original article enhanced proliferation and migration ... · original article enhanced...

Int J Clin Exp Pathol 2017;10(8):8804-8812www.ijcep.com /ISSN:1936-2625/IJCEP0042318

Original ArticleEnhanced proliferation and migration capability of epidermal stem cells by PRP and PDGF stimulation

Yanhong Wu1, Jufeng Fan2, Bin Zhang2, Jianbin Tang1, Qin Li1

1Laser Plastic and Aesthetic Center, PLA General Hospital of Guangzhou Region, Guangzhou, China; 2Department of Plastic and Reconstructive Surgery, Beijing Chao-Yang Hospital, Capital Medical University, Chaoyang District, Beijing, China

Received October 12, 2016; Accepted December 5, 2016; Epub August 1, 2017; Published August 15, 2017

Abstract: Adult epidermal stem cells renew the epithelial compartment of the skin throughout life and are the most accessible of all adult stem cells. Isolation and culture epidermal stem cells in vitro can both benefit stem cell therapy and tissue-engineering study. In this study, we successfully isolated and cultured epidermal stem cells in vitro, and demonstrated for the first time that PRP and PDGF could promote epidermal stem cell proliferation and migration, which meant PRP and PDGF addition during epidermal stem cell culture can be regarded as an optimized condition to improve the number of harvested cells in a short time. Compared with epidermal stem cells treated with K-SFM medium and platelet-poor plasma, the differences showed in cells treated with PRP and PDGF includ-ing: CCK-8 assay showed that cell proliferation was enhanced about twice; the cell cycle analysis showed 7%~12% less cells were arrested in S and G2 phases; western blot assay suggested that PCNA expression was 7-10 times higher; transwell assay also demonstrated that cell migration capability enhanced 8-11 times. These results all proved that the application of PRP and PDGF in epidermal stem cell culture may promote stem cell therapy and tissue-engineered skin study by providing high quantity of seeded cells.

Keywords: PRP, PDGF, epidermal stem cell, cell proliferation, cell migration

Introduction

The epidermis is the outermost layer of our skin that protects our bodies from the external environment thus facing permanent wearing. Benefit to the stem cells within skin, epidermis and its appendages contribute to epidermal repair and regeneration [1, 2]. Like other adult stem cells, epidermal stem cells are best de- fined by their capacity to self-renew and to generate large amounts of tissue for an extend-ed period of time or even a lifetime [3]. In addi-tion, epidermal stem cell is important because they not only play a central role in homeostasis and wound repair, but also represent a major target of tumor initiation and gene therapy, which made it a good seeded cell for tissue-engineering study.

Tissue-engineered skin has always been the hot spot of wound repair study [4-7]. How to acquire good initial cells is a key problem [5, 8]. In recent years, the development of stem cell

technique brings new hopes for tissue engi-neering study. Some researchers have been in attempt to in vitro isolate and culture epidermal stem cells to construct tissue-engineered skin [9]. However, different isolation and culture method of epidermal stem cells can result in the variety of cell quality, which block the deep study of tissue-engineered skin.

Recently, platelet-rich plasma (PRP) and plate-let-derived growth factor (PDGF) were intro-duced in tissue engineering as a source of large quantities of growth factors, and these materi-als have been applied as a novel strategy to enhance the properties of transplanted cells. PRP has been used clinically in humans since the 1970s for its wound-healing properties, which are attributed to its high levels of growth factors and secretory proteins [10]. The growth factors in PRP, including platelet-derived growth factor (PDGF), transforming growth fac-tor β (TGF-β), insulin-like growth factor I, epider-mal growth factor and endothelial cell growth

Proliferative role of PRP and PDGF in stem cells

8805 Int J Clin Exp Pathol 2017;10(8):8804-8812

factor, can promote the recruitment, prolifera-tion, and differentiation of cells involved in tis-sue regeneration [11, 12]. Preclinical studies using PRP and PDGF in combination with mes-enchymal stem cells (MSCs) have been con-ducted in the contexts of periodontal tissue engineering [13, 14], wound healing [15], ten-don repair [16], and bone regeneration [17]. These reports demonstrated that PRP and PDGF could increase the potential of the trans-planted cells used in stem cell therapies. There- fore, it is possible that PRP and PDGF can con-tribute to stem cell therapies. Although there are many studies showed that PRP and PDGF play an important role in cell proliferation and differentiation of mesenchymal stem cells from different sources [18-21], there are no report demonstrates the role of PRP and PDGF play on epidermal stem cells.

The present study primarily investigated human epidermal stem cells in vitro isolation and culture, and the effect of PRP and PDGF on epi-dermal stem cell proliferation and migration. And the results indicate that PRP and PDGF may accelerate the development of tissue-engi-neered skin study.

Methods and materials

Isolation of epidermal stem cells

Human skin samples were obtained from heal- thy adult after written consent, with approval of the ethics commission of PLA General Hospital of Guangzhou District and in accordance with the Declaration of Helsinki protocols. Freshly obtained human skin was washed with D-Hanks for at least twice, and then washed with bal-anced salt solution containing 200 U/mL peni-cillin and 200 U/mL streptomycin (Hyclone) for 30 min under aseptic condition. The skin was then cut into pieces of 0.5 cm×0.5 cm after disinfecting and removing the appendant tis-sues. Epidermis and dermis were separated by mechanical and enzymatic digestion as pre-viously described by Ponec et al. [22]. Briefly, the skin pieces were incubated with 0.5% dis-pase solution (GIBCO) overnight in 4°C and con-tinued the incubation for 30 min at 37°C the next day. The epidermal sheet was then sepa-rated from the dermis as a whole sheet. After removing the epidermis from the dermis, the tissue was washed three times with phosphate-buffered saline (PBS, Hyclone) at room temper-

ature. Afterwards, the pieces were incubated with 0.25% trypsin solution (Hyclone) for 15 min at 37°C with gentle agitation to prepare single cell suspensions. The enzymatic reaction was inactivated with Dulbecco’s modified eagle’s medium (DMEM, Hyclone) containing 20% fetal bovine serum (FBS, Hyclone) and fil-tered through a 70 um filter mesh (Millipore). The cell suspension was centrifuged at 200 g for 5 min. Afterwards, the cell pellet was gently resuspended in complete medium (DMEM con-taining 20% FBS) and plated onto conventional tissue culture flasks T75 (BD Falcon) coated with collagen IV (Sigma) at a density of 3×106/mL. Cells were put still in cell culture incubator for 15 min and then aspirate the floating cells while the attached cells were continued cultur-ing in complete medium for several days, and then changed the culture medium into Kerati- nocyte-SFM medium (K-SFM medium, GIBCO). When the cells come to confluence, digest the cells with 0.25% trypsin solution and passaged at 1:3.

Preparation of PRP

RPR collection was performed as previously described with minor modifications [23, 24]. The protocol was based on immediate use of blood collection, following consent and under the PLA General Hospital of Guangzhou District Ethics Committee approval. Briefly, peripheral blood from donors was collected into 50-mL centrifuge tube (Corning) filled with EDTA (Sig- ma) and centrifuged at 150 g for 5 min at 4°C. The buffy coat and the red blood cell layer remained intact, while the upper fraction was transferred to new tubes. The upper fraction was then centrifuged at 300 g for 20 min at 4°C and the platelet pellet after centrifugation was collected. The supernant was collected and centrifuged again at 4000 g for 20 min at 4°C, and the upper fraction (platelet-poor plas-ma) was collected. After the third centrifugation step, platelets pellet from the second centrifugation was resuspended with the plate-let-poor plasma, forming the inactivated PRP. The platelet activation was achieved by addi-tion of calcium and bovine thrombin as previ-ously described.

Cell culture and treatment

The isolated epidermal stem cells were firstly cultured in complete medium for about one



week, and then cultured K-SFM medium and changed the medium every two days. Group I was cultured with K-SFM medium as blank con-trol group, Group II was treated with 100 ng/mL PDGF (Sigma), Group III was treated with 10% PRP, Group IV was treated with 10% platelet-poor plasma as the negative control.

Preparation of cells on coverslip and cell im-munohistochemical analysis

500 µL of cell culture media containing approxi-mately 5000 cells were plated to the wells of a cell culture plate containing gelatin-coated cov-erslips. When cells have reached the desired density, remove the culture media from each well and wash twice with PBS. Cells were then fixed in 4% cold paraformaldehyde for 30 min at room temperature. After washing with PBS, cells were permeablised with 0.2% Triton X-100 in PBS for 5 min and then blocked with 10% goat serum for 30 min. The primary antibodies used for immunodetection including anti-Cyto-keratin19 (Abcam, diluted 1:100) and anti-β1 integrin (Abcam, diluted 1:500). The primary antibodies were diluted in PBS containing 0.5% BSA and aliquoted onto parafilm with sufficient space between coverslip. The coverslips were placed down onto the antibody drop and incu-bated overnight at 4°C. After washing with PBS for three times (at 5 min each) the next day, the coverslips were incubated with the HRP marked second antibody for 1 hour at room temperature avoiding light. The color reaction was realized by the incubation with DBA and hematoxylin, and finally fixed the coverslip with neutral resin.

Cell proliferation assay

Cell proliferation was determined using Cell Counting Kit-8 (CCK-8, Beyotime Biotechno- logy). Cells with different treatment were seed-ed in 96-well plate at a density of 1×104 cells/well. After attachment, CCK-8 assay was per-formed at 0 h, 24 h, 48 h and 72 h separately. Cells with different treatment were incu-bated with 10% CCK8 and the absorbance was recorded at 450 nm with a microplate read- er (Thermo Fisher Scientific) after four hours incubation.

Analysis of cell migration by transwell assay

Cell migration assay was performed in a trans-well chamber (Corning). Cells with different

treatment were collected separately and resus-pended with serum-free medium. 0.5 mL of serum-free medium was placed in the upper chamber; complete medium was added to the bottom chambers. Equal numbers (1×105) of cells were plated in the upper chambers of the quadruplicate wells and incubated at 37°C for 12-48 h. For quantification of migrated cells, the upper surface of the upper chamber was fixed with 4% paraformaldehyde (PFA) and stained with crystal violet for 10 min, and the cells remaining on the upper surface were scraped off with a cotton swab. After flushing the excess dye with PBS, the remaining dye was eluted with 33% acetic acid. The optical density (OD) was read on an automatic photometer at a wavelength of 570 nm. The results of three independent experiments were averaged. Im- age Processing and Analysis in Java was used to measure the trans-well assay.

Analysis of cell cycle by flow cytometry

The effect of PRP and PDGF on cell cycle was detected on actively growing cells: cells were seeded in 6 cm dishes for 24 h and treated the cells with different conditions (K-SFM medi-um, platelet-poor plasma, PRP and PDGF) for 48 h. Then 1×106 cells from different treatment were collected separately and fixed using cold 70% ethanol and stored overnight at 4°C. The fixed cells were washed with PBS and stained with Propidium Iodide (PI) for analysis. Cell cycle analysis was performed using flow cytom-eter (BD Calibur).

Western blot

Western blotting was conducted as previously described [25]. In brief, cell protein was prom- ptly homogenized in a homogenization buffer containing 1 M Tris-HCl, pH 7.5, 1% Triton X-100, 1% Nonidet p-40 (NP-40), 10% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 0.5 M EDTA, 10 μg/mL leupeptin, 10 μg/mL apro-tinin and 1 mM PMSF. The solution was then removed to an EP tube and centrifuged at 10,000 g for 15 min to collect the supernatant liquid. Total protein concentrations were detected by using the Bio-Rad protein assay (Bio-Rad). The supernatant was mixed with loading buffer at a ratio of 4:1 and boiled for 5 min before loading into the gel. An equivalent amount of protein from each sample was electro-



phoresed by using sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE) and then transferred to a PVDF membrane (Milli- pore). After blocking with TBS containing 5% nonfat milk and 0.1% Tween-20 for 1 h at room temperature, the membrane was incubated overnight at 4°C with a primary polyclonal anti-body separately as following: anti-PCNA (Cell Signaling, diluted in 1:400) and anti-glyceralde-hyde-3-phosphate dehydrogenase (anti-GAP-DH, Sigma, diluted in 1:1000). After washing with TBS containing 0.1% Tween 20 three times (at 5 min each), the membrane was then incu-bated with appropriate secondary antibody for another 2 h at room temperature. The mem-brane was then washed with distilled water three times (at 2 min each). Immunoreactive bands were visualized using the enhanced che-miluminescence detection reagents (Immobil- on, Millipore Co.). Adobe Photoshop CS5 (Adobe Systems) was used in the computer-assisted image analysis for band density evaluation. The experiments were carried out on at least three separate occasions.

Epidermal stem cells isolated from human skin were firstly cultured in complete medium. After removing non-adherent cells, the adher-ent cells were cultured in the complete media for about 1 week. Under an inverted micro-scope, it was found that after 24 hours of cul-ture, the attached epidermal stem cells showed polygonal or round shape, the cell volume was small (Figure 1A) and grew slowly. Cells grew gradually faster and colonies formed after seven days’ culture in complete medium (Figure 1B), although each colony contained only sev-eral cells. In the following days, cells were cul-tured in K-SFM medium and after continued 7 days of culture, round, oval or irregular big colonies formed (Figure 1C). When the colonies became bigger, colonies were digested with trypsin and passaged at 1:3.

Epidermal stem cells presented as round and with different sizes. Immunohistochemistry was performed on the isolated cells growing on coverslip to investigate the expression of cyto-keratin 19 (CK19) and β1-integrin. The results

Figure 1. The morphology of the isolated epidermal stem cells. After 24 hours of culture, cells attached to the flask and showed polygonal and round shape (A); colonies formed after 7 days’ culture in complete medium (B) and the colonies became round, oval or irregular big after the following 7 days’ culture in K-SFM medium (C).

Figure 2. Immunohistochemical analysis of the isolated epidermal stem cells. The isolated cells positively expressed CK19 and the membrane of the cell showed brown-stained (A); and β1 integrin expression of the iso-lated cells was also positive and the cytoplasm was brown-stained (B). The nucleus of the isolated cells was blue-stained by hematoxylin.

Statistical analysis

All measurements in this study were performed blindly. Results were expressed as mean ± SE- M. Student’s t-tests were used to evaluate mean differences between the control and the treated group. P value <0.05 was considered statistically significant.

Results

Isolation and characterization of epidermal stem cells



demonstrated that CK19 expression was posi-tive (Figure 2A). Cell membrane was brown-stained and toroidal ring-like nucleus was blue-stained, while cytoplasm was not stained. β1-integrin expression was also positive (Figure 2B). Cytoplasm was brown-stained and nucleus was blue-stained.

PRP and PDGF promote proliferation of epider-mal stem cells

The effect of PRP and PDGF on epidermal stem cells was evaluated by CCK-8 assay. Cells were treated with platelet-poor plasma, PRP, PDGF and K-SFM medium alone for the indicated time, and then incubated with CCK-8. The results showed that epidermal stem cells were sensitive to PRP and PDGF indicating promo-tion effect of PRP and PDGF in epidermal stem

to exclude the interference on PI detection, thus verified the method feasibility. Cell cycle analysis of the cells with different treatment was presented in Figure 4A, which demonstrat-ed that for all groups, epidermal stem cells in G0/G1 phase accounted for no more than 65% of all cells (Figure 4B). For cells treated with K-SFM medium alone and platelet-poor plas-ma, there was not significant elevation of cell cycle progression from G0/G1 to S and G2 phases which showed 64.98% and 64.96% G0/G1 phase arrest, respectively, indicating that platelet-poor plasma have no influence on cells during G0/G1 stage. For cells treated with PRP and PDGF, cell cycle exhibited apparent differences compared to the cells treated with K-SFM medium alone or platelet-poor plasma. Cells in G0/G1 phase got great promotion to S and G2 phases showing 57.38% and 52.96%

Figure 3. PRP and PDGF promote proliferation of the isolated epidermal stem cells. CCK-8 assay showed that cell number increased significantly after 24 hours of PRP or PDGF addition (A), and the PRP and PDGF works by enhancing cell proliferation (B) and reducing the inhibitory effect (C). *P<0.05, **P<0.01.

cells (Figure 3A). Moreover, cells treated with PDGF grow fast-er than that of PRP. Analyzing the cell proliferation rate according to the cell proliferation rate formula and cell inhibition rate formula, we found that cells treated with PRP and PDGF got higher proliferation rate (Figure 3B) and lower inhibition rate than platelet-poor plasma and K-SFM treated cells, while the inhibition rate were all below zero (Figure 3C). The results indicating that PRP and PDGF have no cytotoxicity to the epi-dermal stem cells and can pro-mote cell proliferation.

PRP and PDGF can shorten the cell cycle of epidermal stem cells

To reveal the influence of PRP and PDGF on epidermal stem cells, cell cycle of epidermal stem cells with different treat-ment were analyzed by flow cytometry using propidium iodide (PI) staining method. Cells cul-tured with K-SFM medium alone were treated with PI and set as control group to disclose the premier cell cycle pattern. Another epidermal stem cells treated with platelet-poor plas-ma were set as negative group



G0/G1 arrest with PRP and PDGF treatment, which were lower than that of K-SFM medium and platelet-poor plasma treatment and meant PRP and PDGF can promote cells in G0/G1 to go into S phase and G2 phase faster. Otherwise, the effect of PDGF alone was stronger than that of PRP (Figure 4B).

PRP and PDGF promote migration of epider-mal stem cells

We conducted trans-well assays to evaluate the cell migration capabilities of epidermal stem cells (Figure 5A). The results showed sig-nificantly higher migration potential of epider-mal stem cells treated with PRP and PDGF com-

pared with that of cultured in K-SFM medium alone or in platelet-poor plasma (Figure 5B). Moreover, cells treated with PDGF showed high-er migration ability than that of treated with PRP (Figure 5B).

PRP and PDGF can improve PCNA expression level of epidermal stem cells

To further determine the proliferation profile of epidermal stem cells with different treat-ment, western blots prepared from each group were probed for PCNA (Figure 6A). As shown in Figure 6, cells treated with PRP and PDGF showed enhanced expression of PCNA when compared with K-SFM or platelet-poor plasma

Figure 4. PVGF and PRP can promote cells enter S phase and G2 phase.



treatment (Figure 6B). In addi-tion, more PCNA protein can be detected after treated with PDGF compared with that of PRP (Figure 6B). The results further indicated that PRP and PDGF could promote cell prolif-eration and PDGF has stronger effect.

Discussion

In this study, we aimed to exam-ine the features of the epider-mal stem cells we isolated and the ability of the PRP and PDGF to enhancing proliferation and migration. We produced PRP and isolated epidermal stem cells according to the previous studies [22-25]. CCK-8 assay, cell cycle analysis, western blot analysis and transwell assay demonstrated that PRP and PDGF contributed to epidermal stem cell proliferation and migration. PRP and PDGF thus improved the viability of epider-mal stem cells and may have applications as promotive fac-tors in the epidermal stem cell culture in vitro.

Recent in vivo studies report that the systemic concentra-

tion of growth factors, insulin-like growth factor (IGF-1), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), PD- GF, epidermal growth factor (EGF), and mRNA expression of pro-inflammatory biomarkers IL-1b, IL-6, IL-8, and inducible nitric oxide syn-thetase (iNOS) change significantly following PRP application [26-28]. Furthermore, it has been reported that the release of growth fac-tors from platelet granules is directly correlated with PRP platelet concentration [24, 29]. The alteration of these biomarkers indicates that PRP appears to trigger multiple biological path-ways and also delivers multiple biological fac-tors directly to the injection site in vivo.

Activated PRP contains PDGF, transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), and oth-ers [11, 23, 30, 31]. Among all the growth fac-tors, PDGF is one of the typical grow factor with

Figure 5. Detection of cell migration ability of the isolated epidermal stem cells by transwell assay. Less cells cultured in K-SFM medium and treated with platelet-poor plasma migrated to the lower surface of the upper clam-ber than that treated with PRP (A). The result was concluded in the histo-gram graph (B).

Figure 6. Western blot analysis of PCNA protein lev-els in epidermal stem cells with different treatment. PCNA levels were detected (A) in cells treated with K-SFM medium alone, platelet-free plasma, PRP and PDGF. The levels of GAPDH are shown to dem-onstrate the amount of protein loaded in each lane. And the result was summarized in the histogram graph (B). *P<0.05, **P<0.01.



the highest amounts and previous study showed that PRP was beneficial for skin wound repair [32]. Considering our results, the mecha-nism of PRP participate in skin wound repair may be that PDGF act as positive stimuli to the epidermal stem cells. Due to the exists of other growth factors in PRP, the interaction between different factors may influence the effect of PDGF and resulted in the lower promo-tion of PRP to cell proliferation and migration than that of PDGF alone.

In conclusion, the present study primarily dem-onstrated that the epidermal stem cells could be purified and screened by means of their rapid adherence to extracellular matrix. More- over, we successfully isolated epidermal stem cells from adult skin at very high purity and characterized the isolated cells by perform- ing immunohistochemical assay for epidermal stem cell markers CD19 and β1-integrin which were both positively expressed in the isolated cells, proving that the isolated cells are epider-mal stem cells. On the other hand, we tested the effect of PRP and PDGF on epidermal stem cells at first time by using CCK-8 assay, cell cycle analysis, trans-well assay and western blot for PCNA. All the results showed that PRP and PDGF could promote epidermal stem cell proliferation and cell migration capability. In addition, PDGF had higher ability to promote cell proliferation than PRP while PRP had higher capability of promoting cell migration and PCNA expression. The addition of PDGF and PRP can be considered according to the experimental requirement during epidermal stem cell culture. Benefit to the promotion effect of PRP and PDGF, they can be used as an optimized condi-tion for epidermal stem cell culture, which will promote the development of tissue-engineered skin by providing enough seeded cells.

Acknowledgements

This work was financially supported by the Research on Early Intervention of Critical Trau- ma Surface and Its Supporting Technology (CGZ14J002).

Disclosure of conflict of interest

None.

Address correspondence to: Qin Li, Laser Plastic and Aesthetic Center, PLA General Hospital of

Guangzhou Region, 111 Liuhua Road, Guangzhou 510010, China. E-mail: [email protected]

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