DSPP, POSTN, CP23, and Col XII., Results: In DPSC transplants, the amount of new tissues was similar in all groups, whereas in predifferentiated transplants the OC and DSPP expression were higher than undifferentiated transplants. Predifferentiated PDLSC transplants generated more hard tissue and expressed higher alkaline phosphatase activity than undifferentiated transplants. In particular, 8-day predifferentiated PDLSC transplants formed tissue closer to the cementum/PDL complex in vivo as confirmed by the higher expression levels of POSTN, CP23, and Col XII., Conclusions: Although there was no significant increase in tissue-forming ability among DPSCs after predifferentiation, predifferentiated DPSCs generated hard tissue closer to dentin. Also, predifferentiated PDLSCs appeared to be able to generate higher-quality and greater amounts of tissue for dental regeneration than undifferentiated PDLSCs., (Copyright © 2015 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)