Introduction

The word wound is referred to any defect created in the skin that can be caused by different reasons such as trauma or medical conditions [1]. From the past, there are several different combinations, including honey, fibers, trees, animal fats, flowers and textile fibers to treat wounds as wound dressings. Some of these compounds are useful and some others are sometimes poisonous and dangerous [2]. To ensure the effective healing of wound, the wound dressing should be non-toxic and bioactive and having the abilities of exchanging gas and protecting the wound from external mechanical stress. Additionally, the wound dressing should provide and maintain a wet environment on the surface of the damaged tissues, because it has been proven that under such moist conditions, the healing process of the wound becomes more accelerated [3]. Among all types of wound dressings, special attention has been recently paid to hydrogels which can meet some of the essential requirements of ideal wound dressings. Hydrogel wound dressings have the unique ability of keeping the wound moisture and also absorption the wound secretion. These wound dressings can cool the surface of the wound and reduce the patient's pain [2, 4, 5]. Furthermore, biocompatibility, ability of swelling by immersing in body fluids, desirable viscoelastic properties and stability in the body are other advantages of the hydrogel wound dressings [6, 7]. However, the low mechanical strength of hydrogels in some cases [8] prevents their use as wound dressing in applications with pressure bearing [9]. In order to overcome the weaknesses of hydrogel wound dressings, a novel class of wound dressings, called nanocomposite hydrogel wound dressings, was introduced by Kokabi et al. in 2007 on the basis of polyvinyl alcohol (PVA) and montmorillonite (MMT) nanoclay [10]. Since then different types of nanocomposite hydrogel wound dressings have been prepared and their performances as wound dressing investigated [11,12,13].

The application of most nanocomposite materials in the biomedical fields is restricted due to the challenge of non-biocompatibility. Therefore, in recent years, special attention has been drawn to the production of nanocomposite materials on the basis of natural biocompatible materials, so called bionanocomposites, for biomedical applications [14]. Recently, we prepared the novel biocompatible wound dressings on the basis of bionanocomposite hydrogels composed of egg white and PVA (as the matrix phase of bionanocomposite) and MMT (as the reinforcing phase) by a freezing–thawing method. A comprehensive study on the structural, physical and mechanical properties of the prepared wound dressings was conducted along with the in vivo assays on the animal model. It was found that the bionanocomposite wound dressings had the exfoliated morphology. The results also indicated the reduction in the average pore size of bionanocomposite hydrogels with an increase in the MMT loading level. The prepared bionanocomposite hydrogels were non-toxic and had no negative effect on cell death, but also increased the number of live cells in vitro. The in vivo results confirmed the positive effect of these wound dressing on the healing process of non-infected animal wounds [15, 16].

The entry, growth and development of microbes in the wound are called wound infection which may cause the delay in wound healing, increased exudate formation, improper collagen sedimentation, increasing the length of confinement and even death [3, 17, 18]. Wound infection is usually caused by microorganisms such as Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli [19]. Recently, with the advancements in biomaterial fabrication techniques, different types of wound dressings with antibacterial properties have been developed for accelerated and effective healing of different kinds of infected wounds [20, 21]. Many types of wounds and ulcers, such as bed sores, burns and diabetic wounds are responsive to honey [22]. Studies have also shown that more than 80 types of bacteria and yeast are effectively controlled by honey [23]. The growth of bacteria requires a water activity level of 0.94–0.99, while honey has a small amount of water activity (0.56–0.62), which prevents bacterial growth. Additionally, honey acidity (with pH of 2.3–4.5) prevents more microorganisms from growing, because optimum pH for most of these organisms is 4.7–7.2 [24]. The use of honey in wound healing helps to stimulate the treatment process, reconstruct the tissue and reduce pain and inflammation of the wound. It provides a wetting environment treatment and prevents the growth of bacteria even in the wound with chronic infections [22]. In the past researches, honey-loaded wound dressings have been prepared successfully and their performance evaluated in practical applications. Afshari et al. [22] prepared honey-loaded hydrogel wound dressings with three components, including the PVA, carboxy methylate chitosan and honey. The dressings were prepared using a radiation method followed by a cyclic freeze-thawing technique and the inhibition of the growth of Escherichia coli using the prepared wound dressings was investigated. Park et al. [25] prepared chestnut honey-impregnated carboxymethyl cellulose sodium hydrogel paste as a therapeutic dressing for inhibiting the growth of Staphylococcus aureus and Escherichia coli. The result was provided good evidence, suggesting that the prepared hydrogel paste has potential as a competitive candidate for diabetic ulcer wound healing. Tavakoli et al. [26] prepared highly concentrated honey/PVA hybrid hydrogel with borax as the crosslinking agent and the morphology, swelling kinetics, permeability, bio-adhesion, mechanical characteristics, cytotoxicity, antibacterial property, cell proliferation ability and controlling release properties were investigated as functions of crosslinking density. El-Malek et al. [27] prepared a hydrogel sheet composed of chitosan and gelatin-loaded with a new formula extracted from manuka honey. The result showed the antibacterial activity of the prepared system against Staphylococcus aureus, Streptococcus pyogenes, Acinetobacter baumannii, Pseudomonas aeruginosa and Proteus mirabilis. Recently, Nouri et al. [28] synthesized honey-loaded hydrogels based on PVA, chitosan and MMT by a freezing–thawing method and investigated their potential as wound dressing materials by studying their physical, mechanical, swelling, and release behavior.

In the current work, following the past researches performed in our research group on non-infected wounds [15, 16], a novel series of the honey-loaded bionanocomposite hydrogel wound dressings based on egg white, PVA and MMT nanoparticles was prepared for the treatment of infected wounds. The swelling and dehydration capabilities, honey release behavior, biocompatibility and optical transparency of the prepared bionanocomposite hydrogel wound dressings were investigated. The in vivo assays were performed to study the performances of the prepared egg white/PVA/MMT/honey wound dressings on the infected and non-infected animal wounds.

Experimental

Materials

PVA with a saponification value greater than 98%, a density of 1.25 g/cm3 and a degree of polymerization of 1700 was purchased from Nippon Gohsei, Japan. Natural hydrophilic MMT ((Na,Ca)0.33(Al,Mg)2Si4O10(OH)2, nH2O) with cation exchange capacity of 92.6 meq/100 g, a molecular weight of 540.46 and a density of 2.86 g/cm3 was purchased from Southern Clay Products Inc, USA. Natural honey was obtained from the Mirnajmi Honey Company (Urmia, Iran). Eggs were obtained from the local market (Urmia, Iran). They were broken and the white separated from the yolk and used without further treatments in the production of bionanocomposite hydrogel wound dressings. The humidity and pH of egg whites were measured, on average, as 86.4 wt% and 4.7, respectively. The annexin V/PI staining kit was purchased from BioVision, USA. The RPMI-1640 medium, penicillin, and streptomycin combined product (Pen/Strep) and fetal bovine serum (FBS) were purchased from GIBCO, USA. Chitopad, commercial wound dressing was purchased from Chitotech Company, Iran. Hematoxylin and eosin stains and Masson’s trichrome staining kit were purchased from Sigma, U.S.A. All other chemicals were purchased from Merck, Germany.

Preparation of Wound Dressings

Two types of wound dressings, including the non-freeze-thawed hydrogel wound dressing, as a topical gel and the freeze-thawed hydrogel films, as wound covering pads were prepared. For the preparation of each wound dressing, a preset value of MMT in a predetermined value of double distilled water (DDW) was mixed at a mixing rate of 500 rpm at room temperature for 10 h. The temperature of the MMT suspension was increased to 90 °C and preset value of PVA was added step by step to the suspension and concurrently mixed with the mixing rate of 50 for another 4 h. Finally, the temperature of the achieved suspension was decreased to room temperature. Separately, another MMT suspension in egg white was prepared by mixing a preset value of MMT in a preweighted value of egg white at a mixing rate of 50 rpm at room temperature for 2 h. Eventually, the achieved two suspensions (PVA/MMT and egg white/MMT suspensions) were mixed at room temperature by a mixing rate of 50 rpm for 2 h. The prepared suspensions had 0, 5 and 10 wt% of MMT, based on the mass of the completely dried sample. Subsequently, an adequate amount of honey was added to each egg white/PVA/MMT suspension by a mixing rate of 50 rpm for 2 h at room temperature to achieve a suspension containing 10 wt% honey, based on the total weight of each suspension. A part of the prepared sample containing 5 wt% of MMT was kept at the refrigerator until performing the in vivo assays (this sample was named as G5 wound dressing and used as a topical gel). The prepared suspensions were then poured in the flat plastic molds and subjected to a cyclic freezing–thawing process to induce crosslinking and hydrogel formation. The samples were frozen at – 15 °C for 24 h and then placed at room temperature for 24 h. This freezing–thawing cycle was repeated three times for each sample. The prepared films were named as Sx wound dressing, which x shows the weight percentage of MMT in the honey-free completely dried sample. The designations and detailed compositions of the prepared honey-loaded bionanocomposite hydrogel wound dressings (on the basis of the honey-free dried state) are summarized in Table 1.

Table 1 Designations and chemical compositions of the honey-free dried bionanocomposite hydrogel wound dressings
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Swelling and Dehydration Experiments

The swelling and dehydration tests were utilized to investigate the abilities of the bionanocomposite hydrogel wound dressings to absorb exudates of wounds and their unintentionally drying during the wound healing process. For swelling experiments, 3 pieces of each honey-free bionanocomposite hydrogel wound dressings (S0, S5 and S10) with the same weights (ca. 1 g) were immersed in an excess amount of DDW for a week. Each extracted sample was then dried under vacuum until to reach a constant weight. Each piece of dried samples was then immersed in 500 cc of DDW. The swelling was measured by determining the weights of the swollen samples eight hours after initiating the swelling process at 37 °C. The swelling ratio of each sample at t = 8 h (q(8)) was calculated from Eq. (1):

$${\text{q}}\left( 8 \right) \, = {\text{ m}}\left( 8 \right)/{\text{m}}\left( 0 \right)$$
(1)

where, m(0) is the initial weight of dried sample and m(8) indicates the weight of swollen hydrogel eight hours after initiating the swelling process.

The dehydration abilities of the S0, S5 and S10 honey-free bionanocomposite hydrogel wound dressings (previously equilibrated in DDW at 37 °C) was determined gravimetrically by measuring their weight loss at eight hours after starting the dehydration operation. Dehydration test was performed on 3 species of each sample at a constant temperature of 37 °C. The fraction of water removed from each bionanocomposite hydrogel wound dressings at t = 8 h, i.e. M(8)/M(∞) was determined, where M(8) and M(∞) are the mass of removed water from the sample at t = 8 h and the total removable mass of water in the sample, respectively. The average value obtained from three experiments for both the swelling and dehydration tests were reported.

Honey Release

Honey release tests were performed on S0, S5 and S10 bionanocomposite hydrogel wound dressings at different release temperatures to investigate the potential of the prepared wound dressings, as drug (honey) delivery systems, for treatment of infection in infected wounds.

First, the calibration curves of honey were plotted at 25, 37 and 50 °C by preparing a set of the honey solution in DDW in the concentration range of ca. 0.1–0.9 g/100cc. The measurements were performed with a one touch glucometer (Accu-Chek Performa, Germany) by measuring the concentration of glucose as the key component of honey. In order to perform the honey release tests, approximately 2 g of each prepared honey-loaded wound dressings was individually placed in closed containers containing 50 g of DDW, as the release medium. The honey release measurements for each sample were carried out at 25, 37 and 50 °C. At predetermined time intervals, the concentration of honey in the release medium was measured using the glucose monitoring system. The honey release kinetics was determined for each sample at any desired release temperature by plotting the fractional release curves, i.e. Mt/M∞ values versus the release time. Mt is the cumulative amount of released honey at time t and M∞ is the mass of initially loaded honey inside each sample or the same total amount of releasable honey from the sample.

In order to determine the mechanism of honey release from the bionanocomposite hydrogel wound dressings, the following power law equation was used [29]:

$${\text{M}}_{{\text{t}}} /{\text{M}}_{\infty } = {\text{ Kt}}^{{\text{n}}}$$
(2)

where, K is the honey release characteristic constant and n is the characteristic exponent of the mode transport of honey molecules. According to the classification of the diffusion mechanism; n = 0.5, n = 1, and 0.5 < n < 1 indicate the Fickian, case II transport and non-Fickian diffusion mechanisms, respectively [30].

Equation (2) could be re-written in logarithmic form as follows:

$$\log \left( {{\text{M}}_{{\text{t}}} /{\text{M}}_{\infty } } \right) \, = \, \log \left( {\text{K}} \right) \, + {\text{ n }}\log \left( {\text{t}} \right)$$
(3)

Plotting the log(Mt/M∞) versus log(t) for Mt/M∞ < 0.6 would give the value of n as the slope of the plotted linear curve and the log(K) as the intercept.

Flow Cytometry

In vitro cytotoxicity assay was performed to present the biocompatibility level of the prepared honey-loaded bionanocomposite hydrogel wound dressings. The flow cytometry was used because this method is generally faster, less expensive, safer and more sensitive to the cytotoxic events than conventional methods [29]. In this assay, the S5 bionanocomposite hydrogel wound dressing along with a control (witness) sample was investigated. To perform the test, the human peripheral blood mononuclear cells (PBMC) were maintained in 1 ml of RPMI-1640 medium, containing 10% FBS and 1% Pen/Strep and the number of the existing cells was determined. Then 50,000 cells contained in 200 μL medium were transferred to each well of the 96-well microplate. Afterward, a piece of S5 sample was added to the related wells. Some other wells were kept intact as control. Two days after the proximity, cells were harvested and stained with the annexin V/PI staining kit according to the manufacturer’s instructions. Resulted suspensions were then transferred to the flow cytometry tubes and analyzed by a FACScan flow cytometer (Becton Dickinson, USA).

In Vivo Assays

Animals

Fifteen female BALB/c mice with age of 8 weeks were purchased from the Royan Institute, Iran. During the study, all animals were kept in individual cages and were maintained under standard laboratory conditions and had adlibitum access to food and water at all times except during the experiments. The study was approved by the Ethical Committee of the Faculty of Medical Sciences, Tarbiat Modares University.

Surgery and Wound Healing

The animals were randomly separated into 5 groups including the CN, SN, SI, GN, and GI (3 mice/per group). All animals were anesthetized by injection of the xylazine and ketamine solution (1.5 cc of ketamine and 0.5 cc of xylazine in 8 cc of distilled water) with a dose of 4 µL/g mouse body weight and then the dorsal fur of each animal was shaved. On each animal's surface, a cut to the deep fascia was made using sterile scissors and forceps. The wounds of animals of SN and SI groups were infected with Staphylococcus aureus. In the CN group (as the control group), the wounds were covered with an advanced commercial wound dressing, i.e. Chitopad. In GN and GI groups, the wounds of animals were covered by rubbing the adequate amount (2 g/day) of the G5 bionanocomposite hydrogel wound dressing. On the other hand, the wounds of animals of SN and SI groups were covered with a sterilized film of S5 bionanocomposite hydrogel wound dressing having a thickness of ca. 3 mm. Up to 10 days after surgery and creation of wounds, the sizes of the wounds of all animals were measured by digital caliper and the wound dressing was changed every day with the respective dressing. The gross view of the wound of a selected animal from each group was obtained by a digital camera after wound creation (0th day) and on 2nd, 5th and 10th day after surgery. The wound size reduction (WSR) was calculated using the below equation:

$${\text{WSR }}\left( \% \right) \, = \, \left( {\left( {{\text{A}}_{0} - {\text{A}}_{{\text{t}}} } \right)/{\text{A}}_{0} } \right) \, \times 100$$
(4)

where, A0 is the initial wound area on the 0th day (immediately after wound creation) and At is the wound area at any time of the healing process.

Tensile Test

One of the most important criteria of wound healing is the stretchability of the healed wound, which determines the amount of healed tissues and the skin's elasticity. Tensile strength and elongation at maximum stress are two important mechanical properties that were assessed directly through the tensile test. Ten days after the injury, all animals were killed and a strip of each animal’s healed skin (ca. 13 × 35 mm) was cut with the wound remaining in the center of the strip. One strip of the healed wound of animals of CN, SN, SI, GN and GI groups was randomly selected for the histological study and the rest strips of each group were used in the tensile test. Prior to the test, the thickness of each strip was measured using a digital caliper. The tensile test was performed using a universal testing machine (Zwick, Germany) with a crosshead speed of 5 mm/min at the ambient conditions.

Histological Observations

The selected strips of the healed wounds of animals were fixed in 10% formalin solution for 72 h to protect their physical structure. Tap water was used to wash the samples then the strips were cleared by increasing the concentrations of alcohol (methyl, ethyl and absolute ethyl), then embedded in paraffin. After molding, tissue blocks were cut at 5 µm. Prepared tissue sections then were collected on glass slides and stained with hematoxylin–eosin and Masson’s trichrome method and exclusive staining of collagen. The semi-quantitative method was used to evaluate the histological processes and structures and the examined sections were evaluated according to the score: 0, 1, 2, 3 and 4 (Table 2) [31]. The histological observations were carried out using an Olympus X51 microscope (Olympus Corporation, Japan). AxioVision LE software was used for the histological study.

Table 2 Explanation of used scale in the semi-quantitative evaluation of histological sections (ST Surrounding tissue, DL demarcation line, SCT Subcutaneous tissue, GT granulation tissue) [32]
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Statistical Analysis

Statistical analysis was performed using GraphPad Prism 6.1 software. The p value was calculated for the indicated groups by using ANOVA. A p value of less than 0.05 was considered to be statistically significant.

Results and Discussion

Swelling and Dehydration Abilities

Swelling is one of the features that can be used to determine the ability of wound dressings to absorb wound exudates. The swelling ratios of the S0, S5 and S10 honey-free bionanocomposite hydrogel wound dressings in the absorption of water were measured as described in the previous section. The swelling ratios of samples at 37 °C at the 8th h of the swelling process are shown in Fig. 1. As seen, by adding MMT nanoparticles to the bionanocomposite hydrogel wound dressing, the swelling ratio is reduced and more reduction can be observed by increasing the MMT content. The results show that up to 25% decrease in the swelling ratio of wound dressing could be achieved by incorporating 10 wt% of MMT to the matrix of the wound dressing. This is related to the role of the MMT as a crosslinker in the matrix of the egg white/PVA hydrogel which creates more crosslinking points and increases the gel fraction and strength of hydrogel. Obviously, by increasing the amount of MMT layers in bionanocomposite hydrogel wound dressings, the entanglements of PVA polymeric chains and the egg white protein chains are increased, while the free space inside the network is reduced and hence the permeability and also the ability of the hydrogel to absorb the water molecules into the network is reduced.

Fig. 1
figure 1

Swelling and dehydration capabilities of the bionanocomposite hydrogel wound dressings

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Since a dried hydrogel wound dressing is useless, maintaining moisture in the wound surface and preventing it from undesired drying is another important feature of wound dressings. Dehydration rate of the S0, S5, and S10 wound dressings at 37 °C were measured and the obtained results are demonstrated in Fig. 1 in the form of the fraction of water removed from the wound dressings at 8th hour after the bingeing of the drying operation. The results show that by increasing the amount of MMT in the wound dressings, the fraction of water removed from the wound dressings was decreased. This can be considered as an advantage for the MMT-loaded bionanocomposite hydrogel wound dressings and is attributed to the increase in the crosslinking density and the reduction of free space for the penetration of water molecules during the dehydration process by increasing the MMT loading level in wound dressings.

Honey Release Kinetics and Mechanism

The calibration curves of honey at temperatures of 25, 37 and 50 °C were determined using a procedure described in the experimental section and plotted in Fig. 2. Excellent linearity was observed for all three calibration curves with coefficients of determination (R2) higher than 0.99. The calculated calibration equations at three examined temperatures have been shown in Fig. 2.

Fig. 2
figure 2

Calibration curves of honey at temperatures of open circle, 25; open square, 37; and open triangle, 50 °C

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Honey release tests were performed on S0, S5 and S10 bionanocomposite hydrogel wound dressings to investigate the effects of MMT nanoparticles contents and release temperature on the honey release kinetics and mechanism. The honey release kinetics curves were plotted for all samples at release temperatures of 25, 37 and 50 °C, as shown in Fig. 3. Nearly identical patterns for all the honey release kinetics were observed regardless of the content of the incorporated MMT into the bionanocomposite hydrogel wound dressings and also the release temperature. However, the results showed that the amount of fractional release of honey was decreased with increasing the content of MMT in wound dressings at all release temperatures. Furthermore, the results demonstrated a more prolonged release of honey for the bionanocomposite hydrogel wound dressings containing higher loading levels of MMT. For instance, the required time to release ca. 80% of honey from MMT-free wound dressing at 37 °C was about 160 min, while this duration for the wound dressings containing 5 and 10 wt% of MMT was increased to 220 and 260 min, respectively. More controlled and prolonged release of honey from the MMT-loaded bionanocomposite hydrogel wound dressings compared with the MMT-free wound dressing could be attributed to their increased gel fraction values [15] and decreased swelling abilities. In addition, the silicate layers of MMT inside the honey-loaded wound dressings may act as physical barriers against the mass transfer and entrap the honey molecules, which create a slower release pattern for honey molecules in MMT-loaded wound dressings. The more controlled or slower release of honey in MMT-loaded bionanocomposite hydrogel wound dressings compared to MMT-free wound dressing is recognized as a major advantage in practical applications, which may prolong the replacement intervals of the wound dressing. Furthermore, the results showed that the amount of released honey from the wound dressings was increased by increasing the release temperature in all samples. This increment in the honey release is due to the increase in the diffusion coefficient of honey molecules with increasing the temperature of the release medium.

Fig. 3
figure 3

The honey release curves for wound dressings at release temperatures of a 25, b 37 and c 50 °C

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The mechanism of the mass transfer in the honey release from bionanocomposite hydrogel wound dressings was determined using the power law equation according to the Eq. (2). The log (Mt/M∞) versus log(t) curves for all samples at different release temperatures have been presented in Fig. 4. A good linear relation was observed between log (Mt/M∞) and log(t) for all samples. The values of constants n and K for all samples were calculated from the slopes and intercepts of lines represented in Fig. 4 and tabulated in Table 3. As seen, regardless of the amount of MMT nanoparticles incorporated into the wound dressings and the release temperature, the mechanism of release of honey in all cases is non-Fickian diffusion. The non-Fickian diffusion mechanism means that during the honey release process, the relaxation rate of the PVA polymeric chains and the egg white proteins in the three-dimensional network of wound dressings and the diffusion rate of honey molecules are comparable and both processes could be recognized as controlling steps against the mass transfer. Table 3 also shows that the values of constant K, which could be considered as a criterion for the honey release rate in bionanocomposite hydrogel wound dressings, were decreased in all cases by increasing the MMT content in wound dressings and decreasing the release medium temperature.

Fig. 4
figure 4

The log (Mt/M∞) versus log(t) curves for all wound dressings at release temperatures of a 25, b 37 and c 50 °C

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Table 3 The calculated values for n and K for wound dressings at three different release temperatures
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Cytotoxicity

Results of the flow cytometry assay on the cell viability of PBMC cells in the vicinity of S5 bionanocomposite hydrogel wound dressing along with the control cells are shown in Fig. 5. The flow cytometry graphs have four quadrants including LL (low left), LR (low right), UR (upper right) and UL (upper left) quadrants which denote viable (alive), early apoptotic, late apoptotic and necrotic cells, respectively. The extracted results from Fig. 4 in the form of the percentages of cells in four quadrants of the flow cytometry graphs were listed in Table 4. The results showed that the proportion of the viable cells in the S5 sample was more than 33% and a part of cells have been apoptized and necrotized in contact with the S5 sample. Since a very high value for the proportion of viable cells has been reported for the honey-free egg white/PVA/MMT bionanocomposite hydrogel wound dressings [15], therefore the reduced cell viability in the present honey-loaded wound dressings could be attributed to the presence of honey molecules inside the wound dressings. Similar results have been reported regarding the effect of the honey molecules in the accelerated apoptosis of cells in vitro [32, 33].

Fig. 5
figure 5

Flow cytometry graphs for a control cells and b the cells in the vicinity with the S5 bionanocomposite hydrogel wound dressing

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Table 4 The percentages of cells detected in four quadrants of the flow cytometry graphs
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Optical Transparency

One of the most important properties of the prepared bionanocomposite hydrogel wound dressings is their acceptable light transmission. Figure 6 demonstrates the transparency of a sheet of a typical bionanocomposite hydrogel wound dressing (S5) with a thickness of 3 mm. It was expected that the wound dressing transparency may be greatly reduced due to the presence of the pigments of the honey, but with regard to Fig. 6, it was observed that the prepared wound dressing has acceptable transparency. Acceptable transparency of the prepared bionanocomposite hydrogel wound dressings allows the patient or physician to observe the wound surface conditions (bleeding, infection, etc.) continuously during the wound treatment without moving or replacing the wound dressing.

Fig. 6
figure 6

Qualitative images demonstrating the transparency of a typical honey-loaded bionanocomposite hydrogel wound dressing (S5)

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In Vivo Assays

Wound Healing Progress

In order to observe and evaluate the quality of wound healing and closure of wounds of the animals treated with the bionanocomposite hydrogel wound dressings, the locations of the wounds were photographed on different healing days. Figure 7 shows the wound healing progress and the qualitative level of wound healing in the selected animals representing the five test groups. As seen, the surfaces of the wounds of all animals were reduced as time goes on; but the healing process of wound and reduction of its area in groups based on the bionanocomposite hydrogel wound dressings was more favorable than the control group.

Fig. 7
figure 7

Macroscopic appearances of the wounds of selected animals of CN, SN, SI, GN and GI groups during the wound healing process

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For a quantitative comparison, the values of WSR for animals of all groups were measured and the obtained results reported versus the healing time in Fig. 8. It can be seen that during the second day, there is no significant difference among the groups. It can be also observed that the mean reduction in the area of the wounds in the SI and GI groups is lower than that of the CN group, which can be attributed to the presence of the infection in the wounds of these groups. Obviously, the infection can disrupt the healing process and postpone the healing phases. After the second day, the average WSR in all groups was increased, but the rate of increase in the SN, SI, GN and GI groups was higher than the CN group and there was a significant difference between these groups and the CN group with P value less than 0.05. In most wounds, two days after the creation of the wound, fibroblasts migrate and collagen is formed and the contraction of the wound begins from the fifth day [34]. Comparing the SN and SI groups, it can be seen that wound healing occurs faster in the SN group due to the absence of infection in this group. However, considering the GN and GI groups, it can be observed that the healing progress rate is nearly comparable. This may be due to the usage mode of these types of wound dressing which can completely cover the surface of the wound and keep a permanent and perfect moist environment on the wound surface. Perfect isolating the wound surface may protect the wounds of animals of the GI group from the secondary infection and accelerate the healing process. This is also the main reason for the observed difference between the mean WSR values of the SI and GI groups during the second to eighth healing days. On the tenth day of healing, the average WSR values of the SN, SI, GN and GI groups were 91.1, 88.6, 94.6 and 93.8, respectively, which were higher than the mean WSR value of the CN group (c.a. 40.7).

Fig. 8
figure 8

WSR values for animals of all groups against healing time (nsp > 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

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The faster healing rates of the prepared hydrogel wound dressings compared to a typical advanced wound dressing (i.e. Chitopad as a control) could be related to their excellent abilities to create and maintain a continuous wet condition on the top of the wound during the healing process. In addition to the moisture, factors such as the presence of honey as a natural antibiotic and a rich source of organic and amino acids, proteins, minerals, vitamins and fats, as well as the presence of egg white as a rich source of different proteins, such as albumin, in the wound dressing structure are some other factors in this respect. In addition, the existence of the proteins of egg white in bionanocomposite hydrogel wound dressings leads to more collagen building and as a result, accelerates the wound healing process.

Comparing to the honey-free hydrogel wound dressing prepared by Sirousazar et al. [16] it was found that the percentages of wounds area reduction, as well as the rates of healing of wounds have been significantly improved using the prepared honey-loaded hydrogel wound dressings in this work.

Tensile Properties of Healed Wounds

Stress–strain curves for the healed wounds of animals ten days after the creation of wounds were determined using the tensile test. Tensile strength and elongation at maximum stress for all animals were extracted from the related stress–strain curves. The calculated values for the mean tensile strength and elongation at maximum stress for all test groups were tabulated in Table 5. The results show that the tensile strength and the elongation at maximum stress of all groups treated with the prepared bionanocomposite hydrogel wound dressings are comparable and even higher than the control group in most cases. The main reason for improving the tensile strength and elongation at maximum stress of the wounds treated by the bionanocomposite hydrogel wound dressings can be related to the significant increase in the production of collagen in the injured tissues due to creation of a suitable moist condition on the surface of wound healed by the bionanocomposite hydrogel wound dressings. Furthermore, the increased collagen formation is attributed to the presence of egg white as a unique source of proteins in the structure of the prepared bionanocomposite hydrogel wound dressings. There is a significant correlation between the collagen content and tensile strength of the wound in a manner which the high tensile strength is due to good collagenization in the affected tissues [35].

Table 5 The average tensile strength and elongation at maximum stress values of the healed wounds of animals on the 10th day of treatment
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Histological Study

Images obtained from the histological observations of the healed wounds of the selected animals of CN, SN, SI, GN, and GI groups stained with the H & E, trichrome and collagen are shown in Figs. 9,10, 11, 12, and 13, respectively. Figure 9 shows the histological images of the healed wound tissues of the selected animal of the control group. There was a lack of epithelization, thin collagen, angiogenesis of three to four vessels per microscope, fibroblast, and inflammatory cells in three to five microscopes. As a result, according to Table 2, the control group was ranked with a score of four. The histological images of the healed tissues of the selected animal of the SN group are demonstrated in Fig. 10. For this sample, a complete epithelization, failure to see inflammatory cells, fibroblasts, lack of angiogenesis and thick collagen can be seen, which according to Table 2, this group was ranked with a score of zero. Figure 11 demonstrates the captured histological images for the healed tissues of the wound of the selected animal of the SI group. No epithelization but cell migration of more than 50%, an inflammatory cell in two microscopes, thick collagen but with a lot of fibroblasts, angiogenesis one to two blood vessels in each microscope were observed in this sample which was ranked with a score of two. Figure 12 shows the histological images for the selected animal of the GN group. In this sample, lack of epithelization, cell migration more than 60%, an inflammatory cell in two microscopes, fibroblast and angiogenesis zero to two blood vessels in each microscope were observed ranking with a score of two. The images obtained from the histological observation of the healed tissues of the selected animal of the GI group are exhibited in Fig. 13. As seen, lack of epithelization, more than four blood vessels in each microscope, fibroblasts and inflammatory cells in more than five microscopes are clear. According to the histological images and Table 2, the GI group was ranked with a score of four. The results of the histological examination showed that the quality of the wound healing using the bionanocomposite hydrogel wound dressings in the form of the sheet was better than the gel form. Furthermore, the performances of the dressings of the SI and GN groups were superior in improving damaged tissue than the wound dressings used for the GI and CN groups. Ultimately, comparing the performances of the wound dressings of the GI and CN groups showed the superiority of the wound dressing of the GI group in healing the wounds. In general, the histological observations exhibited the excellent ability of the prepared honey-loaded bionanocomposite hydrogel wound dressings in healing the infectious and non-infectious animal wounds. Comparing the obtained results for the honey-loaded bionanocomposite hydrogel wound dressings with the similar honey-free bionanocomposite hydrogel wound dressings [16], it can be concluded that the rate of tissue repair progression using the current wound dressings are much better than the similar honey-free wound dressings.

Fig. 9
figure 9

Histological images of the healed wound of the selected animal of CN group stained with a H & E, b trichrome, and c collagen

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Fig. 10
figure 10

Histological images of the healed wound of the selected animal of SN group stained with a H & E, b trichrome, and c collagen

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Fig. 11
figure 11

Histological images of the healed wound of the selected animal of SI group stained with a H & E, b trichrome, and c collagen

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Fig. 12
figure 12

Histological images of the healed wound of the selected animal of GN group stained with a H & E, b trichrome, and c collagen

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Fig. 13
figure 13

Histological images of the healed wound of the selected animal of GI group stained with a H & E, b trichrome, and c collagen

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Conclusion

In this work, a series of honey-loaded bionanocomposite hydrogels based on egg white, PVA and MMT was prepared and their performances as the novel wound dressings were evaluated in vitro and in vivo. The results showed that the presence of MMT layers in the structure of bionanocomposite hydrogel wound dressings may reduce their swelling ability, as well as the rate of dehydration. It was also found that the release of honey from the MMT-loaded bionanocomposite wound dressings was prolonged and happened in a longer period of time with a more controlled release pattern compared with the MMT-free wound dressing. The obtained in vivo results showed the excellent ability of honey-loaded bionanocomposite hydrogel wound dressings in the creation and keeping a moist region on the surface of the infected and non-infected wounds and their capability in accelerating the healing process of wounds. Beside the moisture, factors such as the presence of honey as a natural antibiotic and a rich source of organic and amino acids, proteins, minerals, vitamins and fats, as well as the presence of egg white as a rich source of different proteins in the wound dressing structure are some other factors accelerating the healing process in wounds treated with the prepared bionanocomposite hydrogel wound dressings. The results of the tensile test performed on the treated skin of the animals showed that the tensile strength and elongation at maximum stress in the groups treated with the honey-loaded bionanocomposite hydrogel wound dressings were comparable and in most cases higher than the control group due to better collagenization. The unique ability of the prepared bionanocomposite hydrogel wound dressings in wound healing was also confirmed on the basis of the histological observations. In general, it was concluded that the prepared honey-loaded bionanocomposite hydrogel wound dressings were suitable materials for the management of the infected and non-infected wounds and they were suggested as novel functional wound dressings for practical applications.