Biological effects of plasma rich in growth factors PRGF on human

European Journal of Obstetrics & Gynecology and Reproductive Biology 206 (2016) 125–130 Contents lists available at ScienceDirect European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb Biological effects of plasma rich in growth factors (PRGF) on human endometrial fibroblasts Eduardo Anitua a,b, Marı´a de la Fuente a, Marcos Ferrando c, Fernando Quintana c, Zaloa Larreategui c, Roberto Matorras c,d, Gorka Orive a,b,* a Biotechnology Institute (BTI), Vitoria, Spain Eduardo Anitua Foundation for Biomedical Research, Vitoria, Spain IVI Clinic Bilbao, Leioa, Bizkaia, Spain d Human Reproduction Unit, Cruces Hospital, BioCruces, University of the Basque Country, Basque Country, Spain b c A R T I C L E I N F O A B S T R A C T Article history: Received July 2016 Received in revised form September 2016 Accepted 13 September 2016 Objectives: To evaluate the biological outcomes of plasma rich in growth factors (PRGF) on human endometrial fibroblasts in culture Study design: PRGF was obtained from three healthy donors and human endometrial fibroblasts (HEF) were isolated from endometrial specimens from five healthy women The effects of PRGF on cell proliferation and migration, secretion of vascular endothelial growth factor (VEGF), procollagen type I and hyaluronic acid (HA) and contractility of isolated and cultured human endometrial fibroblasts (HEF) were analyzed Statistical analysis was performed in order to compare the effects of PRGF with respect to control situation (T-test or Mann–Whitney U-test) Results: We report a significantly elevated human endometrial fibroblast proliferation and migration after treatment with PRGF In addition, stimulation of HEF with PRGF induced an increased expression of the angiogenic factor VEGF and favored the endometrial matrix remodeling by the secretion of procollagen type I and HA and endometrial regeneration by elevating the contractility of HEF These results were obtained for all PRGF donors and each endometrial cell line Conclusions: The myriad of growth factors contained in PRGF promoted HEF proliferation, migration and synthesis of paracrine molecules apart from increasing their contractility potential These preliminary results suggest that PRGF improves the biological activity of HEF in vitro, enhancing the regulation of several cellular processes implied in endometrial regeneration This innovative treatment deserves further investigation for its potential in ‘‘in vivo’’ endometrial development and especially in human embryo implantation ß 2016 Elsevier Ireland Ltd All rights reserved Keywords: Plasma rich in growth factors (PRGF) Endometrial fibroblasts Endometrial regeneration Remodeling Introduction Endometrium is a thin uterine lining undergoing cyclic growth, regeneration, differentiation and shedding in response to the rise and fall in ovarian hormones throughout the reproductive life [1] During each proliferative phase of the menstrual cycle, endometrium grows 4–10 mm under the influence of increasing circulating estrogen levels The implantation of a fertilized embryo will probably not occur if the lining of the uterus does not achieve a certain thickness Low estrogen levels, an inadequate blood flow, * Corresponding author at: BTI, Jacinto Quincoces, 39, 01007 Vitoria, Spain Fax: +34 945154187 E-mail address: gorka.orive@bti-implant.es (G Orive) http://dx.doi.org/10.1016/j.ejogrb.2016.09.024 0301-2115/ß 2016 Elsevier Ireland Ltd All rights reserved an unhealthy endometrial tissue with scar tissue or adhesion tissue and the prolonged use of birth control pills can cause a thin uterine lining The nonresponsive thin endometrium has become an unsolved in vitro fertilization (IVF) problem Generally, embryos are transferred into an endometrial cavity with an optimal endometrial thickness of mm or more but approximately 0.6–0.8% of patients not reach this minimum thickness [2] Several approaches are used when the uterine lining is thin, including prolonging the duration of estrogen stimulation, vaginal administration of sildenafil, increasing the capillary blood flow to the uterus by using low-dose aspirin or a combination of vitamin E and pentoxifylline and the uterine perfusion of granulocyte colonystimulating factor (G-CSF) combined or not with endometrial scratching, among others [3] Many of them are controversial or of 126 E Anitua et al / European Journal of Obstetrics & Gynecology and Reproductive Biology 206 (2016) 125–130 limited efficacy and despite of all this strategies, a number of women remains unresponsive, maintaining a thin endometrium Plasma rich in growth factors (PRGF) technology is a relatively new biological approach consisting on an autologous plateletenriched plasma free of leucocytes After activation, a-granules of platelets release a pool of biologically active proteins that influence and stimulate several biological processes such as cell proliferation, recruitment, growth, and differentiation The biological cocktail from PRGF is rich including morphogens like plateletderived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF-b), fibroblast growth factor (FGF), epidermal growth factor (EGF), insulin-like growth factor (IGF-1) and hepatocyte growth factor (HGF) [4,5] Human endometrial tissue contains receptors for PDGF and EGF and both molecules are mitogenic for endometrial stromal cells [6] In fact, PDGF isoforms promote endometrial tissue repair through the proliferation and migration of endometrial stem cells and favors tissue contraction and remodeling [7] whereas HGF stimulates endometrial epithelial cell proliferation and migration via paracrine pathways [8] Furthermore, TGF-b isoforms promote endometrial tissue remodeling [9] PRGF technology has provided significant clinical advances in terms of wound healing and tissue regeneration in several medical fields including dentistry and oral implantology [10], neurobiology [11], orthopedics and sport medicine [12], and ophthalmology [13] among others However, the implication of PRGF in the treatment of gynecological complications has not been widely investigated Assuming that the role of endometrial stromal fibroblasts is crucial in the development of a responsive endometrium and its posterior remodeling, the present study analyzes the in vitro effects of PRGF on proliferation, migration and gel contractility of endometrial cells in culture Moreover, the effects on tissue remodeling were assessed by measuring the secretion of extracellular matrix (EM) components by endometrial cells Materials and methods Human endometrial cells Endometrial samples were obtained from young women (24– 32 years old) undergoing oocyte donation The biopsies were collected after each patient in accordance with the requirements of the Ethical Committee of Cruces Hospital signed informed consent Endometrial tissue was rinsed with phosphate-buffered saline (PBS) supplemented with antibiotics and minced into 1–2 mm3 pieces The fragments were digested with 0.3% collagenase II (Gibco Life Technologies, Gaithersburg, MD, USA) in fibroblast medium (FM, ScienCell Research Laboratories, San Diego, CA, USA) with antibiotics at 37 8C The resulting cell suspension was seeded into culture flasks and maintained with FM supplemented with Fibroblast Growth Supplement, 2% fetal bovine serum (FBS) and antibiotics (penicillin/streptomycin) (termed complete FM) (ScienCell Research Laboratories, San Diego, CA, USA) in a humidified atmosphere at 37 8C with 5% CO2 Cell viability was assessed by trypan blue dye exclusion Passage 3–4 cells were used in all experiments The fibroblast-like morphology of cells isolated was checked by phase contrast microscopy Characterization of human endometrial cells To confirm that the isolated cells were endometrial stromal fibroblasts, the expression of collagen type I (Chemicon – Millipore, Billerica, MA, USA), alpha-smooth muscle actin (a-SMA), pancytokeratin (Pan-CK), fibronectin and vimentin (Sigma–Aldrich, St Louis, MO, USA) was analyzed Briefly, cells were plated on 24-well plates for 48 h and then were fixed and incubated with the aforementioned primary antibodies Next, cells were incubated with their appropriate secondary antibodies and cell nuclei were counterstained with Hoechst 33342 (Molecular Probes-Invitrogen, Grand Island, NY, USA), mounted, and visualized under a fluorescence microscope (Leica DM IRB, Leica Microsystems, Wetzlar, Germany) Cells positive for each marker were counted with Image J Software (NIH, Bethesda, MD, USA) PRGF preparations Blood from three healthy donors (37–47 year old) was collected after informed consent into 9-ml TB9 tubes with 400 ml of sodium citrate (3.8%, w/v) The study was performed following the principles of the Declaration of Helsinki Samples were centrifuged at 580 Â g for at room temperature in a PRGF System ˜ ano, A´lava, Spain) centrifuge (BTI Biotechnology Institute, S.L., Min The whole plasma column called PRGF was aspirated avoiding the buffy coat containing the leucocytes Platelets and leucocytes counts were performed with a hematology analyzer (Micros 60, Horiba ABX, Montpellier, France) The preparations were incubated ˜ ano, with PRGF activator (BTI Biotechnology Institute, S.L., Min A´lava, Spain) and the released supernatants were collected by centrifugation, filtered, aliquoted and stored at À80 8C until their use Quantikine colorimetric sandwich enzyme-linked immunosorbent ELISA Assay kits (R&D Systems, Minneapolis, MN, USA) were employed to quantify PDGF-AB, TGF-b1, VEGF, EGF, and IGF-I concentrations in PRGF from the three donors following the manufacturer’s recommendations Proliferation assay Human endometrial fibroblasts (HEF, n = 5) were seeded at a density of 10000 cells per cm2 on 96 well optical bottom black plates in FM supplemented with 20% PRGF from the three donors during 72 h; cells maintained with 0.1% FBS in FM during the same days were employed as control of non-stimulation condition (control) All samples were assayed in triplicate Final cell number in culture was estimated as DNA amount using the CYQUANT Cell Proliferation Assay (Invitrogen, Carlsbad, CA, USA) Sample fluorescence was measured using a fluorescence microplate reader (Twinkle LB 970, Berthold Technologies) A DNA standard curve ranging from 7.8 to 1000 ng/ml was included in all fluorescence quantifications to correlate fluorescence with concentration of DNA Migration assay in response to PRGF HEF were plated in culture inserts (Ibidi, GmbH, Martinsried, Germany) placed on a 24-well plate at high density and were grown with complete FM until confluence Then inserts were carefully removed leaving two separated cell monolayers with a cell-free gap The cells were washed with PBS and incubated in triplicate for approximately 22 h with FM plus 0.1% FBS or 20% PRGF of each donor Treatments were removed and cells were incubated with Hoechst 33342 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 were measured using the Image J Software (NIH, Bethesda, MD, USA) obtaining the number of migrated cell per mm2 of area Synthesis of VEGF and extracellular matrix (EM) components In order to examine the effect of PRGF on VEGF, procollagen type I and hyaluronic acid (HA) synthesis, HEF were seeded on 12 E Anitua et al / European Journal of Obstetrics & Gynecology and Reproductive Biology 206 (2016) 125–130 well plates at a density of 6000 cells/cm2 in complete FM and maintained until confluence Then cells were rinsed with PBS and incubated with FM + 0.1% FBS or FM + 20% PRGF from the three donors during 72 h in triplicate Additional wells with the same treatments but without cells were included to determine the initial amount of growth factors provided by each PRGF Culture mediums were collected, aliquoted and stored at À80 8C until assayed VEGF (R&D Systems, Minneapolis, MN, USA), procollagen type-I C-peptide (Takara Bio Inc, Otsu, Shiga, Japan) and HA (Corgenix, Broomfield, CO, USA) concentrations were determined in the conditioned culture mediums using ELISA kits following manufacturer’s instructions Collagen matrix contraction assay Collagen solution was prepared by mixing chilled collagen solution from bovine skin (Sigma–Aldrich, St Louis, MO, USA) with 10Â PBS as manufacturer’s instructions The pH of the mixture was carefully adjusted to 7.2–7.6 maintaining temperature of the solution at 2–8 8C to prevent gelation HEF were then resuspended in the collagen solution and plated in 24 well culture plates at high density (40,000 cells/cm2), incubating the plates until formation of the collagen matrices with the fibroblast embedded Then, they were incubated with FM culture medium supplemented with 0.1% (v/v) FBS as a control of non-stimulation or 20% (v/v) PRGF in triplicate Images from the collagen matrices were taken at time 0, 24, 48 and 72 h with a digital camera (Lumix, Panasonic DCM-FZ28, Osaka, Japan) The contractile activity of the fibroblasts was measured with the Image J Software analysis (NIH, Bethesda, MD) expressing the results as percentage (%) of contraction compared to time h Statistical analysis Descriptive statistics were performed using absolute and relative frequency distributions for qualitative variables and mean values and standard deviations for quantitative variables Different normality tests (Kolmogorov–Smirnov and Shapiro–Wilk) were performed on each sample After testing normality, different 127 comparisons on proliferation, migration, GF synthesis and % of contraction between distinct treatment groups (Control versus PRGF) were performed using T-test or Mann–Whitney nonparametric test depending on the normality test results Statistical significance level was set on p < 0.05 SPSS v15.0 for Windows statistical software package (SPSS Inc., Chicago, IL, USA) was used for all statistical analysis Results Endometrial fibroblast characterization Fig shows HEF elongated and spindle-shaped aspect in routine culture; cells were strongly positive for all the fibroblast markers (99.8 Ỉ 0.3% for Collagen Type I, 98.9 Ỉ 1.4% for vimentin and 98.5 Ỉ 2.5% for fibronectin) and negative for pan-CK (1.4 Ỉ 1.1%) showing no presence of contamination with epithelial cells There was a 18.4 Ỉ 7.1% of cells positive for a-SMA showing the usual presence of some myofibroblasts PRGF characterization The concentration of platelets and leucocytes together with the level of some of the most important GFs present in PRGF of the three donors are shown in Table Mean platelet enrichment of the PRGF preparation was 1.74-fold over whole blood and there were no detectable levels of leucocytes PRGF augments HEF proliferation Proliferation of HEF increased significantly after 72 h treatment with PRGF compared with the control (p < 0.01) Fig 2A shows the mean DNA content (ng/ml) from the donors and the primary endometrial cell lines Stimulation of migration by PRGF Fig 2B exposes that the number of migrating cells was significantly higher after treating with every PRGF compared to Fig Representative phase contrast photomicrographs illustrating the typical spindle shape of fibroblasts isolated from human endometrial tissue (scale bar 300 mm) Immunofluorescence microscopy confirmed that the fibroblasts were uniformly positive for vimentin, collagen type I and fibronectin as revealed by immunostaining; there were few cells positive for alpha-smooth actin (a-SMA) showing the presence of some myofibroblasts and there was practically no expression of pan cytokeratin (scale bar 200 mm) confirming the absence of epithelial cells E Anitua et al / European Journal of Obstetrics & Gynecology and Reproductive Biology 206 (2016) 125–130 128 Table Characterization of PRGF The concentration of platelets and leucocytes and concentration of some representative growth factors are shown as mean Ỉ standard deviation from the donors Platelet count (Â106/ml) PRGF (n = 3) 319 Ỉ 29 Leukocyte count (Â106/ml) 0.1 Ỉ Platelet enrichment over peripheral blood 1.74Â Ỉ 0.3 Growth factor levels PDGF-AB (pg/ml) TGF-b1 (ng/ml) VEGF (pg/ml) EGF (pg/ml) IGF-I (ng/ml) 9600 Ỉ 1000 18.1 Ỉ 3.4 152 Ỉ 36 490 Ỉ 113 73 Æ 18 cells cultured with FM medium plus 0.1% FBS (control, p < 0.05) Hoechst images of HEF after a 22-h period showed that there were more HEF invading the yellow-bordered gap after the PRGF treatment Secretion of VEGF, procollagen type I and HA from HEF Synthesis of VEGF and EM components was significantly increased when HEF were treated with every PRGF compared to 0.1% FBS (Fig 3A, p < 0.01) after days of culture The secretion of matrix components measured as synthesis of procollagen type I and hyaluronic acid was up-stimulated in a significant manner in the presence of PRGF compared to the control (p < 0.01) Collagen gel contraction enhancement The effect of PRGF on HEF collagen gel contractile activity was evaluated as it is showed in Fig Cells treated with PRGF showed significantly higher contractility than those cultured with 0.1% FBS (p < 0.01) at every studied time (24, 48 and 72 h) and for every cell line and donor as it can be observed in Fig 3B Time h was considered starting point and 0% of contraction Comment Human endometrium is a dynamic tissue that undergoes regular cycles of regeneration and remodeling Thus, the organization of several locally produced growth factors and cytokines is needed This process involves an orchestrated actuation of autocrine, paracrine and endocrine factors with biological effects mediated by specific cell surface receptors; they act as communicators between endometrial cells and embryo [14,15] Endometrial wound healing is mediated by GF and cytokines, EM molecules as hyaluronan and pro-collagen type I and their cellular receptors There is also a balance between matrix degrading enzymes (matrix metalloproteinases) and their inhibitors for an optimal remodeling [16] In the present study we show that PRGF stimulates human endometrial fibroblast proliferation, migration and paracrine secretion of biomolecules PRGF contains a cocktail of growth factors and cytokines that exert trophic effects over a great variety of primary cells [17–19] Platelet derived growth factor promotes endometrial tissue repair through the stimulation of proliferation, chemotactic migration and invasion of endometrial stromal cells [7]; TGF-b1 also present in PRGF may protect the endometrium Fig (A) Phase contrast photomicrographs of human endometrial fibroblasts (HEF) cultured with 0.1% FBS (control) or 20% PRGF (PRGF) Fibroblasts showed an augmented proliferation after treatment with PRGF and maintained spindle-shaped morphology Bar graph confirms that 20% PRGF significantly increased proliferation of HEF compared to control situation, measured as DNA content (ng/mL) * Statistically significances between PRGF and control (p < 0.001) (B) Immunofluorescence Hoechst images of HEF after 22 h-migration with 0.1% FBS (control) or 20% PRGF (PRGF) show that cell migration was significantly higher when cells were cultured with PRGF Migration gap area is identified by a bordered yellow rectangle included in each image *Statistically significant differences between control and PRGF (p < 0.001) Scale bar 300 mm E Anitua et al / European Journal of Obstetrics & Gynecology and Reproductive Biology 206 (2016) 125–130 129 Fig (A) Synthesis of VEGF, Procollagen type I and Hyaluronic acid by HEF The secretion of angiogenic factor VEGF was significantly stimulated by PRGF after 72 h of culture; *statistically significant differences between control and PRGF (p < 0.01); also, PRGF enhanced significantly the synthesis of extracellular matrix components measured by the secretion of procollagen type I and hyaluronic acid (p < 0.01) (B) The effect of PRGF on the collagen gel contractility of endometrial fibroblasts was measured after 24, 48 and 72 h in culture and the contraction was up-stimulated in a significant way; the graph shows the data as percentages of contraction relative to initial time (time h) *Statistically significant differences between control and PRGF (p < 0.01) from extensive fibrosis and scarring [9] EGF regulates the production of VEGF by endometrial cells and this last factor contributes to neovascularization by the promotion of endometrium proliferation [20,21] In epithelial endometrial cells, HGF acts as a morphogen and mitogen stimulating cell proliferation [8,22] Enhancement of HEF migration by PRGF may be critical to facilitate the arrival of the adequate cells to the required place, earlier than in an unstimulated condition therefore promoting an accelerated wound healing Treatment of endometrial cells with PRGF increased significantly their VEGF secretion VEGF contributes to neovascularization, a key process in the repair and proliferation of the endometrium and in successful embryonic implantation [20] We carried out an in vitro collagen gel contraction assay, in which the cells are cultured in a 3D model mimicking the wound contraction process in vivo [23] Endometrial wound healing is managed by endometrial stromal fibroblasts that contract the connective tissue as it has been described elsewhere [7,9] Our results showed a significant promotion of the contractile activity of HEF after PRGF treatment at every studied time leading to speculate that PRGF could prevent an excessive fibroplasia and could drive an adequate endometrial regeneration Our results are also similar to those obtained by others testing platelet rich plasmas in other fibroblastic phenotypes [24,25] Endometrial extracellular matrix comprises a multifunctional macromolecular network implied in almost all cellular processes; endometrial wound healing requires synthesis and remodeling of the EM consisting mainly on collagens and glycosaminoglycans (as HA) and proteoglycans Treatment of HEF with PRGF enhanced significantly the synthesis of procollagen type I and HA, allowing the renewal and remodeling of EM for a satisfactory restoration Hyaluronan offers beneficial effects in the endometrium maybe due to its physical properties providing a provisional matrix for supporting cellular migration and proliferation Moreover, endometrial stromal cells express the principal receptor for HA, CD44 involved in the implantation [26] If confirmed, this technology could represent a new potential tool in IVF cycles for the treatment of deficient endometrial growth, favoring the thickening of the endometrial lining by stimulating resident cells proliferation and migration and in embryo implantation failures as it has been suggested in a recent case report [27] According to our preliminary results we can postulate that the myriad of GF contained in PRGF could promote HEF proliferation, migration and synthesis of paracrine molecules together with an increased contractility potential If confirmed, these results suggest that PRGF could improve the thickening of endometrium as well as the regulation of several cellular processes implied in endometrial regeneration Conflict of interest The authors declare the following competing financial interest(s): E.A is the Scientific Director of and M.F and G.O are scientists at BTI Biotechnology Institute, a dental implant company that investigates in the fields of oral implantology and PRGFEndoret technology Acknowledgement This study received funding from the GAITEK program from the Basque Government 130 E Anitua et al / European Journal of Obstetrics & Gynecology and Reproductive Biology 206 (2016) 125–130 References [1] Jabbour HN, Kelly 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platelet-rich plasma promotes endometrial growth and improves pregnancy outcome during in vitro fertilization Int J Clin Exp Med 2015;8:1286–90  ... endometrium Plasma rich in growth factors (PRGF) technology is a relatively new biological approach consisting on an autologous plateletenriched plasma free of leucocytes After activation, a-granules of. .. remodeling [16] In the present study we show that PRGF stimulates human endometrial fibroblast proliferation, migration and paracrine secretion of biomolecules PRGF contains a cocktail of growth factors. .. remodeling, the present study analyzes the in vitro effects of PRGF on proliferation, migration and gel contractility of endometrial cells in culture Moreover, the effects on tissue remodeling were