Accepted Manuscript A biocompatible sodium alginate/povidone iodine film enhances wound healing Maria Summa, Debora Russo, Ilaria Penna, Natasha Margaroli, Ilker S. Bayer, Tiziano Bandiera, Athanassia Athanassiou, Rosalia Bertorelli PII: DOI: Reference:
S0939-6411(17)30775-0 https://doi.org/10.1016/j.ejpb.2017.10.004 EJPB 12609
To appear in:
European Journal of Pharmaceutics and Biopharmaceutics
Received Date: Revised Date: Accepted Date:
29 June 2017 6 October 2017 6 October 2017
Please cite this article as: M. Summa, D. Russo, I. Penna, N. Margaroli, I.S. Bayer, T. Bandiera, A. Athanassiou, R. Bertorelli, A biocompatible sodium alginate/povidone iodine film enhances wound healing, European Journal of Pharmaceutics and Biopharmaceutics (2017), doi: https://doi.org/10.1016/j.ejpb.2017.10.004
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.
A biocompatible sodium alginate/povidone iodine film enhances wound healing Maria Summa#1, Debora Russo#1, Ilaria Penna1, Natasha Margaroli1, Ilker S. Bayer2, Tiziano Bandiera1, Athanassia Athanassiou*2, Rosalia Bertorelli*1 1
PharmaChemistry, Drug Discovery and Development, Istituto Italiano di Tecnologia, Via Morego
30, 16163, Genoa, Italy. 2
Smart Materials, Nanophysics, Istituto Italiano di Tecnologia, via Morego 30, 16163 Genoa, Italy.
#
: MS and DR contributed equally to this work
*E-mail addresses: [email protected] (R. Bertorelli)
Keywords: biomaterial, wound dressing, inflammation, sodium alginate, PVPI, human foreskin fibroblasts; mice.
1
Abstract
In the last few years, there has been an increasing tendency to use natural polymers for the fabrication of dressings for wound and burn management. Among them, alginate, a polysaccharide extracted primarily from marine algae, exhibits attractive properties being non-toxic, hydrophilic and biodegradable. The aim of this study was to characterize the in vitro biocompatibility and the efficacy of a composite polymeric material based on sodium alginate (NaAlg) and povidone iodine (PVPI) complex in a mouse model of wound healing. The developed material combines the excellent wound healing properties of alginates with the bactericidal and fungicidal properties of PVPI, providing a controlled antiseptic release. We demonstrated that the NaAlg/PVPI films are able to reduce
the inflammatory response
both in human foreskin fibroblasts
after
lipopolysaccharide (LPS) stimulus and in rodents after wound induction. Furthermore, the NaAlg/PVPI film-treated animals showed a significantly higher wound closure compared to untreated animals at each time point considered. Interestingly, the complete wound closure was achieved within 12 days only in the film-treated group, indicating that the full-thickness wounds healed more rapidly in these animals. The results demonstrate that the NaAlg/PVPI films are biocompatible and possess healing properties that accelerate the wound closure.
2
1. Introduction
Wound healing is a complex and highly regulated physiological process that involves various cell types (i.e. immune cells, endothelial cells, keratinocytes, and fibroblasts) and pathways, activated and coordinated in order to restore tissue integrity and homeostasis (Gurtner et al., 2008). This dynamic process can be divided into four, partly overlapping, phases: hemostasis, inflammation, proliferation and maturation (Boateng et al., 2008). After injury, bleeding occurs and this vascular response helps to cleanse the wound of surface contaminants (e.g. bacteria and/or antigens). Hemostasis results from the activation of platelets, which starts the coagulation cascade and platelet aggregation to form the hemostatic blood clot that acts as a temporarily protective barrier for the injured skin (Sinno & Prakash, 2013). The inflammatory phase occurs almost simultaneously with hemostasis, within 24 hours and lasts for four – six days. This phase is mediated by neutrophils and monocytes (that differentiate into macrophages) (Broughton et al., 2006). Neutrophils are involved in infection control, cleaning of the wound from foreign particles (e.g. debris, bacteria) together with macrophages. Moreover, both these cell types release various cytokines, chemokines, and enzymes that activate fibroblasts and myofibroblasts in the proliferation phase of wound healing (Das & Baker, 2016). These molecules, together with a variety of nutrients, are contained in the wound exudate, which provides the moist environment important for effective healing. In the proliferation phase, epithelialization occurs and newly formed granulation tissue, consisting of endothelial cells, macrophages, and fibroblasts begin to cover and fill the wound area by producing new extracellular matrix (ECM) required for sustaining cells and blood vessels, which provide nutrients needed to restore tissue integrity. Finally, during the maturation (also called “remodeling phase”), collagen forms tight cross-links with other collagen and protein molecules, increasing the tensile strength of the scar (Sinno & Prakash, 2013). Delayed or impaired wound healing has a significant socio-economic burden for patients and health-care systems worldwide (Sen et al., 2009). Multiple factors (e.g. desiccation, oxygenation, transpiration, and infections) and pathological conditions (e.g. diabetes, obesity, and cancer) can lead to impaired wound healing, increasing the incidence of chronic wounds (Guo & Dipietro, 2010). In particular, some common features shared by these conditions include a prolonged or excessive inflammation (Eming et al., 2007), persistent infections (Edwards & Harding, 2004), formation of drug-resistant microbial biofilms (Wolcott et al., 2008), and the inability of dermal and/or epidermal cells to respond to reparative stimuli. Moreover, the excessive exudate production associated to abnormal inflammation can cause skin sensitivities and tissue maceration and infection (Cutting, 2003). Elimination or reduction of prolonged inflammation revitalizes tissue healing, reduces exudate and is usually associated with bioburden reduction.
3
For these reasons, the design of advanced therapeutic dressing, that play an active role in promoting healing of both acute and chronic wounds, represents a promising approach to fulfil the requirements needed for the next generation wound care. Bioactive natural polymers are commonly used as wound dressings because of their biocompatibility, biodegradability, mechanical properties, bioresorption ability, and relatively low-cost (Mayet et al., 2014). Among them, NaAlg, a polysaccharide derived from brown seaweed, is the most widely investigated and used in biomedical applications. Alginate is highly hydrophilic and able to absorb wound exudate, maintaining a moist microenvironment. Moreover, the anti-inflammatory activity of NaAlg electrospun nanofibers was recently reported (Hajiali et al., 2016). Alginate dressings are also useful as delivery platform in order to provide a controlled release of therapeutic substances (e.g. pain-relieving, antibacterial and anti-inflammatory agents) to exuding wounds (Lin et al., 1999; Cho et al., 2002; Maver et al., 2015). PVPI is one of the topical antiseptic agents most commonly used in wound care (Atiyeh et al., 2009). PVPI is a water-soluble complex of iodine and polyvinylpyrrolidone, a broad-spectrum antiseptic agent effective against a wide variety of bacteria, viruses, fungi, protozoa and yeasts. Despite its diffusion as topical antiseptic agent, PVPI solution showed toxic effects on human skin fibroblast, totally inhibiting cell growth at 0.1% and 1.0% concentrations (Balin & Pratt, 2002), suggesting a potential damaging effect towards cells involved in wound repair. In order to overcome this harmful effect, we have incorporated PVPI into alginate matrix to produce NaAlg/PVPI composite films, allowing its time controlled release at the wound surface. Moreover, we have demonstrated that these films have antimicrobial and antifungal activities against Escherichia coli and Candida albicans, respectively (Liakos et al., 2013). In the choice of a wound dressing material, its biocompatibility represents one of the most important features, in particular for bioactive polymers that can interact with the wound environment and influence cell functions (Paul & Sharma, 2004). To this purpose, we evaluated NaAlg/PVPI films biocompatibility on human foreskin fibroblast (HFF-1) and assessed their potential anti-inflammatory effect in a Lipopolysaccharide (LPS)-stimulated inflammatory model, previously developed with the same cell line (Hajiali et al., 2016). Dexamethasone, a synthetic glucocorticoid compound with potent anti-inflammatory activities, was used as positive control for interleukins reduction after LPS-stimulation (Mianji et al., 1996). We then went on to study the effect of NaAlg/PVPI films in vivo, applying them to a murine full-excisional skin wound model and exploring their efficiency in wound healing, measured as time and percentage of decrease of open wound area and as migration of epithelial tongue in the wound bed. We also evaluated the antiinflammatory effect and hydroxyproline levels as an indicator of collagen content in the extracts of healing skin tissues. In vitro and in vivo studies were conducted in parallel on a commercial
4
Povidone Iodine Non-Adherent Dressing product, used as standard of care for wound management.
5
2. Materials and methods
2.1. Materials and cell culture Sodium alginate, PVPI (Poly(vinylpyrrolidone)–Iodine complex), lipopolysaccharides (LPS) from Escherichia coli (serotype 026:B6), dexamethasone (DXM) and Trypan Blue 0.4% solution (T8154) were purchased from Sigma Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), L-glutamine and penicillin–streptomycin were from Euroclone (Milan, Italy). Human foreskin fibroblasts (HFF-1) were from ATCC®. A commercial Povidone Iodine Non-Adherent Dressing product (named as Product A throughout the text) was used as standard of care for wound management. This dressing consists in a knitted viscose fabric impregnated with a polyethylene glycol (PEG) base containing 10% Povidone Iodine equivalent to 1% available iodine, which is released in the presence of wound exudate. RNA extraction kit (PureLink RNA Mini Kit) and Super-Scripts® VILOTM cDNA Synthesis Kit were purchased from Ambion by Life Technologies (USA). Gene-specific primers using fluorogenic probes (TaqMan) and TaqMan ® Universal PCR Master Mix, No AmpErase ® UNG were from Applied Biosystems (USA). 2.2. Preparation of the NaAlg/PVPI films NaAlg/PVPI films were prepared as previously described (Liakos et al., 2013). Briefly, 3 g of NaAlg were dissolved in 100 mL of distilled water under constant stirring for 1 h at 100°C. Then, 0.3 g of PVPI were added to NaAlg solution and dissolved under constant stirring for 1 h at room temperature, to obtain a final concentration of 10% by wt. PVPI. Finally, a volume of glycerol (corresponding to 5% of the starting NaAlg weight) was added to the NaAlg/PVPI solution and dissolved under constant stirring for 1 h at room temperature. Each film was cast with 10 mL of solution and dried for 24 h under a chemical hood. 2.3. Film characterization and extraction for in vitro studies According to ISO10993-12 (sample preparation), an extraction ratio of 6 cm 2/mL (surface area/volume) should be used for preparation of test samples. To standardize the treatments, NaAlg/PVPI films (55 cm2/film) were weighted and their thickness was measured using a digital micrometer. Five measurements were performed on different parts of the films. Average values with the corresponding standard deviation (S.D.) were calculated. Based on the average values obtained, 318.7 mg of films, corresponding to a surface area of 55 cm2, were extracted in 9.2 mL of cell culture medium (containing 3.5 mg PVPI/mL) to perform both cytotoxicity and anti-inflammatory experiments. Extraction medium from NaAlg/PVPI films was prepared adapting the procedure described in ISO10993-5 standard test. Briefly, films were sterilized under a germicidal UV lamp
6
(wavelength 253.7 nm) for 1 h (30 min/side), then samples were cut into small pieces to enhance submersion in the extraction medium. Extraction was performed in cell culture medium for 24 h at 37°C and was used immediately after preparation. Product A was cut into squares according to the following extraction ratios: 0.7 – 1.0 - 1.5 cm2/mL, corresponding to 2.7 - 3.9 - 5.8 mg/mL PVPI, respectively. Extraction was performed in cell culture medium for 24 h at room temperature.
2.4. In vitro biocompatibility assay HFF-1 cells were grown in DMEM supplemented with 10% FBS, 2 mmol L -1 L-glutamine, 100 IU per mL penicillin and 0.1 mg mL-1 streptomycin, in a humidified incubator at 37°C with 5% CO 2. For biocompatibility experiments, HFF-1 cells were seeded in 60 mm cell culture dishes at a density of 3.5 X 105. After 24 h of culture, medium was replaced with the extraction one (control samples were treated with medium processed as the extractions) and cells were incubated for additional 24 h. Cell viability was assessed using the trypan blue exclusion assay. Based on dye uptake, viable (clear) and nonviable (blue) cells were counted using a hemocytometer and a light microscope. Cell viability was expressed as a percentage relative to control cells. 2.5. In vitro evaluation of anti-inflammatory properties HFF-1 cells were seeded in 60 mm cell culture dishes at a density of 4 X 10 5 and cultured for 24 h. Then, cell culture medium was removed and four different types of culture dishes were prepared: dishes containing medium processed as the extractions (control) or extraction media from NaAlg/PVPI films and Product A dressing. Product A extraction was performed using an extraction ratio of 0.7 cm2/mL (corresponding to 2.7 mg PVPI), which showed a percent survival comparable to NaAlg/PVPI films (SI). For each set of dishes, half of them were treated with 1 µg mL -1 LPS to induce inflammatory response. Culture medium with LPS and 1 µM of DXM (a corticosteroid known to decrease inflammation), dissolved in serum free medium, was used as a positive control. After 6 h, cells were harvested and total RNA was extracted by spin column-based purification method, in order to quantify human IL-1β and IL-6 expression by qRT-PCR. 2.6. qRT-PCR cytokine mRNA quantification 1 µg of total RNA was reverse transcribed into first-strand cDNA by using Super-Scripts® VILOTM cDNA Synthesis Kit in a final volume of 20 µL. HPRT-1 was used as the reference housekeeping gene in RT-PCR assays. Amplification of cytokines target genes and HPRT-1 was conducted with 50 ng of cDNA in 20 µL of the reaction mixture by gene-specific primers using fluorogenic TaqMan probes and TaqMan® Universal PCR Master Mix. TaqMan primer/probes sets, spanning exon-exon junctions, for human IL-1β (Hs00174097_m1), IL-6 (Hs00985639_m1) and the housekeeping gene
7
HPRT-1 (Hs02800695_m1) were used in PCR reactions. They were run in 96-well format on ViiATM 7 Real-Time PCR System (Applied Biosystems) using universal cycling conditions (95°C, 10 min; 95°C, 15 s; and 60°C, 1 min for 40 cycles). Finally, cycle threshold (CT) values were determined by QuantStudio™ Real-Time PCR software v1.1. 2.7. Animals Male C57BL/6J mice, 8 weeks old (Charles River, Calco, Italy), were used for in vivo studies. Animals were group-housed in ventilated cages and had free access to food and water. They were maintained under a 12-hour light/dark cycle (lights on at 8:00 am) at a controlled temperature of (21 ± 1°C) and relative humidity of (55 ± 10%). All experiments were carried out in accordance with the guidelines established by the European Communities Council Directive (Directive 2010/63/EU of 22 September 2010) and approved by the National Council on Animal Care of the Italian Ministry of Health. All efforts were made to minimize animal suffering and to use the minimal number of animals required to produce reliable results. 2.8. Wound healing model Mice were anesthetized with an intraperitoneal injection of ketamine (10%) and xylazine (5%), and their dorsal surface was shaved and rinsed with an alcohol swab. A full-thickness excisional wound of 1 cm2 was induced in the center of the back of each animal. A photo of the wound was taken immediately afterwards the biopsy generation. Both dressings were applied three times throughout the experiment, and specifically, immediately soon, 3 and 4 days after wound generation (n = 5 mice each experimental group). After the first application, all treated mice were covered with TegadermTM. However, during the first three-day observation, and when we changed the dressings, we realized that the TegadermTM covering application was highly uncomfortable for animals. Then, wounds were covered only with dressings, but since animals removed Product A during the night, we reapplied both dressings the day after and they were left in place until day 7. Before taking pictures, Product A was removed, while, on the contrary, NaAlg/PVPI was already reabsorbed. No other application was done up to the end of experiments (Day 12). In conclusion, patches have been applied on day 0, 3, and 4 after wound induction. On day 0, 3, 7, 9 and 12 pictures were collected in order to measure wound closure (ImageJ software), which was calculated as percentage based on wound size relative to control group. During the experiments, mice were housed individually, with water and food ad libitum.
2.9. In vivo cytokine expression measurements Skin samples from sham, wound, NaAlg/PVPI and Product A treated animals were collected 2 days post wound induction and snap-frozen in liquid nitrogen (n = 5 mice each experimental
8
group). Cytokines (IL-6 and IL-1β) expression were measured using ELISA quantikine kit (R&D system), according to the manufacturer’s instructions (Romano et al., 2015). The cytokine concentration was normalized against the total protein content for a given sample, as measured using the bicinchoninic acid (BCA) assay (Thermo Scientific, Rockford, IL, USA). 2.10. Hydroxyproline production measurements Hydroxyproline (4-hydroxyproline) production was evaluated in skin biopsy from untreated, NaAlg/PVPI and Product A treated animals collected 7 days post wound induction and snap-frozen in liquid nitrogen (n = 5 mice each experimental group). By using hydroxyproline assay kit (Sigma Aldrich), according to the manufacturer’s instructions, the amount of hydroxyproline was calculated as µg of hyroxyproline per full thickness skin biopsy. Hydroxyproline content variation was expressed as percent vs. the control group. 2.11. Histology After 7 days from wound induction, an area of 1.5 cm diameter was excised for the three different groups of treatment. The histological specimens were fixed in 4% paraformaldehyde, embedded in paraffin and 5 µm serial skin sections were collected. Sections were stained with hematoxylin and eosin (H&E) and analyzed with an Olympus BX51 microscope (tip distance measured with Neurolucida software, MBF Bioscience). Migration tongue distance variation was expressed as an absolute distance in µm vs control group (n = 5 mice each experimental group). 2.12. Data analysis
2.12.1. In vitro studies The amplification of the PCR product was expressed as the number of amplification cycles at which this particular product was first detected above the background (CT). The analysis of relative gene expression was performed by the comparative 2 -ΔΔCT method. The average CT from three replicates was calculated for both target and HPRT-1 genes. The sample target quantity was corrected by the respective value of HPRT-1. The ΔCT was determined to normalize for amounts of RNA used in reverse transcription reactions; the data are presented as fold change (2 -ΔΔCT ± S.D.). One-way ANOVA was used to evaluate statistical significance, followed by Bonferroni’s post-hoc test. GraphPad Prism 5 was used for all statistical analysis (GraphPad Software Inc. San Diego, CA, USA). P values less than 0.05 were considered significant.
9
2.12.2. In vivo studies All data were expressed as means ± S.E.M. For ELISA experiments, the value obtained from each mouse was calculated as pg/mg of protein. One way ANOVA followed by Dunnett's test multiple comparison was used to evaluate statistical significance. For wound closure analysis, two-way ANOVA followed by Bonferroni’s post-hoc test was applied GraphPad Prism 5 was used for all statistical analysis (GraphPad Software Inc. San Diego, CA, USA). P values less than 0.05 were considered significant.
3. Results
3.1. In vitro studies
3.1.1. NaAlg/PVPI film characterization and biocompatibility studies Treatment of HFF-1 cells for 24 h with NaAlg/PVPI extraction medium did not affect viability since 93.2% of cells remained viable (Fig. 1). No statistically significant differences (p>0.05) were observed in the cell survival in comparison with the control. According to ISO10993-5 guidelines, a reduction of cell viability by less than 30% indicates no toxicity of NaAlg/PVPI sample. Product A was tested in parallel for its biocompatibility at different extraction ratios (0.7 – 1.0 - 1.5 cm2/mL, corresponding to 2.7 - 3.9 - 5.8 mg/mL PVPI, respectively) (see Supporting Information), in order to determine a safe dose to use in anti-inflammatory in vitro experiments. The lowest ratio tested (0.7 cm2/mL), that gave a survival higher than 70%, was selected. 3.1.2. Anti-inflammatory properties of NaAlg/PVPI film HFF-1 cells stimulated with LPS were treated with DXM (positive control) or extraction media obtained from NaAlg/PVPI and Product A dressing and of mRNA levels of IL-6 and IL-1β were investigated (Fig. 2A-B and Table 1). DXM, a well-known and largely used anti-inflammatory drug, induced a very high reduction of IL-6 and IL-1β mRNA, accounting for 95.1% (Fig. 2A) and 97.6% (Fig. 2B) of control, respectively. NaAlg/PVPI extraction medium effectively reduced the mRNA levels of about 96.4% and 92.7% for IL-6 and IL-1β (p
S0939-6411(17)30775-0 https://doi.org/10.1016/j.ejpb.2017.10.004 EJPB 12609
To appear in:
European Journal of Pharmaceutics and Biopharmaceutics
Received Date: Revised Date: Accepted Date:
29 June 2017 6 October 2017 6 October 2017
Please cite this article as: M. Summa, D. Russo, I. Penna, N. Margaroli, I.S. Bayer, T. Bandiera, A. Athanassiou, R. Bertorelli, A biocompatible sodium alginate/povidone iodine film enhances wound healing, European Journal of Pharmaceutics and Biopharmaceutics (2017), doi: https://doi.org/10.1016/j.ejpb.2017.10.004
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.
A biocompatible sodium alginate/povidone iodine film enhances wound healing Maria Summa#1, Debora Russo#1, Ilaria Penna1, Natasha Margaroli1, Ilker S. Bayer2, Tiziano Bandiera1, Athanassia Athanassiou*2, Rosalia Bertorelli*1 1
PharmaChemistry, Drug Discovery and Development, Istituto Italiano di Tecnologia, Via Morego
30, 16163, Genoa, Italy. 2
Smart Materials, Nanophysics, Istituto Italiano di Tecnologia, via Morego 30, 16163 Genoa, Italy.
#
: MS and DR contributed equally to this work
*E-mail addresses: [email protected] (R. Bertorelli)
Keywords: biomaterial, wound dressing, inflammation, sodium alginate, PVPI, human foreskin fibroblasts; mice.
1
Abstract
In the last few years, there has been an increasing tendency to use natural polymers for the fabrication of dressings for wound and burn management. Among them, alginate, a polysaccharide extracted primarily from marine algae, exhibits attractive properties being non-toxic, hydrophilic and biodegradable. The aim of this study was to characterize the in vitro biocompatibility and the efficacy of a composite polymeric material based on sodium alginate (NaAlg) and povidone iodine (PVPI) complex in a mouse model of wound healing. The developed material combines the excellent wound healing properties of alginates with the bactericidal and fungicidal properties of PVPI, providing a controlled antiseptic release. We demonstrated that the NaAlg/PVPI films are able to reduce
the inflammatory response
both in human foreskin fibroblasts
after
lipopolysaccharide (LPS) stimulus and in rodents after wound induction. Furthermore, the NaAlg/PVPI film-treated animals showed a significantly higher wound closure compared to untreated animals at each time point considered. Interestingly, the complete wound closure was achieved within 12 days only in the film-treated group, indicating that the full-thickness wounds healed more rapidly in these animals. The results demonstrate that the NaAlg/PVPI films are biocompatible and possess healing properties that accelerate the wound closure.
2
1. Introduction
Wound healing is a complex and highly regulated physiological process that involves various cell types (i.e. immune cells, endothelial cells, keratinocytes, and fibroblasts) and pathways, activated and coordinated in order to restore tissue integrity and homeostasis (Gurtner et al., 2008). This dynamic process can be divided into four, partly overlapping, phases: hemostasis, inflammation, proliferation and maturation (Boateng et al., 2008). After injury, bleeding occurs and this vascular response helps to cleanse the wound of surface contaminants (e.g. bacteria and/or antigens). Hemostasis results from the activation of platelets, which starts the coagulation cascade and platelet aggregation to form the hemostatic blood clot that acts as a temporarily protective barrier for the injured skin (Sinno & Prakash, 2013). The inflammatory phase occurs almost simultaneously with hemostasis, within 24 hours and lasts for four – six days. This phase is mediated by neutrophils and monocytes (that differentiate into macrophages) (Broughton et al., 2006). Neutrophils are involved in infection control, cleaning of the wound from foreign particles (e.g. debris, bacteria) together with macrophages. Moreover, both these cell types release various cytokines, chemokines, and enzymes that activate fibroblasts and myofibroblasts in the proliferation phase of wound healing (Das & Baker, 2016). These molecules, together with a variety of nutrients, are contained in the wound exudate, which provides the moist environment important for effective healing. In the proliferation phase, epithelialization occurs and newly formed granulation tissue, consisting of endothelial cells, macrophages, and fibroblasts begin to cover and fill the wound area by producing new extracellular matrix (ECM) required for sustaining cells and blood vessels, which provide nutrients needed to restore tissue integrity. Finally, during the maturation (also called “remodeling phase”), collagen forms tight cross-links with other collagen and protein molecules, increasing the tensile strength of the scar (Sinno & Prakash, 2013). Delayed or impaired wound healing has a significant socio-economic burden for patients and health-care systems worldwide (Sen et al., 2009). Multiple factors (e.g. desiccation, oxygenation, transpiration, and infections) and pathological conditions (e.g. diabetes, obesity, and cancer) can lead to impaired wound healing, increasing the incidence of chronic wounds (Guo & Dipietro, 2010). In particular, some common features shared by these conditions include a prolonged or excessive inflammation (Eming et al., 2007), persistent infections (Edwards & Harding, 2004), formation of drug-resistant microbial biofilms (Wolcott et al., 2008), and the inability of dermal and/or epidermal cells to respond to reparative stimuli. Moreover, the excessive exudate production associated to abnormal inflammation can cause skin sensitivities and tissue maceration and infection (Cutting, 2003). Elimination or reduction of prolonged inflammation revitalizes tissue healing, reduces exudate and is usually associated with bioburden reduction.
3
For these reasons, the design of advanced therapeutic dressing, that play an active role in promoting healing of both acute and chronic wounds, represents a promising approach to fulfil the requirements needed for the next generation wound care. Bioactive natural polymers are commonly used as wound dressings because of their biocompatibility, biodegradability, mechanical properties, bioresorption ability, and relatively low-cost (Mayet et al., 2014). Among them, NaAlg, a polysaccharide derived from brown seaweed, is the most widely investigated and used in biomedical applications. Alginate is highly hydrophilic and able to absorb wound exudate, maintaining a moist microenvironment. Moreover, the anti-inflammatory activity of NaAlg electrospun nanofibers was recently reported (Hajiali et al., 2016). Alginate dressings are also useful as delivery platform in order to provide a controlled release of therapeutic substances (e.g. pain-relieving, antibacterial and anti-inflammatory agents) to exuding wounds (Lin et al., 1999; Cho et al., 2002; Maver et al., 2015). PVPI is one of the topical antiseptic agents most commonly used in wound care (Atiyeh et al., 2009). PVPI is a water-soluble complex of iodine and polyvinylpyrrolidone, a broad-spectrum antiseptic agent effective against a wide variety of bacteria, viruses, fungi, protozoa and yeasts. Despite its diffusion as topical antiseptic agent, PVPI solution showed toxic effects on human skin fibroblast, totally inhibiting cell growth at 0.1% and 1.0% concentrations (Balin & Pratt, 2002), suggesting a potential damaging effect towards cells involved in wound repair. In order to overcome this harmful effect, we have incorporated PVPI into alginate matrix to produce NaAlg/PVPI composite films, allowing its time controlled release at the wound surface. Moreover, we have demonstrated that these films have antimicrobial and antifungal activities against Escherichia coli and Candida albicans, respectively (Liakos et al., 2013). In the choice of a wound dressing material, its biocompatibility represents one of the most important features, in particular for bioactive polymers that can interact with the wound environment and influence cell functions (Paul & Sharma, 2004). To this purpose, we evaluated NaAlg/PVPI films biocompatibility on human foreskin fibroblast (HFF-1) and assessed their potential anti-inflammatory effect in a Lipopolysaccharide (LPS)-stimulated inflammatory model, previously developed with the same cell line (Hajiali et al., 2016). Dexamethasone, a synthetic glucocorticoid compound with potent anti-inflammatory activities, was used as positive control for interleukins reduction after LPS-stimulation (Mianji et al., 1996). We then went on to study the effect of NaAlg/PVPI films in vivo, applying them to a murine full-excisional skin wound model and exploring their efficiency in wound healing, measured as time and percentage of decrease of open wound area and as migration of epithelial tongue in the wound bed. We also evaluated the antiinflammatory effect and hydroxyproline levels as an indicator of collagen content in the extracts of healing skin tissues. In vitro and in vivo studies were conducted in parallel on a commercial
4
Povidone Iodine Non-Adherent Dressing product, used as standard of care for wound management.
5
2. Materials and methods
2.1. Materials and cell culture Sodium alginate, PVPI (Poly(vinylpyrrolidone)–Iodine complex), lipopolysaccharides (LPS) from Escherichia coli (serotype 026:B6), dexamethasone (DXM) and Trypan Blue 0.4% solution (T8154) were purchased from Sigma Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), L-glutamine and penicillin–streptomycin were from Euroclone (Milan, Italy). Human foreskin fibroblasts (HFF-1) were from ATCC®. A commercial Povidone Iodine Non-Adherent Dressing product (named as Product A throughout the text) was used as standard of care for wound management. This dressing consists in a knitted viscose fabric impregnated with a polyethylene glycol (PEG) base containing 10% Povidone Iodine equivalent to 1% available iodine, which is released in the presence of wound exudate. RNA extraction kit (PureLink RNA Mini Kit) and Super-Scripts® VILOTM cDNA Synthesis Kit were purchased from Ambion by Life Technologies (USA). Gene-specific primers using fluorogenic probes (TaqMan) and TaqMan ® Universal PCR Master Mix, No AmpErase ® UNG were from Applied Biosystems (USA). 2.2. Preparation of the NaAlg/PVPI films NaAlg/PVPI films were prepared as previously described (Liakos et al., 2013). Briefly, 3 g of NaAlg were dissolved in 100 mL of distilled water under constant stirring for 1 h at 100°C. Then, 0.3 g of PVPI were added to NaAlg solution and dissolved under constant stirring for 1 h at room temperature, to obtain a final concentration of 10% by wt. PVPI. Finally, a volume of glycerol (corresponding to 5% of the starting NaAlg weight) was added to the NaAlg/PVPI solution and dissolved under constant stirring for 1 h at room temperature. Each film was cast with 10 mL of solution and dried for 24 h under a chemical hood. 2.3. Film characterization and extraction for in vitro studies According to ISO10993-12 (sample preparation), an extraction ratio of 6 cm 2/mL (surface area/volume) should be used for preparation of test samples. To standardize the treatments, NaAlg/PVPI films (55 cm2/film) were weighted and their thickness was measured using a digital micrometer. Five measurements were performed on different parts of the films. Average values with the corresponding standard deviation (S.D.) were calculated. Based on the average values obtained, 318.7 mg of films, corresponding to a surface area of 55 cm2, were extracted in 9.2 mL of cell culture medium (containing 3.5 mg PVPI/mL) to perform both cytotoxicity and anti-inflammatory experiments. Extraction medium from NaAlg/PVPI films was prepared adapting the procedure described in ISO10993-5 standard test. Briefly, films were sterilized under a germicidal UV lamp
6
(wavelength 253.7 nm) for 1 h (30 min/side), then samples were cut into small pieces to enhance submersion in the extraction medium. Extraction was performed in cell culture medium for 24 h at 37°C and was used immediately after preparation. Product A was cut into squares according to the following extraction ratios: 0.7 – 1.0 - 1.5 cm2/mL, corresponding to 2.7 - 3.9 - 5.8 mg/mL PVPI, respectively. Extraction was performed in cell culture medium for 24 h at room temperature.
2.4. In vitro biocompatibility assay HFF-1 cells were grown in DMEM supplemented with 10% FBS, 2 mmol L -1 L-glutamine, 100 IU per mL penicillin and 0.1 mg mL-1 streptomycin, in a humidified incubator at 37°C with 5% CO 2. For biocompatibility experiments, HFF-1 cells were seeded in 60 mm cell culture dishes at a density of 3.5 X 105. After 24 h of culture, medium was replaced with the extraction one (control samples were treated with medium processed as the extractions) and cells were incubated for additional 24 h. Cell viability was assessed using the trypan blue exclusion assay. Based on dye uptake, viable (clear) and nonviable (blue) cells were counted using a hemocytometer and a light microscope. Cell viability was expressed as a percentage relative to control cells. 2.5. In vitro evaluation of anti-inflammatory properties HFF-1 cells were seeded in 60 mm cell culture dishes at a density of 4 X 10 5 and cultured for 24 h. Then, cell culture medium was removed and four different types of culture dishes were prepared: dishes containing medium processed as the extractions (control) or extraction media from NaAlg/PVPI films and Product A dressing. Product A extraction was performed using an extraction ratio of 0.7 cm2/mL (corresponding to 2.7 mg PVPI), which showed a percent survival comparable to NaAlg/PVPI films (SI). For each set of dishes, half of them were treated with 1 µg mL -1 LPS to induce inflammatory response. Culture medium with LPS and 1 µM of DXM (a corticosteroid known to decrease inflammation), dissolved in serum free medium, was used as a positive control. After 6 h, cells were harvested and total RNA was extracted by spin column-based purification method, in order to quantify human IL-1β and IL-6 expression by qRT-PCR. 2.6. qRT-PCR cytokine mRNA quantification 1 µg of total RNA was reverse transcribed into first-strand cDNA by using Super-Scripts® VILOTM cDNA Synthesis Kit in a final volume of 20 µL. HPRT-1 was used as the reference housekeeping gene in RT-PCR assays. Amplification of cytokines target genes and HPRT-1 was conducted with 50 ng of cDNA in 20 µL of the reaction mixture by gene-specific primers using fluorogenic TaqMan probes and TaqMan® Universal PCR Master Mix. TaqMan primer/probes sets, spanning exon-exon junctions, for human IL-1β (Hs00174097_m1), IL-6 (Hs00985639_m1) and the housekeeping gene
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HPRT-1 (Hs02800695_m1) were used in PCR reactions. They were run in 96-well format on ViiATM 7 Real-Time PCR System (Applied Biosystems) using universal cycling conditions (95°C, 10 min; 95°C, 15 s; and 60°C, 1 min for 40 cycles). Finally, cycle threshold (CT) values were determined by QuantStudio™ Real-Time PCR software v1.1. 2.7. Animals Male C57BL/6J mice, 8 weeks old (Charles River, Calco, Italy), were used for in vivo studies. Animals were group-housed in ventilated cages and had free access to food and water. They were maintained under a 12-hour light/dark cycle (lights on at 8:00 am) at a controlled temperature of (21 ± 1°C) and relative humidity of (55 ± 10%). All experiments were carried out in accordance with the guidelines established by the European Communities Council Directive (Directive 2010/63/EU of 22 September 2010) and approved by the National Council on Animal Care of the Italian Ministry of Health. All efforts were made to minimize animal suffering and to use the minimal number of animals required to produce reliable results. 2.8. Wound healing model Mice were anesthetized with an intraperitoneal injection of ketamine (10%) and xylazine (5%), and their dorsal surface was shaved and rinsed with an alcohol swab. A full-thickness excisional wound of 1 cm2 was induced in the center of the back of each animal. A photo of the wound was taken immediately afterwards the biopsy generation. Both dressings were applied three times throughout the experiment, and specifically, immediately soon, 3 and 4 days after wound generation (n = 5 mice each experimental group). After the first application, all treated mice were covered with TegadermTM. However, during the first three-day observation, and when we changed the dressings, we realized that the TegadermTM covering application was highly uncomfortable for animals. Then, wounds were covered only with dressings, but since animals removed Product A during the night, we reapplied both dressings the day after and they were left in place until day 7. Before taking pictures, Product A was removed, while, on the contrary, NaAlg/PVPI was already reabsorbed. No other application was done up to the end of experiments (Day 12). In conclusion, patches have been applied on day 0, 3, and 4 after wound induction. On day 0, 3, 7, 9 and 12 pictures were collected in order to measure wound closure (ImageJ software), which was calculated as percentage based on wound size relative to control group. During the experiments, mice were housed individually, with water and food ad libitum.
2.9. In vivo cytokine expression measurements Skin samples from sham, wound, NaAlg/PVPI and Product A treated animals were collected 2 days post wound induction and snap-frozen in liquid nitrogen (n = 5 mice each experimental
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group). Cytokines (IL-6 and IL-1β) expression were measured using ELISA quantikine kit (R&D system), according to the manufacturer’s instructions (Romano et al., 2015). The cytokine concentration was normalized against the total protein content for a given sample, as measured using the bicinchoninic acid (BCA) assay (Thermo Scientific, Rockford, IL, USA). 2.10. Hydroxyproline production measurements Hydroxyproline (4-hydroxyproline) production was evaluated in skin biopsy from untreated, NaAlg/PVPI and Product A treated animals collected 7 days post wound induction and snap-frozen in liquid nitrogen (n = 5 mice each experimental group). By using hydroxyproline assay kit (Sigma Aldrich), according to the manufacturer’s instructions, the amount of hydroxyproline was calculated as µg of hyroxyproline per full thickness skin biopsy. Hydroxyproline content variation was expressed as percent vs. the control group. 2.11. Histology After 7 days from wound induction, an area of 1.5 cm diameter was excised for the three different groups of treatment. The histological specimens were fixed in 4% paraformaldehyde, embedded in paraffin and 5 µm serial skin sections were collected. Sections were stained with hematoxylin and eosin (H&E) and analyzed with an Olympus BX51 microscope (tip distance measured with Neurolucida software, MBF Bioscience). Migration tongue distance variation was expressed as an absolute distance in µm vs control group (n = 5 mice each experimental group). 2.12. Data analysis
2.12.1. In vitro studies The amplification of the PCR product was expressed as the number of amplification cycles at which this particular product was first detected above the background (CT). The analysis of relative gene expression was performed by the comparative 2 -ΔΔCT method. The average CT from three replicates was calculated for both target and HPRT-1 genes. The sample target quantity was corrected by the respective value of HPRT-1. The ΔCT was determined to normalize for amounts of RNA used in reverse transcription reactions; the data are presented as fold change (2 -ΔΔCT ± S.D.). One-way ANOVA was used to evaluate statistical significance, followed by Bonferroni’s post-hoc test. GraphPad Prism 5 was used for all statistical analysis (GraphPad Software Inc. San Diego, CA, USA). P values less than 0.05 were considered significant.
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2.12.2. In vivo studies All data were expressed as means ± S.E.M. For ELISA experiments, the value obtained from each mouse was calculated as pg/mg of protein. One way ANOVA followed by Dunnett's test multiple comparison was used to evaluate statistical significance. For wound closure analysis, two-way ANOVA followed by Bonferroni’s post-hoc test was applied GraphPad Prism 5 was used for all statistical analysis (GraphPad Software Inc. San Diego, CA, USA). P values less than 0.05 were considered significant.
3. Results
3.1. In vitro studies
3.1.1. NaAlg/PVPI film characterization and biocompatibility studies Treatment of HFF-1 cells for 24 h with NaAlg/PVPI extraction medium did not affect viability since 93.2% of cells remained viable (Fig. 1). No statistically significant differences (p>0.05) were observed in the cell survival in comparison with the control. According to ISO10993-5 guidelines, a reduction of cell viability by less than 30% indicates no toxicity of NaAlg/PVPI sample. Product A was tested in parallel for its biocompatibility at different extraction ratios (0.7 – 1.0 - 1.5 cm2/mL, corresponding to 2.7 - 3.9 - 5.8 mg/mL PVPI, respectively) (see Supporting Information), in order to determine a safe dose to use in anti-inflammatory in vitro experiments. The lowest ratio tested (0.7 cm2/mL), that gave a survival higher than 70%, was selected. 3.1.2. Anti-inflammatory properties of NaAlg/PVPI film HFF-1 cells stimulated with LPS were treated with DXM (positive control) or extraction media obtained from NaAlg/PVPI and Product A dressing and of mRNA levels of IL-6 and IL-1β were investigated (Fig. 2A-B and Table 1). DXM, a well-known and largely used anti-inflammatory drug, induced a very high reduction of IL-6 and IL-1β mRNA, accounting for 95.1% (Fig. 2A) and 97.6% (Fig. 2B) of control, respectively. NaAlg/PVPI extraction medium effectively reduced the mRNA levels of about 96.4% and 92.7% for IL-6 and IL-1β (p
