Research Article www.acsami.org
Stimulation of Wound Healing by Electroactive, Antibacterial, and Antioxidant Polyurethane/Siloxane Dressing Membranes: In Vitro and in Vivo Evaluations Reza Gharibi,† Hamid Yeganeh,*,† Alireza Rezapour-Lactoee,‡ and Zuhair M. Hassan§ †
Department of Polyurethane, Iran Polymer and Petrochemical Institute, P.O. Box 14965-115, Tehran, Iran Department of Tissue Engineering, School of Advanced Medical Technologies, Tehran University of Medical Sciences, 14177-55469 Tehran, Iran § Department of Immunology, School of Medical Sciences, Tarbiat Modares University, P.O. Box 14115-331, Tehran, Iran
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S Supporting Information *
ABSTRACT: A series of novel polyurethane/siloxane-based wound dressing membranes was prepared through sol−gel reaction of methoxysilane end-functionalized urethane prepolymers composed of castor oil and ricinoleic methyl ester as well as methoxysilane functional aniline tetramer (AT) moieties. The samples were fully characterized and their physicochemical, mechanical, electrical, and biological properties were assayed. The biological activity of these dressings against fibroblast cells and couple of microbes was also studied. It was revealed that samples that displayed electroactivity by introduction of AT moieties showed a broad range of antimicrobial activity toward different microorganisms, promising antioxidant (radical scavenging) efficiency and significant activity for stimulation of fibroblast cell growth and proliferation. Meanwhile, these samples showed appropriate tensile strength and ability for maintaining a moist environment over a wound by controlled equilibrium water absorption and water vapor transmission rate. The selected electroactive dressing was subjected to an in vivo assay using a rat animal model and the wound healing process was monitored and compared with analogous dressing without AT moieties. The recorded results showed that the electroactive dressings induced an increase in the rate of wound contraction, promoted collagen deposition, and encouraged vascularization in the wounded area. On the basis of the results of in vitro and in vivo assays, the positive influence of designed dressings for accelerated healing of a wound model was confirmed. KEYWORDS: polyurethane/siloxane, wound healing, antimicrobial, antioxidant, electroactivity
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INTRODUCTION A wound can be defined as an injury or tear on the skin surface by physical, chemical, mechanical, and thermal damages.1 When skin is damaged, the resulting wound should be protected from further contamination or trauma by covering with a proper dressing. To ensure effective wound healing, the dressing should be nontoxic and biocompatible, promote gaseous exchange, and protect the wound from external mechanical stress. Dressings should also provide and maintain a moist environment over the wounded skin, because it is widely accepted that under such conditions acceleration of the healing process can occur.2 To construct a dressing with the maximum number of ideal factors, several synthetic and natural polymers © 2015 American Chemical Society
can be utilized. Among synthetic materials, polyurethanes with high biocompatibility, good permeability to oxygen and carbon dioxide, excellent mechanical strength, and proper flexibility have been widely used for wound dressing applications.3 The attractive characteristics of polyurethanes for wound dressing application are mainly determined by the nature of the starting materials in their synthesis. Preparation of polyurethanes from renewable resources, especially vegetable-oil-based polyols, has become an area of intensive interest, due to their attractive Received: September 7, 2015 Accepted: October 16, 2015 Published: October 16, 2015 24296
DOI: 10.1021/acsami.5b08376 ACS Appl. Mater. Interfaces 2015, 7, 24296−24311
Research Article
ACS Applied Materials & Interfaces physicomechanical and excellent biocompatibility.4,5 Nowadays, these materials have found applications in bone substitutes,6 nerve regeneration,7 and wound dressing.8 Castor oil (CO) is a very special material among vegetable oils. The major constituent of CO is ricinoleic acid (12hydroxy-cis-9-octadecenoic acid), a hydroxyl-containing fatty acid that provides the conditions for direct synthesis of polyurethane without any prior functionalization reactions. Interesting biological properties are also reported for CO and its derivatives such as high biocompatibility, remarkable analgesic and anti-inflammatory effects,9,10 as well as positive influence on the epithelialization process.11 Therefore, CO has been selected as the main starting material for preparation of polyurethane dressings in the present work. Close inspection of advanced wound dressings recently introduced into the market revealed that these materials have undergone significant changes. In addition to physical protection of wounds, the advanced dressing materials should stimulate tissue regeneration and the healing process actively. Among several possibilities for active involvement in the healing process, we have focused on three characteristic features, i.e., antimicrobial activity, electroactivity, and antioxidant properties. Below, the logics behind these prerequisites are described. Bacterial infections delay the healing process, increase exudate formation, and facilitate improper collagen deposition.12 Wound dressing itself can also cause infection if it is improperly sterilized.12 There are interesting reports in the literature that reveal that antimicrobial agents like silver nanoparticles have the ability of reducing the number of pathogens and the inflammatory response of the wound site can promote the wound healing process.13 Therefore, utilization of antimicrobial wound dressings is a good method to avoid the above-mentioned problems. Some recent studies have confirmed the broad spectrum of antimicrobial activity of polyaniline and its oligomeric analogous.14,15 To take advantage of these characteristics, oligoaniline was examined in the present work. There are interesting research findings regarding the significant role electroactive materials, such as conductive polymers, play in cellular activities such as cell adhesion, proliferation, migration, and differentiation of electrically excitable cells such as nerve, bone, muscle, keratinocytes, fibroblasts, cardiac, and mesenchymal stem cells.16 Because the regulation of cellular behavior is critical for the regeneration of new tissue, utilization of this advantage in dressing membrane is considered in this study. Reactive oxygen species (ROS) are important inflammatory mediators under pathological conditions and overproduction of these species can disrupt the cellular oxidant/antioxidant balance leading to tissue damage, infection, and slow wound healing.17 The conducting polymers have been widely studied to scavenge free radicals;18,19 therefore, wound dressings containing conducting polymers can reduce the excessive levels of free radicals and consequently protect tissue from oxidative damage during the healing process. Utilizing radical scavenging activity of oiligoaniline for modification of dressing membranes was considered in the present work. In our previous work, we have shown that simultaneous utilization of these features (antimicrobial activity, electroactivity, and antioxidant property) in a wound dressing material could have a positive influence on the wound healing process under in vitro conditions.20 To expand this strategy and widen
the wound types that can be healed by this active category of dressings, new polyurethane/siloxane membranes were prepared using a renewable resource raw material, CO, and its derivative ricinoleic methyl ester (RM). An oligoaniline material, aniline tetramer (AT), was also properly functionalized and incorporated into the dressing formulations as an active ingredient. These novel materials with tailor-made physical and mechanical properties were fully characterized by conventional methods. Their biological activities including their influence on proliferation of fibroblast cells and inhibition of microbial growth were examined under in vitro conditions. Also, the designed active dressing was applied on a full thickness skin wound created on a rat animal model to acquire a better insight regarding its potential influence on the wound healing process. For comparison, a similar dressing without active AT moieties was prepared and its performance on a similar wound model was explored.
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EXPERIMENTAL SECTION
Materials. CO with hydroxyl number 154.5 mg KOH/g was purchased from Sigma. It was dried at 80 °C under vacuum for 24 h just before use. Isophorone diisocyanate (IPDI) from Merck was purified via vacuum distillation. (3-Aminopropyl)trimethoxysilane (APS), N-phenyl-1,4-phenylenediamine, methanol, dibutyltindilaurate (DBTDL), ammonium persulfate, glutaraldehyde (GA) (50 wt % in H2O), sodium metal, diethyl ether, and camphorsulfonic acid (CSA) were purchased from Aldrich. N,N-Dimethylformamide (DMF), chloroform, and methyl ethyl ketone (MEK) were distilled over CaH2. All other chemicals were of analytical grade and used as received. Staphylococcus aureus (ATCC 6538) and Pseudomonas aeruginosa (ATCC 15449) bacteria and Candida albicans (ATCC 10231) fungi were purchased from Iranian Research Organization for Science and Technology (IROST). Mouse L929 fibroblast cells were also received from Pasteur Institute of Iran and used as obtained. Synthesis of Methoxysilane-Terminated CO-based Polyurethane Prepolymer (Si-CPU). A three-necked polymerization reactor equipped with a mechanical stirrer, condenser, dropping funnel, and a nitrogen inlet was charged with IPDI (37.36 g) and CHCl3 (100 mL). A solution of CO (40.00 g) in CHCl3 (100 mL) was slowly dropped into the reactor during 30 min under ambient temperature. After that, a drop of DBTDL catalyst was introduced into the system and the temperature increased. The reaction continued under reflux conditions until the free NCO content of the product reached the calculated theoretical value as determined by a backtitration method (ASTMD-2572) using a standard solution of dibutylamine. The heating mantle was removed, and the reactor content was cooled to 5 °C. Then, APS (60.23 g) was dropped into the reactor. The mixture was stirred at the same temperature for 30 min, and then the temperature increased to 50 °C for 3 h. The reaction product with predetermined solid content (30% w/w) was transferred to a glass bottle, sealed, and kept in a refrigerator. Synthesis of Methoxysilane-Terminated Ricinoleic Methyl Ester Urethane Prepolymer (Si-RM). First, ricinoleic methyl ester (RM) was synthesized and characterized according to the procedure reported by Romero21 with some modifications. A 250 mL two-necked round-bottomed flask, equipped with condenser and magnetic stirrer, was charged with CO (20.0 g) and methanol (64.0 g). Then, a solution of sodium methoxide in methanol (0.6 mL, 16% w/v) was added to the above solution and the reaction mixture was heated to 65−70 °C and stirring was continued for 2 h. After this stage, the excess amount of methanol was removed by using a rotary evaporator. The crude product was dissolved in diethyl ether (40 mL) and transferred into a separating funnel. The ether solution was repeatedly washed with distilled water, until the pH value of the water phase was neutral. The solvent was evaporated and the final product was subjected to high vacuum at 85 °C for 12 h to ensure complete removal of any residual solvent and water molecules. The OH number of synthesized RM was measured as 168.5 mg KOH/g. 24297
DOI: 10.1021/acsami.5b08376 ACS Appl. Mater. Interfaces 2015, 7, 24296−24311
Research Article
ACS Applied Materials & Interfaces Table 1. Formulation of Sample with Different Amounts of Si-RMa samples NESiPU1 NESiPU2 NESiPU3 NESiPU4
Si-CPU solution (g)
Si-RM solution (g)
3.33 3.33 3.33 3.33
H2O (g)
gel content (%)
0.10 0.10 0.10 0.10
± ± ± ±
1.66 2.50 3.33
99.5 99.6 99.3 98.7
υc (mol/cm3)b
a
1.3 6.7 1.9 6.5
0.2 0.1a 0.4a 0.2a
× × × ×
10−1 10−2 10−2 10−3
appearance brittle brittle semiflexible flexible
a
According to analysis of variances, P-values of
