Human Dental Pulp Cell Cultures Research Articles

A critical review of in vitro research methodologies used to study mineralization in human dental pulp cell cultures.

BackgroundThe pulp contains a resident population of stem cells which can be stimulated to differentiate in order to repair the tooth by generating a mineralized extracellular matrix. Over recent decades there has been considerable interest in utilizing in vitro cell culture models to study dentinogenesis, with the aim of developing regenerative endodontic procedures, particularly where some vital pulp tissue remains.ObjectivesThe purpose of this review is to provide a structured oversight of in vitro research methodologies which have been used to study human pulp mineralization processes.MethodThe literature was screened in the PubMed database up to March 2021 to identify manuscripts reporting the use of human dental pulp cells to study mineralization. The dataset identified 343 publications initially which were further screened and consequently 166 studies were identified and it was methodologically mined for information on: i) study purpose, ii) source and characterization of cells, iii) mineralizing supplements and concentrations, and iv) assays and markers used to characterize mineralization and differentiation, and the data was used to write this narrative review.ResultsMost published studies aimed at characterizing new biological stimulants for mineralization as well as determining the effect of scaffolds and dental (bio)materials. In general, pulp cells were isolated by enzymatic digestion, although the pulp explant technique was also common. For enzymatic digestion, a range of enzymes and concentrations were utilized, although collagenase type I and dispase were the most frequent. Isolated cells were not routinely characterized using either fluorescence‐activated cell sorting (FACS) and magnetic‐activated cell sorting (MACS) approaches and there was little consistency in terming cultures as dental pulp cells or dental pulp stem cells. A combination of media supplements, at a range of concentrations, of dexamethasone, ascorbic acid and beta‐glycerophosphate, were frequently applied as the basis for the experimental conditions. Alizarin Red S (ARS) staining was the method of choice for assessment of mineralization at 21‐days. Alkaline phosphatase assay was relatively frequently applied, solely or in combination with ARS staining. Further assessment of differentiation status was performed using transcript or protein markers, with dentine sialophosphoprotein (DSPP), osteocalcin and dentine matrix protein‐1 (DMP ‐1), the most frequent.DiscussionWhile this review highlights variability among experimental approaches, it does however identify a consensus experimental approach.ConclusionStandardization of experimental conditions and sustained research will significantly benefit endodontic patient outcomes in the future.

P8 - Polycaprolactone/Nano-Hydroxyapatite Nanofibrous Scaffold as a Cell Homing-Based Therapy

This study investigated the behavior of human dental pulp cells (HDPCs) seeded on the surface of polycaprolactone/nano-hydroxyapatite nanofibrous scaffolds, aiming a dentin tissue engineering-based vital pulp therapy (VPT). Polycaprolactone (PCL)-based solutions (10% w/v in 8:2 chloroform and dimethylformamide) containing nano-hydroxyapatite (nHA) particles (0.5; 1.0; or 2.0% w/v) were electrospun into nanofibrous scaffolds. PCL scaffolds without nHA were used as control. The scaffolds were blindly characterized for morphology and composition (SEM/EDS; n=12), solubility (n=6), the release of calcium/phosphate (n=6), and change of medium pH (n=6). A culture of HDPCs was obtained from sound third molars and characterized for the presence of a subpopulation of mesenchymal stem cells. Then, HDPCs were seeded on the surface of the scaffolds and evaluated for cell viability (alamarBlue; n=8), cell proliferation (Live/Dead assay; n=4), adhesion and spreading (F-actin; n=4), total protein content (TP; Lowry method; n=8), alkaline phosphatase activity (ALP; thymolphthalein assay; n=8), expression of odontogenic genes (RT-qPCR; DSPP, DMP1, COL1A1, and ALPL; n=6), and formation of a mineralized matrix (Alizarin Red staining; n=8). All experiments were repeated at least twice. Sample sizes were calculated using G-Power. Data were analyzed with confidence intervals and one- or two-way ANOVA followed by Tukey, Sidak, or Games-Howell post-hocs (?=5%). All formulations generated randomly arranged fibers ranging from 600 to 900 nm in diameter. Higher nHA concentrations roughened the surface of the nanofibers, while PCL+2%nHA increased the interfibrillar spaces. PCL+1%nHA and PCL+2%nHA significantly released calcium/phosphate over a period of 28 days in comparison with PCL+0.5%nHA. Nonetheless, the medium pH was maintained below 8.0. HDPCs viability and proliferation were not affected by the incorporation of nHA, while cell adhesion/spreading was stimulated, especially for PCL+2%nHA. Higher total protein content and increased ALP activity were seen in the presence of nHA. PCL+1%nHA and PCL+2%nHA upregulated the expression of DSPP and DMP1 in 14 days; and COL1A1, ALPL, and DMP1 in 21 days. The formation of a mineralized matrix after 21 days was concentration-dependent, and it was about 9× higher for PCL+2%nHA formulation compared to control.

Inorganic Phosphate Effect of on Human Dental Pulp Cell Cultures

This study was designed to investigate the effect of inorganic phosphate (Pi) at different concentrations on odontogenesis of the normal human dental pulp cells (hDPCs). Normal human dental pulp cells derived from extracted pristine teeth were cultured in growth medium with supplements of inorganic phosphate (Pi) in 0 ppm, 2 ppm, 4 ppm, 5 ppm and 8 ppm, for the time intervals of 16 hours, 7, 14, and 21 days. Cell proliferation rates were measured by the optical density of crystal violet dye stained cells. ALP activity was measured by fluorometric assay. Expression of Dentin Sialoprotein (DSP) was measured by ELISA. The data were presented as the mean of triplicates. Statistical analysis was conducted using JMP Pro 12 (ver. 12.1.0) in one-way ANOVA and Tukey HSD post-hoc tests. Cell attachment efficiency was reduced significantly by additional Pi of 2, 4 and 5 ppm (P<0.05). At 21 days, cultures with 2, 4 and 5 ppm supplemental Pi displayed significantly higher cell proliferation rates compared to the control group at day 14 (P<0.05) and at day 21 (P<0.05). At day 7, cultures with 2, 4, 5 and 8 ppm supplemental Pi yield significantly higher levels of ALP activity (P<0.05) compared to the control group. At day 7, cultures with 5 ppm Pi supplement showed significantly higher levels of DSP expression (P<0.05) compared to the control group and the rest of the other groups. Supplemental Pi in concentration of 5 ppm could significantly induce proliferation and odontogenesis of hDPCs. This is the first report to demonstrate Pi-induced odontogenesis, leading to potential development and clinical application of future Pi containing dental pulp capping or root canal filling materials.

Effect of Silicon and Calcium on Human Dental Pulp Cell Cultures

This study was designed to investigate the odontogenic effects of Silicon, Calcium and Phosphorous on human dental pulp cells. Human dental pulp cells derived from extracted pristine teeth were cultured in growth media with supplements of Si 25ppm, Si 25ppm+Ca 8.3ppm, Si 25ppm+Ca 8.3ppm+P 4.16ppm, Si 50ppm, Si 50ppm+Ca 16.7ppm, Si 50ppm+Ca 16.7ppm+P 8.3ppm and media without additional supplement as control, for the time intervals of 16 hours, 7, 12, and 21 days. Cell proliferation rates were measured by the optical density of crystal violet dye stained cells. ALP activity was measured by fluorometric assay. Expression of Dentin Sialoprotein (DSP) was measured by ELISA. Mineralization of cultures was measured by Alizarin Red staining. The data were presented as the mean of triplicates and normalized on a per million cell basis. Statistical analysis was conducted using ANOVA and Tukey HSD post-hoc tests. Culture with 50ppm supplemental Si at day 21 yield significantly higher levels of ALP activity, DSP expression and mineralization (<i>P</i>&lt;0.05) compared to the control group and other supplemented groups. Cultures with Si 25ppm+Ca 8.3ppm supplemental and Si 50ppm+Ca 16.7ppm supplemental displayed significantly higher cell proliferation rates compared to the control group at day 12 (<i>P</i>&lt;0.05) and at day 21 (<i>P</i>&lt;0.05). Supplemental silicon in concentration of 50 ppm could significantly induce differentiation and mineralization of normal human dental pulp cells. Calcium has a synergetic effect in up-regulating the proliferation rates. This is the first report to demonstrate the silicon- and calcium-induced mineralized tissue formation of human dental pulp cell cultures, leading to the potential development and clinical application of a future novel dental pulp capping material.

Changes in SIRT gene expression during odontoblastic differentiation of human dental pulp cells.

ObjectivesThe aim of this study was to investigate the expression of 7 different sirtuin genes (SIRT1-SIRT7) in human dental pulp cells (HDPCs), and to determine the role of SIRTs in the odontoblastic differentiation potential of HDPCs.Materials and MethodsHDPCs were isolated from freshly extracted third molar teeth of healthy patients and cultulred in odontoblastic differentiation inducing media. Osteocalcin (OCN) and dentin sialophosphoprotein (DSPP) expression was analyzed to evaluate the odontoblastic differentiation of HDPCs by reverse transcription-polymerase chain reaction (RT-PCR), while alizarin red staining was used for the mineralization assay. To investigate the expression of SIRTs during odontoblastic differentiation of HDPCs, real time PCR was also performed with RT-PCR.ResultsDuring the culture of HDPCs in the differentiation inducing media, OCN, and DSPP mRNA expressions were increased. Mineralized nodule formation was also increased in the 14 days culture. All seven SIRT genes were expressed during the odontogenic induction period. SIRT4 expression was increased in a time-dependent manner.ConclusionsOur study identified the expression of seven different SIRT genes in HDPCs, and revealed that SIRT4 could exert an influence on the odontoblast differentiation process. Further studies are needed to determine the effects of other SIRTs on the odontogenic potential of HDPCs.

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.

The Involvement of Mitoge-activated Protein Kinases and Transcriptional Factors in Substance P-induced IL-6 Production in Human Dental Pulp Cell Cultures

The Involvement of Mitoge-activated Protein Kinases and Transcriptional Factors in Substance P-induced IL-6 Production in Human Dental Pulp Cell Cultures

Expression and Characterization of Vanilloid Receptor Subtype 1 in Human Dental Pulp Cell Cultures

The expression of the vanilloid receptor subtype 1 (VR1, TRPV1) was detected in human dental pulp fibroblasts (PF-10) using RT-PCR, Western blotting, and immunocytochemical analysis. As revealed by ELISA, capsaicin induced IL-6 expression in PF-10 cells, and the VR1 antagonist capsazepine dose-dependently inhibited capsaicin-induced IL-6 production, indicating that capsaicin-induced IL-6 expression is related to VR1 activation. The interaction between capsaicin and mitogen-activated protein kinases (MAPKs) was investigated. The phosphorylation of p38 MAPK and c-Jun NH 2-terminal kinase (JNK) were detected after capsaicin stimulation. p38 MAPK is involved in capsaicin-induced IL-6 production, as shown by the use of specific inhibitors of this kinase. The result of EMSA showed that capsaicin inhibited tumor necrosis factor-alpha (TNF-α)-induced nuclear factor-kappa B (NF-κB) activation in PF-10 cell cultures. These results suggest that the activation of VR1 plays an important role in dental pulp inflammation.

Substance P Enhances Expression of Lipopolysaccharide-induced Inflammatory Factors in Dental Pulp Cells

To examine how substance P (SP) is related with dental pulp inflammation, we examined the effects of SP on expression of genes for inflammatory factors in human dental pulp cell cultures. Using reverse transcriptase-polymerase chain reaction, we found that Prevotella intermedia lipopolysaccharide (LPS) induced expression of SP and SP-receptor mRNAs, and that somatostatin inhibited the LPS-induced expression of SP mRNA. We also found that SP enhanced LPS-induced stimulation of NF-kappaB binding activity. In addition, SP induced expression of cyclooxygenase-2 and interleukin-10 receptor mRNAs. In contrast, SP inhibited expression of interferon-gamma receptor mRNA. These results suggest that SP may play a regulatory role in the immunological response of dental pulp tissue to pathogenic bacteria.

Interleukin-10 Inhibits Expression of Interleukin-6 and -8 mRNA in Human Dental Pulp Cell Cultures via Nuclear Factor-κB Deactivation

The effects of interleukin (IL)-10 on the expression of IL-6 and IL-8 mRNA in human dental pulp cell cultures were investigated by using the Northern blot analysis. On stimulation with Prevotella intermedia lipopolysaccharide (PiLPS), IL-10 was produced in peripheral blood but was not detected in human dental pulp cell culture supernatants. IL-10 inhibited IL-8 mRNA expression, which is normally stimulated by PiLPS, IL-1alpha, and tumor necrosis factor-alpha and inhibited IL-6 mRNA expression, which is normally stimulated by IL-1alpha. In addition, IL-10 inhibited the activation of nuclear factor-kappaB, which is normally induced by PiLPS. We conclude that IL-10 inhibits expression of IL-6 and IL-8 mRNA in dental pulp cell cultures by inhibiting the activation of nuclear factor-kappaB.

Regulation of Interleukin-6 Expression in Human Dental Pulp Cell Cultures Stimulated with Prevotella intermedia Lipopolysaccharide

Interleukin (IL)-6 expression in human dental pulp cell cultures after stimulation with Prevotella intermedia lipopolysaccharide (LPS) was investigated by Northern blot analysis, enzyme immunoassay, and bioassay. The IL-6 mRNA expression began to increase after 1 hr and continued after up to 8 hr of exposure on stimulation with 10 microg/ml of P. intermedia LPS. The bioactivity was dose-dependent on the concentration of P. intermedia LPS (0 to 100 microg/ml). The IL-6 mRNA expression was inhibited by actinomysin D and super-induced by cycloheximide. Anti-CD14 monoclonal antibody (MY4) inhibited the IL-6 mRNA expression when administered at a 0.5 microg/ml concentration before stimulation with P. intermedia LPS at 1 microg/ml. The immunoregulatory cytokines (interferon-gamma, IL-10, and IL-4) inhibited LPS-induced IL-6 production with a combined treatment. These results suggest the IL-6 expression by pulp cell cultures is CD14-dependent and regulated at the transcriptional level, and a combined treatment with immunoregulatory cytokines may be effective for control of pulpal inflammation due to P. intermedia LPS.

Serine protease activity is essential for thrombin-induced protein synthesis in cultured human dental pulp cells: modulation roles of prostaglandin E2.

Irritations and injuries to the dental pulp usually lead to different degrees of pulpal inflammation. To investigate the roles of thrombin and prostaglandins in the healing and inflammatory processes of dental pulp as well as their effects on pulpal protein synthesis, human dental pulp cell cultures were established and their protein production was measured with or without the presence of exogenous thrombin and prostaglandins. At concentrations of 1-25 U/ml, alpha-thrombin increased the protein synthesis to 1.4-2.3 fold over the vehicle control. On the contrary, 0.1 microg/ml of prostaglandin E1 (PGE1) suppressed protein synthesis by 60%. Prostaglandin E2 (PGE2) also inhibited protein synthesis with an IC50 of 0.4 microg/ml. The stimulatory effects of thrombin (10 U/ml) can be inhibited by antithrombin III (2 U/ml) (a natural thrombin inhibitor) with heparin (2 U/ml), PPACK (D-Phe-Pro-ArgCH2Cl) (20-50 microg/ml) (a serine protease inhibitor), and PGE2 (0.5-1.0 microg/ml). Moreover, TRAP (20-40 microg/ml), a thrombin receptor agonist peptide, also exerted a stimulatory effect (1.21-1.37 fold). In conclusion, thrombin-induced protein synthesis by pulp cells is dependent on proteolytic activity, but not on binding to receptors. Both PGE1 and PGE2 exert suppressive effects on protein synthesis, indicating that interactions between thrombin and prostaglandins are important in regulating the inflammation, repair and regeneration of pulp tissue following injury.

Collagen gene expression in human dental pulp cell cultures

Pulp cells from human permanent molars were isolated and established in culture; 40% showed positive alkaline phosphatase staining. When incubated with 50 μ/ml of ascorbic acid and 10 mM of β-glycerophosphate, the cells formed a mineralized extracellular matrix; they could thus have the potential to differentiate into odontoblast-like cells in vitro. Collagen synthesis was analysed by SDS interrupted gel electrophoresis, Northern blot and slot blot: the cells produced predominantly (~99%) type I collagen and only trace amount of type III collagen. The ratio of α1(I) to α2(I) procollagen chains was about 68:32, indicating that no significant amount of collagen type I trimer was synthesized in this system. The ratios of α1(I), α2(I) and α1(III) procollagen mRNAs were about 61:25:1; these were compatible with the ratios of corresponding procollagen a chains. In addition, a novel 5.8 kb proα1(III) mRNA was detected. These observations indicate that collagen synthesis in these cultured pulp cells was regulated at the transcriptional level.