PLGA/gelatin hybrid nanofibrous scaffolds encapsulating EGF for skin regeneration Mohammad Norouzi,1 Iman Shabani,2 Hana H. Ahvaz,3 Masoud Soleimani4 1

Department Department 3 Department 4 Department 2

of of of of

Nanotechnology and Tissue Engineering, Stem Cell Technology Research Center, Tehran, Iran Biomedical Engineering, Amirkabir University of Technology, Tehran, Iran Stem Cell Biology, Stem Cell Technology Research Center, Tehran, Iran Hematology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran

Received 26 June 2014; revised 20 September 2014; accepted 9 October 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35355 Abstract: The novel strategies of skin regenerative treatment are aimed at the development of biologically responsive scaffolds capable of delivering multiple bioactive agents and cells to the target tissues. In this study, nanofibers of poly(lacticco-glycolic acid) (PLGA) and gelatin were electrospun and the effect of parameters viz polymer concentration, acid concentration, flow rate and voltage on the morphology of the fibers were investigated. PLGA nanofibers encapsulating epidermal growth factor were also prepared through emulsion electrospinning. The core–sheath structure of the nanofibers was verified by transmission electron microscopy. The hemostatic attributes and the biocompatibility of the scaffolds for human

fibroblast cell were scrutinized. Furthermore, gene expression of collagen type I and type III by the cells on the scaffolds was quantified using real-time reverse transcriptase polymerase chain reaction. The results indicated desirable bioactivity and hemostasis of the scaffolds with the capability of encapsulation and controlled release of the protein which can be served as skin tissue engineering scaffolds and wound dressC 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A: ings. V 00A:000–000, 2014.

Key Words: nanofibrous scaffold, epidermal growth factor, skin tissue engineering, wound dressing, PLGA, gelatin

How to cite this article: Norouzi M, Shabani I, Ahvaz HH, Soleimani M. 2014. PLGA/gelatin hybrid nanofibrous scaffolds encapsulating EGF for skin regeneration. J Biomed Mater Res Part A 2014:00A:000–000.

INTRODUCTION

A wound is referred to as a defect in the skin’s structural or operational impeccability. The damage to the skin fall into several levels. The slightest damage associates with the injuries happening at the epidermis, the most external skin’s layer, in which the wound healing transpires through reepithelialization without any skin transplantation. A partial or complete injury to both dermal and subdermal tissues may be caused by more critical traumas. In this case, there is no residual origin of the cells for regeneration but the wound boundaries. Accordingly, the re-epithelialization is accomplished over a long period of time and may be problematic through the formation of scar. Recently, noticeable attempts have been made in tissue engineering to produce substitutes resembling human skin.1–4 The principal approach in tissue engineering involves fabrication of cell-possessed biodegradable polymeric scaffolds in order to prepare a three-dimensional temporal support for the cells and adjust their growth to the corresponding tissues. A desired scaffold in tissue engineering yields to simulate the extracellular matrix (ECM) characteristics. To this end, besides the simulation of the ECM topography and composition, the scaffold systems are also a

prerequisite to be integrated with macromolecular bioactive agents in order to control the processes of cell namely, migration, proliferation, and differentiation.5,6 The electrospun fibrous mats offer a spectrum of effective attributes as high-specific surface area, high aspect ratio, and high porosity along with small sized pore which are architecturally similar to the ECM structure.7,8 Wound healing is an interactive constant process consisting diverse mechanisms namely coagulation, inflammation, proliferation, tissue regeneration, and tissue remodeling. A great number of cells and factors influence the wound rehabilitation process and facilitate the tissue healing procedure.9,10 Along with these factors, growth factors (GFs) are crucial modulators orchestrating all wound healing occurrences. Cellular survival factors, namely, the epidermal growth factor (EGF), basic fibroblast GF, plateletderived GF, and vascular EGF incite cellular migration, proliferation, and angiogenesis which are indispensable for favorable wound rehabilitation and tissue regeneration.10,11 Amidst the aforementioned GFs being applied clinically, EGF has been the first and foremost factor used to deal with the wounds. EGF, a polypeptide made of 53 amino acids, possesses high-affinity receptors expressed in both

Correspondence to: M. Soleimani; e-mail: [email protected]

C 2014 WILEY PERIODICALS, INC. V

1

fibroblast and keratinocyte and increases the epidermal and mesenchymal regeneration, cell mobility, proliferation as well as synthesis of the ECM and consequently facilitates wound healing procedure.9,11 Notwithstanding, owing to the degradation in wound boundaries, enzymatically digested or deactivated, as well as the fast diffusion and dryness of the used GFs in the wound periphery; a direct topical use of GFs or cytokines has been found apart from outstanding improvements. Therefore, a variety of the GF topical delivery systems around the lesions have been proposed in order to enhance the wound rehabilitation.10,12 An efficient approach to apply EGF locally, preserve its bioactivity and also protect the tissue in the reconstruction phase can be the introduction of this molecule into a suitable dressing with the capability of sustained release of EGF, applied on top of the wound.9,11 Several studies have used nanofibrous scaffolds as delivery system for GFs. Schneider et al.11 reported that bioactive EGF was released from electrospun silk nanofibers prepared by blend electrospinning. Jin et al.13 encapsulated epidermal induction factors (EIF) within nanofibers through blend and core–sheath electrospinning. Superior proliferation and differentiation of adipose-derived stem cells were observed on the core–sheath nanofibers on account of the sustained release of EIF. In another study, EGF was conjugated on the surface of electrospun poly(E-caprolactone) nanofibers functionalized with amine groups and it was reported that the EGF-nanofibers improved the in vivo wound healing processes significantly, in comparison to the direct application of EGF solutions.14 Poly(lactic-co-glycolic acid) (PLGA) is an Food and Drug Administration-approved biocompatible polymer with appropriate physical and mechanical proprieties which have found wide application in tissue engineering and drug distribution systems.15,16 Moreover, gelatin, a hydrolyzed form of collagen, contains arginine-glycine-aspartic (RGD) acid sequences which enhance cell adhesion and migration and has some hemostatic attributes facilitating wound healing.15,17 The main purpose of the present study is to fabricate and evaluate the bioactive hybrid nanofibrous scaffolds of gelatin and PLGA, encapsulating recombinant human EGF, appropriate for skin tissue engineering and wound dressing applications. MATERIALS AND METHODS

Materials PLGA (PurasorbV, PLG8523) was acquired from Purac (The Netherlands). Chloroform, acetone, bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), glutaraldehyde, ethanol and span 80 were all purchased from Merck (Germany). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum, phosphate-buffered saline (PBS), trypsin, streptomycin, and amphotericin were obtained from Gibco. The recombinant human epidermal growth factor (rhEGF) was supplied from PeproTech. The enzyme-linked immunosorbent assay (ELISA, QuantikineV) Kit for EGF was purchased from R&D systems. The lactate dehydrogenized assay kit R

R

2

NOROUZI ET AL.

was acquired from BioVision. Gelatin derived from porcine skin, MTT (3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) reagent, 40 ,6-diamidino-2-phenylindole dihydrochloride (DAPI), and penicillin were obtained from Sigma-Aldrich. The HansaplastV wound dressing was provided from Beiersdorf (Germany). R

Preparation of electrospun nanofibers The PLGA solution was produced utilizing a polymer concentration of 4–6 wt % in a blend of chloroform/acetone (2/1 volume ratio). Although chloroform is an independent suitable solvent for PLGA, the system of the blended chloroform/acetone, each with respective dielectric constant of 4.81 and 21, facilitates the electrospinning process stemming from the high dielectric constant of acetone.16 The effects of electrospinning parameters including flow rate (0.4, 0.8, and 1.2 mL/h) and voltage (12, 16, and 20 kV) on the morphology of PLGA fibers were studied. The gelatin nanofibers were also electrospun from an aqueous acetic acid solution. Likewise, the effects of the polymer concentration (10, 15, and 20 wt %), concentration of acetic acid solution (20, 40, and 60%, v/v), flow rate (0.4, 0.8, and 1.2 mL/h), and voltage (12, 16, and 20 kV) on morphology of the gelatin nanofibers were investigated. The EGF solution with a concentration of 0.1 wt % considering the polymer weight in PBS (50 mM, BSA 1 wt %, pH 7.4) was added dropwise into the PLGA solution while the polymer solution was being stirred at 1000 rpm in order to prepare a water-in-oil (W/O) emulsion. Span 80 (5 wt %) was used as a nonionic surfactant to stabilize the emulsion. Several constituents including a high voltage power supply, a polymer solution reservoir, a digital syringe pump and a conductive collection device are involved in the electrospinning system. In order to subdue the liquid surface tension and provide the formation of polymer jet, a positive voltage is applied. The electrostatic repulsion extends and whips the resulting jet proceeding toward the collector and simultaneously the solidification is met. The electrospun nanofibers were gathered on an aluminum foil covering the cylindrical collector with a rotation speed of 100 rpm which was set at a distance of 10 cm from the tip of the 21-gauge needles. The process was conducted at ambient conditions (25 6 2 C and 50 6 5% relative humidity). In order to fabricate hybrid scaffold, the polymer solutions of PLGA/EGF and gelatin were electrospun simultaneously from two syringe needles in opposite directions facing a collector at the optimized condition of the process. A low frequency plasma generator set on 40 kHz (Diener Electronics, Germany) was employed for surface treatment of the scaffolds. The reaction chamber was exposed to the pure oxygen under a 0.4-mbar pressure, then the glow discharge was set on ignition for 5 min. The plasma treatment is one of the most efficient methods of enhancing the surface hydrophilicity which results in the improvement in cell attachment, expansion, and proliferation on the surface.18

PLGA/GELATIN HYBRID NANOFIBROUS SCAFFOLDS

ORIGINAL ARTICLE

Scaffold characterization A field emission scanning electron microscope (FESEM, Hitachi, S4160, Japan) with an accelerating voltage of 15 kV was employed in order to observe the morphology of the fibers. Prior to the observation, the specimens were sputter-coated with gold. The Image-J analysis software (National Institute of Health) was used to measure the average diameter of the fibers (approximately 100 fibers were measured for each sample). Also, the core–sheath structure of the emulsion electrospun PLGA/EGF composite nanofibers was observed with a transmission electron microscope (TEM; Zeiss, EMLOC, Germany) at an 80 kV accelerating voltage. The measurement of the tensile characteristics of the scaffolds was carried out using an Instron universal testing machine (STM-20; Santam Company, Iran) at ambient conditions. Three specimen of the each scaffold with length 3 width 5 50 mm 3 10 mm dimensions were prepared and elongated at a rate of 10 mm/min and the stress-strain curves were plotted. To evaluate hydrophilicity of the scaffolds, predetermined weight of the scaffolds were placed into 20 mL of PBS (pH 5 7.4) and incubated at 37 C for 1 h. After removing the membranes from PBS and blotting them with a filter paper to absorb excess water on the surface, the wet weight of the membranes was measured. The water absorption of the membranes in PBS was calculated by the following formula: ðW1 2W0 Þ Að%Þ5 3100% W0 where A is the PBS absorption, W1 and W0 are the weights of the membranes, before and after immersion in the medium, respectively. Blood collection and platelet isolation The fresh human blood was acquired from healthy volunteers and anticoagulated with acid citrate dextrose (20 mM citric acid, 110 mM sodium citrate, and 5 mM D-glucose) at a ratio of 9:1 v/v. The whole blood was centrifuged at 180g for 20 min and the platelet-rich plasma was separated from the red blood cell (RBC) fraction and further centrifuged at 1500g for 15 min to isolate the platelets. The platelet pellet was taken and resuspended in a buffer (140 mM NaCl, 3 mM KCl, 12 mM NaHCO3, 0.4 mM NaH2PO4, 0.1% glucose, pH 7.4, regulated with 4% HEPES). The concentration of the platelet suspension was ascertained using an inverted microscope and the suspensions with >100,000 platelets/ mL were used in the forthcoming trials. Blood clotting The blood clotting assay was proposed by Shih et al.19 Briefly, the scaffolds were pre-heated to 37 C. Then, the entire citrated blood (0.2 mL) was applied on the mats and subsequently 20 mL of CaCl2 (0.2 M) solution was added to commence the coagulation. The samples were incubated at 37 C and shaken at 30 rpm for 10 min. The RBCs, not entrapped in the clot, were hemolyzed by 5 mL of water

and the optical density (OD) of the resulting hemoglobin solution was recorded at 540 nm. Platelet adhesion The platelet adhesion test was adopted from Vanickova et al.20 At first, the platelets were reconstituted with CaCl2 (2.5 mM) and MgCl2 (1.0 mM). Then, perdressing, 1.5 mL of the platelet suspension was added and they were incubated at 37 C for 1 h. Afterward, the dressings were dip-rinsed twice in PBS to eliminate the unattached platelets. In order to lyse the adhered platelets, the samples were placed in PBS containing 0.9% Triton-X100 for 1 h at 37 C. The released lactate dehydrogenase enzyme was measured using the kit according to the instructions of the manufacturer. Protein release in vitro The hybrid scaffolds with 10 6 1 mg weight were immersed in 1 mL of PBS containing 1% penicillin/streptomycin and 1% amphotericin as biocides and were then incubated at 37 C, in order to measure the amount of the released EGF. A 100 lL of the medium was recovered at the outlined intervals and an equal volume of the fresh medium was replenished. The EGF concentration was quantified using ELISA kit, specific for human EGF, on the basis of the manufacturer’s instruction. Cell isolation and culture Postnatal human foreskin tissue was obtained from Sarem hospital concerning the bioethics germane to the possession and usages of human tissue. The tissue was washed with PBS. After mincing the tissue into tiny pieces, they were suspended in PBS containing 0.5% trypsin and incubated along with gentle shaking at 37 C for 30 min and then centrifuged to collect and disperse the cells from the undigested tissue and debris. Subsequently, the cells were cultured and extended in the DMEM complemented with 10% FBS. Afterward, the cells at the second passage were seeded on the surface of the UV-sterilized scaffolds at a 10,000 cells/cm2 concentration. The cultured specimens were incubated at 37 C with 5% CO2 and humidity of 95%. The cell culture medium was replaced every 2 days. MTT assay In order to assess the cell viability and proliferation, the MTT colorimetric assay was applied. The premise of MTT assay is based on the assessment of the cellular metabolic activities through the cleavage of the MTT tetrazolium ring and formation of the formazone blue crystals. To this end, 300 mL of MTT solution was added to each well at specific times, then incubated for 3 h. Subsequently, in order to dissolve the formazone crystals, the solution was removed and each well was supplied with 1000 mL of DMSO. After shaking, the solution was recovered and the OD540 of the solution was recorded using an ELISA Reader (BioTek). Histological analysis On day 5 of cell culture, the seeded scaffolds were prepared for histological analyses. Briefly, the samples were fixed

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2014 VOL 00A, ISSUE 00

3

FIGURE 1. Effect of electrospinning parameters on diameter of PLGA nanofibers. (a) Polymer concentration, (b) flow rate, and (c) voltage. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

with formaldehyde and embedded in paraffin. Then, they were sectioned and stained with hematoxylin and eosin (H&E) or Masson’s trichrome. The sections were mounted on glass slides and analyzed by a light microscope (Labomed). Real-time reverse transcriptase polymerase chain reaction The effects of the scaffolds and the released rhEGF on the expression of the ECM proteins, namely collagen type I and collagen type III were quantified using the real-time reverse transcriptase polymerase chain reaction (RT-PCR) method. The total RNA extraction was performed using Qiazole reagent (Qiagen, Germany) according to the manufacturer’s protocol. The reverse transcription was carried out using the M-MulV reverse transcriptase (Vivantis, Malaysia) and random hexamer (Fermentas). The real-time RT-PCR reactions were conducted in the rotor gene 6000 (Corbett life science). The gene expression was ascertained by the SYBRGreen for RT-PCR (Rotor-Gene Q, Qiagen). The statistical analyses were calculated by the relative expression software tool (RestV) and all the data was normalized with the housekeeping gene of b2M. C

RESULTS AND DISCUSSION

Effects of electrospinning parameters on fiber morphology There are a number of factors which influence the cell adhesion, migration, and proliferation, namely diameter and

4

NOROUZI ET AL.

orientation of nanofibers, porosity, pore size, mechanical properties, and biodegradability which must be taken into consideration in scaffold design. The geometrical and structural properties of the scaffolds in electrospinning can easily be controlled by altering the attributes of the polymer solution and the processing parameters.4,21 Effect of polymer and acid concentration The spinnability of a solution is dependent on the polymer concentration which by turns is based on the solution viscosity. As an increase in the concentration occurred, the viscosity of the polymer solution raised. Hence, an optimum concentration for the occurrence of an entanglement in the polymer chain and also the formation of a stable polymer jet is required.22,23 In order to study the effect of the polymer concentration on the morphology of the electrospun fibers, polymer concentration of PLGA was altered from 4 to 6 wt %, while, the other parameters, that is, voltage (16 kV), flow rate (0.4 mL/h) and collecting distance (10 cm) remained constant. As shown in Figure 1(a), the polymer concentration plays a dominant role in determining the fiber morphology. Increasing the polymer concentration, a significant rise happened in the average diameter of the fibers. Similarly, various concentrations of gelatin solution in the range of 5–20 wt % were electrospun while the other parameters were kept constant. As the polymer concentration increased, as a result of an increase in the solution viscosity,23 the average diameter of the fibers raised significantly [Fig. 2(a)]. Gelatin microspheres were formed

PLGA/GELATIN HYBRID NANOFIBROUS SCAFFOLDS

ORIGINAL ARTICLE

FIGURE 2. Effect of electrospinning parameters on diameter of gelatin nanofibers. (a) Polymer concentration, (b) concentration of acetic acid solution, (c) flow rate, and (d) voltage. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

at a low concentration (less than 10%) because the gelatin solution was electrosprayed [Fig. 3(c)]. In fact, having a greater effect of the surface tension than the viscoelastic force, the microspheres or beads were formed at a lower concentration of the polymer solution. As a matter of fact, a competition between the surface tension and viscosity may result in the evolution of smooth fibers from the beads or

beads-on-string structures. However, at higher concentrations of the polymer solution, in which viscoelastic forces preceded the surface tension, the formation of beads diminished and eventually halted.23 Additionally, in order to study the effect of acetic acid concentration on the electrospinnability and morphology of the fibers, gelatin solutions were electrospun in various acid

FIGURE 3. FESEM images of: (a) PLGA scaffold; (b) gelatin scaffold, (c) gelatin microspheres, and (d) hybrid scaffold (PLGA/EGF/gelatin).

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2014 VOL 00A, ISSUE 00

5

In summary, polymer concentration of 4.5 and 15 wt %, applied voltage of 16 kV and flow rate of 0.4 mL/h, were selected as the optimized conditions to produce uniform nanofibers of PLGA and gelatin, respectively.

FIGURE 4. TEM image of PLGA/EGF nanofiber.

concentrations, namely 20, 40, and 60% (v/v). It can be concluded that a higher concentration of acetic acid solution results in an increased average diameter of the fibers [Fig. 2(b)]. This may be related to the more volatility of acetic acid leading to a faster solidification of the polymer jet before it is exposed to a more elongational force and also, the higher viscosity of acetic acid (1.22 mPa s) compared to the viscosity of water (1.00 mPa s) resulting in the higher viscosity of the polymer jet. Uniform fibers were formed in acidic solutions with concentrations higher than 10% (v/v), and in this study 20% was selected as the optimized concentration. Effect of flow rate Another parameter which influences the morphology of the electrospun fibers is the flow rate of the polymer solution. The formation and maintenance of the Taylor cone at the top of the capillary as well as evaporation of the solvent before reaching the collector require sufficient time.22 The relationship between the average diameter of the fibers and flow rate of the polymer solution was studied. As the feeding rate increased from 0.4 to 1.2 mL/h, average diameter of PLGA and PLGA/EGF fibers was also raised on the account of the fact that a greater volume of the solution was ejected from the spinneret [Fig. 1(b)]. The same behavior was observed for the gelatin nanofibers. As the flow rate of the polymer increased from 0.4 to 1.2 (mL/h), average diameter of the fibers grew briefly from 175 6 15 to 208 6 22 nm [Fig. 2(c)]. Effect of voltage The applied voltage is the other parameter which affects the morphology of the fibers formed during the electrospinning. Low applied voltage leads to a failure in the jet formation or a beaded morphology.22 In order to study the effect of voltage, it was increased from 12 to 20 kV. By increasing the voltage, the average diameter of PLGA [Fig. 1(c)] and gelatin [Fig. 2(d)] fibers increased briefly which can be related to a rise in the ejected volume of the polymer solution from the spinneret.

6

NOROUZI ET AL.

Characterization of scaffolds The morphology of PLGA and gelatin nanofibers as well as the hybrid scaffold (PLGA/EGF/gelatin) in the optimized conditions of the process is illustrated in Figure 3. Each PLGA and gelatin scaffold was formed of randomly oriented fibers with a uniform and smooth morphology. Average diameter of PLGA fibers [Fig. 3(a)] was 630 6 80 nm, while it decreased to 390 6 75 nm as the protein and span 80 were introduced into the solution. In fact, polymer jet which carries an extra charge density, faces more elongational force, leading to formation of finer fibers.24 Moreover, gelatin nanofibers held the average diameter of 175 6 45 nm [Fig. 3(b)]. The morphology of the hybrid scaffold of PLGA/ EGF/gelatin (PLGA:gelatin 50:50) is shown in Figure 3(d). The core–sheath structure of the PLGA/EGF nanofibers is depicted in Figure 4. The dark phase implies the protein encapsulated in the core of the nanofibers.25–27 In fact, the immiscible nature of the two organic and aqueous phases and also, the fast solidification process of the jet prohibiting a significant mixing of the two fluids, results in the development of the core–sheath structure. Since chloroform and acetone evaporate quickly, the viscosity of the jet’s outer layer increases faster than the inner one. Consequently, the droplets of the aqueous phase migrate to the jet’s core as a result of the viscosity gradient. On the other hand, throughout the electrospinning, the emulsion droplets with a spherical form, undergo substantial elongation and their spherical layout is broadly disappeared during their elongation and a continuous aqueous phase is formed.25,28 The mechanical properties are considered as one of the main factors in the application of nanofibrous scaffolds. PLGA and PLGA/EGF nanofibrous scaffolds showed ultimate tensile stresses of 1.3 6 0.2 and 0.8 6 0.2 MPa, respectively. In fact, adding the protein to the nanofibers led to a tensile strength reduction. The weak interfaces of the protein and

FIGURE 5. Representative tensile stress–strain curves of PLGA, PLGA/ EGF, gelatin, and hybrid nanofibrous scaffolds. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary. com.]

PLGA/GELATIN HYBRID NANOFIBROUS SCAFFOLDS

ORIGINAL ARTICLE

FIGURE 6. Blood clotting properties of the scaffolds. All the error bars represent one standard deviation from three replicate experiments. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

polymer resulted in the formation of voids and cracks in the fiber structure under tension and consequently failure in the fiber (Fig. 5). The ultimate tensile strength of the gelatin and hybrid scaffolds was significantly higher than PLGA, while their elongation was lower than that, and this is attributed to the different mechanical behavior of gelatin and PLGA. The hybrid scaffold offers ultimate tensile strength and strain of 1 6 0.2 MPa and 120 6 15%, respectively, which

desirably mimics the mechanical attributes of human skin’s fibrillar structure. Collagen fibers which provide strength of the human skin are known by their high strength (tensile strength of 1.5–3.5 MPa) while elastin fibers, the second main component of the dermis, which provide the elasticity of the skin, show reversible strain of more than 100%.29 The exudate drainage ability of the scaffolds was evaluated through the water absorption in PBS. The water absorption of PLGA/gelatin and PLGA scaffolds was 130 6 10 and 23 6 4%, respectively. The higher ability of the hybrid scaffold to absorb water, is attributed to the hydrophilicity of the gelatin which offers the potential ability of gelatin to absorb the exudates of the wound. Additionally, the capillary effect of nanofibrous scaffolds results in the water absorption.15 It was not possible to measure the PBS uptake of the gelatin scaffold due to the high swelling and partially solving. Blood clotting and platelet adhesion One of the most usual causes of the patient’s death is uncontrolled bleeding of wounds. Therefore, in order to control the wound bleeding with large amounts of skin loss, as well as protect the wound and accelerate its healing, immediate coverage with a suitable dressing is required.30,31 Hemostatic agents are also used extensively in surgical procedures and postoperative hemorrhage.32 The hemostatic

FIGURE 7. Clotted blood on the membranes after washing. (a) Hansaplast, (b) PLGA, (c) hybrid, and (d) gelatin. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2014 VOL 00A, ISSUE 00

7

FIGURE 8. Number of platelet adhered to the scaffolds. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

feature of the scaffolds is shown in Figure 6. The higher absorbance of the hemoglobin solution indicates the slower clotting rate. Therefore, the gelatin nanofibrous scaffold showed the best blood clotting performance, followed by PLGA/gelatin hybrid scaffold, while Hansaplast, a commercial wound dressing, was not able to induce a significant blood clotting. Also, Figure 7 shows the clotted blood on the scaffolds after washing which indicates that the blood was absorbed and clotted efficiently on the gelatin and hybrid scaffolds, while PLGA scaffold ascribed to its hydrophobic nature as well as Hansaplast did not absorb the blood effectively. Additionally, Figure 8 demonstrates the platelet adhesion to the scaffolds. Gelatin scaffolds showed the highest amount of the adhered platelets followed by PLGA/gelatin scaffold, while Hansaplast indicated the lowest amount of that. Hence, it is concluded that the gelatin and the hybrid scaffolds can be good candidates as the hemostatic bandages in bleeding. In fact, gelatin induces hemostasis through accelerated formation of thrombus and providing structural support for the forming clot.33 In vitro protein release Figure 9 shows the cumulative release of EGF from PLGA/ gelatin scaffold. The release profile consists of two stages:

FIGURE 9. Release profiles of EGF from the hybrid scaffold. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

8

NOROUZI ET AL.

FIGURE 10. MTT assay of human fibroblast. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary. com.]

the burst release in the first day, followed by the sustained release over the next days. The first stage is explainable by diffusion of the protein located on or near the surface of the fibers, while the second one is ascribed to the diffusion of the protein, which is in the interior of the fibers. Generally, two possible approaches have been suggested for drug release, namely diffusion depending on concentration gradient and polymer degradation which occurs in a longer period of time.34,35 While the gradient of EGF concentration decreases between the core and the surface, the rate of the protein release reduces gradually. Moreover, it was expected to take a longer time for the remaining encapsulated EGF to be released. In the current study, the effective released concentration of EGF was estimated to be 4.2 6 0.2 ng/mL on the ninth day. The relationship between the optimal EGF concentration and matrix strength as well as thickness was studied by Throm et al.36 It was proved that the formation of a strong ECM can be efficiently promoted by 0.5–5.0 ng/mL of EGF in 3 weeks. Furthermore, in order to protect the body specifically against microbes and water loss, the reepithelialization of wounds is required to be started with a significant rate immediately after an injury occurs. Therefore, the burst release of EGF from the scaffold is of great importance in stimulating the keratinocytes on the edge of the ulcer.10,11

Cell attachment and metabolic activity PLGA is a biocompatible and biodegradable polymer which owns some suitable mechanical characteristics. However, due to the lack of cell binding sites and hydrophobicity, cell adherence on PLGA is insufficient which necessities additional modifications such as introducing ECM proteins in the scaffold.15,21,37 Therefore, gelatin, which contains RGD (Arg-Gly-Asp) sequence, was inserted to recognize the cell’s integrin receptors and facilitate cell attachment, migration as well as proliferation. Moreover, the gelatin constitution of the hybrid scaffold is gradually dissolved throughout the time of cell culture providing more space for cell

PLGA/GELATIN HYBRID NANOFIBROUS SCAFFOLDS

ORIGINAL ARTICLE

FIGURE 11. FESEM images of cell morphology on (a) PLGA and (b) hybrid nanofibrous scaffolds.

migration.15,38 As shown in Figure 10 the hybrid scaffold which releases EGF in a sustained manner seemed more beneficial than the other scaffolds and TCPs in which it proved a better cell proliferation. The effect of EGF on increased cell proliferation and mitosis has been reported.9,14,39 Also, gelatin increased cell proliferation in the hybrid scaffolds as mentioned before. Therefore, the PLGA and hybrid scaffold were selected for the microscopic and histological analyses. The morphology of the cells cultured on PLGA and PLGA/EGF/gelatin scaffolds was observed by FESEM on the fifth day. More flattering and polygonal extensions of the fibroblasts were perceived on the hybrid scaffold (Fig. 11). In addition, the fluorescence microscopic images of the stained cells with DAPI nuclear stain are illustrated in Figure 12 which indicates extensive cell attachment and confluent coverage on the entire scaffold surface. The histological analysis of the cell-seeded scaffolds, stained with H&E, proves the confluent coverage of the cells on the surface in addition to the proliferation throughout the PLGA/EGF/gelatin scaffold in comparison to PLGA (Fig. 13). As mentioned earlier, the gelatin nanofibers in the hybrid scaffold is gradually dissolved during the time of cell culture providing more space for cell migration and infiltration. Other studies have reported that collagen can enhance cell infiltration into the composite scaffolds to form a 3D structure of cells and nanofibers.40,41 Moreover, the blue color in the sample stained with Masson’s trichrome

(Fig. 14) represents collagen fibrils produced by fibroblasts which are more abundant on the surface of the hybrid scaffold, while no collagen fibril was observed on the surface of PLGA scaffold (data not shown). In fact, EGF is known to increase the synthesis of the ECM proteins by fibroblast.39,42 Real time RT-PCR The expression of collagen type I and III by the human fibroblast cultured on PLGA/EGF/gelatin scaffold showed significantly higher values than that cultured on PLGA/gelatin scaffold at day 7 (Fig. 15). The expression level of target genes on the scaffolds were calibrated to gene expression of cells on the TCPS. In fact, during the early phases of wound healing, the expression of collagen type I and type III is required to increase through the dermis. EGF stimulates fibroblasts to synthesize new collagen and deposit ECM in the wound site.43 The significant increase in expression of collagen type I in comparison to the control, will have positive effects on the matrix deposition and wound healing. Additionally, notable increase in expression of collagen type III is an indication for the formation of granulated tissue during the process of wound healing.43 CONCLUSION

In this study, nanofibers of PLGA and gelatin were electrospun and the effects of operational parameters on

FIGURE 12. Fluorescence microscopic images of cells on (a) PLGA and (b) hybrid nanofibrous scaffolds. 403 magnification. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2014 VOL 00A, ISSUE 00

9

FIGURE 13. H&E-stained cross-sections of the scaffolds (a and b) PLGA and (c and d) PLGA/EGF/gelatin. (a and c) 203 and (b and d) 403. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

morphology of the nanofibers were investigated. In the optimum conditions, average diameter of PLGA, PLGA/EGF and gelatin fibers was 630 6 80, 390 6 75, and 175 6 45 nm, respectively. The core–sheath structure of the nanofibers encapsulating EGF was confirmed by TEM. The hybrid scaffold showed the similar mechanical behavior of the fibrillar collagen and elastin in the human skin. The release profile of EGF demonstrated a sustained release of the protein in the optimum range of concentration required for wound healing. The gelatin and hybrid scaffolds exhibited excellent blood clotting and platelet

adhesion in comparison to the commercial wound dressing. MTT results of human fibroblast indicated an increased cell proliferation on the PLGA/EGF/gelatin scaffold. Also, histological analyses confirmed cell infiltration into the hybrid scaffold as well as collagen synthesis. Furthermore, the hybrid scaffold exhibited a significantly higher gene expression of collagen type I and III. The capability of encapsulation and controlled release of the protein with desirable bioactivity and hemostasis of the scaffolds show their potential applications in skin tissue engineering and wound dressing.

FIGURE 14. Masson’s Trichrome-stained cross-section of the PLGA/ EGF/gelatin scaffold. 203. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

FIGURE 15. Relative gene expression on the scaffolds. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

10

NOROUZI ET AL.

PLGA/GELATIN HYBRID NANOFIBROUS SCAFFOLDS

ORIGINAL ARTICLE

REFERENCES 1. Singer AJ, Hollander JE, Blumm RM. Skin and Soft Tissue Injuries and Infections: A Practical Evidence Based Guide. Shelton, CT: PMPH-USA; 2010. 2. Groeber F, Holeiter M, Hampel M, Hinderer S, Schenke-Layland K. Skin tissue engineering—In vivo and in vitro applications. Adv Drug Deliv Rev 2011;63:352–366. 3. Ravichandran R, Venugopal JR, Sundarrajan S, Mukherjee S, Sridhar R, Ramakrishna S. Composite poly-L-lactic acid/poly-(a, b)-DL-aspartic acid/collagen nanofibrous scaffolds for dermal tissue regeneration. Mater Sci Eng C 2012;32:1443–1451. 4. Chong E, Phan T, Lim I, Zhang Y, Bay B, Ramakrishna S, Lim C. Evaluation of electrospun PCL/gelatin nanofibrous scaffold for wound healing and layered dermal reconstitution. Acta Biomater 2007;3:321–330. 5. Meng D, Erol M, Boccaccini AR. Processing technologies for 3D nanostructured tissue engineering scaffolds. Adv Eng Mater 2010; 12:B467–B87. 6. Lu Y, Jiang H, Tu K, Wang L. Mild immobilization of diverse macromolecular bioactive agents onto multifunctional fibrous membranes prepared by coaxial electrospinning. Acta Biomater 2009; 5:1562–1574. 7. Babaeijandaghi F, Shabani I, Seyedjafari E, Naraghi ZS, Vasei M, Haddadi-Asl V, Hesari KK, Soleimani M. Accelerated epidermal regeneration and improved dermal reconstruction achieved by polyethersulfone nanofibers. Tissue Eng Part A 2010;16:3527–3536. 8. Liao Y, Zhang L, Gao Y, Zhu Z-T, Fong H. Preparation, characterization, and encapsulation/release studies of a composite nanofiber mat electrospun from an emulsion containing poly(lactic-coglycolic acid). Polymer 2008;49:5294–5299. 9. Choi JK, Jang J-H, Jang W-H, Kim J, Bae I-H, Bae J, Park Y-H, Kim BJ, Lim K-M, Park JW. The effect of epidermal growth factor (EGF) conjugated with low-molecular-weight protamine (LMWP) on wound healing of the skin. Biomaterials 2012;33:8579–8590. 10. Yang Y, Xia T, Zhi W, Wei L, Weng J, Zhang C, Li X. Promotion of skin regeneration in diabetic rats by electrospun core–sheath fibers loaded with basic fibroblast growth factor. Biomaterials 2011;32:4243–4254. 11. Schneider A, Wang X, Kaplan D, Garlick J, Egles C. Biofunctionalized electrospun silk mats as a topical bioactive dressing for accelerated wound healing. Acta Biomater 2009;5:2570–2578. 12. Biondi M, Ungaro F, Quaglia F, Netti PA. Controlled drug delivery in tissue engineering. Adv Drug Deliv Rev 2008;60:229–242. 13. Jin G, Prabhakaran MP, Kai D, Ramakrishna S. Controlled release of multiple epidermal induction factors through core–shell nanofibers for skin regeneration. Eur J Pharm Biopharm 2013;85:689–698. 14. Choi JS, Leong KW, Yoo HS. In vivo wound healing of diabetic ulcers using electrospun nanofibers immobilized with human epidermal growth factor (EGF). Biomaterials 2008;29:587–596. 15. Meng Z, Wang Y, Ma C, Zheng W, Li L, Zheng Y. Electrospinning of PLGA/gelatin randomly-oriented and aligned nanofibers as potential scaffold in tissue engineering. Mater Sci Eng C 2010;30: 1204–1210. 16. You Y, Lee SJ, Min BM, Park WH. Effect of solution properties on nanofibrous structure of electrospun poly(lactic-co-glycolic acid). J Appl Polym Sci 2006;99:1214–1221. 17. Domb AJ, Kumar N. Biodegradable Polymers in Clinical Use and Clinical Development. Hoboken, New Jersey: John Wiley & Sons; 2011. 18. Chandrasekaran AR, Venugopal J, Sundarrajan S, Ramakrishna S. Fabrication of a nanofibrous scaffold with improved bioactivity for culture of human dermal fibroblasts for skin regeneration. Biomed Mater 2011;6:015001. 19. Shih M-F, Shau M-D, Chang M-Y, Chiou S-K, Chang J-K, Cherng J-Y. Platelet adsorption and hemolytic properties of liquid crystal/ composite polymers. Int J Pharm 2006;327:117–125. 20. Vanickova M, Suttnar J, Dyr JE. The adhesion of blood platelets on fibrinogen surface: Comparison of two biochemical microplate assays. Platelets 2006;17:470–476. 21. Heydarkhan-Hagvall S, Schenke-Layland K, Dhanasopon AP, Rofail F, Smith H, Wu BM, Shemin R, Beygui RE, MacLellan WR. Threedimensional electrospun ECM-based hybrid scaffolds for cardiovascular tissue engineering. Biomaterials 2008;29:2907–2914.

22. Dhandayuthapani B, Krishnan UM, Sethuraman S. Fabrication and characterization of chitosan-gelatin blend nanofibers for skin tissue engineering. J Biomed Mater Res Part B Appl Biomater 2010;94:264–272. 23. Gu S-Y, Wang Z-M, Ren J, Zhang C-Y. Electrospinning of gelatin and gelatin/poly(L-lactide) blend and its characteristics for wound dressing. Mater Sci Eng C 2009;29:1822–1828. 24. Yan S, Xiaoqiang L, Lianjiang T, Chen H, Xiumei M. Poly(L-lactide-co-E-caprolactone) electrospun nanofibers for encapsulating and sustained releasing proteins. Polymer 2009;50:4212–4219. 25. Norouzi M, Soleimani M, Shabani I, Atyabi F, Ahvaz HH, Rashidi A. Protein encapsulated in electrospun nanofibrous scaffolds for tissue engineering applications. Polym Int 2013;62:1250–1256. 26. Zhang H, Zhao Y, Han F, Wang M, Yuan X. Controlled release of bovine serum albumin from electrospun fibrous membranes via an improved emulsion-core technique. J Controlled Release 2011; 152:e181–e2. 27. Yang Y, Li X, Cui W, Zhou S, Tan R, Wang C. Structural stability and release profiles of proteins from core–shell poly(DL-lactide) ultrafine fibers prepared by emulsion electrospinning. J Biomed Mater Res Part A 2008;86:374–385. 28. Yan S, Xiaoqiang L, Shuiping L, Xiumei M, Ramakrishna S. Controlled release of dual drugs from emulsion electrospun nanofibrous mats. Colloids Surf B: Biointerfaces 2009;73:376–381. 29. Hendriks F. Mechanical behaviour of human skin in vivo. Biomed Eng 1969;4:322–327. 30. Gupta B, Edwards J. Textile materials and structures for wound care products. Adv Text Wound Care 2009;85. 31. Spasova M, Paneva D, Manolova N, Radenkov P, Rashkov I. Electrospun chitosan-coated fibers of poly(L-lactide) and poly(L-lactide)/poly(ethylene glycol): Preparation and characterization. Macromol Biosci 2008;8:153–162. 32. Barnard J, Millner R. A review of topical hemostatic agents for use in cardiac surgery. Ann Thorac Surg 2009;88:1377–1383. 33. Totre J, Ickowicz D, Domb AJ. Properties and hemostatic application of gelatin. In: Domb AJ, Neeraj Kumar, editors. Biodegradable Polymers in Clinical Use and Clinical Development. NJ: Wiley; 2011. p 91–109. 34. Szentivanyi A, Chakradeo T, Zernetsch H, Glasmacher B. Electrospun cellular microenvironments: Understanding controlled release and scaffold structure. Adv Drug Deliv Rev 2011;63:209– 220. 35. Hong KH, Woo SH, Kang TJ. In vitro degradation and drugrelease behavior of electrospun, fibrous webs of poly(lactic-coglycolic acid). J Appl Polym Sci 2012;124:209–214. 36. Throm AM, Liu WC, Lock CH, Billiar KL. Development of a cellderived matrix: Effects of epidermal growth factor in chemically defined culture. J Biomed Mater Res Part A 2010;92:533–541. 37. Wu L, Li H, Li S, Li X, Yuan X, Li X, Zhang Y. Composite fibrous membranes of PLGA and chitosan prepared by coelectrospinning and coaxial electrospinning. J Biomed Mater Res Part A 2010;92: 563–574. 38. Zhang Y, Ouyang H, Lim CT, Ramakrishna S, Huang ZM. Electrospinning of gelatin fibers and gelatin/PCL composite fibrous scaffolds. J Biomed Mater Res Part B: Appl Biomater 2005;72:156– 165. 39. Yu A, Matsuda Y, Takeda A, Uchinuma E, Kuroyanagi Y. Effect of EGF and bFGF on fibroblast proliferation and angiogenic cytokine production from cultured dermal substitutes. J Biomater Sci Polym Ed 2012;23:1315–1324. 40. Shabani I, Haddadi-Asl V, Soleimani M, Seyedjafari E, Babaeijandaghi F, Ahmadbeigi N. Enhanced infiltration and biomineralization of stem cells on collagen-grafted three-dimensional nanofibers. Tissue Eng Part A 2011;17:1209–1218. 41. Chiu JB, Liu C, Hsiao BS, Chu B, Hadjiargyrou M. Functionalization of poly(L-lactide) nanofibrous scaffolds with bioactive collagen molecules. J Biomed Mater Res Part A 2007;83:1117– 1127. 42. Moreno-Layseca P, Streuli CH. Signalling pathways linking integrins with cell cycle progression. Matrix Biol 2014;34:144–153. 43. Kumbar SG, Nukavarapu SP, James R, Nair LS, Laurencin CT. Electrospun poly(lactic acid-co-glycolic acid) scaffolds for skin tissue engineering. Biomaterials 2008;29:4100–4107.

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2014 VOL 00A, ISSUE 00

11

gelatin hybrid nanofibrous scaffolds encapsulating EGF for skin regeneration.

The novel strategies of skin regenerative treatment are aimed at the development of biologically responsive scaffolds capable of delivering multiple b...
924KB Sizes 3 Downloads 8 Views