Human Dental Pulp Research Articles

Therapeutic potential of melatonin-pretreated human dental pulp stem cells (hDPSCs) in an animal model of spinal cord injury.

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.

Divergent Proteomic Profiles and Uptake Mechanisms of Exosomes Derived from Human Dental Pulp Stem Cells, Endothelial Cells, and Fibroblasts.

Effective intercellular communication is crucial for tissue repair and regeneration, with exosomes playing a key role in mediating these processes by transferring proteins, lipids, and nucleic acids between cells. This study explored the mechanisms underlying the uptake of exosomes derived from human dental pulp stem cells (hDPSCs), human umbilical vein endothelial cells (HUVECs), and human fibroblasts (HFBs). Our findings revealed that hDPSCs exhibited the greatest capacity for exosome uptake across all three cell types. Moreover, exosomes originating from hDPSCs were also taken up in the highest amounts by all three cell types. Proteomic analysis uncovered significant differences in protein expression among exosomes from these different cell types, particularly in proteins related to endocytosis. Clathrin-dependent endocytosis emerged as the primary pathway for exosome uptake in hDPSCs and HUVECs, while HFBs appeared to use a different mechanism. Additionally, proteins such as fibronectin and tetraspanins were found to be highly expressed in hDPSC-derived exosomes, suggesting their potential involvement in exosome-cell interactions. This study offers new insights into exosome uptake mechanisms and highlights the potential of exosomes in advancing tissue engineering and regenerative medicine.

“Young-Mechanical Niche” biomimetic hydrogel promotes dental pulp regeneration through YAP-dependent mechanotransduction

Aging leads to the irreversible deterioration of the extracellular matrix (ECM), compromising cell self-renewal, differentiation, and tissue regeneration. However, the specific mechanisms by which age-related changes in ECM-mediated cell mechanical microenvironment impair regeneration remain elusive. Human dental pulps (hDPs) provide an ideal model to explore this issue due to their accessibility and distinct mechanical properties. In this study, we discovered that young hDPs exhibit higher stiffness and viscoelasticity (i.e., faster stress relaxation time) compared to aged hDPs. Leveraging these findings, we engineered three groups of biomimetic hydrogels recapitulating the native mechanical microenvironment of young and aged hDPs. Our results demonstrated that Y-Gel, which replicates the high stiffness and viscoelasticity of young hDPs, significantly enhances the proliferation and odontogenic differentiation of human dental pulp stem cells (hDPSCs) in vitro and promotes dental pulp regeneration in vivo more effectively than hydrogels mimicking either aged stiffness (AS-Gel) or aged viscoelasticity (AV-Gel) alone. Moreover, YAP-dependent mechanotransduction, particularly through the YAP/TEAD1/CTGF/Cyr61 signaling pathway, plays a critical role in mediating these regenerative effects, with stiffness emerging as a more dominant factor than viscoelasticity. This study provides new insights into the synergistic role of mechanical factors in regulating cell behavior and offers a promising strategy for optimizing mechanical microenvironment to enhance dental pulp regeneration and potentially other tissue regeneration applications, especially from the mechanomedicine perspective.

Tertiary Lymphoid Structure in Dental Pulp: The Role in Combating Bacterial Infections.

Tertiary lymphoid structure (TLS) is associated with various pathologies, including those of cancers and chronic infections. Depending on the organ, multiple factors regulate the formation of TLS. However, the role of TLS in immune response and the molecules that drive its formation remain uncertain. The dental pulp, includes a few immune cells surrounded by rigid mineralized tissue, and opens to the outside through the apical foramen. Owing to this special organization, the dental pulp generates a directional immune response to bacterial infection. Considering this aspect, the dental pulp is an ideal model for comprehensively studying the TLS. In the present study, single-cell RNA sequencing of healthy and inflamed human dental pulp reveals known markers of TLS, including C-C motif chemokine ligand 19 (CCL19), lysosome-associated membrane glycoprotein 3 (LAMP3), CC chemokine receptor 7 (CCR7), and CD86, present in inflamed dental pulp. Compared with the healthy pulp, types and proportions of immune cells increase, along with enhanced cellular communication. Multiple immunofluorescence staining reveals that typical TLS emerges in dental pulp with pulpitis, consistent with the high expression of CC chemokine ligand 3 (CCL3), which may be a key driver of TLS formation. Moreover, TLS is also observed in a mouse model of pulpitis. These findings collectively offer insights into the formation and function of TLS in response to infection.

A transcriptomic analysis of dental pulp stem cell senescence in vitro

Background/purposeThe use of human dental pulp stem cells (hDPSCs) as autologous stem cells for tissue repair and regenerative techniques is a significant area of global research. The objective of this study was to investigate the effects of long-term in vitro culture on the multidifferentiation potential of hDPSCs and the potential molecular mechanisms involved.Materials and methodsThe tissue block method was used to extract hDPSCs from orthodontic-minus-extraction patients, which were then expanded and cultured in vitro for 12 generations. Stem cells from passages three, six, nine, and twelve were selected. Flow cytometry was used to detect the expression of stem cell surface markers, and CCK-8 was used to assess cell proliferation. β-Galactosidase staining was employed to detect cellular senescence, Alizarin Red S staining to assess osteogenic potential, and Oil Red O staining to evaluate lipogenic capacity. RNA sequencing (RNA-seq) was conducted to identify differentially expressed genes in DPSCs and investigate their potential mechanisms.ResultsWith increasing passage numbers, pulp stem cells showed an increase in senescence and a decrease in proliferative capacity and osteogenic–lipogenic multidifferentiation potential. The expression of stem cell surface markers CD34 and CD45 was stable, whereas the expression of CD73, CD90, and CD105 decreased with increasing passages. According to the RNA-seq analysis, the differentially expressed genes CFH, WNT16, HSD17B2, IDI1, and COL5A3 may be associated with stem cell senescence.ConclusionIncreased in vitro expansion induced cellular senescence in pulp stem cells, which resulted in a reduction in their proliferative capacity and osteogenic–lipogenic differentiation potential. The differential expression of genes such as CFH, WNT16, HSD17B2, IDI1, and COL5A3 may represent a potential mechanism for the induction of cellular senescence in pulp stem cells.

Limited Adipogenic Differentiation Potential of Human Dental Pulp Stem Cells Compared to Human Bone Marrow Stem Cells.

Bone marrow and teeth contain mesenchymal stem cells (MSCs) that could be used for cell-based regenerative therapies. MSCs from these two tissues represent heterogeneous cell populations with varying degrees of lineage commitment. Although human bone marrow stem cells (hBMSCs) and human dental pulp stem cells (hDPSCs) have been extensively studied, it is not yet fully defined if their adipogenic potential differs. Therefore, in this study, we compared the in vitro adipogenic differentiation potential of hDPSCs and hBMSCs. Both cell populations were cultured in adipogenic differentiation media, followed by specific lipid droplet staining to visualise cytodifferentiation. The in vitro differentiation assays were complemented with the expression of specific genes for adipogenesis and osteogenesis-dentinogenesis, as well as for genes involved in the Wnt and Notch signalling pathways. Our findings showed that hBMSCs formed adipocytes containing numerous and large lipid vesicles. In contrast to hBMSCs, hDPSCs did not acquire the typical adipocyte morphology and formed fewer lipid droplets of small size. Regarding the gene expression, cultured hBMSCs upregulated the expression of adipogenic-specific genes (e.g., PPARγ2, LPL, ADIPONECTIN). Furthermore, in these cells most Wnt pathway genes were downregulated, while the expression of NOTCH pathway genes (e.g., NOTCH1, NOTCH3, JAGGED1, HES5, HEY2) was upregulated. hDPSCs retained their osteogenic/dentinogenic molecular profile (e.g., RUNX2, ALP, COLIA1) and upregulated the WNT-specific genes but not the NOTCH pathway genes. Taken together, our in vitro findings demonstrate that hDPSCs are not entirely committed to the adipogenic fate, in contrast to the hBMSCs, which are more effective to fully differentiate into adipocytes.

Inhibitory effects of chlorhexidine-loaded calcium carbonate nanoparticles against dental implant infections

This study aimed to design sustained released biodegradable calcium carbonate nanoparticles loaded with chlorhexidine (CHX-loaded NPs) and to investigate the early osteogenic differentiation and antimicrobial effects on the important bacteria involved in infections of dental implants. The microemulsion method was used to prepare the calcium carbonate nanoparticles loaded with chlorhexidine. The prepared nanoparticles were characterized using conventional methods. The release pattern determination and the biodegradation test were performed for the prepared nanoparticles. For the early osteogenic differentiation test of the prepared nanoparticles, alkaline phosphatase (ALP) activity was detected in human dental pulp stem cells (HDPSCs). The antimicrobial effects of the nanoparticles were evaluated against Escherichia coli, Streptococcus mutans, Enterococcus faecalis, Staphylococcus aureus, and Pseudomonas aeruginosa. The sizes of free calcium carbonate nanoparticles and CHX-loaded NPs were 105 ± 1.63 and 118 ± 1.47 nm and their zeta potentials were − 27 and − 36, respectively. A 50% degradation of nanoparticles was achieved after 100 days. These nanoparticles showed a two-stage sustained release pattern in vitro. Microscopic images revealed that the morphology of free calcium carbonate nanoparticles primarily took on a spherical calcite form, while CHX-loaded NPs predominantly exhibited a cauliflower-like vaterite polymorph. The nanoparticles increased the activity of ALP in cells in two weeks significantly (p < 0.05). Antimicrobial and antibiofilm results showed an efficient effect of the prepared nanoparticle against the studied bacteria. Calcium carbonate nanoparticles are an efficient multifunctional vector for chlorhexidine and can be used as a bioactive antibacterial agent against various oral microorganisms to prevent implant infections.

Effect of three different root canal sealants on human dental pulp stem cells

The cytotoxic effects of three root canal sealers with different bases on human dental pulp stem cells were assessed in this study using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test. The cytotoxic effects of three root canal sealers with different bases on human dental pulp stem cells (DPSCs) were assessed in this study using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test. The cytotoxicity of the sealers was tested after one, 4, and 7 d. Human dental pulp stem cell proliferation was concluded using an MTT assay. Cells not treated with sealer extract were used as controls. The absorption levels were measured using an Eliza spectrophotometer. P was set at 0.05 when the percentage of cell proliferation was matched between groups and observation times using one-way analysis of variance (ANOVA).During the second passage (P2), human dental pulp stem cells displayed a single morphological and phenotypic trait, with fibroblast morphology being the most common. There were no appreciable variations between the four groups after a day. There was a notable variation in the average percentage of cell proliferation between the groups after 4 and 7 days. The control group had the highest percentage, followed by the GuttaFlow Bioseal group, the Well Root St group, and the AH-Plus group, which had the lowest percentage. For every sealing group, after one day, the highest mean percentage of cell proliferation was recorded, followed by day four, and after day seven, the lowest mean percentage. The observation periods showed minimal cytotoxic effects of GuttaFlow Bioseal, whereas AH-Plus was the most cytotoxic to human dental pulp stem cells. The highest mean percentage of cell proliferation for all sealers was recorded on day one.

Investigation of the cytotoxic effect of current dentine bonding agents on human dental pulp cells

BackgroundAn ideal aesthetic restorative material should be attached to the tooth tissues by adhesion, have a smooth surface as possible, should not cause toxic reactions in the pulp and discoloration and microleakage. This study aims at comparatively assess the cytotoxicity of current adhesive systems on human dental pulp cells.Materials and methodsThe adequate density of human pulp cells was observed from the ready cell line. The passaging was performed and the 3rd passage cells were selected. Adhesive systems and MTA were used on the cultures. Trypan blue staining was conducted on the cells at the 1st, 2nd, 3rd days and a count of live and dead cells using a light microscope. The dead cells whose membrane integrity was impaired by staining with trypan blue and the viability rate was determined using live and dead cell numbers. Data analysis was performed using IBM SPSS Statistics 22.ResultsA significant difference in vialibity rates between adhesive systems was observed on the first day. No significant statistical differences were observed on the 2nd and 3rd days (p < 0.05).ConclusionFuturabond M showed similar biocompatibility with MTA on human pulp cells and it can be applied in cavities with 1–1.5 mm hard tissue between pulp and dentine.

Human dental pulp MSCs attenuated motor neuron dysfunction and prolonged lifespan in ALS murine model

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that causes skeletal muscle weakness and atrophy, resulting in respiratory failure and a short lifespan. Considering the lack of effective treatment, this study investigated the effects of human dental pulp stem cells (hDPSCs) on the clinical symptoms and potential mechanisms in a mouse model of ALS superoxide dismutase 1 (SOD1-G93A). Neurological assessments, including neurological scoring, rotarod testing, and 7-T magnetic resonance imaging, were conducted to evaluate neurological impairments. Survival rates and body weight of the mice were also recorded. Immunofluorescence staining and flow cytometry analyses were performed to investigate the number of neurons and infiltrated inflammatory cells in the spinal cord as well as the central nervous system. The results indicate that infusion of hDPSCs increased the body weight, mitigated motor neuron dysfunction, and extended the lifespan of SOD1-G93A mice by approximately 15 days. Moreover, hDPSCs infusion reduced the degree of spinal cord atrophy. Results suggested that the number of neurons in the central nervous system of SOD1-G93A mice was significantly decreased, but hDPSCs infusion resulted in an increase in these numbers. However, hDPSCs infusion had no obvious effect on microglia phenotypes in SOD1-G93A mice. This study emphasizes the potential of hDPSCs to mitigate neuronal loss in an ALS mouse model, suggesting a promising therapeutic avenue for ALS.

Integrating Phosphate Enhances Biomineralization Effect of Methacrylate Cement in Vital Pulp Treatment with Improved Human Dental Pulp Stem Cells Stimulation.

Vital pulp treatment (VPT) is crucial for preserving the health and function of the tooth in cases where the pulp tissue remains vital despite exposure. Various materials are introduced for this purpose. However, challenges such as low strength, high solubility, and tooth discoloration persist. Methylmethacrylate-based cement (MC) offers excellent sealing ability, feasibility, and mechanical properties, making it a promising alternative for VPT. Phosphate-based glass (PBG) has the potential to promote hard tissue regeneration by releasing key inducers, phosphorus (P) and calcium (Ca), for reparative odontogenesis. This study investigates PBG-integrated MC (PIMC) by characterizing its properties, assessing human dental pulp stem cell activity related to initial inflammatory adaptation and odontogenic differentiation, and evaluating hard tissue formation using an in vivo dog pulpotomy model. Results indicate that a 5% PBG-integrated MC (5PIMC) maintains the physicochemical properties of MC. Furthermore, 5PIMC demonstrates cytocompatibility, excellent expression of osteo/odontogenic markers, and resistance to inflammatory markers, significantly outperforming MC. Enhanced hard tissue formation is observed in the dental pulp of mongrel dog teeth treated with 5PIMC. These findings suggest that 5PIMC could be an optimal and suitable material for reparative odontogenesis through VPT.

Flavonoids modulate regenerative-related cellular events in LPS-challenged dental pulp cells

ObjectiveTo investigate the effects of quercetin (QU), hesperetin (HT), and taxifolin (TX) on human dental pulp cells (hDPCs) chronically exposed to lipopolysaccharide (LPS). MethodsFirst, the cytotoxicity (alamarBlue) and bioactivity (biomineralization, Alizarin Red) of QU, HT, and TX concentrations were evaluated on healthy hDPCs. Then, the effects of non-cytotoxic and bioactive concentrations were investigated on hDPCs after previous stimulation with E. coli LPS (10 µg/mL) for 7 days. Cell culture media with and without LPS were used as positive and negative controls, respectively. Cell viability (alamarBlue), NF-κB activation (immunofluorescence), reactive oxygen species production (ROS, H2DCFDA probe), cell migration (Transwell), inflammatory gene expression (RT-qPCR), and odontogenic differentiation (RT-qPCR and alizarin red) were evaluated (n=8). Data were analyzed using confidence intervals and ANOVA (α=5%). ResultsThe concentrations of 20 µM QU, 20 µM HT, and 200 µM TX reduced cell viability by more than 30%. The 5 µM QU, 10 µM HT, and 100 µM TX concentrations were cytocompatible and stimulated biomineralization by healthy hDPCs. These concentrations were tested under the LPS challenge, and cell viability and odontogenic differentiation were significantly increased, while ROS production and inflammatory response were significantly decreased. In addition, the flavonoids significantly stimulated cell migration, reduced NF-κB activation, and increased biomineralization by LPS-challenged hDPCs compared to cells exposed to LPS alone and without any other treatment. ConclusionFlavonoids can modulate the metabolism of hDPCs chronically exposed to LPS in vitro, stimulating cellular events compatible with stem cell-based regenerative processes. Clinical SignificanceFlavonoids may be explored as adjuvant therapeutic agents during pulp capping to counteract chronic inflammatory conditions and stimulate regeneration of the dentin-pulp complex in caries-affected teeth, thereby preserving tooth vitality.

Effect of interleukin-17A on inflammatory mediator production in interleukin-1β-stimulated human dental pulp fibroblasts.

In response to pro-inflammatory cytokines such as interleukin (IL)-1β, dental pulp fibroblasts produce various inflammatory mediators, including IL-6, IL-8, CC chemokine ligand 20 (CCL20), and CXC chemokine ligand 10 (CXCL10), leading to the progression of pulpitis. IL-17/IL-17A (IL-17A) is a pro-inflammatory cytokine secreted by T helper (Th) 17 cells following their recruitment to inflamed sites; however, the roles of IL-17A during pulpitis remain unclear. The purpose of this study was to investigate the effect of IL-17A on IL-6, IL-8, CCL20 and CXCL10 production by human dental pulp fibroblasts (HDPFs) in vitro. IL-17A at a concentration of 100ng/ml induced the production of 10 times more IL-8 and 4 times more CXCL10, but not IL-6 and CCL20, compared to controls. Co-stimulation of HDPFs with IL-17A and IL-1β synergistically enhanced the production of IL-6, CCL20, IL-8 and CXCL10. IL-1β increased expression of IL-17 receptor/IL-17RA (IL-17R) on HDPFs. Moreover, the cell signal pathways of p38 mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) were more potently activated by simultaneous stimulation with IL-17A and IL-1β. These findings suggest that IL-17A participates in the progression of dental pulp inflammation through the enhanced production of inflammatory mediators in HDPFs.

Role of Pyrrolidine dithiocarbamate in inducing odontoblastic differentiation of dental pulp stem cells

Abstract Background: Pyrrolidine dithiocarbamate (PDTC) is an efficient, reproducible, and biological antioxidant of clinical utility, which may also be preferred for obtaining human dental pulp stem cells (hDPSCs) for the purpose of tissue engineering and regenerative medicine. Aim and Objectives: The study was conducted to evaluate the effects of PDTC on the propagation and differentiation of hDPSCs. Materials and Methods: hDPSCs were isolated by explant culture method and characterized for stem cell properties using flow cytometry method. The effects of PDTC-induced odontoblastic differentiation of hDPSC at different concentrations (0.5, 1.0, and 5.0 mM) were determined by staining for mineralization. Results: Mineralization was more prominent and significantly higher in the induced hDPSCs treated with 1.0 mM concentration of PDTC. Conclusion: PDTC at 1.0 mM concentration could have a significant pharmacological role in activating and enhancing odontogenic differentiation of dental stem cells and make it an important step in regenerative dentistry.

The construction of an inflammation classification model for HDPCs applied in guiding pulpotomy based on single-cell Raman spectroscopy

The immune defense and the repair function of the pulp tissue serve as the biological foundation of pulpotomy. The precise evaluation of the pulp inflammation extent and determining its reversibility are essential for the success of pulpotomy. The objective of this study was to classify the molecular-level of dental pulp cell physiology and inflammatory state based on the biochemical changes obtained by single-cell Raman spectroscopy. Firstly, we differentiated the growth of HDPCs (human dental pulp cells) under physiological states by employing Raman spectroscopy with multivariate statistical analysis. Raman spectroscopy reflected the biochemical changes at different growth phases, including the lag phase, log phase, and stationary phase. Secondly, we evaluated the optimal concentration and duration of Porphyromonas gingivalis lipopolysaccharide (P.g.LPS) stimulation to establish a six-level inflammation classification model of HDPCs. Thirdly, we performed label-free characterization of biological component changes in cells of different inflammation grades by Raman spectroscopy. As a result, the differences of peaks in the region 600–1800 cm−1 demonstrated the biochemical molecular alterations in the different inflammation grades of HDPCs. As inflammation progresses in steps, protein peaks increased first and then decreased, while lipid and nucleic acid peaks gradually decreased compared to unstimulated cells. However, when the inflammatory stimulation reached grade V, the changes in the biological properties were characterized by a recovery in protein and lipid content, and a decrease in nucleic acid content. We then established the diagnostic model using the Raman spectra of HDPCs in physiological and inflammatory states, which had a prediction accuracy of 100 % and 97.4 %, respectively. Finally, we determined the reversibility threshold of HDPCs at different grades of inflammation. We observed that the inflammation of grade I and II cells had potential reversibility and could be attempted to be retained. In conclusion, Raman spectroscopy combined with multivariate statistical analysis has potential possibility to effectively distinguish the degree of inflammation in the dental pulp, thus providing new tools and perspectives on pulpotomy in clinical practice.

Hypoxia-induced NLRP3 inflammasome activation via the HIF-1α/NF-κB signaling pathway in human dental pulp fibroblasts

BackgroundPrevious studies have reported the link between hypoxic conditions and NLRP3 inflammasome-mediated pulpal inflammation in the progression of pulpitis. However, the underlying mechanism has not been fully elucidated. This study aimed to investigate the role of HIF-1α in the regulation of NLRP3 inflammasome pathway via NF-κB signaling under hypoxic conditions with or without LPS in human dental pulp fibroblasts (HDPFs) during the progression of pulpitis.MethodsHIF-1α plasmids or siRNAs were used to upregulate or downregulate HIF-1α in HDPFs, respectively. The effect of hypoxia with or without LPS on the NF-κB signaling and NLRP3 inflammasome pathway was analyzed by immunofluorescence staining, qRT-PCR, western blotting and ELISA.ResultsThe hypoxic conditions alone induced ASC oligomerization and NLRP3/CASP1 inflammasome pathway activation via NF-κB signaling in a time-dependent manner in HDPFs. The upregulation of HIF-1α further promoted hypoxia-induced ASC oligomerization and NLRP3/CASP1 inflammasome pathway activation via NF-κB signaling compared to the hypoxia-induced group. In comparison, downregulation of HIF-1α inhibited ASC oligomerization and NLRP3/CASP1 inflammasome pathway activation via NF-κB signaling compared to the hypoxia-induced group. Additionally, LPS plus hypoxia further promoted HIF-1α expression and NLRP3/ASC/CASP1 inflammasome pathway activation via NF-κB signaling compared to the hypoxia-induced group.ConclusionsHIF-1α served as a positive regulator of NLRP3/ASC/CASP1 inflammasome pathway activation via NF-κB signaling in HDPFs in the sterile pulpal inflammation and caries-related pulpitis microenvironment. The finding of a novel functional HIF-1α-NF-κB-NLRP3 axis provides insight into the link between the hypoxic microenvironment and pulpal inflammation, thus supporting a promising therapeutic strategy for the control of pulpal inflammation.

Integrated MicroRNA-mRNA Analyses of the Osteogenic Differentiation of Human Dental Pulp Stem Cells by a Helioxanthin Derivative.

Dental pulp stem cells (DPSCs) demonstrate high proliferative and multilineage differentiation potential. As previously reported, the helioxanthin derivative 4-(4-methoxyphenyl)pyrido[40,30:4,5]thieno[2,3-b]pyridine-2-carboxamide (TH) has been demonstrated to induce the osteogenic differentiation of DPSCs. However, the mechanism of osteogenesis induced by TH in DPSCs remains unknown. The objective of this study was to identify functional extracellular vesicle (EV) microRNAs (miRNAs), and the principal genes involved in the TH-induced osteogenesis of DPSCs. DPSCs were derived from dental pulp extracted from the third molars of three healthy subjects, and were cultured with or without TH. miRNAs were extracted from DPSC-derived EVs. The gene expression patterns of mRNA and miRNA were compared using RNA-Seq and miRNA-Seq. To investigate miRNA/mRNA interacting networks, functional analyses were performed by Ingenuity Pathway Analysis. Alkaline phosphatase (ALP) staining demonstrated that treatment with TH resulted in enhanced ALP activity in DPSCs after 7 days. The expression levels of ALP and type 1 collagen alpha 1 were significantly higher in TH-induced DPSCs on day 7. RNA-Seq and miRNA-Seq analyses identified 869 differentially expressed genes (DEGs) and 18 miRNA-DEGs. Gene Ontology analysis of the mRNA-Seq results showed that TH induced several biological activities associated with signal transduction, cell adhesion, and cell differentiation. Integrated miRNA-mRNA analyses showed that these miRNAs contain the targeting information of 277 mRNAs of the DEGs. Among them, 17 target genes known to be involved in the differentiation of osteoblasts, and 24 target genes known to be involved in the differentiation of bone cells were identified. Quantitative real-time PCR showed that WNT5a expression in DPSCs was upregulated by 48 h of TH treatment. Upstream regulator analysis indicated that WNT3a, FOS, and RAC1 may be responsible for gene expression changes in DPSCs after TH treatment. EV miRNA regulatory networks might play crucial roles in TH-induced osteogenic differentiation of DPSCs. Our results presented herein offer valuable insights that will facilitate further research into the mechanism of osteogenesis of DPSCs, which is expected to lead to the clinical application of TH-induced DPSCs for bone regeneration. Furthermore, EVs derived from TH-induced DPSCs might be useful as therapeutic tools for bone defects.

Biocompatibility and Enamel Remineralization Potential of Titanium-Doped Phosphate Glasses With/Without Diode Laser Irradiation

This study investigates the biocompatibility and enamel remineralization potential of titanium-doped phosphate glasses (TDPG), third-generation materials with promising applications in dentistry. The biocompatibility of TDPG paste and its components (TDPG powder and polyacrylic acid liquid) was assessed using human gingival fibroblast cells (hGF) and human dental pulp stem cells (hDPSC). Cell attachment and viability were examined at 1, 3, and 7 days, comparing results with Nupro® Prophy paste and Teethmate, commonly used desensitizers, and positive control cells in normal tissue culture medium. Furthermore, the remineralization potential of these glasses with or without the use of diode laser irradiation on white spot lesions in enamel was explored at 12- and 21-days using Raman Spectroscopy and Field Emission Scanning Electron Microscopy (FESEM). The remineralization potential of TDPG was compared with Nupro® Prophy paste and Teethmate. Furthermore, both sound and demineralized enamel were also used as other controls.Results indicated that cells exposed to TDPG extracts displayed similar morphology and viability to positive control cells. While all tested pastes did not show remineralizing action after 12 days, Nupro® Prophy and Teethmate exhibited such action after 21 days. However, laser application enhanced remineralization potential for all pastes at both time points, with TDPG showing comparable results to Nupro® Prophy paste and significantly higher potential than Teethmate when used with laser at 21 days.In summary, this study reveals the biocompatibility and enhanced remineralization potential of TDPG when combined with diode laser irradiation. These findings signify innovative strides in dental materials and treatment modalities, offering new possibilities for advanced dental applications.

Unveiling the Neurodegenerative Alterations through Oral Stem Cells

Parkinson’s disease (PD) is a neurodegenerative condition characterized by the progressive and selective loss of dopaminergic (DAergic) neurons in the midbrain. The replacement of neuromelanin (NM)–containing DAergic neurons in the substantia nigra and the enhancement of NM concentration could offer a promising and safe approach to treating PD symptoms. The objective of this study was to investigate and compare the potential of human periapical-cysts mesenchymal stem cells (hPCy-MSCs) and dental pulp stem cells (DPSCs) to differentiate into DAergic NM-producing neurons and to generate functional 3-dimensional (3D) midbrain-like organoids in vitro. We assessed the changes in morphology and behavior of neuron-like cells (NLCs) as well as the expression of molecular markers characterizing the DAergic neurons. Furthermore, we observed electrically active and functionally mature DAergic neurons by means of electrophysiological assays, NM dosage assays, and the quantification of dopamine release by high-performance liquid chromatography. Our results demonstrate for the first time that both hPCy-MSCs and DPSCs are capable of differentiating into NLCs, further confirmed by the increase in lactate levels in the medium of cells exposed to neurogenic conditions. Importantly, we have induced such NLCs to further differentiate into functional DAergic NM-producing neurons. Finally, 3D midbrain-like organoids have been produced from oral stem cells: they appear as neurosphere-like structures diffusely expressing the neural marker β-III tubulin and containing NM-like granules. Our findings open up a novel and fascinating opportunity to rethink oral stem cells, and the derived 3D disease models, as a strategic and reliable tool for unveiling the neurodegenerative alterations.

Hybrid scaffolds for bone tissue engineering: Integration of composites and bioactive hydrogels loaded with hDPSCs

Bone tissue regeneration remains a significant challenge in clinical settings due to the complexity of replicating the mechanical and biological properties of bone environment. This study addresses this challenge by proposing a hybrid scaffold designed to enhance both bioactivity and physical stability for bone tissue regeneration. This research is the fisrt to develop a rigid 3D structure composed of polycaprolactone (PCL) and hydroxyapatite nanoparticles (nHA) integrated with a bioink containing human dental pulp stem/stromal cells (hDPSCs), alginate, nHA and collagen (Col). The biofabricated constructs were extensively characterized through cytocompatibility tests, osteogenic differentiation assessment, and biocompatibility evaluation in a rat model. In vitro results demontrated that the hybrid scaffolds presented significantly higher cell viability after 168 h compared to the control group. Furthermore, the hybrid scaffolds showed increased osteogenic differentiation relative to other groups. In vivo evaluation indicated good biocompatibility, characterized by minimal inflammatory response and successful tissue integration. These findings highlight the scaffold's potential to support bone tissue regeneration by combining the mechanical strength of PCL and nHA with the biological activity of the alginate-nHA-Col and hDPSCs bioink. The current study provides a promising foundation for the development of biomaterials aimed at improving clinical outcomes in bone repair and regeneration, particulary for the treatment of critical-size bone defects, targeted drug administration, and three-dimensional models for bone tissue engineering.