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Dental pulp stem cells (DPSCs) show potential for treating neurodegenerative and traumatic diseases due to their neural crest origin. Melatonin (MT), an endogenous neurohormone with well-documented anti-inflammatory and antioxidant properties, has shown promising results with MSCs in terms of engraftment, proliferation, and neuronal differentiation in animal SCI models. However, the effects of melatonin preconditioning on human dental pulp stem cells (hDPSCs) for SCI treatment remain unclear. This study investigates the impact of melatonin preconditioning on hDPSCs engraftment, neural differentiation, and neurological function in rats with SCI. Forty-two male Sprague–Dawley rats were divided into six groups: Control, Sham, Model, Vehicle, Lesion Treatment A (SCI + hDPSCs), and Lesion Treatment B (SCI + MT-hDPSCs). After obtaining hDPSCs, stem cells were evaluated using flow cytometry. Cell viability was assessed using the MTT assay. SCI was induced in the Model, Vehicle, Lesion Treatment A, and Lesion Treatment B groups. The Lesion Treatment A and B groups received hDPSCs and hDPSCs pretreated with melatonin, respectively, 1 week after SCI, while the Vehicle group received only an intravenous injection of DMEM to simulate treatment. The other groups were used for behavioral testing. Immunohistochemistry (IHC) was employed to assess hDPSCs engraftment and differentiation at the SCI site. Motor function across the six groups was evaluated using the Basso, Beattie, and Bresnahan (BBB) score. Histological studies and cell counts confirmed hDPSCs implantation at the injury site, with a significantly higher presence in the MT-hDPSCs compared to hDPSCs (p < 0.01). IHC revealed that hDPSCs and MT-hDPSCs differentiated into neurons and astrocytes, with greater differentiation observed in the MT-hDPSCs compared to the hDPSCs (p < 0.01 and p < 0.05, respectively). Functional improvement was noted in both SCI + hDPSCs and SCI + MT-hDPSCs groups compared to SCI and Vehicle groups from Week 4 onward (p < 0.001). Significant differences were also observed between the SCI + hDPSCs and SCI + MT-hDPSCs groups starting from Week 7 (p < 0.01). Preconditioning hDPSCs with melatonin enhances engraftment, neuronal differentiation, and greater performance improvement compared to hDPSCs alone in the SCI animal model. [ABSTRACT FROM AUTHOR]

This study investigated the impact of some specific experimental calcium phosphate cements doped with different fluoride salts (FDCPCs) concentrations on the basal functions of human Dental Pulp Stem Cells (hDPSCs). Furthermore, this study also examined the migration, as well as the mineralisation through osteogenic differentiation. Experimental FDCPCs were formulated using different concentrations of calcium/sodium fluoride salts [(5 wt%: VS5F), (10 wt%: VS10F), (20 wt%: VS20F)]. A fluoride-free calcium phosphate (VS0F) was used as a control. The hDPSCs were assessed to evaluate their self-renewal and migration activity in the presence of eluates of the different FDCPCs. A viability assay in osteogenic conditions was carried out, along with the differentiation potential through Alkaline Phosphatase Activity (ALP), and Alizarin Red Staining (ARS). Moreover, the gene expression of specific markers (RUNX2, ALP, COL1α1, OCN, OPN, DSPP, MEPE, and DMP-1) was also evaluated. All the tested FDCPD had no influence on cell migrations, but they caused a decrease in cell viability in osteogenic conditions when not diluted. Conversely, the eluants of VS20F showed a positive effect on stem cell differentiation. This result was corroborated through ALP activity, ARS assay. Moreover, upregulation of specific gene markers such as RUNX2, DMP-1, and DSPP was observed in hDPSCs, especially when treated with VS20F. The experimental FDCPC tested in this study exhibits a dose-dependent capacity to promote mineralisation in osteogenic environment. The FDCPC-VS20F seems to be the most promising experimental material suitable for developing of pulp-capping materials with osteogenic and bioactive properties. • The experimental fluoride-doped calcium phosphate cements exhibit a dose-dependent capacity to promote osteogenesis. • Gene expression markers RUNX2, OCN, and MEPE resulted particularly upregulated in VS20F. • Cells treated with 1:50 dilution extracts exhibited elevated ALP activity on day 7, especially in the presence of VS20F. • The experimental calcium phosphate containing 20 % fluoride may be the most promising cements for pulp-capping application. [ABSTRACT FROM AUTHOR]

Objective To observe the promoting effects of mineral trioxide aggregate (MTA) and three modified pulp-capping agents (namely nRoot, Vitapex, and iRoot BP Plus) on the proliferation and differentiation of human dental pulp stem cells (hDPSCs) into odontoblasts. Methods ① Observation on the promoting effects of four different concentrations of pulp-capping agents on proliferation of hDPSCs: In this study, the fifth-passage hDPSCs were divided into the MTA, iRoot BP Plus, nRoot, Vitapex, and control groups. The MTA, iRoot BP Plus, nRoot, and Vitapex groups were treated with various concentrations (0. 02, 0. 2, 1, and 2 mg/mL) of MTA, iRoot BP Plus, nRoot, and Vitapex culture media, respectively; the control group was treated with DMEM/F12 culture medium containing 10% fetal bovine serum (FBS). Cell proliferation activity was assessed using the CCK-8 method at 24 and 48 h after incubation. ② Observation on the promoting effects of four pulp-capping agents on differentiation of hDPSCs into odontoblasts: The optimal inducing concentrations of the four materials for hDPSCs were screened through alkaline phosphatase (ALP) activity detection. The fourth-passage hDPSCs were divided into five groups: control, MTA, iRoot BP Plus, nRoot, and Vitapex groups. The cells in the MTA, iRoot BP Plus, nRoot, and Vitapex groups were treated with osteogenic media containing 0. 2 mg/mL of MTA, iRoot BP Plus, nRoot, and Vitapex, respectively, while the control group was treated with normal osteogenic media. On day 21 of culture, alizarin red staining and semi-quantitative analysis were used to observe the formation of mineralized nodules in each group. On day 7 of culture, Western blotting was employed to detect the expression levels of runt-related transcription factor 2 (RUNX-2), osteocalcin (OCN), dentin sialophosphoprotein (DSPP), and dentin matrix protein 1 (DMP-1). Results On the second day of culture, compared with the control group, the cell proliferation activity was significantly higher in the MTA group, iRoot BP Plus group and nRoot group at concentrations of 0. 02, 0. 2, and 1 mg/mL (all P<0. 05). Additionally, compared with that of the first day of culture, the cell proliferation activity decreased in the MTA group and Vitapex group at 2 mg/mL on the second day (all P<0. 05). Consequently, 0. 2 mg/mL was selected as the subsequent test concentration. In comparison to the nRoot and Vitapex groups, the iRoot BP Plus and MTA groups exhibited higher cellular calcium deposition (all P<0. 05). Furthermore, compared with the control group, the relative expression levels of OCN, RUNX-2, DSPP, and DMP-1 were up-regulated in the MTA, iRoot BP Plus, nRoot, and Vitapex groups (all P<0. 05). Notably, the iRoot BP Plus group demonstrated higher relative expression levels of OCN, RUNX-2, DSPP, and DMP-1 as compared with both the nRoot and Vitapex groups (all P<0. 05). Conclusion Compared with MTA and Vitapex, nRoot and iRoot BP Plus are more effective in promoting the proliferation of hDPSCs and their differentiation into odontoblasts. [ABSTRACT FROM AUTHOR]

Various materials have been used to promote human dental pulp stem cells (hDPSCs) differentiation to produce dentin bridge formation with less-than-optimal results. Polydatin (PD), a naturally present material with osteogenic properties can be a promising material in the pulp regeneration/repair process. The aim of this study was to evaluate the effect of (PD) on the viability and differentiation of human dental pulp stem cells. PD effect on hDPSCs in terms of cellular viability, alkaline phosphatase (ALP) production, and messenger RNAs (mRNA) of odontogenic markers production using quantitative reverse transcription polymerase chain reaction (RT-qPCR) were evaluated. In addition, mineral deposits were detected with Alizarin red stain. The viable hDPSCs in the presence of 0.01 μM and 0.1 μM PD were significantly higher than the control on days 3 and 7, respectively. In addition, ALP activity of hDPSCs was significantly increased with 0.01, 0.1, and 1 μM of PD. In addition, increased expression mRNAs of ALP, osteocalcin (OC), osteonectin (ON), osteopontin (OP), Runt-related transcription factor-2 (RUNX-2), dentin sialophosphoprotein (DSPP), and dentin matrix protein-1 (DMP-1) was observed after PD treatment, however, the difference was not statistically significant. Furthermore, increased size of mineral deposits was observed with PD. PD promoted the expression of markers associated with odontogenic differentiation and mineralized tissue deposition in hDPSCs. [ABSTRACT FROM AUTHOR]

Abstract Dental pulp stem cells (DPSCs) show potential for treating neurodegenerative and traumatic diseases due to their neural crest origin. Melatonin (MT), an endogenous neurohormone with well-documented anti-inflammatory and antioxidant properties, has shown promising results with MSCs in terms of engraftment, proliferation, and neuronal differentiation in animal SCI models. However, the effects of melatonin preconditioning on human dental pulp stem cells (hDPSCs) for SCI treatment remain unclear. This study investigates the impact of melatonin preconditioning on hDPSCs engraftment, neural differentiation, and neurological function in rats with SCI. Forty-two male Sprague–Dawley rats were divided into six groups: Control, Sham, Model, Vehicle, Lesion Treatment A (SCI + hDPSCs), and Lesion Treatment B (SCI + MT-hDPSCs). After obtaining hDPSCs, stem cells were evaluated using flow cytometry. Cell viability was assessed using the MTT assay. SCI was induced in the Model, Vehicle, Lesion Treatment A, and Lesion Treatment B groups. The Lesion Treatment A and B groups received hDPSCs and hDPSCs pretreated with melatonin, respectively, 1 week after SCI, while the Vehicle group received only an intravenous injection of DMEM to simulate treatment. The other groups were used for behavioral testing. Immunohistochemistry (IHC) was employed to assess hDPSCs engraftment and differentiation at the SCI site. Motor function across the six groups was evaluated using the Basso, Beattie, and Bresnahan (BBB) score. Histological studies and cell counts confirmed hDPSCs implantation at the injury site, with a significantly higher presence in the MT-hDPSCs compared to hDPSCs (p

Background/purpose: Various materials have been used to promote human dental pulp stem cells (hDPSCs) differentiation to produce dentin bridge formation with less-than-optimal results. Polydatin (PD), a naturally present material with osteogenic properties can be a promising material in the pulp regeneration/repair process. The aim of this study was to evaluate the effect of (PD) on the viability and differentiation of human dental pulp stem cells. Materials and methods: PD effect on hDPSCs in terms of cellular viability, alkaline phosphatase (ALP) production, and messenger RNAs (mRNA) of odontogenic markers production using quantitative reverse transcription polymerase chain reaction (RT-qPCR) were evaluated. In addition, mineral deposits were detected with Alizarin red stain. Results: The viable hDPSCs in the presence of 0.01 μM and 0.1 μM PD were significantly higher than the control on days 3 and 7, respectively. In addition, ALP activity of hDPSCs was significantly increased with 0.01, 0.1, and 1 μM of PD. In addition, increased expression mRNAs of ALP, osteocalcin (OC), osteonectin (ON), osteopontin (OP), Runt-related transcription factor-2 (RUNX-2), dentin sialophosphoprotein (DSPP), and dentin matrix protein-1 (DMP-1) was observed after PD treatment, however, the difference was not statistically significant. Furthermore, increased size of mineral deposits was observed with PD. Conclusion: PD promoted the expression of markers associated with odontogenic differentiation and mineralized tissue deposition in hDPSCs.

Abstract Background Insulin has been known to regulate bone metabolism, yet its specific molecular mechanisms during the proliferation and osteogenic differentiation of dental pulp stem cells (DPSCs) remain poorly understood. This study aimed to explore the effects of insulin on the bone formation capability of human DPSCs and to elucidate the underlying mechanisms. Methods Cell proliferation was assessed using a CCK-8 assay. Cell phenotype was analyzed by flow cytometry. Colony-forming unit-fibroblast ability and multilineage differentiation potential were evaluated using Toluidine blue, Oil red O, Alizarin red, and Alcian blue staining. Gene and protein expressions were quantified by real-time quantitative polymerase chain reaction and Western blotting, respectively. Bone metabolism and biochemical markers were analyzed using electrochemical luminescence and chemical colorimetry. Cell adhesion and growth on nano-hydroxyapatite/collagen (nHAC) were observed with a scanning electron microscope. Bone regeneration was assessed using micro-CT, fluorescent labeling, immunohistochemical and hematoxylin and eosin staining. Results Insulin enhanced the proliferation of human DPSCs as well as promoted mineralized matrix formation in a concentration-dependent manner. 10− 6 M insulin significantly up-regulated osteogenic differentiation-related genes and proteins markedly increased the secretion of bone metabolism and biochemical markers, and obviously stimulated mineralized matrix formation. However, it also significantly inhibited the expression of genes and proteins of receptors and receptor substrates associated with insulin/insulin-like growth factor-1 signaling (IIS) pathway, obviously reduced the expression of the phosphorylated PI3K and the ratios of the phosphorylated PI3K/total PI3K, and notably increased the expression of the total PI3K, phosphorylated AKT, total AKT and mTOR. The inhibitor LY294002 attenuated the responsiveness of 10− 6 M insulin to IIS/PI3K/AKT/mTOR pathway axis, suppressing the promoting effect of insulin on cell proliferation, osteogenic differentiation and bone formation. Implantation of 10− 6 M insulin treated DPSCs into the backs of severe combined immunodeficient mice and the rabbit jawbone defects resulted in enhanced bone formation. Conclusions Insulin induces insulin resistance in human DPSCs and effectively promotes their proliferation, osteogenic differentiation and bone formation capability through gradually inducing the down-regulation of IIS/PI3K/AKT/mTOR pathway axis under insulin resistant states.

Abstract Most of the conditions involving cartilaginous tissues are irreversible and involve degenerative processes. The aim of the present study was to fabricate a biocompatible fibrous and film scaffolds using electrospinning and casting techniques to induce chondrogenic differentiation for possible application in cartilaginous tissue regeneration. Polycaprolactone (PCL) electrospun nanofibrous scaffolds and PCL film were fabricated and incorporated with multi-walled carbon nanotubes (MWCNTs). Thereafter, coating of chondroitin sulfate (CS) on the fibrous and film structures was applied to promote chondrogenic differentiation of human dental pulp stem cells (hDPSCs). First, the morphology, hydrophilicity and mechanical properties of the scaffolds were characterized by scanning electron microscopy (SEM), spectroscopic characterization, water contact angle measurements and tensile strength testing. Subsequently, the effects of the fabricated scaffolds on stimulating the proliferation of human dental pulp stem cells (hDPSCs) and inducing their chondrogenic differentiation were evaluated via electron microscopy, flow cytometry and RT‒PCR. The results of the study demonstrated that the different forms of the fabricated PCL-MWCNTs scaffolds analyzed demonstrated biocompatibility. The nanofilm structures demonstrated a higher rate of cellular proliferation, while the nanofibrous architecture of the scaffolds supported the cellular attachment and differentiation capacity of hDPSCs and was further enhanced with CS addition. In conclusion, the results of the present investigation highlighted the significance of this combination of parameters on the viability, proliferation and chondrogenic differentiation capacity of hDPSCs seeded on PCL-MWCNT scaffolds. This approach may be applied when designing PCL-based scaffolds for future cell-based therapeutic approaches developed for chondrogenic diseases.

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The potential therapeutic benefits of human dental pulp stem cells (HDPSCs) in dental regenerative medicine have been demonstrated. However, little is known about the molecular mechanisms regulating the biological characteristics of HDPSCs. The experiment aims to explore whether VEGF activates signaling pathways such as FAK, PI3K, Akt, and p38 in HDPSCs, and to investigate the molecular mechanisms by which VEGF influences proliferation and migration of HDPSCs. Normal and inflamed human dental pulp (HDP) samples were collected, and the levels of VEGF in HDP were assessed. HDPSCs were cultured and purified. HDPSCs were stimulated with lipopolysaccharide (LPS) at gradient concentrations, and real-time quantitative polymerase chain reaction (qPCR) was used to assess changes in VEGF mRNA. Gradient concentrations of VEGF were used to stimulate HDPSCs, and cell migration ability was evaluated through scratch assays and Transwell chamber experiments. Phosphorylation levels of FAK, AKT, and P38 were assessed using Western blotting. Inhibitors of VEGFR2, FAK, AKT, P38, and VEGF were separately applied to HDPSCs, and cell migration ability and phosphorylation levels of FAK, AKT, and P38 were determined. The results indicated significant differences in VEGF levels between normal and inflamed HDP tissues, with levels in the inflamed state reaching 435% of normal levels (normal: 87.91 ng/mL, inflamed: 382.76 ng/mL, P < 0.05). LPS stimulation of HDPSCs showed a significant increase in VEGF mRNA expression with increasing LPS concentrations (LPS concentrations of 0.01, 0.1, 1, and 10 μg/mL resulted in VEGF mRNA expressions of 181.2%, 274.2%, 345.8%, and 460.9%, respectively, P < 0.05). VEGF treatment significantly enhanced the migration ability of HDPSCs in Transwell chamber experiments, with migration rates increasing with VEGF concentrations (VEGF concentrations of 0, 1, 10, 20, 50, and 100 ng/mL resulted in migration rates of 8.41%, 9.34%, 21.33%, 28.41%, 42.87%, and 63.15%, respectively, P < 0.05). Inhibitors of VEGFR2, FAK, AKT, P38, and combined VEGF stimulation demonstrated significant migration inhibition, with migration rates decreasing to 8.31%, 12.64%, 13.43%, 18.32%, and 74.17%, respectively. The migration rate with combined VEGF stimulation showed a significant difference (P < 0.05). The analysis of phosphorylation levels revealed that VEGF stimulation significantly activated phosphorylation of FAK, AKT, and P38, with phosphorylation levels increasing with VEGF concentrations (P < 0.05). The VEGF/VEGFR2 signaling axis regulated the migration ability of HDPSCs through the FAK/PI3K/AKT and P38MAPK pathways. This finding highlighted not only the crucial role of VEGF in injury repair of HDPSCs but also provided important clues for a comprehensive understanding of the potential applications of this signaling axis in dental regenerative medicine. [ABSTRACT FROM AUTHOR]

Background: Insulin has been known to regulate bone metabolism, yet its specific molecular mechanisms during the proliferation and osteogenic differentiation of dental pulp stem cells (DPSCs) remain poorly understood. This study aimed to explore the effects of insulin on the bone formation capability of human DPSCs and to elucidate the underlying mechanisms. Methods: Cell proliferation was assessed using a CCK-8 assay. Cell phenotype was analyzed by flow cytometry. Colony-forming unit-fibroblast ability and multilineage differentiation potential were evaluated using Toluidine blue, Oil red O, Alizarin red, and Alcian blue staining. Gene and protein expressions were quantified by real-time quantitative polymerase chain reaction and Western blotting, respectively. Bone metabolism and biochemical markers were analyzed using electrochemical luminescence and chemical colorimetry. Cell adhesion and growth on nano-hydroxyapatite/collagen (nHAC) were observed with a scanning electron microscope. Bone regeneration was assessed using micro-CT, fluorescent labeling, immunohistochemical and hematoxylin and eosin staining. Results: Insulin enhanced the proliferation of human DPSCs as well as promoted mineralized matrix formation in a concentration-dependent manner. 10− 6 M insulin significantly up-regulated osteogenic differentiation-related genes and proteins markedly increased the secretion of bone metabolism and biochemical markers, and obviously stimulated mineralized matrix formation. However, it also significantly inhibited the expression of genes and proteins of receptors and receptor substrates associated with insulin/insulin-like growth factor-1 signaling (IIS) pathway, obviously reduced the expression of the phosphorylated PI3K and the ratios of the phosphorylated PI3K/total PI3K, and notably increased the expression of the total PI3K, phosphorylated AKT, total AKT and mTOR. The inhibitor LY294002 attenuated the responsiveness of 10− 6 M insulin to IIS/PI3K/AKT/mTOR pathway axis, suppressing the promoting effect of insulin on cell proliferation, osteogenic differentiation and bone formation. Implantation of 10− 6 M insulin treated DPSCs into the backs of severe combined immunodeficient mice and the rabbit jawbone defects resulted in enhanced bone formation. Conclusions: Insulin induces insulin resistance in human DPSCs and effectively promotes their proliferation, osteogenic differentiation and bone formation capability through gradually inducing the down-regulation of IIS/PI3K/AKT/mTOR pathway axis under insulin resistant states. [ABSTRACT FROM AUTHOR]

Most of the conditions involving cartilaginous tissues are irreversible and involve degenerative processes. The aim of the present study was to fabricate a biocompatible fibrous and film scaffolds using electrospinning and casting techniques to induce chondrogenic differentiation for possible application in cartilaginous tissue regeneration. Polycaprolactone (PCL) electrospun nanofibrous scaffolds and PCL film were fabricated and incorporated with multi-walled carbon nanotubes (MWCNTs). Thereafter, coating of chondroitin sulfate (CS) on the fibrous and film structures was applied to promote chondrogenic differentiation of human dental pulp stem cells (hDPSCs). First, the morphology, hydrophilicity and mechanical properties of the scaffolds were characterized by scanning electron microscopy (SEM), spectroscopic characterization, water contact angle measurements and tensile strength testing. Subsequently, the effects of the fabricated scaffolds on stimulating the proliferation of human dental pulp stem cells (hDPSCs) and inducing their chondrogenic differentiation were evaluated via electron microscopy, flow cytometry and RT‒PCR. The results of the study demonstrated that the different forms of the fabricated PCL-MWCNTs scaffolds analyzed demonstrated biocompatibility. The nanofilm structures demonstrated a higher rate of cellular proliferation, while the nanofibrous architecture of the scaffolds supported the cellular attachment and differentiation capacity of hDPSCs and was further enhanced with CS addition. In conclusion, the results of the present investigation highlighted the significance of this combination of parameters on the viability, proliferation and chondrogenic differentiation capacity of hDPSCs seeded on PCL-MWCNT scaffolds. This approach may be applied when designing PCL-based scaffolds for future cell-based therapeutic approaches developed for chondrogenic diseases. [ABSTRACT FROM AUTHOR]

Objectives: This study developed a sol–gel tricalcium silicate/graphene oxide (TCS-GO) composite and examined its physicochemical properties, antimicrobial activity, and osteo/odontogenic effect on dental pulp stem cells. Methods: Tricalcium silicate was synthesized and combined with graphene oxide at three different concentrations, namely 0.02%, 0.04%, and 0.08% w/w, while tricalcium silicate and mineral trioxide aggregate served as controls. The setting time, compressive strength, pH, and calcium ion release of the composites were evaluated, as well as antimicrobial properties against Streptococcus mutans and Lactobacillus acidophilus. Additionally, the viability of dental pulp stem cells; apatite forming ability; and the gene expression of Alkaline phosphatase, Dentin sialophosphoprotein, and Runt-related transcription factor 2 were assessed. Results: TCS-GO (0.08%) showed a significantly shorter setting time and higher compressive strength when compared to MTA (p < 0.05). Additionally, tricalcium silicate and TCS-GO groups showed a higher release of Ca ions than MTA, with no significant difference in pH values among the different groups. TCS-GO (0.08%) also demonstrated a significantly stronger antimicrobial effect against Lactobacillus acidophilus compared to MTA (p < 0.05). ALP expression was higher in TCS-GO (0.08%) than MTA on days 3 and 7, while DSPP expression was higher in TCS-GO (0.08%) than MTA on day 3 but reversed on day 7. There was no significant difference in RUNX2 expression between TCS-GO (0.08%) and MTA on days 3 and 7. Conclusions: The TCS-GO (0.08%) composite demonstrated superior physicochemical characteristics and antimicrobial properties compared to MTA. Moreover, the early upregulation of ALP and DSPP markers in TCS-GO (0.08%) indicates that it has the potential to promote and enhance the osteo/odontogenic differentiation of DPSCs. [ABSTRACT FROM AUTHOR]

Objective: Despite that, the odontoblasts of the dental pulp are considered a terminally differentiated type of cell. We were interested in investigating if they express any embryonic, mesenchymal, or neural stem cell markers, along with other differentiation markers they were reported to express previously. Methods: An immunohistochemistry study was performed on wisdom teeth extracted from healthy donors aged between 17 and 19 for dental reasons. Nine markers were tested: c-Myc, SOX2, MCAM, CD73, NCAM1, STRO1, osteocalcin, S100, and Thy1. Results: Odontoblasts expressed the following markers: embryonic stem cell markers SOX2, c-Myc, mesenchymal stem cell marker MCAM, the neural differentiation marker S100, and the osteogenic differentiation marker osteocalcin. Odontoblasts did not express the following markers: mesenchymal stem cell markers CD73, STRO1, Thy1, and neural stem cell marker NCAM1. Conclusion: These findings suggest that odontoblasts’ expression of these stem cell markers may enable them to dedifferentiate under certain conditions. Further investigation is needed into whether dental materials could induce such dedifferentiation for functional dentin regeneration.

Human dental pulp stem cells are a potentially useful resource for cell‐based therapies and tissue repair in dental and medical applications. However, the primary culture of isolated dental pulp stem cells has notably been limited. A major requirement of an ideal human dental pulp stem cell culture system is the preservation of efficient proliferation and innate stemness over prolonged passaging, while also ensuring ease of handling through standard, user‐friendly culture methods. In this study, we have engineered a novel human dental pulp stem cell line, distinguished by the constitutive expression of telomerase reverse transcriptase (TERT), and the conditional expression of the R24C mutant cyclin‐dependent kinase 4 (CDK4R24C) and Cyclin D1. We have named this cell line Tet‐off K4DT hDPSCs. Furthermore, we have conducted a comprehensive comparative analysis of their biological attributes in relation to a previously immortalized human dental pulp stem cells, hDPSC‐K4DT, which were immortalized by the constitutive expression of CDK4R24C, Cyclin D1 and TERT. In Tet‐off K4DT cells, the expression of the K4D genes can be precisely suppressed by the inclusion of doxycycline. Remarkably, Tet‐off K4DT cells demonstrated an extended cellular lifespan, increased proliferative capacity, and enhanced osteogenic differentiation potential when compared to K4DT cells. Moreover, Tet‐off K4DT cells had no observable genomic aberrations and also displayed a sustained expression of stem cell markers even at relatively advanced passages. Taken together, the establishment of this new cell line holds immense promise as powerful experimental tool for both fundamental and applied research involving dental pulp stem cells. Significance Statement: We have engineered a novel human dental pulp stem cell line, distinguished by the constitutive expression of telomerase reverse transcriptase (TERT), and the conditional expression of the R24C mutant cyclin‐dependent kinase 4 (CDK4R24C) and Cyclin D1 (hereafter referred to as Tet‐off K4DT). In Tet‐off K4DT cells, the expression of the K4D genes can be precisely suppressed by the inclusion of doxycycline. Tet‐off K4DT cells have demonstrated an extended cellular lifespan, increased proliferative capacity, and had no observable genomic aberrations and displayed a sustained expression of stem cell markers even at relatively advanced passages. [ABSTRACT FROM AUTHOR]

Despite that, the odontoblasts of the dental pulp are considered a terminally differentiated type of cell. We were interested in investigating if they express any embryonic, mesenchymal, or neural stem cell markers, along with other differentiation markers they were reported to express previously. Methods : An immunohistochemistry study was performed on wisdom teeth extracted from healthy donors aged between 17 and 19 for dental reasons. Nine markers were tested: c-Myc, SOX2, MCAM, CD73, NCAM1, STRO1, osteocalcin, S100, and Thy1. Results : Odontoblasts expressed the following markers: embryonic stem cell markers SOX2, c-Myc, mesenchymal stem cell marker MCAM, the neural differentiation marker S100, and the osteogenic differentiation marker osteocalcin. Odontoblasts did not express the following markers: mesenchymal stem cell markers CD73, STRO1, Thy1, and neural stem cell marker NCAM1. Conclusion : These findings suggest that odontoblasts' expression of these stem cell markers may enable them to dedifferentiate under certain conditions. Further investigation is needed into whether dental materials could induce such dedifferentiation for functional dentin regeneration. [ABSTRACT FROM AUTHOR]

In recent years, there has been a surge in demand for and research focus on cell therapy, driven by the tissue-regenerative and disease-treating potentials of stem cells. Among the candidates, dental pulp stem cells (DPSCs) or human exfoliated deciduous teeth (SHED) have garnered significant attention due to their easy accessibility (non-invasive), multi-lineage differentiation capability (especially neurogenesis), and low immunogenicity. Utilizing these stem cells for clinical purposes requires careful culture techniques such as excluding animal-derived supplements. Human platelet lysate (hPL) has emerged as a safer alternative to fetal bovine serum (FBS) for cell culture. In our study, we assessed the impact of hPL as a growth factor supplement for culture medium, also conducting a characterization of SHED cultured in hPL-supplemented medium (hPL-SHED). The results showed that hPL has effects in enhancing cell proliferation and migration and increasing cell survivability in oxidative stress conditions induced by H2O2. The morphology of hPL-SHED exhibited reduced size and elongation, with a differentiation capacity comparable to or even exceeding that of SHED cultured in a medium supplemented with fetal bovine serum (FBS-SHED). Moreover, no evidence of chromosome abnormalities or tumor formation was detected. In conclusion, hPL-SHED emerges as a promising candidate for cell therapy, exhibiting considerable potential for clinical investigation. [ABSTRACT FROM AUTHOR]

Repair strategies for injured peripheral nerve have achieved great progresses in recent years. However, the clinical outcomes remain unsatisfactory. Recent studies have found that exosomes secreted by dental pulp stem cells (DPSC-exos) have great potential for applications in nerve repair. In this study, we evaluated the effects of human DPSC-exos on improving peripheral nerve regeneration. Initially, we established a coculture system between DPSCs and Schwann cells (SCs) in vitro to assess the effect of DPSC-exos on the activity of embryonic dorsal root ganglion neurons (DRGs) growth in SCs. We extracted and labeled human DPSC-exos, which were subsequently utilized in uptake experiments in DRGs and SCs. Subsequently, we established a rat sciatic nerve injury model to evaluate the therapeutic potential of DPSC-exos in repairing sciatic nerve damage. Our findings revealed that DPSC-exos significantly promoted neurite elongation by enhancing the proliferation, migration, and secretion of neurotrophic factors by SCs. In vivo, DPSC-exos administration significantly improved the walking behavior, axon regeneration, and myelination in rats with sciatic nerve injuries. Our study underscores the vast potential of DPSC-exos as a therapeutic tool for tissue-engineered nerve construction. [ABSTRACT FROM AUTHOR]

Abstract Reduced graphene oxide (rGO) is an graphene oxide (GO) derivative of graphene, which has a large specific surface area and exhibited satisfactory physicochemical characteristics. In this experiment, GO was reduced by PDA to generate PDA-GO complex, and then PDA-GO was combined with Chitosan (CS) to synthesize PDA-GO/CS composite scaffold. PDA-GO was added to CS to improve the degradation rate of CS, and it was hoped that PDA-GO/CS composite scaffolds could be used in bone tissue engineering. Physicochemical and antimicrobial properties of the different composite scaffolds were examined to find the optimal mass fraction. Besides, we examined the scaffold’s biocompatibility by Phalloidin staining and Live and Dead fluorescent staining. Finally, we applied ALP staining, RT-qPCR, and Alizarin red S staining to detect the effect of PDA-GO/CS on the osteogenic differentiation of human dental pulp stem cells (hDPSCs). The results showed that PDA-GO composite was successfully prepared and PDA-GO/CS composite scaffold was synthesized by combining PDA-GO with CS. Among them, 0.3%PDA-GO/CS scaffolds improves the antibacterial activity and hydrophilicity of CS, while reducing the degradation rate. In vitro, PDA-GO/CS has superior biocompatibility and enhances the early proliferation, migration and osteogenic differentiation of hDPSCs. In conclusion, PDA-GO/CS is a new scaffold materialsuitable for cell culture and has promising application prospect as scaffold for bone tissue engineering.

Human dental pulp stem cells (hDPSCs) play a vital role in the regeneration of the pulp-dentin complex after pulp disease. While the regeneration efficiency relies on the odontoblastic differentiation capacity of hDPSCs, this is difficult to regulate within the pulp cavity. Although nicotinamide riboside (NR) has been found to promote tissue regeneration, its specific role in pulp-dentin complex regeneration is not fully understood. Here, we aimed to explore the role of NR in the odontoblastic differentiation of hDPSCs and its underlying molecular mechanism. It was found that NR enhanced the viability and retarded senescence in hDPSCs with higher NAD+/NADH levels. In contrast to the sustained action of NR, the multi-directional differentiation of hDPSCs was enhanced after NR pre-treatment. Moreover, in an ectopic pulp regeneration assay in nude mice, transplantation of hDPSCs pretreated with NR promoted the formation of a dentin-like structure surrounded by cells positively expressing DMP-1 and DSPP. RNA-Seq demonstrated inhibition of the HIF-1 signaling pathway in hDPSCs pretreated with NR. The number of HIF-1α-positive cells was significantly decreased in hDPSCs pretreated by NR in vivo. Similarly, NR significantly downregulated the expression of HIF-1α in vitro. The findings suggested that NR could potentially regulate hDPSC odontoblastic differentiation and promote the development of innovative strategies for dental pulp repair. [ABSTRACT FROM AUTHOR]

Aim: This study aimed to investigate the upstream regulators and specific mechanisms of metastasis‐associated lung adenocarcinoma transcript 1 (MALAT1) in the odontoblastic differentiation of human dental pulp stem cells (hDPSCs). Methodology: Human dental pulp stem cells were isolated and cultured, followed by conducting loss‐ or gain‐of‐function experiments on ATF4 and loss experiments on MALAT1 to elucidate their respective biological functions in odontoblastic differentiation. Chromatin immunoprecipitation assays and RNA immunoprecipitation were performed to uncover the interaction between ATF4‐MALAT1 and MALAT1‐JMJD3, respectively. The odontoblastic differentiation was estimated by the mRNA and protein of DSPP and DMP1, as well as alkaline phosphatase staining. Results: Expression of MALAT1 was upregulated in the hDPSCs cultured in an odontoblastic medium, and MALAT1 downregulation suppressed the odontoblastic differentiation of the hDPSCs. Subsequent experiments confirmed that ATF4 promoted odontoblastic differentiation and induced MALAT1 expression by binding to the MALAT1 promoter region. Further experiments revealed that nuclear MALAT1 interacted with JMJD3. MALAT1 knockdown decreased the JMJD3 protein level and demethylase activity, and it enhanced H3K27me3 occupancy of the promoter region of DSPP and DMP1, resulting in the inhibition of DSPP and DMP1 transcription. Importantly, JMJD3 overexpression significantly attenuated the inhibition of odontoblastic differentiation induced by MALAT1 knockdown. Conclusions: ATF4‐regulated MALAT1 plays a positive regulatory role in odontoblastic differentiation of hDPSCs through JMJD3‐mediated H3K27me3 modifications of the DSPP and DMP1 promoters. [ABSTRACT FROM AUTHOR]

Aging severely impairs the beneficial effects of human dental pulp stem cells (hDPSCs) on cartilage regeneration. Lysine demethylase 3A (KDM3A) is involved in regulating mesenchymal stem cells (MSCs) senescence and bone aging. In this study, we investigated the role of KDM3A in hDPSCs aging and whether KDM3A could rejuvenate aged hDPSCs to enhance their chondrogenic differentiation capacity. The cellular aging of hDPSCs was evaluated by senescence-associated β-galactosidase (SA-β-gal) staining. Protein levels were determined using Western blot analysis. KDM3A was overexpressed in aged hDPSCs by lentivirus infection. Quantitative reverse-transcription polymerase chain reaction (RT-qPCR) were used to determine the mRNA levels of stemness markers. Toluidine blue staining was used to evaluate the effect of KDM3A overexpression on the chondrogenic differentiation of aged hDPSCs. hDPSCs at passage 12 or treated with etoposide exhibited augmented cellular senescence as evidenced by increased SA-β-gal activity. KDM3A was significantly increased during senescence of hDPSCs. Overexpression of KDM3A did not affect the stemness properties but significantly promoted the chondrogenic differentiation of aged hDPSCs. Our findings indicate that KDM3A plays an important role in the maintenance of the chondrogenic differentiation capacity of aged hDPSCs and suggest that therapies targeting KDM3A may be a novel strategy to rejuvenate aged hDPSCs. [ABSTRACT FROM AUTHOR]

Critical-size defects (CSDs) of the craniofacial bones cause aesthetic and functional complications that seriously impact the quality of life. The transplantation of human dental pulp stem cells (hDPSCs) is a promising strategy for bone tissue engineering. Chirality is commonly observed in natural biomolecules, yet its effect on stem cell differentiation is seldom studied, and little is known about the underlying mechanism. In this study, supramolecular chiral hydrogels were constructed using L/d-phenylalanine (L/D-Phe) derivatives. The results of alkaline phosphatase expression analysis, alizarin red S assay, as well as quantitative real-time polymerase chain reaction and western blot analyses suggest that right-handed D-Phe hydrogel fibers significantly promoted osteogenic differentiation of hDPSCs. A rat model of calvarial defects was created to investigate the regulation of chiral nanofibers on the osteogenic differentiation of hDPSCs in vivo. The results of the animal experiment demonstrated that the D-Phe group exhibited greater and faster bone formation on hDPSCs. The results of RNA sequencing, vinculin immunofluorescence staining, a calcium fluorescence probe assay, and western blot analysis indicated that L-Phe significantly promoted adhesion of hDPSCs, while D-Phe nanofibers enhanced osteogenic differentiation of hDPSCs by facilitating calcium entry into cells and activate the MAPK pathway. These results of chirality-dependent osteogenic differentiation offer a novel therapeutic strategy for the treatment of CSDs by optimising the differentiation of hDPSCs into chiral nanofibers.

Objective To study the effect of light-emitting diode (LED) red light on the osteogenic/odontogenic differentiation of human dental pulp stem cells (hDPSCs) and its mechanism were discussed. Methods This study has been reviewed and approved by the Ethics Committee. hDPSCs were cultured by tissue block enzyme digestion. The proliferative capacity of hDPSCs was detected by the CCK-8 at days 1, 3, 5 and 7 under stimulation with 0, 1, 5 and 10 μg/mL lipopolysaccharide (LPS), and the LPS stimulatory concentration was screened. The CG group (mineralization induction), LPS+CG group, and LPS+CG+ (2, 4, 6, 8, and 10 J/cm2) LED red light groups were set. On day 7, alkaline phosphatase (ALP) staining and ALP activity were determined. Relative expression levels of the ALP, osterix (OSX), dentin matrix protein-1 (DMP-1) and dentin sialophosphoprotein (DSPP) genes were measured by qRT-PCR. On day 21, alizarin red staining and calcium nodule quantitative determination were performed to screen the best light energy. The LPS+CG group and LPS+CG+LED group (optimal energy) were set up, and the secretion and expression levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were detected by ELISAs on days 1, 3, 5 and 7. The relative expression levels of the extracellular regulated protein kinases 1/2 (ERK1/2), p38, c-Jun N-terminal kinase (JNK), and extracellular regulated protein kinases 5 (ERK5) proteins and their phosphorylated proteins in the MAPK signaling pathway were detected by Western blots. After the pathway was blocked, the relative expression levels of the ALP, OSX, DMP-1, and DSPP proteins after LED red light irradiation on day 7 were detected by Western blots. Results CCK-8 assays showed that the proliferation of hDPSCs induced by 10 μg/mL LPS was lower than that of the 0, 1, and 5 μg/mL groups on the 5th and 7th days (P0.05). Conclusion In inflammatory conditions, LED red light promotes osteogenic/odontogenic differentiation of hDPSCs. This effect may be attributed to enhancement of the ERK1/2, JNK, and ERK5 signaling pathways, which reduces the production of the inflammatory cytokines TNF-α and IL-1β.

Objectives: This study developed a sol–gel tricalcium silicate/graphene oxide (TCS-GO) composite and examined its physicochemical properties, antimicrobial activity, and osteo/odontogenic effect on dental pulp stem cells. Methods: Tricalcium silicate was synthesized and combined with graphene oxide at three different concentrations, namely 0.02%, 0.04%, and 0.08% w/w, while tricalcium silicate and mineral trioxide aggregate served as controls. The setting time, compressive strength, pH, and calcium ion release of the composites were evaluated, as well as antimicrobial properties against Streptococcus mutans and Lactobacillus acidophilus. Additionally, the viability of dental pulp stem cells; apatite forming ability; and the gene expression of Alkaline phosphatase, Dentin sialophosphoprotein, and Runt-related transcription factor 2 were assessed. Results: TCS-GO (0.08%) showed a significantly shorter setting time and higher compressive strength when compared to MTA (p < 0.05). Additionally, tricalcium silicate and TCS-GO groups showed a higher release of Ca ions than MTA, with no significant difference in pH values among the different groups. TCS-GO (0.08%) also demonstrated a significantly stronger antimicrobial effect against Lactobacillus acidophilus compared to MTA (p < 0.05). ALP expression was higher in TCS-GO (0.08%) than MTA on days 3 and 7, while DSPP expression was higher in TCS-GO (0.08%) than MTA on day 3 but reversed on day 7. There was no significant difference in RUNX2 expression between TCS-GO (0.08%) and MTA on days 3 and 7. Conclusions: The TCS-GO (0.08%) composite demonstrated superior physicochemical characteristics and antimicrobial properties compared to MTA. Moreover, the early upregulation of ALP and DSPP markers in TCS-GO (0.08%) indicates that it has the potential to promote and enhance the osteo/odontogenic differentiation of DPSCs.

Abstract Background Redox signaling and energy metabolism are known to be involved in controlling the balance between self-renewal and proliferation/differentiation of stem cells. In this study we investigated metabolic and redox changes occurring during in vitro human dental pulp stem cells (hDPSCs) osteoblastic (OB) differentiation and tested on them the impact of the reactive oxygen species (ROS) signaling. Methods hDPSCs were isolated from dental pulp and subjected to alkaline phosphatase and alizarin red staining, q-RT-PCR, and western blotting analysis of differentiation markers to assess achievement of osteogenic/odontogenic differentiation. Moreover, a combination of metabolic flux analysis and confocal cyto-imaging was used to profile the metabolic phenotype and to evaluate the redox tone of hDPSCs. Results In differentiating hDPSCs we observed the down-regulation of the mitochondrial respiratory chain complexes expression since the early phase of the process, confirmed by metabolic flux analysis, and a reduction of the basal intracellular peroxide level in its later phase. In addition, dampened glycolysis was observed, thereby indicating a lower energy-generating phenotype in differentiating hDPSCs. Treatment with the ROS scavenger Trolox, applied in the early-middle phases of the process, markedly delayed OB differentiation of hDPSCs assessed as ALP activity, Runx2 expression, mineralization capacity, expression of stemness and osteoblast marker genes (Nanog, Lin28, Dspp, Ocn) and activation of ERK1/2. In addition, the antioxidant partly prevented the inhibitory effect on cell metabolism observed following osteogenic induction. Conclusions Altogether these results provided evidence that redox signaling, likely mediated by peroxide species, influenced the stepwise osteogenic expansion/differentiation of hDPSCs and contributed to shape its accompanying metabolic phenotype changes thus improving their efficiency in bone regeneration and repair.

Background/purpose: Human dental pulp stem cells (hDPSCs) possess excellent proliferative and osteogenic differentiation potentials. This study aimed to elucidate the role of lysophosphatidic acid (LPA) signaling in the proliferation and osteogenic differentiation of hDPSCs. Materials and methods: hDPSCs were treated with LPA and proliferation was measured using the cell counting kit-8 assay. Following the osteogenic differentiation of hDPSCs using osteogenic medium in the presence or absence of LPA, alkaline phosphatase (ALP) staining, ALP activity measurements, and RT-qPCR were performed to analyze the osteoblast differentiation. Small interfering RNA (siRNA)-mediated LPAR3 silencing and extracellular signal-regulated (ERK)/mitogen-activated protein (MAP) kinase inhibitors were used to elucidate the molecular mechanisms underlying LPA-induced proliferation and differentiation of hDPSCs. Results: LPA treatment significantly induced proliferation and osteogenic differentiation of hDPSCs. The depletion of LPAR3 expression by LPAR3-speicifc siRNA in hDPSCs diminished LPA-induced proliferation and osteogenic differentiation. The LPAR3-mediated proliferation and osteogenic differentiation of hDPSCs in response to LPA were significantly suppressed by U0126, a selective inhibitor of ERK. Conclusion: These findings suggest that LPA induces the proliferation and osteogenic differentiation of hDPSCs via LPAR3-ERK-dependent pathways.

Introduction:Dental pulp stem cells from humans possess self-renewal and versatile differentiation abilities. These cells, known as DPSC, are promising for tissue engineering due to their outstanding biological characteristics and ease of access without significant donor site trauma. Existing methods for isolating DPSC mainly include enzyme digestion and explant techniques. Compared with the enzymatic digestion technique, the outgrowth method is less prone to cell damage and loss during the operation, which is essential for DPSC with fewer tissue sources.Methods:In order to maximize the amount of stem cells harvested while reducing the cost of DPSC culture, the feasibility of the optimized explant technique was evaluated in this experiment. Cell morphology, minimum cell emergence time, the total amount of cells harvested, cell survival, and proliferative and differentiation capacity of DPSC obtained with different numbers of explant attachments (A1-A5) were evaluated.Results:There was a reduction in the survival rate of the cells in groups A2-A5, and the amount of harvested DPSC decreased in A3-A5 groups, but the DPSC harvested in groups A1-A4 had similar proliferative and differentiation abilities. However, starting from group A5, the survival rate, proliferation and differentiation ability of DPSC decreased significantly, and the adipogenic trend of the cells became more apparent, indicating that the cells had begun to enter the senescence state.Discussion:The results of our study demonstrated that the DPSC obtained by the optimized explant method up to 4 times had reliable biological properties and is available for tissue engineering.

This study aimed to clarify the effects of strontium (Sr2+), borate (BO33−), and silicate (SiO32−) on cell proliferative capacity, the induction of differentiation into odontoblast-like cells (OLCs), and substrate formation of human dental pulp stem cells (hDPSCs). Sr2+, BO33−, and SiO32− solutions were added to the hDPSC culture medium at three different concentrations, totaling nine experimental groups. The effects of these ions on hDPSC proliferation, calcification, and collagen formation after 14, 21, and 28 days of culture were evaluated using a cell proliferation assay, a quantitative alkaline phosphatase (ALP) activity assay, and Alizarin Red S and Sirius Red staining, respectively. Furthermore, the effects of these ions on hDPSC differentiation into OLCs were assessed via quantitative polymerase chain reaction and immunocytochemistry. Sr2+ and SiO32− increased the expression of odontoblast markers; i.e., nestin, dentin matrix protein-1, dentin sialophosphoprotein, and ALP genes, compared with the control group. BO33− increased the ALP gene expression and activity. The results of this study suggested that Sr2+, BO33−, and SiO32− may induce hDPSC differentiation into OLCs. [ABSTRACT FROM AUTHOR]

Aim: The objective of this study was to assess and compare the cytocompatibility of decellularized porcine small intestine submucosal dural graft from Biodesign (BD) and polyester urethane‐based Neuro‐Patch (NP) dural substitute with the mineral trioxide aggregate (MTA) and the cyanoacrylate‐based Histoacryl surgical adhesive. Furthermore, the study evaluated the inflammatory response and osteogenic differentiation of human dental pulp stem cells (hDPSCs) when cultured in direct contact with the dural substitutes in comparison with MTA. Methodology: The viability of hDPSCs in direct contact with the tested materials was investigated in vitro by a CCK‐8 assay. Additionally, the effects of dural substitutes and MTA on the expression of the inflammatory mediator interleukin‐6 (IL‐6) was investigated via enzyme‐linked immunosorbent assay (ELISA), whilst effects on the differentiation were evaluated using alizarin red staining, alkaline phosphatase staining, ELISA and energy‐dispersive X‐ray elemental mapping. Results: The dural substitutes were cytocompatible and promoted cellular adhesion. The Histoacryl and MTA demonstrated cytotoxicity in fresh preparations but showed a more favourable cellular reaction when set. Investigations of biological activity indicated that dural substitute membranes did not induce an inflammatory response or osteogenic differentiation of hDPSCs. In contrast, MTA induced the expression of IL‐6 and alkaline phosphatase activity contributing to enhanced differentiation and mineralization. Conclusions: The dural substitute membranes showed cytocompatibility, did not provoke an inflammatory response and maintained the stemness of hDPSCs better than MTA. Additionally, the set Histoacryl surgical adhesive demonstrated good biocompatibility. Taken together, these results highlight the potential use of dural substitutes in regenerative endodontic procedures as coronal barriers alternative to MTA to reduce the incidence of intracanal calcifications. [ABSTRACT FROM AUTHOR]

In this study we examine the influence of wool-derived keratin intermediate filament proteins (kIFPs) on human dental pulp-derived stem cells (hDPSCs). kIFPs were diluted (10 mg/mL to 0.001 mg/mL) in cell culture media. Effects on hDPSCs proliferation were measured using Alamar blue assay. Keratin concentrations of 1 mg/mL and 0.1 mg/mL were tested for odontogenic differentiation and mineralisation. Alkaline phosphatase (ALP) quantification (7th, 14th, and 21st days), alizarin red S (AR-S) staining and calcium quantification (21st day), reverse transcription polymerase chain reaction (RT-PCR, collagen expression), and immunocytochemical staining for dentin matrix protein (DMP) were performed. hDPSCs showed higher proliferation with kIFPs of 0.1 mg/mL or less (p < 0.0001). The 0.1 mg/mL keratin concentration promoted odontogenic differentiation, confirmed by increased ALP activity, significant calcium deposits (AR-S staining, p < 0.05), up-regulated collagen expression (RT-PCR, p < 0.05), and positive DMP staining. These results suggest that kIFPs could be a potential biomaterial for pulp-dentin regeneration. [ABSTRACT FROM AUTHOR]

Introduction: Stem cells and scaffolds are an important foundation and starting point for tissue engineering. Human dental pulp stem cells (DPSC) are mesenchymal stem cells with self-renewal and multi-directional differentiation potential, and are ideal candidates for tissue engineering due to their excellent biological properties and accessibility without causing major trauma at the donor site. Tetracycline hydrochloride (TCH), a broad-spectrum antibiotic, has been widely used in recent years for the synthesis of cellular scaffolds to reduce the incidence of postoperative infections. Methods: In order to evaluate the effects of TCH on DPSC, the metabolism of DPSC in different concentrations of TCH environment was tested. Moreover, cell morphology, survival rates, proliferation rates, cell migration rates and differentiation abilities of DPSC at TCH concentrations of 0-500 µg/ml were measured. Phalloidin staining, live-dead staining, MTS assay, cell scratch assay and real-time PCR techniques were used to detect the changes in DPSC under varies TCH concentrations. Results: At TCH concentrations higher than 250 µg/ml, DPSC cells were sequestered, the proportion of dead cells increased, and the cell proliferation capacity and cell migration capacity decreased. The osteogenic and adipogenic differentiation abilities of DPSC, however, were already inhibited at TCH concentrations higher than 50 µg/ml. Here, the expression of the osteogenic genes, runt-related transcription factor 2 (RUNX2) and osteocalcin (OCN), the lipogenic genes lipase (LPL), as well as the peroxisome proliferator-activated receptor-γ (PPAR-γ) expression were found to be down-regulated. Discussion: The results of the study indicated that TCH in concentrations above 50 µg/ml negatively affects the differentiation capability of DPSC. In addition, TCH at concentrations above 250 µg/ml adversely affects the growth status, percentage of living cells, proliferation and migration ability of cells. [ABSTRACT FROM AUTHOR]

Human dental pulp stem cells (DPSCs) exhibit multilineage differentiation capabilities and superior clonogenic and proliferative properties. However, the use of animal-derived components such as FBS raises concerns regarding the clinical application of stem-cell-based therapies. Platelet-rich fibrin (PRF) derived from human blood is rich in fibrin, platelets, and growth factors and acts as a bioactive scaffold for grafting with biomaterials. In this study, we assessed the efficacy of PRF-conditioned medium (CM) in promoting DPSCs proliferation and osteogenic differentiation compared with the standard culture medium supplemented with FBS. A comparison of DPSCs cultured in FBS and PRF-CM revealed no differences in characteristics or morphology. However, cells cultured with PRF-CM exhibited inferior proliferation rates and cell numbers during passage in comparison with those cultured with FBS. In contrast, DPSCs cultured in PRF-CM showed significantly higher levels of calcification, and RT-PCR confirmed that the gene expression levels of markers associated with osteoblast differentiation were significantly increased. The PRF-CM approach offers a convenient, straightforward, and advantageous method for culturing DPSCs, without relying on animal-derived components. In summary, this study introduces a novel application of PRF-CM for enhancing the osteogenesis of DPSCs, which provides an alternative to FBS culture medium and addresses concerns associated with the use of animal-derived components in clinical settings. [ABSTRACT FROM AUTHOR]

Dental pulp is built by proteins that have various roles in the biological process of pulp, such as structural protein, regulation protein, and catalytic protein. L-arginine, an amino acid and one of the building blocks of proteins, regulates pro-inflammatory and anti-inflammatory activity. Therefore, L-arginine-based culture has potential to promote dental pulp regeneration. This study aimed to investigate the potential of L-arginine-based culture in improving the viability of human dental pulp stem cells (hDPSCs). We evaluated the viability of hDPSCs in culture media supplemented with different concentrations of L-arginine amino acid (250, 300, 350, and 400 μmol/L) and Dulbecco's Modified Eagle Medium plus fetal bovine serum 10% (control) using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay after 24-h incubation time. Statistical analysis was conducted using a one-way analysis of variance and post hoc least significant difference test. In qualitative analysis, the 4´, 6-diamidino-2-phenylindole staining method was used. The evaluation has shown a significant result when 250, 300, and 350 μmol/L concentration of L-arginine amino acid culture media compared with control, and 400 μmol/L has the best result and was not significantly different with control toward viability of hDPSCs. [ABSTRACT FROM AUTHOR]

Abstract Background Human dental pulp stem cells (hDPSCs) may be the best choice for self-repair and regeneration of teeth and maxillofacial bone tissue due to their homogeneous tissue origin, high proliferation and differentiation rates, and no obvious ethical restrictions. Recently, several studies have shown that extracellular matrix (ECM) proteins can effectively regulate the proliferation and differentiation fate of mesenchymal stem cells (MSCs). However, the role of elastin microfibril interface-located protein-1 (EMILIN-1), a new ECM glycoprotein, in osteo/odontogenic differentiation of hDPSCs has not been reported. The aim of this study was to explore the effect of EMILIN-1 during osteo/odontogenic differentiation of hDPSCs. Methods hDPSCs were cultured in osteo/odontogenic induction medium. qPCR and Western blot analysis were performed to detect osteo/odonto-specific genes/proteins expression as well as the expression of EMILIN-1. After knockdown of Emilin-1 in hDPSCs with small interfering RNA and exogenous addition of recombinant human EMILIN-1 protein (rhEMILIN-1), Cell Counting Kit-8 assay, alkaline phosphatase staining, alizarin red S staining, qPCR and Western blot were performed to examine the effect of EMILIN-1 on proliferation and osteo/odontogenic differentiation of hDPSCs. Results During the osteo/odontogenic induction of hDPSCs, the expression of osteo/odonto-specific genes/proteins increased, as did EMILIN-1 protein levels. More notably, knockdown of Emilin-1 decreased hDPSCs proliferation and osteo/odontogenic differentiation, whereas exogenous addition of rhEMILIN-1 increased them. Conclusions These findings suggested that EMILIN-1 is essential for the osteo/odontogenic differentiation of hDPSCs, which may provide new insights for teeth and bone tissue regeneration.

Background/purpose: Naringenin, a naturally occurring flavanone in citrus fruits, regulates bone formation by bone marrow-derived mesenchymal stem cells. The purpose of this study was to characterize the effects of naringenin on some biological behaviors of human dental pulp stem cells (HDPSCs). Materials and methods: HDPSCs were cultured in osteogenic differentiation medium and osteo/odontogenic differentiation and mineralization were analyzed by alkaline phosphatase (ALP) staining and Alizarin Red S (ARS) staining. The migration of HDPSCs was evaluated by transwell chemotactic migration assays and scratch wound healing migration assay. Using tooth slice/scaffold model, we assessed the in vivo odontogenic differentiation potential of HDPSCs. Results: We have demonstrated that naringenin increases the osteogenic/odontogenic differentiation of HDPSCs through regulation of osteogenic-related proteins and the migratory ability of HDPSCs through stromal cell derived factor-1 (SDF-1)/C-X-C chemokine receptor type 4 (CXCR4) axis. Moreover, naringenin promotes the expression of dentin matrix acidic phosphoprotein-1 (DMP-1) and dentin sialophosphoprotein (DSPP) in HDPSCs seeded on tooth slice/scaffolds that are subcutaneously implanted into immunodeficient mice. Conclusion: Our present study suggests that naringenin promotes migration and osteogenic/odontogenic differentiation of HDPSCs and may serve as a promising candidate in dental tissue engineering and bone regeneration.

In recent years, there has been a surge in demand for and research focus on cell therapy, driven by the tissue-regenerative and disease-treating potentials of stem cells. Among the candidates, dental pulp stem cells (DPSCs) or human exfoliated deciduous teeth (SHED) have garnered significant attention due to their easy accessibility (non-invasive), multi-lineage differentiation capability (especially neurogenesis), and low immunogenicity. Utilizing these stem cells for clinical purposes requires careful culture techniques such as excluding animal-derived supplements. Human platelet lysate (hPL) has emerged as a safer alternative to fetal bovine serum (FBS) for cell culture. In our study, we assessed the impact of hPL as a growth factor supplement for culture medium, also conducting a characterization of SHED cultured in hPL-supplemented medium (hPL-SHED). The results showed that hPL has effects in enhancing cell proliferation and migration and increasing cell survivability in oxidative stress conditions induced by H2O2. The morphology of hPL-SHED exhibited reduced size and elongation, with a differentiation capacity comparable to or even exceeding that of SHED cultured in a medium supplemented with fetal bovine serum (FBS-SHED). Moreover, no evidence of chromosome abnormalities or tumor formation was detected. In conclusion, hPL-SHED emerges as a promising candidate for cell therapy, exhibiting considerable potential for clinical investigation.

Background: Redox signaling and energy metabolism are known to be involved in controlling the balance between self-renewal and proliferation/differentiation of stem cells. In this study we investigated metabolic and redox changes occurring during in vitro human dental pulp stem cells (hDPSCs) osteoblastic (OB) differentiation and tested on them the impact of the reactive oxygen species (ROS) signaling. Methods: hDPSCs were isolated from dental pulp and subjected to alkaline phosphatase and alizarin red staining, q-RT-PCR, and western blotting analysis of differentiation markers to assess achievement of osteogenic/odontogenic differentiation. Moreover, a combination of metabolic flux analysis and confocal cyto-imaging was used to profile the metabolic phenotype and to evaluate the redox tone of hDPSCs. Results: In differentiating hDPSCs we observed the down-regulation of the mitochondrial respiratory chain complexes expression since the early phase of the process, confirmed by metabolic flux analysis, and a reduction of the basal intracellular peroxide level in its later phase. In addition, dampened glycolysis was observed, thereby indicating a lower energy-generating phenotype in differentiating hDPSCs. Treatment with the ROS scavenger Trolox, applied in the early-middle phases of the process, markedly delayed OB differentiation of hDPSCs assessed as ALP activity, Runx2 expression, mineralization capacity, expression of stemness and osteoblast marker genes (Nanog, Lin28, Dspp, Ocn) and activation of ERK1/2. In addition, the antioxidant partly prevented the inhibitory effect on cell metabolism observed following osteogenic induction. Conclusions: Altogether these results provided evidence that redox signaling, likely mediated by peroxide species, influenced the stepwise osteogenic expansion/differentiation of hDPSCs and contributed to shape its accompanying metabolic phenotype changes thus improving their efficiency in bone regeneration and repair. [ABSTRACT FROM AUTHOR]

Human dental pulp stem cells (hDPSCs) possess excellent proliferative and osteogenic differentiation potentials. This study aimed to elucidate the role of lysophosphatidic acid (LPA) signaling in the proliferation and osteogenic differentiation of hDPSCs. hDPSCs were treated with LPA and proliferation was measured using the cell counting kit-8 assay. Following the osteogenic differentiation of hDPSCs using osteogenic medium in the presence or absence of LPA, alkaline phosphatase (ALP) staining, ALP activity measurements, and RT-qPCR were performed to analyze the osteoblast differentiation. Small interfering RNA (siRNA)-mediated LPAR3 silencing and extracellular signal-regulated (ERK)/mitogen-activated protein (MAP) kinase inhibitors were used to elucidate the molecular mechanisms underlying LPA-induced proliferation and differentiation of hDPSCs. LPA treatment significantly induced proliferation and osteogenic differentiation of hDPSCs. The depletion of LPAR3 expression by LPAR3-speicifc siRNA in hDPSCs diminished LPA-induced proliferation and osteogenic differentiation. The LPAR3-mediated proliferation and osteogenic differentiation of hDPSCs in response to LPA were significantly suppressed by U0126, a selective inhibitor of ERK. These findings suggest that LPA induces the proliferation and osteogenic differentiation of hDPSCs via LPAR3-ERK-dependent pathways. [ABSTRACT FROM AUTHOR]

Background: Human dental pulp stem cells (HDPSCs) may be differentiated into neural lineages. The main aim of the study was to assess the DNA demethylation and histone deacetylation inhibition on the differentiation of HDPSCs into neural progenitor-like cells (NPCs). Methods: HDPSCs were treated with 5-aza2′-deoxycytidine (AZA), DNA methylation inhibitor, and the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) for 3 and 5 days followed by their differentiation into NPCs. The efficiency of the differentiation was evaluated by apoptosis, cellular proliferation, and relative expression of Nestin among the NPCs derived with the different treatments. Results: Five-day treatment of AZA was crucial for the more efficient demethylation of the HDPSCs. Analysis of the proliferation, apoptosis, and relative expression of the Nestin indicated that the AZA and SAHA neither enhance nor inhibit the differentiation of the HDPSCs into NPCs. Howevere, the expression of Nestin decreased at day 7 in NPCs derived with SAHAH treatment compared with NPCs derived with AZA treatmement. However, there was no difference in Nestin expression in any treatment-derived NPCs compared with control NPCs. All of the NPCs derived from all of the groups were able to differentiate into terminal neurons. Conclusion: Neither DNA demethylation nor the histone deacetylation has any main effects on proliferation and apoptosis during the differentiation of HDPSCs into NPCs. The only significant effect of the treatments was on the size of the NPCs at day 7; the SAHAH treatment had the smallest NPCs.

Dental pulp is built by proteins that have various roles in the biological process of pulp, such as structural protein, regulation protein, and catalytic protein. L-arginine, an amino acid and one of the building blocks of proteins, regulates pro-inflammatory and anti-inflammatory activity. Therefore, L-arginine-based culture has potential to promote dental pulp regeneration. This study aimed to investigate the potential of L-arginine-based culture in improving the viability of human dental pulp stem cells (hDPSCs). We evaluated the viability of hDPSCs in culture media supplemented with different concentrations of L-arginine amino acid (250, 300, 350, and 400 μmol/L) and Dulbecco's Modified Eagle Medium plus fetal bovine serum 10% (control) using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay after 24-h incubation time. Statistical analysis was conducted using a one-way analysis of variance and post hoc least significant difference test. In qualitative analysis, the 4´, 6-diamidino-2-phenylindole staining method was used. The evaluation has shown a significant result when 250, 300, and 350 μmol/L concentration of L-arginine amino acid culture media compared with control, and 400 μmol/L has the best result and was not significantly different with control toward viability of hDPSCs.

Salivary glands (SG) are exocrine organs with secretory units commonly injured by radiotherapy. Bio-engineered organoids and extracellular vesicles (EV) are currently under investigation as potential strategies for SG repair. Herein, three-dimensional (3D) cultures of SG functional organoids (SGo) and human dental pulp stem cells (hDPSC) were generated by magnetic 3D bioassembly (M3DB) platforms. Fibroblast growth factor 10 (FGF10) was used to enrich the SGo in secretory epithelial units. After 11 culture days via M3DB, SGo displayed SG-specific acinar epithelial units with functional properties upon neurostimulation. To consistently develop 3D hDPSC in vitro, 3 culture days were sufficient to maintain hDPSC undifferentiated genotype and phenotype for EV generation. EV isolation was performed via sequential centrifugation of the conditioned media of hDPSC and SGo cultures. EV were characterized by nanoparticle tracking analysis, electron microscopy and immunoblotting. EV were in the exosome range for hDPSC (diameter: 88.03 ± 15.60 nm) and for SGo (123.15 ± 63.06 nm). Upon ex vivo administration, exosomes derived from SGo significantly stimulated epithelial growth (up to 60%), mitosis, epithelial progenitors and neuronal growth in injured SG; however, such biological effects were less distinctive with the ones derived from hDPSC. Next, these exosome biological effects were investigated by proteomic arrays. Mass spectrometry profiling of SGo exosomes predicted that cellular growth, development and signaling was due to known and undocumented molecular targets downstream of FGF10. Semaphorins were identified as one of the novel targets requiring further investigations. Thus, M3DB platforms can generate exosomes with potential to ameliorate SG epithelial damage.

Objective: The buccal fat pad is a source of adipose stem cells accessible from the oral cavity and dental pulp is a common dental source of stem cells. This study aimed to assess the biological behaviors and osteogenic differentiation of human buccal fat pad adipose-derived stem cells (hBFP-ADSCs) and human dental pulp derived stem cells (hDPSCs). Material and Methods: Buccal fat pads (595.4±80 mg) and dental pulp from 2 wisdom teeth were obtained from 3 matched donors and hBFP-ADSCs and hDPSC cells, respectively, and isolated. The isolated cells were sequentially expanded through passages 5, 10, and 15 for analysis. Cell growth, osteogenic differentiation, and cell senescence were investigated. Results: HBFP-ADSCs and hDPSCs exhibited different cell morphologies and behaviors. Cell expansion was associated with the decrease of cell growth and in vitro mineralization of hDPSCs. The cell yields of hBFP-ADSCs and hDPSCs at the primary passage were 3.2x10^5 ±9.9x10^3 cells/100 mg and 1.13x10^6 ±2.4x10^5 cells/tooth, respectively. The expanded cells exhibited a limited life span and maintained normal karyotypes at the late cell expansion stage. Conclusion: HBFP-ADSCs showed steady cell growth, high osteogenic differentiation potential, tissue availability, and minimal in vitro cell expansion. The availability and accessibility of hBFP-ADSCs would enable clinical applicability as a stem cell source for bone regeneration.

Abstract Background The repair of cranio-maxillofacial bone defects remains a formidable clinical challenge. The Ets variant 2 (ETV2) transcription factor, which belongs to the E26 transformation-specific (ETS) family, has been reported to play a key role in neovascularization. However, the role of ETV2 in the osteogenesis of human dental pulp stem cells (hDPSCs) remains unexplored. Methods Transgenic overexpression of ETV2 was achieved using a lentiviral vector, based on a Dox-inducible system. The effects of Dox-induced overexpression of ETV2 on the osteogenesis of hDPSCs were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR), western blot, immunofluorescence staining, alkaline phosphatase (ALP) staining, and Alizarin Red S (ARS) staining. Additionally, RNA-sequencing (RNA-Seq) analysis was performed to analyze the underlying mechanisms of ETV2-induced osteogenesis. Additionally, the role of ETV2 overexpression in bone formation in vivo was validated by animal studies with a rat calvarial defect model and a nude mice model. Results Our results demonstrated that ETV2 overexpression significantly upregulated the mRNA and protein expression levels of osteogenic markers, markedly enhanced ALP activity, and promoted matrix mineralization of hDPSCs. Moreover, the results of RNA-Seq analysis and western blot showed that the ERK/MAPK and PI3K-Akt signaling pathways were activated upon transgenic overexpression of ETV2. The enhanced osteogenic differentiation of hDPSCs due to ETV2 overexpression was partially reversed by treatment with inhibitors of ERK/MAPK or PI3K-AKT signaling. Furthermore, the results of in vivo studies demonstrated that ETV2 overexpression improved bone healing in a rat calvarial defect model and increased ectopic bone formation in nude mice. Conclusions Collectively, our results indicated that ETV2 overexpression exerted positive effects on the osteogenesis of hDPSCs, at least partially via the ERK/MAPK and PI3K/AKT signaling pathways.

Objectives: This study aims to investigate the anti-inflammatory effect of curcumin and underlying mechanisms regarding the modulation of the nod-like receptor pyrin domain containing 3 (NLRP3) inflammasome in human dental pulp stem cells (hDPSCs). Materials and methods: The impact of curcumin on the viability of hDPSCs was evaluated. The effect of curcumin on the expression of IL-1β and NLRP3 in hDPSCs stimulated by lipopolysaccharide (LPS) was assessed. Then, LPS-primed hDPSCs were pre-treated with curcumin before ATP triggering NLRP3 inflammasome activation, and NLRP3 inflammasome-related mediators were assessed. The mechanism of curcumin inactivation of LPS plus ATP-induced inflammasome associated with NF-κB pathway was explored. The NF-κB pathway related pro-inflammatory mediators at mRNA and protein levels were evaluated. The expression of NF-κB p65 and phosphorylation p65 was visualized after curcumin or NF-κB inhibitor administrating respectively in hDPSCs with an activated NLRP3 inflammasome. Statistical analysis was performed. Results: While curcumin at the concentration of 0.5–5 μM showed no obvious impact on the viability of hDPSCs, it significantly decreased IL-1β and NLRP3 mRNA expression in LPS-induced hDPSCs in a dose-dependent manner. Curcumin significantly inhibited the LPS plus ATP-primed NLRP3 inflammasome activation in hDPSCs (NLRP3, ASC, caspase-1, and IL-1β). Curcumin evidently attenuated the LPS plus ATP-induced expression of NF-κB pathway-related pro-inflammatory mediators (IL-6, IL-8, TNF-α, and COX-2). Furthermore, curcumin effectively reduced p65 phosphorylation, which acts as an NF-κB inhibitor in hDPSCs with an activated NLRP3 inflammasome. Conclusions: Curcumin pre-treatment may exert an anti-inflammatory role via inactivation of the NLRP3 inflammasome by inhibiting NF-κB p65 phosphorylation in cultured hDPSCs. Clinical relevance: Curcumin may have therapeutic potential in pulp inflammation. [ABSTRACT FROM AUTHOR]