Pectin-honey hydrogel: Characterization,antimicrobial activity and biocompatibility

2.1. Materials

Honey was purchased from Manuka Health (66 Weona Court,Te Awamutu 3800, New Zealand) and pectin from Ardet s.r.l. (Torino, Italy). Culture media, that is, tryptone soy agar (TSA), tryptone soy broth (TSB), peptone water, and 5% sheep blood agar, were purchased from Oxoid (Milan, Italy). Escherichia coli (Turin strain) and Staphylococcus aureus (Turin strain) isolated from canine wounds were used for this work.

2.2. Preparation of PHHs

The preparation method was modified after the procedure described by Walker (Walker, 1942) [18]. Briefly, the pectin-honey hydrogels were prepared starting from a solution (1:1 v/v) of liquid honey (Manuka Health, New Zealand) and sterile deionized water. The same volume of pectin powder (ARDET s.r.l., Italy) was then added and with continuous stirring until the mixture was homogeneous. The resulting gel was spread into 2 mm thick films and hot air dried at 40 + / − 0.5 °C for 6 h. Then it was cut into squares of 5 × 5 cm and further conditioned in an air drier at 25 + / − 1 °C for 5 days. The films were then collected and packed in polyethylene under vacuum conditions. All membranes were sterilized by gamma-irradiation at 25 KGray at the Sterigenics Italy (Sterigenics Italy, Minerbio (BO), Italy) [19].

2.3. Fluid uptake test (swelling test)

To investigate the fluid swelling ratio of PHHs, samples were cut into disks with a diameter of approximately 25 mm. The dry weights (Wdry) of the membranes were measured and recorded. Afterwards, pre-weighed dry samples were immersed in PBS solution, pH 7.4, at 37°C. The weights of the swollen PHHs were determined every 5 min subsequently by sandwiching the membranes between two paper towels to remove excess water on the surface, and then wet weights (Wwet) were measured. All experiments were performed in triplicate. The swelling ratios were calculated as the average value according to the following formula: DS = [ ( Ww − Wd ) / Wd ] × 100 . Where Ww and Wd represent the weights of wet and dry samples, respectively.

2.4. Water vapour transmission rate (WVTR)

The moisture permeability of the PHH was determined by measuring the WVTR. A piece of the specimen was fixed over the top of a tube (diameter, 34 mm) containing 10 mL PBS. The tube was then placed in an incubator at 37°C and 35% relative humidity. The membranes were weighed at regular intervals of time and the weight loss was recorded and plotted on a graph versus time. The WVTR was calculated from the slope of the graph by the following formula: WVTR g ( m 2 / day ) = ( slope × 24 ) / A . Where A is area of the sample (m²). Experiments were performed in triplicate.

2.5. H₂O₂ analysis

The analysis of H₂O₂ from honey and PHHs was carried out according to the method reported by Long (1999) [20] (ferrous ion oxidation-xylenol orange [FOX] assay), with minor modifications. The stock reagents were as follows: reagent 1, 4.4 mM butylated hydroxy toluene (BHT) in HPLC-grade methanol; reagent 2, 1 mM xylenol orange and 2.56 mM ammonium ferrous sulphate in 250 mM H₂SO₄; working reagent: one volume of reagent 2 added to nine volumes of reagent 1. Approximately 200 mg of honey were diluted with deionized water to the ratio 20% w/w and immediately kept in a thermostat at T = 37 °C, and 100-μL aliquots were added to 900 μl of FOX working reagent. After 20 min of incubation at room temperature, absorbance was measured at λ = 560 nm against a blank comprising 900 μL of working reagent and 100-μL aliquots of distilled water (absorbance of diluted honey was negligible at the operative wavelength). H₂O₂ was monitored at 5, 10, 15, 20, 30, 60, and 120 min and up to 24 and 48 h of incubation.

To reduce the processing time required to extract the water-soluble components from PHHs, their size was reduced prior to incubation by cutting the specimens into small pieces with the aid of a small and clean knife. Aliquots of 100 μL of the aqueous layer of the final mixture (40% w/w) were subjected to FOX analysis as described above for honey. Monitoring of H₂O₂ was carried out at 60 min, 90 min, 24 h, and 48 h. The FOX assay was calibrated using standard H₂O₂ (molar extinction coefficient, 43 M–1 cm–1; absorbance wave length of H₂O₂ λ = 240 nm; linear range, 0–50 μM). Ten units of catalase were sufficient to destroy all the H₂O₂ immediately. Controls with catalase were used to exclude interference due to honey constituents.

2.6. Methylglyoxal (MGO) analysis

MGO was evaluated by the method proposed by Wild (2012) [21] with slight modifications. The method is based on the reaction between N-acetyl-L-cysteine (Sigma Aldrich) and MGO at room temperature. Samples were diluted in water (340 mg/ml) and the reaction was performed in 100 mM sodium dihydrogen phosphate buffer (adjusted to pH 7.0 with 10 M NaOH) at 22°C. For the standard curve of the reaction, different concentrations of MGO (0.5, 1, 2, and 5 mM) were used. MGO solutions (Sigma Aldrich) equivalent to 0.5, 2, and 5 mM and 10 μl of each honey solution (170 mg HBM/ml water) were added up to a volume of 980 μL with sodium dihydrogen phosphate. The reaction was started by adding 20 μL 500 mM N-acetyl-L-cysteine, and the absorption was recorded after 7 min. The condensation product, N-α-acetyl-S-(1-hydroxy-2-oxo-prop-1-yl) cysteine, was determined by recording the absorption at 288 nm (UVIKON 923, Bio-Tek Instrument). Results are given in μmol/mg of honey.

2.7. Protein content analysis of honeys

Honey samples were diluted in water and the protein content was determined using the BCA protein assay kit according to the manufacturer’s instructions (Pierce, BCA Protein Assay).

2.8. Microbiological analysis

The success of sterilization was verified by the absence of bacterial growth on solid medium (5% sheep blood agar) at 37°C for 24 h, both in aerobiosis and anaerobiosis. Subsequently, the antibacterial activity of PHHs was determined against S. aureus and E. coli, which had been previously isolated from canine wound infections. The agar good diffusion method was used to screen the antimicrobial activity of the Manuka PHHs [22]. Clinical strains were grown overnight in TSB at 37°C and adjusted to 0.5 McFarland standard. Each culture was inoculated on the surface of Petri plates. Subsequently, wells with 6-mm diameter were bored into the surface of the agar. The wells were filled with 6 mm of a Manuka honey-based patch, 80 μl of a Manuka honey sample 100% v/v (as positive control), 80 μl of a Manuka honey sample 50% v/v (as a positive control that resembles the concentration of honey present in PHHs), and 6 mm of pectin (as negative control). Plates were incubated at 37°C, and after 24 h, the diameters of the inhibition zones were measured. Each assay was carried out in triplicate.

2.9. In vitro cytotoxicity assay

The cytocompatibility of PHHs was evaluated using L929 cells (mice fibroblasts) (ECACC Cell Lines-Sigma Aldrich, Milan, Italy) that were cultured in 75 mL flasks containing Modified Eagles Medium (MEM; Sigma Aldrich, Milan, Italy), 10% fetal bovine serum (FBS; Sigma Aldrich, Milan, Italy), 2% L-glutamine (Sigma Aldrich, Milan, Italy), and 2% penicillin-streptomicin-amphotericin B solution (Sigma Aldrich) at 37°C with 5% CO₂, 95% air, and complete humidity. When a confluence of 80% was reached, cells were detached using 0.1% trypsin/EDTA solution (Sigma Aldrich, Milan, Italy), centrifuged, and counted using Trypan Blue solution (Sigma Aldrich) with a Burker chamber. Cells were either resuspended at a concentration of 5 × 105 cells/mL or stored at − 80 °C for further analysis. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay, a routine method for quantifying cell viability, was used [23].

Cells ( 5 × 10 5 cells / 100 μ L ) were transferred into 96-well plates in order to perform the MTT assay at 24, 48, and 72 hours after seeding. After an overnight incubation at 37°C and 5% CO₂, the medium was changed (MEM, FBS 10%, antibiotic and antimycotic solution 2%, L-glutamine 2%), and serial decreasing concentrations of honey membranes dissolved in phosphate buffered saline (PBS) solution, starting from 0.1 g/ml, were added. After adding MTT (Sigma Aldrich, Milan, Italy) solution (4 mg/ml in PBS), the plates were incubated at 37°C and 5% CO₂ for 4 hours. At the end of incubation, the medium was removed, and 100 μL of dimethyl sulfoxide (DMSO; Sigma Aldrich, Milan, Italy) was added in order to lysate the formazan crystals. The plates were further incubated for 10 minutes on a continuous shaker at room temperature, and the optical density of the wells was determined using a plate reader (Poverwave x; Bio-Tek Instruments Inc., Winooski, USA) at a wavelength of 570 nm.

2.10. In vivo biocompatibility study

All procedures were approved by the Bioethical Committee of the University of Turin and by the Italian Ministry of Health (protocol number 304/2015-PR, 20/04/2015).

A total of 39 adult male Sprague-Dawley rats, weighing 225–250 grams, were purchased by Harlan Laboratories (Italy). All rats were housed in single cages for 7 days prior to the beginning of the experiment. The room temperature was set at 23°C throughout the duration of the experiment and cages were cleaned daily. Animals were fed with a commercial diet and water was given ad libitum. Anaesthesia was induced by administering 5 mg/kg of xylazine (Rompum^®, Bayer Animal Health, Italy) and 50 mg/kg of tiletamine and zolazepam (Zoletil 50, Virbac, Italy) intramuscularly. Anaesthesia lasted for approximately 1 hour. Under anaesthesia, blood samples were collected from the caudal vein to perform biochemical analysis. Soon after, the ventral hair was shaved and the skin was prepared using a 3-step iodopovidone–chlorhexidine scrub. The animals were randomly assigned to the treatment or control groups. A 4-cm midline incision was made in the abdominal wall. In the treatment group, subcutaneous and intraperitoneal implantation of PHH was performed as previously described [24]. Briefly, one square each of PHH measuring 1 × 1 cm was implanted intraperitoneally under the left abdominal wall and subcutaneously at ∼1 cm left to the midline between the muscle and skin. In the control group, only the surgical procedure was performed without PHH implantation. In both groups, the midline incision was closed in two layers, with 3–0 USP glycomer 631 for the fascia, and with 3–0 USP nylon for the skin. Each surgical procedure lasted about ∼20 minutes.

Blood samples for the biochemical analysis were collected from the caudal vein. The blood was collected into tubes before surgery and at 6 h (T6), 24 h (T24), and 72 h (T72) post-surgery and at the time of euthanasia. Plasma levels of IL-1β, IL-6, TNF-α and PG(E2) were measured by using a commercial ELISA kit (Rat IL1 beta ELISA kit, Booster Biological Technology; Rat Il-6 ELISA, AB Frontier; Rat TNF alpha ELISA, AB Frontier; Prostaglandin E2 Express EIA kit, Cayman Chemical).

At 0, 6, 24, 72 hour’s post-surgery, three rats from each group were euthanized and target organs (liver, kidney, left abdominal wall) were collected and fixed in 4% formaldehyde. These tissues were sectioned, stained in H&E, and observed by two pathologists in a blinded manner [24].

2.11. Statistical analysis

Normality of the data was evaluated using the Shapiro–Wilk normality test. For the in vitro cytotoxicity assay, all experiments were performed in triplicate, and the data are representative of at least three independent experiments. The results, expressed as mean ± SEM values, were analysed using the Kruskal–Wallis test and a Dunn’s post-test.

For plasma cytokine levels, all experiments were performed in duplicate. For IL-1β, IL-6, and TNF-α, the results have been expressed as median (95% IC) values and were analysed using the one-way ANOVA test. For PG(E2), the results are expressed as median (95% IC) and were analysed using the Friedman test. Statistical analysis was performed with the GraphPad Prism 6.01 software. Values with p < 0.05 were considered significant.

3.1. Fluid uptake test (swelling test)

The weight of PHHs immersed in PBS solution under physiological mimicking conditions (pH 7.4, 37°C). The fluid content increased to about 150% after 180 minutes. The results from the fluid uptake experiment revealed that the dressing has wide capacity to prevent fluid accumulation if used on wound.

3.2. WVTR

The transmission of water vapour through the membranes is an important parameter for the evaluation of their effectiveness as a hydration factor when placed on a wound. The WVTR recommended for wound dressing is 2000–2500 g/m²/day in order to ensure proper wound moisture without risk of dehydration or excessive production of exudates [1,4]. A good WVTR facilitates the healing process because it improves cell migration and promotes re-epithelialization.

The water loss from a fully hydrated dressing on exposure to air was evaluated. The mean evaporative water loss from PHHs was 2689.8 ± 158.5 g/m²/day.

3.3. H₂O₂-producing activity

All the honey samples, before inclusion in the membranes, were able to produce significant amounts of H₂O₂ while no H₂O₂ development was observed in the case of corresponding PHHs (data not shown). In Manuka honey, at shorter incubation times up to 90 min of incubation, the concentration of H₂O₂ generated by honey glucose oxidase was in the range reported in the literature for other types of honey with different methods of analyses (1–2 mM at 30 min of incubation) [16,25–27]. By contrast, Manuka honey showed the lowest H₂O₂ production at all incubation times, probably because of its high content of MGO, which has previously been suggested to be a glucose oxidase-inhibitory agent [16,25]. At longer incubation times (24 and 48 h), the tested honey samples displayed a significantly different behaviour: The dramatic loss of H₂O₂ producing activity found for PHHs indicated that their production procedure, which included a heating step at 80°C and exposure to γ-rays for final sterilization, induced complete loss of glucose oxidase activity. This demonstrated that the preserved antibacterial activity in the microbiological testing was generated solely by the action of nonperoxide agents.

3.4. MGO analysis

Dihydroxyacetone (DHA) is a direct precursor of MG in Manuka honey [15]. The MGO concentration in PHHs, determined by the N-acetyl-L-cysteine assay, was 0.26 ± 0.07 μmol/mg of proteins. The PHHs had higher MG concentration than bulk honey. MGO content is important because it can serve as a suitable quality and cost parameter for Manuka honey. The H₂O₂ and MG content is responsible for the antibacterial activity of honey [28] and PHHs maintain antibacterial activity similar to that of bulk honey.

3.5. Microbiological analysis

Manuka membranes did not show bacterial contamination after sterilization by gamma-irradiation. Table 1 outlines the antibacterial activity based on the clear zone that was produced.

Results concerning the effects induced by different concentrations of dissolved honey membrane on viability of L929 cells are represented in Fig. 1.

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Fig. 1.

MTT assay ( N = 8 ) to evaluate the modulation of L292 cells growth after the treatment with decreasing concentrations of dissolved honey membrane in PBS at different experimental time points (24, 48, and 72 hours).

Three rats from the treated group died of ascites in the first 24 hours. The gross evaluation in 36 rats showed no wound site infection or presence of adhesions. On performing histological analysis 24 and 72 hours’ post-surgery, the tissue near the implant was found to be characterized by the presence of fibroblasts with some cellular response, including lymphocytes, macrophages, and neovascularisation. No reaction was observed in distant organs. Thus, the PHHs did not induce a foreign body reaction. The differences in the blood levels of the IL-1β, IL-6, TNF-α and PG(E2) at the 0, 6, 24, and 72 hours’ time points were not statistically significant. The results are summarized in Table 2.