Controlled release of rhEGF and rhbFGF from electrospun scaffolds for skin regeneration Omolbanin Mirdailami1,3, Masoud Soleimani2, Rassoul Dinarvand1,3, Mohammad Reza Khoshayand4, Mohammad Norouzi5, Athena Hajarizadeh6, Masumeh Dodel5 and Fatemeh Atyabi1,3* 1 Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Sciences, P.O. Box 141556451, Tehran, Iran 2 Department of Hematology, Faculty of Medical Sciences, University of Tarbiat Modares, Tehran, Iran 3 Nanotechnology Research Centre, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran 4 Food and Drug Control Laboratory, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran 5 Department of Nanotechnology and Tissue Engineering, Stem Cell Technology Research Center, Tehran, Iran 6 Department of Molecular Biology and Genetic Engineering, Stem Cell Technology Research Center, Tehran, Iran

∗Corresponding author: F. Atyabi Fax: +98 21 66959052 E-mail address: [email protected]

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/jbm.a.35479 This article is protected by copyright. All rights reserved.

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ABSTRACT Controlled delivery of multiple therapeutic agents can be considered as an effective approach in skin tissue engineering. In this study, recombinant human epidermal growth factor (rhEGF) and recombinant human basic fibroblast growth factor (rhbFGF) encapsulated in PLGA microspheres were loaded in hybrid scaffolds of PLGA and PEO. The scaffolds with various formulations were fabricated through electrospinning in order to maintain dual, individual or different release rate of rhEGF and rhbFGF. Morphological, physical and mechanical properties of the scaffold were investigated. The scaffold possessed uniform morphology with an average diameter of 280 nm for PLGA and 760 nm for PEO nanofibers. Furthermore, the mechanical properties of the scaffolds were shown to be akin to those of human skin. Bioactivity of the scaffolds for human skin fibroblasts was evaluated. The HSF acquired significant proliferation and well-spread morphology on the scaffolds particularly in the case of different release rate of rhEGF and rhbFGF which implies the synergistic effect of the growth factors. Additionally, collagen and elastin gene expression was significantly up-regulated in the HSF seeded on the scaffolds in the case of individual delivery of rhEGF and dual delivery of rhEGF and rhbFGF. In conclusion, the prepared scaffolds as a suitable supportive substrate and multiple growth factor delivery system can find extensive utilization in skin tissue engineering. Keywords: Nanofibrous scaffold, rhEGF, rhbFGF, Controlled delivery, Skin tissue engineering

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1. Introduction Skin is the largest tissue in mammals and functions as a defensive shield at the interface between the human body and the surrounding world. The skin protects all other tissues and organs beneath it and prevents pathogens and microorganisms from entering the body. In the last decades, there has been plenty of effort to produce substitutes that mimic human skin. These skin substitutes which are produced by applying advanced tissue engineering approaches, have found extensive utilizations for clinical applications, promoting acute and chronic wound healing as well as burn treatment [1, 2]. The ideal scaffolds in tissue engineering should simulate the extracellular matrix (ECM) in structure and function. In fact, there are a number of tasks for the scaffolds to undertake; they are meant to maintain the necessary mechanical support, deliver inductive biomolecules and cells to the reconstruction site, and supervise the structure and function of the tissue being regenerated [3-6]. A largely favored tissue engineering technique which has been becoming increasingly popular in generating suitable scaffolds is electrospinning. This practical approach produces structures of nanoscale fibers with microscale-interconnected pores, which topographically simulates the ECM. This nanofibrous structure, produced through electrospinning, can support cell proliferation, enhance cell attachment, and determine their growth based on nanofiber orientation [3, 7, 8]. Furthermore, a great deal of effort has been recently made in the realm of bioactive scaffolds in order to find new strategies to deliver therapeutic agents. It is noticeable that the high specific surface area and the stereological porous structure of electrospun nanofibers lead to the 3|Page John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.

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enhancement of biomolecules delivery. It is also possible to integrate various biomolecules into the scaffolds in order to improve their biomedical functional properties. Some commonly used biomolecules include proteins, such as growth factors (GFs) or cytokines, and growth factor encoding genes [3, 9]. There are two approaches to deliver biomolecules; directly from the electrospun scaffolds, or from micro/nanospheres embedded in the scaffolds which possess independent release mechanisms. The latter approach, in which the electrospun scaffolds merely function as a support, can overcome the disadvantages of uncontrolled release rates and loss of bioactivity during the preparation processes [3]. This innovative strategy enables multiple introductions of particulates with different release profiles into the scaffolds by encapsulating each biomolecules into a specific particulate system, in which several specified release kinetics will be achieved. The sequential delivery of several therapeutic factors, as a revolutionary and efficient method of drug delivery, is made possible by distinct tailored release kinetics in these particulate delivery systems [10]. Wound healing, as an intricate procedure, comprises a number of phases, including as inflammation, cells proliferation and tissue regeneration. Furthermore, a complex network of signaling, subsuming several GFs, cytokines, and chemokines, is involved to administer and regulate this healing procedure [10-12]. GFs are endogenous proteins with the feature of binding to the receptors on the surface of the cells and regulating cellular activities involved in the regeneration process of new tissue. Therefore, localized delivery of GFs is considered therapeutically influential in tissue regeneration and healing processes. However, it should be mentioned, due to short half-life, 4|Page John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.

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toxicity in high concentration, physical- and chemical instability and enzymatic degradation of the proteins in physiological conditions, devising an efficient delivery method, plays a vital role in the application of GFs [3, 13]. Owing to the fact that tissue regeneration is a complex process, in which, a variety GFs play an important role and they illustrate synergistic effects with each other, multiple GFs delivery can be considered as an auspicious and effective strategy in tissue engineering. Integrated utilization of multiple GFs is believed to show incremental and synergistic results in different aspects including cell proliferation, motility and differentiation. This particular advantage leads to accelerated and improved tissue regeneration [10, 14]. For instance, Basmanav et al. [15] used nanofibrous scaffolds of PLGA with the capability of sequentially delivering two bone morphogenetic proteins (BMP-2 and BMP-7,) loaded in microspheres.

The co-administration of two sorts of BMPs showed a significant raise in

osteogenic differentiation compared with that of a single delivery. In another study, Elisseeff et al. [16] produced TGF-β1 and IGF-I loaded- PLGA microspheres which were embedded in PEO scaffolds and they reported a 10-fold increase in chondrocyte proliferation as a result of simultaneous utilization delivery of TGF-β1 and IGF-I. Furthermore, it has been reported that the combined utilization of bFGF and growth and differentiation factor (GDF-5) can improve medial collateral ligament (MCL) healing [17]. Generally, there are a variety of GFs which play important roles in skin regeneration. Amidst those, Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) are regarded as two prominent GFs. EGF, as a polypeptide comprising 53 different amino acids, has been the first GF successfully employed in wound treatment [18]. The unique structure of this polypeptide

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largely enhances epidermal as well as mesenchymal regeneration, cell motility, proliferation, and synthesis of extracellular matrix [18, 19]. Moreover, bFGF, as a multifunctional polypeptide, is capable of enhancing the growth and differentiation of a variety of cells such as dermal fibroblasts, keratinocytes, endothelial cells and melanocytes. It can positively affect tissue remodeling, wound healing and neovascularization based on its mitogenic and angiogenic properties. bFGF, available in a great proportion of normal and transformed cells and tissues, is a single chain peptide with 146 amino acids. Generally, it has been shown that the application of exogenous bFGF has positive effects on the healing of acute and chronic wounds in animals and clinical experiments [11, 20, 21]. To date, EGF [2, 18, 22] and bFGF [11, 23] have been incorporated into the electrospun scaffolds by some researchers for skin regeneration and wound healing purposes. In the present study, the aim is to draw a comparison between the dual as well as different release rate of bFGF and EGF together and the single application of them in skin tissue engineering. For this purpose, hybrid membranes were fabricated by electrospinning PLGA and PEO loaded with microspheres containing GFs. This system of multiple factors delivery, may find broad utilization in tissue engineering. 2. Materials and methods 2.1.Materials PLGA (LA:GA 50:50, MW 13 600 Da.) and PLGA (LA:GA 85:15) were supplied from Boehringer Ingelheim, (Ingelheim, Germany) and Purac (Gorinchem, The Netherlands), respectively. BSA and PVA (88% hydrolyzed, MW 13 000–23 000) were provided from SigmaAldrich (MO, USA). Sodium hydroxide, sodium chloride, Tween 80, dichloromethane (DCM), 6|Page John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.

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dimethylformamide (DMF), chloroform, and dimethyl sulfoxide (DMSO) were all purchased from Merck (Darmstadt, Germany). The rhEGF and rhbFGF were provided from PeproTech (NJ, USA). Human EGF Standard ELISA Development Kit and human bFGF Standard ELISA Development Kit were purchased from R&D Systems (Minneapolis, MN, USA). Dulbecco’s modified Eagle’s medium (DMEM), trypsin, ethylene diamine tetra acetic acid solution and fetal bovine serum (FBS) were supplied by Gibco (Grand Island, NY, USA). 3-[4,5-dimethylthiazol2-yl]- diphenyl tetrazolium bromide (MTT) was obtained from Sigma-Aldrich, (MO, USA). 2.2.Preparation of PLGA microspheres PLGA microspheres encapsulating rhEGF and rhbFGF were prepared through the double emulsion (W/O/W) solvent evaporation/extraction technique. Briefly, BSA solution in PBS (13.83 mg, 12.15%, w/w) as the internal aqueous phase, with or without poly vinyl alcohol (166 l, 0.1% w/v), which was supplemented with either rhEGF or rhbFGF (20 g, 0.02 % w/w), was mixed with 100 mg of PLGA solution in DCM (3.33 ml, 3%w/v) as the oil phase. The mixture was sonicated in ice water for 30 seconds, and then introduced into 40 ml of an aqueous solution of PVA (0.7 % w/v) to form a W/O/W emulsion, and was then emulsified by a high-speed homogenizer (IKA T18 basic ULTRA TURRAX Homogenizer, Wilmington, NC) at 16000 rpm for 5 minutes. Afterwards, the emulsion was constantly stirred overnight to promote solvent evaporation. Subsequently, the solidified microspheres were collected by centrifugation and were washed three times with deionized water, followed by lyophilization. The prepared microspheres were stored at -20 °C. Four different formulations were prepared to deliver the GFs (Table 1). Formulations A and B for rhEGF, as well as, formulations C and D for rhbFGF were all prepared under the same

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conditions with the exception of that, the formulations A and C contained PVA in the internal water phase and formulations B and D were prepared without PVA, as mentioned above in detail.

2.3.Preparation/fabrication of hybrid scaffolds Composite nanofibrous scaffolds of PLGA/PEO containing PLGA microspheres were fabricated via dual-electrospinning. Electrospinning of PEO solution supplemented by PLGA microspheres led to the formation of nanofibers, impregnated with the microspheres. PLGA solution with a polymer concentration of 5% w/w in a mixed solvent system of chloroform/DMF (in a volume ratio of 3:1) and PEO solution with polymer concentration of 3% w/w in water containing 20% w/w PLGA microspheres were prepared. The electrospinning system consisted of a high voltage power supply, a grounded electrode covered by an aluminum foil, and a polymer solution reservoir. High voltage was applied to overcome the liquid surface tension and enable injection of the polymer through the capillary tip with a diameter of 0.5 mm. Under the influence of the electrical charge, the ejected solution was elongated and whipped towards the collector and solidified instantaneously forming continuous fibers with diameters of a few nanometers to micrometers which collected on the target. PLGA and PEO/microsphere solutions were separately electrospun through two opposing spinnerets onto a common rotating drum under optimized conditions of the flow rate: 0.3 and 0.5 mL/h, voltage: 25 and 20 kV while the distances between the collector and the needles were 15 and 18 cm, for each of the polymers, respectively. Furthermore, plasma treatment was performed by a low frequency plasma generator of 40 kHz (Diener Electronics, Ebhausen, Germany) in order to modify surface characteristics of the 8|Page John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.

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scaffolds. For this purpose, pure oxygen was introduced into the reaction chamber with a pressure of 0.4 mbar and then the glow discharge was ignited for 3 minutes. In this study, six different scaffolds were designed (Table 2). Scaffold 1 contains microspheres without the GFs. Scaffold 2 and 3 contain rhEGF and rhbFGF respectively, with low and high burst release of each of them. Scaffold 4 contains both GFs with both low and high release approaches. Scaffold 5 has been designed for the high burst release of rhEGF with low burst release of rhbFGF, while scaffold 6, on the other hand, releases rhbFGF with a high burst release and rhEGF with a low burst release.

2.4.Characterization of Electrospun Scaffolds The morphology of the microspheres and the electrospun scaffolds were observed by field emission scanning electron microscopy (FESEM, S-4160, Hitachi, Japan). The specimens were sputter coated with gold prior to observation. The average diameter of the electrospun fibers and the pore size of the scaffolds were measured at 100 random locations using Image-J analysis software (NIH, Bethesda, MD, USA). Fourier transform infrared spectroscopic analysis of the scaffolds was conducted in the range of 400-4000 cm-1 using a Shimadzu FT-IR 4300 spectrometer (Shimadzu, Kyoto, Japan). Thermal characteristics of the scaffolds were studied using a differential scanning calorimetry (DSC, Mettler Toledo, Greifensee, Switzerland) equipped with a Julabo thermocryostate model FT100Y (Julabo Labortechnik, Seelbach, Germany). The equipment was calibrated with indium. The sample with a weight of approximately 5 mg was heated from 40 to 600 °C, at a constant temperature increase of 10◦C/min under nitrogen atmosphere.

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Tensile properties of the scaffolds were characterized by Instron Universal Testing Machine (STM-20, Santam, Tehran, Iran). The samples were cut into rectangular shapes with the dimensions of 6×1 cm and elongated at a rate of 10 mm/min and consequently their representative stress-strain curves were plotted.

2.5.Encapsulation efficiency of microspheres To evaluate the efficiency of protein encapsulation, total content of rhEGF and rhbFGF in the microspheres were determined through the extraction method. For this purpose, 10 mg of the freeze-dried microspheres were dissolved in 1 ml of DCM for 20 min, followed by adding 1ml of PBS into the solution and agitating it for 10 min in order to extract the protein into PBS from the organic phase. Afterwards, the solutions were centrifuged and the concentration of rhEGF and rhbFGF in the supernatant were assessed using rhEGF and rhbFGF enzyme-linked immunosorbent assay kits, respectively. The ratio of actual to theoretical protein loading was defined as encapsulation efficiency of the microspheres. 2.6.Dual release of proteins from microspheres and hybrid scaffold In order to determine the release profile of rhEGF and rhbFGF, the microspheres with a weight of 5 mg were immersed into 1 ml of PBS containing sodium azide and Tween 80, and the samples were placed in a shaker incubator at 100 rpm and 37 °C. At predetermined intervals, the medium was retrieved and an equal volume of fresh medium was replenished. The amounts of rhEGF and rhbFGF were ascertained by rhEGF and rhbFGF enzyme-linked immunosorbent assay kit, respectively. Similarly, in order to evaluate the release profile of the GFs from the

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scaffolds, the hybrid scaffolds with a weight of 50 mg were immersed into 1 ml of the medium and the procedure was pursued as mentioned above. 2.7.Cell seeding Human skin fibroblast (HSF) cell line (AGO1522) was provided from the National Cell Bank of Iran, Pasteur Institute. The cells were grown to confluence for 7 days (37 °C, 5% CO2) in the culture medium (high glucose DMEM, containing 10% fetal bovine serum albumin, 1% penicillin/streptomycin and 1% amphotericine). Afterwards, the cells were trypsinized, centrifuged and resuspended in DMEM, and the viable cells were counted. The surface treated scaffolds were cut into circular discs with an area of approximately 1.9 cm2 and immersed in 70% ethanol for 40 minutes and then washed with the culture medium to remove the alcohol residue. The cells were seeded on the sterilized scaffolds with a density of 5×103 cells /scaffold and were incubated at 37 °C for 1 h to promote cell attachment/adhesion. Then, 1 ml of culture medium was added into each well. The culture medium was changed every two days.

2.8.Cell viability assay The cell viability and proliferation on the scaffolds was assessed using MTT assay. This approach utilizes the evaluation of the metabolism of the cells during the cleavage of tetrazolium ring of MTT and the formation of blue crystals. At predetermined time intervals (2, 7, 14, 21 days), the scaffolds were washed with PBS to remove non-adherent cells and 10% MTT solution in DMEM was added followed by incubation for 3 h. Afterwards, the formazone crystals were dissolved in DMSO. The differential absorbance of the solutions at 540 and 630 nm were recorded. Furthermore, the calibration curve of the cell number and the absorbance was plotted at cell density of 1, 5, 25, 50, 70, 100, and 500 103×cell/ml/well. 11 | P a g e John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.

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2.9.Cell morphology Morphology of the cells on the scaffolds was observed at time points of 2, 7, 14 and 21 days of the culture by FESEM. The cell-seeded scaffolds were washed with PBS and fixed in 2.5% glutaraldehyde followed by dehydration in incremental concentrations of ethanol (50%, 70%, 80%, 90%, and 100%) for 15 min. Then, the specimens were dried at room temperature and coated with gold to be observed.

2.10. Cell staining The fibroblast cells on the scaffold on the 7th day of culture were stained with fluorescent 5chloromethylfluorescein diacetate (CMFDA, Cambrex, USA). Briefly, the cells were incubated with 5 mM of CMFDA for 120 min at 37 °C, followed by removing the dye and washing it with PBS. Then, the cells were incubated in the culture medium for 24 h, washed with PBS and observed by confocal laser scanning microscopy (Nikon Ti Eclipse, Tokyo, Japan) at a wavelength of 490 nm. Furthermore, for nuclear staining, the cultured cells on the scaffolds were fixed in 3.7% paraformaldehyde solution and were then stained with 4-6-diamidino-2phenylindole (DAPI) and observed by a fluorescent microscope (Nikon Eclipse TE2000-S, Tokyo, Japan) at 405 nm.

2.11. Real-time RT-PCR The effect of scaffold construction, as well as, rhEGF and rhbFGF release on the expression of ECM proteins including collagen type I, III, IV and elastin were assessed using real-time RTPCR. The total RNA was extracted from different lysed samples using Qiazol (Qiagen, Hilden,

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Germany) according to the manufacturer’s protocol of RNA extraction. Transcription of RNA to DNA was conducted using M-MulV Reverse Transcriptase kit (Vivantis, CA, USA) and random hexamer. The real-time RT-PCR reactions were performed based on the standard procedure in Rotor Gene 6000 (Corbett life science, Sydney, Australia): 95°C for 2 min (for initial enzyme activation) , thermal cycling (40 repeats, 95 °C for 5 sec and 62 °C for 20 sec). Gene expression was determined using SYBR-Green for RT-PCR (Rotor-Gene Q, Qiagen, Hilden, Germany). Statistical analyses were done by relative expression software tool (Rest©) and all the data were normalized with the house keeping gene of β actin (ACTB). The primers and product lengths are illustrated in Table 3. 2.12.

Statistical Analysis

All pairwise multiple comparison procedures with Holm-Sidak method followed by one way analysis of variance (one way ANOVA) have been done to show the differences between group means in MTT assay.

3. Results 3.1.Characterization of microspheres and scaffolds FESEM images showed the spherical shape of the microspheres with a smooth surface (Fig. 1 a). Moreover, Fig. 1. b-d illustrates the electrospun nanofibers with a uniform morphology. The average diameter of PLGA fibers and PEO/microsphere fibers were determined to be 280 and 760 nm, respectively. Electrospun fibers loaded with microspheres demonstrated thickened regions around the microspheres. The required space for the cells to reside is provided by the porous structure of the scaffolds made by tissue engineering. Therefore, the efficiency of the scaffold is largely dependent on 13 | P a g e John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.

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the attributes of this porous structure, including porosity, dimension, and volume of the pores [8]. The average pore diameter of the scaffolds was measured to be 2.7±0.8 µm. The FTIR spectrum of protein-encapsulated PLGA microspheres and microsphere-loaded scaffolds were compared with pure BSA as a model protein for the GFs, PLGA and PEO (Fig. 2). The characteristic peaks of carbonyl groups of PLGA near 1760 cm-1, amide bond and double bonded carbons of BSA at 1657 and 1540 cm-1, respectively, as well as, carbon bending of PEO near 840 cm-1 are all remained unchanged in PLGA/PEO nanofibers and microspheres. This implies the fact that there is no physicochemical interaction between the polymers and the protein. To assess the thermal characteristics of the scaffolds, differential scanning calorimetry (DSC) was utilized. A comparison between the DSC thermograms of scaffolds, polymers and microspheres corroborated the results that the peaks observed in case of PLGA/PEO nanofibers corresponded to the ones in the pure and mixed forms of the materials (Fig. 3). Fig. 4 illustrates the stress–strain curves of PLGA/PEO microsphere-loaded scaffold under tensile loading. Tensile modulus, ultimate tensile stress, and ultimate strain of the scaffold were 53.57±5.05 (MPa), 2.64±0.07 (MPa) and 91.75±8.11 (%), respectively. Generally, the results imply desirable mechanical properties of the electrospun scaffolds compared to natural skin which possess tensile modulus of 15 to 150 MPa and ultimate strain of 35 to 115% in order to be utilized as suitable skin graft [24]. 3.2.Release Study of Growth factors

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In the present study, PLGA microspheres were utilized as a vehicle for delivering rhEGF and rhbFGF. The size of the microspheres and their encapsulation efficiency have been summarized in Table 1. Cumulative release kinetics of rhEGF and rhbFGF have been presented in Fig. 5. In case of formulations A and C (rhEGF and rhbFGF), whose internal water phase contained PVA, an ultimately low burst accompanies the release on the first day (13.06±1.35 for rhEGF and 7.18±4.79 for rhbFGF) followed by the continuous release of approximately 22.56±2.41% and 32.48±4.48% over the following weeks. Nevertheless, in case of formulations B and D (rhEGF and rhbFGF, without PVA) the initial burst on day 1 was 49.07±2.34 for rhEGF and 55.82±0.80 for rhbFGF, while the continuous releases of 25.44±4.96% and 33.88±3.64% were obtained respectively, within the following three weeks. In case of all formulations, release measurement continued as long as three weeks, during which the microspheres were not totally degraded. Since the ultimate goal of this study was to prepare bioactive scaffolds with capability of different release rate of GFs, finally, the release profile of the GFs from the scaffolds embedded with the microspheres was investigated. For this purpose scaffolds with six different formulations were prepared. The cumulative percentages of the GFs release have been shown in Fig. 6 A-E. Respectively, for scaffolds 2 and 3 (Fig. 6 a and b), over 25.5±0.6% of rhEGF and 28.3±0.81% of rhbFGF were released in the first day and the scaffolds continued to release the GFs at an almost identical rate. Similarly, more than 26.6±0.34% of rhEGF and 27.6±1.52% of rhbFGF were released simultaneously from scaffold 4 (Fig. 6 c) in the first day, at almost the same rate. Scaffold 5 (Fig. 6 d) shows high initial burst releases of rhEGF, and low initial burst releases of rhbFGF, in the first day. Scaffold 6 illustrated the inverse behavior as compared to scaffold 5, through the 15 | P a g e John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.

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high initial burst release of rhbFGF and a trifle initial release of rhEGF (Fig. 6 e). Hence, in addition to scaffold 1 as a control with no GFs, and scaffolds 2 and 3, releasing rhEGF and rhbFGF alone, scaffold 4 was able to release two GFs simultaneously, while scaffolds 5 and 6 released them at different release rate. In fact, the gradients of GFs and cell competencies enable the regulation of tissue regeneration and recovery and therefore, these scaffolds simulate biological procedures in GF delivery. As a result, the approaches for delivering GFs mentioned here, have important implications in optimizing therapeutic interventions in tissue regeneration. 3.3.Cell Attachment and Metabolic Activity In this study, the viability and proliferation of HSF on PLGA/PEO scaffolds on days 2, 7, 14 and 21 was evaluated by MTT assay (Fig. 7). Generally, statistical analysis of data with Holm-Sidak followed by one way ANOVA showed that the proliferation of HSF was noticeably higher in case of PLGA/PEO scaffolds supplemented by rhEGF in the cases of singularly (scaffold 2), dual (scaffold 4) and different release rate (scaffold 5) [p3.5, >5 and >14 fold, respectively). The MTT assay of scaffold 5 demonstrated a 96% and 220% increase in the cell proliferation as compared to tissue culture polystyrene (TCPS) and scaffold 1, respectively on the 21st day [p In vivo and< i> in vitro applications. Advanced drug delivery reviews 2011;63:352-66. [2] Norouzi M, Shabani I, Ahvaz HH, Soleimani M. PLGA/gelatin hybrid nanofibrous scaffolds encapsulating EGF for skin regeneration. Journal of Biomedical Materials Research Part A 2014. [3] Ji W, Sun Y, Yang F, van den Beucken JJ, Fan M, Chen Z, et al. Bioactive electrospun scaffolds delivering growth factors and genes for tissue engineering applications. Pharmaceutical research 2011;28:1259-72. [4] Prabaharan M, Jayakumar R, Nair S. Electrospun nanofibrous scaffolds-current status and prospects in drug delivery. Biomedical applications of polymeric nanofibers: Springer; 2012. p. 241-62. [5] Li C, Vepari C, Jin H-J, Kim HJ, Kaplan DL. Electrospun silk-BMP-2 scaffolds for bone tissue engineering. Biomaterials 2006;27:3115-24. 21 | P a g e John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.

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[6] Norouzi M, Soleimani M, Shabani I, Atyabi F, Ahvaz HH, Rashidi A. Protein encapsulated in electrospun nanofibrous scaffolds for tissue engineering applications. Polymer International 2013;62:1250-6. [7] Yang D, Jin Y, Ma G, Chen X, Lu F, Nie J. Fabrication and characterization of chitosan/PVA with hydroxyapatite biocomposite nanoscaffolds. Journal of Applied Polymer Science 2008;110:3328-35. [8] Li WJ, Laurencin CT, Caterson EJ, Tuan RS, Ko FK. Electrospun nanofibrous structure: a novel scaffold for tissue engineering. Journal of biomedical materials research 2002;60:613-21. [9] Selcan Gungor‐Ozkerim P, Balkan T, Kose GT, Sezai Sarac A, Kok FN. Incorporation of growth factor loaded microspheres into polymeric electrospun nanofibers for tissue engineering applications. Journal of Biomedical Materials Research Part A 2014;102:1897-908. [10] Chen F-M, Zhang M, Wu Z-F. Toward delivery of multiple growth factors in tissue engineering. Biomaterials 2010;31:6279-308. [11] Yang Y, Xia T, Zhi W, Wei L, Weng J, Zhang C, et al. Promotion of skin regeneration in diabetic rats by electrospun core-sheath fibers loaded with basic fibroblast growth factor. Biomaterials 2011;32:424354. [12] Ojeh NO, Navsaria HA. An in vitro skin model to study the effect of mesenchymal stem cells in wound healing and epidermal regeneration. Journal of Biomedical Materials Research Part A 2014;102:2785-92. [13] He J-T, Su H-B, Li G-P, Tao X-M, Mo W, Song H-Y. Stabilization and encapsulation of a staphylokinase variant (K35R) into poly (lactic-< i> co-glycolic acid) microspheres. International journal of pharmaceutics 2006;309:101-8. [14] Ker ED, Chu B, Phillippi JA, Gharaibeh B, Huard J, Weiss LE, et al. Engineering spatial control of multiple differentiation fates within a stem cell population. Biomaterials 2011;32:3413-22. [15] Buket Basmanav F, Kose GT, Hasirci V. Sequential growth factor delivery from complexed microspheres for bone tissue engineering. Biomaterials 2008;29:4195-204. [16] Elisseeff J, McIntosh W, Fu K, Blunk T, Langer R. Controlled-release of IGF-I and TGF-β1 in a photopolymerizing hydrogel for cartilage tissue engineering. Journal of orthopaedic research 2001;19:1098-104. [17] Saiga K, Furumatsu T, Yoshida A, Masuda S, Takihira S, Abe N, et al. Combined use of bFGF and GDF5 enhances the healing of medial collateral ligament injury. Biochemical and biophysical research communications 2010;402:329-34. [18] Schneider A, Wang X, Kaplan D, Garlick J, Egles C. Biofunctionalized electrospun silk mats as a topical bioactive dressing for accelerated wound healing. Acta biomaterialia 2009;5:2570-8. [19] Choi JK, Jang J-H, Jang W-H, Kim J, Bae I-H, Bae J, et al. The effect of epidermal growth factor (EGF) conjugated with low-molecular-weight protamine (LMWP) on wound healing of the skin. Biomaterials 2012. [20] Tiede S, Ernst N, Bayat A, Paus R, Tronnier V, Zechel C. Basic fibroblast growth factor: a potential new therapeutic tool for the treatment of hypertrophic and keloid scars. Annals of AnatomyAnatomischer Anzeiger 2009;191:33-44. [21] Schweigerer L. Basic fibroblast growth factor as a wound healing hormone. Trends in pharmacological sciences 1988;9:427-8. [22] Gümüşderelioğlu M, Dalkıranoğlu S, Aydın R, Çakmak S. A novel dermal substitute based on biofunctionalized electrospun PCL nanofibrous matrix. Journal of Biomedical Materials Research Part A 2011;98:461-72. [23] Zou J, Yang Y, Liu Y, Chen F, Li X. Release kinetics and cellular profiles for bFGF-loaded electrospun fibers: Effect of the conjugation density and molecular weight of heparin. Polymer 2011;52:3357-67.

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[24] Kim G, Ahn S, Kim Y, Cho Y, Chun W. Coaxial structured collagen–alginate scaffolds: fabrication, physical properties, and biomedical application for skin tissue regeneration. Journal of Materials Chemistry 2011;21:6165-72. [25] Park CJ, Clark SG, Lichtensteiger CA, Jamison RD, Johnson AJW. Accelerated wound closure of pressure ulcers in aged mice by chitosan scaffolds with and without bFGF. Acta biomaterialia 2009;5:1926-36. [26] Ye M, Kim S, Park K. Issues in long-term protein delivery using biodegradable microparticles. Journal of Controlled Release 2010;146:241-60. [27] Mirdailami O, Khoshayand MR, Soleimani M, Dinarvand R, Atyabi F. Release optimization of epidermal growth factor from PLGA microparticles. Pharmaceutical development and technology 2013:1-9. [28] Yang Y-Y, Chung T-S, Ping Ng N. Morphology, drug distribution, and in vitro release profiles of biodegradable polymeric microspheres containing protein fabricated by double-emulsion solvent extraction/evaporation method. Biomaterials 2001;22:231-41. [29] Sun D, Bullock MR, Altememi N, Zhou Z, Hagood S, Rolfe A, et al. The effect of epidermal growth factor in the injured brain after trauma in rats. Journal of neurotrauma 2010;27:923-38. [30] Mujaj S, Manton K, Upton Z, Richards S. Serum-free primary human fibroblast and keratinocyte coculture. Tissue Engineering Part A 2010;16:1407-20. [31] Choi JS, Choi SH, Yoo HS. Coaxial electrospun nanofibers for treatment of diabetic ulcers with binary release of multiple growth factors. Journal of Materials Chemistry 2011;21:5258-67. [32] Norouzi M, Boroujeni SM, Omidvarkordshouli N, Soleimani M. Advances in Skin Regeneration: Application of Electrospun Scaffolds. Advanced Healthcare Materials 2015. [33] Kumbar SG, Nukavarapu SP, James R, Nair LS, Laurencin CT. Electrospun poly (lactic acid-< i> coglycolic acid) scaffolds for skin tissue engineering. Biomaterials 2008;29:4100-7. [34] Ulubayram K, Cakar AN, Korkusuz P, Ertan C, Hasirci N. EGF containing gelatin-based wound dressings. Biomaterials 2001;22:1345-56. [35] Huang S, Xu Y, Wu C, Sha D, Fu X. < i> In vitro constitution and< i> in vivo implantation of engineered skin constructs with sweat glands. Biomaterials 2010;31:5520-5. [36] Lee DY, Yang JM, Park KH. A dermal equivalent developed from fibroblast culture alone: Effect of EGF and insulin. Wound Repair and Regeneration 2007;15:936-9. [37] Lefler A, Ghanem A. Development of bFGF-chitosan matrices and their interactions with human dermal fibroblast cells. Journal of Biomaterials Science, Polymer Edition 2009;20:1335-51. [38] Sahoo S, Toh SL, Goh JC. A bFGF-releasing silk/PLGA-based biohybrid scaffold for ligament/tendon tissue engineering using mesenchymal progenitor cells. Biomaterials 2010;31:2990-8. [39] Tajima S, Izumi T. Differential in vitro responses of elastin expression to basic fibroblast growth factor and transforming growth factor β1 in upper, middle and lower dermal fibroblasts. Archives of dermatological research 1996;288:753-6.

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Fig. 1. FESEM images of microspheres (A) and PLGA/PEO microsphere-loaded scaffolds at a magnification of 500X(B), 2500X(C), and 5000X(D). 150x101mm (300 x 300 DPI)

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Fig.2. FTIR spectra of PLGA/PEO microsphere-loaded nanofibrous scaffolds 316x364mm (300 x 300 DPI)

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Fig.3. DSC profiles of PLGA/PEO microsphere-loaded scaffolds 163x147mm (300 x 300 DPI)

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Fig. 4. Representative Stress–strain curve of PLGA/PEO microsphere-loaded scaffolds 142x120mm (300 x 300 DPI)

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Fig. 5. Cumulative release of growth factors from different microspheres formulations. The results are presented as means ± standard deviation for n=3. 178x106mm (300 x 300 DPI)

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Fig. 6. Cumulative release of rhEGF( ◊) and rhbFGF(□)determined by ELISA, from scaffold 2(A),scaffold3(B),scaffold4(C),scaffold5(D) and scaffold6(E). The results are presented as means ± standard deviation for n=3. 233x162mm (300 x 300 DPI)

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Fig. 7. Proliferation of HSF seeded on PLGA/PEO scaffolds of varying growth factor loading. At each time point triplicate samples were measured and the average of HSF proliferation on all samples and tissue culture poly(styrene) (TCPS) on all days (A), on day-2 (B), on day-7 (C), on day-14 (D), and on day-21 (E) were determined and presented as means ± standard deviation (* and # indicate the statistical significance at p < 0.05). 248x135mm (300 x 300 DPI)

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Fig. 8. FESEM images of HSF seeded on PLGA/PEO scaffolds at the time points of 1 day and 10 days. Scaffold 1 at day 1 (A), scaffold 5 at day 1(B), Scaffold 1 at day 10 (C), and Scaffold 5 at day 10 (D), at a magnification of 400X. 165x122mm (300 x 300 DPI)

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Fig. 9. Attachment and proliferation of seeded HSF on scaffold 5 at various time points of day 2 (A), day 7 (B), day 14 (C), and day 21 (D). 165x108mm (300 x 300 DPI)

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Fig. 10. CSLM imaging (A) and fluorescent microscopy of DAPI-stained cells (B) and CMFDAstained cells (C), seeded on the surface of the scaffolds, after 3 days. The blue and green colors represent the nucleus and cytoplasm, respectively. 454x135mm (150 x 150 DPI)

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Fig. 11. Gene expression by HSF seeded on the scaffolds at day 7 of culture. Collagen I (A), collagen III (B), collagen IV (C), and elastin (D). The error bars represent one standard deviation from three replicate experiments and comparisons were made within the group (* indicates the statistical significance at p < 0.05). Scaffold without growth factor considered as the control group and the expression levels were normalized to the control group. 547x281mm (300 x 300 DPI)

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Table 1. Microspheres encapsulating growth factors with different formulations

rhbFGF(ug in

PVA in internal

rhEGF(ug in 100 100 mg

Formulation

Microsphere Microsphere

aqueous phase %

mg PLGA)

encapsulation size(um)

PLGA)

(w/v)

efficiency(%)

A

20

0.1%

6.44±2.45

97.04±1.13

B

20



5.05±0.26

77.69±4.06

C

20

0.1%

6.07±1.19

98.87±0.15

D

20



4.79±0.28

82.15±1.69

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Table 2. Composition of scaffolds with different growth factor delivery

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Scaffold

Growth Factor Delivery

Microsphere

Percent of

Formulation

Microspheres

Scaffold 1

No Delivery(as Control Group)

N‫٭‬

100%

Scaffold 2

Singular Delivery of rhEGF

A

50%

B

50%

C

50%

D

50%

A

25%

B

25%

C

25%

D

25%

B

50%

C

50%

A

50%

D

50%

Scaffold 3

Scaffold 4

Singular Delivery of rhbFGF

Dual Delivery of rhEGF & rhbFGF

Scaffold 5

Sequential Delivery of rhEGF then rhbFGF

Scaffold 6

Sequential Delivery of rhbFGF then rhEGF

‫٭‬Microspheres with no growth factor .

Table 3. Human primers used in RT-PCR

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Gene name

Accession number

Sequence

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Product size (bp)

1

Beta actin(ACTB)

NM_001101.3

F: CTT CCT TCC TGG GCA TG

85

R: GTC TTT GCG GAT GTC CAC 2

Collagen1a1 (COL1A1)

NM_000088.3

F: TGG AGC AAG AGG CGA GAG

121

R: CAC CAG CAT CAC CCT TAG C 3

Collagen3a1(COL3A1)

NM_000090.3

F: CCA GGT GCT GAT GGT GTC

158

R: ACC TCT CTC ACC AGG GCT 4

Collagen4a1(COL4A1)

NM_001845.4

F: AGA GCC TGG AGT TGG TCT

153

AC R: TTC ACC TCT GAT CCC CTG 5

Elastin(ELN)

NM_000501.2

F: AGG CTC CAG GTG TAG GTG R: TGT GGT GTA GGG CAG TCC

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136

Controlled release of rhEGF and rhbFGF from electrospun scaffolds for skin regeneration.

Controlled delivery of multiple therapeutic agents can be considered as an effective approach in skin tissue engineering. In this study, recombinant h...
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