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

Abstract

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.

Fig. 1

Workflow of the isolation of the thrombin-activated platelet-derived exosome (T-aPDE). The exosome derived from the activated platelet-rich plasma-thrombin (PRP-T) was stated as T-aPDE isolated by the total exosome isolation method, and then, it was divided into three concentrations.

Fig. 2

Determination of the size and morphology of the T-aPDE.

Fig. 3

Results of the (A) harvesting of the human dental pulp stem cells (hDPSCs) between the third and fourth passages (P3 – P4) ( left ) followed by their 24-hour starvation in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 1% fetal bovine serum (FBS; right) and of the (B) flow cytometry mesenchymal stem cells marker expression of the hDPSCs used in this study, showing a positive cocktail of CD90+ (97.9%), a negative cocktail of Lin ^Neg (0.5%), CD105+ (97.7%), and CD73+ (98.6%).

Fig. 4

Sizes and morphologies of T-aPDE by transmission electron microscopy: particle size analyzer results. (A) A double lipid layer of exosome vesicles was seen at the outer part: vesicles ( white arrow ) and exosomes ( white arrow ). (B) Results of the physical characterization analysis of PRP-E protein using the specific immunophenotyping flow cytometry test for plasma (ab267479 Exosome Isolation and Analysis Kit—Flow Cytometry, Plasma, Abcam) for the T-aPDE of CD63+ and CD81+ ( C – D ).

Fig. 5

Viability mean rate of the viable hDPSCs (%) cultured in PRP-T and T-aPDE (0.5, 1, and 5%) after 24, 48, and 72 hours of observation (and after conversion of the absorbances values of the 3-(4,5-dimethiazole-2-yl)2,5-diphenyltetrazolium bromide assay to the percent of viable cells).

Fig. 6

Qualitative results of the migration activity of the hDPSCs showing that the 5% T-aPDE group had the highest wound closure area (97.52%) and the fastest speed rate (3.069 μm/h) compared with the other groups ( white arrow ) after 24 hours of observation.

Fig. 7

Quantitative results of the post hoc vascular endothelial growth factor A expressions of the hDPSCs after their culture in T-aPDE groups (0.5, 1, and 5%) and comparison with 10% PRP-T at 24, 48, and 72 hours of observation. The post hoc analysis of the T-aPDE groups showed statistically significant differences between 5% T-aPDE and the control group and other concentrations of T-aPDE after 24 and 72 hours of observation ( p  < 0.05) ( post hoc Mann–Whitney test). * p  < 0.05; ns = not significant.