Platelet-rich plasma promotes cell viability of human hair dermal papilla cells (HHDPCs) in vitro

Abstract

Purpose: This study aimed to clarify the effect of platelet-rich plasma (PRP) on human hair dermal papilla cell (HHDPC) viability and expression levels of alkaline phosphatase (ALP) and versican. Methods: Eight healthy volunteers were enrolled, and different concentrations of PRP and platelet-poor plasma (PPP) were preparedby 2-step centrifugation method. HHDPCs were thencultivated. To identify the best incubating time of HHDPCs and plasmaconcentration, cell viability was investigated by MTT assays. In PRP- or PPP-treated HHDPCs, the mRNA levels of versican and ALP were checked by real-time quantitative PCR (qRT-PCR). Results: Both PRP (5% and 10%) and PPP (5% and 10%) could significantly promote cell viability of the HHDPCs (p4) in 72 h group, while the effect of 10% PRP on HHDPC viability was less than that of 5% PRP. qRT-PCR showed that PRP could significantly increase mRNA levels of ALP and versican in HHDPCs (p4) when compared with the control group. Meanwhile,Western blot indicated an obviously increased protein level of ALP and versican in PRP-treated HHDPCs (p4). Conclusion: PRP could promote cell viability of HHDPCs. Besides, 5% PRP could significantly increase ALP and versican expression in both mRNA and protein levels in HHDPCs (p4).

Introduction

Hair follicleregeneration, which is composed by anagen, catagen, telogen and exogen [1], is of great importance in treating dermal wound and alopecia [2,3]. Alopecia (also known as hair loss) has many risk factors, such asnutritional deficiencies, traumatic damage and fungal infection [4]. Besides, dermal wound can result in significant disability or even lead to death [5,6]. Along with increased mechanical trauma, burns, skin disease and ageing, dermal wound healing is becoming more and more urgent [5,7].

Compared with whole blood, platelet-rich plasma (PRP) is blood plasma that enriched with platelets, and platelet-poor plasma (PPP) is blood plasma with low-level platelets. Alpha granules in platelets contain plenty of growth factors that can promote cell proliferation and differentiation, thus PRP has more growth factors than PPP [8]. Researchers have found that PRP can stimulate mesenchymal stem cell (MSC) proliferation and induce MSC differentiating to chondrocyte in vitro [9-11]. Previous studies report that PRP has a considerable effect on hair induction and hair follicle regeneration [12,13]. In addition, dermal papilla cells (DPCs, a kind of dermal cells in hair follicle) can induce hair growth and hairfollicleregeneration [14]. However, the effect of PRP on human hair dermal papilla cells (HHDPCs) viability and hair follicle regeneration were stillunclear.

In this study, different concentrations of PRP and PPP were prepared, and HHDPCs were cultivated. Afterwards, cell viability was investigated to identify the best incubating time of HHDPCs and plasmaconcentration. Furthermore, the expression levels of versican and alkaline phosphatase (ALP) in PRP- or PPP-treated HHDPCs were checked.

Materials and methods

Sample preparation

A total of 8 healthy volunteers (8 males, mean age = 24.9 years) were enrolled and provided their inform consent. About 10 ml whole blood was drawn from cubitalvein of each volunteer and stored in centrifuge tubes containing 3.2% sodium citrate (vol/vol = 10:1, BD). Then, PRP and the PPP were prepared using 2-step centrifugation method as described previously [15]. After PRP was activated by adding 1000 U bovine thrombin and 0.2 ml of 10% CaCl₂, it was placed at room temperature for about 10 min and then recentrifuged (3200 r/min, 5 min, r = 13.5 cm). Finally, the supernatant was collected and stored at -80°C.

HHDPCs cultivation

The second-generation HHDPCs were purchased from Shanghai Huzheng Industrial CO. LTD. The cell were resuspended in the mixture of RPMI 1640 medium (GIBCO), 10% fetal bovine serum (FBS, GIBCO) and 1% double antibody (GIBCO). Followed bythe suspension was cultivated at 37°C in a humidified 5% CO₂ incubator (Thermo). Then, the second-generation HHDPCs were spread on Petri dish for passage. When 80%~90% of the Petri dish were covered, HHDPCs were digested with pancreatin (GIBCO). After centrifugation, the supernatant was removed. Subsequently, the HHDPCs were added with the frozen stock solution made from 50% RPMI 1640 medium (GIBCO), 40% FBS (GIBCO) and 10% dimethyl sulfoxide (DMSO). Finally, the HHDPCs were preserved in a program frozen box.

MTT assay

The fourth- (p4) and eighth-generation (p8) HHDPCs were counted, and then each well of 96-well plates (ABI) was added with 1×10⁴ cells. After the HHDPCs were starved for 24 h, their medium was replaced. In experimental group 1, the medium was replaced by medium containing 5% or 10% PRP. In experimental group 2, the medium was changed into medium containing equal PPP. Meanwhile, the medium in control groupwere replaced by normal medium. There were six repeatsfor each group. After HHDPCs being incubated for 24 h, 48 h and 72 h, 10 μl MTT reagent (0.5 mg/ml) was added to each well and thenthe 96-well plate was incubated at 37°C for 4 h. Subsequently, the medium of HHDPCs was replaced by 100 μl DMSO. Followed bythe plate was shook gently for 10 min to dissolvecrystals thoroughly. Finally, the optical densities (ODs) of HHDPCs solution in each well were measured at 570 nm by spectrophotometer (Merinton). Each assay was repeated for three times.

RNA extraction and first-strand cDNA synthesis

After the HHDPCs (p4 and p8) were counted, 2×10⁵ cells were added into each well of 6-well plates (ABI) and starved for 24 h. In experimental group 1, the medium of HHDPCs was changed into medium containing 5% PRP. In experimental group 2, the medium was replaced by medium containing equal PPP. In control group, the medium were replaced by normal medium. Each group had two repeats. After incubation for 72 h, HHDPCs were collected for RNA extraction and the following western blot. Total RNA was extracted using Trizol-based procedure as described previously [16]. The purity and concentration of total RNA were measured using spectrophotometer (Merinton) and described by A260/A280. Using a reverse transcription kit (TaKaRa), reverse transcription experiment was performed for total RNA to synthesize first-strand cDNA. In addition, the synthesized cDNA was stored at -20°C.

Real-time quantitative PCR (qRT-PCR) analysis

The primers for qRT-PCR were quoted from the study of Yamauchi et al. [17]. Versican and ALP expression levels were evaluated using 100 ng/µl of cDNA template. The reaction systemcontained 10 µl SYBR Premix Ex Taq (2×, ABI), 1 µl cDNA, 1 µl forward primer (10 mM), 1 µl reverse primer (10 mM) and ddH₂O to reach a total volume of 20 µl. The reaction program was as follows: 50°C for 3 min, 95°C for 3 min, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s. After the results were obtained, a melting curve was created. The β-actin was used as reference gene, and all samples had three repeats.

Western blot analysis

Cell solution was drawn to prepare protein sample. The HHDPCs were washed with 2 ml precooled PBS and then digested by pancreatin. After being resuspended in 1 ml PBS, the HHDPCs were centrifuged at 600 g for 5 min at 4°C. Subsequently, the cells were dissolved by lysates (Sangon) containing PMSF (Sangon) and centrifuged at 12000 rpm for 10 min at 4°C. Using bicinchoninic acid (BCA), concentrations of the protein were determined. The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with a 5% stacking gel and a 10% separatinggel. Afterwards, the separated proteins were transferred to polyvinylidene fluoride membranes (Merck Millipore). The membranes were blocked with sealing solution (containing 5% non-fat milk) for 1 h at room temperature, and incubated with primary antibodies (anti-versican, 1:50 dilution, Boster and anti-ALP, 1:1000 dilution, ABSCI) for a night at 4°C. After being washed in 1×TBST for three times, the membranes were incubated by secondary antibody (1:10000 dilution, Jackson) for 1 h. Then, the membranes were washed in 1×TBST for three times again. Using ECL chemoluminescence kit (Merck Millipore), the membranes were developed in gel document (Bio-Rad).

Statistical analysis

After qRT-PCR being performed, the mRNA levels of ALP and versicanwere calculated bythe 2^-ΔΔCt method [18]. The data were analyzed by SPSS 13.0 (SPSS Inc) and exhibited as mean ± standard error of mean (SEM). A P < 0.05 was used as the cut-off criterion.

Results

The cell viability of HHDPCs treated by PRP and PPP

To evaluate the cell viability of HHDPCs treated by PRP and PPP, MTT assays were performed. Compared with the control group, the cell viability in the groups treated with PRP were significantly higher, especially for the HHDPCs (p4) in 24 h (Figure 1A) and 72 h groups (Figure 1E) (P < 0.05). Meanwhile, 10% PPP could significantly promote cell viability of the HHDPCs (p4) in 24 h (Figure 1A) and 72 h (Figure 1E) groups (P < 0.05). And 5% PPP could also significantly accelerate cell viability of the HHDPCs (p4) in 72 h group (Figure 1E) (P < 0.05). However, the effect of 10% PRP on HHDPC viability was less than that of 5% PRP.

Figure 1.

Cell viability investigated by MTT assays. A. The cell viability of the HHDPCs (p4) in 24 h group. B. The cell viability of the HHDPCs (p8) in 24 h group. C. The cell viability of the HHDPCs (p4) in 48 h group. D. The cell viability of the HHDPCs (p8) in 48 h group. E. The cell viability of the HHDPCs (p4) in 72 h group. F. The cell viability of the HHDPCs (p8) in 72 h group. The error bars stand for standard deviations from three samples. *P < 0.05 vs. the control group.

Analysis of ALP and versican expression

PRP could significantly increase the expression levels of ALP (Figure 2A) and versican (Figure 2B) in HHDPCs (p4) compared with the control group (P < 0.05). However, PRP and PPP had no significant effect on ALP and versican expression in HHDPCs (p8) and HHDPCs (p4 or p8), respectively.

Figure 2.

Expression of ALP and versican in HHDPCs detected by real-time quantitative PCR. A. The mRNA level of ALP in the HHDPCs (p4). B. The mRNA evel of versicanin the HHDPCs (p4). C. The mRNA level of ALP in the HHDPCs (p8). D. The mRNA level of versicanin the HHDPCs (p8). The error bars stand for standard deviations from three samples. *p ; 0.05 vs. the control group.

Western blot analysis

Compared with the control group, PRP could obviously increase the protein levels of ALP and versican in HHDPCs (p4) (Figure 3A). However, PRP had no remarkable influence on the protein levels of ALP and versican in HHDPCs (p8). Moreover, PPP had obvious impact on the ALP expression in HHDPCs (p4) (Figure 3A), but had no prominent effect on the versican expression in HHDPC cells (p4 or p8) and the ALP expression in HHDPCs (p8).

Figure 3.

Protein levels of ALP and versican in HHDPCs investigated using western blot. A. The protein level of ALP and versican in HHDPCs (p4). B. The protein level of ALP and versican in HHDPCs (p8).

Discussion

In this study, both PRP (5% and 10%) and PPP (5% and 10%) could significantly promote the cell viability of the HHDPCs (p4) in 72 h group. However, the effect of 10% PRP on HHDPC viability was less than that of 5% PRP. The 10% PRP did not provide a better environment for HHDPCs viability, indicating that 10% PRP might mainly contribute to HHDPC differentiation. The results of the three assays were different in HHDPCs (p4) and HHDPCs (p8), suggesting that the viability of HHDPCs (p8) was worse than that of HHDPCs (p4).

Versican, which belongs to the family of large aggregating chondroitin sulfate proteoglycan, functions in cell proliferation, differentiation, migration and adhesion [19,20]. For example, versican V1 isoform can promote cell proliferation, control cell cycle progression, and protect the cells from apoptosis [21]. Through functioning in cell adhesion, as well as matrix assembly and structure, versican is involved in hair follicle morphogenesis and cycling [22]. The expression of ALP may be associated with dermal papilla (DP)’s functions in hair formation and structural reconstruction of bulbar during hair follicle regeneration [23]. Results of qRT-PCR showed that PRP could significantly increase the mRNA levels of ALP and versican in HHDPCs (p4) compared with the control group. This was in accordance with the results of western blot, which showed obviously increased protein levels of ALP and versican in PRP-treated HHDPCs (p4). Thus, PRP hadan effect of promoting cell proliferation and differentiation on HHDPCs as indicated by the versican and ALP expression.

Platelet-released growth factors, including platelet-derived growth factor (PDGF), platelet-derived epidermal growth factor (PDEGF), transforming growth factor (TGF), insulin growth factor-1 (IGF-1), platelet-derived angiogenesis factor (PDAF) and platelet factor-4 (PF-4), can be advantageous for cell proliferation, cell differentiation, and even tissue regeneration [24,25]. Additionally, some other factors including interleukin-1 (IL-1), epithelial cell growth factor (ECGF) and basic fibroblast growth factor (bFGF) have been found in the a-granules of the platelets [26-28]. As an antilogoussource of growth factors, PRP might stimulate cell proliferation and differentiation of HHDPCs through the factors in it.

Previous study reports that PRP can help DPC proliferation, promote hair growth and be used as a promising therapeutic tool [29]. Also, dalteparin and protamine microparticles (D/P MPs) can carry growth factors in PRP effectively, and PRP-containing D/P MPs (PRP & D/P MPs) can better contribute to hair growth [30]. Through chamber assay, Miao et al. find that PRP can help hair formation and induce hair follicle reconstitution in vivo [13]. PRP is reported to be able topromote hair growth in patients with alopecia through DPC proliferation, as well as keratinocyte growth factor (KGF) and B-cell leukemia/lymphoma 2 (BCl-2) up-regulation [31]. These are in keeping with our results. The increase of HHDPC proliferation contributes to clinical applications of HHDPCs in hair follicle regeneration. Thus, our research provided an experimental evidence for the clinical application of PRP as a potential tool to promote HHDPC proliferation.

In conclusion, the results of our study indicated that PRP can stimulate HHDPCs viability. Besides, 5% PRP could significantly increase the ALP and versican expression in both mRNA and protein levels in HHDPCs (p4). However, this is an in vitro study, and the factors that play important roles in PRP-induced cell viability haven’t been identified. There are many growth factors in PRP; hence, the identification of the exact factors inducing proliferation will cost much time and effort.

Disclosure of conflict of interest

None.