Potential Use of L-Arginine Amino Acids towards Proliferation and Migratory Speed Rate of Human Dental Pulp Stem Cells (hDPSCs).

OBJECTIVE: Inflamed pulp has a decreased amount of protein and amino acids. L-Arginine is a semi-essential amino acid, the production of which is insufficient under oxidative stress and inflammation. L-Arginine is the sole substrate for nitric oxide synthase (NOS) to produce nitric oxide (NO), which plays an important role in cell proliferation and migration. This study aims to evaluate the proliferation and migration rate of hDPSCs after L-Arginine supplementation. METHODS: Serum-starved hDPSCs were divided into four groups: control: hDPSCs in Dulbecco's modified Eagle medium; hDPSCs in 300 µmol/L of the L-Arginine based culture media group; hDPSCs in 400 µmol/L of the L-Arginine based culture media group; and hDPSCs in 500 µmol/L of the L-Arginine based culture media group, which were planted in two separate 24-well-plates (5x104 cell/well) for proliferation and migration evaluation. The proliferation of all groups was measured by using a cell count test (haemacytometer and manual checker) after 24 h. The migratory speed rate of all groups was measured by using cell migration assay (scratch wound assay) after 24 h. Cell characteristics were evaluated under microscope (Inverted microscope, Zeiss®, Observer Z1, UK) that can be read through image-J® interpretation. This image J represented the measurement of migratory speed rate (nm/h) data. Statistical analysis was conducted using one-way ANOVA and post hoc Bonferroni (p<0.05) for proliferation and post hoc LSD (p<0.05) for migration (IBM SPSS Statistics Software, version 22.0). RESULTS: There was a statistically significant difference in hDPSC proliferation among various concentration groups of the L-Arginine based solution (300, 400 and 500 µmol/L) compared to the control group. There was a statistically significant difference in the migratory speed rate of hDPSCs in 500 µmol/L of the L-Arginine based solution group compared to lower concentrations and control group. CONCLUSION: The highest potency of hDPSC proliferation and migration was observed in the 500 µmol/L group. (EEJ-2023-01-08).

Viability of human dental pulp stem cells: The potential of L-arginine-based culture media.

Dental pulp is built by proteins that have various roles in the biological process of pulp, such as structural protein, regulation protein, and catalytic protein. L-arginine, an amino acid and one of the building blocks of proteins, regulates pro-inflammatory and anti-inflammatory activity. Therefore, L-arginine-based culture has potential to promote dental pulp regeneration. This study aimed to investigate the potential of L-arginine-based culture in improving the viability of human dental pulp stem cells (hDPSCs). We evaluated the viability of hDPSCs in culture media supplemented with different concentrations of L-arginine amino acid (250, 300, 350, and 400 µmol/L) and Dulbecco's Modified Eagle Medium plus fetal bovine serum 10% (control) using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay after 24-h incubation time. Statistical analysis was conducted using a one-way analysis of variance and post hoc least significant difference test. In qualitative analysis, the 4´, 6-diamidino-2-phenylindole staining method was used. The evaluation has shown a significant result when 250, 300, and 350 µmol/L concentration of L-arginine amino acid culture media compared with control, and 400 µmol/L has the best result and was not significantly different with control toward viability of hDPSCs.

Changes in Migratory Speed Rate of Human Dental Pulp Stromal Cells Cultured in Advanced Platelet-Rich Fibrin.

OBJECTIVE: Migratory speed rate evaluation of human dental pulp stromal cells (hDP-SCs) is one of the important steps in dental pulp regeneration. Therefore, the aim of the study is to analyze various concentrations of advanced platelet-rich fibrin (A-PRF) culture media toward hDP-SCs' migratory speed rate evaluations. MATERIALS AND METHODS: The hDP-SCs were divided into four groups: control: hDP-SCs in Dulbecco's modified Eagle medium + 10% fetal bovine serum group; hDP-SCs in 1% A-PRF group; hDP-SCs in 5% A-PRF group; and hDP-SCs in 10% A-PRF group, which were planted in 24-well (5 × 104 cell/well). The migratory speed rate of all groups was measured by using cell migration assay (scratch wound assay) after 24 hours. Cell characteristics were evaluated under microscope (Inverted microscope, Zeiss, Observer Z1, UK) that can be read through image-J interpretation. This image J represented the measurement of migratory speed rate (nm/h) data. Statistical analysis was conducted using one-way analysis of variance and post hoc Tamhane's test (p < 0.05) (IBM SPSS Statistics Software, version 22.0). RESULTS: There was a statistically significant difference in the migratory speed rates of hDP-SCs among various concentration groups of A-PRF (1, 5, and 10%) compared with the control group. CONCLUSION: The increase in the migratory speed rate of hDP-SCs was highest in 10% A-PRF group.

Analysis of Thrombin-Activated Platelet-Derived Exosome (T-aPDE) Potential for Dental Pulp Regeneration: In-Vitro Study.

OBJECTIVE: This study analyzed the potential of various concentrations of the thrombin-activated platelet-derived exosome (T-aPDE) to regenerate the dental pulp by performing an in-vitro analysis of the cell viability, migration activity, and vascular endothelial growth factor A (VEGF-A) expression of human dental pulp stem cells (hDPSCs). MATERIAL AND METHODS: The hDPSCs were collected from nine third molar teeth of nine healthy donors and were isolated and cultured using the explant method. They were harvested between the third and fourth passages and starved, after which they were seeded in the following treatments: Dulbecco's Modified Eagle Medium and 10% platelet-rich plasma-thrombin as the control groups, and 0.5, 1, and 5% T-aPDE as the experimental groups. All groups had three biological triplicates (Triplo) and two number of experiments. The T-aPDE was analyzed using transmission electron microscopy testing, particle size analyzer, and CD63 + and CD81 + specific immune phenotyping flow cytometry tests for plasma exosomes. The cell viability was evaluated using the colorimetric assay of activity cellular enzymes (MTT assay); the migration activity, using scratch assay; and the VEGF-A expression, using enzyme-linked immunosorbent assay. RESULTS: The highest viability absorbance value of hDPSCs after 24, 48, 72 hours of observation was in the 5% T-aPDE group (p<0.05). Whereas, the closest distance result of migratory activation hDPSCs was also in the same group (p<0.05). However the highest VEGF-A expression of hDSPCs was noted in the same group at 72 hours observation (p<0.05). STATISTICAL ANALYSIS: The data were analyzed using one-way analysis of variance and the Kruskal-Wallis test. The statistical power was set at p <0.05 CONCLUSION: The 5% T-aPDE had a higher potential to induce dental pulp regeneration than the other groups.

Characterization, Physical Properties, and Biocompatibility of Novel Tricalcium Silicate-Chitosan Endodontic Sealer.

OBJECTIVE: The purpose of this study was to compare the characteristics, physical properties, and biocompatibility of the novel tricalcium silicate-chitosan (TCS-C) sealer with AH Plus and Sure-Seal Root. MATERIALS AND METHODS: The TCS-C powder was prepared by mixing tricalcium silicate with 2% water-soluble chitosan at a 5:1 ratio, followed by sufficient addition of 10 g/mL ratio of double-distilled water to form a homogeneous cement. Material characterizations (the Fourier Transform InfraRed [FTIR] and X-ray diffraction [XRD]), physical property investigations (flow and film thickness), and cytotoxicity tests in 3T3 mouse embryo fibroblast cell (MTT assay method) were performed on sealers, and the results were compared with those of the commercial products. STATISTICAL ANALYSIS: Statistical analysis was performed on flow and film thickness. The normality of the data was tested using the Shapiro-Wilk test. Statistical analysis was performed with one-way analysis of variance (ANOVA). The level of significance was set at p < 0.05. RESULTS: The TCS-C showed a mean flow of 31.98 ± 0.68 mm, compared with Sure Seal Root at 26.38 ± 0.69 mm and AH Plus at 26.50 ± 0.12 mm. The TCS-C showed a mean film thickness of 60 ± 10.0 mm compared with Sure-Seal Root at 50 ± 10.0 mm and AH Plus at 40 ± 15.8 mm. The TCS-C exhibited low to no cytotoxicity in fibroblast cell at all concentrations and exposure times. CONCLUSION: Adding water-soluble chitosan may improve the physical and biologic properties of tricalcium silicate cement. The novel TCS-C sealer did not fully meet the physical properties of an endodontic sealer, but it was not cytotoxic to fibroblast cells.

The Effect of Hydrogel Hyaluronic Acid on Dentine Sialophosphoprotein Expression of Human Dental Pulp Stem Cells.

OBJECTIVE: Hyaluronic acid (HA) is glycosaminoglycan and one of important factors in extracellular matrix. In an inflamed pulp, when niche biology is conducive, the recruitment of human dental pulp stem cells (hDPSCs) will take place and differentiate into odontoblast like cell, creating reparative dentine and expressing dentine sialophosphoprotein (DSPP). Therefore, the purpose of this study was to analyze the potential of hydrogel HA in various concentration towards hDPSCs differentiation via DSPP expression at day 7 and 14. METHODS: After hDPSCs incubation reaching 80% confluence, cells were then starved for 24 hours. Then, culture media were supplemented with osteogenic media. hDPSCs planted into 96 well plate and HA 10 µg/mL, 20 µg/mL, and 30 µg/mL were added. DSPP expression was analysed using elisa reader at day 7 and 14, qualitative result was analysed using alizarin red at day 21. Data was analysed using one-way ANOVA. RESULTS: At day 7, there was a statistically significant different potential of HA conditioned media in various concentration (p<0.05) towards hDPSCs differentiation via expression of DSPP with HA 30 µg/mL being the most potential concentration to increase DSPP expression. CONCLUSION: HA have the potential to increase odontoblast differentiation process via expression of DSPP, with HA 30 µg/mL being the optimum concentration for hDPSCs. (EEJ-2022-12-169).

Effect of etch-and-rinse and self-etch modes in universal adhesives on push-out bond strength of fiber post.

Background: Nowadays, the universal adhesive become more popular among clinicians due to its simple procedure. The application of universal adhesive on root canal dentin prior the self-adhesive resin cement may increase bond-strength between fiber post and dentin. The objective of this study was to evaluate the effect of different etching modes (etch-and-rinse and self-etch) to universal adhesives on push-out bond strength of fiber post. Material and Methods: Thirty extracted sound human lower premolars were randomly divided into three groups based on adhesives systems prior to fiber post cementation; two-step etch-and-rinse (group A, Adper Scotchbond), etch-and-rinse universal (group B, Prime & Bond Universal), and self-etch universal (group C, Prime & Bond Universal). After adhesive application, self-adhesive resin cement was filled to the prepared root canal (Smart CEM2, Dentsply) for fiber post cementation. The adhesion between the fiber post and root canal walls was investigated using push-out test after 24 h water storage at 37◦C and the modes of failure were determined under SEM. Data were analyzed using two-way ANOVA test and the Bonferroni test was used to compare values among the three adhesives groups, followed by Tukey HSD for multiple comparisons. Furthermore, Weibull parameters were calculated for tested groups. Results: Universal adhesive with self-etch mode significantly improved bond-strength compared to the two-step etch-and-rinse group (p<0.05). The coronal part has higher bond strength than the apical section (p<0.05). However, the bond-strength in two-step etch-and-rinse and etch-and-rinse universal was not significantly different. Self-etch mode showed higher bond strength compared to etch-and-rinse universal adhesive in the apical root section (p<0.05). SEM revealed that all tested groups predominantly had an adhesive failure (p>0.05). Conclusions: Self-etch mode in universal adhesive system were effectively improved the push-out bond strength of fiber post to root canal dentin, especially in apical root section. Key words:Push-out bond-strength, self-adhesive resin cement, self-etch adhesive, total-etch.

The Effect of Calcium Gluconate on Platelet Rich Plasma Activation for VEGF-A Expression of Human Dental Pulp Stem Cells.

OBJECTIVE: Platelet-rich plasma (PRP) activation is an important factor in triggering the initial release of blood-derived growth factors from platelets. Vascular endothelial growth factor-A (VEGF-A) can be expressed by human dental pulp stem cells (hDPSCs) and plays an important role in dental pulp angiogenesis. The aim of this study is to analyze the effects of calcium gluconate on PRP activation in hDPSC VEGF-A expression. MATERIALS AND METHODS: Two types of PRP and their corresponding activators were analyzed in this study: PRP (activated using calcium chloride/CaCl2) and PRP-T (activated using CaCl2 with the addition of 10% calcium gluconate). hDPSCs were obtained by using an out-growth method (DPSCs-OG), and harvest between the fifth and sixth passages, then cultured in three different media groups: control, PRP, and PRP-T, which were planted in 96 wells (5 × 103 each well). The VEGF-A expression of hDPSCs was analyzed by using an ELISA test and observed at 24, 48, and 72 hours. STATISTICAL ANALYSIS: This study was performed by using one-way ANOVA (p < 0.05) test. RESULTS: There were significant differences between all groups (p < 0.05) at 48 and 72 hours of observations, and no significant differences in the PRP and PRP-T groups at 48 and 72 hours of observations (p > 0.05). CONCLUSION: PRP and PRP-T were equally effective in inducing VEGF-A expression of hDPSCs.

Comparison of Transforming Growth Factor 1 (TGF-β 1) Expression in Various Lysate Platelet-Rich Fibrin (L-PRF) Concentrations on Human Dental Pulp Stem Cell Differentiation

ABSTRACT Objective: To compare Transforming growth factor-β1 (TGF-β1) expression in various L-PRF concentrations on the hDPSC differentiation process. Material and Methods: hDPSC cell cultures were subjected to serum starvation by reducing FBS levels in the hDPSC culture media. Lysate PRF was obtained from the PRF gel, which was then incubated at 4°C for 24 h. The supernatant was dried, transferred to a 2-ml Eppendorf tube, and stored at −20°C. The evaluation of TGF-β1 expression in 1%, 5%, 10%, and 25% L-PRF samples and 10% FBS (control) during the process of hDPSC differentiation was quantified using an ELISA reader on day 7. The expression of TGF-β1 was subjected to a one-way ANOVA test, followed by Bonferroni's post hoc test with significant values (p<0.05). Results: Significant differences were noted in TGF-β1 expression between 1%, 5%, 10%, and 25% L-PRF and the control group (10% FBS). The highest TGF-β1 expression occurred with 25% L-PRF (0.645 ± 0.048), followed by 10% L-PRF (0.461 ± 0.035), 10% FBS (0.374 ± 0.013), 5% L-PRF (0.275 ± 0.045), and the lowest expression was with 1% L-PRF (0.160 ± 0.045). Conclusion: The best result of TGF-B1 expression in hDPSC differentiation was in the 25% L-PRF group.

Identification of active compounds in ethyl acetate, chloroform, and N-hexane extracts from peels of Citrus aurantifolia from Maribaya, West Java, Indonesia.

Lime peel contains essential oils are used as anti-oxidants or anti-cancer compounds. As a traditional medicine, lime has been widely used as a substitute for antibiotics. This study aimed to identify active compounds in peel extracts from Citrus aurantifolia that grows in Indonesia. Extraction was carried out by maceration using three different solvents: ethyl acetate, chloroform, and n-hexane. The extracts were analyzed using gas chromatography-mass spectrometry. The results showed that lime peel contained many important compounds and that 28, 27, and 24 different chemical compounds, both minor and major constituents, were extracted by ethyl acetate, chloroform, and n-hexane, respectively. Four compounds were found in all three solvent extracts, namely, D-limonene, phytol, α-tocopherol, and 5, 7-dimethoxycoumarin. Forty-seven compounds were uniquely present in one solvent, including 17 in ethyl acetate, 17 in chloroform, and 13 in n-hexane. Among the active compounds extracted, several are of biological importance, for example, stigmasterol, D-limonene, Vitamin E, and α-tocopherol. It can be concluded that a variety of distinct compounds are extracted from the same lime peel sample when different solvents are used and that some of these are bioactive compounds with anti-oxidant, anti-microbial, or anti-cancer properties.

Antibacterial Effects of Cuminum cyminum Extract Against Enterococcus Faecalis Biofilms From Clinical Isolates

Abstract Objective: To compare the antibacterial efficacy of Cuminum cyminum (cumin) extract and 2% chlorhexidine. Material and Methods: E. faecalis was isolated from non-vital teeth with chronic apical abscess. Samples were then bred in the ChromAgar medium. Subsequently, E. faecalis bacteria's DNA extraction was performed. DNA was then amplified by conventional PCR, and the product was run on an electrophoresis gel. Subsequently, we extracted Cuminumcyminum seeds using the steam distillation technique. The extract was diluted at various concentrations: 0.2, 0.5, 0.7, 1.0, and 1.2 mg/mL.The extract's antibacterial effect was evaluated using an ELISA reader with optical density. Specifically, we assessed the turbidity of E. faecalis in biofilms following immersion in antibacterial agents Results: In the clinically isolated E. faecalis group, the OD values of 0.7 and 1.0 mg/mL cumin extracts were significantly different from that of 0.2 mg/mL cumin extract. A significant difference was also observed between the OD values of 1.0 mg/mL cumin extract and 2% CHX (p<0.05) Conclusion: The antibacterial effect of 1.0 mg/mL Cuminum cyminum extract had higher efficiency than 2% chlorhexidine against E. faecalis biofilms from clinical isolates.

Ideal Concentration of Advanced-Platelet Rich Fibrin (A-PRF) Conditioned Media for Human Dental Pulp Stem Cells Differentiation

Abstract Objective: To discover the ideal concentration of Advanced Platelet Rich Fibrin (A-PRF) as modification of PRF, for human Dental Pulp Stem Cells (hDPSCs) differentiation. Material and Methods: hDPSCs were devided into five experimental groups: Group I (control group) consist of hDPSCs cultured in 10% FBS, Group II consist of hDPSCs cultured in 1% A-PRF, Group III consist of hDPSCs cultured in 5% A-PRF, Group IV consist of hDPSCs cultured in 10% A-PRF and Group V consist of hDPSCs cultured in 25% A-APRF. All group have been observed for 7 and 14 days and each group had three biological replicates (triplo). Formation of the mineralized nodules was detected after 7 days by Alizarin red-based assay and Dentin Sialophosphoprotein (DSPP) expression after 7 and 14 days quantified by ELISA reader. Statistical analysis was proven with Kruskal-Wallis and post hoc Mann-Whitney test Results: The differentiation of hDPSCs in all A-PRF groups was significantly different on day-7 (p<0.05) compare to control group (Group I). There were no significant differences between all groups on day-14 (p>0.05). Significantly differences between Group II (1% A-PRF) and Group I (control), Group II (1% A-PRF) and Group III (5% A-PRF), also Group II (1% A-PRF) and Group V (25% A-PRF) was found from post hoc test analysis Conclusion: The ideal conditioned media concentration for differentiation of human dental pulp stem cells was on 1% up to 5% A-PRF group.