Effects of Platelet-Rich Plasma on Proliferation, Viability, and Odontogenic Differentiation of Neural Crest Stem-Like Cells Derived from Human Dental Apical Papilla

Objective This study is aimed at evaluating the effects of platelet-rich plasma (PRP) on proliferation, viability, and odontogenic differentiation of neural crest stem-like cells (NCSCs) derived from human dental apical papilla. Materials and Methods Cells from apical papillae were obtained and then induced to form neural spheres. The expression of NCSC markers p75NTR and HNK-1 in neural sphere cells was detected by immunofluorescence staining. Human PRP was prepared by a 2-step centrifugation method and activated by CaCl2 and thrombin. The concentrations of PDGF-BB and TGF-β1 in whole blood and PRP were measured by an ELISA kit. PRP in five different concentrations (0%, 2.5%, 5%, 10%, and 25%) was applied to culture NCSCs. On the 1st, 3rd, 5th, and 7th days, cell proliferation was evaluated by CCK8. Cell viability was tested by a live/dead staining kit. mRNA and protein expression of DSPP and BMP4 were analyzed by RT-qPCR and western blot, respectively. Statistical analysis was performed by a one-way analysis of variance (ANOVA) test or t-test. Results Dental apical papilla cells formed neural spheres, from which cells displayed positive expression of p75NTR and HNK-1. The concentrations of PDGF-BB and TGF-β1 in PRP were about 3.5-fold higher than those in whole blood. 5% and 10% PRP significantly promoted proliferation of NCSCs, while 25% and 50% PRP inhibited cell proliferation from Day 3 to Day 7. Low-concentration (2.5%, 5%, and 10%) PRP slightly improved viability of NCSCs on Day 7. On the other hand, high-concentration (25% and 50%) PRP significantly inhibited viability of NCSCs from Day 3 to Day 7. RT-qPCR and western blot results indicated that 10% PRP could promote odontogenic differentiation of NCSCs on Day 7. mRNA and protein expression of DSPP and BMP4 were significantly upregulated in the 10% PRP group compared to those in the control group (P < 0.05). Conclusions PRP is a simply acquirable blood derivative which contains high concentration of growth factors like PDGF-BB and TGF-β1. PRP in a proper concentration could promote proliferation, viability, and odontogenic differentiation of NCSCs derived from human dental apical papilla.


Introduction
The American Association of Endodontics recommends using regenerative endodontic therapy (RET) in immature teeth with necrotic pulp [1,2]. For dental pulp treatment, "RET," "revascularization," and "revitalization" are three synonyms, which are interchangeable [1]. The critical proce-dure of RET is to induce bleeding in periapical tissue. The blood clots can serve as scaffolds and release growth factors to guide stem cells from apical papilla (SCAP) into root canals for pulp regeneration [2][3][4]. It was reported that platelet-rich plasma (PRP) could be applied in RET to promote apical closure and root growth in immature permanent teeth [5][6][7][8].
Both dental pulp and apical papilla develop from a cranial neural crest [14]. Some studies reported that NCSCs existed in dental pulp and apical papilla [15,16]. When cultured under neural sphere-forming condition, dental pulp cells formed spheres in which cells expressed NCSC markers (p75NTR, HNK-1, Slug, Snail, Nestin, and Musashi1) [15]. These NCSCs in spheres had multiple differentiation capabilities, as they could differentiate into adipocytes, chondrocytes, osteoblasts, neurons, and smooth muscle cells. NCSCs obtained from apical papilla of immature teeth could differentiate into odontoblasts [16]. However, the influence of PRP on NCSCs is still unclear. Here, we studied effects of PRP on proliferation, viability, and odontogenic differentiation of NCSCs induced from human dental apical papilla.

Materials and Methods
2.1. Obtain NCSCs from Dental Apical Papilla. The experiment protocol of the current research was approved by the Ethical Review Committee of Sun Yat-sen University. Immature teeth were extracted from patients (aged 16-18 years) and then washed three times with saline to remove residual blood [17][18][19]. Dental papillae were separated from roots by a dental probe and placed in α-MEM (Gibco, Thermo Fisher Scientific, USA) with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin. Subsequently, dental apical papillae were cut into small tissue pieces and then treated with collagenase type I and dispase type II for 30 minutes at 37°C. The tissue blocks were filtrated. After that, remaining liquid was centrifuged at 1000 rpm for 5 minutes. The supernatant was removed, and remaining cells were resuspended. The cells isolated from papillae were seeded on cell culture dishes and cultured in a condition of 5% CO 2 at 37°C for two weeks. Next, these cells were cultured with neural sphere medium, i.e., DMEM/F12 was supplemented with N-2 (100x), B-27 (50x), 100 U/ml penicillin, 100 μg/ml streptomycin, 20 ng/ml recombinant human EGF, and 20 ng/ml recombinant human FGF2 (Gibco, Thermo Fisher Scientific, USA). One week later, neural spheres were collected and detached into single cells by a StemPro Accutase Cell Dissociation Reagent (Life Technologies, Thermo Fisher Scientific) [20,21]. These cells were seeded onto 1% Matrigel (Corning, Bedford, MA, USA) precoated dishes for subculture.

Immunofluorescence
Assay. NCSCs at the 2 nd passage were digested, centrifuged, and placed on a 6-well culture plate at a density of 2 × 10 4 per well. After 24 hours, 4% paraformaldehyde was used to fix the cells for 30 minutes, and 0.5% Triton X-100 was used to permeabilize the cells for 20 minutes. Later, the cells were blocked in 5% bovine serum albumin (BSA) for one hour and incubated with primary antibodies against p75NTR (8238S, Cell Signaling) and HNK-1 (ab187274, Abcam) overnight at 4°C. After 12 hours, the cells were probed with Goat Anti-Rabbit Alexa Fluor 488 (ab150077, Abcam) and Goat Anti-Mouse Alexa Fluor 647 (ab150115, Abcam) antibodies for one hour and then washed with phosphate buffer solution (PBS) for three times. Additionally, cell nuclei were counterstained with DAPI. Images were captured with an inverted fluorescence microscope.

Preparation of Human PRP.
A total of four students from Sun Yat-sen University, China, donated their blood. The preparation of human PRP followed a standard protocol [22]. Briefly, whole blood was collected by a 100 ml syringe and centrifuged for 10 minutes at 180 g to separate plasma and blood cells. Next, plasma and blood cells were centrifuged again to three layers: the lower layer was blood cells. The upper layer was poor platelet plasma, and between the two layers was PRP. PRP was activated by adding CaCl 2 (22 mM) and thrombin at 37°C for 40 minutes, and then, blood clots were removed. The exudate was centrifuged at 890 g for 10 minutes, and then, supernatant which contained growth factors was cryopreserved at −80°C for further application.

Growth Factor Concentration Measurement.
The concentration of two growth factors (PDGF-BB and TGF-β1) in PRP and whole blood was tested by enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, USA). Three repeats were carried out for each test; after that, the absorbance at 450 nm was detected and analyzed.
2.5. Cell Proliferation Assay. The 3 rd passage NCSCs were placed on 96-well cell culture plates with initial density of 2 × 10 3 cells per well. Cell Counting Kit 8 (CCK8) (Dojindo, Tokyo, Japan) was applied to evaluate proliferation of those cells on the 1 st , 3 rd , 5 th , and 7 th days after culture [17]. To evaluate the impacts of PRP on NCSC proliferation, five PRP concentrations (0%, 2.5%, 5%, 10%, and 25% PRP) were applied. The optical density (OD) was measured at 450 nm by a microplate reader (Thermo Scientific, UK).

Dental Apical Papilla Cells Formed Neural Spheres.
The pink apical papillae were gently separated from teeth (Figure 1(a)). The dental apical papilla cells formed colonies one week after culture (Figure 1(b)). In sphere-forming medium containing FGF2, EGF, B-27, and N-2 supplements, the 3 rd passage dental papilla cells formed spheres within one week (Figure 1(c)).

Expression of NCSC Markers in Sphere-Forming Cells.
Sphere-forming cells were visualized by immunofluorescence and verified positive expression of NCSC surface markers, including p75NTR and HNK-1 ( Figure 2). These results indicated that the sphere-forming cells were NCSCs.

Influence of PRP on Viability of NCSCs. The fluorescent images of live/dead cells are shown in
3.6. 10% PRP Promoted Odontogenic Differentiation of NCSCs. 10% PRP was selected to assess NCSC differentiation because it achieved optimal cell proliferation and viability as mentioned above. RT-qPCR was conducted to analyze key gene expression (DSPP and BMP4) involved in odontogenic differentiation of NCSCs ( Figure 5(a)). Both DSPP and BMP4 mRNA expressions were significantly upregulated in the 10% PRP group compared with those in the control group (P < 0:05). Similarly, western blot results displayed that 10% PRP significantly promoted DSPP and BMP4 expression in the protein level (P < 0:05) ( Figure 5(b)). Both RT-qPCR and western blot results suggested that 10% PRP promoted odontogenic differentiation of NCSCs in vitro.

Discussion
SCAP are progenitor cells of odontoblasts, which are crucial for dentin formation and root completion [23]. SCAP obtained from human third molars exhibited characteristics of neural crest-derived progenitor cells [16]. In the current study, the results displayed that SCAP formed neural spheres after directional induction using neural sphere-forming medium for one week. The similar phenomenon was observed in dental pulp cells using neural sphere-forming medium [15]. In our study, the neural sphere cells derived from apical papillae expressed p75NTR and HNK-1 which were classic markers of NCSCs [24,25]. These results indicated that neural sphere cells derived from apical papillae were NCSCs.
Various studies reported that proliferation of dental cells could be influenced by different concentrations of PRP. A researcher pointed out that 1%-20% PRP significantly enhanced human DPSC proliferation in six days [26]. 2% to 10% PRP derived from human umbilical cord blood significantly stimulated proliferation of human DPSCs, while 2% PRP led to the highest proliferation in three days [27]. In another study, 0.5 and 1% PRP promoted proliferation of human DPSCs, but 5% PRP inhibited cell proliferation in five days [28]. Similar results were also observed in rat DPSCs;

BioMed Research International
1% and 10% PRP enhanced proliferation of DPSCs in ten days, but 50% PRP inhibited cell proliferation [12]. In the current experiment, 2.5%-10% PRP increased proliferation of NCSCs derived from the dental apical papilla, while 25% and 50% PRP inhibited cell proliferation in seven days. This result explained that low-concentration (2.5%-10%) PRP could promote NCSC proliferation, while high-concentration (25% and 50%) PRP gave an opposite result. The effects of PRP concentration on NCSCs are similar to DPSCs as mentioned above, but variance of PRP concentration might be attributed to the source of PRP and cell types. There is only one direct evidence about the role of PRP on cell viability in the dental field. DPSCs exposed to 10% and 20% PRP acquired significantly (P < 0:05) higher     [29]. Similar results were also observed in adipose tissue-derived mesenchymal stem cells, of which viability significantly increased when cultured with 5% and 20% PRP for 16 hours [30]. For Schwann cells, 2.5-10% PRP significantly promoted cell viability on Day 3 and Day 7 [31]. Besides, 5% PRP significantly increased viability of periodontal cells, while 50% PRP inhibited viability on Day 2 and Day 3 [32]. Creeper et al. pointed out that the high concentration of PRP could cause cytotoxic effect on periodontal ligament cells [33]. In our study, high-concentration (25% and 50%) PRP resulted in obvious low-level cell viability, while low-concentration (2.5%-10%) PRP slightly improved viability of NCSCs.
A few studies pointed out that PRP could improve odontogenic/osteogenic differentiation [13,34,35] and mineralization [11,12] of dental stem cells. PRP promoted mRNA and protein expression of DSPP and DMP-1 in SCAP [13]. Because DSPP and DMP-1 are markers of odontoblasts, this result indicated that PRP could be a candidate for inducing SCAP into odontoblasts. Previous studies also reported that BMP4 acted as a crucial mesenchymal odontogenic signal during early tooth development [36]. Our results demonstrated that 10% PRP-treated NCSCs obtained higher expression of odontogenic differentiation markers, such as DSPP and BMP4. This result was consistent with the previous studies, which means NCSCs treated by certain concentration of PRP could obtain better odontogenic differentiation potential.

Conclusions
In this study, we have elucidated that PRP isolated from humans is a simply acquirable blood derivative which contains high concentration of growth factors like PDGF-BB and TGF-β1. Moreover, we found that PRP in proper concentration (2.5%-10%) could promote cell proliferation, viability, and odontogenic differentiation of NCSCs derived from human dental apical papilla. Therefore, these findings provide considerable evidence and valuable information to apply PRP in regenerative endodontic therapy.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
There are no conflicts of interest from any author.