Reza Gharibia,
Hamid Yeganeh*a,
Hoshyar Gholamia and
Zuhair M. Hassanb
aIran Polymer and Petrochemical Institute, P.O. Box: 14965/115, Tehran, Iran. E-mail: h.yeganeh@ippi.ac.ir; Fax: +98 2144787023; Tel: +98 2148662447
bDepartment of Immunology, School of Medical Science, Tarbiat Modares University, P.O. Box: 14115-331, Tehran, Iran. E-mail: hasan_zm@modares.ac.ir
First published on 13th November 2014
The preparation of functional intelligent wound dressing membranes with features including: excellent dimensional stability in both dry and hydrated states, preserving a moist environment over the wounded area by self-adjustment of their permeability in response to wound moisture level, a broad spectrum of antimicrobial activity towards different microorganisms, promising antioxidant (radical scavenging) efficiency and significant activity for stimulation of fibroblast cells growth and proliferation is reported in this work. For the preparation of dressing membranes, NCO-terminated polyurethane prepolymers and a CHO-terminated aniline tetramer are functionalized with silane groups. Polyurethane/siloxane networks are prepared through a sol–gel reaction of these components at different weight ratios. The corresponding nanosilver composites are simply prepared by a redox reaction through impregnating the membranes with a silver salt solution. Based on various in vitro tests, it is revealed that some of the prepared membranes have the basic characteristics of ideal dressings, while they can actively alleviate the healing of tissue in the wounded area.
Accelerating the wound healing processes is an important issue that has attracted much attention in recent years. It is anticipated that modern wound dressings should change from traditional materials to active and functional materials. It is expected that these dressings not only protect wounded area, but also interact with the wound bed appropriately to encourage the healing process.3 Stimulation of proliferation and migration of keratinocytes by transdermal delivery of growth factors like epidermal growth factor (EGF) is an example of accelerated wound healing process.4 Indeed, the wound healing process is complex and involves the interactions of many different types of cells and matrix components to establish a provisional tissue and eventually a complete regenerated epidermis. Therefore, application of growth factors may suffer from some limitations such as high manufacturing costs, low bioavailability and imperfect effect of a single growth factor on wound healing processes. As well, inactivation of growth factors at the chronic wound bed due to proteolytic degradation by protease activity is another concern in this method.3
It seems that constructing wound dressing materials that can help body to heal itself is the preferred choice in this emerging subject. To full fill this goal; three simultaneous strategies are followed in the present study. The first concern is preventing the effect of free radicals. Free radicals are present to a great extent in all types of trauma.5,6 The process of wound healing can be hampered by the presence of free radicals; therefore, quenching radical activity can prevent further damage to wound surrounding cells and consequently increase the rate of healing process.7 Some of recent studies have shown promising performance of antioxidant and radical scavenging properties of conducting polymers such as polyaniline and oligoaniline derivatives.8,9 Utilization of these unique aspects of oligoaniline is examined in the present work.
The second concern is reducing the bacteria influence on damaged tissue. It is confirmed that reducing infection and subsequently decreasing the wound exudates by application of effective antimicrobial agents on infected wounds can accelerate the healing process.10
In recent studies it is emerged that polyaniline and its derivatives may exhibit broad spectrum of biocidal activity.11 Gizdavic-Nikolaidis and co-workers12 have shown that in addition to polyaniline and its functionalized analogous, the low molecular weight oligomeric types of these materials have antibacterial activity as well. Therefore, utilization of this inherent property of oligomeric aniline moieties (aniline tetramer) is considered in the present work. The common practice for utilization of dressing materials with antibacterial activity is loading of different antimicrobial agents such as iodine,13 quaternary ammonium salts,14,15 zinc oxide16 and silver17 in wound dressings. Due to a broad spectrum of antimicrobial activity and low bacterial resistance, application of silver nanoparticles has currently attracted considerable attention. To broaden and increase the efficiency of biocidal activity of dressing membranes, the incorporation and utilization of silver nanoparticles has also been considered in the present study. Two approaches are commonly employed for the preparation of polymer–silver nanocomposites. In the first method, as-prepared silver nanoparticles can be mixed with polymeric matrix. Agglomeration and improper distribution of silver nanoparticles are the main drawbacks of this approach.18 In the second approach, the polymeric matrix is swollen with the solution of silver salt. In situ reduction of silver ions by a reducing agent like NaBH4 leads to homogeneous dispersion of silver nanoparticles into the polymeric matrix. Utilization of reducing power of embedded oiligoaniline19,20 into the polymeric matrix is a fascinating method that has been used in the present work for rapid and efficient production and distribution of silver nanoparticles into polymeric matrix of the dressing.
Electrical stimulation has been found to be an effective technique to promote wound healing process.21 Recent studies have shown that the electrical stimulation may increase protein, collagen and DNA synthesis. Cellular activities involving cell adhesion, migration, proliferation and differentiation can be also regulated by this type of stimulation.22–24 To have these advantages, many researchers have tried to incorporate different kinds of conducting polymers into biomaterials, mainly for tissue engineering application. In this regard, use of oligoaniline with accept' electrical conductivity and good processability has attracted extensive interest.25–27 It is worth mentioning that these advantages can be attained even in the absence of an external electrical stimulation.28–30 Accelerated proliferation, adhesion and growth of different cells such as osteoblasts and preosteoblastic MC3T3-E1 cells on polymeric materials containing electroconductive oligoaniline were reported by Yen Wei and co-workers.31–33 Neurite extension and nerve repair accelerating without any need of external electrical signals have also been reported for matrix-containing electroactive polymers.23 Primary dermal fibroblast and keratinocytes are other important cells, especially for skin wounds, have been examined for their accelerated growth on electroactive matrix by Ateh and Shin groups.34–36 Therefore, utilization of this possible advantage is another goal of this study.
Synthesis, characterization and assessment of hybrid organic–inorganic polyurethane networks embedded with aniline tetramer and corresponding nanocomposites containing silver nanoparticles are described in the present study. Based on different in vitro methods it is tried to show that regulation of wound exudates, facilitation of cell proliferation, combating bacterial contamination and having radical scavenging ability are versatile means to boost wound healing process.
Sample | Si-PU (g) | Si-AT (g) | TEOS (g) | H2O (g) | Gel Content (%) |
---|---|---|---|---|---|
a According to analysis of variances P-values of <0.05 were considered significant. The difference between quantities with similar superscripts is not significant (p ≥ 0.05) for data of each column. | |||||
NEPU | 1.00 | — | 0.20 | 0.15 | 97.7 ± 0.7a |
EAPU1 | 1.00 | 0.18 | 0.20 | 0.15 | 97.8 ± 0.4a |
EAPU2 | 1.00 | 0.30 | 0.20 | 0.15 | 97.6 ± 0.1a |
EAPU3 | 1.00 | 0.42 | 0.20 | 0.15 | 97.6 ± 0.6a |
For comparison, a none-electroactive polyurethane membrane (NEPU) was also prepared through the similar procedure using all ingredients except Si-AT component.
It is worth to mention that the final dressing membranes were subjected to the solvent extraction/purification procedure by immersion into ethanol (70% w/w) solvent for 24 h and subsequently into distilled water for another 24 h before further characterization and assays.
To mimic the typical environment of serum and wound exudate, a solution of sodium/calcium chloride containing 142 mmol l−1 of sodium ions and 2.5 mmol l−1 of calcium ions was used.
For measuring the AC of samples a completely dried membrane with thickness of (0.1 mm) was soaked into a beaker containing the test solution. The weight of swelled membrane was determined after being gently wiped with filter paper to remove the surface liquid. AC was calculated using the following equation:
WVTR of membranes was determined under two different states of conditions. In the first state the membrane was not in direct contact with liquid and it was just exposed to moisture vapour. In the second state the membrane was placed in direct contact with the test solution.
Five samples of each dressing of known weight were applied on the cup mouth containing test fluid. The cups were weighed and placed in an incubator at 37 ± 2 °C for a period of 24 h. At the end of the test, the cups were removed from the incubator, allowed to equilibrate to room temperature and reweighed. The loss in weight due to the passage of moisture vapour through the dressing was determined using the following equation:
The samples were sterilized by incubation at 120 °C for 15 min before each test. In “direct contact test”, the evaluation of cells morphology present on the interface of samples and culture plate was performed after incubation time of 48 h.
To check whether the cytotoxic agents may possibly leach out from the wound dressing membranes, the freshly synthesized samples were immersed in the culture medium for 3 days at 37 °C and then MTT assay was performed on extracted leachates using L929 fibroblast cells.
The percentage of relative cell viability was calculated according to following equation:
The spatial distribution and morphology of the L929 cells attached onto the surface of the membranes were assessed after staining of cells by a fluorescent dye. For this purpose, the above mentioned procedure was followed. After seeding of cells on the samples surface and their incubation for specifically determined times of 1, 3, and 5 days, the samples were washed three times with PBS. The adhered cells were fixed with 2.5% glutaraldehyde at room temperature for 8 min. The cells were dyed with DMSO containing 2% of curcumin for 8 min; followed by washing the samples with PBS several times. The stained cells were observed by an inverted microscope (TE2000-U, Nikon).
For those samples containing Ag NPs, the “shaking flask” method was followed. The membranes (triplicate samples) were seeded with 10 ml determined colony forming units per ml (CFU ml−1) of either E. coli, S. aureus, P.aeruginosa and C. albicans. The solutions containing the samples were incubated in a shaking incubator at 37 °C for 24 h. For correct counting of the grown bacterial colonies, 0.1 ml of the treated solution was diluted with double distilled water to a suitable volume and then, the diluted solution was spread on a nutrient agar plate and incubated at 37 °C for 12 h. A NEPU sample (blank sample) was also studied as positive control. The counts were used to calculate the surviving number of bacteria. The percentage of bacterial reduction was calculated using the following equation:
For the measurement of the electrical conductivity, the membranes, at both dry and hydrated states, were used in circular shape with thickness of about 1 mm. The conductivity was measured using a home-made four-point probe instrument utilizing following equation:
Dynamic mechanical thermal analysis (DMA) was carried out with a Tritec 2000 DMA instrument in the temperature range −100 to 150 °C at a heating rate of 3 °C min−1 and a frequency of 1 Hz. The dimension of each sample was 30 × 10 × 1 mm3. The values of storage modulus, loss modulus and tanδ versus temperature were recorded for each sample.
The measurement of tensile strength, elongation-at-break, and modulus of samples under dry and hydrated states were carried out using a tensile tester (Instron 6025) with a crosshead speed 50 mm min−1. Samples were cut into bars of each 50 mm length and 5 mm width. The test was performed at room temperature. For each sample five specimens were tested.
The gel content of cured membranes was evaluated. For this purpose the membranes were dried under vacuum for 24 h at room temperature and weighed. Then, the samples were extracted by THF in a Soxhlet extractor for 24 h. The insoluble part was dried at 50 °C and weighed. The gel content was defined as follows:
The size of Ag NPs was evaluated by atomic force microscopy (AFM) in contact mode. For this purpose, the EAPU membrane containing Ag NPs was placed in distilled water for 72 h. A few drops of the extracted solution were placed on a mica slide. The residue, which was obtained after slow evaporation of solvent, was examined by AFM. The silver atom content and its distribution map were evaluated by a Tescan scanning electron microscope (Vega II, Czech) equipped with an energy dispersive X-ray analyzer system (EDX, Oxford Instrument, INCA, England).
Surface hydrophilicity of membranes was determined by measuring the water droplet contact angle at six different positions of membrane surface for each sample. The contact angle was determined by running Image J 1.44p software on digital pictures taken from interfaces of membranes and droplets. The values reported are an average of six measurements.
AT was synthesized via oxidative coupling of N-phenyl-1,4-phenylenediamine in the presence of ammonium persulfate. To incorporate AT segments into the final networks silane-functional aniline tetramer was prepared as shown in Scheme 2. GA was used as the coupling agent between AT and APS. FTIR spectra of AT, GA-AT and Si-AT were shown in Fig. S2a–c.†
To confirm that the functionalization reactions performed on AT would not affect its electroactivity, the UV-visible spectra of AT, GA-AT and Si-AT were recorded in DMF (Fig. S3†).
The wound dressing membranes with different concentrations of electroactive AT counterpart were prepared through mixing of ingredients as tabulated in Table 1. The reaction of alkoxysilane moieties and formation of siloxane network (Si–O–Si
) through hydrolysis and condensation reaction was triggered by addition of water under acidic condition. The probable structure of final network is shown in Scheme S1.† To control the crosslink density and tune the mechanical property of final networks through formation of siloxane domain, a constant weight of TEOS was added to all formulations. The high value for gel content of the prepared networks (Table 1) showed successful progress of sol–gel reaction. The presence of Si–O–Si peak at about 1050 cm−1 in the ATR-FTIR spectra of the cured membranes (Fig. S4a and b†) was also a confirmation in the formation of desired networks.
The dressing materials applied to the wound bed should preserve their dimensional stability during the healing period. To acquire an insight into this important factor, tensile strength of the membranes was assessed at both dry and wet conditions. It is well known that plasticization effect of the absorbed water molecules can dramatically reduce the intermolecular interaction of polar polymeric chains and consequently reduce the overall mechanical strength. Therefore, the tensile test was also performed on fully hydrated samples to confirm the extended applicability of these membranes for the protection of high exuding wounds. The tensile strength, initial modulus, and elongation-at-break of samples were extracted from their stress–strain curves (Fig. 2a and b) and corresponding data are summarized in Table S1.† All the prepared membranes showed general behavior of lightly crosslinked elastomers with suitable mechanical property for wound dressing application. The reinforcing effect of polysiloxane domain produced through hydrolysis and condensation of methoxysilane moieties is responsible for such observed behavior. The tensile strength and initial modulus were increased with increasing the concentration of Si-AT in the formulation of membranes, whereas an opposite trend was detected for the elongation-at-break values. This phenomenon was in accordance with DMA data and it was related to the presence of Si-AT moieties. As it was stated by Wei,45 the aromatic rings of aniline oligomers can stack over each other through strong π–π interactions and consequently increase the strength of the network. It is interesting to note that the reinforcing effect of oligoaniline moieties is more evident in tensile properties at large deformation. In fact, alignment and extension of polyetherurethane chains in rubbery state contribute to the improved performance of AT moieties.
As expected, the tensile strength of membranes was reduced under wet condition due to plasticizing effect of the absorbed water molecules. Samples with higher concentration of Si-AT (EAPU2, EAPU3) showed lower drop in tensile strength under wet condition in comparison to NEPU and EAPU1 as a result of the reinforcing effect of AT molecules. Another reason for higher tensile strength for EAPU2 and EAPU3 was lower water absorption of these samples (Table 2) and consequently, lower deterioration of intermolecular interactions due to presence of water molecules between the chain segments. The range of tensile properties and elongation-at-break values in this study implied that most of the prepared membranes were considered suitable materials for wound dressings with appropriate level of mechanical properties.
Sample | EWA (%) | EWC (%) | Contact angle (°) | WVTR (g/10 cm2 per day) | |
---|---|---|---|---|---|
Dry | Wet | ||||
a According to analysis of variances P-values of <0.05 were considered significant. The difference between quantities with similar superscripts is not significant (p ≥ 0.05) for data of each column. | |||||
NEPU | 299.3 ± 2.3a | 196.6 ± 4.5a | 59.2 ± 2.3a | 1.15 ± 0.03a | 10.3 ± 0.21a |
EAPU1 | 277.6 ± 2.5b | 178 ± 2.1b | 61.3 ± 2.1a,b | 1.01 ± 0.02b | 9.8 ± 0.11b |
EAPU2 | 242.6 ± 7.6c | 141.3 ± 5.8c | 68.4 ± 1.2c | 0.92 ± 0.02c | 9.1 ± 0.22c |
EAPU3 | 217 ± 1.0d | 116.9 ± 0.5d | 73 ± 1.5d | 0.84 ± 0.01d | 8.4 ± 0.18d |
To evaluate the ability of the prepared membranes for absorbing and retaining wound exudates, their EWA and EWC were measured (Table 2). It was found that NEPU membrane have EWA and EWC around 300% and 200%, respectively. High water absorption ability of this membrane is attributed to the hydrogen bonding type interaction of PEG segment with water molecules. Electroactive membranes with embedded AT moieties showed lower EWA and EWC compared to the NEPU. Sample EAPU3 with highest concentration of Si-AT showed the lowest EWA of EAPU. This behaviour was related to the hydrophobic nature of aromatic AT molecules. Comparison of recorded data with some of important commercial film dressings such as Tegaderm (3 M), Bioclusive (Johnson & Johnson) and Op Site (Smith & Nephew) with EWA of 21, 24 and 35, respectively,46 showed superior performance of the membranes prepared in this study.
The same trend was observed for the surface hydrophilicity of membranes as determined by measuring the contact angle of water droplets on the surface of samples. As shown on Table 2, by increasing the hydrophobic Si-AT content, the contact angle increased from 59° to 73°. Previous studies revealed that the fibroblast cells (as one of important cells that have critical role in wound healing process) show maximum adhesion to the surface of materials with contact angle in the range of 60° and 80°.47 Therefore, the prepared membranes have suitable surface hydrophilicity for effective contribution in cellular activity.
To prevent excessive dehydration as well as build up of exudates, the ideal wound dressing should have adequate WVTR. Commonly used dressings have fixed WVTR regardless of exudate level. Not only different wounds have different exudate levels, but they may vary with time for a specific type of wound. It is expected that modern dressings should have the ability to respond to a wounds exudate level and raise or lower their WVTR to maintain a constant moist environment over wound bed. To estimate the ability of dressing material for proper handling of wound exudates, a test methodology was developed, which involved direct and indirect fluid contact with the dressings.48 The data of WVTR for the prepared membranes are summarized in Table 2.
In dry state, when membranes are not in contact with fluid but just exposed to vapour the NEPU membrane showed higher WVTR than those samples containing Si-AT. This phenomenon can be attributed to either hydrophobic nature of AT segments, or barrier effect of π–π stacked AT moieties against water molecules. The recorded values are much higher than some of commercially available membrane dressings made from dense polyurethane films such as Bioclusive (Johnson–Johnson, 0.394 g/10 cm2 per day), Tegaderm (3 M, 0.492 g/10 cm2 per day) and Op Site (Smith & Nephew, 0.792 g/10 cm2 per day).46 In hydrated state, when membranes are in contact with fluid, much higher values may be recorded for the prepared membranes. The higher WVTR of membranes at wet state can be attributed to the association of chains with water molecules creating structures that facilitate water transportation across the dressing membrane. In fact, by structural expansion or contraction due to changing moisture level the prepared membranes could be able to control their WVTR. This intelligent ability of the prepared materials for self-adjusting of their WVTR in response to exudate level of wound enables them to be applied for management of exudates over a wide range of wounds and different stages during healing process.
To find better perspective regarding practical applicability of the prepared dressing membranes for different wound types and comparison with commercially available dressings another parameter, fluid handling capacity, was calculated (Fig. 3).49,50 This parameter constituted both AC and WVTR and provided better view for exudates managing power of dressings.
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Fig. 3 FHC of the membranes at (a) dry and (b) wet states; (c) exudates produced by different wound types (data were collected from ref. 50). (According to analysis of variances P-values of <0.05 were considered significant. The difference between quantities with similar superscripts is not significant (p ≥ 0.05)). |
In dry state the calculated FHC values for the prepared membranes were in the range of 1.51 to 1.93 g/10 cm2/24 h. The recorded data was comparable with some of related commercially available thin absorbent wound dressing such as BeneHold (1.97 g/10 cm2/24),51 and hydrocolloid dressings such as Askina Biofilm Transparent (0.51 g/10 cm2/24), Granuflex (1.78 g/10 cm2/24), Algoplaque (1.54 g/10 cm2/24) and Varihesive E (0.51 g/10 cm2/24).52 In hydrated state the FHC values of dressings were increased up to 11.1g/10 cm2/24 h. For comparison, data for some related commercial dressings were as follows: ActivHeal dressing (FHC = 4.6 g/10 cm2/24 h), Allevyn dressing (FHC = 16 g/10 cm2/24 h).50
The intelligent behaviour of the prepared dressing suggests the possible application of these materials for various types of wounds with different levels of exudates (Fig. 3c). These materials can prevent dryness of tissue over lightly exuding wound, and also prevents wound maceration due to accumulation of exudates over heavily exuding wounds.
The size of Ag NPs was also measured, since it has determining effect on antibacterial activity.55 For this purpose, the extracted solution containing Ag NPs was deposited on a freshly cleaved Mica film and the AFM analysis was performed on the film after slow evaporation of water. The visualized Ag NPs (Fig. 4) had an average size at about 45 nm. Furthermore, the AFM micrograph exposed the homogeneous size distribution of these nanoparticles.
The weight percent and distribution of Ag NPs within the membranes were also estimated via EDX analysis (Fig. S6†). The analysis was carried out on the cross section of each membrane. A good distribution of Ag NPs (red dots) with higher population near the surface of membranes was detected. According to EDX data, the amount of in situ generated Ag NPs was in the range of 0.06–0.46 w%. This value showed dependency to the concentration of chemically embedded AT moieties. The overall concentration of Ag NPs was increased by higher redox active AT segment concentration in the formulation of membranes. Inspection of Fig. S6† showed very good distribution of the siloxane network (green dots) throughout the membrane, therefore, an isotropic tensile property was expected for these materials.
Sample | Conductivity in dry state (S cm−1) | Conductivity in wet state (S cm−1) |
---|---|---|
NEPU | 6.5 × 10−11 | — |
Doped EAPU1 | 2.6 × 10−7 | 2.7 × 10−3 |
Doped EAPU2 | 4.5 × 10−6 | 8.5 × 10−3 |
Doped EAPU3 | 1.3 × 10−5 | 1.2 × 10−2 |
NEPU-Ag | 6.7 × 10−11 | — |
EAPU1-Ag | 3.4 × 10−7 | 3.4 × 10−3 |
EAPU2-Ag | 7.8 × 10−6 | 1.1 × 10−2 |
EAPU3-Ag | 3.5 × 10−5 | 4.5 × 10−2 |
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Fig. 5 Optical microscopy of L-929 cells in direct contact with the membranes (a) negative control (b) NEPU (c) EAPU2 (d) doped EAPU2 9 (e) doped EAPU3 and 9 (f) EAPU2-Ag. |
To further confirm this phenomenon, the MTT assay was performed on leachates extracted from membranes after 3 days. Results are collected in Fig. 6. Cells remained viable in contact with extracted leachates from membranes without Ag NPs. Due to the absence of labile linkages in the structure of the prepared membranes; there was no chance for the chemical bond cleavage and consequently, release of possibly toxic materials during the time scale of using such materials for wound dressing application. As well, the purification procedures applied for treatment of final networks (solvent extraction) was successful for expelling any possible residues may remain during preparation of dressing membranes.
After incorporation of Ag NPs in the EAPU membranes, a very low reduction in L929 cells viability was detected that was associated to the toxic nature of the silver nanoparticles released from the membranes. Optimization of Ag NPs concentration has prime importance in the present work since high concentration of these particles can directly improve the antimicrobial activity, but at the same time it can reduce the viability of cells. Fortunately, the reduction in cells viability for EAPU1-Ag and EAPU2-Ag samples was not very high, but still these samples showed perfect antimicrobial property (discussed in next section). In contrast, the viability of cells was significantly reduced for EAPU3-Ag in comparison to two other silver containing samples. In this sample high concentration of AT moieties led to higher in situ reduction of silver ions to Ag NPs. Although such higher Ag NPs may be advantageous for their antimicrobial activity but living cells cannot also tolerate such high concentration. Therefore, this sample was not suitable for the intended application as wound dressing membrane. Fortunately, the versatility of our methodology enable us to tune the desired level of AT and consequently the Ag NPs concentration.
Affinity of cells for adhesion to the biomaterial surface is an excellent indication for evaluation of cytocompatibility of biomaterials. Due to dark colour of the prepared electroactive membranes, it was impossible to follow adhered cells by direct observation of samples surface by optical microscopy. For quantification of cells populated on the prepared membranes, the membranes were cultured with fibroblast cells (type of cell that engage in synthesis of extracellular matrix and collagen and plays a critical role in wound healing process). After certain time intervals (1, 3 and 5 days) the samples were removed from culture media, washed and placed into the new culture media with no cells. Then, the numbers of adhered cells were quantified via MTT assy. Results are collected in Fig. 7.
As it was depicted in Fig. 7, after 1 day culture time, there was no significant difference between non-electroactive and electroactive membranes prior to doping with CSA. However, there was a significant increase (p < 0.05) in the numbers of adhered cells onto the doped EAPU2. With increasing culture time to 3 and 5 days a distinct increase (p < 0.05) was detected for number of adhered and proliferated cells to doped electroactive samples (EAPU1 and EAPU2) in comparison to either non-electroactive, electroactive samples in undoped form and doped EAPU3 with highest concentration of AT moieties. It seems that after attachment of cells to the doped elctroconductive membranes with proper concentration of AT moieties, they showed greater tendency for entering into a rapid proliferative growth phase, establish critical cell–cell interactions and grow to confluence on the membranes. In fact, after doping with CSA, AT moieties of EAPU membranes were able to display their electroactivity and induced the chemical and energy exchange between the cells and its surroundings, which benefit the cell growth. It is well known that charge can influence on the adsorption of proteins and consequently affect cell viability and cell proliferation.61 Meanwhile, toxicity may also develop by introduction of charge on the material. As it was shown in Fig. 7, the developed charge on AT moieties of membranes after doping with CSA exhibited a strong influence on cell growth. The proper concentration of AT moieties in EAPU1 and EAPU2 led to excellent proliferative cell growth as good as tissue culture plate (TCP). The high concentration of AT in doped EAPU3 resulted in reduction in cell viability; however, the cell viability on this sample is still comparable with NEPU and EAPU1-3 in undoped state. As expected, introduction of Ag NPs in surface and bulk of membranes led to slight reduction of cells viability. The EAPU1-Ag and EAPU2-Ag with proper concentration of Ag NPs still showed acceptable cell viability and could support cell growth and proliferation.
For finding better insight regarding the state of adhered cells, the morphology of stained fibroblast cells cultured for 3 days on different membranes was investigated by fluorescence microscopy. The results are collected in Fig. S8.† Fortunately, most of the fibroblast cells were grown properly. They were flat and elongated with spindle-shaped morphology on the surface of the prepared membranes which confirmed their excellent ability to support cell growth and proliferation.
Based on the above discussion regarding mode of interaction of dressing membranes with fibroblast cells, there is promising sign for positive influence of optimum dressing membranes on accelerated healing of the wounded tissue.
The aforementioned in vitro cytocompatibility studies showed desired properties of these materials. These results confirmed the proper selection of starting materials, and indicated the suitability of reaction conditions selected for the preparation of final networks through the sol–gel process. However, judgments regarding biocompatibility of the prepared dressings were made based on data obtained through in vitro study of fibroblast cells behaviour. Evaluating the interaction of other cells involve in healing of dermal wounds such as Keratinocytes and extending these studies on animal models can help to find deeper insight regarding practical usage of such dressing materials.
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Fig. 8 Antibacterial activity of the prepared membranes against (a) S. aureus, (b) C. albicans, (c) P. aeruginosa and (d) E. coli. |
At least three mechanistic pathways were reported for the bactericidal effect of Ag NPs on different bacteria. Ag NPs can join to the surface of the cell membrane and disturb some basic functions, such as permeability and respiration. For this mode of action binding of the Ag NPs onto the bacteria directly depends on their available surface area. Since the smaller particles have a larger available surface area for interaction with bacteria they will exhibit a stronger bactericidal effect. Penetration of Ag NPs through cell wall and damaging bacteria, possibly by interacting with sulphur and phosphorus-containing compounds found in abundance throughout the cell membrane is another mechanistic pathway for bactericidal effect of Ag NPs. The smaller Ag NPs penetrate easer through cell wall. Penetrated particles can also inactivate enzymes, generating hydrogen peroxide and causing bacterial cell death.63 Silver ions released from Ag NPs make an additional contribution to the bactericidal effect since DNA loses its replication ability and denaturation of cellular proteins occur upon Ag+ treatment.64
The simple method developed in the present study enabled us to produce spherical or near spherical Ag NPs in an average size of about 45 nm, which is a suitable dimension for having promising antibacterial properties.65 Results of bactericidal activity of Ag NPs containing dressings are collected in Table S2.† According to the recorded data even for EAPU1-Ag sample with the lowest concentration of AT moieties and consequently the lowest amount of generated Ag NPs, a significant bactericidal activity was detected. It is well documented that Ag NPs have lower bactericidal activity on gram positive bacteria.66 This phenomenon can be explained by the presence of thick peptidoglycan and teichoic acids in the gram-positive bacteria cell wall. With increasing the Ag NPs concentration in EAPU2-Ag and EAPU3-Ag almost complete bactericidal activity was detected for all of strains under study.
From antimicrobial point of view, the doped electroconductive membranes (EAPU1-3) have moderate to good antimicrobial activity. However, from cells viability and consequently cells proliferation point of view which is a good indication for possible participation in accelerating of healing process, these membranes have superior property. Antimicrobial activity could be improved to the almost complete killing ability of microorganisms, simply, via immersion of membranes into the silver salt solution and generation of silver nanoparticles on the surface and bulk of the membranes. However, this was associated with reduction in cells viability and proliferation. These broad properties for prepared membranes enabled us to choose the proper dressings based on wound state.
There are various reports concerning preparation of wound dressings impregnated with antioxidants, since the effectiveness of the antioxidants is much higher when they are included into a dressing material.69 Previous studies proved that the aniline and its polymeric analogous have radical scavenging effect.8 Therefore, it was anticipated that the embedded AT moieties of the prepared dressing material as an oligomeric analogous of aniline should also have antioxidant property. To evaluate this property of dressing membranes, the common assessment method based on electron transfer reaction to DPPH radicals was utilized. The behavior of AT containing membranes was compared with NEPU sample (Fig. S9†). No reaction occurred between NEPU sample with DPPH solution, since no change was detected in the intensity of peak at 516 nm associated to DPPH radicals. However, a progressive decrease in intensity of this peak was detected, when CSA doped EAPU samples with increasing amount of AT moieties were tested. This phenomenon could be attributed to the electron transfer or hydrogen atom donation from AT segments to DPPH free radicals. Elimination of these radicals led to reduction of the peak intensity at 516 nm. This decrease in peak intensity was more predominant for samples with higher concentration of AT moieties. The efficiency for DPPH scavenging activity of samples is collected in Table 4. The antioxidant activity of EAPU2-Ag was also measured under the same condition. Fortunately, no considerable reduction in antioxidant activity of this sample was recorded in comparison to the corresponding parent membrane (EAPU2). The small decrease in DPPH scavenging effect of silver-containing membrane may be related to partial conversion of AT segment at emeraldine form to pernigraniline state during redox reactions leading to the production of Ag NPs.
Sample | DPPH scavenged (%) |
---|---|
a According to analysis of variances P-values of <0.05 were considered significant. The difference between quantities with similar superscripts is not significant (p ≥ 0.05) for data of each column. | |
NEPU | 2.1 ± 0.8a |
EAPU1 | 36.9 ± 1.5b |
EAPU2 | 65.4 ± 1.1c |
EAPU3 | 79.2 ± 2.1d |
EAPU2-Ag | 56.7 ± 1.9e |
Fibroblast cells viability and proliferation rate were significantly improved for dressing membranes containing proper concentration of AT moieties. Meanwhile, these samples showed moderate to good biocidal activity for gram positive, gram negative and fungal strains. Sample EAPU2 showed the best biological properties, i.e. highest cell compatibility and strongest antimicrobial activity. Introduction of Ag NPs into the dressing membranes simply through immersion of membranes into the silver salt solution offered perfect antimicrobial activity; however, partial reduction in cytocompatibility was detected. Sample EAPU2-Ag showed the balanced properties among silver containing membranes.
All of the electroconductive membranes (with and without Ag NPs) showed promising radical scavenging property, therefore, it is expected that these membranes can participate in regulating the concentration of reactive radical species present in wound site during inflammatory stage of healing process.
In vivo evaluation of selected dressing membranes is under consideration in our lab.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra11454j |
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