ANTIBACTERIAL EFFICACY OF GRAPE SEED EXTRACT CONTAINING 2.9% TANNIN AGAINST ENTEROCOCCUS FAECALIS BIOFILM

Objective: The aim of this study was to observe the antibacterial efficacy of grape seed extract (GSE) against Enterococcus faecalis biofilm.Methods: A biofilm of E. faecalis American Type Culture Collection (ATCC) 29212 strain was prepared using sterile cellulose nitrate filter membraneincubated on brain heart infusion agar at 37°C for 72 h under aerobic condition. Each membrane containing E. faecalis biofilm was added to threetubes of phosphate-buffered saline (control), three tubes of GSE, and three tubes of 2% clorhexidine. The number of viable DNA cells was measuredusing real-time polymerase chain reaction. The data were statistically analyzed using non-parametric Kruskal–Wallis test and Mann–Whitney U-test.Results: GSE had antibacterial efficacy against E. faecalis biofilm. The difference in the amount of E. faecalis DNA between all groups was statisticallysignificant (p=0.05).Conclusion: GSE has antibacterial efficacy against E. faecalis biofilm

EFFECT OF GRAPE SEED EXTRACT SOLUTION ON THE MICROHARDNESS OF THE ROOT CANAL DENTIN: AN IN VITRO STUDY

Objective: Grape seed extract (GSE) containing proanthocyanidin as a root canal irrigation solution has its antibacterial effects and ability to eliminatethe smear layer. In addition, proanthocyanidin acts as a cross-linking agent of collagen, which adds to dentin’s mechanical properties. This studyanalyzed the effect of GSE containing 2.9% proanthocyanidin on the microhardness of the dentin in the root canal.Methods: Fifty teeth were divided into three groups and immersed in GSE solution, 3% NaOCl solution, or distilled water (controls) for 30 min for 3consecutive days. The microhardness was measured using the Vickers method. Data were analyzed using the Kruskal–Wallis test.Results: The GSE group presented the highest microhardness values, whereas the lowest values in the NaOCl group. No significant difference inmicrohardness observed between the GSE and distilled water groups.Conclusion: The GSE solution maintains the microhardness of the root canal dentin

MICROLEAKAGE EVALUATION OF MODIFIED MINERAL TRIOXIDE AGGREGATE EFFECT TOWARD MARGINAL ADAPTATION ON CERVICAL DENTIN PERFORATION

Objective: To analyze the marginal adaptation of conventional MTA (ProRoot®MTA) and modified MTA (MTA Flow™) on perforated dentin.Methods: Forty specimens of human's premolar teeth with lateral perforations were sealed by conventional MTA (n=20) and modified MTA (n=20). After 24 hours, the specimens were immersed in Indian ink for 24 hours, then embedded in resin and sectioned longitudinally in mesial-distal direction. The score of microleakage was determined using stereo microscope (SteREO Discovery. V12, Carl Zeiss) with 20x magnification. Statistical analysis was done by Chi Square (p<0,05).Results: Less microleakage score (0,5-1mm) was detected in modified MTA (25%) compared to conventional MTA (45%), although not statistically significant.Conclusion: Microleakage were detected in both conventional and modified MTA as material for cervical dentin perforation treatment, although modified-MTA group showed less microleakage score

ANALYSIS OF LYSATE PLATELET-RICH FIBRIN EFFECTS ON HUMAN DENTAL PULP STEM CELL DIFFERENTIATION THROUGH DENTIN SIALOPHOSPHOPROTEIN EXPRESSION

Objective: In vitro, the culture media in which human dental pulp stem cells (hDPSCs) are grown are supplemented with specific growth factors thatinduce cell cycle entry and differentiation. Lysate platelet-rich fibrin (L-PRF) is a unique and stable growth factor supplement produced from plateletslysed by freezing-thawing. In this study, we aimed to analyze the potential effects of L-PRF on hDPSC differentiation.Methods: We divided hDPSCs isolated from human third molars at the second passage into five culture media groups treated with 1%, 5%, 10%,and 25% L-PRF or 10% fetal bovine serum (control). After 7 days, we evaluated hDPSC differentiation using an enzyme-linked immunosorbent assayspecific for dentin sialophosphoprotein and Alizarin-Red staining.Results: None of our analyses revealed any significant differences between the L-PRF- and control-treated cells.Conclusion: L-PRF could potentially induce the differentiation of hDPSCs in vitro

COMPARISON OF HUMAN PLATELET LYSATE AND FETAL BOVINE SERUM IN CULTURE MEDIA FOR HUMAN DENTAL PULP STEM CELL PROLIFERATION

Objective: Ex vivo and in vitro cell cultures require a basal medium with added supplements containing growth factors, proteins, and enzymes tosupport attachment, growth, and proliferation. Fetal bovine serum (FBS) is used to supplement cell culture media. However, human platelet lysate(hPL) represents an attractive alternative as it is nonxenogeneic.Methods: Human third molars were collected from six healthy donors (19–35 years old) with no history of regular alcohol consumption or smoking.Human dental pulp stem cells (hDPSCs) at the second passage were divided into two culture media groups, 10% FBS and 5% hPL, as well as a controlgroup after 24 h of serum starvation. A flow cytometry analysis was conducted to measure CD90, CD105, CD73, CD34, CD45, and Human LeukocyteAntigen-DR isotype (HLA-DR). Cellular proliferation was evaluated on days 1, 3, and 5.Results: The flow cytometry analysis revealed that the majority of the cells expressed positive mesenchymal stem cell surface markers, includingCD73 (98.5%), CD90 (98.3%), and CD105 (71.0%), and lacked CD34, CD45, and HLA-DR. There were significant differences among the 5% hPL, 10%FBS, and control groups on days 1, 3, and 5.Conclusion: For a nonxenogeneic culture, 5% hPL can be used as an alternative in culture media for hDPSC proliferation

Comparison of Transforming Growth Factor 1 (TGF-β1) Expression in Various Lysate Platelet-Rich Fibrin (L-PRF) Concentrations on Human Dental Pulp Stem Cell Differentiation

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

TRANSFORMING GROWTH FACTOR-β1 EXPRESSION IN VARIOUS CONCENTRATIONS OF ADVANCED PLATELET-RICH FIBRIN MODULATING HUMAN DENTAL PULP STEM CELL DIFFERENTIATION

Objective: Modification of the speed and time of centrifugation based on the low-speed centrifugation concept for platelet-rich fibrin (PRF) has resulted in a new type of PRF known as advanced PRF (A-PRF). A-PRF can release several types of growth factors (GFs) that participate in the process of differentiation, such as transforming GF-β1 (TGF-β1). The aim of this study was to analyze TGF-β1 expression in various concentrations of A-PRF in the differentiation process of human dental pulp stem cells (hDPSCs).Methods: hDPSC cultures were obtained from those of previous research (ethical approval form has been attached). These hDPSCs were in the 2nd–3rd passage, and serum starvation was done by reducing fetal bovine serum (FBS) levels in the hDPSC culture media. A-PRF was obtained using 10 ml blood collected from the cubital vein, which was centrifuged at 1500 rpm for 14 min and then divided into four concentration groups. TGF-β1 expression in 1%, 5%, and 25% A-PRF as well as in 10% FBS (control) was analyzed by ELISA on day 7.Results: Although no significant differences were observed in TGF-β1 expression between 1%, 5%, and 25% A-PRF, and 10% FBS, it was observed that the higher the concentration of A-PRF, the greater the TGF-β1 expression.Conclusion: The expression of TGF-β1 was consistent with the increase in A-PRF concentration. The highest TGF-β1 expression was detected in 25% A-PRF among all concentrations in the differentiation process of hDPSCs

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

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 (p0.05). 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

The effect of hyaluronic acid conditioned media on hDPSCs differentiation through CD44 and transforming growth factor-β1 expressions

Hyaluronic acid (HA) has the capability to influence dentin niche which is important in regenerative process. The CD44 as a specific receptor of HA was found to be related to dentin mineralization process. Meanwhile, transforming growth factor β1 (TGF-β1) has a vital role in the transition from proliferation into the differentiation of human dental pulp stem cell human dental pulp stem cells (hDPSCs) to become odontoblast cells and dentin mineralization. This study aims to analyzed HA's effect on dentin mineralization through CD44 and TGF-β1 expressions. Stem cells were cultured in four different supplemented conditioned media (control, +10 μg/mL, +20 μg/mL, and + 30 μg/mL of HA). Evaluation of CD44 expression was analyzed using flow cytometry and TGF-β1 was analyzed using enzyme-linked immunosorbent assay reader. Qualitative result using Alizarin red test after 21 days was done to confirm the formation of mineralization nodules. It was shown that HA expression of CD44 and TGF-β1 on hDPSCs were higher in AH groups compared to the control group and 30 μg/mL HA induced the highest TGF-β1 expression on hDPSCs. Alizarin red test also showed the highest mineralization nodules in the same group. Therefore, from this study, we found that supplemented 30 μg/mL of HA was proved in initiating hDPSCs differentiation process and promote dentin mineralization