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.

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.

Objective: To compare the potency of fibroblast cells proliferation in 12.5% and 25% Culture Media Conditioned Warton's Jelly (CMCWJ) and Advanced Platelet Rich Fibrin (A-PRF) cultured medium. Material and Methods: Fibroblast cells were divided into five groups: Group I (Control Group): serum-starved fibroblast without any treatment as a negative control; Group II: fibrolast that supplemented in 12.5% CMCWJ medium; Group III: fibrolast that supplemented 12.5% A-PRF medium; Group IV: fibrolast that supplemented 25% CMCWJ medium, and Group V: fibrolast that supplemented 25% A-PRF medium. The fibroblasts proliferation was counted by an automated cell counter. Statistical analysis was performed using One-way ANOVA and Post hoc Tamhane test was conducted to analyze the potential fibroblast proliferation differences in different concentration of CMCWJ and A-PRF group. Results: There were no significant differences in the fibroblast cell proliferation between GI and GIV, GII and GIV, GII and GIII, GII and GV, also GIV and GV. There were significant differences between GI and GII, GI and GIII, GI and GV, also GIII and GIV. Conclusion: The 12.5% CMCWJ group, 12.5% A-PRF group and 25% A-PRF group has excellent potential ability of fibroblast cells proliferation, meanwhile 25% CMCWJ group has the lowest mean potency of fibroblast cells proliferation compared to other groups. The 12.5% A-PRF Group has the highest mean of fibroblast cell proliferation amongst other groups.

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.

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.