Growth Factors Released from Advanced Platelet-Rich Fibrin in the Presence of Calcium-Based Silicate Materials and Their Impact on the Viability and Migration of Stem Cells of Apical Papilla

Abstract

Advanced platelet-rich fibrin (A-PRF) provides the scaffold and growth factors necessary for stem cells to proliferate and differentiate in successful regenerative endodontic procedures. This study investigates the release of transforming growth factor-β1 (TGF-β1), platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF) from A-PRF in cell culture media in the presence and absence of mineral trioxide aggregate (MTA) or Biodentine. Additionally, this research assesses the viability and migration of stem cells of the apical papilla (SCAP) in previously conditioned media. A-PRF obtained from 14 participants were incubated for 7 days in cell culture media alone or via layering with MTA or Biodentine discs and the release of selected growth factors in the media was evaluated using ELISA. The viability of SCAP grown in conditioned media was measured using the CCK8 assay, while SCAP migration was assessed via a transwell assay by counting migrated cells. The release of TGF-β1, PDGF, and VEGF was significantly higher in media with A-PRF alone than in the presence of either calcium-based silicate material (p < 0.05), which showed no difference from the no-A-PRF control (p < 0.05). None of the tested growth factors released in the A-PRF-conditioned media correlated with clot weight. A-PRF-conditioned media, both with and without calcium-based silicate materials, did not impact SCAP viability and migration (p > 0.05). This study shows that SCAP behavior is not impacted by the decrease in growth factor released in the presence of calcium-based silicate materials and that their role in REPs warrants further investigation.

Keywords: advanced platelet-rich fibrin; calcium-based silicate materials; growth factors; regenerative endodontic procedures; stem cells of the apical papilla.

Figure 1

Quantification of growth factors. The release of growth factors from A-PRF incubated for 7 days in cell culture medium alone or in the presence of MTA or BD was evaluated by growth factor specific ELISA kits for (A) VEGF (a: p > 0.05, b: p < 0.05) (B) PDGF-AB (c: p > 0.05, d: p < 0.001), and (C) TGF-β1 (e: p > 0.05, f: p < 0.001). Absolute growth factor levels were measured in duplicate and represented as means of the 14 samples +/− standard deviation. One-way ANOVA and post hoc Tukey’s multiple comparison test were performed. CM (control medium); PRF (A-PRF-conditioned medium); PRF + BD (A-PRF- and Biodentine-conditioned medium); PRF + MTA (A-PRF- and MTA-conditioned medium).

Figure 2

Correlation between A-PRF weight and growth factor levels in the A-PRF-only group. A linear regression analysis was performed to correlate the A-PRF weight for each of the 14 samples with the respective release of growth factors assessed by ELISA: VEGF (A); PDGF (B); and TGF-β1 (C). A Spearman correlation analysis was used to determine the strength of the correlation for each growth factor. r = correlation coefficient.

Figure 3

SCAP viability and migration analysis. The impact of conditioned media with A-PRF in the presence or absence of calcium silicate-based materials on SCAP was evaluated via specific assays. (A) Viability of SCAP after 48 h growth in conditioned media was assessed via CCK-8. Absorbance was measured at 450 nm and results were reported as the percent of viable cells when the control group was set to 100%. (B) Migration of SCAP was analyzed using a Transwell assay where cells that migrated toward conditioned media after 24 h were stained with crystal violet and counted in five fields of view per sample. All data were reported as mean of the 14 samples +/− standard deviation and statistical significance among groups was determined using one-way ANOVA with post hoc Tukey’s multiple comparison tests. CM (control medium); PRF (A-PRF-conditioned medium); PRF + BD (A-PRF and BD-conditioned medium); PRF + MTA (A-PRF and MTA-conditioned medium). ns, not significant (p > 0.05).