Proliferation Of Dental Pulp Cells Research Articles

Oscillatory fluid flow enhanced mineralization of human dental pulp cells

The purpose of this study is to evaluate the optimum frequency of oscillatory fluid flow (OFF) for increasing osteogenesis in human dental pulp cells (DPCs) in an incubating rocking shaker. DPCs from 3 donors were cultured in an osteogenic induction medium (OIM) and mechanical stimulation was applied using an incubating rocking shaker at frequencies of 0 (control), 10, 20, 30, and 40 round per minute (RPM) for 1 h/day, 5 days/week. Cell proliferation was measured using total protein quantification, and osteogenic activity was measured by alkaline phosphatase (ALP) activity, calcium deposition, and collagen production on days 7, 14, and 21 of culture. Results of DPCs morphology in the 30 RPM group were more clustered and formed interconnections between cells. Results of DPC proliferation and collagen production showed no significant differences between the experiment groups. The ALP activity on day 7 and 14, and calcium deposition on day 21 of the 30 RPM group were significantly higher than the control groups. Thus 30 RPM is likely an effective frequency for increasing calcium deposition. This study uses strategies in Tissue Engineering followed the research topic about an application of human cells to stimulate oral and maxillofacial hard tissue regeneration. In the future, the mineralization of DPCs could be enhanced by using an incubating rocking shaker at 30 RPM in the lab to create a cell sheet. The mineralized cell sheet could then be implanted into the patient for bone repair of orofacial defects.

Mesenchymal stromal cell exosomes enhance dental pulp cell functions and promote pulp-dentin regeneration

Mesenchymal stromal/stem cell (MSC) therapies are currently being explored for dental pulp regeneration. As the therapeutic effects of MSCs in tissue repair are mediated mainly through the release of extracellular vesicles (EVs) including exosomes, we investigated here the cellular processes and molecular mechanisms modulated by MSC exosomes in dental pulp regeneration. Using dental pulp cell (DPC) cultures, we demonstrated that MSC exosomes could increase DPC migration, proliferation, and odontogenic differentiation. The enhancement of these cellular processes was mediated through exosomal CD73-mediated adenosine receptor activation of AKT and ERK signaling. Consistent with these observations, MSC exosomes increased the expression of dentin matrix proteins and promoted the formation of dentin-like tissue and bridge-like structures in a rat pulp defect model. These effects were comparable to that of mineral trioxide aggregate (MTA) treatment. MSC exosomes also yielded recellularized pulp-dentin tissues in the root canal of endodontically-treated human premolars, following subcutaneous implantation in the mouse dorsum. Together, our findings suggest that MSC exosomes could exert a multi-faceted effect on DPC functions including migration, proliferation and odontogenic differentiation to promote dental pulp regeneration. This study provides the basis for development of MSC exosomes as a cell-free MSC therapeutic alternative for pulp-dentin regeneration.

Characterisation of miRNA Expression in Dental Pulp Cells during Epigenetically-Driven Reparative Processes.

Within regenerative endodontics, exciting opportunities exist for the development of next-generation targeted biomaterials that harness epigenetic machinery, including microRNAs (miRNAs), histone acetylation, and DNA methylation, which are used to control pulpitis and to stimulate repair. Although histone deacetylase inhibitors (HDACi) and DNA methyltransferase inhibitors (DNMTi) induce mineralisation in dental pulp cell (DPC) populations, their interaction with miRNAs during DPC mineralisation is not known. Here, small RNA sequencing and bioinformatic analysis were used to establish a miRNA expression profile for mineralising DPCs in culture. Additionally, the effects of a HDACi, suberoylanilide hydroxamic acid (SAHA), and a DNMTi, 5-aza-2'-deoxycytidine (5-AZA-CdR), on miRNA expression, as well as DPC mineralisation and proliferation, were analysed. Both inhibitors increased mineralisation. However, they reduced cell growth. Epigenetically-enhanced mineralisation was accompanied by widespread changes in miRNA expression. Bioinformatic analysis identified many differentially expressed mature miRNAs that were suggested to have roles in mineralisation and stem cell differentiation, including regulation of the Wnt and MAPK pathways. Selected candidate miRNAs were demonstrated by qRT-PCR to be differentially regulated at various time points in mineralising DPC cultures treated with SAHA or 5-AZA-CdR. These data validated the RNA sequencing analysis and highlighted an increased and dynamic interaction between miRNA and epigenetic modifiers during the DPC reparative processes.

Biomechanical characterization of a fibrinogen-blood hydrogel for human dental pulp regeneration.

In dental practice, Regenerative Endodontic Treatment (RET) is applied as an alternative to classical endodontic treatments of immature necrotic teeth. This procedure, also known as dental pulp revitalization, relies on the formation of a blood clot inside the root canal leading to the formation of a reparative vascularized tissue similar to dental pulp, which would provide vitality to the affected tooth. Despite the benefit of this technique, it lacks reproducibility due to the fast degradation and poor mechanical properties of blood clots. This work presents a method for constructing a fibrinogen-blood hydrogel that mimics the viscoelastic properties of human dental pulp while preserving the biological properties of blood for application in RET. By varying the blood and fibrinogen concentrations, gels with different biomechanical and biological properties were obtained. Rheology and atomic force microscopy (AFM) were combined to study the viscoelastic properties. AFM was used to evaluate the elasticity of human dental pulp. The degradation and swelling rates were assessed by measuring weight changes. The biomimetic properties of the gels were demonstrated by studying the cell survival and proliferation of dental pulp cells (DPCs) for 14 days. The formation of an extracellular matrix (ECM) was assessed by multiphoton microscopy (MPM). The angiogenic potential was evaluated by an ex vivo aortic ring assay, in which the endothelial cells were observed by histological staining after migration. The results show that the Fbg-blood gel prepared with 9 mg ml-1 fibrinogen and 50% blood of the Fbg solution volume has similar elasticity to human dental pulp and adequate degradation and swelling rates. It also allows cell survival and ECM secretion and enhances endothelial cell migration and formation of neovessel-like structures.

Dental Pulp Cell Conditioning through Polyinosinic-Polycytidylic Acid Activation of Toll-like Receptor 3 for Amplification of Trophic Factors

IntroductionRegeneration of the pulp-dentin complex hinges on functionally diverse growth factors, cytokines, chemokines, signaling molecules, and other secreted factors collectively referred to as trophic factors. The delivery of exogenous factors and the induced release of endogenous dentin-bound factors by conditioning agents have been explored toward these goals. The aim of this study was to investigate a promising regeneration strategy based on the conditioning of dental pulp cells (DPCs) with polyinosinic-polycytidylic acid (poly[I:C]) for the amplification of endogenous trophic factors. MethodsDPCs were isolated from human dental pulps, propagated in culture, and treated with an optimized dose of poly(I:C). The 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide assay and metabolite analysis were conducted to monitor the cytotoxicity of poly(I:C). Enzyme-linked immunosorbent assays and quantitative polymerase chain reaction assays were performed to quantify the induction of trophic factors in response to DPC conditioning. Statistical significance was P < .05. ResultsThe analysis of 32 trophic factors involved in Wnt signaling, cell migration and chemotaxis, cell proliferation and differentiation, extracellular matrix remodeling and angiogenesis, and immunoregulation revealed that DPCs abundantly express many trophic factors including AMF, BDNF, BMP2, FGF1, FGF2, FGF5, HGF, MCP1, NGF, SDF1, TGFβ1, TIMP1, TIMP2, TIMP3, and VEGFA, many of which were further induced by DPC conditioning; induction was significant for BDNF, EGF, HGF, LIF, MCP1, SDF1, IL6, IL11, MMP9, and TIMP1. Both DPC proliferation and lactate production (P < .05) were inhibited by 8 μg/mL poly(I:C) relative to the control. ConclusionsIn vitro DPC conditioning through poly(I:C) activation of toll-like receptor 3 led to the amplification of trophic factors involved in tissue repair. The strategy offers promise for endodontic regeneration and tooth repair and warrants further investigation.

Blue light photobiomodulation of dental pulp cells

PurposeBlue light has been reported to enhance the differentiation and mineralisation potential of mesenchymal stem cells. No studies have investigated the photobiomodulation effects of blue light on the odontogenic differentiation of dental pulp stem cell-containing populations. This study aimed to determine the effect of 405-nm wavelength on the mineralisation response in these cells.MethodsHuman dental pulp cells were irradiated using a calibrated 405 nm light source at an irradiance of 57.7 mW/cm2 for 35, 69, 90 and 104 s; delivering energy densities of 2, 4, 5 and 6 J/cm2. Cell proliferation was investigated on days 2 and 4. Alkaline phosphatase activity and dentine matrix protein-1 levels were investigated on days 3 and 5. Mineralised nodule formation was investigated on days 7, 4 and 21.ResultsThe 405-nm blue light had significant inhibitory effects on dental pulp cell proliferation. Simultaneously, blue light irradiation enhanced the mineralisation response, as determined by the upregulation of odontogenic markers. Irradiated cultures also demonstrated a significant increase in mineralised nodule formation compared with the non-irradiated controls at all time points.ConclusionData indicate that phototherapy utilising a 405-nm wavelength may have therapeutic potential in stimulating odontogenic responses of pulp cells.

Nestin and NG2 transgenes reveal two populations of perivascular cells stimulated by photobiomodulation.

Pericytes and glial cells are known to collaborate in dental pulp tissue repair. Cell-based therapies that stimulate these stromal components may be of therapeutic relevance for partially vital dental pulp conditions. This study aimed to examine the early effect of photobiomodulation (PBM) in pericytes from experimentally injured pulp tissue. To accomplish this, we used the Nestin-GFP/NG2-DsRed mice, whichcould allow the identification of distinct pericyte phenotypes. We discovered the presence of two pericytes subsets within the dental pulp, the Nestin + NG2+ (type-2) and Nestin- NG2+ (type-1). Upon injury, PBM treatment led to a significant increase in Nestin+ cells and pericytes. This boost was mainly conferred by the more committed pericyte subset (NestinNG2+ ). PBM also stimulated terminal blood vessels sprouting adjacent to the injury site while maintaining signs of pulp vitality. In vitro, PBM induced VEGF upregulation, improved dental pulp cells proliferation and migration, and favored their mineralization potential. Herein, different subsets of perivascular cells were unveiled in the pulp tissue. PBM enhanced not only NG2+ cells but nestin-expressing progenitors in the injured dental pulp.

Insulin Effect on Gene Expression of Dental Pulp Cell during Osteodifferentiation

Insulin, an old but still actual molecule, has many roles in various cellular processes including bone formation and also angiogenesis. Insulin effects on different cell types were investigated, and we intended to check its effect on dental pulp cells (DPC) during osteoblast differentiation. First, bone differentiation ability of isolated dental pulp cells was assessed by alizarin red (AR) staining. Insulin increased dental pulp cell proliferation after 25 hours of culture. It increased mRNA expression of osteogenic markers such as Col1, RunX2, ALP, Osc, Mef2C and expression of genes involved in TGF b pathway such as Smad3, TSP1, VEGF at different time points.

Effect of erythropoietin on angiogenic potential of dental pulp cells.

Erythropoietin (EPO) is a 34-kDa glycoprotein that possesses the potential for angiogenesis, as well as anti-inflammatory and anti-apoptotic properties. The present study aimed to examine the effect of EPO on the angiogenesis of dental pulp cells (DPCs) and to explore the underlying mechanisms of these effects. It was demonstrated that EPO not only promoted DPCs proliferation but also induced angiogenesis of DPCs in a paracrine fashion. EPO enhanced the angiogenic capacity by stimulating DPCs to secrete a series of angiogenic cytokines. ELISA confirmed that high concentrations of EPO increased the production of MMP-3 and angiopoietin-1 but decreased the secretion of IL-6. Furthermore, EPO activated the ERK1/2 and p38 signaling pathways in DPCs, while inhibition of these pathways diminished the angiogenesis capacity of DPCs. The present study suggested that EPO may have an important role in the repair and regeneration of dental pulp.

The molecular functions of Biodentine and mineral trioxide aggregate in lipopolysaccharide-induced inflamed dental pulp cells.

To explore the proliferation, adhesion and differentiation response and the underlying mechanisms that occur in lipopolysaccharide (LPS)-induced inflamed dental pulp cells (DPCs) in contact with Biodentine and mineral trioxide aggregate (MTA). The DPCs were isolated from three healthy donors and named DPC-H1 to DPC-H3. The DPCs were pre-cultured with 2 or 5μgmL-1 LPS for 24h to induce inflammation. The expression of inflammation marker miR-146a was detected by q-PCR. The normal and LPS-induced DPCs were further treated with 0.14mgmL-1 Biodentine or 0.13mgmL-1 MTA for 24h. MTT assay and adhesion assay were used to analyse the changes of cell phenotypes. DSPP, AKT and ERK expressions were detected by Western blotting. The data were analysed by Mann-Whitney test or two-way anova. Differences were considered statistically significant when P<0.05. In LPS-induced DPCs, Biodentine and MTA treatment neither induced nor aggravated LPS-induced inflammation, but their presence did increase the expression of the odontogenic differentiation marker DSPP. Under 2 or 5μgmL-1 LPS-induced inflammation, Biodentine and MTA promoted the proliferation of DPC cells, and significantly in DPC-H2 (P<0.0001 for both reagents). With the treatment of 2μgmL-1 LPS, the cell adhesion of DPCs on the fibronectin-coated culture plates was increased significantly by Biodentine (P=0.0413) and MTA (P<0.0001). Biodentine and MTA regulated cell adhesion on the fibronectin-coated culture plates (P<0.0001 for both reagents) and proliferation (P<0.0001 for both reagents) via the AKT pathway. However, the AKT pathway was not involved in the expression of DSPP induced by Biodentine and MTA. Biodentine and MTA enhanced the proliferation, adhesion and differentiation of LPS-induced DPCs. The proliferation and adhesion process induced by Biodentine and MTA was via the AKT pathway. However, the cellular differentiation process might not use the same pathway, and this needs to be explored in future studies.

Pluripotency of Dental Pulp Cells and Periodontal Ligament Cells Was Enhanced through Cell-Cell Communication via STAT3/Oct-4/Sox2 Signaling.

Alternation in culture environment due to cell-cell communications can rejuvenate the biological activity of aged/differentiated cells and stimulate the expression of pluripotency markers. Dental pulp cells (DPCs) and periodontal ligament cells (PDLCs) are promising candidates in dental tissue regeneration. However, the molecular network that underlies cell-cell communications between dental-derived cells and the microenvironment remains to be identified. To elucidate the signaling network that regulates the pluripotency of DPCs and PDLCs, proliferation, apoptosis, cell cycle, and the expression of Oct-4/Sox2/c-Myc in DPCs and PDLCs with indirect/direct coculture were examined. PCR arrays were constructed to identify genes that were differentially expressed, and the results were confirmed by a rat model with injury. Further research on the mechanism of the related signaling pathways was investigated by overexpression/silence of STAT3, ChIP, the dual-luciferase reporter assay, and EMSA. We found that the proliferation and apoptosis of DPCs and PDLCs were inhibited, and their cell cycles were arrested at the G0/G1 phase after coculture. Oct-4, Sox2, and STAT3 expression significantly increased and PAX5 expression decreased in the coculture systems. Oct-4/Sox2/STAT3/PAX5 was actively expressed in the rat defect model. Moreover, STAT3 was directly bound to the Oct-4 and Sox2 gene promoter regions and activated the expression of those genes. Our data showed that the pluripotency of DPCs and PDLCs was enhanced through cell-cell communication. STAT3 plays essential roles in regulating the pluripotency of DPCs and PDLCs by targeting Oct-4 and Sox2 both in vitro and in vivo.

Reparative Dentin Formation by Dentin Matrix Proteins and Small Extracellular Vesicles

IntroductionVital pulp therapy aims at preserving pulp vitality and regenerating dentin. Therefore, the purpose of this study was to explore the effects of a combination of treated dentin matrix (TDM) proteins and dental pulp cell (DPC)-derived small extracellular vesicles (sEVs) on pulp-dentin complex repair. MethodsWe prepared TDM by chemical demineralization and mechanical disruption of teeth to a powder preparation. The sEVs were isolated from culture supernatants of DPCs and identified by nanoparticle tracking analysis, Western blotting, and transmission electron microscopy. The effect of a combination of TDM proteins and DPC-derived sEVs on DPC proliferation, migration, and odontogenic differentiation was evaluated in vitro. A minipig model of pulp injury was used to compare the clinical outcomes and tissue responses attributed to 4 materials including TDM, sEV-TDM, sEVs, and mineral trioxide aggregate. ResultsThe sEV isolated from the cell supernatant promoted DPC proliferation and migration. The combination of TDM extracts and sEV synergistically promoted the migration of DPCs but suppressed their proliferation. Real-time polymerase chain reaction and Western blot revealed that sEV-TDM enhanced the odontoblast-related protein expressions in DPCs. In in vivo studies, TDM and sEV-TDM promoted the formation of continuous reparative dentin. Furthermore, odontoblastlike high columnar cells were observed on the pulp side of the dentin bridge. ConclusionsThe sEV-TDM complex exhibits intrinsic biological activities, which has potential applications as a bioactive pulp-capping material.

Effect of the Soluble Factors Released by Dental Apical Papilla-Derived Stem Cells on the Osteo/Odontogenic, Angiogenic, and Neurogenic Differentiation of Dental Pulp Cells.

Stem cells derived from dental apical papilla (SCAPs) can secrete various soluble factors, which may stimulate tissue repair and regeneration in vivo. The aim of this study was to elucidate the effect of the soluble factors released by SCAPs on the proliferation and differentiation of dental pulp cells (DPCs). We compared the osteo/odontogenic, angiogenic, and neurogenic effects of soluble factors released from SCAPs and bone marrow mesenchymal stem cells (BMSCs) in vitro. Conditioned media (CM) were collected from human SCAPs and BMSCs cultures, and their effects on human DPCs proliferation and differentiation were evaluated. Cellular proliferation was unaffected by SCAPs-CM and was inhibited by BMSCs-CM. Cells treated with osteo/odontogenic inducing medium (OM) plus SCAPs-CM showed higher alkaline phosphatase activity than did cells in the OM group. The expression level of osteo/odontogenic markers were higher in the SCAPs-CM plus OM group than in the BMSCs-CM plus OM and OM groups. SCAPs-CM and BMSCs-CM significantly promoted DPCs migration. DPCs angiogenic differentiation was not affected by SCAPs-CM but was significantly enhanced by BMSCs-CM. In DPCs cultured in media optimized for neural stem cell growth for 2 weeks, the expression levels of neurogenic markers were significantly enhanced by the addition of SCAPs-CM. Neuronal markers expression was significantly reduced, while neurotrophic marker expression significantly increased by the addition of BMSCs-CM. In conclusion, SCAPs-CM significantly enhanced osteo/odontogenic differentiation, migration, and neurogenic differentiation potential of DPCs, but have no effect on DPCs proliferation and angiogenic differentiation in vitro. CM released from SCAPs have a greater osteo/odontogenic and neurogenic inductive effect on DPCs than BMSCs-CM. It indicates that SCAPs-CM can serve as additive to improve pulp tissue repair and regeneration.

Biological responses of dental pulp cells to surfaces modified by collagen 1 and fibronectin.

Collagen 1 (COL1) and fibronectin (FN) are extracellular matrix proteins that contribute in cell activity and involve in regulating dental pulp cells (DPCs). The purpose of this study was to investigate the effect of COL1 and FN on the behavior of DPCs. Here, DPCs were grown under three different conditions: COL1 coating, FN coating, and control group without coating. The proliferation and differentiation of DPCs were investigated. DPCs in osteogenic media were able to differentiate into osteoblastic phenotype. The morphological analysis revealed no obvious difference on the shape of cells. Cells had spread well on both coated and noncoated culture plates with slightly more spreading in the coated plates after 24 hr. The MTT analysis did not demonstrate a significant difference at 1 and 3 hr among the groups, but interestingly, the analysis disclosed more cells on the coated plates after longer cultures, which indicated a higher proliferative capacity in response to COL1 and FN. RT-PCR, Western Blotting and mineralization assays did not reveal significant differences between the coated and noncoated surfaces in relation to osteogenic differential potential. Our data suggested that the surface coating of COL1 and FN were able to promote cellular proliferation and the osteogenic differentiation tendency of DPCs was also observed in vitro.

Catechins reduce inflammation in lipopolysaccharide-stimulated dental pulp cells by inhibiting activation of the NF-κB pathway.

To ascertain the anti-inflammation mechanism of catechins in lipopolysaccharide-treated human dental pulp cells (HDPCs). Expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 was measured using quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assays. The anti-inflammatory mechanism was explored by examining activation of nuclear factor-kappa B (NF-κB) signaling using qPCR, Western blotting, and immunofluorescence staining. Human dental pulp cells proliferation was not affected by treatment with epigallocatechin (ECG) or epigallocatechin 3-gallate (EGCG). mRNA expression of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 was decreased significantly in ECG- and EGCG-treated HDPCs. Subsequently, the effects of ECG and EGCG upon activation of NF-κB signaling were evaluated by Western blotting and immunofluorescence staining. Expression of p-p65 protein in HDPCs treated with ECG, EGCG, or an NF-κB inhibitor (Bay 11-7082) was lower than that in HDPCs treated with lipopolysaccharide, data that were consistent with the location of p65 protein according to immunofluorescence staining. Catechin could reduce lipopolysaccharide-stimulated inflammation in HDPCs by inhibiting activation of the NF-κB pathway.

Concentrated Growth Factor Promotes Dental Pulp Cells Proliferation and Mineralization and Facilitates Recovery of Dental Pulp Tissue.

BackgroundDental pulp cells (DPCs) play vital roles in the recovery of dental pulp tissue. Concentrated growth factor (CGF) can promote proliferation and mineralization of various cells. However, the functions of CGF on DPCs and dental pulp tissue are unclear. The object of our study was to identify the roles of CGF in DPCs proliferation and mineralization in vitro and to assess the effects of CGF on direct pulp capping in vivo.Material/MethodsWe performed CCK-8 and Transwell assay to detect proliferation and migration activity of DPCs. Alizarin Red staining was performed to examine mineralized nodules. Alkaline phosphatase activity test was used to measure the mineralization capacity of DPCs. We assessed the odontogenic differentiation gene expression level by Western blot and qPCR. The effect of CGF on direct pulp capping in vivo were evaluated by radiography and histopathology.ResultsCGF increased the number of proliferative and migratory DPCs. CGF enhanced DPCs mineralized nodules and improved the gene expression levels of DSPP, DMP-1, BSP, and ALP. CGF upregulated the protein levels of ALP, BMP2, SMAD5, Runx2, and p-Smad, and the effect could be partially reversed by Noggin. CGF promoted pulp recovery and kept its vitality in directly pulp capping.ConclusionsCGF promotes DPCs proliferation and mineralization. It regulates the mineralization of DPCs via the BMP2/SMAD5/Runx2 signaling pathway. CGF can be used as the effective graft for direct pulp capping.

Interaction of Nel-like molecule 1 with apoptosis related protein 3 with its influence on human dental pulp cells proliferation and differentiation into odontoblasts

Nel-like molecule 1 (Nell-1) is an essential positive regulator of tooth development and odontoblast differentiation. However, its precise mechanism remains undetermined. This study aims to explore the possible receptor or binding protein of Nell-1. Results showed that Nell-1 and Apoptosis related protein 3(APR3) expression levels were high in odontoblasts and inversely correlated. Endogenous Nell-1 co-immunoprecipitated with APR3, and this co-IP was reciprocal. Double immunofluorescence staining revealed that Nell-1 and APR3 colocalized on the nuclear envelope of human dental pulp cells. Nell-1 inhibited the proliferation of these cells co-infected with APR3 through Cyclin D1 downregulation. The interaction of Nell-1 with APR3 stimulated alkaline phosphatase (ALP) activity and promoted the expression and mineralization of DSPP, ALP, OPN, and BSP. The shRNA of APR3 decreased cell differentiation and mineralization. Nell-1 could reciprocally interact with APR3 and stimulate the differentiation and mineralization of human dental pulp cells. Future studies should explore the potential functional connection and the molar mechanism of such interaction.

Regulation of IGF-I by IGFBP3 and IGFBP5 during odontoblast differentiation in mice

ObjectivesAlthough intracellular signaling pathways of insulin-like growth factor I (IGF-I) related to the proliferation of dental pulp cells have been investigated, the switching mechanism from cell proliferation to differentiation during odontogenesis remains elusive. This study aimed to elucidate the role of IGF binding protein (IGFBP) 3 and 5 in regulation of IGF-I during odontoblast differentiation in mouse incisors. MethodsThe detailed expression patterns of IGF-I, IGF-I receptor (IGF-IR), IGFBP3, and IGFBP5 together with that of an odontoblast differentiation marker, nestin, were examined by immunohistochemistry and/or in situ hybridization using paraffinized sections of TetOP-H2B-GFP mouse incisors at postnatal 4 weeks. ResultsUndifferentiated dental papilla cells and preodontoblasts (preOB) showed intense IGF-I- and IGF-IRα-positive reactions, and the expression was observed in differentiated odontoblasts, such as immature odontoblasts (iOB) and mature odontoblasts (mOB). IGFBP3/Igfbp3 was transiently expressed in preOB and early iOB, and the intensity of expression gradually reduced with the progression of odontoblast differentiation. In contrast, immunohistochemical analysis for IGFBP5 identified a positive reaction in the undifferentiated dental papilla cells and differentiated odontoblasts, and the expression of Igfbp5 was reduced in the differentiated odontoblasts. ConclusionThe present study demonstrated the expression patterns of IGF-I, IGF-IR, IGFBP3, and IGFBP5 during odontoblast differentiation in mouse incisors. These results suggested that IGFBP3 regulates the transition from the proliferative to differentiation stage by inhibiting the action of IGF-I on the proliferation of dental papilla cells, and that IGFBP5 plays an important role in the maintenance of the differentiated odontoblasts during tooth development.

Lin28 promotes dental pulp cell proliferation via upregulation of cyclin-dependent proteins and interaction with let-7a/IGF2BP2 pathways

Caries, pulpitis, and trauma are the main causes of dental pulp damage. The regeneration capacity of dental pulp declines with age. Lin28 is a conserved RNA-binding protein in higher eukaryotes that regulates several important cellular functions associated with development, glucose metabolism, differentiation, and pluripotency. Conditional reactivation of Lin28 gene in adult mice markedly accelerates the wound-healing process in injured digits. However, little is known about its functions and molecular mechanism in human dental pulp. The aim of this study was to investigate the effects and mechanism of overexpression of Lin28 gene on the proliferation of human dental pulp cells (HDPCs). For this purpose, a number of molecular and biochemical analytical techniques, including the ethynyl-2ʹ-deoxyuridine (EdU) incorporation assay, RNA-protein immunoprecipitation (RIP) analysis, and luciferase assays, were used for detailed characterization. In addition, factors regulating HDPCs activation were explored through gain-of-function and loss-of-function analyses. The results demonstrate that Lin28 promotes cell proliferation and the S-G2/M transition of HDPCs and directly binds to a group of cell cycle regulatory mRNAs in HDPCs. Through bioinformatics analysis and luciferase assays, we confirmed that let-7a targets IGF2BP2. Silencing of IGF2BP2 showed similar cellular and molecular effects as let-7a. Similarly, restoration of IGF2BP2 counteracted the effects of let-7a expression. In conclusion, Lin28 promotes cell proliferation by regulation of both mRNA translation (let-7-independent) and miRNA biogenesis (let-7-dependent). Lin28 can promote the expression of pro-proliferative genes by directly enhancing their translation to maintain a tight control over HDPC proliferation.

Biological properties of modified bioactive glass on dental pulp cells

Dental caries is a bacteria-caused condition classified among the most common chronic diseases worldwide. Treatment of dental caries implies the use of materials having regenerative and anti-bacterial properties, and controlling inflammation is critical for successful endodontic regeneration. ObjectivesThe aim of this study was to fabricate and characterize a novel composite incorporating sol-gel derived silver-doped bioactive glass (BG) in a chitosan (CS) hydrogel at a 1:1 wt ratio(Ag-BG/CS). MethodsThe effect of Ag-BG/CS on dental pulp cells (DPCs) proliferation was analyzed by CCK-8 assay, whereas the adhesion of DPCs was evaluated by confocal microscopy. The physical morphology of Ag-BG/CS was analyzed by scanning electron microscope. The anti-inflammatory effect of Ag-BG/CS was investigated by quantitative polymerase chain reaction (qPCR). Moreover, the effect of Ag-BG/CS on odontogenic differentiation of DPCs was studied by immunochemical staining, tissue-nonspecific alkaline phosphatase staining, qPCR, and western blot analyses. The antibacterial activity against dental caries key pathogenic bacteria was also evaluated. ResultsThe results of this study showed that Ag-BG/CS did not affect the proliferation of DPCs, it down-regulated the inflammatory-associated markers (IL-1β, IL-6, IL-8, TNF-α) of DPCs treated with Escherichia coli lipopolysaccharide (LPS) by inhibiting NF-κB pathway, and enhanced the in vitro odontogenic differentiation potential of DPCs. Furthermore, Ag-BG/CS strongly inhibited Streptococcus mutans and Lactobacillus casei growth. ConclusionsThis novel biomaterial possessed antibacterial and anti-inflammatory activity, also enhanced the odontogenic differentiation potential of LPS-induced inflammatory-reacted dental pulp cells. The material introduced in this study may thus represent a suitable dental pulp-capping material for future clinical applications.