Dental pulp tissues were collected from healthy caries-free premolars or third molars from 13-to-25-year-old patients. Informed consent was obtained from the patients and the protocol was approved by the Ethics Committee of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology. Briefly, under the sterile condition, tooth surfaces were cleaned by 75% ethanol; debris and periodontal ligament were then cleaned off by a scalpel blade. The tooth was slowly cut along cemento-enamel junction and pulp tissue was gently separated from the crown and root. Pulp tissue was then cut into small pieces and digested in a solution containing 3 mg/ml collagenase type I (Invitrogen) and 4 mg/ml dispase (Roche) for 1 h at 37°C. Digested pieces were cultured in DMEM supplemented with 20% FBS (Fetal Bovine Serum) and 1% penicillin-streptomycin (Huang et al., 2008; Karamzadeh et al., 2012). Cells were passaged three to four times and used in subsequent experiments.

Human whole blood samples were harvested with informed consent from 6 (mean age 20 ± 15 years) healthy volunteer donors. The blood sample collection was also approved by the Institutional Research Ethics Committee of Union Hospital. PRP was separated from the blood samples by secondary centrifugation. The blood collection was firstly centrifugalized at 550 g for 10 min; the top plasma and middle platelet layers were then carefully transferred to a new tube. After secondly centrifugalizing the blood collection at 1,230 g for 15 min, the upper layer was discarded carefully and 1 ml of PRP containing precipitated platelets was obtained. Before each use, calcium chloride was added to PRP at a ratio of 1:9, and thrombin was added at 500 IU per 1 ml volume of PRP (Marx et al., 1998; Mizushima, 2018).

Cell migration was firstly assessed by scratch assay. In short, hDPCs (7 × 10⁵) were seeded into 6-well plates and incubated for 24 h. The cells were scored with a sterile pipette tip to leave a plus shape scratch of approximately 0.4–0.5 mm in width. The culture medium was then changed into DMEM containing 1 and 10% FBS and 1, 5, 10, and 20% PRP separately in different groups. The cell migration was observed with an inverted microscope at 24 h.

In transwell assay, hDPCs (6 × 10⁴) were suspended in 200 uL of serum-free DMEM and were added to the upper chamber of the insert (transwell plates are 6.5 mm in diameter with 8 µm pore filters; Corning Costar, Cambridge, MA). The culture medium in the lower chamber was changed into 500 uL of DMEM with 1 and 10% FBS and 1, 5, 10, and 20% PRP, respectively. After 24 h, the upper chambers were washed with PBS and the cells inside the chamber were gently wiped off by a cotton swab. The cells beneath the filter were fixed with 4% paraformaldehyde and then stained with crystal violet for 30 min. The number of the migration cells was observed and counted under an inverted microscope. All experiments were done in triplicate.

The effect of PRP on the proliferation of hDPCs was evaluated by using a cell-counting kit 8 assay (CCK-8, Dojindo, Japan). Briefly, hDPCs (3 × 10³) were seeded into 96-well plates and divided into six groups. After overnight incubation, the medium was changed into different concentrations of FBS (1 and 10%) and PRP (1, 5, 10, and 20%) respectively. At the indicated time points (1, 3, 5 days), cells were quantified using CCK-8 assay and absorbance was measured at 450 nm in quintuplicate wells per condition.

To further explore the effect of autophagy on cell migration and proliferation in PRP-treated hDPCs, autophagy modulation was employed. Cells were pre-treated with rapamycin (RAP) (100 nM, MCE, China) or 3-methyladenine (3-MA) (5 mM, MCE, China) before incubation with 5% PRP in scratch assay, transwell assay, and CCK-8 assay.

For immunofluorescence detection, hDPCs were seeded on top of coverslips in 12-well plates. After overnight incubation, the culture medium was changed into DMEM with 1 and 10% FBS and 1, 5, 10, and 20% PRP separately in different groups. After incubation for 24h, the cells were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 1% BSA. LC3B (1:500, Abclone, China) was added as primary antibody and incubated overnight at 4°C. Alexa Fluor 488 (Proteintech) green fluorescence conjugate goat anti-mouse was used as a secondary antibody (1:200). Alexa Fluor cy3 phalloidin red fluorescence conjugate (1:500) was then applied to mark the actin cytoskeleton, and subsequently, the DPSCs were treated with 4′, 6-diamidino-2-phenylindole (DAPI, 1:1,000, Biosharp, China). The coverslips were mounted on a microscope slide with an embedding medium and observed under a laser scanning confocal microscope. To investigate the effect of autophagy on immunofluorescence, cells were cultured in the presence of an inhibitor or agonist of autophagy.

hDPCs (5 × 10⁶) were cultured with 10% FBS or 5% PRP for 24 h, digested with 2.5% trypsin/EDTA (HyClone, America), centrifuged, and fixed with 2.5% glutaraldehyde (Sigma) overnight at 4°C. The samples were examined using a transmission electron microscope (Hitachi HT7700-SS, Hitachi, Japan) operating at 75 kV.

hDPCs were cultivated in mineralized medium containing 10% FBS and 1, 5, 10, and 20% PRP for 7 days and fixed with 4% paraformaldehyde. The cells were stained with an ALP assay kit (Jiancheng Bioengineering Institute, China) according to the manufacturer’s instructions. To investigate the role of autophagy in PRP-induced cell osteogenic differentiation, autophagy inhibitor or enhancer was also introduced.

hDPCs (4 × 10⁴) were seeded into 24-well culture plates and cultured in osteogenic induction medium (Cyagen, China). The cells were divided into five groups and in each group 10% FBS and 1, 5, 10, and 20% PRP were added, respectively. Cultures were stained with Alizarin Red (Servicebio, China) on day 21 to show deposits of calcium phosphate according to the manufacturer’s instructions. Shortly, the cells were fixed with 4% paraformaldehyde for 30 min and stained by the Alizarin Red (Servicebio, China) for 15 min.

Total RNA was isolated from hDPCs after 3 weeks of incubation (LC3B, Beclin-1 for 24 h, ALP for 7 days) and reversely transcribed into complementary DNA using the RNA Isolation Total RNA Extraction Reagent (Vazyme, China) and HiScript III RT SuperMix for qPCR (Vazyme, China). qRT-PCR was performed in triplicate using an AceQ Universal SYBR qPCR Master Mix (Vazyme, China). The reaction conditions were as follows: polymerase activation at 95°C for 10 min, 40 cycles of denaturation at 95°C for 15 s, and annealing and extension at 60°C for 1 min. The primers for the reference gene β-actin and for the target genes ALP, collagen-1(COL 1), osteocalcin (OCN), dentin matrix protein 1 (DMP-1), dentin sialophosphoprotein (DSPP), LC3B, and Beclin-1 are shown in Table 1.

hDPCs were cultured in 10% PBS, 5%PRP, RAP, or 5%3-MA for 24 h; protein was extracted using RIPA buffer with fresh protease inhibitors (InvivoGen). Protein concentration was measured by the BCA kit (Beyotime). The proteins were separated by electrophoresis on an SDS-PAGE gel and then transferred onto a PVDF membrane. After blocking for 1 h with skim milk, the membranes were incubated with primary antibodies against LC3B (1:1,000; Abclonal), Beclin-1 (1:1,000; Abclonal), and GAPDH (1:1,0000; Proteintech) overnight at 4°C. Then, the membranes were incubated with secondary antibodies for more than 1 h. The protein-antibody complexes were then visualized using the enhanced chemiluminescence detection system.

Data from different experiments were represented as mean ± standard deviation and analyzed by SPSS 20.0. Student’s t-test or one-way ANOVA was used to analyze the significant difference. p < 0.05 was considered statistically significant.

In scrape assay, the results showed that 5, 10, and 20% PRP significantly promoted cell migration of hDPCs (Figure 1A) and 5% PRP showed the best effect. Simultaneously, in the transwell assay (Figures 1A,B), the number of cells that migrated toward 10% FBS and 1, 5, 10, and 20% PRP was significantly higher than the control group (1% FBS) (Figures 1A,B). The number of migrating cells toward 5% PRP was also the highest (Figure 1B).

To examine the effect of PRP on cell proliferation, a CCK-8 test was performed. The results demonstrated that there were no significant differences in cell proliferation rates between groups on day 1. On day 3, PRP at 5, 10, and 20% concentration significantly increased cell proliferation rates. On day 5, the cell cultured in 1, 5, and 10% PRP showed significantly higher proliferation rates, while a significant reduction in cell proliferation was observed with 20% PRP concentration than the control group (1% FBS) (Figure 1C).

To explore the effects of PRP on osteogenic differentiation, ALP and alizarin red staining were analyzed. The hDPCs cultured in 5, 10, and 20% PRP increased the level of ALP staining than the control group (10% FBS) (Figure 2A). The alizarin red results showed that PRP increased mineralized nodule formation (5, 10, and 20%) (Figure 2B). The group treated with 20% PRP demonstrated the highest osteogenic induction capacities.

To determine the transcriptomic changes accompanied by PRP treatment, we performed qRT-PCR. ALP, Col-1, OCN, DMP-1, and DSPP are important genes for osteogenic differentiation. The expression levels of ALP and Col-1 were highly elevated in 5, 10, and 20% PRP-treated group (Figure 2C). The expression level of OCN was dramatically increased in the 20% PRP group, while DSPP and DMP-1 expression were significantly induced by 10% PRP (Figure 2C).

To investigate the activation of autophagy by PRP treatment, immunofluorescence staining was firstly employed to detect the expression of the autophagy-associated protein (LC3B). As shown in Figures 3A,B, the expression of LC3B was increased significantly by PRP at 5% concentration. The results of the transmission electron microscope showed that a large number of autophagic vacuoles (arrows in Figure 3C) were observed in hDPCs incubated with 5% PRP (Figure 3D). Consistent with the immunofluorescence staining results, the western blotting and qRT-PCR results showed that expression of autophagy-associated proteins, LC3B and Beclin-1, were increased by 5% PRP (Figures 3G–I). The result suggested that autophagy was strongly activated by PRP treatment.

After the agonist and inhibitor of autophagy were added, the immunofluorescence staining of LC3B was increased in the presence of rapamycin and decreased by 3-MA (Figures 3E,F). The expression level of autophagy-related genes and protein expressions, LC3B and Beclin-1, was also elevated by rapamycin and reduced by 3-MA (Figures 3G–I).

In the presence of an autophagy activator (RAP), cell migration was promoted in both transwell (Figures 4A,B) and scratch assay (Figure 4C). In the presence of inhibitor (3-MA), the migration of hDPCs was weakened in both scrape and transwell assay (Figures 4A–C). In the CCK-8 assay, the proliferation of hDPCs was enhanced after treatment with autophagy activator at 12 and 24 h time points. Cell proliferation was weakened in the presence of autophagy inhibitor at 24 h (Figure 4D). The ALP staining and quantitative-PCR results revealed that autophagy inhibition suppressed PRP-induced hDPCs osteogenic differentiation, whereas autophagy activator augmented PRP-stimulated differentiation (Figures 4E,F). These data suggested that autophagy was involved in cell migration, proliferation, and differentiation induced by PRP.