August 2011
Volume 52, Issue 9
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Cornea  |   August 2011
Plasma Rich in Growth Factors (PRGF-Endoret) Stimulates Proliferation and Migration of Primary Keratocytes and Conjunctival Fibroblasts and Inhibits and Reverts TGF-β1–Induced Myodifferentiation
Author Affiliations & Notes
  • Eduardo Anitua
    From the Biotechnology Institute (BTI), Vitoria, Spain;
  • Mikel Sanchez
    Unidad de Cirugía Artroscópica “Mikel Sánchez”, Vitoria, Spain; and
  • Jesus Merayo-Lloves
    Fundación de Investigación Oftalmológica, Instituto Oftalmológico Fernández-Vega, Oviedo, Spain.
  • Maria De la Fuente
    From the Biotechnology Institute (BTI), Vitoria, Spain;
  • Francisco Muruzabal
    From the Biotechnology Institute (BTI), Vitoria, Spain;
  • Gorka Orive
    From the Biotechnology Institute (BTI), Vitoria, Spain;
  • *Each of the following is a corresponding author: Eduardo Anitua, Instituto Eduardo Anitua, c/ Jose Maria Cagigal 19, Vitoria, Spain; eduardoanitua@eduardoanitua.com. Gorka Orive, Instituto Eduardo Anitua, c/ Jose Maria Cagigal 19, Vitoria, Spain; gorka.orive@ehu.es
Investigative Ophthalmology & Visual Science August 2011, Vol.52, 6066-6073. doi:10.1167/iovs.11-7302
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      Eduardo Anitua, Mikel Sanchez, Jesus Merayo-Lloves, Maria De la Fuente, Francisco Muruzabal, Gorka Orive; Plasma Rich in Growth Factors (PRGF-Endoret) Stimulates Proliferation and Migration of Primary Keratocytes and Conjunctival Fibroblasts and Inhibits and Reverts TGF-β1–Induced Myodifferentiation. Invest. Ophthalmol. Vis. Sci. 2011;52(9):6066-6073. doi: 10.1167/iovs.11-7302.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: Plasma rich in growth factors (PRGF-Endoret) technology is an autologous platelet-enriched plasma obtained from patient's own blood, which after activation with calcium chloride allows the release of a pool of biologically active proteins that influence and promote a range of biological processes including cell recruitment, and growth and differentiation. Because ocular surface wound healing is mediated by different growth factors, we decided to explore the potential of PRGF-Endoret technology in stimulating the biological processes related with fibroblast-induced tissue repair. Furthermore, the anti-fibrotic properties of this technology were also studied.

Methods.: Blood from healthy donors was collected, centrifuged and, whole plasma column (WP) and the plasma fraction with the highest platelet concentration (F3) were drawn off, avoiding the buffy coat. Primary human cells including keratocytes and conjunctival fibroblasts were used to perform the “in vitro” investigations. The potential of PRGF-Endoret in promoting wound healing was evaluated by means of a proliferation and migration assays. Fibroblast cells were induced to myofibroblast differentiation after the treatment with 2.5 ng/mL of TGF-β1. The capability of WP and F3 to prevent and inhibit TGF-β1–induced differentiation was evaluated.

Results.: Results show that this autologous approach significantly enhances proliferation and migration of both keratocytes and conjunctival fibroblasts. In addition, plasma rich in growth factors prevents and inhibits TGF-β1–induced myofibroblast differentiation. No differences were found between WP and F3 plasma fractions.

Conclusions.: These results suggest that PRGF-Endoret could reduce scarring while stimulating wound healing in ocular surface. F3 or whole plasma column show similar biological effects in keratocytes and conjunctival fibroblast cells.

Two tissues compose the ocular surface: the cornea and the conjunctiva. Both provide important functions to the eye including ocular protection, lubrication, and refractive power. After an injury, the main layers affected in both tissues are the stratified epithelium, the basement membrane, and the stroma. The stroma is one of the most important layers involved in wound healing, and it is composed mainly of fibroblasts. Keratocytes are the specialized corneal fibroblasts characterized by their low activity (quiescent) and their distribution through the stroma. Some of their main functions include maintaining the corneal transparency and producing stromal components such as collagen fibers and extracellular matrix. It has been reported that to effectively repair a damaged area after an injury, the fibroblasts adjacent to the injury need to proliferate and migrate to repopulate the area. 1,2 These processes are in part mediated by different growth factors such as epithelial growth factor (EGF), fibroblast growth factor (FGF), and transforming growth factor β1 (TGF-β1), 3 6 among others. 
In some types of wounds these fibroblasts may develop actin contractile filaments, being differentiated into myofibroblasts. 7,8 During injury repair, myofibroblasts are responsible for wound contraction and extracellular matrix (ECM) deposition and organization. TGF-β has been identified as one of the main inductors of fibroblast differentiation into myofibroblasts. 9,10 However, the persistence of myofibroblastic cells after wound healing has been identified as the primary biological episode responsible for the development of scarring tissue. 11,12 The presence of fibrotic tissue at the anterior surface of the eye after an injury or a surgery may induce the opacification of the cornea (corneal haze), 13,14 or may lead to surgical failure. 15,16  
Different approaches to regenerate the ocular surface injury 17,18 and to treat the scar formation have been attempted. 19 21 One interesting alternative is to evaluate the potential of autologous plasma and platelet-derived growth factors in stimulating fibroblast-modified wound healing. The technology of plasma rich in growth factors (PRGF-Endoret, trademarks for Europe and USA, respectively) consists of the elaboration and use of a platelet-enriched plasma obtained from patient's own blood, which after activation with calcium chloride allows the in situ formation of a biodegradable fibrin scaffold and the release of a pool of biologically-active proteins that influence and promote a range of biological process including cell recruitment, growth, and differentiation. 22,23 Interestingly, some of the proteins secreted from α-granules of platelets, including EGF, platelet-derived growth factor (PDGF), and nerve growth factor (NGF) are necessary to promote wound repair and to maintain well-preserved ocular surface. 
PRGF-Endoret technology has provided significant clinical advances in terms of wound healing and tissue regeneration in dentistry and oral implantology, 24,25 orthopedics, sport medicine, 26 and ulcer treatment, 27 among others. In all these situations, small volumes of plasma rich in growth factors and reduced number of doses are needed to achieve therapeutic efficacy. According to the technique, the fraction with the highest platelet concentration (also known as fraction 3) should be used to promote tissue regeneration as this plasma volume contains the higher amount of proteins. However, this may hamper its use as an autologous eye drop, due to the elevated number of doses necessary per day and the long period of treatment required to complete healing in several pathologies. 
Assuming that function of fibroblasts is critical during ocular surface healing, the purpose of the present study is to assess the potential of PRGF-Endoret technology as an innovative approach for enhancing ocular repair and regeneration. To address these issues, the effects of fraction 3 (F3) or whole plasma (WP) column (with lower amount of proteins than F3) obtained from PRGF-Endoret technology were assessed over primary keratocytes and conjunctival fibroblast proliferation and migration. In addition, the potential of WP and F3 to inhibit and revert TGF-β1–stimulated myodifferentiation was also evaluated. 
Materials and Methods
Cells
Primary human cells including keratocytes (HK) and conjunctival fibroblasts (HConF; ScienCell Research Laboratories, San Diego, CA), were cultured according to manufacturer's instructions. Briefly, cells were cultured until confluence in fibroblast medium supplemented with fibroblast growth supplement (Complete FM; ScienCell Research Laboratories) and then were detached with animal origin-free trypsin-like enzyme (TrypLE Select, Gibco-Invitrogen, Grand Island, NY). Cell viability was assessed by trypan blue dye exclusion. Passage 3 to 6 cells were used in all experiments. 
Immunolabeling of Cells
The fibroblast-like morphology of cells and the absence of dedifferentiation were confirmed by phase-contrast microscopy and immunolabeling for collagen type I (Chemicon-Millipore, Billerica, MA), and fibronectin and vimentin (Sigma-Aldrich, St. Louis, MO). The cells were also tested against typical endothelial cells and hematopoietic progenitor cell markers: CD105 and CD34 (BD Biosciences, San Jose, CA) and against α-Smooth muscle actin (Sigma-Aldrich) to check the spontaneous differentiation to myofibroblasts in culture. 
Briefly, 9500 cells per well were plated on a 24-well plate with poly-l-lysine-coated glass coverslips (BD BioCoat, BD Biosciences). Cells were fixed for 10 minutes in 4% formaldehyde for CD34, CD105, and type I collagen antigens, in methanol:acetic acid (3:1) for vimentin antigen and in methanol precooled at −20°C for fibronectin and α-smooth muscle actin (α-SMA). Cells for type I collagen staining were permeabilized with 1% Triton X-100, in phosphate-buffered saline (PBS) for 10 minutes. After that, cells were blocked with fetal bovine serum (FBS) (10% in PBS) for 30 minutes, and incubated for 1 hour with the primary antibodies in the dilutions: 1:20 for type I collagen, 1:30 for CD34 and CD105; 1:50 for vimentin, and 1:800 for fibronectin and α-SMA. Next, cells were incubated with their appropriate secondary antibodies, goat anti-mouse IgG conjugated with Alexa Fluor 488 or goat anti-rabbit IgG conjugated with Alexa Fluor 594 (both from Molecular Probes-Invitrogen, Grand Island, NY). Finally, cell nuclei were stained with Hoechst 33342 (Molecular Probes-Invitrogen), mounted, and visualized under a fluorescence microscope (Leica DM IRB, Leica Microsystems, Wetzlar, Germany). 
PRGF-Endoret Preparations
Blood from one healthy young male donor was collected after informed consent into 9-mL tubes with 3.8% (wt/vol) sodium citrate. The study was performed following the principles of the Declaration of Helsinki. Samples were centrifuged at 580g for 8 minutes at room temperature in a PRGF-Endoret system centrifuge (BTI Biotechnology Institute, S.L., Miñano, Álava, Spain). Half of the tubes were used to separate the whole plasma column (WP) over the buffy coat and the other half to take the immediately upper milliliter over the buffy coat called Fraction 3 (F3)—the platelet-enriched fraction (see Fig. 1). In both cases, care was taken to avoid the buffy coat containing the leukocytes. Platelet and leukocyte counts were performed with a hematology analyzer (Micros 60; Horiba ABX, Montpelier, France). Both preparations were incubated with PRGF-Endoret activator (BTI Biotechnology Institute) at 37°C in glass tubes for 1 hour. The released supernatants were collected by aspiration after centrifugation at 1000g for 20 minutes at 4°C. Finally, plasma obtained from WP and F3 was aliquoted and stored at −80°C until use. Growth factors (TGF-β1, PDGF-AB, vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), EGF, insulin-like growth factor I (IGF-1), and Thrombospondin 1 (TSP-1)) were measured in the supernatants using commercially available colorimetric sandwich enzyme-linked immunosorbent assay (ELISA) kits (Quantikine; R&D Systems, Minneapolis, MN; Table 1). 
Figure 1.
 
Scheme of the different plasma fractions obtained with the PRGF-Endoret technology. In all the different plasma preparations, care was taken to avoid the buffy coat containing leukocytes. WP, whole plasma obtained with PRGF-Endoret System. F1, fraction 1; F2, fraction 2; F3, fraction 3.
Figure 1.
 
Scheme of the different plasma fractions obtained with the PRGF-Endoret technology. In all the different plasma preparations, care was taken to avoid the buffy coat containing leukocytes. WP, whole plasma obtained with PRGF-Endoret System. F1, fraction 1; F2, fraction 2; F3, fraction 3.
Table 1.
 
Platelet and Leukocyte Count and Concentrations of Several Growth Factors in the Two Different Plasma Preparations (WP and F3) of the Blood Donor
Table 1.
 
Platelet and Leukocyte Count and Concentrations of Several Growth Factors in the Two Different Plasma Preparations (WP and F3) of the Blood Donor
Plasma Preparation Leukocyte Count (×106/mL) Platelet Count (×106/mL) Growth Factor Levels
TGF-β1 (ng/mL) PDGF-AB (ng/mL) IGF-I (ng/mL) VEGF (pg/mL) HGF (pg/mL) EGF (pg/mL) TSP-1 (μg/mL)
WP <0.2 481 63 19 83 568 400 508 29
F3 <0.3 663 81 30 86 791 491 779 50
Proliferation Assay
Keratocytes and conjunctival fibroblasts were seeded at a density of 10,000 cells per cm2 on 96-well optical bottom black plates and maintained with serum-free medium for 48 hours. Then, culture medium was replaced by serum-free medium supplemented with either the culture medium alone (FM) with 0.1% FBS as a control of nonstimulation (NS); 20% (vol/vol) WP; or 20% (vol/vol) F3. The study period was 48 hours. Density of cells in culture was estimated (CYQUANT Cell Proliferation Assay; Invitrogen, Carlsbad, CA). Briefly, medium was removed and wells were washed carefully with PBS. Then microplate was freezed at –80°C for efficient cell lysis in the assay. After thawing the plates at room temperature, samples were incubated with RNase A (1.35 Kunitz Units [KU]/mL) diluted in cell lysis buffer for 1 hour at room temperature. Then 2× dye/cell lysis buffer (CyQUANT GR; Invitrogen) was added to each sample well, mixed gently, and incubated for 5 minutes at room temperature protected from light. Sample fluorescence was measured using a fluorescence microplate reader (Twinkle LB 970, Berthold Technologies, Bad Wildbad, Germany). A DNA standard curve ranging from 7.8 to 1000 ng/mL was included in all fluorescence quantifications. As an index of cell number, calibration curves ranging from 2500 to 90,000 cells per cm2 were established (CyQUANT assay; Invitrogen). 
Migration Assay in Response to WP and F3
To quantify the migratory potential of conjunctival fibroblasts and keratocytes, they were plated in culture inserts (Ibidi GmbH, Martinsried, Germany) placed on a 24-well plate at high density and were grown with fibroblast growth supplement (Complete FM; ScienCell Research Laboratories) until confluence. After carefully remove the inserts, two separated cell monolayers leaving a cell-free gap of approximately 500 μm thickness were created. The cells were washed with PBS and incubated with the same treatments as in the proliferation assay (0.1% FBS, 20% WP, or 20% F3) in quintuplicate for 24 hours. After this period, the different culture mediums were removed and cells were incubated with 1/500 Hoechst 33,342 in PBS for 10 minutes. To quantify the number of migratory cells, phase contrast images of the central part of the septum before treatment and phase contrast and fluorescence photographs after the treatment time were captured with a digital camera coupled to an inverted microscope (Leica DFC300 FX and Leica DM IRB, Leica Microsystems). The gap area and the migratory cells found in this gap after the 24 hours of treatment were measured using ImageJ software (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD; available at http://rsb.info.nih.gov/ij/index.html). The results were expressed as number of cell migrated per mm2 of area. 
Myofibroblast Differentiation
Differentiation of conjunctival fibroblasts and keratocytes to myofibroblasts was induced by 2.5 ng/mL of TGF-β1 (Chemicon-Millipore) for 72 hours. Passage 4 cells were plated at a density of 5000 cells per cm2 in 48-well tissue culture plates and maintained with serum-free culture medium for 48 hours. After this period, the medium was replaced by FM and keratocytes and conjunctival fibroblasts were stimulated with either 2.5 ng/mL TGF-β1, 2.5 ng/mL TGF-β1 plus 20% (vol/vol) WP, or 2.5 ng/mL TGF-β1 plus 20% (vol/vol) F3 supernatant for 72 hours. In the following experiments, cells were initially pretreated with 2.5 ng/mL TGF-β1 for 3 days. After this pretreatment, culture medium was replaced by FM and supplemented with either one of three treatments: 2.5 ng/mL TGF-β1, 2.5 ng/mL TGF-β1 plus 20% (vol/vol) WP supernatant, or 2.5 ng/mL TGF-β1 plus 20% (vol/vol) F3 supernatant for 3 days. In both protocols, a supplement of 0.1% FBS was added to the 2.5 ng/mL TGF-β1 treatment to maintain cell viability in the control group. Experiments were performed in quintuplicate. 
After incubation time, medium was removed and cells were fixed for 10 minutes in methanol. Cells then were blocked with FBS (10% in PBS) for 30 minutes, and incubated for 1 hour with mouse anti-α-SMA antibody at 1:800, followed by incubation with goat anti-mouse IgG conjugated with Alexa Fluor 488 at 1:100 for 1 hour. Finally, cell nuclei were counterstained with Hoechst 33342 mounted using an anti-fade solution (SouthernBiotech, Birmingham, AL) and visualized under a fluorescence microscope (Leica DM IRB, Leica Microsystems). Control isotype was performed by substituting the primary antibodies with 10% of FBS diluted in PBS. 
For myofibroblast cell counting, two random 10× microscopic fields were photographed on each well. The digitalized images were analyzed (Image J software). Hoechst (+) cells were counted to obtain the total cell number. Hoechst (+) and α-SMA (+) cells were counted as myofibroblasts. Cells showing any kind of greenish staining were considered α-SMA–positive. Expression of α-SMA was also evaluated on cells treated with 20% (vol/vol) WP or F3 to check the myofibroblast differentiation. 
Inhibition of Myofibroblastic Differentiation
Fibroblasts were seeded at a density of 5000 cell per cm2 in a 48-well plate and were pretreated with 2.5 ng/mL of TGF-β1 plus 0.1% FBS as differentiation medium. After 72 hours, one part of the cells were fixed and stained for α-SMA and Hoechst 33342 as a positive control of differentiation. The remaining cells were then treated with FM plus 0.1% (vol/vol) FBS plus 2.5 ng/mL TGF-β1, 20% (vol/vol) plasma rich in growth factors plus 2.5 ng/mL TGF-β1, or 20% (vol/vol) F3 plus 2.5 ng/mL TGF-β1 for 3 days. All samples were performed in quintuplicate. The immunolabeling for α-SMA and Hoechst and cell counting were performed as previously described. 
Statistical Analysis
Means and their respective 95% confidence intervals were calculated for each of the treatments, proceeding to review the potential differences between treatments for each experimental process (proliferation, migration, and protective and reversible effect of WP and F3). Differences between treatments were considered to be significant in cases where the boundaries of the respective 95% did not overlap. 
Results
The human primary conjunctival fibroblasts (HConF) and keratocytes (HK) showed the typical spindle-shaped aspect in culture and did not spontaneously differentiate into myofibroblasts as confirmed by the absence of α-SMA expression (data not shown). Cells were positive for all the fibroblast markers (Collagen Type I, vimentin, and fibronectin) and negative for markers of hematopoietic and endothelial cells (data not shown). 
Platelet enrichment of the PRGF-Endoret preparations were 2.6-fold for WP (481 × 106 platelets/mL) and 3.6-fold for F3 (663 × 106 platelets/mL) over the baseline concentration in whole blood. None of the preparations contained detectable levels of leukocytes. Table 1 shows platelet and leukocyte concentration for each sample (WP and F3) and the levels of some of the most important growth factors. 
Cell Proliferation
Proliferation of conjunctival fibroblasts and keratocytes significantly increased after treatment with both PRGF-Endoret preparations (20% WP or 20% F3) as shown in Figure 2. In fact, conjunctival fibroblasts proliferated 3.1-fold and 3.4-fold after treatment with WP and F3 respectively. On the other hand, keratocytes showed a significant increase over basal conditions of 2.8-fold with WP and 2.7-fold with F3. No significant differences were found between the autologous treatments. 
Figure 2.
 
(A) Proliferation of HConF cells after culturing with 0.1% FBS as a control of nonstimulation (N.S.), 20% WP, or 20% F3 for 2 days. (B) Proliferation of keratocyte (HK) cells after culturing with 0.1% FBS (N.S.), 20% WP, or 20% F3 for 2 days. WP and F3 significantly increased proliferation of both cells compared with nonstimulatory conditions; *, 95% confidence interval. No statistical differences were found between WP and F3.
Figure 2.
 
(A) Proliferation of HConF cells after culturing with 0.1% FBS as a control of nonstimulation (N.S.), 20% WP, or 20% F3 for 2 days. (B) Proliferation of keratocyte (HK) cells after culturing with 0.1% FBS (N.S.), 20% WP, or 20% F3 for 2 days. WP and F3 significantly increased proliferation of both cells compared with nonstimulatory conditions; *, 95% confidence interval. No statistical differences were found between WP and F3.
Migration Assay
WP and F3 significantly stimulated the migratory capacity of both HConF and HK. In particular, migration of HConF increased 1.8-fold and 1.7-fold over the nonstimulatory situation for WP and F3 respectively (Fig. 3A) whereas migration of keratocytes increased 2.3-fold for WP and 1.8-fold for F3 (Fig. 3B). The number of migrating cells was significantly higher with plasma preparations than with nonstimulation cells. No statistical differences were found between plasma rich in growth factors treatments. Figure 3C shows phase contrast images of HConF and HK cells after a 24-hour period of migration and highlights the potent stimulatory effect of WP and F3 over the treated cells. 
Figure 3.
 
(A) Migration rate of HConF cells after culturing with 0.1% FBS (N.S.), 20% WP, or 20% F3 for 24 hours. (B) Migration rate of keratocyte (HK) cells after culturing with 0.1% FBS (N.S.), 20% WP, or 20% F3 for 24 hours. WP and F3 significantly increased migration of both cells compared with nonstimulatory conditions; *, 95% confidence interval. No statistical differences were found between WP and F3. (C) Phase contrast photomicrographs illustrating the migration rate of HConF and HK cells. Scale bar: 300 μm.
Figure 3.
 
(A) Migration rate of HConF cells after culturing with 0.1% FBS (N.S.), 20% WP, or 20% F3 for 24 hours. (B) Migration rate of keratocyte (HK) cells after culturing with 0.1% FBS (N.S.), 20% WP, or 20% F3 for 24 hours. WP and F3 significantly increased migration of both cells compared with nonstimulatory conditions; *, 95% confidence interval. No statistical differences were found between WP and F3. (C) Phase contrast photomicrographs illustrating the migration rate of HConF and HK cells. Scale bar: 300 μm.
Protective Effect of Plasma Preparations
The effects of plasma preparations (WP and F3) on the prevention of the TGF-β1–stimulated myofibroblastic differentiation were evaluated. Cells treated with either 20% WP or 20% F3 alone were not differentiated into myofibroblasts (data not shown). HConF and HK cells showed a spontaneous differentiation to myofibroblasts in a percentage of 16% ± 2% and 14% ± 8% respectively after 72-hour culture with 0.1% of FBS. After 3 days of stimulation with 2.5 ng/mL of TGF-β1, conjunctival fibroblasts and keratocytes showed a 61% ± 32% and a 48% ± 23% of α-SMA–positive cells respectively (Figs. 4A and 4B). The immunofluorescence for α-SMA exhibited that after culturing the cells 3 days either with 20% of WP plus 2.5 ng/mL TGF-β1 or with 20% of F3 plus 2.5 ng/mL TGF-β1, the percentage of positive HConF cells decreased drastically to 0.2% ± 0.1% and 0.1% ± 0.3%, respectively. This decrease was statistically significant for both types of fibroblasts with respect to the spontaneous transformation in culture and also with respect to the condition of TGF-β1 alone. Figure 4C shows the protective role of WP and F3 against the effect of TGF-β1 over HConF and HK cells. No significant differences were found between the responses induced by WP or F3 on fibroblast transformation to myofibroblasts. 
Figure 4.
 
When cells were treated simultaneously with TGF-β1 and WP or TGF-β1 and F3, (A) conjunctival fibroblasts (HConF) and (B) keratocytes (HK), number of α-SMA–positive cells were significantly lower compared with the TGF-β1 treatment group; 95% confidence interval. There is also a significant difference between spontaneous myotransformation and the number of α-SMA–positive cells after treatment with 2.5 ng/mL TGF-β1 plus 0.1% FBS, or plus 20% WP, or plus 20% F3. (C) Immunofluorescence for detection of α-SMA protein in HConF and HK cultured cells. α-SMA–positive and Hoechst-positive cells are considered as myofibroblasts. Scale bar: 200 μm.
Figure 4.
 
When cells were treated simultaneously with TGF-β1 and WP or TGF-β1 and F3, (A) conjunctival fibroblasts (HConF) and (B) keratocytes (HK), number of α-SMA–positive cells were significantly lower compared with the TGF-β1 treatment group; 95% confidence interval. There is also a significant difference between spontaneous myotransformation and the number of α-SMA–positive cells after treatment with 2.5 ng/mL TGF-β1 plus 0.1% FBS, or plus 20% WP, or plus 20% F3. (C) Immunofluorescence for detection of α-SMA protein in HConF and HK cultured cells. α-SMA–positive and Hoechst-positive cells are considered as myofibroblasts. Scale bar: 200 μm.
Inhibition of Myofibroblastic Phenotype
The effects of both plasma fractions (WP and F3) on the inhibition of the TGF-β1–stimulated myofibroblast differentiation were evaluated. After pretreating the cells during 3 days with 2.5 ng/mL of TGF-β1, a 60% ± 25% of α-SMA–positive HConF cells and a 77% ± 7% of α-SMA–positive HK were found (Figs. 5A and 5B). Then cells were treated for another 3 days with either TGF-β1, a combination of TGF-β1 plus 20% WP, or a combination of TGF-β1 plus 20% F3. Interestingly, the percentage of myofibroblasts in the HConF population was reduced significantly to 7% ± 2% when WP was added and to 11% ± 3% when F3 was added (Fig. 5A). In the case of HK cells, the number of α-SMA–positive cells decreased drastically to 8% ± 2% and to 12% ± 2% when WP and F3 were added respectively (Fig. 5B). Therefore, the use of PRGF-Endoret treatments reduced significantly (with a 95% confidence interval) the number of myofibroblasts. As counterpoint, cells treated with 0.1% FBS plus 2.5 ng/mL TGF-β1 for 3 days maintained the myofibroblastic phenotype. In fact, 86% ± 8% of the HConF cells and 71% ± 7% of the HK cells were positive for α-SMA. 
Figure 5.
 
Capacity of reversion of PRGF-Endoret technology over the myofibroblastic phenotype. HConF (A) and HK (B) cells were treated for 3 days with 2.5 ng/mL TGF-β1 as a previous stimulation to get a population of myofibroblasts. Then they were cultured with 2.5 ng/mL TGF-β1 simultaneously with 20% WP or 20% F3 for another 3 days to prove the capacity of WP and F3 to dedifferentiate the cells. There is no statistical difference between the response induced by WP and F3 but there is with respect to starting point (2.5 ng/mL); *, 95% confidence interval. (C) Immunofluorescence of α-SMA showing positive cells before and after treatment with plasma preparations. Myofibroblasts are α-SMA–positive and Hoechst-positive cells. Scale bar: 200 μm.
Figure 5.
 
Capacity of reversion of PRGF-Endoret technology over the myofibroblastic phenotype. HConF (A) and HK (B) cells were treated for 3 days with 2.5 ng/mL TGF-β1 as a previous stimulation to get a population of myofibroblasts. Then they were cultured with 2.5 ng/mL TGF-β1 simultaneously with 20% WP or 20% F3 for another 3 days to prove the capacity of WP and F3 to dedifferentiate the cells. There is no statistical difference between the response induced by WP and F3 but there is with respect to starting point (2.5 ng/mL); *, 95% confidence interval. (C) Immunofluorescence of α-SMA showing positive cells before and after treatment with plasma preparations. Myofibroblasts are α-SMA–positive and Hoechst-positive cells. Scale bar: 200 μm.
The immunofluorescence detection of α-SMA revealed that both WP and F3 inhibited the differentiation of the different populations of fibroblasts into myofibroblasts (Fig. 5C). 
Discussion
Several groups have focused their studies in understanding the different processes related to wound healing of the ocular surface. There is a general agreement that one of the main events involved in wound healing is cell proliferation as well as the migration of these cells to the damaged area. 1,28,29 There are also reports showing that different growth factors and cytokines and an intricate network of signals are implicated in the modulation of wound healing. 30  
PRGF-Endoret is an autologous platelet-rich plasma technology by which it is possible to obtain different growth factor-enriched formulations that can be used in the treatment of several disorders. 31 The effects of PRGF-Endoret on tissue regeneration have been demonstrated in dentistry, oral implantology, orthopedics, sports medicine, and treatment of skin disorders. 32  
In this study, we report for the first time the effects of PRGF-Endoret on the proliferation, migration, and differentiation of human keratocytes and conjunctival fibroblasts in vitro. Results demonstrate that the different plasma formulations evaluated (WP and F3) enhance proliferation and migration of both types of fibroblast populations and significantly protect and inhibit TGF-β1–induced myofibroblast differentiation. Interestingly, there are not significant differences between the effects induced by the platelet-enriched plasma fraction (F3) and the whole plasma fraction (WP). Although preliminary, these results may help to understand the potential of the autologous formulations in corneal wound healing. 
One important concern in ocular wound healing is scar formation. TGF-β has been identified as one of the most potent inductors of fibroblast differentiation into α-SMA-expressing myofibroblasts. 9,22 According to Masur et al., 33 a spontaneous fibroblast differentiation to myofibroblasts is observed when cells are cultured at low cell density or at low concentration of FBS. In our study, a spontaneous differentiation of 16% for HConF cells and 14% for HK cells was detected. No additional differentiation was observed after adding the plasma rich in growth factors formulations (WP and F3). 
Our current findings confirm that PRGF-Endoret technology protects TGF-β1–induced α-SMA–expression of keratocytes and conjunctival fibroblasts. In fact, when cells were cocultured either with 20% WP or F3 and TGF-β1 simultaneously, α-SMA–expression was under 0.3%. In addition, both WP and F3 significantly inhibited myofibroblast differentiation even when a previous 3-day culture with TGF-β1 had been carried out. 
Although further research is needed to clarify the molecular events that regulate PRGF-Endoret biological activity, it seems reasonable that some of the proteins present in both WP and F3 may have played key roles in cell proliferation, migration, and differentiation. Some growth factors present in PRGF-Endoret preparations have been described as key regulators of corneal wound healing. For example, EGF, HGF, and keratocyte growth factor (KGF) stimulate epithelial cell proliferation and migration, while in the case of stroma, these processes are mediated by TGF, PDGF, and FGF. 34 38  
Some studies have discussed the mechanisms by which the myofibroblastic phenotype disappears from corneal tissue or cultured cells. Interestingly, it has been observed that FGF-1 and -2, some proteins present in plasma rich in PRGF-Endoret formulations, promote the fibroblast phenotype and reverse the myofibroblast phenotype. 11 Some other studies suggest that myofibroblast apoptosis may be one of the initial mechanisms of myofibroblast disappearance; although myofibroblasts transdifferentiation to keratocytes or corneal fibroblasts should also be considered. 39  
Another important conclusion from this study is that WP and F3 exert similar in vitro biological effects. It has to be assumed that until now, F3, that is, the plasma fraction with the highest platelet concentration, has been widely used in several medical areas 28 31 including the treatment of ocular diseases. 40 43 The data presented herein may modify the current clinical protocols as WP shows similar biological effects to F3 but represents an improvement in the yield of 400%. In fact, while only 1 mL of F3 can be obtained from 9 mL of blood, almost 4 mL of WP can be obtained from the same blood volume. 
In summary, the different formulations of PRGF-Endoret enhance proliferation and migration of keratocytes and conjunctival fibroblasts while they protect and inhibit TGF-β1–induced myofibroblast differentiation. Although further studies are needed to determine the exact mechanisms underlying the effects of this autologous technology, results from this study suggest that the different PRGF-Endoret formulations (WP and F3) could improve the wound healing in ocular surface. 
Footnotes
 Supported by Customized Eye Care, CEYEC (No. CEN-20091021) project, which has been supported by the Centre for Industrial Technological Development (CDTI) in the fifth edition of the CENIT program. The aim of this program is to promote the public-private stable cooperation in research, development and innovation (R+D+i), which is part of the Spanish government initiative INGENIO 2010.
Footnotes
 Disclosure: E. Anitua, BTI Technology Institute (E), P; M. Sanchez, None; J. Merayo-Lloves, None; M. De la Fuente, BTI Technology Institute (E); F. Muruzabal, BTI Technology Institute (E); G. Orive, BTI Biotechnology Institute (E)
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Figure 1.
 
Scheme of the different plasma fractions obtained with the PRGF-Endoret technology. In all the different plasma preparations, care was taken to avoid the buffy coat containing leukocytes. WP, whole plasma obtained with PRGF-Endoret System. F1, fraction 1; F2, fraction 2; F3, fraction 3.
Figure 1.
 
Scheme of the different plasma fractions obtained with the PRGF-Endoret technology. In all the different plasma preparations, care was taken to avoid the buffy coat containing leukocytes. WP, whole plasma obtained with PRGF-Endoret System. F1, fraction 1; F2, fraction 2; F3, fraction 3.
Figure 2.
 
(A) Proliferation of HConF cells after culturing with 0.1% FBS as a control of nonstimulation (N.S.), 20% WP, or 20% F3 for 2 days. (B) Proliferation of keratocyte (HK) cells after culturing with 0.1% FBS (N.S.), 20% WP, or 20% F3 for 2 days. WP and F3 significantly increased proliferation of both cells compared with nonstimulatory conditions; *, 95% confidence interval. No statistical differences were found between WP and F3.
Figure 2.
 
(A) Proliferation of HConF cells after culturing with 0.1% FBS as a control of nonstimulation (N.S.), 20% WP, or 20% F3 for 2 days. (B) Proliferation of keratocyte (HK) cells after culturing with 0.1% FBS (N.S.), 20% WP, or 20% F3 for 2 days. WP and F3 significantly increased proliferation of both cells compared with nonstimulatory conditions; *, 95% confidence interval. No statistical differences were found between WP and F3.
Figure 3.
 
(A) Migration rate of HConF cells after culturing with 0.1% FBS (N.S.), 20% WP, or 20% F3 for 24 hours. (B) Migration rate of keratocyte (HK) cells after culturing with 0.1% FBS (N.S.), 20% WP, or 20% F3 for 24 hours. WP and F3 significantly increased migration of both cells compared with nonstimulatory conditions; *, 95% confidence interval. No statistical differences were found between WP and F3. (C) Phase contrast photomicrographs illustrating the migration rate of HConF and HK cells. Scale bar: 300 μm.
Figure 3.
 
(A) Migration rate of HConF cells after culturing with 0.1% FBS (N.S.), 20% WP, or 20% F3 for 24 hours. (B) Migration rate of keratocyte (HK) cells after culturing with 0.1% FBS (N.S.), 20% WP, or 20% F3 for 24 hours. WP and F3 significantly increased migration of both cells compared with nonstimulatory conditions; *, 95% confidence interval. No statistical differences were found between WP and F3. (C) Phase contrast photomicrographs illustrating the migration rate of HConF and HK cells. Scale bar: 300 μm.
Figure 4.
 
When cells were treated simultaneously with TGF-β1 and WP or TGF-β1 and F3, (A) conjunctival fibroblasts (HConF) and (B) keratocytes (HK), number of α-SMA–positive cells were significantly lower compared with the TGF-β1 treatment group; 95% confidence interval. There is also a significant difference between spontaneous myotransformation and the number of α-SMA–positive cells after treatment with 2.5 ng/mL TGF-β1 plus 0.1% FBS, or plus 20% WP, or plus 20% F3. (C) Immunofluorescence for detection of α-SMA protein in HConF and HK cultured cells. α-SMA–positive and Hoechst-positive cells are considered as myofibroblasts. Scale bar: 200 μm.
Figure 4.
 
When cells were treated simultaneously with TGF-β1 and WP or TGF-β1 and F3, (A) conjunctival fibroblasts (HConF) and (B) keratocytes (HK), number of α-SMA–positive cells were significantly lower compared with the TGF-β1 treatment group; 95% confidence interval. There is also a significant difference between spontaneous myotransformation and the number of α-SMA–positive cells after treatment with 2.5 ng/mL TGF-β1 plus 0.1% FBS, or plus 20% WP, or plus 20% F3. (C) Immunofluorescence for detection of α-SMA protein in HConF and HK cultured cells. α-SMA–positive and Hoechst-positive cells are considered as myofibroblasts. Scale bar: 200 μm.
Figure 5.
 
Capacity of reversion of PRGF-Endoret technology over the myofibroblastic phenotype. HConF (A) and HK (B) cells were treated for 3 days with 2.5 ng/mL TGF-β1 as a previous stimulation to get a population of myofibroblasts. Then they were cultured with 2.5 ng/mL TGF-β1 simultaneously with 20% WP or 20% F3 for another 3 days to prove the capacity of WP and F3 to dedifferentiate the cells. There is no statistical difference between the response induced by WP and F3 but there is with respect to starting point (2.5 ng/mL); *, 95% confidence interval. (C) Immunofluorescence of α-SMA showing positive cells before and after treatment with plasma preparations. Myofibroblasts are α-SMA–positive and Hoechst-positive cells. Scale bar: 200 μm.
Figure 5.
 
Capacity of reversion of PRGF-Endoret technology over the myofibroblastic phenotype. HConF (A) and HK (B) cells were treated for 3 days with 2.5 ng/mL TGF-β1 as a previous stimulation to get a population of myofibroblasts. Then they were cultured with 2.5 ng/mL TGF-β1 simultaneously with 20% WP or 20% F3 for another 3 days to prove the capacity of WP and F3 to dedifferentiate the cells. There is no statistical difference between the response induced by WP and F3 but there is with respect to starting point (2.5 ng/mL); *, 95% confidence interval. (C) Immunofluorescence of α-SMA showing positive cells before and after treatment with plasma preparations. Myofibroblasts are α-SMA–positive and Hoechst-positive cells. Scale bar: 200 μm.
Table 1.
 
Platelet and Leukocyte Count and Concentrations of Several Growth Factors in the Two Different Plasma Preparations (WP and F3) of the Blood Donor
Table 1.
 
Platelet and Leukocyte Count and Concentrations of Several Growth Factors in the Two Different Plasma Preparations (WP and F3) of the Blood Donor
Plasma Preparation Leukocyte Count (×106/mL) Platelet Count (×106/mL) Growth Factor Levels
TGF-β1 (ng/mL) PDGF-AB (ng/mL) IGF-I (ng/mL) VEGF (pg/mL) HGF (pg/mL) EGF (pg/mL) TSP-1 (μg/mL)
WP <0.2 481 63 19 83 568 400 508 29
F3 <0.3 663 81 30 86 791 491 779 50
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