Human Pulp Cells Research Articles

Dentin-like tissue formation and biomineralization by multicellular human pulp cell spheres in vitro.

IntroductionMaintaining or regenerating a vital pulp is a preferable goal in current endodontic research. In this study, human dental pulp cell aggregates (spheres) were applied onto bovine and human root canal models to evaluate their potential use as pre-differentiated tissue units for dental pulp tissue regeneration.MethodsHuman dental pulp cells (DPC) were derived from wisdom teeth, cultivated into three-dimensional cell spheres and seeded onto bovine and into human root canals. Sphere formation, tissue-like and mineralization properties as well as growth behavior of cells on dentin structure were evaluated by light microscopy (LM), confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX).ResultsSpheres and outgrown cells showed tissue-like properties, the ability to merge with other cell spheres and extra cellular matrix formation; CLSM investigation revealed a dense network of actin and focal adhesion contacts (FAC) inside the spheres and a pronounced actin structure of cells outgrown from the spheres. A dentin-structure-orientated migration of the cells was shown by SEM investigation. Besides the direct extension of the cells into dentinal tubules, the coverage of the tubular walls with cell matrix was detected. Moreover, an emulation of dentin-like structures with tubuli-like and biomineral formation was detected by SEM- and EDX-investigation.ConclusionsThe results of the present study show tissue-like behavior, the replication of tubular structures and the mineralization of human dental pulp spheres when colonized on root dentin. The application of cells in form of pulp spheres on root dentin reveals their beneficial potential for dental tissue regeneration.

Three-Dimensional Human Cell Cultures for Cytotoxicity Testing of Dental Filling Materials

So far, bovine immortalized pulp cells have been used as three dimensional cultures for cytotoxicity testing of filling materials in the dentin barrier test (DBT). In this study, the use of human pulp-derived cells was evaluated, which would better simulate the clinical situation, and a composite material with a new resin base was tested. SV40-transfected human pulp cells (tHPC) were cultured in hydrogels (fibrin, peptide, collagen) and mechanical properties and cell viability (MTT or WST-1) were determined. For cell cultures in collagen, a four week - proliferation assay was performed (WST-1). After 14 days of three-dimensional culture in collagen, tHPC were introduced into the DBT with 200 µm dentin disks. After a 24-hour incubation under perfusion (0.3 ml/h), the following materials were applied according to the manufacturers' instructions (1) President (Coltene): negative (non-toxic) control, (2) CaGPG14 (ISO 7405): positive (toxic) control, (3) Tetric EvoCeram (Ivoclar Vivadent) with Clearfil SEBond (Kuraray, reference material), (4) N´Durance (Sepodont, test material), (5) N´Durance with Clearfil SEBond. Cell viability was determined after 24-hour incubation (WST-1). The percentage of relative viability was calculated (negative control=100%) and statistically analyzed (Kruskal-Wallis-test, p<0.05). Fibrin and peptide gels had insufficient mechanical properties for the DBT Collagen and appeared suitable for three-dimensional cell culture of tHPC for up to 21 days. The cultures could be transferred to the DBT device and results for controls were similar to previous tests with bovine cells. The DBT using tHPC in the collagen showed no statistically significant difference between the test material with and without the adhesive and the reference resin composite. tHPC in collagen can replace bovine cells in the DBT. The tested filling material is not likely to cause pulp damage, if the pulp is covered by an intact dentin layer.

Enamel matrix derivative promote primary human pulp cell differentiation and mineralization.

Enamel matrix derivative (EMD) has been found to induce reactive dentin formation; however the molecular mechanisms involved are unclear. The effect of EMD (5–50 μg/mL) on primary human pulp cells were compared to untreated cells and cells incubated with 10−8 M dexamethasone (DEX) for 1, 2, 3, 7, and 14 days in culture. Expression analysis using Affymetrix microchips demonstrated that 10 μg/mL EMD regulated several hundred genes and stimulated the gene expression of proteins involved in mesenchymal proliferation and differentiation. Both EMD and DEX enhanced the expression of amelogenin (amel), and the dentinogenic markers dentin sialophosphoprotein (DSSP) and dentin matrix acidic phosphoprotein 1 (DMP1), as well as the osteogenic markers osteocalcin (OC, BGLAP) and collagen type 1 (COL1A1). Whereas, only EMD had effect on alkaline phosphatase (ALP) mRNA expression, the stimulatory effect were verified by enhanced secretion of OC and COL1A from EMD treated cells, and increased ALP activity in cell culture medium after EMD treatment. Increased levels of interleukin-6 (IL-6), interleukin-8 (IL-8), and monocyte chemoattractant proteins (MCP-1) in the cell culture medium were also found. Consequently, the suggested effect of EMD is to promote differentiation of pulp cells and increases the potential for pulpal mineralization to favor reactive dentine formation.

Expression profiles and bioinformatic analysis of miRNA in human dental pulp cells during endothelial differentiation

To investigate the differential expression profile and bioinformatic analysis of microRNA (miRNA) in human dental pulp cells (DPC) during endothelial differentiation. DPC were cultured in endothelial induction medium (50 µg/L vascular endothelial growth factor, 10 µg/L basic fibroblast growth factor and 2% fetal calf serum) for 7 days. Meanwhile non-induced DPC were used as control.Quantitative real-time PCR (qRT-PCR) was applied to detect vascular endothelial marker genes [CD31, von Willebrand factor (vWF) and vascular endothelial-cadherin (VE-cadherin)] and in vitro tube formation on matrigel was used to analyze the angiogenic ability of differentiated cells. And then miRNA expression profiles of DPC were examined using miRNA microarray and then the differentially expressed miRNA were validated by qRT-PCR. Furthermore, bioinformatic analysis was employed to predict the target genes of miRNA and to analyze the possible biological functions and signaling pathways that were involved in DPC after induction. The relative mRNA level of CD31, vWF and VE-cadherin in the control group were (3.48 ± 0.22) ×10(-4), (3.13 ± 0.31) ×10(-4) and (39.60 ± 2.36) ×10(-4), and (19.57 ± 2.20) ×10(-4), (48.13 ± 0.54) ×10(-4) and (228.00 ± 8.89) ×10(-4) in the induced group. The expressions of CD31, vWF and VE-cadherin were increased significantly in endothelial induced DPC compared to the control group (P < 0.05). For in vitro tube formation assay, tubular structures were formed on the matrigel by differentiated DPC. A total of 47 miRNA were differentially expressed, in which 15 miRNA were up-regulated and 32 miRNAs down-regulated in differentiated DPC compared with the control. Of these, 4 miRNA were confirmed by qRT-PCR. The target genes of differential miRNA were predicted to associate with several biological functions, such as the regulation of transcription, cell motion, blood vessel morphogenesis, angiogenesis and cytoskeletal protein, and signaling pathways including the mitogen-activated protein kinase (MAPK) and the Wnt signaling pathway. The differential miRNA expression identified in this study may be involved in governing DPC endothelial differentiation, thus contributing to the future research on regulatory mechanisms in dental pulp angiogenesis.

Evaluation of antibacterial photodynamic therapy effects on human dental pulp cell cultures

The antibacterial photodynamic therapy (aPDT) has been used in dentistry against oral microorganisms because of its excellent biocide effect. However, for carious lesions applications, there is little evidence that this therapy is safe for the pulp tissue. This study evaluates the effects of an aPDT protocol on human pulp cells in vitro. Pulp cells isolated from dental pulp were exposed to an aPDT protocol associating methylene blue (MB) at concentrations of 0.0125, 0.025 and 0.050mg/ml and red laser irradiation using a continuous-wave indium-gallium-aluminum-phosphide (InGaAlP) diode laser (λ=660nm, 40mW, 2.4J, 60J/cm(2) for 1min). Pre-irradiation time was 5min for each MB concentration. Cell viability was determined by MTT assay and activity of alkaline phosphatase was assessed by BCIP-NBT assay. Type of aPDT-induced cell death was assessed by flow cytometry. Data was statistically compared (ANOVA followed by Tukey' or Bonferroni's post hoc tests). aPDT was able to kill pulp cells in a dye concentration-dependent manner. The cellular viability was significantly reduced when used MB at 0.025 or 0.050mg/ml concentrations (p<0.0001). At these concentrations, aPDT-induced cell death occurred mostly by necrosis. Alkaline phosphatase activity was significantly reduced in all experimental groups (p<0.001). Pulp cells showed suitable viability when MB at 0.0125mg/ml was exposed to laser irradiation. aPDT with MB at 0.0125mg/ml may represent a low-risk therapy for restorative dentistry applications. aPDT protocol using concentrations above 0.025mg/ml of MB associating red laser irradiation may be harmful for dental pulp cells.

Effects of Pulpectomy on the Amount of Root Resorption during Orthodontic Tooth Movement

IntroductionPrevious studies have revealed that orthodontic force affects dental pulp via the rupture of blood vessels and vacuolization of pulp tissues. We hypothesized that pulp tissues express inflammatory cytokines and regulators of odontoclast differentiation after excess orthodontic force. The purpose of this study was to investigate the effects of tensile force in human pulp cells and to measure inflammatory root resorption during tooth movement in pulpless rat teeth. MethodsAfter cyclic tensile force application in human pulp cells, gene expression and protein concentration of macrophage colony-stimulating factor, receptor activator of nuclear factor kappa-B ligand, interleukin-1 beta, and tumor necrosis factor alpha were determined by real-time polymerase chain reaction and enzyme-linked immunoassay. Moreover, the role of the stretch-activated channel was evaluated by gadolinium (Gd3+) treatment. The upper right first molars of 7-week Wistar rats were subjected to pulpectomy and root canal filling followed by mesial movement for 6 months. ResultsThe expression of cytokine messenger RNAs and proteins in the experimental group peaked with loading at 10-kPa tensile force after 48 hours (P < .01). Gd3+ reduced the expression of these cytokine messenger RNAs and protein concentrations (P < .01). The amount of inflammatory root resorption was significantly larger in the control teeth than the pulpectomized teeth (P < .05). ConclusionsThis study shows that tensile forces in the pulp cells enhance the expression of various cytokines via the S-A channel, which may lead to inflammatory root resorption during tooth movement. It also suggests that root canal treatment is effective for progressive severe inflammatory root resorption during tooth movement.

Effects of Dental Methacrylates on Oxygen Consumption and Redox Status of Human Pulp Cells

Several studies have already demonstrated that the incomplete polymerization of resin-based dental materials causes the release of monomers which might affect cell metabolism. The aim of this study was to investigate the effects of triethylene glycol dimethacrylate, 1,4-butanediol dimethacrylate, urethane dimethacrylate, and 2-hydroxyethyl methacrylate on (1) cellular energy metabolism, evaluating oxygen consumption rate, glucose consumption, glucose 6-phosphate dehydrogenase activity, and lactate production, and (2) cellular redox status, through the evaluation of glutathione concentration and of the activities of enzymes regulating glutathione metabolism. Methods. Human pulp cells were used and oxygen consumption was measured by means of a Clark electrode. Moreover, reactive oxygen species production was quantified. Enzymatic activity and glucose and lactate concentrations were determined through a specific kit. Results. Triethylene glycol dimethacrylate, 1,4-butanediol dimethacrylate, and 2-hydroxyethyl methacrylate induced a decrease in oxygen consumption rate, an enhancement of glucose consumption, and lactate production, whilst glucose 6-phosphate dehydrogenase and glutathione reductase activity were not significantly modified. Moreover, the monomers induced an increase of reactive oxygen species production with a consequent increase of superoxide dismutase and catalase enzymatic activities. A depletion of both reduced and total glutathione was also observed. Conclusion. The obtained results indicate that dental monomers might alter energy metabolism and glutathione redox balance in human pulp cells.

Improved platelet-rich plasma effects on the proliferation and immunogenicity of human bone marrow mesenchymal stem cells

Previous experiments have shown that improved platelet-rich plasma can promote the proliferation of human dental pulp cells in a concentration-dependent manner, particularly when the concentration is 10%. To investigate the effect of platelet-rich plasma at different concentrations on the proliferation and immunogenicity of human bone marrow mesenchymal stem cells. Human bone marrow mesenchymal stem cells from healthy donors were cultured and passaged for 3-4 passages, identified by flow cytometry and differentiation inductions. Platelet-rich plasma samples which were manufactured from the venous blood of the same donor were used for culturing human bone marrow mesenchymal stem cells. The proliferation of human bone marrow mesenchymal stem cells was measured by cell counting kit-8 method and the growth curves were drawn. The most suitable concentration of platelet-rich plasma was selected to culture human bone marrow mesenchymal stem cells for three generations and the Stro-1 expression rate on the surface of human bone marrow mesenchymal stem cells was determined through flow cytometry. Platelet-rich plasma at the concentration of 5%-10% evidently promoted the proliferation of human bone marrow mesenchymal stem cells on the 6th and 8th days. The most effective concentration to promote the proliferation was 10%. Platelet-rich plasma at the concentration of 10% still promoted the proliferation of human bone marrow mesenchymal stem cells on the 10th day, and maintained a better immunogenicity of human bone marrow mesenchymal stem cells compared to the control group. These findings indicate that platelet-rich plasma can promote the proliferation of human bone marrow mesenchymal stem cells in a concentration-dependent manner, and 10% platelet-rich plasma is better to maintain the immunogenicity of human bone marrow mesenchymal stem cells.

Interference of octamer-binding transcription factor 4A and its effect on the proliferation and multiple differentiation of human dental pulp cells

To investigate the expression of octamer-binding transcription factor 4A(Oct4A) in human dental pulp cells(DPC)and the effect of Oct4A on the proliferation and multiple differentiation ability of DPC. Expression of Oct4A in DPC was detected by realtime quantitative PCR(RT-qPCR). siRNA-Oct4A was constructed and transfected (50 nmol/L) into DPC with Lipofectamine(TM) RNAiMAX for 24, 48, 72, 96 and 120 h. The proliferation rate of DPC was examined using cell counting kit 8 (CCK-8) assay. The alizarin red staining was used to observe the formation of calcification nodules in DPC with 14 d of osteogenic induction, and oil red O staining to observe the formation of lipid droplet in DPC with 14 d of adipogenic induction. The expression of osteogenesis-related genes dentin sialophosphoprotein (DSPP) and adipogenesis-related genes lipoprotein lipase (LPL) was detected using RT-qPCR and Western blotting. The expression of Oct4A reached the peak in P3 DPC (2.10 ± 0.10), which was 2.10 times as much as that in P1 (P = 0.000 vs. P1 and P7), and decreased along passages. The interference efficiency of DPC transfection peaked at 72 h (69.7%). Compared with control group and negative control group (IR-siRNA), the proliferation rate and multiple differentiation ability of DPC in interference group (Oct4A-siRNA) were downregulated (P = 0.000), and DSPP and LPL in DPC from interference group significantly decreased (P = 0.000). The interference of Oct4A significantly downregulated the cell proliferation rate and multilineage differentiation capability of DPC.

Emdogain regulates the expression of bone sialoprotein gene in human dental pulp cells

To analyze the effects of emdogain(EMD) on the expression of the bone sialoprotein(BSP) gene in human dental pulp cells and to elucidate the molecular mechanism of BSP gene regulated by EMD. Human dental pulp was harvested from premolars freshly extracted for orthodontic purpose and cultured. Cells were divided into different concentrations (25, 50, 100 and 250 mg/L) of EMD and control groups (Dulbecco's modified Eagle's medium). Total RNA of cells was extracted. Human BSP mRNA levels was detected with the real-time PCR. Regulations of EMD on human BSP protein levels were detected with Western blotting. In the real-time PCR, at the same time point, there were significant differences on BSP mRNA levels between 25, 50, 100 and 250 mg/L EMD groups (7 d:1.79 ± 0.03, 2.03 ± 0.10, 2.67 ± 0.08, 2.94 ± 0.07) and control group (7 d:1.06 ± 0.11) (P < 0.001); at the different time point (1, 3, 5 and 7 d), the same dose(250 mg/L) of EMD stimulated human dental pulp cells, BSP mRNA (2.30 ± 0.06, 2.65 ± 0.05, 2.76 ± 0.05, 2.94 ± 0.07) was increased (P < 0.05). Treatment of human dental pulp cells with EMD (250 mg/L) increased the protein levels. EMD increases BSP mRNA and protein levels in human dental pulp cells.

The role of preameloblast-conditioned medium in dental pulp regeneration

Pulp regeneration using human dental pulp stem cells (hDPSCs) maintains tooth vitality compared with conventional root canal therapy. Our previous study demonstrated that preameloblast-conditioned medium (PA-CM) from murine apical bud cells induces the odontogenic differentiation of hDPSCs and promoted dentin formation in mouse subcutaneous tissue. The purpose of the present study is to evaluate the effects of PA-CM with human whole pulp cells on pulp regeneration in an empty root canal space. Human pulp cells were seeded in the pulp cavities of 5 mm-thick human tooth segments with or without PA-CM treatment, and then transplanted subcutaneously into immunocompromised mice. In the pulp cell-only group, skeletal muscle with pulp-like tissue was generated in the pulp cavity. A reparative dentin-like structure with entrapped cells lined the existing dentin wall. However, in the PA-CM-treated group, only pulp-like tissue was regenerated without muscle or a reparative dentin-like structure. Moreover, human odontoblast-like cells exhibited palisade arrangement around the pulp, and typical odontoblast processes elongated into dentinal tubules. The results suggest that PA-CM can induce pulp regeneration of human pulp cells with physiological structures in an empty root canal space.

Mineralization of Dense Collagen Hydrogel Scaffolds by Human Pulp Cells

While advances in biomineralization have been made in recent years, unanswered questions persist on bone- and tooth-cell differentiation, on outside-in signaling from the extracellular matrix, and on the link between protein expression and mineral deposition. In the present study, we validate the use of a bioengineered three-dimensional (3D) dense collagen hydrogel scaffold as a cell-culture model to explore these questions. Dental pulp progenitor/stem cells from human exfoliated deciduous teeth (SHEDs) were seeded into an extracellular matrix-like collagen gel whose fibrillar density was increased through plastic compression. SHED viability, morphology, and metabolic activity, as well as scaffold mineralization, were investigated over 24 days in culture. Additionally, measurements of alkaline phosphatase enzymatic activity, together with immunoblotting for mineralized tissue cell markers ALPL (tissue-non-specific alkaline phosphatase), DMP1 (dentin matrix protein 1), and OPN (osteopontin), demonstrated osteo/odontogenic cell differentiation in the dense collagen scaffolds coincident with mineralization. Analyses of the mineral phase by electron microscopy, including electron diffraction and energy-dispersive x-ray spectroscopy, combined with Fourier-transform infrared spectroscopy and biochemical analyses, were consistent with the formation of apatitic mineral that was frequently aligned along collagen fibrils. In conclusion, use of a 3D dense collagen scaffold promoted SHED osteo/odontogenic cell differentiation and mineralization.

Effect of bioactive glasses on angiogenic growth factors of human dental pulp cells

To investigate the effects of bioactive glasses (BG) including 45S5 and nano-58S on proliferation, angiogenic markers vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) secretion and gene expression of human dental pulp cells (HDPC). HDPC of 4th passage were cultured in Dulbecco's modified Eagle's medium (DMEM) which contained 0.1 g/L 45S5 or nano-58S ionic dissolution products. Meanwhile HDPC were cultured in DMEM without BG as control group. Proliferation of the cells was evaluated with methyl thiazolyl tetrazolium (MTT) assay on day 1, 2, 3. Quantitative real-time PCR and quantitative sandwich enzyme immunoassays were used to test VEGF and bFGF gene expression and protein secretion of HDPC on day 1, 2, 3. The relative growth rate (RGR) of 45S5 and nano-58S groups were (134.5 ± 5.0)% and (146.3 ± 19.8)%, which was significantly different from that of control group (P < 0.05). The quantity of VEGF secretion of two experimental groups were (189.29 ± 4.64) and (216.18 ± 14.67) ng/L, respectively, significantly higher than that of the control group [(159.03 ± 11.69) ng/L] (P < 0.05). Furthermore, the nano-58S group secreted much more VEGF than 45S5 group (P < 0.05).bFGF secretion of HDPC was also enhanced by both 45S5 and nano-58S bioactive glasses. The VEGF gene expression of 45S5 and nano-58S on day 1 were (1.70 ± 0.19) and (1.63 ± 0.42), while the bFGF gene expressin on day 3 were (1.49 ± 0.02) and (2.30 ± 0.04), all significantly higher than that of control group (P < 0.05). Bioactive glasses can enhance the proliferation, VEGF and bFGF secretion and gene expression of human dental pulp cells. Compared with 45S5, nano-58S showed a higher activation.

Stimulation of Matrix Metalloproteinases by Tumor Necrosis Factor–α in Human Pulp Cell Cultures

IntroductionThe purpose of this study was to investigate whether in vitro stimulation of pulp cells leads to increased secretion of matrix metalloproteinases (MMPs) and, if so, to identify which MMPs are affected. MethodsCells cultured from dental pulp were stimulated with tumor necrosis factor–α (TNF-α) (10 ng/mL) for 24 hours, and lysates were analyzed with an antibody array (Bio-Rad Laboratories, Hercules, CA). The mRNA and protein levels of MMP-3, -10, and -13 were measured by real-time polymerase chain reaction (real-time PCR), enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and zymography. In addition, tumor necrosis factor receptors in the pulp cells were assayed by flow cytometry. The ELISA and real-time PCR results were analyzed by paired t tests. ResultsThe expression of MMP-3, -10, and -13 was up-regulated in the pulp cells after 24 hours of stimulation with TNF-α (10 ng/mL) as seen in the antibody array, real-time PCR, and ELISA results, but MMP-10 was not detected by Western blotting or casein zymography. Flow cytometry analysis showed that the majority of the pulp cells expressed tumor necrosis factor receptor 1. ConclusionsIn regions of inflammation, TNF-α may initiate the degradation of dental connective tissue by activating MMP-3 and MMP-13. These proteins may play an important pathologic role in the inflammation of dental pulp.

Usefulness of Controlled Release of Growth Factors in Investigating the Early Events of Dentin-pulp Regeneration

IntroductionLittle information is yet available on the signals involved in progenitor cell migration that precede reparative dentin synthesis. Our aim was to investigate the effect of the controlled release of fibroblast growth factor (FGF)-2 and transforming growth factor β1 (TGF-β1) on permanent teeth pulp cell proliferation and progenitor cell migration. MethodsFGF-2 and TGF-β1 were encapsulated into a biodegradable polymer matrix of lactide and glycolide. Human pulp cells were prepared from third molars, and progenitor cells were sorted by STRO-1. The synthesized microsphere toxicity was checked with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test. The growth factor release kinetics were checked by an enzyme-linked immunosorbent assay while maintaining their biological activity and were evaluated by investigating their effects on pulp cell proliferation. Their chemotactic potential was investigated on STRO-1–sorted cells in a migration chamber on Matrigel (Cambrex Bio Science, Walkersville, MD). ResultsThe cell viability was unaffected by the presence of microspheres. The released amount of FGF-2 and TGF-β1 from the microspheres was maintained after 21 days. Increasing the FGF-2–loaded microsphere concentration or the release period significantly increased dental pulp cell proliferation. TGF-β1 acted as a potent chemotactic factor of STRO-1–sorted cells. ConclusionsEncapsulating TGF-β1 and FGF-2 in a biodegradable polymer of lactide and glycolide microsphere allowed a sustained release of growth factors and provided a protection to their biological activities. Our results clearly show the usefulness of growth factor controlled release in investigating the early events of pulp/dentin regeneration. It provides additional data on the signals required for vital pulp therapy and future tissue engineering.

Endoplasmic reticulum stress and mineralization inhibition mechanism by the resinous monomer HEMA

To investigate the expression of two endoplasmic reticulum (ER)-resident key chaperone proteins, ERdj5 and BiP, under the influence of resinous monomers and its relationship with the inhibition of mineralization caused by the monomer 2-hydroxyethyl methacrylate (HEMA). The ERdj5 and BiP expression was studied in vitro, in primary human pulp cell cultures after treatment with three different HEMA concentrations at different time periods. Subsequently, the expression of both the odontoblast markers dentine sialoprotein (DSP) and osteonectin (OSN) was studied in human pulp cells under the same conditions. The ERdj5 and BiP expression was upregulated in the pulp cells. DSP and OSN were largely dispersed in the cytoplasm in control cell cultures but accumulated in a perinuclear area after exposure to HEMA. Their expression levels were not affected. The increased expression of ERdj5 and BiP may reflect activation of ER stress. DSP and OSN accumulation into the cells may lead to their secretion arrest and inhibition of dentine matrix formation. These events may elucidate the mechanism by which HEMA inhibits the mineralization process.

In vitro effects of ascorbic acid and β-glycerophosphate on human gingival fibroblast cells

Ascorbic acid (AA) and β-glycerophosphate (βG) are considered in vitro osteogenic factors important to the differentiation of osteoblastic progenitor and dental pulp cells into mineralized tissue-forming cells. So, the present study investigated in vitro if these mineralizing inducible factors (AA and βG) could influence differentiation of human gingival fibroblasts when compared with human pulp cells and osteogenic cells derived from rat calvaria cultured. The expression of osteopontin (OPN) and osteoadherin (OSAD) was analyzed by indirect immunofluorescence, immunocytochemistry as well as Western-blotting. In addition, the main ultrastructural aspects were also investigated. No mineralized matrix formation occurred on gingival fibroblasts induced with AA+βG. On these cells, no expression of OPN and OSAD was observed when compared with pulp cells, pulp cells induced with AA+βG as well as osteogenic cells. Ultrastructure analysis additionally showed that gingival fibroblasts exhibited typical fibroblast morphology with no nodule formation. The present findings showed that AA and βG could not promote a mineralized cell differentiation of human gingival fibroblasts and confirm that human dental pulp cells, as the osteogenic cells, are capable to form a mineralized extracellular.

A novel furanone-modified antibacterial dental glass ionomer cement

A novel furanone derivative and a polyacid constructed from it were synthesized, characterized and formulated into experimental high strength cements. The compressive strength (CS) and Streptococcus mutans viability were used to evaluate the mechanical strength and antibacterial activity of the cements. The effect of human saliva and aging were investigated. The antibacterial activity against Lactobacillus sp. and cytotoxicity to human pulp cells were also evaluated. The results show that all the formulated furanone-containing cements showed antibacterial activity, with an initial reduction in CS. The effect of the furanone derivative loading was significant. Increasing loading enhanced the antibacterial activity but reduced the initial CS of the formed cements. The derivative showed antibacterial activity against both S. mutans and Lactobacillus sp. Human saliva did not affect the antibacterial activity of the cement. The cytotoxicity study with human dental pulp cells shows that the furanone-modified cement was biocompatible. A 30day aging study indicated that the cements may have long-lasting antibacterial activity. Within the limitations of this study it appears that the experimental cement could be a clinically attractive dental restorative due to its high mechanical strength and antibacterial function.

DMP1 and DSP localization during in vitro odontoblast like cell differentiation

The aim of this study was to analyze the temporal and spatial localization of dentin matrix protein 1 (DMP1) and dentin sialoprotein (DSP) in human dental pulp cells differentiated in vitro towards odontoblast-like cells. Following to a notable injury to the tooth, the odontoblasts die and a new generation of odontoblast-like cells differentiate from pulp progenitor cells [1]. They are responsible for the secretion of the reparative dentin matrix. In this study, morphological changes and the analysis of DMP1 and DSP were investigated starting from human dental pulp cells differentiated in vitro. Alizarin red staining, transmission and scanning electron microscopy in combination with Real Time PCR and immunofluorescence techniques were performed to demonstrate the odontoblast-differentiated phenotype and the specific expression and localization of DMP1 and DSP. The results showed a gradual calcium deposition to demonstrate a mineralization matrix. Electron microscopy analysis showed fibroblast-like cells characterized by a large number of mitochondria and a developed rough endoplasmic reticulum and Golgi apparatus involved in intense protein synthesis. A large extracellular matrix was detectable in which collagen type I fibers were detected. The DMP1 and DSP showed low expression and nuclear localization at the beginning of the odontoblast-like cell differentiation. After 21 days, elevated cytoplasm expression was detectable for both odontogenic markers.

A novel glass ionomer cement containing MgCO3 apatite induced the increased proliferation and differentiation of human pulp cells in vitro

This study aimed to investigate the in vitro biological response of human dental pulp cells to glass ionomer cement (GIC, Fuji IX GP(®)) containing 2.5% magnesium carbonate apatite (MgCO(3)Ap). MgCO(3)Ap was synthesized by wet method and characterized using FT-IR, XPS, and SEM. Fuji IX GP(®) served as a control. Test and control cements were prepared by encapsulated mixing the powder with Fuji IX-liquid (P/L=3.6:1). Eluates from cements extracted by 1 mL culture medium were collected at day 1, 7 and 14, and used for WST-1 proliferation assay. For ALPase activity, cells were maintained with cements in transwells, harvested and enzyme activity was measured at day 1, 4, 7, 14, and 21. We found a higher cell proliferation and increased ALPase activity by pulp cells in the test group compared to the control. This suggests the potential of GIC containing this novel biological apatite as a restorative material for pulp-dentin regeneration.