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Abstract Calcium phosphate-based materials (CaP) are introduced as potential dental pulp capping materials for deciduous teeth. The present study investigated the influence of inorganic phosphate (Pi) on regulating stem cells isolated from human exfoliated deciduous teeth (SHED). SHEDs were treated with Pi. Cell cycle progression and apoptosis were examined using flow cytometry analysis. Osteo/odontogenic and adipogenic differentiation were analyzed using alizarin red S and oil red O staining, respectively. The mRNA expression profile was investigated using a high-throughput RNA sequencing technique. Pi increased the late apoptotic cell population while cell cycle progression was not altered. Pi upregulated osteo/odontoblastic gene expression and enhanced calcium deposition. Pi-induced mineralization was reversed by pretreatment of cells with Foscarnet, or p38 inhibitor. Pi treatment inhibited adipogenic differentiation as determined by decreased PPARγ expression and reduced intracellular lipid accumulation. Bioinformatic analysis of gene expression profiles demonstrated several involved pathways, including PI3K/AKT, MAPK, EGFR, and VEGF signaling. In conclusion, Pi enhanced osteo/odontogenic but inhibited adipogenic differentiation in SHED.

Abstract Background Decellularized extracellular matrix (dECM) has been proposed as a useful source of biomimetic materials for regenerative medicine due to its biological properties that regulate cell behaviors. The present study aimed to investigate the influence of decellularized ECM derived from dental pulp stem cells (DPSCs) on gingival fibroblast (GF) cell behaviors. Cells were isolated from dental pulp and gingival tissues. ECM was derived from culturing dental pulp stem cells in growth medium supplemented with ascorbic acid. A bioinformatic database of the extracellular matrix was constructed using Metascape. GFs were reseeded onto dECM, and their adhesion, spreading, and organization were subsequently observed. The migration ability of the cells was determined using a scratch assay. Protein expression was evaluated using immunofluorescence staining. Results Type 1 collagen and fibronectin were detected on the ECM and dECM derived from DPSCs. Negative phalloidin and nuclei were noted in the dECM. The proteomic database revealed enrichment of several proteins involved in ECM organization, ECM–receptor interaction, and focal adhesion. Compared with those on the controls, the GFs on the dECM exhibited more organized stress fibers. Furthermore, cultured GFs on dECM exhibited significantly enhanced migration and proliferation abilities. Interestingly, GFs seeded on dECM showed upregulation of FN1, ITGB3, and CTNNB1 mRNA levels. Conclusions ECM derived from DSPCs generates a crucial microenvironment for regulating GF adhesion, migration and proliferation. Therefore, decellularized ECM from DPSCs could serve as a matrix for oral tissue repair.

Abstract Background Osseointegrated dental implants are widely established as a first-choice treatment for the replacement of missing teeth. Clinical outcomes are however often compromised by short or longer-term biological complications and pathologies. Nanoparticle-coated materials represent a very active research area with the potential to enhance clinical outcomes and reduce complications of implant therapy. This scoping review aimed to summarize current research on various types of nanoparticles (NPs) used as surface modifiers of dental implants and their potential to promote biological and clinical outcomes. Methods A systematic electronic search was conducted in SCOPUS, PubMed and Google Scholar aiming to identify in vivo, in situ, or in vitro studies published between 2014 and 2024. Inclusion and exclusion criteria were determined and were described in the methods section. Results A total of 169 articles (44 original papers from Scopus and PubMed, and 125 articles from Google Scholar) were identified by the electronic search. Finally, 30 studies fit the inclusion criteria and were further used in this review. The findings from the selected papers suggest that nanoparticle-coated dental implants show promising results in enhancing bone regeneration and promoting angiogenesis around the implant site. These effects are due to the unique physicochemical properties of nanoparticle-coated implants and the controlled release of bioactive molecules from nanoparticle-modified surfaces. Conclusion Nanoscale modifications displayed unique properties which could significantly enhance the properties of dental implants and further accelerate revascularization, and osseointegration while facilitating early implant loading. Yet, since many of these findings were based on in-vitro/in-situ systems, further research is required before such technology reaches clinical application.

Abstract Background The integration of stem cells, signaling molecules, and biomaterial scaffolds is fundamental for the successful engineering of functional bone tissue. Currently, the development of composite scaffolds has emerged as an attractive approach to meet the criteria of ideal scaffolds utilized in bone tissue engineering (BTE) for facilitating bone regeneration in bone defects. Recently, the incorporation of polycaprolactone (PCL) with hydroxyapatite (HA) has been developed as one of the suitable substitutes for BTE applications owing to their promising osteogenic properties. In this study, a three-dimensional (3D) scaffold composed of PCL integrated with HA (PCL/HA) was prepared and assessed for its ability to support osteogenesis in vitro. Furthermore, this scaffold was evaluated explicitly for its efficacy in promoting the proliferation and osteogenic differentiation of canine bone marrow-derived mesenchymal stem cells (cBM-MSCs) to fill the knowledge gap regarding the use of composite scaffolds for BTE in the veterinary orthopedics field. Results Our findings indicate that the PCL/HA scaffolds substantially supported the proliferation of cBM-MSCs. Notably, the group subjected to osteogenic induction exhibited a markedly upregulated expression of the osteogenic gene osterix (OSX) compared to the control group. Additionally, the construction of 3D scaffold constructs with differentiated cells and an extracellular matrix (ECM) was successfully imaged using scanning electron microscopy. Elemental analysis using a scanning electron microscope coupled with energy-dispersive X-ray spectroscopy confirmed that these constructs possessed the mineral content of bone-like compositions, particularly the presence of calcium and phosphorus. Conclusions This research highlights the synergistic potential of PCL/HA scaffolds in concert with cBM-MSCs, presenting a multidisciplinary approach to scaffold fabrication that effectively regulates cell proliferation and osteogenic differentiation. Future in vivo studies focusing on the repair and regeneration of bone defects are warranted to further explore the regenerative capacity of these constructs, with the ultimate goal of assessing their potential in veterinary clinical applications.

Abstract Objectives The study explored the expression profile of miRNAs in Notch-activated periodontal ligament stem cells (PDLSCs) and examined their potential cellular targets. Methods PDLSCs were cultured and treated with indirect immobilized Jagged1. The miRNA expression profile was examined using NanoString analysis. Bioinformatic analysis was performed together with enrichment, and miRNA expression was evaluated and validated using a quantitative polymerase chain reaction (qPCR). Results A total of 26 miRNAs were differentially expressed in Jagged1 treated PDLSCs compared with the controls. Pathway analysis revealed that altered miRNAs were significantly associated with the transforming growth factor β (TGF-β) signaling pathway. Target prediction analysis demonstrated that 11,170 genes as predictable targets of these altered miRNAs. Enrichment of predicted target genes revealed that they were related to ErbB, Ras and MAPK signaling pathways and small GTPase transduction. Conclusions The research concludes that several miRNAs are differentially expressed in jagged-1 treated PDLSCs. In translational terms the differential functionality of these miRNAs offer promise for the development of targeted regenerative materials that are necessary for managing lost tissue replacement in periodontal diseases.

Peri-implantitis, a prevalent complication in dental implant therapy, poses a significant threat to long-term implant success. The identification of reliable biomarkers for the early detection and monitoring of peri-implantitis is crucial for timely intervention and improved treatment outcomes. Salivary and peri-implant sulcular fluid (PISF) biomarkers have become promising diagnostic tools in the field of implant dentistry. This scoping review aims to explore current studies in the literature on salivary and PISF biomarkers for peri-implantitis. A systematic search was conducted on 2 databases (PubMed and Scopus) to identify relevant studies published up to January 2023. A total of 86 articles were included, which underwent data extraction and analysis. Several biomarkers have been investigated in salivary and PISF samples for association with peri-implantitis. Investigations included a wide range of biomarkers, including inflammatory markers, matrix metalloproteinases and bone loss markers. The findings suggested that certain salivary and PISF biomarkers demonstrated potential in distinguishing healthy peri-implant conditions from peri-implantitis. Elevated levels of proinflammatory cytokines, such as interleukin-1β (IL-1β) and interleukin-6 (IL-6), tumour necrosis factor-alpha (TNF-α), and matrix metalloproteinases, have been consistently associated with peri-implantitis. Additionally, alterations in bone loss markers have shown potential as indicators of disease progression and treatment response. In conclusion, this scoping review provides an overview of current knowledge on salivary and PISF biomarkers for peri-implantitis. The identified biomarkers are promising as noninvasive diagnostic tools for early detection, monitoring, and personalised management of peri-implantitis. Future studies should focus on establishing standardised protocols and conducting well-designed clinical trials to validate the diagnostic accuracy and clinical relevance of these biomarkers.

The decellularised extracellular matrix (dECM) of in vitro cell culture is a naturally derived biomaterial formed by the removal of cellular components. The compositions of molecules in the extracellular matrix (ECM) differ depending on various factors, including the culture conditions. Cell-derived ECM provides a 3-dimensional structure that has a complex influence on cell signalling, which in turn affects cell survival and differentiation. This review describes the effects of dECM derived from mesenchymal stem cells (MSCs) on cell responses, including cell migration, cell proliferation, and cell differentiation in vitro. Published articles were searched in the PubMed databases in 2005 to 2022, with assigned keywords (MSCs and decellularisation and cell culture). The 41 articles were reviewed, with the following criteria. (1) ECM was produced exclusively from MSCs; (2) decellularisation processes were performed; and (3) the dECM production was discussed in terms of culture systems and specific supplementations that are suitable for creating the dECM biomaterials. The dECM derived from MSCs supports cell adhesion, enhances cell proliferation, and promotes cell differentiation. Importantly, dECM derived from dental MSCs shows promise in regenerative dentistry applications. Therefore, the literature strongly supports cell-based dECMs as a promising option for innovative tissue engineering approaches for regenerative medicine.

Dental unit waterlines (DUWLs) are the conduits within the dental chair through which water derived from the municipal or other peripheral supply systems flows through the dental chair to reach the dental patient. The quality of the water so delivered must have a low pathogenic microbial burden so as to be safe for the patient and the dental personnel. Regulatory bodies have therefore set minimum standards for the quality of water that exits from DUWLs as part of routine infection control. Adopting a comprehensive approach that combines physical, chemical, and automated methodologies is recommended to effectively decontaminate DUWLs. This review discusses the complexities of DUWL decontamination in terms of (1) Best Practice Guidelines in dental healthcare settings to mitigate DUWL contamination, (2) origins of DUWL contamination and biofilm formation and the associated infectious risks, (3) contemporary approaches for minimizing DUWL contamination, and (4) constraints in combating biofilms in DUWLs. Compliance with regional and national regulations on DUWL decontamination is a legal obligation for all dental practitioners and fundamental to protecting public health.

ABSTRACT: Aim: Simvastatin has emerged as having a promising role in controlling stem cell behaviours. This study aimed to evaluate the effects of simvastatin on the viability, growth, and migration of stem cells isolated from apical papillae (SCAPs) in vitro. Methods: SCAPs were isolated and characterised. The viability and proliferation were assessed using live/dead and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays, respectively. Cell migration was evaluated using scratch assays. Cell cycle progression and apoptosis were examined using flow cytometry analysis. Results: Simvastatin at a concentration of 100 to 1000 nM did not exhibit cytotoxicity. Simvastatin reduced cell numbers at days 3 and 7. In addition, simvastatin markedly decreased colony formation in both colony number and cell density in a dose-dependent manner. An increase in apoptosis was observed at day 7. There was statistically significant increased in sub G0 population. An in vitro cell migration was attenuated in a dose-dependent manner. Conclusion: Simvastatin affects SCAPs’ viability, proliferation, and cell migration. The reduction of cell viability at day 7 could be due to apoptotic induction.

Abstract Extracellular matrix (ECM) is an intricate structure providing the microenvironment niche that influences stem cell differentiation. This study aimed to investigate the efficacy of decellularized ECM derived from human dental pulp stem cells (dECM_DPSCs) and gingival-derived mesenchymal stem cells (dECM_GSCs) as an inductive scaffold for osteogenic differentiation of GSCs. The proteomic analysis demonstrated that common and signature matrisome proteins from dECM_DPSCs and dECM_GSCs were related to osteogenesis/osteogenic differentiation. RNA sequencing data from GSCs reseeded on dECM_DPSCs revealed that dECM_DPSCs upregulated genes related to the Hippo and Wnt signaling pathways in GSCs. In the inhibitor experiments, results revealed that dECM_DPSCs superiorly promoted GSCs osteogenic differentiation, mainly mediated through Hippo and Wnt signaling. The present study emphasizes the promising translational application of dECM_DPSCs as a bio-scaffold rich in favorable regenerative microenvironment for tissue engineering.

Abstract Background This study aimed to investigate the effects of various toll-like receptor (TLR) and C-type lectin receptor (CLR) ligands on osteogenic differentiation in human dental pulp stem cells (hDPSCs). Methods hDPSCs were cultured and treated with various concentrations (0.01, 0.1, 1.0, and 10 µg/mL) of TLR or CLR agonists (PG-LPS, E.coli LPS, poly(I:C), Pam3CSK4, Furfurman, and Zymosan). Cell viability was determined by MTT assay. The effects of TLR and CLR agonists on osteogenic differentiation of hDPSCs were measured by alkaline phosphatase (ALP) activity, Alizarin Red S staining, and Von Kossa staining. In addition, the mRNA expression of osteogenesis-related genes (ALP, COL1A1, RUNX2, OSX, OCN and DMP1) was examined by RT-qPCR. A non-parametric analysis was employed for the statistical analyses. The statistically significant difference was considered when p

Betaine, known as trimethylglycine, is a non-toxic natural substance reported to affect cancer cell responses. This study delves into the impact of betaine on the survival, proliferation, and invasion of oral squamous cell carcinoma (OSCC) cells in vitro. Human OSCC cells (HSC-4 and HSC-7) were subjected to varying concentrations of betaine, and their viability and proliferation were assessed through colourimetric MTT and colony-forming unit assays. Cell cycle progression and cell apoptosis were also investigated using flow cytometry, while cell migration and invasion were examined using a transwell migration assay, and the mRNA expression was evaluated by a quantitative polymerase chain reaction. Finally, proteomic analysis was conducted through liquid chromatography-tandem mass spectrometry on the extracted protein component of the cells. Results indicate that betaine effectively suppressed OSCC proliferation and colony formation. It triggered early apoptosis without disrupting cell cycle progression, reduced cell migration, and inhibited invasion. Betaine exposure led to significantly decreased mRNA levels of MMP1, MMP2, and MMP9 while downregulating FN1, a gene linked to epithelial-to-mesenchymal transition. Proteomic analysis revealed 9240 differentially expressed up/downregulated proteins in cells treated with betaine. The significantly upregulated proteins were associated with ATP-binding cassette (ABC) transporters, while the down-regulated proteins were associated with G protein-coupled receptors (GPCR) ligand binding. In conclusion, betaine exhibits potent anti-cancer properties by attenuating OSCC cell proliferation and mitigating invasion. Exploring this natural product as an adjunct for managing oral squamous cell carcinoma shows promise, although further investigations are needed to fully elucidate its functionality.

Abstract Objective This study aimed to develop enamel substitute material using a mechanochemical technique. Materials and Methods Hydroxyapatite was synthesized with and without tricalcium phosphate under uniaxial pressing of 10 and 17 MPa (HA10, HA17, BCP10, and BCP17), followed by sintering at 1250 °C for 2 h. Human enamel and dentin blocks were used as control groups. The mechanical properties were determined by compressive strength test and Vickers microhardness. The data were analyzed with one-way ANOVA and LSD post-hoc test (α = 0.05). The phase formation and morphology of the specimens were characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM). Results HA17 and HA10 had compressive strength values comparable to enamel and dentin, respectively (p > 0.05). The microhardness of all synthesized groups was significantly higher than that of tooth structures (p

Abstract Asiatic acid (AA) and asiaticoside, pentacyclic triterpenoid compounds derived from Centella asiatica, are known for their biological effects in promoting type I collagen synthesis and inducing osteogenesis of stem cells. However, their applications in regenerative medicine are limited due to their low potency and poor aqueous solubility. This work aimed to evaluate the osteogenic induction activity of AA derivatives in human periodontal ligament stem cells (hPDLSCs) in vitro. Four compounds were synthesised, namely 501, 502, 503, and 506. AA was used as the control. The 502 exhibited low water solubility, while the 506 compound showed the highest. The cytotoxicity analysis demonstrated that 503 caused significant deterioration in cell viability, while other derivatives showed no harmful effect on hPDLSCs. The dimethyl aminopropyl amine derivative of AA, compound 506, demonstrated a relatively high potency in inducing osteogenic differentiation. An elevated mRNA expression of osteogenic-related genes, BMP2, WNT3A, ALP, OSX and IBSP was observed with 506. Additionally, the expression of BMP-2 protein was enhanced with increasing dose of 506, and the effect was pronounced when the Erk signalling molecule was inhibited. The 506 derivative was proposed for the promotion of osteogenic differentiation in hPDLSCs by upregulating BMP2 via the Erk signalling pathway. The 506 molecule showed promise in bone tissue regeneration.

Abstract Objectives The aim of this study was to investigate the effect of mechanical force on possible dynamic changes of the matrix proteins deposition in the PDL upon in vitro mechanical and in vivo occlusal forces in a rat model with hypofunctional conditions. Materials and methods Intermittent compressive force (ICF) and shear force (SF) were applied to human periodontal ligament stem cells (PDLSCs). Protein expression of collagen I and POSTN was analyzed by western blot technique. To establish an in vivo model, rat maxillary molars were extracted to facilitate hypofunction of the periodontal ligament (PDL) tissue of the opposing mandibular molar. The mandibles were collected after 4-, 8-, and 12-weeks post-extraction and used for micro-CT and immunohistochemical analysis. Results ICF and SF increased the synthesis of POSTN by human PDLSCs. Histological changes in the hypofunctional teeth revealed a narrowing of the PDL space, along with a decreased amount of collagen I, POSTN, and laminin in perivascular structures compared to the functional contralateral molars. Conclusion Our results revealed that loss of occlusal force disrupts deposition of some major matrix proteins in the PDL, underscoring the relevance of mechanical forces in maintaining periodontal tissue homeostasis by modulating ECM composition.

Abstract Objective Adenosine triphosphate (ATP) is an essential nucleotide that is normally present in both intracellular and extracellular compartments. Extracellular ATP (eATP) has a pivotal role in both physiological and pathological processes of periodontal ligament tissues. Here, this review aimed to explore the various functions of eATP that are involved in the control of behaviours and functions of periodontal ligament cells. Methods To identify the included publications for review, the articles were searched in PubMed (MEDLINE) and SCOPUS with the keywords of adenosine triphosphate and periodontal ligament cells. Thirteen publications were used as the main publications for discussion in the present review. Results eATP has been implicated as a potent stimulator for inflammation initiation in periodontal tissues. It also plays a role in proliferation, differentiation, remodelling, and immunosuppressive functions of periodontal ligament cells. Yet, eATP has diverse functions in regulating periodontal tissue homeostasis and regeneration. Conclusion eATP may provide a new prospect for periodontal tissue healing as well as treatment of periodontal disease especially periodontitis. It may be utilized as a useful therapeutic tool for future periodontal regeneration therapy.

Abstract Periodontal ligament stem cells (PDLSCs) play a significant role on periodontal tissue and alveolar bone homeostasis. During inflammation, interleukin (IL)-6 serves as one of key cytokine players controlling tissue reaction as well as alveolar bone tissue remodeling. It is believed that periodontal tissue inflammation causes periodontium degradation, especially alveolar bone. However, in this study, we show that an inflammatory mediator, IL-6, may serve another direction on alveolar bone homeostasis during inflammatory condition. We found that, IL-6 at 10 and 20 ng/mL was not cytotoxic and dose-dependently exerted beneficial effects on osteogenic differentiation of human PDLSCs (hPDLSCs), as demonstrated by increased alkaline phosphatase activity, mRNA expression of osteogenic markers, and matrix mineralization. The presence of physiological and inflammatory level of IL-6, the osteogenic differentiation potential by hPDLSCs was enhanced by several possible mechanisms including transforming growth factor (TGF), Wnt, and Notch pathways. After in-depth and thorough exploration, we found that Wnt pathway serves as key regulator controlling osteogenic differentiation by hPDLSCs amid the IL-6 presentation. Surprisingly, apart from other mesenchymal stem cells, distinct Wnt components are employed by hPDLSCs, and both canonical and non-canonical Wnt pathways are triggered by different mechanisms. Further validation by gene silencing, treatment with recombinant Wnt ligands, and β-catenin stabilization/translocation confirmed that IL-6 governed the canonical Wnt/β-catenin pathway via either WNT2B or WNT10B and employed WNT5A to activate the non-canonical Wnt pathway. These findings fulfill the homeostasis pathway governing periodontal tissue and alveolar bone regeneration and may serve for further therapeutic regimen design for restoring the tissues.

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.

Abstract The self-organizing potential of induced pluripotent stem cells (iPSCs) represents a promising tool for bone tissue engineering. Shear stress promotes the osteogenic differentiation of mesenchymal stem cells, leading us to hypothesize that specific shear stress could enhance the osteogenic differentiation of iPSCs. For osteogenesis, embryoid bodies were formed for two days and then maintained in medium supplemented with retinoic acid for three days, followed by adherent culture in osteogenic induction medium for one day. The cells were then subjected to shear loading (0.15, 0.5, or 1.5 Pa) for two days. Among different magnitudes tested, 0.5 Pa induced the highest levels of osteogenic gene expression and greatest mineral deposition, corresponding to upregulated connexin 43 (Cx43) and phosphorylated Erk1/2 expression. Erk1/2 inhibition during shear loading resulted in decreased osteogenic gene expression and the suppression of mineral deposition. These results suggest that shear stress (0.5 Pa) enhances the osteogenic differentiation of iPSCs, partly through Cx43 and Erk1/2 signaling. Our findings shed light on the application of shear-stress technology to improve iPSC-based tissue-engineered bone for regenerative bone therapy.

Abstract Objectives This study aimed to evaluate the effect of betaine (BET) on immortalized human dental pulp stem cell (ihDP) osteogenic differentiation. Materials and methods hDPs were immortalized using SV40 T-antigen transfection. Characterization, multilineage differentiation, proliferation, cell cycle, colony-forming unit, and cellular senescence were evaluated (n = 4). The effect of BET on ihDP response was assessed (n = 4). Osteogenic differentiation was detected using ALP, ARS staining, and RT-qPCR (n = 4). To investigate the involvement of calcium signaling, the cells were pretreated with either 8-(NN-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) or thapsigargin before BET treatment (n = 6). Results ihDPs retained similar phenotypic characteristics presented in hDPs but exhibited an increase in cell proliferation and extended culture to passage 25. An increased proportion of cells in S and G2/M phases without senescence was observed in ihDPs. BET (50 mM) treatment significantly increased mineral deposition at 14 days and upregulated ALP, MSX2, BMP2, and RUNX2 expression. TMB-8 pretreatment reduced the effect of BET-induced ihDP osteogenic differentiation, whereas thapsigargin promoted osteogenic differentiation in ihDPs synergistically with BET. Conclusion ihDPs showed superior proliferation ability and a longer life span, which could serve as a promising cell for regenerative dentistry. BET promoted odonto/osteogenic differentiation via intracellular calcium regulation.

Abstract Canine mesenchymal stem cells (cMSCs) have potential applications for regenerative therapy, including the generation of insulin-producing cells (IPCs) for studying and treating diabetes. In this study, we established a useful protocol for generating IPCs from canine adipose mesenchymal stem cells (cAD-MSCs). Subsequently, in vitro preservation of pluronic F127-coated alginate (ALGPA)-encapsulated cAD-MSC-derived IPCs was performed to verify ready-to-use IPCs. IPCs were induced from cAD-MSCs with the modulated three-stepwise protocol. The first step of definitive endoderm (DE) induction showed that the cooperation of Chir99021 and Activin A created the effective production of Sox17-expressed DE cells. The second step for pancreatic endocrine (PE) progenitor induction from DE indicated that the treatment with taurine, retinoic acid, FGF2, EGF, TGFβ inhibitor, dorsomorphin, nicotinamide, and DAPT showed the significant upregulation of the pancreatic endocrine precursor markers Pdx1 and Ngn3. The last step of IPC production, the combination of taurine, nicotinamide, Glp-1, forskolin, PI3K inhibitor, and TGFβ inhibitor, yielded efficiently functional IPCs from PE precursors. Afterward, the maintenance of ALGPA-encapsulated cAD-MSC-derived IPCs with VSCBIC-1, a specialized medium, enhanced IPC properties. Conclusion, the modulated three-stepwise protocol generates the functional IPCs. Together, the encapsulation of cAD-MSC-derived IPCs and the cultivation with VSCBIC-1 enrich the maturation of generated IPCs.

Purpose: There are two commonly cited modulus of elasticity of the human periodontal ligament (EPDL), i.e., 6.89 ✕ 10−5 GPa (E1) and 6.89 ✕ 10−2 GPa (E2), which are exactly 1000-fold different from each other. This study aims to clarify the ambiguity of the two EPDL used for simulations and determine a more accurate EPDL value of human first premolars using experimental and simulation approaches. Methods: Numerical simulations using finite element analysis were performed to analyze PDL deformation under an average Asian occlusal force. To confirm the results, simple and multi-component, true-scale 3D models of a human first premolar were used in the simulations. Finally, a compression test using a universal testing machine on PDL specimens was conducted to identify the compressive EPDL of human first premolars. Results: The simulation results from both models revealed that E1 was inaccurate, because it resulted in excessive PDL deformation under the average occlusal force, which should not occur during mastication. Although the E2 did not lead to excessive PDL deformation, it was obtained by an error in unit conversion with no scientific backing. In contrast, the compression test results indicated that the compressive EPDL was 9.64 ✕ 10−4 GPa (E3). In the simulation, E3 did not cause excessive PDL deformation. Conclusion: The simulation results demonstrated that both commonly cited EPDL values (E1 and E2) were incorrect. Based on the experimental and simulation results, the average compressive EPDL of 9.64 ✕ 10−4 GPa is proposed as a more accurate value for human first premolars. Clinical significance: The proposed more accurate EPDL would contribute to more precise and reliable FEA simulation results and provide a better understanding of the stress distribution and deformation of dental materials, which will be beneficial to precision dentistry, orthodontics and restoration designs.

Abstract Osteoblast differentiation requires the interaction of various cell signaling pathways to modulate cell responses. Notch and Wnt signaling are among the crucial pathways that control numerous biological processes, including osteo/odontogenic differentiation. The aim of the present study was to examine the involvement of Wnt signaling in the Jagged1-induced osteo/odontogenic differentiation in human dental pulp stem cells (hDPSCs). The Wnt-related gene expression was analyzed from publicly available data of Jagged1-treated human dental pulp cells. The mRNA expression of Wnt ligands (WNT2B, WNT5A, WNT5B, and WNT16) and Wnt inhibitors (DKK1, DKK2, and SOST) were confirmed using real-time polymerase chain reaction. Among the Wnt ligands, WNT2B and WNT5A mRNA levels were upregulated after Jagged1 treatment. In contrast, the Wnt inhibitors DKK1, DKK2, and SOST mRNA levels were downregulated. Recombinant WNT5A, but not WNT2B, significantly promoted in vitro mineral deposition by hDPSCs. Wnt signaling inhibition using IWP-2, but not DKK1, inhibited Jagged1-induced alkaline phosphatase (ALP) activity, mineralization, and osteo/odontogenic marker gene expression in hDPSCs. In conclusion, Jagged1 promoted hDPSC osteo/odontogenic differentiation by modulating the non-canonical Wnt pathway.

Abstract In vitro manipulation of induced pluripotent stem cells (iPSCs) by environmental factors is of great interest for three-dimensional (3D) tissue/organ induction. The effects of mechanical force depend on many factors, including force and cell type. However, information on such effects in iPSCs is lacking. The aim of this study was to identify a molecular mechanism in iPSCs responding to intermittent compressive force (ICF) by analyzing the global gene expression profile. Embryoid bodies of mouse iPSCs, attached on a tissue culture plate in 3D form, were subjected to ICF in serum-free culture medium for 24 h. Gene ontology analyses for RNA sequencing data demonstrated that genes differentially regulated by ICF were mainly associated with metabolic processes, membrane and protein binding. Topology-based analysis demonstrated that ICF induced genes in cell cycle categories and downregulated genes associated with metabolic processes. The Kyoto Encyclopedia of Genes and Genomes database revealed differentially regulated genes related to the p53 signaling pathway and cell cycle. qPCR analysis demonstrated significant upregulation of Ccnd1, Cdk6 and Ccng1. Flow cytometry showed that ICF induced cell cycle and proliferation, while reducing the number of apoptotic cells. ICF also upregulated transforming growth factor β1 (Tgfb1) at both mRNA and protein levels, and pretreatment with a TGF-β inhibitor (SB431542) prior to ICF abolished ICF-induced Ccnd1 and Cdk6 expression. Taken together, these findings show that TGF-β signaling in iPSCs enhances proliferation and decreases apoptosis in response to ICF, that could give rise to an efficient protocol to manipulate iPSCs for organoid fabrication.

Background and Aim: Fibrin forms in the coagulation process, enhancing local hemostatic properties and promoting wound healing. The study aimed to evaluate the efficacy of bubaline-derived fibrin glue in silk ligature-induced periodontitis rats. Materials and Methods: Bubaline blood–derived fibrin glue was prepared using cryoprecipitation and cryocentrifugation. Periodontitis was induced in rats by placing 5-0 silk ligatures around the mandibular first molars. The animals were divided into two groups: (1) Non-treatment and (2) bubaline fibrin glue–treated groups. Plaque, gingival inflammation, and mobility index were scored on days 1, 7, and 14 after intervention. Histological examinations were performed. The mRNA expression of inflammatory cytokines and growth factors was evaluated using a real-time polymerase chain reaction. Ligature-induced periodontitis was confirmed by the increase in inflammatory cell infiltration as well as histological bone and attachment loss. Results: Compared to the non-treatment group, bubaline fibrin glue application reduced mononuclear cell infiltration into periodontal tissues corresponding to the reduction of collagen destruction. On days 7 and 14 after intervention, the inflammatory score and histological attachment loss were significantly lower in the bubaline fibrin glue–treated group than in the non-treatment group. A significant reduction in histological bone loss was observed in the treated group on day 7. Bubaline fibrin glue application led to a significant reduction of Tnfa and Il1b mRNA levels, while an increased expression of Pdgfa, Tgfb1, and Il10 was observed compared with the control. Conclusion: Bubaline fibrin glue could be beneficial in periodontitis treatment aiming to reduce inflammation and delay the progression of periodontal disease.

Potential aerosols containing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles can be generated during dental treatment. Hence, patient triage is essential to prevent the spread of SARS-CoV-2 in dental clinical settings. The present study described the use of rapid antigen tests for SARS-CoV-2 screening prior to dental treatment in an academic dental clinical setting in Thailand during the pandemic. The opinions of dental personnel toward the use of rapid antigen test screening prior to dental treatment were also assessed. From August 25 to October 3, 2021, dental patients who were expected to receive aerosols generating dental procedures were requested to screen for SARS-CoV-2 using a rapid antigen test before their treatment. A total of 7,618 cases completed the screening process. The average was 212 cases per day. Only five patients (0.07%) were positive for SARS-CoV-2 in the rapid antigen screening tests. All positive cases exhibited mild symptoms. For the questionnaire study, experienced dental personnel frequently and consistently agreed with the use of the rapid antigen test for SARS-CoV-2 screening, which made them feel safer during their patient treatment. However, implementing rapid antigen tests for SARS-CoV-2 may increase the total time spent on a dental appointment. In conclusion, a rapid antigen test could detect the infected individual prior to dental treatment. However, the specificity of rapid antigen tests for SARS-CoV-2 must be taken into account for consideration as a screening process before dental treatment. The enhanced infection control protocols in dental treatment must be consistently implemented.

Abstract Platelet-rich fibrin (PRF) promotes wound healing by providing the release of growth factors. Here, the influence of Thai and Murrah bubaline blood derived PRF on canine periodontal ligament cells (cPDLs) was investigated. PRF was prepared from Thai and Murrah buffaloes with single centrifugation. Results demonstrated that Thai bubaline blood derived PRF exhibited fiber-mesh like morphology and contained more platelet entrapment than Murrah bubaline blood derived PRF. Both bubaline PRFs were able to degrade in vitro under condition with trypsin. Thai but not Murrah bubaline blood derived PRF promoted cPDLs proliferation in serum free and 2% serum culture conditions. Correspondingly, the significant upregulation of KI67 mRNA expression was observed in those cells treated with Thai bubaline blood derived PRF. However, both Thai and Murrah bubaline blood derived PRF accelerated cell migration in an in vitro wound healing assay and facilitated cell spreading. Further, cPDLs cultured in osteogenic induction medium supplemented with Thai bubaline blood derived PRF exhibited the increased mineral deposition in vitro. Frozen Thai bubaline blood derived PRF also promoted cell proliferation, KI67 mRNA expression, cell migration, and cell spreading in cPDLs. Taken these evidence together, bubaline blood derived PRF could provide potential benefits for canine periodontal tissue healing.

Abstract The trend of regenerative therapy for diabetes in human and veterinary practices has conceptually been proven according to the Edmonton protocol and animal models. Establishing an alternative insulin-producing cell (IPC) resource for further clinical application is a challenging task. This study investigated IPC generation from two practical canine mesenchymal stem cells (cMSCs), canine bone marrow-derived MSCs (cBM-MSCs) and canine adipose-derived MSCs (cAD-MSCs). The results illustrated that cBM-MSCs and cAD-MSCs contain distinct pancreatic differentiation potential and require the tailor-made induction protocols. The effective generation of cBM-MSC-derived IPCs needs the integration of genetic and microenvironment manipulation using a hanging-drop culture of PDX1-transfected cBM-MSCs under a three-step pancreatic induction protocol. However, this protocol is resource- and time-consuming. Another study on cAD-MSC-derived IPC generation found that IPC colonies could be obtained by a low attachment culture under the three-step induction protocol. Further, Notch signaling inhibition during pancreatic endoderm/progenitor induction yielded IPC colonies through the trend of glucose-responsive C-peptide secretion. Thus, this study showed that IPCs could be obtained from cBM-MSCs and cAD-MSCs through different induction techniques. Also, further signaling manipulation studies should be conducted to maximize the protocol’s efficiency.

Objective: Indirect Jagged1 immobilization efficiently activates canonical Notch signaling in human dental pulp stem cells (hDPSCs). This study aimed to investigate the characteristics of the Jagged1-treated hDPSC-derived decellularized extracellular matrix (dECM) and its biological activity on odonto/osteogenic differentiation of stem cells isolated from apical papilla (SCAPs).Methods: Bioinformatic database of Jagged1-treated hDPSCs was analyzed using NetworkAnalyst. hDPSCs seeded on the Jagged1 immobilized surface were maintained with normal or osteogenic induction medium (OM) followed by decellularization procedure, dECM-N, or dECM-OM, respectively. SCAPs were reseeded on each dECM with either the normal medium or the OM. Cell viability was determined by MTT assay. Characteristics of dECMs and SCAPs were evaluated by SEM, EDX, immunofluorescent staining, and alcian blue staining. Mineralization was assessed by alizarin red S, Von Kossa, and alkaline phosphatase staining. Statistical significance was considered at p < 0.05.Results: RNA-seq database revealed upregulation of several genes involved in ECM organization, ECM–receptor interaction, and focal adhesion in Jagged1-treated hDPSCs. Immobilized Jagged1 increased the osteogenesis of the hDPSC culture with OM. dECMs showed fibrillar-like network structure and maintained major ECM proteins, fibronectin, type I-collagen, and glycosaminoglycans. A decrease in calcium and phosphate components was observed in dECMs after the decellularized process. Cell viability on dECMs did not alter by 7 days. Cell attachment and f-actin cytoskeletal organization of SCAPs proliferated on Jagged1-treated dECMs were comparable to those of the control dECMs. SCAPs exhibited significantly higher mineralization on dECM-N in OM and markedly enhanced on dECM-OM with normal medium or OM conditions.Conclusion: Jagged1-treated hDPSC-derived dECMs are biocompatible and increase odonto/osteogenic differentiation of SCAPs. The results suggested the potential of Jagged1 dECMs, which could be further developed into ECM scaffolds for application in regenerative medicine.

Bone tissue engineering consists of three major components namely cells, scaffolds, and signaling molecules to improve bone regeneration. These integrated principles can be applied in patients suffered from bone resorption diseases, such as osteoporosis and periodontitis. Osteogenic growth peptide (OGP) is a fourteen-amino acid sequence peptide that has the potential to regenerate bone tissues. This study aimed to disseminate the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) with OGP treatment. OGP was elaborated for proliferation, cytotoxicity, osteogenic differentiation effects, and the involvement of osteogenic related signaling pathways in vitro. This study found that OGP at lower concentration shows better effects on cytotoxicity and proliferation. Moreover, OGP at concentration 0.01 nM had the most potential to differentiate hPDLSCs toward osteogenic lineage comparing with higher concentrations of OGP. The phenomenon was mainly involving transforming growth factor-beta (TGF-β), bone morphogenetic protein (BMP), Hedgehog, and Wingless-related (Wnt) pathways. Further, SB-431542 treatment demonstrated the partial involvement of OGP in regulating osteogenic differentiation of hPDLSCs. In conclusion, OGP at low concentration enhances osteogenic differentiation of hPDLSCs by governing TGF-β signaling pathway.

Abstract Background Direct pulp capping is a vital pulp therapy for a pin-point dental pulp exposure. Applying a pulp capping material leads to the formation of a dentin bridge and protects pulp vitality. The aim of this study was to compare the effects of four dental materials, DyCal®, ProRoot® MTA, Biodentine™, and TheraCal™ LC in vitro. Methods Human dental pulp stem cells (hDPs) were isolated and characterized. Extraction medium was prepared from the different pulp capping materials. The hDP cytotoxicity, proliferation, and migration were examined. The odonto/osteogenic differentiation was determined by alkaline phosphatase, Von Kossa, and alizarin red s staining. Osteogenic marker gene expression was evaluated using real-time polymerase chain reaction. Results ProRoot® MTA and Biodentine™ generated less cytotoxicity than DyCal® and TheraCal™ LC, which were highly toxic. The hDPs proliferated when cultured with the ProRoot® MTA and Biodentine™ extraction media. The ProRoot® MTA and Biodentine™ extraction medium induced greater cell attachment and spreading. Moreover, the hDPs cultured in the ProRoot® MTA or Biodentine™ extraction medium migrated in a similar manner to those in serum-free medium, while a marked reduction in cell migration was observed in the cells cultured in DyCal® and TheraCal™ LC extraction media. Improved mineralization was detected in hDPs maintained in ProRoot® MTA or Biodentine™ extraction medium compared with those in serum-free medium. Conclusion This study demonstrates the favorable in vitro biocompatibility and bioactive properties of ProRoot® MTA and Biodentine™ on hDPs, suggesting their superior regenerative potential compared with DyCal® and TheraCal™.

Abstract Background Current approach for diabetes treatment remained several adverse events varied from gastrointestinal to life-threatening symptoms. Regenerative therapy regarding Edmonton protocol has been facing serious limitations involving protocol efficiency and safety. This led to the study for alternative insulin-producing cell (IPC) resource and transplantation platform. In this study, evaluation of encapsulated human dental pulp-derived stem cell (hDPSC)-derived IPCs by alginate (ALG) and pluronic F127-coated alginate (ALGPA) was performed. Results The results showed that ALG and ALGPA preserved hDPSC viability and allowed glucose and insulin diffusion in and out. ALG and ALGPA-encapsulated hDPSC-derived IPCs maintained viability for at least 336 h and sustained pancreatic endoderm marker (NGN3), pancreatic islet markers (NKX6.1, MAF-A, ISL-1, GLUT-2 and INSULIN), and intracellular pro-insulin and insulin expressions for at least 14 days. Functional analysis revealed a glucose-responsive C-peptide secretion of ALG- and ALGPA-encapsulated hDPSC-derived IPCs at 14 days post-encapsulation. Conclusion ALG and ALGPA encapsulations efficiently preserved the viability and functionality of hDPSC-derived IPCs in vitro and could be the potential transplantation platform for further clinical application.

Background: Extracellular matrix (ECM) plays a pivotal role in many physiological processes. ECM macromolecules and associated factors differ according to tissues, impact cell differentiation, and tissue homeostasis. Dental pulp ECM may differ from other oral tissues and impact mineralization. Thus, the present study aimed to identify the matrisome of ECM proteins derived from human dental pulp stem cells (DPSCs) and its ability to regulate mineralization even in cells which do not respond to assaults by mineralization, the human gingival fibroblasts (GF).Methods: ECM were extracted from DPSCs cultured in normal growth medium supplemented with L-ascorbic acid (N-ECM) or in osteogenic induction medium (OM-ECM). ECM decellularization (dECM) was performed using 0.5% triton X-100 in 20 mM ammonium hydroxide after 21 days. Mass spectrometry and proteomic analysis identified and quantified matrisome proteins.Results: The dECM contained ECM proteins but lacked cellular components and mineralization. Interestingly, collagens (COL6A1, COL6A2, and COL6A3) and elastic fibers (FBN1, FBLN2, FN1, and HSPG2) were significantly represented in N-ECM, while annexins (ANXA1, ANXA4, ANXA5, ANXA6, ANXA7, and ANXA11) were significantly overdetected in OM-ECM. GF were reseeded on N-dECM and OM-dECM and cultured in normal or osteogenic medium. GF were able to attach and proliferate on N-dECM and OM-dECM. Both dECM enhanced mineralization of GF at day 14 compared to tissue culture plate (TCP). In addition, OM-dECM promoted higher mineralization of GF than N-dECM although cultured in growth medium.Conclusions: ECM derived from DPSCs proved to be osteoinductive, and this knowledge supported cell-derived ECM can be further utilized for tissue engineering of mineralized tissues.

FAM83H mutations lead to autosomal dominant hypocalcified amelogenesis imperfecta (ADHCAI). However, the biological role of FAM83H remains unclear. The present study aimed to characterize the alveolar bone cells isolated from a patient with ADHCAI having the mutation, c.1261G > T, p.E421*, in FAM83H. We showed that FAM83H mutant cells had proliferation ability and morphology similar to the controls. The F-actin staining revealed that FAM83H mutant cells were remained in the earlier stages of cell spreading compared to the controls at 30 min, but their spreading was advanced comparable to the controls at later stages. After osteogenic induction, a significant decrease in mRNA levels of RUNX2 and ALP was observed in FAM83H mutant cells at day 7 compared with day 3 while their expressions were increased in the controls. The OPN levels in FAM83H mutant cells were not significantly changed at day 7 compared to day 3 while the controls showed a significant increase. After 14 days, the mineral deposition of FAM83H mutant cells was slightly lower than that of the controls. In conclusion, we identify that FAM83H bone cells have lower expression of osteogenic marker genes and mineralization while they maintain their morphology, proliferation, and spreading. Consistent with previous studies in the ameloblasts and periodontal ligamental cells, these evidences propose that FAM83H influences osteogenic differentiation across different cell types in oral cavity. Keywords: Autosomal dominant inheritance, Enamel hypoplasia, Hypocalcified enamel, Mineralization, Osteogenic differentiation, Periodontium

SARS-CoV-2 can transmit undetected from asymptomatic and pre-symptomatic patients in dental clinics. Triaging dental patients using temperature and questionnaire screening cannot completely exclude asymptomatic SARS-CoV-2 infected individuals. Hence, asymptomatic SARS-CoV-2 infected individuals might visit dental hospitals/clinics seeking dental treatment without knowing that they are infected and might infect others, especially in a pandemic area. Ideally, a nasopharyngeal swab for real-time polymerase chain reaction or rapid antigen screening for dental personnel and patients prior to their appointment should be done. However, the implementation of this approach is impractical in some situations. Here, we describe the procedures for dental hospitals/clinics in case of an asymptomatic SARS-CoV-2 infected individual involved in dental service/treatment and later after testing positive for SARS-CoV-2. Potential closely contacted individuals were traced and classified according to their exposure risk. The recommended course of action is to identify individuals based on their risk and take the risk-appropriate action. We also discuss the implementation of these procedures in a dental setting during the COVID-19 pandemic in our school as a case study.

The indirect immobilisation of Jagged-1 (Jagged-1) promoted osteogenic differentiation of human dental pulp cells (hDPs). Furthermore, the analysis of the Reactome pathway of RNA sequencing data indicates the upregulated genes involved with the extracellular matrix (ECM). Hence, our objective was to investigate the effects of Jagged-1 on proteomic profiles of human dental pulp stem cells (hDPSC). hDPSCs were cultured on the surface coated with human IgG Fc fragment (hFc) and the surface coated with rhJagged1/Fc recombinant protein-coated surface. Cells were differentiated to the osteogenic lineage using an osteogenic differentiation medium (OM) for 14 days, and cells cultured in a growth medium were used as a control. The protein component of the cultured cells was extracted into the cytosol, membrane, nucleus, and cytoskeletal compartment. Subsequently, the proteomic analysis was performed using liquid chromatography–tandem mass spectrometry (LC-MS). Metascape gene list analysis reported that Jagged-1 stimulated the expression of the membrane trafficking protein (DOP1B), which can indirectly improve osteogenic differentiation. hDPSCs cultured on Jagged-1 surface under OM condition expressed COL27A1, MXRA5, COL7A1, and MMP16, which played an important role in osteogenic differentiation. Furthermore, common matrisome proteins of all cellular components were related to osteogenesis/osteogenic differentiation. Additionally, the gene ontology categorised by the biological process of cytosol, membrane, and cytoskeleton compartments was associated with the biomineralisation process. The gene ontology of different culture conditions in each cellular component showed several unique gene ontologies. Remarkably, the Jagged-1_OM culture condition showed the biological process related to odontogenesis in the membrane compartment. In conclusion, the Jagged-1 induces osteogenic differentiation could, mainly through the regulation of protein in the membrane compartment.

Objective: The aim of this study was to compare five toothpastes in terms of alteration of surface roughness and colour of red-wine-stained human enamel over time after brushing simulation. Methods: Stained specimens were randomly divided into five groups (n = 8): Oral-B Gum and Enamel (C), ZACT Stain Fighter (ZW), Colgate Optic White Volcanic Mineral (CW), Oral-B 3D White Luxe Fresh Breath (3DW), and Thepthai (TW). The colour and surface roughness of the specimens were measured after brushing simulation for four durations; two weeks, one month, six months, and twelve months. Abrasive particles in toothpaste were inspected under a scanning electron microscope. Results: Surface roughness was increased in the specimens that underwent brushing simulation in all groups (p < 0.05). ZW (6.33 ± 0.98 μm) exerted the most changes in surface roughness for all durations. Other groups showed similar surface roughness at each time point. ΔE00 and ΔL were increased in all groups until twelve months; however, there were no significant differences between C (ΔE00 = 30.17 ± 4.16, ΔL = 38.19 ± 4.34), CW (ΔE00 = 24.25 ± 10.52, ΔL = 31.12 ± 11.00), and TW (ΔE00 = 29.00 ± 3.96, ΔL = 36.68 ± 4.53) at any time period. Moreover, ZW (ΔE00 = 45.6 ± 8.01, ΔL = 53.03 ± 6.71) and 3DW (ΔE00 = 43.6 ± 7.33, ΔL = 51.03 ± 6.47) exhibited a substantial change and were statistically differed from the other groups after four-weeks. Various characteristics of abrasive particles were inspected under SEM. Conclusion: All five toothpastes increased the surface roughness altered the colour of red wine-stained human enamel over time.

6-bromoindirubin-3′-oxime (BIO) is a candidate small molecule that effectively modulates Wnt signalling owing to its stable property. The present study investigated the influence of BIO on the odonto/osteogenic differentiation of human dental pulp stem cells (hDPSCs). hDPSCs were treated with 200, 400, or 800 nM BIO, and the effects on hDPSC responses and osteogenic differentiation were assessed. BIO-mediated Wnt activation was confirmed by β-catenin nuclear translocation detected by immunofluorescence staining. BIO attenuated colony formation and cell migration determined by in vitro wound-healing assay. BIO increased early apoptotic cell population evaluated using flow cytometry. For osteogenic induction, BIO promoted alkaline phosphatase (ALP) activity and mineralisation in a dose-dependent manner. ALP, RUNX2, OCN, OSX, ANKH, DMP1, and DSPP mRNA expression were significantly upregulated. The OPG/RANKL expression ratio was also increased. Further, BIO attenuated adipogenic differentiation as demonstrated by decreased lipid accumulation and adipogenic-related gene expression. Bioinformatic analysis of RNA sequencing data from the BIO-treated hDPSCs revealed that BIO modulated pathways related to autophagy and actin cytoskeleton regulation. These findings demonstrated that BIO treatment promoted hDPSC osteogenic differentiation. Therefore, this small molecule is a strong candidate as a bioactive molecule to enhance dentin repair.

Notch signaling is associated with many human malignancies, including oral squamous cell carcinoma (OSCC). However, the exact function of Notch signaling in OSCC remains unclear. Here, we investigated the effect of Notch signaling inhibition using a γ-secretase inhibitor (DAPT) on OSCC behaviours in vitro. Bioinformatic analysis of public-available gene expression profiles revealed the dysregulation of the Notch signaling pathway in OSCC compared with normal tissues, indicating the role of Notch signaling in OSCC regulation. RNA sequencing analysis of DAPT-treated human OSCC cells revealed the dysregulation of genes related to cell cycle-related pathways. Blocking Notch signaling significantly inhibited cell proliferation. DAPT-induced G0/G1 cell cycle arrest induced cell apoptosis. Furthermore, cell migration and invasion were also reduced in DAPT-treated cells. These findings indicate that Notch signaling activation participates in OSCC regulation by promoting cell growth, cell cycle progression, cell migration, and invasion. These mechanisms could facilitate OSCC progression. These results imply the potential use of Notch signaling inhibitors as a candidate adjuvant treatment in OSCC patients.

Relevant immunomodulatory effects have been proposed following allogeneic cell-based therapy with human periodontal ligament stem cells (hPDLSCs). This study aimed to examine the influence of shear stress on the immunosuppressive capacity of hPDLSCs. Cells were subjected to shear stress at different magnitudes (0.5, 5 and 10 dyn/cm2). The expression of immunosuppressive markers was evaluated in shear stress-induced hPDLSCs using qRT-PCR, western blot, enzyme activity and enzyme-linked immunosorbent assays. The effects of a shear stress-derived condition medium (SS-CM) on T cell proliferation were examined using a resazurin assay. Treg differentiation was investigated using qRT-PCR and flow cytometry analysis. Our results revealed that shear stress increased mRNA expression of IDO and COX2 but not TGF-β1 and IFN-γ. IDO activity, kynurenine and active TGF-β1 increased in SS-CM when compared to the non-shear stress-derived conditioned medium (CTL-CM). The amount of kynurenine in SS-CM was reduced in the presence of cycloheximide and ERK inhibitor. Subsequently, T cell proliferation decreased in SS-CM compared to CTL-CM. Treg differentiation was promoted in SS-CM, indicated by FOXP3, IL-10 expression and CD4+CD25hiCD127lo/− subpopulation. In conclusion, shear stress promotes kynurenine production through ERK signalling in hPDLSC, leading to the inhibition of T cell proliferation and the promotion of Treg cell differentiation.

Abstract Background Basic fibroblast growth factor (bFGF) regulates maintenance of stemness and modulation of osteo/odontogenic differentiation and mineralization in stem cells from human exfoliated deciduous teeth (SHEDs). Mineralization in the bones and teeth is in part controlled by pericellular levels of inorganic phosphate (Pi), a component of hydroxyapatite, and inorganic pyrophosphate (PPi), an inhibitor of mineralization. The progressive ankylosis protein (gene ANKH; protein ANKH) and ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1/ENPP1) increase PPi and inhibit mineralization, while tissue-nonspecific alkaline phosphatase (ALPL; TNAP) is a critical pro-mineralization enzyme that hydrolyzes PPi. We hypothesized that regulation by bFGF of mineralization in SHEDs occurs by modulation of Pi/PPi-associated genes. Methods Cells were isolated from human exfoliated deciduous teeth and characterized for mesenchymal stem cell characteristics. Cells were treated with bFGF, and the osteogenic differentiation ability was determined. The mRNA expression was evaluated using real-time polymerase chain reaction. The mineralization was examined using alizarin red S staining. Results Cells isolated from primary teeth expressed mesenchymal stem cell markers, CD44, CD90, and CD105, and were able to differentiate into osteo/odontogenic and adipogenic lineages. Addition of 10 ng/ml bFGF to SHEDs during in vitro osteo/odontogenic differentiation decreased ALPL mRNA expression and ALP enzyme activity, increased ANKH mRNA, and decreased both Pi/PPi ratio and mineral deposition. Effects of bFGF on ALPL and ANKH expression were detected within 24 h. Addition of 20 mM fibroblast growth factor receptor (FGFR) inhibitor SU5402 revealed the necessity of FGFR-mediated signaling, and inclusion of 1 μg/ml cyclohexamide (CHX) implicated the necessity of protein synthesis for effects on ALPL and ANKH. Addition of exogenous 10 μm PPi inhibited mineralization and increased ANKH, collagen type 1a1 (COL1A1), and osteopontin (SPP1) mRNA, while addition of exogenous Pi increased mineralization and osterix (OSX), ANKH, SPP1, and dentin matrix protein 1 (DMP1) mRNA. The effects of PPi and Pi on mineralization could be replicated by short-term 3- and 7-day treatments, suggesting signaling effects in addition to physicochemical regulation of mineral deposition. Conclusion This study reveals for the first time the effects of bFGF on Pi/PPi regulators in SHEDs and implicates these factors in how bFGF directs osteo/odontogenic differentiation and mineralization by these cells.

Background: Basic fibroblast growth factor (bFGF) regulates cell proliferation, migration, and differentiation in various cell types. The aim of the present study was to determine the bFGF target genes in stem cells isolated from human exfoliated deciduous teeth (SHEDs). Methods: Cells were isolated from pulp tissue obtained from exfoliated deciduous teeth. Mesenchymal stem cell surface markers and the differentiation potential toward adipogenic and neurogenic lineages were characterized. The bFGF-treated SHED transcriptome was examined using a high throughput RNA sequencing technique. The mRNA and protein expression of selected genes were evaluated using real-time polymerase chain reaction and immunofluorescence staining, respectively. Cell cycle analysis was performed by flow cytometry. The colony forming unit number was also examined. Results: The isolated cells expressed CD44, CD90, CD105, but not CD45. The upregulation of adipogenic and neurogenic marker genes was observed after culturing cells in the appropriate induction medium. Transcriptome analysis of the bFGF treated cells revealed that the upregulated genes were in the cell cycle related pathways, while the downregulated genes were in the extracellular matrix related pathways. Correspondingly, bFGF induced MKI67 mRNA expression and Ki67 protein expression. Furthermore, bFGF treatment significantly decreased the G0/G1, but increased the G2/M, population in SHEDs. Colony formation was markedly increased in the bFGF treated group and was attenuated by pretreating the cells with FGFR or PI3K inhibitors. Conclusion: bFGF controls cell cycle progression in SHEDs. Thus, it can be used to amplify cell number to obtain the amount of cells required for regenerative treatments.

The data contained within this article relate to experimental data on mechanical behavior of an in-house cast hyperelastic Polydimethysiloxane (PDMS) based membrane (SilasticⓇ T-4, Dow Corning) for cell culture, which was used as a tool for applying tensile stress on cells and tissues. With the experimentally obtained material constants, tensile stress distribution over the membrane surface was numerically assessed using a finite element analysis (FEA). The membrane area having a uniform tensile stress distribution under different strain loading conditions of 1–20% was suggested for cell culture.

Microspace culture is promising for self-organization of induced pluripotent stem cells (iPSCs). However, the optimal size of microspaces for osteogenic differentiation is unclear. We hypothesized that a specific microspace size could facilitate self-organizing iPSC differentiation to form bone-like tissue in vitro. The objectives of this study were to investigate such effects of microspace size and to evaluate bone regeneration upon transplantation of the resulting osteogenic constructs. Dissociated mouse gingival fibroblast-derived iPSCs were plated in ultra-low-attachment microspace culture wells containing hundreds of U-bottom-shaped microwell spots per well to form cell aggregates in growth medium. The microwells had different aperture diameters/depths (400/560 μm (Elp400), 500/700 μm (Elp500), and 900/700 μm (Elp900)) (Kuraray; Elplasia). After 5 days of aggregation, cells were maintained in osteogenic induction medium for 35 days. Only cells in the Elp500 condition tightly aggregated and maintained high viability during osteogenic induction. After 10 days of induction, Elp500 cell constructs showed significantly higher gene expression of Runx2, Osterix, Collagen 1a1, Osteocalcin, Bone sialoprotein, and Osteopontin compared to constructs in Elp400 and Elp900. In methylene blue-counterstained von Kossa staining and Movat’s pentachrome staining, only Elp500 constructs showed robust osteoid formation on day 35, with high expression of type I collagen (a major osteoid component) and osteocalcin proteins. Cell constructs were transplanted into rat calvarial bone defects, and micro-CT analysis after 3 weeks showed better bone repair with significantly higher bone mineral density in the Elp500 group compared to the Elp900 group. These results suggest that microspace size affects self-organized osteogenic differentiation of iPSCs. Elp500 microspace culture specifically induces mouse iPSCs into osteoid-rich bone-like tissue possessing high bone regeneration capacity.

Indirect immobilized ligand has been shown as an effective technique to activate Notch signalling in vitro. The data presented in this article are related to the published article entitled “Indirect immobilized Jagged1 suppresses cell cycle progression and induces odonto/osteogenic differentiation in human dental pulp cells” (Manokawinchoke et al. 2017) [1]. This data article describes gene expression in indirect immobilized Jagged1 treated human dental pulp cells (hDPs) using high throughput RNA sequencing technique. These data are valuable to analyze the regulation of Notch signalling in hDPs for understanding its molecular mechanism(s). Raw RNA sequencing data were deposited in the NCBI Sequence Read Archive (SRP100068) and NCBI Gene Expression Omnibus (GSE94989).

Periodontal disease is the most common oral disease in dogs. Platelet-rich fibrin (PRF) is widely utilized to facilitate soft and hard tissue healing and has been proposed in periodontal healing in small animal treatment. However, the quality and amount of autologous PRF is compromised in animals with systemic diseases. The present study aimed to evaluate the efficacy of xenologous bubaline blood-derived PRF (bPRF) on periodontal tissue healing in canine periodontal defects. Split-mouth design was employed in twenty dogs diagnosed with periodontal disease. The defects were divided randomly into two groups: the open-flap debridement (OFD)-treated group and the OFD with bPRF (OFD+bPRF) application group. Results demonstrated that gingival index and periodontal probing depth decreased significantly in the OFD+bPRF group compared with those treated with OFD alone. Application of bPRF in periodontal defects also promoted fibrous tissue formation, as confirmed by the marked increase in fibrosis score. bPRF application significantly increased COL1A1 and PDGFB mRNA levels at day 14 compared with the baseline. Taking this evidence together, bPRF provided a favorable therapeutic modality in canine periodontal defects. bPRF could be an alternative biomaterial for the treatment of periodontal defects in dogs.

Objective: To investigate a dysregulation of Notch signaling in oral lichen planus (OLP) using public available microarray dataset. Methods: A mRNA expression profiling dataset from Gene Expression Omnibus was downloaded. Differential gene expression between OLP and normal oral epithelium was examined using NetworkAnalyst. The dysregulated genes related to Notch signaling were identified. Results: Thirteen genes in Notch signaling pathway were significantly differential expressed between OLP and normal epithelium. OLP samples significantly increased the mRNA levels of HEYL, APH1B, CNTN1 and PSEN2. Whilst, ITCH, HES1, TLE2, DLK2, DTX2, NOTCH3, JAG2, RFNG, and SPEN were downregulated in OLP groups. Conclusions: Notch signaling was dysregulated and may participate in pathophysiologic process in OLP.

Bone tissue engineering has been widely studied and proposed as a promising platform for correcting the bone defects. The applications of mesenchymal stem cell (MSC)-based bone tissue engineering have been investigated in various in vitro and in vivo models. In this regard, the promising animal bone defect models have been employed for illustrating the bone regenerative capacity of MSC-based bone tissue engineering. However, most studies aimed for clinical applications in human. These evidences suggest a knowledge gap to fulfill the accomplishment for veterinary implementation. In this review, the fundamental concept, knowledge, and technology of MSC-based bone tissue engineering focusing on veterinary applications are summarized. In addition, the potential canine MSCs resources for veterinary bone tissue engineering are reviewed, including canine bone marrow-derived MSCs, canine adipose-derived MSCs, and canine dental tissue-derived MSCs. This review will provide a basic and current information for studies aiming for the utilization of MSC-based bone tissue engineering in veterinary practice.

Mechanical force regulates numerous biological functions. Application of different force types leads to different cell responses. This data article describes RNA sequencing data identifying gene expression of human periodontal ligament cells (hPDLs) treated with the continuous or intermittent compressive force. These data could be further utilized to investigate the controlling pathways that regulate hPDLs’ behaviors by the different force types. Raw RNA sequencing data were deposited in the NCBI Sequence Read Archive (SRP136155) and NCBI Gene Expression Omnibus (GSE112122). Keywords: Periodontal ligament, Mechanical force, RNA sequencing

Objective: To investigate the influence of Notch signaling on osteoprotegerin (OPG) expression in a human oral squamous cell carcinoma cell line. Methods: Activation of Notch signaling was performed by seeding cells on Jagged1 immobilized surfaces. In other experiments, a γ-secretase inhibitor was added to the culture medium to inhibit intracellular Notch signaling. OPG mRNA and protein were determined by real-time PCR and ELISA, respectively. Finally, publicly available microarray database analysis was performed using connection up- or down-regulation expression analysis of microarrays software. Results: Jagged1-treatment of HSC-4 cells enhanced HES1 and HEY1 mRNA expression, confirming the intracellular activation of Notch signaling. OPG mRNA and protein levels were significantly suppressed upon Jagged1 treatment. Correspondingly, HSC-4 cells treated with a γ-secretase inhibitor resulted in a significant reduction of HES1 and HEY1 mRNA levels, and a marked increase in OPG protein expression was observed. These results implied that Notch signaling regulated OPG expression in HSC-4 cells. However, Jagged1 did not alter OPG expression in another human oral squamous cell carcinoma cell line (HSC-5) or a human head and neck squamous cell carcinoma cell line (HN22). Conclusions: Notch signaling regulated OPG expression in an HSC-4 cell line and this mechanism could be cell line specific.