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arginine–chitosan polymer blends for assembly of nanofibrous membranes for wound regeneration
Accepted Manuscript Title: Chitosan/Arginine-Chitosan polymer blends for assembly of nanofibrous membranes for wound regeneration Author: B.P. Antunes A.F. Moreira V.M. Gaspar I.J. Correia PII: DOI: Reference:
S0144-8617(15)00399-9 http://dx.doi.org/doi:10.1016/j.carbpol.2015.04.072 CARP 9904
To appear in: Received date: Revised date: Accepted date:
9-2-2015 15-4-2015 28-4-2015
Please cite this article as: Antunes, B. P., Moreira, A. F., Gaspar, V. M., and Correia, I. J.,Chitosan/Arginine-Chitosan polymer blends for assembly of nanofibrous membranes for wound regeneration, Carbohydrate Polymers (2015), http://dx.doi.org/10.1016/j.carbpol.2015.04.072 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Highlights
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- Electrospun membranes produced herein are aimed to be used as wound dressings.
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- Produced membranes have an ECM-like structure with a hydrophilic character.
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- Membranes bactericidal activity is crucial for wound healing improvement.
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- Electrospun membranes were suitable to coat wounds and improve healing.
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Chitosan/Arginine-Chitosan polymer blends for assembly of nanofibrous membranes for wound regeneration
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B.P. Antunesa, A.F: Moreiraa, V.M. Gaspara, I.J. Correiaa,*
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* Corresponding author. Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal. Tel.: +351 275 329 002; Fax: +351 275 329 099; e-mail: [email protected].
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CICS-UBI – Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal.
Graphical Abstract
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Abstract
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Frequently, skin is subjected to damaging events, such as deep cuts, burns or ulcers, which may compromise the integrity of this organ. To overcome such lesions, different strategies have been employed. Among them, wound dressings aimed to re-establish skin native properties and decrease patient pain have been pursued for a long time. Herein, an electrospun membrane comprised by deacetylated/arginine modified chitosan (CH-A) was produced to be used as a wound dressing. The obtained results showed that the membrane has a highly hydrophilic and porous three-dimensional nanofibrous network similar to that found in human native extracellular matrix. In vitro data indicate that human fibroblasts adhere and proliferate in contact with membranes, thus corroborating their biocompatibility. This nanofiber-based biomaterial also demonstrated bacteriostatic activity for two bacterial strains. In vivo application of CH-A nanofibers in full thickness wounds resulted in an improved tissue regeneration and faster wound closure, when compared to non-modified membranes. Such findings, support the suitability of using this membrane as a wound dressing in a near future.
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Keywords: Antimicrobial Activity, Arginine, Chitosan, Electrospinning, Wound Healing.
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1. Introduction
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Skin is a relatively soft tissue that covers the body external surface, playing crucial
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functions, such as body protection against external threats, fluid homeostasis and
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sensory detection (Jennemann et al., 2012). Throughout lifetime, wounds may arise
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due to mechanical trauma, surgical procedures, reduced blood circulation, burns, aging
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or simple daily basis activities. Despite the fact that the majority of skin wounds
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possess the capacity of self-healing, extensive or irreversible lesions need medical
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assistance for the regenerative process to take place (Zhong, Zhang & Lim, 2010).
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Wound dressings act as coatings materials that avoid fluid losses and pathogens entry
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(Bottcher-Haberzeth, Biedermann & Reichmann, 2010). Such deleterious scenario,
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could cause severe health problems such as respiratory failure, shock, systemic
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infection and electrolyte imbalances (Bottcher-Haberzeth, Biedermann & Reichmann,
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2010).
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Nanofiber based materials are highly promising alternatives as scaffolds for tissue
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regeneration. Several manufacturing techniques can be employed in its production,
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such as fiber drawing, template synthesis, temperature-induced phase separation,
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molecular self-assembly, and electrospinning (Cui et al., 2013; Pan et al., 2010).
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However, electrospinning was found to be one of the most efficient, simple, cost
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effective and versatile techniques for production of wound dressings (Zhong, Zhang &
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Lim, 2010). Different polymers or polymer blends can be used to produce nanofibers
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that provide protective and structural characteristics similar to those of native human
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skin (Zhong, Zhang & Lim, 2010). In this context, Chen and collaborators have
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described that nanofibrous membranes possess a broad surface area, which in turn
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contributes for fibroblasts migration into the wound location (Chen, Chang & Chen,
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2008). Also, due to their nanosized structure, nanofibers provide additional anchoring
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points that promote cell adhesion and migration (Stevens & George, 2005). Such is
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crucial for wound healing, since these cells are involved in the production of
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extracellular matrix components such as collagen and growth factors that are involved
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in tissue regeneration (Chen, Chang & Chen, 2008). Furthermore, electrospun
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scaffolds are highly porous with a variable pore-size distribution, usually ranging from 1
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to 10 μm, which allow gases, nutrient and fluids exchange between the scaffold and
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the surrounding environment (Li, Laurencin, Caterson, Tuan & Ko, 2002).
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Among the different polymers used for nanofibers production, chitosan (CH) presents
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particularly valuable characteristics (De Vrieze, Westbroek, Van Camp & Van
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Langenhove, 2007). CH is biocompatible, biodegradable, and has been described as
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capable of improving wound healing by enhancing infiltration of inflammatory cells into
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the wound region, by promoting fibroblasts migration and proliferation, as well as,
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collagen deposition (Ueno, Mori & Fujinaga, 2001). Moreover, it has been shown that
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CH can have haemostatic properties promoting erythrocytes aggregation. The
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activation of the coagulation cascade is an important factor in acute full thickness
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wounds as excessive bleeding can result in life-threatening (Paul & Sharma, 2004).
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CH has also shown the ability to inhibit the growth of some bacterial strains (Unnithan,
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Gnanasekaran, Sathishkumar, Lee & Kim, 2014). Since, infected wounds can
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significantly compromise the healing process and in some cases inhibit it. Chitosan
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bacteriostatic activity is highly desirable for its envisioned therapeutic application (Katti,
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Robinson, Ko & Laurencin, 2004). This antimicrobial activity may result from the
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interaction of the positively charged amino groups with the negatively charged groups
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present on the surface of bacterial cells. This interaction leads to microbial membrane
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disruption and, subsequently, to the leakage of proteins and other intracellular
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constituents (Kong, Chen, Xing & Park, 2010). Despite this intrinsic activity, CH
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antimicrobial properties can be improved by increasing the number of positively
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charged groups available on its backbone. This can be accomplished by grafting
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positively charged amino acids, such as L-asparagine, L-arginine or L-lysine (Kim,
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2001; Xiao, Wan, Zhao, Liu & Zhang, 2011). In particular, due to the guanidine group
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(pKa=12.5) the inclusion of L-arginine is expected to increase the number of positively
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charged groups at physiological pH. In turn, this inclusion is expected to enhance the
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CH antibacterial properties (Sokalingam, Raghunathan, Soundrarajan & Lee, 2012).
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Moreover, as demonstrated by Shi et al., L-arginine also possess the capacity to
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improve the collagen deposition, which is fundamental for the wound healing process
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(Shi, Wang, Zhang, Zhang & Barbul, 2007).
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In previous works, our research group already showed the benefits of CH based
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dressings for wound regeneration (Miguel, Ribeiro, Brancal, Coutinho & Correia, 2014;
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Ribeiro et al., 2009; Ribeiro, Morgado, Miguel, Coutinho & Correia, 2013). Herein, a
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novel biocompatible and biodegradable electrospun membrane comprised by
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deacetylated (CH-D) and arginine modified chitosan (CH-Arg) was produced to
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evaluate its suitability for wound healing.
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2. Materials and methods
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2.1 Materials
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Amphotericin B, Bovine serum albumin (BSA), high molecular weight Chitosan (310-
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375
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Ethylenediaminetetraacetic acid (EDTA), L-arginine, L-glutamine, LB Broth (Miller), N-
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Hydroxysuccinimide (NHS), Phosphate-buffered saline (PBS), Trifluoroacetic acid
kDa),
Dulbecco's
modified
Eagle's
medium
(DMEM-F12),
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(TFA) and trypsin were purchased from Sigma–Aldrich (Sintra, Portugal). LB Agar
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(Lennox) was purchased from Conda (Barcelona, Spain). Dichloromethane (DCM),
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glutaraldehyde (GA) and Sodium hydroxide (NaOH) were obtained from Fischer
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Scientific (Loures, Portugal). Primary normal human dermal fibroblasts (FibH) were
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purchased
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Dimethylaminopropyl)-3-ethylcarbodiimide Hydrochloride (EDC) was purchased from
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TCI (Zwijndrecht, Belgium). Fetal bovine serum (FBS) was purchased from Biochrom
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AG (Berlin, Germany). 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
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sulfophenyl)-2H-tetrazolium reagent, inner salt (MTS) and electron coupling reagent
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(phenazine methosulfate; PMS) were purchased from Promega (Madison, WI, USA).
PromoCell
(Labclinics,
S.A.;
Barcelona,
Spain).
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CH was purified and deacetylated through a one-step alkali process previously
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described in our group (Gaspar, Sousa, Queiroz & Correia, 2011). Briefly, CH (500 mg)
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was mixed with 10 mL of NaOH (1 M) for 3 h at 50 ˚C under vigorous stirring. The
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mixture was then filtered with a Whatman® quantitative filter paper, grade 541: 0.22 µm
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(Sigma–Aldrich) and a Buchner funnel recovering the CH-D polymer. Afterwards, the
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CH-D polymer was extensively washed with ultrapure water and dried at 37 ˚C
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overnight.
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2.3 Synthesis of L-arginine grafted chitosan
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L-arginine was coupled to CH (CH-Arg) by amidation of the primary amine groups
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present in CH glucosamine (GlcN) units by using EDC/NHS as coupling agents as
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previously described with slight modifications (Gaspar, Marques, Sousa, Louro,
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Queiroz & Correia, 2013). Briefly, the previously deacetylated CH-D polymer was
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dissolved in acetic acid 1 % (v/v), under stirring, to a final concentration of 1 % (w/v) at
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room temperature (RT). Subsequently, NHS was dissolved in the CH-D solution (0.55
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mol/mol EDC), under intense magnetic stirring. EDC was then added to the reaction
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(1.5 mol/mol L-arginine). Finaly, L-Arginine was incorporated (0.8 mol/mol of CH
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amines) into the mixture and the coupling reaction proceeded for 24 h. To remove
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traces of unreacted components the CH-Arg conjugate was dialyzed for five days
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against deionized water with daily changes of the dialysant (MWCO 12-14 kDa). The
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purified CH-Arg polymer was finaly recovered by freeze-drying (Scanvac CoolSafeTM,
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ScanLaf A/S, Lynge, Denmark) for 24 h.
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2.4 Characterization of deacetylated chitosan and L-arginine grafted deacetylated
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The CH-D polymer deacetylation degree was measured by using the first derivative
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UV-spectroscopy (1DUVS) method described in literature (Muzzarelli & Rocchetti,
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1986). UV–vis CH-D polymer spectrum was obtained using a Shimadzu 1700 UV–vis
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spectrophotometer.
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The L-arginine coupling and the degree of substitution (DS) were evaluated by Fourier
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transform infrared spectroscopy (ATR-FTIR), as previously reported in the literature
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(Moreira, Oliveira, Pires, Simoes, Barbosa & Pego, 2009). The infrared spectra were
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recorded in a Nicolet iS10 spectrometer (Thermo Scientific Inc., MA, USA) by
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acquisition of 256 interferograms with a 4 cm-1 spectral resolution.
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2.5 Production of the electrospun nanofiber membrane
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To produce different nanofibrous membranes, CH-D/CH-Arg polymer blends or CH-D
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polymer solutions were electrospun in a conventional electrospinning apparatus. The
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system setup was comprised by a high voltage source (Spellman CZE1000R, 0–30kV),
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a precision syringe pump (KDS-100), a plastic syringe with a stainless steel needle (21
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Gauge) and an aluminium disk connected to a copper collector, at a working distance
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of 10 cm. CH-D and CH-Arg polymers (combined at 60:40 w/w ratio) were dissolved at
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a final concentration of 8 % (w/v), under stirring, using TFA and DCM (combined at a
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ratio of 70:30 v/v). Different final concentrations of CH-D/CH-Arg polymers were tested
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and 8 % (w/v) was the concentration which presented better results. The CH-D/CH-Arg
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solution was used to produce a nanofibrous membrane at a constant flow rate of 1.2
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mL/h and an applied voltage of 28 kV. Additionally, a different membrane was also
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produced as described above, but the electrospunned solution only contained CH-D
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polymer at a concentration 8 % (w/v), CH-DD membrane.
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The CH-D/CH-Arg membrane (CH-A membrane) and CH-DD membrane were
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subsequently cross-linked by using chemical vapour deposition (CVD) with GA (25 %
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v/v), according to a method previously described elsewhere (Abdelgawad, Hudson &
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Rojas, 2014). Briefly, the samples were exposed to GA vapour for 1 h and then heat-
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treated in an oven at 70 ˚C under vacuum for 24 h to remove unreacted GA.
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2.6 Scanning electron microscopy analysis
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Membranes morphology (before and after crosslinking via CVD), and with adhered
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human fibroblasts seeded on their surface was analysed by scanning electron
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microscopy (SEM) (Ferreira, Carvalho, Correia, Antunes, Correia & Alves, 2013). For
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SEM analysis, electrospun membranes containing cells were fixed overnight with GA
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(2.5 % v/v) in PBS, at 4 ˚C. Afterwards, the samples were dehydrated in ethanol
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solutions with increasing concentrations (70, 80, 90 and 100 % (v/v)). Finally, the 6
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samples were mounted on an aluminium stub using a double-sided adhesive tape and
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sputter coated with gold by using an Emitech K550 (London, England) sputter coater.
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The samples were analysed using a Hitachi S-2700 (Tokyo, Japan) scanning electron
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microscope operated at an accelerating voltage of 20 kV. The nanofiber diameter
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distribution in the membranes was determined by using ImageJ software (National
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Institutes of Health, Bethesda (MD), USA) (Correia et al., 2013).
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The CH-A membrane water contact angle (WCA) was determined by using a Data
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Physics Contact Angle System OCAH 200 apparatus, operating in static mode (Correia
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et al., 2013). For each sample, water drops (double deionised water, dH2O, 0.22 µm
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filtered, ρ=17.9 MΩ/cm) were placed at various locations of the analysed surface, at
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room temperature
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The total porosity of the membranes was measured through the determination of the
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amount of ethanol absorbed by the membranes, after 1 h of immersion in that solvent,
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using Eq. (1) (Correia et al., 2013):
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(1)
where W 1 is the weight of the dry membrane and W 2 is the weight of the wet
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membrane, dethanol the density of the ethanol 100% (v/v) at room temperature, and
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Vmembrane is the volume of the wet membrane. Membrane volume was determined and
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by measuring membrane thickness with a micrometer Adamel Lhomargy M120
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acquired from Testing Machines Inc. (Delaware, USA).
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2.9 Water vapour transmission
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The water vapour diffusion through membranes was evaluated as describe elsewhere
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(Lin, Lien, Yeh, Yu & Hsu, 2013). Briefly, CH-A membranes were used to seal the
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opening of a glass test tube (1.77 cm2) containing 10 mL of ultrapure water, barrier
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tape was used to attach the membrane and prevent water losses. Afterwards, the
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membranes where left in an incubator at 37°C. Non-sealed were used as control. At
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specific time points, water evaporation was determined by weight measurement with an
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OHAUS Adventurer Pro analytical balance (New Jersey, USA). The water vapour
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transmission rate (WVTR) was calculated by Eq.(2):
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(2)
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where W loss is the daily weight loss of water and A the area of tube opening.
222 2.10 Electrospun nanofiber membranes cytotoxic profile
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Membranes biocompatibility was evaluated by using the MTS assay as recommended
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in ISO 10993-5:2009 (Biological evaluation of medical devices - Part 5: Tests for in
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vitro cytotoxicity). Briefly, human fibroblast cells were seeded, at a density of 1x105
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cells/well, in 96-well flat bottom culture plates. After 24 h, CH-DD or CH-A membranes,
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occupying less than 10% of the well area, as recommended in ISO 10993-5:2009
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(Biological evaluation of medical devices - Part 5: Tests for in vitro cytotoxicity), were
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added to each well and incubated for 24, 48 and 72 h. Afterwards, the medium of each
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well was removed and replaced with a mixture of 100 μL of fresh culture medium and
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20 μL of MTS/PMS reagent solution. Then, the cells were incubated for 4 h at 37 °C,
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under a 5 % CO2 humidified atmosphere. The absorbance was measured at 492 nm
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using a microplate reader (Anthos 2020, Biochrom UK). Cells incubated with absolute
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ethanol were used as a positive control (K+), whereas non-incubated cells were used
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as negative controls (K-).
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2.11 Antimicrobial activity characterization
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Microbiological assays were performed with Escherichia Coli DH5α (E. Coli) and
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Staphylococus Aureus clinical isolate (S. Aureus) as models of prokaryotic organisms
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(Torres‐Giner, Ocio & Lagaron, 2008). Briefly, 25 or 50 mg of CH-DD or CH-A
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electrospun mats were added to 10 mL of LB broth at pH 6.2, containing 1 x 105
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CFU/mL of an early mid-log phase culture of S. Aureus incubated at 37 ˚C, for 24 h.
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The same process was conducted for E Coli. After the incubation period, serial
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dilutions were prepared and 100 µL of bacterial samples were transferred into LB agar
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plates. After overnight incubation at 37 ˚C, bacterial colonies were counted and
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expressed as the number of colony forming units per mL. The bacterial concentrations
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were monitored through optical density measurements at 600 nm (Levard et al., 2013).
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To further evaluate membranes antimicrobial properties, the formation of an inhibition
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halo in E. Coli and S. Aureus cultures, in contact with CH-DD or CH-A membranes,
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was assessed. Briefly, 100 µL containing 1 x 108 CFU/mL of E. Coli or S. Aureus were
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spread into agar plates. Subsequently CH-DD or CH-A membranes with ~1cm
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diameter were placed on top of the agar layer and incubated for 24 h, at 37ºC. The
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inhibition halo was photographed with a digital camera (NikonD50) and halo size
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determined with ImageJ (Scion Corp., Frederick, MD), image analysis software.
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256 2.12 In vivo studies
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A total of 25 female Wistar rats (8–10 weeks old) weighing between 150 and 200 g
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were used to evaluate the suitability of CH-D or CH-A nanofibrous membranes to be
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applied in the healing of full thickness wounds. The animal experiments were
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performed according to the protocol approved by the ethics committee and the
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guidelines set forth in the National Institute for the care and use of laboratory animals.
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The animals were housed in individually ventilated cages, Sealsafe Next, Tecniplast
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(Buguggiate, Italy), at 22ºC with a 12 h/12 h light cycle and fed with commercial rat
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food, 4RF 21 Mucedola (Milan, Italy) and water ad libitum.. In the day of experiments
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each rat was individually anesthetized with an intra-peritoneal injection of ketamine (40
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mg/kg) and xylazine (5 mg/kg). Prior to burn induction, the skin from the dorsal area
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was shaved and disinfected using ethanol (96%), subsequently wounds with a
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diameter of approximately 2 cm were created as previously described by our group and
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with no visible bleeding (Ribeiro, Morgado, Miguel, Coutinho & Correia, 2013).
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Afterwards, the animals were randomly divided into three groups (n=5): (i) control
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(group 1), the wounds were washed with PBS pH=7.4; (ii) experimental group 2, the
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wounds were treated with CH-DD membranes and (iii) experimental group 3, the
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wounds were treated with CH-A membrane. Animals were only treated once,
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immediately after wound induction. In order to follow the wound healing process, the
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wounds were posteriorly photographed with a digital camera (NikonD50) along time.
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The wound size (WS) was determined by using the image analysis software ImageJ
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(Scion Corp., Frederick, MD). Changes in animal weight and general animal condition
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were monitored every other day. At predetermined time points animals from different
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groups were euthanized via excess CO2. Necropsy was performed to collect the
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regenerated tissue and major organs for histological analysis.
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2.13 Statistical analysis
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Data is presented as mean ± standard deviation (s.d.). One-way analysis of variance
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(ANOVA) with the Student–Newman–Keuls test was used to compare different groups, 9
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except when stated otherwise. A p value lower than 0.05 was considered statistically
287
significant. Statistical analysis was performed using GraphPad Prism v.5.0 software
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(Trial version, GraphPadSoftware, CA, USA).
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3. Results and discussion
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3.1 Membrane Characterization
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The successful binding of L-arginine to CH-DD backbone via EDC/NHS coupling
293
chemistry was evaluated by FTIR. The infra-red spectra (Figure S1) revealed that CH
294
presents a broad band between 3200-3500 cm-1, that was attributed to the
295
characteristic -NH and –OH stretching vibration. The band at 2879 cm-1 was assigned
296
to –CH– stretching of CH polymer. The characteristic peaks present at 1630 and 1528
297
cm-1 are assigned to the amide I and II bands of CH, respectively (Xiao, Wan, Zhao, Liu
298
& Zhang, 2011). CH polymer can be easily identified through the peaks at 1160 cm-1,
299
regarding the asymmetric stretching of the C-O-C, and at 1060-1026 cm-1, regarding
300
the C-O stretching vibrations of the pyranose ring (Bhattarai, Ramay, Gunn, Matsen &
301
Zhang, 2005; Liu, Zhang, Cao, Xu & Yao, 2004). L-arginine exhibits an absorption
302
band at 1614 cm-1 that was assigned to the guanidine group (Liu, Zhang, Cao, Xu &
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Yao, 2004). The bands at 1419 and 764 cm-1 were ascribed to COO- symmetric and
304
asymmetric bending, respectively. The peak found at 1130 cm-1 is correlated with the
305
C-C-N asymmetric bending (Liu, Zhang, Cao, Xu & Yao, 2004) (Xiao, Wan, Zhao, Liu &
306
Zhang, 2011).
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In the FTIR spectra of CH-Arg polymer, the characteristic peaks of arginine at 1630
308
and 1410 cm-1 (guanidine group and COO- symmetric bending, respectively), as well as
309
both C-O stretching vibrations of CH polymer pyranose (1062-1033 cm-1) could be
310
observed (Liu, Zhang, Cao, Xu & Yao, 2004). Furthermore, the increase in the amide I
311
peak at ~1654 cm-1 suggests that L-Arginine moieties are indeed linked to CH polymer
312
backbone. The substitution degree of CH-D polymer (17.09±0.25 %) is similar to the
313
value reported in literature to similar coupling conditions (Xiao, Wan, Zhao, Liu &
314
Zhang, 2011). The analysis of the CH-A membrane spectra reveals the characteristic
315
peaks of all the components used in membranes production.
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Preliminary tests were performed to evaluate the ability to produce nanofibers with the
317
synthesized amino acid modified CH. However, the polymer solutions were difficult to
318
be electrospun, a fact that limited membranes production. To overcome this issue, a
319
blend of CH-Arg and CH-DD polymers was used to allow nanofiber production. The
320
resulting CH-A membranes morphological characteristics are shown in Figure 1 and
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Figure S2. The CH-A nanofibrous membrane has an opaque appearance, as well as a
322
uniform structure (Figure 1A). A more detailed analysis through SEM (Figure S2B and
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C) reveals a dense 3D nanofiber network comprised by randomly arranged fibrils,
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which results in an irregular surface with an interconnected porous structure. This 3D
325
organization creates a large contact area with anchoring points for cell-nanofiber
326
interactions enhancing cell adhesion, migration and proliferation (Sangsanoh &
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Supaphol, 2006). Furthermore, after the cross-linking process (Figure S2C) the CH-A
328
membrane fiber matrix retained its integrity and randomly oriented fiber organization
329
without a significant changes (Schiffman & Schauer, 2007).
330
Biomaterials porosity has been previously described in the literature as a crucial
331
parameter for tissue engineering applications (Khil, Cha, Kim, Kim & Bhattarai, 2003).
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As demonstrated in Figure 1B, the electrospun CH-A membrane presents a total
333
porosity of 88.25 ± 4.13 %, a value within the preferred range of 60-90%(Chong et al.,
334
2007). The porous nature of these systems is beneficial for cellular infiltration and
335
proliferation. Moreover, porosity also ensures a correct gas, nutrient and fluids
336
exchange, which are essential parameters to attain hemostasis, and ultimately to allow
337
a proper wound healing (Chong et al., 2007). Material surface properties may also
338
influence the host response to the implanted material and the wound healing process
339
(Oliveira, Alves & Mano, 2014). Therefore, the WCA was determined to verify the
340
surface wettability of CH-A membrane (Figure 1C). A WCA value of 25.2±8.4º was
341
obtained, revealing that membrane surface possess a hydrophilic character according
342
to the previously proposed classifications (Oliveira, Alves & Mano, 2014). Hydrophilic
343
surfaces are considered to be the most effective for serum proteins adsorption, which
344
is an important step for subsequent cell adhesion and proliferation (Oliveira, Alves &
345
Mano, 2014). Furthermore, the fiber diameter analysis (Figure 1D) revealed the
346
presence of randomly distributed fibers with diameters ranging from 100 to 400 nm.
347
This is an important parameter, since collagen, the main component of the ECM, has a
348
fibrous-like structure with the collagen fibrils diameters varying from 50 to 500 nm
349
(Jayakumar, Menon, Manzoor, Nair & Tamura, 2010). Therefore, this result suggests
350
that at a structural and morphological level, CH-A electrospun membranes are
351
somehow similar to the human native ECM structure (Stevens & George, 2005).
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Figure 1 – CH-A membrane characterization. (A) Macroscopic image of the produced CH-A membrane. (B) CH-A membrane porosity analysis. Data is represented as mean ± s.d., n=3. (C) CH-A membrane surface contact angle analysis. Data is represented as mean ± s.d., n=3. (D) CH-A membrane fiber diameter distribution. Data is represented as mean ± s.d., n=2.
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The CH-A membrane capacity to maintain moisture in the wound during the healing
360
process was assessed by WVTR assay (Figure S2D). The obtained data shows a
361
nearly constant water weight loss for control and membrane sealed group. The results
362
showed that CH-A membrane has a WVTR of 1713 ± 26 mL/m2/day, a value similar to
363
that of the control, 1801 ± 205 mL/m2/day. Thus, indicating that the CH-A membrane
364
does not limit water vapour exchange. The recommended transmission rate for wound
365
dressings is 2000-2500 mL/m2/day, which is half of the WVTR obtained in granulating
366
tissue (5138±202 mL/m2/day). Therefore, the results indicate that the nanofiber mats
367
could control, at a suitable rate, the water vapour losses and accumulation of exudate
368
fluids, which are common in full thickness wounds (Wu et al., 2004). In fact it is
369
important to mention that exudates accumulation may lead to maceration of healthy
370
skin and inhibit the healing process (Boateng, Matthews, Stevens & Eccleston, 2008).
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3.2 Evaluation of the antimicrobial activity of CH-A membranes
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CH-A membranes bactericidal properties were evaluated using S. Aureus and E. Coli
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bacteria (Karami, Rezaeian, Zahedi & Abdollahi, 2013; Unnithan, Gnanasekaran,
375
Sathishkumar, Lee & Kim, 2014). These bacterial strains were considered appropriate
376
for performing this assay as models of gram-positive and gram-negative pathogens,
377
respectively.
378
The results obtained from the bactericidal activity evaluation showed that the CH-DD
379
membrane, at a concentration of 2.5 mg, presents an inhibitory effect of ~50% for E.
380
Coli and ~16.5% for S. Aureus. Interestingly, increasing the concentration of CH-DD
381
membrane to 5 mg does not result in a significant improvement in the observed
382
bactericidal effect (Table S1).
383
On the other hand E. Coli and S. Aureus bacterial colonies were completely eliminated
384
(> 99.99%) in the presence of CH-A membranes. These results highlight the
385
importance of coupling L-arginine residues to CH polymer for achieving an enhanced
386
bactericidal effect. It is also important to mention that CH-A membranes bactericidal
387
effect is similar to other chitosan nanofibers that were conjugated with antibiotics or
388
silver based materials (Bai, Chou, Tsai & Yu, 2014).
389
To further evaluate the bactericidal activity of the produced membranes, the formation
390
of an inhibition halo was also evaluated (Figure 2). As shown in Figure 2A1 and B1 the
391
CH-A membranes presented the largest inhibitory halo area (bactericidal area) for both
392
bacterial strains tested. This result confirms an improvement in the antibacterial activity
393
of CH by chemically grafting L-arginine residues in the polymer backbone. Interestingly,
394
both membranes have a higher inhibition area for E. Coli bacteria in comparison to S.
395
Aureus, which is in accordance with the results previously reported in literature for a
396
similar L-arginine CH modifications (Xiao, Wan, Zhao, Liu & Zhang, 2011). This strain
397
dependent efficacy can be related to the different cell wall structures or components
398
present in the different bacteria, which can affect the CH mechanism of action (Xiao,
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Wan, Zhao, Liu & Zhang, 2011).
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Figure 2 – Characterization of antimicrobial properties of different CH membranes. Analysis of inhibition halo area in function of the CH-DD or CH-A membrane area for E. Coli (A1) and S. Aureus (B1). Data presented as mean ± s.d., t-student test, *p<0.05, n=3. Macroscopic images of CH-DD membrane inhibition halo in E. Coli (A2) and S. Aureus (B2). Macroscopic images of CH-A membrane inhibition halo in E. Coli (A3) and S. Aureus (B3). The white line delimits the nanofibrous membrane.
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3.3 In vitro biocompatibility of CH nanofibrous membranes
408
The biocompatibility of CH membranes was characterized through the MTS assay. The
409
results obtained showed that FibH cells remained metabolically active in contact with
410
the CH-DD or CH-A membranes even after 72 h of incubation (Figure 3A). These
411
results indicate that L-arginine coupling does not affect membranes biocompatibility,
412
since no differences were observed between CH-DD and CH-A membrane cell viability
413
(Figure 3A). To further assess the applicability of the CH-A membrane for the intended
414
biomedical application, FibH cell adhesion on CH-A membranes and cell morphology in
415
the presence of the membrane was assessed (Figure 3B and C). As demonstrated by
416
Figure 3B, FibH cells already present a typical morphology with various filopodia
417
protrusions after 24 h of incubation, suggesting that skin fibroblasts can adhere and
418
start to proliferate on the CH-A membrane surface. Such can be explained by the
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membrane hydrophilic character and the cells membrane glycosaminoglycans
420
interaction with the amine groups present in CH-A membrane (Chen, Chung, Wang &
421
Young, 2012; Stevens & George, 2005).
422
Furthermore, microscopic images of cells incubated with CH-A membranes (Figure
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3C2, C4 and C6) show no variations on FibH proliferation rate and morphology in
424
comparison to non-incubated cells, thus corroborating MTS results.
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Figure 3 – Characterization of membranes in vitro biocompatibility. (A) Evaluation of the cell viability through MTS assay after 24, 48 and 72 h. Data represented as mean ± s.d., *p<0.05, n=5. (B) Representative pseudo-colored SEM image of FibH cells in contact with CH-A membrane after 24 h. (C) Representative optical microscopy images of FibH cells, incubated with CH-A membranes for 24 (C2), 48 (C4) and 72 h (C6), and negative controls at 24 (C1), 48 (C3) and 72 h (C5).
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3.3 In vivo evaluation of the wound healing process
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To evaluate the suitability of CH-A membranes for wound healing, a full thickness burn
435
was induced in female Wistar rats. All animals showed good general health condition
436
throughout the study with no unregular behaviour. Moreover, as observed in Figure S3,
437
all the three groups gained weight during the 21 days of the study. Macroscopic
438
analysis of wound regeneration revealed different healing patterns between the tested
439
groups (Figure 4A and B). In the initial days after wound induction, the groups treated
440
with the electrospun membranes, CH-DD or CH-A, presented a faster wound closure
441
than the control group (Figure 4A). These results emphasize the need to perform an
442
initial coverage of the damaged tissue in extensive lesions, creating a protective
443
environment and a support for cell migration (Zhong, Zhang & Lim, 2010). After 21
444
days, the group treated with the CH-A membranes demonstrated a significantly higher
445
wound closure than the other two groups, CH-DD and control group (Figure 4A). Also,
446
taking into account the L-arginine improvement in CH bactericidal activity, it is expected
447
that CH-A membrane provide an efficient protective barrier against infection.
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The histological analysis (Figure 5) corroborated the results observed in macroscopic
449
images. In membrane treated groups, at 14 days, negligible scar formation,
450
immunogenic response or capsule formation was observed when compared to control
451
wounds (Figure 5 A1, A2 and A3). Moreover, treated groups presented a higher collagen
452
deposition in the wound site (Figure 5 B1, B2 and B3). At 21 days, the CH-A membrane
453
treated group showed a higher re-epithelialization degree and tissue reorganization
454
(Figure 5 A6 and B6), when compared to the other groups (CH-DD and control). Such
455
data, supports the applicability of the CH-A membrane for wound healing purposes and
456
its biocompatible character.
457
The improved tissue regeneration observed in CH-A treated group may be attributed to
458
the combination of CH intrinsic wound healing properties, to the nanofibers ECM-like
459
structure and to the additional effect of L-arginine. Actually, different reports in the
460
literature describe that L-arginine has the capacity to improve wound healing by
461
enhancing collagen deposition and the wound tensile strength (Shi, Wang, Zhang,
462
Zhang & Barbul, 2007; Witte & Barbul, 2003). Moreover, the positive wound healing
463
effect observed for CH-A nanofibrous membranes is comparable to the one observed
464
in literature for fiber based materials comprised by polymer (e.g., CH, polycaprolactone
465
and PLGA) and expensive ECM constituents (e.g. collagen) (Bai, Chou, Tsai & Yu,
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2014; Chen, Chang & Chen, 2008; Liu, Kau, Chou, Chen, Wu & Yeh, 2010).
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Figure 4 – Evaluation of the healing of a third-degree burn over 21 days. (A) Evolution of wound size along time. Data represented as mean ±s.d., *p<0.05, n=5. (B) Macroscopic images of the wound healing process. Black scale bars correspond to 1cm.
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Figure 5 – Representative images of tissue samples histological analysis in the group treated with PBS (Control), CH-DD membrane and CH-A membrane after 14 and 21 days. Sections stained with Hematoxylin and Eosin (A) and Masson's Trichrome (B). S: scar, M: possible membrane residue, RE: re-epithelialization and C: high collagen density. Black scale bars correspond to 500 µm.
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4. Conclusion
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To the best of our knowledge, this is the first time that the development and
483
applicability of electrospun fibers comprised by L-arginine modified CH and CH-D
484
polymers is evaluated. The developed deacetylated/arginine modified CH polymer
485
blend electrospun membrane presented a highly porous ECM-like nanofiber structure
486
with a hydrophilic character. Furthermore, the membrane demonstrated bactericidal
487
activity for different prokaryotic organisms. The in vitro assays revealed that the
488
membrane is non-cytotoxic, and provides a 3D matrix, with significant contact area, to
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allow cell adhesion and proliferation. In vivo assays demonstrated that the CH-A
490
membrane significantly improves tissue regeneration of full thickness wounds, when
491
compared to CH-DD membrane or the natural healing process. This work shows the
492
applicability of L-arginine to modify CH and improve its bactericidal and wound healing
493
properties. Furthermore it was shown that CH-A membrane can be efficiently applied to
494
improve the wound healing process in a cost-effective way. In a near future, the
495
inclusion of growth factors, proteins, plant extracts, drugs or genetic material in these
496
membranes may enhance even further the wound healing capacity of the system.
497
Moreover, a layer-by-layer approach can be used to mimic the native structural
498
organization of the different skin layers.
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499 Acknowledgments
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The authors would like to thank Eng. Ana Paula Gomes for the help in the acquisition
502
of SEM images, but also to M.Sc. Ricardo Fradique for the pseudo-colored version of
503
SEM FibH adhesion image. This work was supported by the Portuguese Foundation for
504
Science
505
OE/SAU/UI0709/2014)). V. M. Gaspar acknowledges an individual PhD fellowship from
506
FCT (SFRH/BD/80402/2011). A. F: Moreira acknowledges a fellowship from UBI-
507
Banco Santander/Totta
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(PEst-C/SAU/UI0709/2011
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ed
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(FCT),
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Technology
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and
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Page 19 of 22
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S0144-8617(15)00399-9 http://dx.doi.org/doi:10.1016/j.carbpol.2015.04.072 CARP 9904
To appear in: Received date: Revised date: Accepted date:
9-2-2015 15-4-2015 28-4-2015
Please cite this article as: Antunes, B. P., Moreira, A. F., Gaspar, V. M., and Correia, I. J.,Chitosan/Arginine-Chitosan polymer blends for assembly of nanofibrous membranes for wound regeneration, Carbohydrate Polymers (2015), http://dx.doi.org/10.1016/j.carbpol.2015.04.072 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Highlights
2
- Electrospun membranes produced herein are aimed to be used as wound dressings.
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- Produced membranes have an ECM-like structure with a hydrophilic character.
4
- Membranes bactericidal activity is crucial for wound healing improvement.
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- Electrospun membranes were suitable to coat wounds and improve healing.
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Chitosan/Arginine-Chitosan polymer blends for assembly of nanofibrous membranes for wound regeneration
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B.P. Antunesa, A.F: Moreiraa, V.M. Gaspara, I.J. Correiaa,*
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a
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* Corresponding author. Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal. Tel.: +351 275 329 002; Fax: +351 275 329 099; e-mail: [email protected].
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CICS-UBI – Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal.
Graphical Abstract
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Abstract
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Frequently, skin is subjected to damaging events, such as deep cuts, burns or ulcers, which may compromise the integrity of this organ. To overcome such lesions, different strategies have been employed. Among them, wound dressings aimed to re-establish skin native properties and decrease patient pain have been pursued for a long time. Herein, an electrospun membrane comprised by deacetylated/arginine modified chitosan (CH-A) was produced to be used as a wound dressing. The obtained results showed that the membrane has a highly hydrophilic and porous three-dimensional nanofibrous network similar to that found in human native extracellular matrix. In vitro data indicate that human fibroblasts adhere and proliferate in contact with membranes, thus corroborating their biocompatibility. This nanofiber-based biomaterial also demonstrated bacteriostatic activity for two bacterial strains. In vivo application of CH-A nanofibers in full thickness wounds resulted in an improved tissue regeneration and faster wound closure, when compared to non-modified membranes. Such findings, support the suitability of using this membrane as a wound dressing in a near future.
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Keywords: Antimicrobial Activity, Arginine, Chitosan, Electrospinning, Wound Healing.
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1. Introduction
36
Skin is a relatively soft tissue that covers the body external surface, playing crucial
37
functions, such as body protection against external threats, fluid homeostasis and
38
sensory detection (Jennemann et al., 2012). Throughout lifetime, wounds may arise
39
due to mechanical trauma, surgical procedures, reduced blood circulation, burns, aging
40
or simple daily basis activities. Despite the fact that the majority of skin wounds
41
possess the capacity of self-healing, extensive or irreversible lesions need medical
42
assistance for the regenerative process to take place (Zhong, Zhang & Lim, 2010).
43
Wound dressings act as coatings materials that avoid fluid losses and pathogens entry
44
(Bottcher-Haberzeth, Biedermann & Reichmann, 2010). Such deleterious scenario,
45
could cause severe health problems such as respiratory failure, shock, systemic
46
infection and electrolyte imbalances (Bottcher-Haberzeth, Biedermann & Reichmann,
47
2010).
48
Nanofiber based materials are highly promising alternatives as scaffolds for tissue
49
regeneration. Several manufacturing techniques can be employed in its production,
50
such as fiber drawing, template synthesis, temperature-induced phase separation,
51
molecular self-assembly, and electrospinning (Cui et al., 2013; Pan et al., 2010).
52
However, electrospinning was found to be one of the most efficient, simple, cost
53
effective and versatile techniques for production of wound dressings (Zhong, Zhang &
54
Lim, 2010). Different polymers or polymer blends can be used to produce nanofibers
55
that provide protective and structural characteristics similar to those of native human
56
skin (Zhong, Zhang & Lim, 2010). In this context, Chen and collaborators have
57
described that nanofibrous membranes possess a broad surface area, which in turn
58
contributes for fibroblasts migration into the wound location (Chen, Chang & Chen,
59
2008). Also, due to their nanosized structure, nanofibers provide additional anchoring
60
points that promote cell adhesion and migration (Stevens & George, 2005). Such is
61
crucial for wound healing, since these cells are involved in the production of
62
extracellular matrix components such as collagen and growth factors that are involved
63
in tissue regeneration (Chen, Chang & Chen, 2008). Furthermore, electrospun
64
scaffolds are highly porous with a variable pore-size distribution, usually ranging from 1
65
to 10 μm, which allow gases, nutrient and fluids exchange between the scaffold and
66
the surrounding environment (Li, Laurencin, Caterson, Tuan & Ko, 2002).
67
Among the different polymers used for nanofibers production, chitosan (CH) presents
68
particularly valuable characteristics (De Vrieze, Westbroek, Van Camp & Van
69
Langenhove, 2007). CH is biocompatible, biodegradable, and has been described as
70
capable of improving wound healing by enhancing infiltration of inflammatory cells into
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the wound region, by promoting fibroblasts migration and proliferation, as well as,
72
collagen deposition (Ueno, Mori & Fujinaga, 2001). Moreover, it has been shown that
73
CH can have haemostatic properties promoting erythrocytes aggregation. The
74
activation of the coagulation cascade is an important factor in acute full thickness
75
wounds as excessive bleeding can result in life-threatening (Paul & Sharma, 2004).
76
CH has also shown the ability to inhibit the growth of some bacterial strains (Unnithan,
77
Gnanasekaran, Sathishkumar, Lee & Kim, 2014). Since, infected wounds can
78
significantly compromise the healing process and in some cases inhibit it. Chitosan
79
bacteriostatic activity is highly desirable for its envisioned therapeutic application (Katti,
80
Robinson, Ko & Laurencin, 2004). This antimicrobial activity may result from the
81
interaction of the positively charged amino groups with the negatively charged groups
82
present on the surface of bacterial cells. This interaction leads to microbial membrane
83
disruption and, subsequently, to the leakage of proteins and other intracellular
84
constituents (Kong, Chen, Xing & Park, 2010). Despite this intrinsic activity, CH
85
antimicrobial properties can be improved by increasing the number of positively
86
charged groups available on its backbone. This can be accomplished by grafting
87
positively charged amino acids, such as L-asparagine, L-arginine or L-lysine (Kim,
88
2001; Xiao, Wan, Zhao, Liu & Zhang, 2011). In particular, due to the guanidine group
89
(pKa=12.5) the inclusion of L-arginine is expected to increase the number of positively
90
charged groups at physiological pH. In turn, this inclusion is expected to enhance the
91
CH antibacterial properties (Sokalingam, Raghunathan, Soundrarajan & Lee, 2012).
92
Moreover, as demonstrated by Shi et al., L-arginine also possess the capacity to
93
improve the collagen deposition, which is fundamental for the wound healing process
94
(Shi, Wang, Zhang, Zhang & Barbul, 2007).
95
In previous works, our research group already showed the benefits of CH based
96
dressings for wound regeneration (Miguel, Ribeiro, Brancal, Coutinho & Correia, 2014;
97
Ribeiro et al., 2009; Ribeiro, Morgado, Miguel, Coutinho & Correia, 2013). Herein, a
98
novel biocompatible and biodegradable electrospun membrane comprised by
99
deacetylated (CH-D) and arginine modified chitosan (CH-Arg) was produced to
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evaluate its suitability for wound healing.
101 102
2. Materials and methods
103
2.1 Materials
104
Amphotericin B, Bovine serum albumin (BSA), high molecular weight Chitosan (310-
105
375
106
Ethylenediaminetetraacetic acid (EDTA), L-arginine, L-glutamine, LB Broth (Miller), N-
107
Hydroxysuccinimide (NHS), Phosphate-buffered saline (PBS), Trifluoroacetic acid
kDa),
Dulbecco's
modified
Eagle's
medium
(DMEM-F12),
4
Page 4 of 22
108
(TFA) and trypsin were purchased from Sigma–Aldrich (Sintra, Portugal). LB Agar
109
(Lennox) was purchased from Conda (Barcelona, Spain). Dichloromethane (DCM),
110
glutaraldehyde (GA) and Sodium hydroxide (NaOH) were obtained from Fischer
111
Scientific (Loures, Portugal). Primary normal human dermal fibroblasts (FibH) were
112
purchased
113
Dimethylaminopropyl)-3-ethylcarbodiimide Hydrochloride (EDC) was purchased from
114
TCI (Zwijndrecht, Belgium). Fetal bovine serum (FBS) was purchased from Biochrom
115
AG (Berlin, Germany). 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
116
sulfophenyl)-2H-tetrazolium reagent, inner salt (MTS) and electron coupling reagent
117
(phenazine methosulfate; PMS) were purchased from Promega (Madison, WI, USA).
PromoCell
(Labclinics,
S.A.;
Barcelona,
Spain).
1-(3-
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118 2.2 Chitosan Deacetylation
120
CH was purified and deacetylated through a one-step alkali process previously
121
described in our group (Gaspar, Sousa, Queiroz & Correia, 2011). Briefly, CH (500 mg)
122
was mixed with 10 mL of NaOH (1 M) for 3 h at 50 ˚C under vigorous stirring. The
123
mixture was then filtered with a Whatman® quantitative filter paper, grade 541: 0.22 µm
124
(Sigma–Aldrich) and a Buchner funnel recovering the CH-D polymer. Afterwards, the
125
CH-D polymer was extensively washed with ultrapure water and dried at 37 ˚C
126
overnight.
ed
127
M
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2.3 Synthesis of L-arginine grafted chitosan
129
L-arginine was coupled to CH (CH-Arg) by amidation of the primary amine groups
130
present in CH glucosamine (GlcN) units by using EDC/NHS as coupling agents as
131
previously described with slight modifications (Gaspar, Marques, Sousa, Louro,
132
Queiroz & Correia, 2013). Briefly, the previously deacetylated CH-D polymer was
133
dissolved in acetic acid 1 % (v/v), under stirring, to a final concentration of 1 % (w/v) at
134
room temperature (RT). Subsequently, NHS was dissolved in the CH-D solution (0.55
135
mol/mol EDC), under intense magnetic stirring. EDC was then added to the reaction
136
(1.5 mol/mol L-arginine). Finaly, L-Arginine was incorporated (0.8 mol/mol of CH
137
amines) into the mixture and the coupling reaction proceeded for 24 h. To remove
138
traces of unreacted components the CH-Arg conjugate was dialyzed for five days
139
against deionized water with daily changes of the dialysant (MWCO 12-14 kDa). The
140
purified CH-Arg polymer was finaly recovered by freeze-drying (Scanvac CoolSafeTM,
141
ScanLaf A/S, Lynge, Denmark) for 24 h.
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142 143
2.4 Characterization of deacetylated chitosan and L-arginine grafted deacetylated
144
chitosan 5
Page 5 of 22
The CH-D polymer deacetylation degree was measured by using the first derivative
146
UV-spectroscopy (1DUVS) method described in literature (Muzzarelli & Rocchetti,
147
1986). UV–vis CH-D polymer spectrum was obtained using a Shimadzu 1700 UV–vis
148
spectrophotometer.
149
The L-arginine coupling and the degree of substitution (DS) were evaluated by Fourier
150
transform infrared spectroscopy (ATR-FTIR), as previously reported in the literature
151
(Moreira, Oliveira, Pires, Simoes, Barbosa & Pego, 2009). The infrared spectra were
152
recorded in a Nicolet iS10 spectrometer (Thermo Scientific Inc., MA, USA) by
153
acquisition of 256 interferograms with a 4 cm-1 spectral resolution.
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2.5 Production of the electrospun nanofiber membrane
156
To produce different nanofibrous membranes, CH-D/CH-Arg polymer blends or CH-D
157
polymer solutions were electrospun in a conventional electrospinning apparatus. The
158
system setup was comprised by a high voltage source (Spellman CZE1000R, 0–30kV),
159
a precision syringe pump (KDS-100), a plastic syringe with a stainless steel needle (21
160
Gauge) and an aluminium disk connected to a copper collector, at a working distance
161
of 10 cm. CH-D and CH-Arg polymers (combined at 60:40 w/w ratio) were dissolved at
162
a final concentration of 8 % (w/v), under stirring, using TFA and DCM (combined at a
163
ratio of 70:30 v/v). Different final concentrations of CH-D/CH-Arg polymers were tested
164
and 8 % (w/v) was the concentration which presented better results. The CH-D/CH-Arg
165
solution was used to produce a nanofibrous membrane at a constant flow rate of 1.2
166
mL/h and an applied voltage of 28 kV. Additionally, a different membrane was also
167
produced as described above, but the electrospunned solution only contained CH-D
168
polymer at a concentration 8 % (w/v), CH-DD membrane.
169
The CH-D/CH-Arg membrane (CH-A membrane) and CH-DD membrane were
170
subsequently cross-linked by using chemical vapour deposition (CVD) with GA (25 %
171
v/v), according to a method previously described elsewhere (Abdelgawad, Hudson &
172
Rojas, 2014). Briefly, the samples were exposed to GA vapour for 1 h and then heat-
173
treated in an oven at 70 ˚C under vacuum for 24 h to remove unreacted GA.
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155
175
2.6 Scanning electron microscopy analysis
176
Membranes morphology (before and after crosslinking via CVD), and with adhered
177
human fibroblasts seeded on their surface was analysed by scanning electron
178
microscopy (SEM) (Ferreira, Carvalho, Correia, Antunes, Correia & Alves, 2013). For
179
SEM analysis, electrospun membranes containing cells were fixed overnight with GA
180
(2.5 % v/v) in PBS, at 4 ˚C. Afterwards, the samples were dehydrated in ethanol
181
solutions with increasing concentrations (70, 80, 90 and 100 % (v/v)). Finally, the 6
Page 6 of 22
182
samples were mounted on an aluminium stub using a double-sided adhesive tape and
183
sputter coated with gold by using an Emitech K550 (London, England) sputter coater.
184
The samples were analysed using a Hitachi S-2700 (Tokyo, Japan) scanning electron
185
microscope operated at an accelerating voltage of 20 kV. The nanofiber diameter
186
distribution in the membranes was determined by using ImageJ software (National
187
Institutes of Health, Bethesda (MD), USA) (Correia et al., 2013).
ip t
188 2.7 Water contact angle determination
190
The CH-A membrane water contact angle (WCA) was determined by using a Data
191
Physics Contact Angle System OCAH 200 apparatus, operating in static mode (Correia
192
et al., 2013). For each sample, water drops (double deionised water, dH2O, 0.22 µm
193
filtered, ρ=17.9 MΩ/cm) were placed at various locations of the analysed surface, at
194
room temperature
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195 2.8 Determination of membrane porosity
197
The total porosity of the membranes was measured through the determination of the
198
amount of ethanol absorbed by the membranes, after 1 h of immersion in that solvent,
199
using Eq. (1) (Correia et al., 2013):
201 202
M
ed
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an
196
(1)
where W 1 is the weight of the dry membrane and W 2 is the weight of the wet
204
membrane, dethanol the density of the ethanol 100% (v/v) at room temperature, and
205
Vmembrane is the volume of the wet membrane. Membrane volume was determined and
206
by measuring membrane thickness with a micrometer Adamel Lhomargy M120
207
acquired from Testing Machines Inc. (Delaware, USA).
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209
2.9 Water vapour transmission
210
The water vapour diffusion through membranes was evaluated as describe elsewhere
211
(Lin, Lien, Yeh, Yu & Hsu, 2013). Briefly, CH-A membranes were used to seal the
212
opening of a glass test tube (1.77 cm2) containing 10 mL of ultrapure water, barrier
213
tape was used to attach the membrane and prevent water losses. Afterwards, the
214
membranes where left in an incubator at 37°C. Non-sealed were used as control. At
215
specific time points, water evaporation was determined by weight measurement with an
216
OHAUS Adventurer Pro analytical balance (New Jersey, USA). The water vapour
217
transmission rate (WVTR) was calculated by Eq.(2):
7
Page 7 of 22
218 219
(2)
220 221
where W loss is the daily weight loss of water and A the area of tube opening.
222 2.10 Electrospun nanofiber membranes cytotoxic profile
224
Membranes biocompatibility was evaluated by using the MTS assay as recommended
225
in ISO 10993-5:2009 (Biological evaluation of medical devices - Part 5: Tests for in
226
vitro cytotoxicity). Briefly, human fibroblast cells were seeded, at a density of 1x105
227
cells/well, in 96-well flat bottom culture plates. After 24 h, CH-DD or CH-A membranes,
228
occupying less than 10% of the well area, as recommended in ISO 10993-5:2009
229
(Biological evaluation of medical devices - Part 5: Tests for in vitro cytotoxicity), were
230
added to each well and incubated for 24, 48 and 72 h. Afterwards, the medium of each
231
well was removed and replaced with a mixture of 100 μL of fresh culture medium and
232
20 μL of MTS/PMS reagent solution. Then, the cells were incubated for 4 h at 37 °C,
233
under a 5 % CO2 humidified atmosphere. The absorbance was measured at 492 nm
234
using a microplate reader (Anthos 2020, Biochrom UK). Cells incubated with absolute
235
ethanol were used as a positive control (K+), whereas non-incubated cells were used
236
as negative controls (K-).
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2.11 Antimicrobial activity characterization
239
Microbiological assays were performed with Escherichia Coli DH5α (E. Coli) and
240
Staphylococus Aureus clinical isolate (S. Aureus) as models of prokaryotic organisms
241
(Torres‐Giner, Ocio & Lagaron, 2008). Briefly, 25 or 50 mg of CH-DD or CH-A
242
electrospun mats were added to 10 mL of LB broth at pH 6.2, containing 1 x 105
243
CFU/mL of an early mid-log phase culture of S. Aureus incubated at 37 ˚C, for 24 h.
244
The same process was conducted for E Coli. After the incubation period, serial
245
dilutions were prepared and 100 µL of bacterial samples were transferred into LB agar
246
plates. After overnight incubation at 37 ˚C, bacterial colonies were counted and
247
expressed as the number of colony forming units per mL. The bacterial concentrations
248
were monitored through optical density measurements at 600 nm (Levard et al., 2013).
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Page 8 of 22
To further evaluate membranes antimicrobial properties, the formation of an inhibition
250
halo in E. Coli and S. Aureus cultures, in contact with CH-DD or CH-A membranes,
251
was assessed. Briefly, 100 µL containing 1 x 108 CFU/mL of E. Coli or S. Aureus were
252
spread into agar plates. Subsequently CH-DD or CH-A membranes with ~1cm
253
diameter were placed on top of the agar layer and incubated for 24 h, at 37ºC. The
254
inhibition halo was photographed with a digital camera (NikonD50) and halo size
255
determined with ImageJ (Scion Corp., Frederick, MD), image analysis software.
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256 2.12 In vivo studies
258
A total of 25 female Wistar rats (8–10 weeks old) weighing between 150 and 200 g
259
were used to evaluate the suitability of CH-D or CH-A nanofibrous membranes to be
260
applied in the healing of full thickness wounds. The animal experiments were
261
performed according to the protocol approved by the ethics committee and the
262
guidelines set forth in the National Institute for the care and use of laboratory animals.
263
The animals were housed in individually ventilated cages, Sealsafe Next, Tecniplast
264
(Buguggiate, Italy), at 22ºC with a 12 h/12 h light cycle and fed with commercial rat
265
food, 4RF 21 Mucedola (Milan, Italy) and water ad libitum.. In the day of experiments
266
each rat was individually anesthetized with an intra-peritoneal injection of ketamine (40
267
mg/kg) and xylazine (5 mg/kg). Prior to burn induction, the skin from the dorsal area
268
was shaved and disinfected using ethanol (96%), subsequently wounds with a
269
diameter of approximately 2 cm were created as previously described by our group and
270
with no visible bleeding (Ribeiro, Morgado, Miguel, Coutinho & Correia, 2013).
271
Afterwards, the animals were randomly divided into three groups (n=5): (i) control
272
(group 1), the wounds were washed with PBS pH=7.4; (ii) experimental group 2, the
273
wounds were treated with CH-DD membranes and (iii) experimental group 3, the
274
wounds were treated with CH-A membrane. Animals were only treated once,
275
immediately after wound induction. In order to follow the wound healing process, the
276
wounds were posteriorly photographed with a digital camera (NikonD50) along time.
277
The wound size (WS) was determined by using the image analysis software ImageJ
278
(Scion Corp., Frederick, MD). Changes in animal weight and general animal condition
279
were monitored every other day. At predetermined time points animals from different
280
groups were euthanized via excess CO2. Necropsy was performed to collect the
281
regenerated tissue and major organs for histological analysis.
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282 283
2.13 Statistical analysis
284
Data is presented as mean ± standard deviation (s.d.). One-way analysis of variance
285
(ANOVA) with the Student–Newman–Keuls test was used to compare different groups, 9
Page 9 of 22
286
except when stated otherwise. A p value lower than 0.05 was considered statistically
287
significant. Statistical analysis was performed using GraphPad Prism v.5.0 software
288
(Trial version, GraphPadSoftware, CA, USA).
289
3. Results and discussion
291
3.1 Membrane Characterization
292
The successful binding of L-arginine to CH-DD backbone via EDC/NHS coupling
293
chemistry was evaluated by FTIR. The infra-red spectra (Figure S1) revealed that CH
294
presents a broad band between 3200-3500 cm-1, that was attributed to the
295
characteristic -NH and –OH stretching vibration. The band at 2879 cm-1 was assigned
296
to –CH– stretching of CH polymer. The characteristic peaks present at 1630 and 1528
297
cm-1 are assigned to the amide I and II bands of CH, respectively (Xiao, Wan, Zhao, Liu
298
& Zhang, 2011). CH polymer can be easily identified through the peaks at 1160 cm-1,
299
regarding the asymmetric stretching of the C-O-C, and at 1060-1026 cm-1, regarding
300
the C-O stretching vibrations of the pyranose ring (Bhattarai, Ramay, Gunn, Matsen &
301
Zhang, 2005; Liu, Zhang, Cao, Xu & Yao, 2004). L-arginine exhibits an absorption
302
band at 1614 cm-1 that was assigned to the guanidine group (Liu, Zhang, Cao, Xu &
303
Yao, 2004). The bands at 1419 and 764 cm-1 were ascribed to COO- symmetric and
304
asymmetric bending, respectively. The peak found at 1130 cm-1 is correlated with the
305
C-C-N asymmetric bending (Liu, Zhang, Cao, Xu & Yao, 2004) (Xiao, Wan, Zhao, Liu &
306
Zhang, 2011).
307
In the FTIR spectra of CH-Arg polymer, the characteristic peaks of arginine at 1630
308
and 1410 cm-1 (guanidine group and COO- symmetric bending, respectively), as well as
309
both C-O stretching vibrations of CH polymer pyranose (1062-1033 cm-1) could be
310
observed (Liu, Zhang, Cao, Xu & Yao, 2004). Furthermore, the increase in the amide I
311
peak at ~1654 cm-1 suggests that L-Arginine moieties are indeed linked to CH polymer
312
backbone. The substitution degree of CH-D polymer (17.09±0.25 %) is similar to the
313
value reported in literature to similar coupling conditions (Xiao, Wan, Zhao, Liu &
314
Zhang, 2011). The analysis of the CH-A membrane spectra reveals the characteristic
315
peaks of all the components used in membranes production.
316
Preliminary tests were performed to evaluate the ability to produce nanofibers with the
317
synthesized amino acid modified CH. However, the polymer solutions were difficult to
318
be electrospun, a fact that limited membranes production. To overcome this issue, a
319
blend of CH-Arg and CH-DD polymers was used to allow nanofiber production. The
320
resulting CH-A membranes morphological characteristics are shown in Figure 1 and
321
Figure S2. The CH-A nanofibrous membrane has an opaque appearance, as well as a
322
uniform structure (Figure 1A). A more detailed analysis through SEM (Figure S2B and
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Page 10 of 22
C) reveals a dense 3D nanofiber network comprised by randomly arranged fibrils,
324
which results in an irregular surface with an interconnected porous structure. This 3D
325
organization creates a large contact area with anchoring points for cell-nanofiber
326
interactions enhancing cell adhesion, migration and proliferation (Sangsanoh &
327
Supaphol, 2006). Furthermore, after the cross-linking process (Figure S2C) the CH-A
328
membrane fiber matrix retained its integrity and randomly oriented fiber organization
329
without a significant changes (Schiffman & Schauer, 2007).
330
Biomaterials porosity has been previously described in the literature as a crucial
331
parameter for tissue engineering applications (Khil, Cha, Kim, Kim & Bhattarai, 2003).
332
As demonstrated in Figure 1B, the electrospun CH-A membrane presents a total
333
porosity of 88.25 ± 4.13 %, a value within the preferred range of 60-90%(Chong et al.,
334
2007). The porous nature of these systems is beneficial for cellular infiltration and
335
proliferation. Moreover, porosity also ensures a correct gas, nutrient and fluids
336
exchange, which are essential parameters to attain hemostasis, and ultimately to allow
337
a proper wound healing (Chong et al., 2007). Material surface properties may also
338
influence the host response to the implanted material and the wound healing process
339
(Oliveira, Alves & Mano, 2014). Therefore, the WCA was determined to verify the
340
surface wettability of CH-A membrane (Figure 1C). A WCA value of 25.2±8.4º was
341
obtained, revealing that membrane surface possess a hydrophilic character according
342
to the previously proposed classifications (Oliveira, Alves & Mano, 2014). Hydrophilic
343
surfaces are considered to be the most effective for serum proteins adsorption, which
344
is an important step for subsequent cell adhesion and proliferation (Oliveira, Alves &
345
Mano, 2014). Furthermore, the fiber diameter analysis (Figure 1D) revealed the
346
presence of randomly distributed fibers with diameters ranging from 100 to 400 nm.
347
This is an important parameter, since collagen, the main component of the ECM, has a
348
fibrous-like structure with the collagen fibrils diameters varying from 50 to 500 nm
349
(Jayakumar, Menon, Manzoor, Nair & Tamura, 2010). Therefore, this result suggests
350
that at a structural and morphological level, CH-A electrospun membranes are
351
somehow similar to the human native ECM structure (Stevens & George, 2005).
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353
Figure 1 – CH-A membrane characterization. (A) Macroscopic image of the produced CH-A membrane. (B) CH-A membrane porosity analysis. Data is represented as mean ± s.d., n=3. (C) CH-A membrane surface contact angle analysis. Data is represented as mean ± s.d., n=3. (D) CH-A membrane fiber diameter distribution. Data is represented as mean ± s.d., n=2.
359
The CH-A membrane capacity to maintain moisture in the wound during the healing
360
process was assessed by WVTR assay (Figure S2D). The obtained data shows a
361
nearly constant water weight loss for control and membrane sealed group. The results
362
showed that CH-A membrane has a WVTR of 1713 ± 26 mL/m2/day, a value similar to
363
that of the control, 1801 ± 205 mL/m2/day. Thus, indicating that the CH-A membrane
364
does not limit water vapour exchange. The recommended transmission rate for wound
365
dressings is 2000-2500 mL/m2/day, which is half of the WVTR obtained in granulating
366
tissue (5138±202 mL/m2/day). Therefore, the results indicate that the nanofiber mats
367
could control, at a suitable rate, the water vapour losses and accumulation of exudate
368
fluids, which are common in full thickness wounds (Wu et al., 2004). In fact it is
369
important to mention that exudates accumulation may lead to maceration of healthy
370
skin and inhibit the healing process (Boateng, Matthews, Stevens & Eccleston, 2008).
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354 355 356 357 358
371 372
3.2 Evaluation of the antimicrobial activity of CH-A membranes
12
Page 12 of 22
CH-A membranes bactericidal properties were evaluated using S. Aureus and E. Coli
374
bacteria (Karami, Rezaeian, Zahedi & Abdollahi, 2013; Unnithan, Gnanasekaran,
375
Sathishkumar, Lee & Kim, 2014). These bacterial strains were considered appropriate
376
for performing this assay as models of gram-positive and gram-negative pathogens,
377
respectively.
378
The results obtained from the bactericidal activity evaluation showed that the CH-DD
379
membrane, at a concentration of 2.5 mg, presents an inhibitory effect of ~50% for E.
380
Coli and ~16.5% for S. Aureus. Interestingly, increasing the concentration of CH-DD
381
membrane to 5 mg does not result in a significant improvement in the observed
382
bactericidal effect (Table S1).
383
On the other hand E. Coli and S. Aureus bacterial colonies were completely eliminated
384
(> 99.99%) in the presence of CH-A membranes. These results highlight the
385
importance of coupling L-arginine residues to CH polymer for achieving an enhanced
386
bactericidal effect. It is also important to mention that CH-A membranes bactericidal
387
effect is similar to other chitosan nanofibers that were conjugated with antibiotics or
388
silver based materials (Bai, Chou, Tsai & Yu, 2014).
389
To further evaluate the bactericidal activity of the produced membranes, the formation
390
of an inhibition halo was also evaluated (Figure 2). As shown in Figure 2A1 and B1 the
391
CH-A membranes presented the largest inhibitory halo area (bactericidal area) for both
392
bacterial strains tested. This result confirms an improvement in the antibacterial activity
393
of CH by chemically grafting L-arginine residues in the polymer backbone. Interestingly,
394
both membranes have a higher inhibition area for E. Coli bacteria in comparison to S.
395
Aureus, which is in accordance with the results previously reported in literature for a
396
similar L-arginine CH modifications (Xiao, Wan, Zhao, Liu & Zhang, 2011). This strain
397
dependent efficacy can be related to the different cell wall structures or components
398
present in the different bacteria, which can affect the CH mechanism of action (Xiao,
399
Wan, Zhao, Liu & Zhang, 2011).
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Page 13 of 22
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Figure 2 – Characterization of antimicrobial properties of different CH membranes. Analysis of inhibition halo area in function of the CH-DD or CH-A membrane area for E. Coli (A1) and S. Aureus (B1). Data presented as mean ± s.d., t-student test, *p<0.05, n=3. Macroscopic images of CH-DD membrane inhibition halo in E. Coli (A2) and S. Aureus (B2). Macroscopic images of CH-A membrane inhibition halo in E. Coli (A3) and S. Aureus (B3). The white line delimits the nanofibrous membrane.
407
3.3 In vitro biocompatibility of CH nanofibrous membranes
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The biocompatibility of CH membranes was characterized through the MTS assay. The
409
results obtained showed that FibH cells remained metabolically active in contact with
410
the CH-DD or CH-A membranes even after 72 h of incubation (Figure 3A). These
411
results indicate that L-arginine coupling does not affect membranes biocompatibility,
412
since no differences were observed between CH-DD and CH-A membrane cell viability
413
(Figure 3A). To further assess the applicability of the CH-A membrane for the intended
414
biomedical application, FibH cell adhesion on CH-A membranes and cell morphology in
415
the presence of the membrane was assessed (Figure 3B and C). As demonstrated by
416
Figure 3B, FibH cells already present a typical morphology with various filopodia
417
protrusions after 24 h of incubation, suggesting that skin fibroblasts can adhere and
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start to proliferate on the CH-A membrane surface. Such can be explained by the
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membrane hydrophilic character and the cells membrane glycosaminoglycans
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interaction with the amine groups present in CH-A membrane (Chen, Chung, Wang &
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Young, 2012; Stevens & George, 2005).
422
Furthermore, microscopic images of cells incubated with CH-A membranes (Figure
423
3C2, C4 and C6) show no variations on FibH proliferation rate and morphology in
424
comparison to non-incubated cells, thus corroborating MTS results.
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Figure 3 – Characterization of membranes in vitro biocompatibility. (A) Evaluation of the cell viability through MTS assay after 24, 48 and 72 h. Data represented as mean ± s.d., *p<0.05, n=5. (B) Representative pseudo-colored SEM image of FibH cells in contact with CH-A membrane after 24 h. (C) Representative optical microscopy images of FibH cells, incubated with CH-A membranes for 24 (C2), 48 (C4) and 72 h (C6), and negative controls at 24 (C1), 48 (C3) and 72 h (C5).
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an
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3.3 In vivo evaluation of the wound healing process
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To evaluate the suitability of CH-A membranes for wound healing, a full thickness burn
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was induced in female Wistar rats. All animals showed good general health condition
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throughout the study with no unregular behaviour. Moreover, as observed in Figure S3,
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all the three groups gained weight during the 21 days of the study. Macroscopic
438
analysis of wound regeneration revealed different healing patterns between the tested
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groups (Figure 4A and B). In the initial days after wound induction, the groups treated
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with the electrospun membranes, CH-DD or CH-A, presented a faster wound closure
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than the control group (Figure 4A). These results emphasize the need to perform an
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initial coverage of the damaged tissue in extensive lesions, creating a protective
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environment and a support for cell migration (Zhong, Zhang & Lim, 2010). After 21
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days, the group treated with the CH-A membranes demonstrated a significantly higher
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wound closure than the other two groups, CH-DD and control group (Figure 4A). Also,
446
taking into account the L-arginine improvement in CH bactericidal activity, it is expected
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that CH-A membrane provide an efficient protective barrier against infection.
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The histological analysis (Figure 5) corroborated the results observed in macroscopic
449
images. In membrane treated groups, at 14 days, negligible scar formation,
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immunogenic response or capsule formation was observed when compared to control
451
wounds (Figure 5 A1, A2 and A3). Moreover, treated groups presented a higher collagen
452
deposition in the wound site (Figure 5 B1, B2 and B3). At 21 days, the CH-A membrane
453
treated group showed a higher re-epithelialization degree and tissue reorganization
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(Figure 5 A6 and B6), when compared to the other groups (CH-DD and control). Such
455
data, supports the applicability of the CH-A membrane for wound healing purposes and
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its biocompatible character.
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The improved tissue regeneration observed in CH-A treated group may be attributed to
458
the combination of CH intrinsic wound healing properties, to the nanofibers ECM-like
459
structure and to the additional effect of L-arginine. Actually, different reports in the
460
literature describe that L-arginine has the capacity to improve wound healing by
461
enhancing collagen deposition and the wound tensile strength (Shi, Wang, Zhang,
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Zhang & Barbul, 2007; Witte & Barbul, 2003). Moreover, the positive wound healing
463
effect observed for CH-A nanofibrous membranes is comparable to the one observed
464
in literature for fiber based materials comprised by polymer (e.g., CH, polycaprolactone
465
and PLGA) and expensive ECM constituents (e.g. collagen) (Bai, Chou, Tsai & Yu,
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2014; Chen, Chang & Chen, 2008; Liu, Kau, Chou, Chen, Wu & Yeh, 2010).
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Figure 4 – Evaluation of the healing of a third-degree burn over 21 days. (A) Evolution of wound size along time. Data represented as mean ±s.d., *p<0.05, n=5. (B) Macroscopic images of the wound healing process. Black scale bars correspond to 1cm.
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Figure 5 – Representative images of tissue samples histological analysis in the group treated with PBS (Control), CH-DD membrane and CH-A membrane after 14 and 21 days. Sections stained with Hematoxylin and Eosin (A) and Masson's Trichrome (B). S: scar, M: possible membrane residue, RE: re-epithelialization and C: high collagen density. Black scale bars correspond to 500 µm.
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4. Conclusion
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To the best of our knowledge, this is the first time that the development and
483
applicability of electrospun fibers comprised by L-arginine modified CH and CH-D
484
polymers is evaluated. The developed deacetylated/arginine modified CH polymer
485
blend electrospun membrane presented a highly porous ECM-like nanofiber structure
486
with a hydrophilic character. Furthermore, the membrane demonstrated bactericidal
487
activity for different prokaryotic organisms. The in vitro assays revealed that the
488
membrane is non-cytotoxic, and provides a 3D matrix, with significant contact area, to
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allow cell adhesion and proliferation. In vivo assays demonstrated that the CH-A
490
membrane significantly improves tissue regeneration of full thickness wounds, when
491
compared to CH-DD membrane or the natural healing process. This work shows the
492
applicability of L-arginine to modify CH and improve its bactericidal and wound healing
493
properties. Furthermore it was shown that CH-A membrane can be efficiently applied to
494
improve the wound healing process in a cost-effective way. In a near future, the
495
inclusion of growth factors, proteins, plant extracts, drugs or genetic material in these
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membranes may enhance even further the wound healing capacity of the system.
497
Moreover, a layer-by-layer approach can be used to mimic the native structural
498
organization of the different skin layers.
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499 Acknowledgments
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The authors would like to thank Eng. Ana Paula Gomes for the help in the acquisition
502
of SEM images, but also to M.Sc. Ricardo Fradique for the pseudo-colored version of
503
SEM FibH adhesion image. This work was supported by the Portuguese Foundation for
504
Science
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OE/SAU/UI0709/2014)). V. M. Gaspar acknowledges an individual PhD fellowship from
506
FCT (SFRH/BD/80402/2011). A. F: Moreira acknowledges a fellowship from UBI-
507
Banco Santander/Totta
an
(PEst-C/SAU/UI0709/2011
and
PEst-
ed
M
(FCT),
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Technology
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and
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