Identification of DPSCs
Under the inverted microscope, the cells isolated from the rat’s dental pulp tissue were evaluated day after day till subculturing. At first, and on the 4th day, a great number of cells were rounded and nonadherent whereas at the 7th day, there was an increase in adherent cells, and some rounded cells transformed into a fibroblastic spindle-like shape. Cell colonies were observed under the microscope on the 10th day and on the 14th day, the confluence of cells was notified. At this point, all adherent cells transformed into spindle-shaped cells (Figure 2).
Characterization of DPSCs
The findings of the FC showed that DPSCs were negative for CD45 (0.07%), CD 34 (0.00%) and CD14 (0.08%), whereas the clusters of DPSCs were positive to CD90 (98.95%), CD 73 (99.86%) and CD105 (99.90%). These results reflect the high yield of DPSCs isolation that was derived from rats’ dental pulp (Figure 3).
Characterization of DPSCs-sEVs
Transmission electron microscopy was used to confirm the presence of sEVs in the suspensions. SEVs shapes were wrinkled, oval or spherical. The size of sEVs was (152.17±47.5 nm) in a range between 60 to 260 nm in diameter with a mean size of 230 nm measured by Image J software (Figure 4). The tetraspanin proteins CD81 (~26 kDa) and CD63 (~53kDa) and the cytosolic protein syntenin with ~32 kDa were identified using Western immunoblotting (Figure 5). The whole uncropped images of the original Western blots for Figure 5 are provided in Supplementary Figure 10.
Nanoparticle tracking analyses and measuring sEVs concentration.
The NTA profiles of sEVs revealed that the major peak of sEVs was at 225.5 nm i.e., 47% had an average diameter less than 225 nm. Moreover, nanoparticle quantification confirmed that sEVs concentration was 6.2x107 particles/mL. The surface charge of the vehicles revealed that 61.3% of sEVs zeta potential was -6.5 mV while 38.7% exhibited-11.5 mV which indicated to the stability of sEVs. Using BCA protein assay, the total sEVs yield was determined by protein estimation from intact sEVs. Protein concentration from BCA assay: 4.2 mg/mL. The particle concentration from NTA analysis was 6.2 x 107 p/mL, the dilution factor was 1 and the total volume of medium was 400 ml. Therefore, the sEVs purity was calculated as 6.2 x 107 (p/mL) x 1 x 400 (ml) / 4.2 (mg/mL) x 400 (ml) and the result was 1.4588235 x 107 particle/mg protein (Figure 6).
Results of MTT assay
The MTT assay was used to detect the proper concentration of DPSCs-sEVs. DPSCs treated with 50 μg/mL DPSCs-sEVs showed greater cell proliferation than those treated with 20 μg/mL either alone or in combination with MTA, Therefore the optimal sEVs concentration selected in the present study was 50 μg/mL DPSCs-sEVs (Table 3, Figure 7A).
Then, MTT assay was used to determine the effect of sEVs, MTA and, their combination in DPSCs proliferation rate after 7 and 14 days. The results showed that DPSCs-sEVs, MTA and their combination could stimulate proliferation of DPSCs, and the proliferative capacity of DPSCs treated with sEVs/MTA combination was significantly higher when compared with the other groups. At 7 days, the mean values of the MTT assay were 0.11 ± 0.00, 0.26 ±0.02, 0.68 ± 0.04, and 0.82 ± 0.07 in the untreated group, MTA, DPSCs-sEVs and MTA/DPSCs-sEVs treated groups, respectively and there was a statistical difference between all groups (P value <0.001). After 14 days, the mean values of the MTT assay were 0.26 ± 0.03, 0.46 ± 0.03, 0.92 ± 0.04, and 1.39 ±0.02 for the same groups, respectively and there was a statistical difference between all groups (P value <0.001). Moreover, the proliferation of DPSCs after 14 days was greater than that after 7 Days (P value <0.001) (Table 3, Figure 7B).
Results of transwell migration assay
To determine the effect of MTA, sEVs and their combination on the migration ability of DPSCs, a transwell migration assay was performed. The results showed greater potentialities of DPSCs migration in the treated groups compared to the untreated ones. MTA exhibited potentiality in inducing DPSCs migration higher than untreated and sEVs treated groups. Moreover, DPSCs treated with both MTA and DPSCs-sEVs showed a significant increase in migration ability in comparison with other groups. The descriptive statistics for the mean and SD of the number of migrating cells revealed that the greatest mean value was for DPSCs + MTA/50 μg/mL sEVs (79.00 ± 3.22) while the smallest value was for untreated DPSCs (40.00±1.79). The mean number of the migrated cells in the DPSCs + MTA and DPSCs + 50 μg/mL sEVs were 65.00 ± 2.68 and 53.67 ± 4.59, respectively. The one-way ANOVA test results showed that there was a significant difference (P = 0.001) among the various groups. Tukey’s honest significant difference test revealed a significant difference between two groups (P = 0.001) (Table 4, Figures 8).
Results of real-time polymerase chain reaction
Under the inverted microscope, the DPSCs in the four groups were normal spindle-shaped cells without any abnormal observations. The RT-PCR was performed to measure the expression level of OCN, RUNX2, DSPP and Col 1 genes in the untreated and treated groups. Relative to β actin, the mean average 2^-ΔΔCt (fold change/expression) of OCN for the untreated DPSCs, DPSCs + MTA, DPSCs + 50 μg/mL sEVs and DPSCs + MTA/50 μg/mL sEVs were 1.00, 2.50, 2.15 and 2.66 and for RUNX2 were 0.09, 1.39, 1.23 and 1.57 for the same groups, respectively. Whereas the expression patterns for DSPP were 0.84, 1.44, 2.78 and 4.02, respectively. Moreover, the expression level of Col 1, were 0.71, 1.08, 2.76 and 3.28 for the same groups, respectively. One-way ANOVA test revealed a total significant difference between the mean averages of 2^-ΔΔCt (fold change) of untreated DPSCs, DPSCs + MTA, DPSCs + 50 μg/mL sEVs and DPSCs + MTA/50 μg/mL sEVs (P = 0.001) for OCN, RUNX2, DSPP and Col 1. In addition, Tukey’s honest significant difference test revealed a significant difference between each two groups (P = 0.001). MTA treated group showed higher expression of OCN gene than the DPSCs-sEVs treated and untreated groups. MTA/50 μg/mL sEVs treated groups showed significant upregulation of both OCN and RUNX2 gene expression compared with other groups. The expression pattern of DSPP and Col 1 were increased when using MTA or 50 μg/mL sEVs and 50 μg/mL sEVs showed higher expression in both genes compared with MTA treated group. When using MTA/50 μg/mL sEVs, the upregulation level was significantly increased compared with other groups. (Table 5, Figure 9).