Manuka honey and bioactive glass impart methylcellulose foams with antibacterial effects for wound-healing applications

To fabricate the foams, 2.5 wt.% MC (viscosity 4000 cP, Sigma Aldrich) was dissolved in distilled water at 40°C. After cooling the solution to room temperature, 2.5 wt.% MH was added to the solution, which was kept under stirring for at least 15 min. For composite scaffolds, 0.75 wt.% B3 BG (composition in wt.%: 5.5 Na₂O, 11.1 K₂O, 4.6 MgO, 18.5 CaO, 56.6 B₂O₃, and 3.7 P₂O₅) or 0.75 wt.% B3C BG (composition in wt.%: 5.5 Na₂O, 11.1 K₂O, 4.6 MgO, 15.5 CaO, 56.6 B₂O₃,3.7 P₂O₅, and 3.0 CuO) in particulate form was added to achieve a BG content of 15 wt.%. The BGs were produced according to a previous report [44], then crushed using a Jaw Crusher (Retsch, Haan, Germany) and milled using a zirconia planetary mill (Retsch, Haan, Germany) to a fine powder with an average particle size of 5–20 µm. The produced MC-based solutions were then frozen at −20 °C and subsequently moved to the freeze dryer (Alpha 2–4 LSC plus, Christ, Germany). Cylindrical samples of 12 mm height and 9 mm diameter were obtained.

We performed SEM analysis (Auriga 0750, Zeiss, Oberkochen, Germany) to investigate the morphology of the freeze-dried composite foams. Pore sizes were calculated based on SEM images using ImageJ (NIH, Bethesda, MD, USA) based on three foams of each kind of sample [45]. By using energy-dispersive spectroscopy (Oxford Instruments, Abingdon, UK), the presence of BG particles inside the MC–MH foams was assessed. The composite foams were additionally characterized using Fourier transform infrared spectroscopy (FTIR, IRAffinity-1S, Shimadzu Corp). FTIR spectra were collected with a resolution of 4 cm⁻¹, 40 scans, in the range of 4000 to 400 cm⁻¹. In order to evaluate the mechanical properties, compression tests using a universal testing machine (Instron 5960, Germany) were performed, using ten samples of each composition at the speed of 5 mm min⁻¹ and a load cell of 100 N. Moreover, the wettability of the foams was assessed on five samples of each material group by contact angle measurement (DSA 30, Krüss, Germany) using a 3 µl water drop. The porosity of the produced freeze-dried foams was calculated based on the following formula: porosity = 1–(ρ_foam/ρ_material), where ρ_foam and ρ_material are the densities of the fabricated foams and used material, respectively. The mean density of the foam (ρ_foam) was calculated by dividing the mass by the volume of ten different foams of each kind. The density of the material (ρ_material) was measured based on the Archimedes' method (employing an analytical balance BM-252, A&D) using films made by drying the different solutions made for freeze drying in a petri dish at room temperature (10 pieces of 1 cm² were used).

In order to assess the acellular bioactivity of the fabricated freeze-dried foams, samples were immersed in simulated body fluid (SBF) according to the protocol of Kokubo et al [46] for 3, 7, and 14 d. After the different immersion times, the pH was recorded and the samples were collected for further characterization using FTIR and SEM. Moreover, the phase compositions of the freeze-dried foams were additionally characterized by powder x-ray diffraction (XRD) analysis using a diffractometer (Miniflex 600 HR, Rigaku, Japan). Data were collected over a 2θ range from 20° to 60° with a step size of 0.02°. To evaluate the release of MH during immersion in SBF, the collected dissolution medium was analyzed using a UV–Vis spectrophotometer (Specord 40, Analytik Jena, Germany) at 300 nm based on a calibration curve (R² = 0.9989).

Prior to cell culture tests, freeze-dried foams (cut in pieces of 9 mm diameter and 2 mm height) were disinfected for 1 h under UV light. To better understand the effects of the different samples on cell response, samples were immersed in cell culture medium (CCM) composed of Dulbecco's modified eagle medium containing 10% fetal bovine serum and 1% antibiotic (penicillin and streptomycin) in the same ratio as used in the performed cell culture test and incubated in the same atmosphere as used for cell culture experiments. After 24 h incubation time, the concentration of ionic dissolution products (IDPs) was measured by optical emission spectroscopy with inductively coupled plasma (ICP-OES; Vista MPX, Varian). Prior to the ICP–OES analysis, the samples were acidified by nitric acid to pH = 2. A series of four calibration solutions was prepared to obtain a linear correlation between intensity of ions and concentration. The reference standards certified for ICP techniques were diluted to prepare the stock calibration solutions. In order to deal with non-spectral interferences, the internal standardization technique with scandium was used. Precision of the analysis for all required ions, expressed as RSD%, was below 5%. The average values including standard deviations from three replicates for each dissolved ion are reported.

In order to evaluate the possible cytotoxic effect of the fabricated foams, an indirect cell test using mouse embryotic fibroblast (MEF) cells was performed. MEF cells were cultured in a humidified atmosphere of 95% air and 5% CO₂ at 37°C in the just-described CCM. To prepare cell cultures, 100 000 MEF cells were seeded in 1 ml CCM in 24-well plates. Samples, placed in TC-inserts (Sarstedt, Germany), were immersed in 1 ml CCM without making contact with the MEF cells. Cells in indirect contact to samples were incubated for 24 h in the same atmosphere as described. Cells without contact to samples were taken as positive control and cells incubated in CCM containing additionally 6% of Dimethyl sulfoxide were taken as negative control. Four replicates of each sample type were used. After 24 h of incubation, the viability of the cultivated MEF cells was evaluated by using a WST-8 assay (Sigma-Aldrich) [47] and cell morphology was assessed by using rhodamine phalloidin and DAPI based on an established protocol [48].

Moreover, the freeze-dried foams were tested in a direct cell test using human dermal fibroblasts (hDFs). The cells were grown, harvested, and counted as just described. 50 000 cells were seeded directly by drop-seeding using 50 µl CCM drop on each sample, incubated for 15 min and then filled with 1 ml CCM. Samples with hDFs on top were incubated for 24 h and 7 d under described conditions. After 24 h and 7 d, the cell viability was assessed as already described and the morphology was evaluated by SEM analysis after fixing the samples as described elsewhere [48, 49]. Foams were transferred to a new well plate prior to measuring the cell viability in order not to take into account cells attached to the bottom of the well-plate.

Additionally, based on an established protocol, an in vitro scratch test was performed using human keratinocyte-like HaCaT cells. CCM containing IDPs was prepared as described before. HaCaT cells were seeded at 500 000 cells per ml per well in a 24-well plate for 24 h under the same conditions as described before. After 24 h, by using a 200 µl pipette tip, a scratch was created on the grown HaCaT cell monolayer. After washing with PBS, cells were kept in conditioned CCM for 24 h. Additionally, pure CCM was used as positive control and CCM containing 6% dimethyl sulfoxide (Sigma Aldrich, Germany) as negative control. During this period of 24 h, the cell migration (closure of wound) was observed after 2.5, 5, 8, and 24 h using a light microscope (Primo Vert, Carl Zeiss). The test was performed in triplicate and the mean width of the scratch was calculated using ImageJ software [45].

The antibacterial effect of the fabricated samples was evaluated against the gram-positive bacteria S. aureus and the gram-negative bacteria E. coli. Bacteria strains were incubated in lysogeny broth medium at 37°C for 24 h under static conditions. Then, optical density (OD) (600 nm, Thermo ScientificTM GENESYS 30TM, Germany) of the bacteria medium was arranged to reach 0.015, according to turbidity measurement of bacterial cultures. 10 µl of arranged bacteria suspension was placed in a tube containing 1 ml of the previously described CCM and freeze-dried samples prepared as described for the cell culture experiments. After 24 h of incubation, the OD was recorded. Tubes containing pure CCM were taken as positive control and MH (same amount as incorporated in the foams) as control for the honey-containing samples. All samples were performed in triplicate. Relative bacterial viability (%) was then calculated by the following equation:

$$\begin{equation*}Relative\,bacterial\,viability\,\left( \% \right) = \,\frac{{O{D_{sample}}}}{{O{D_{control}}}}\, \times 100\end{equation*}$$

In figure 1, the FTIR spectrum of MH shows the typical bands previously reported in the literature [50]. In fact, OH vibrations are detectable as a broad band centered around 3250 cm⁻¹ (OH stretching) and at 1645 cm⁻¹ (bending deformation). The characteristic FTIR bands related to the presence of sugar (main component of the honey, usually in the range of 60%–75%) and other organic compounds (i.e. organic acids) are noticeable in the range 1500–750 cm⁻¹ [51]. Usually, in the studies reported in the literature related to the use of MH in combination with other compounds for food or biomedical applications, the typical FTIR bands related to the presence of methylglyoxal, responsible for the antibacterial properties of MH, are not reported. A possible reason could be the fact that, being a highly reactive, low molecular mass dialdehyde, the FTIR bands of this compound overlap the other main bands of honey related to sugar and organic acids. Nevertheless, the use of IR spectroscopy has been reported to be a useful tool for the identification of methylglyoxal and the related antibacterial properties of MH, as reported by Sultanbawa et al [52].

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Glyoxal and methylglyoxal are well known for their properties related to the crosslinking of polymers and proteins [20, 53]. The main reactions behind the crosslinking process occur in relation with the hydroxyl and amino groups of the polymer or protein to be crosslinked [54]. Considering that methylglyoxal is an essential component in MH and that the mechanism of interaction between cellulose and glyoxal in acid environment has been established [55, 56], showing the promotion of the crosslinking reaction through hemiacetalization and acetalization processes [57], the authors anticipated that a similar interaction would occur between MH and MC during the preparation of the present scaffolds.

For what concerns the FTIR spectrum of MC, the main bands related to hydroxyl groups stretching vibrations are detectable at 3440 cm⁻¹, while the C–H stretching vibrations are represented by the peaks between 2925 and 2830 cm⁻¹. Other two characteristic peaks of MC are the shoulder at 1150 cm⁻¹ due to the C–O stretching from oxygen bridge and the C–O–C stretching mode of the glucosidic unit at 1050 cm⁻¹. Moreover, the vibration of OCH₃ groups is identified by the peak centered at 940 cm⁻¹.

The crosslinking reaction is suggested to occur among the hydroxyl groups of MC and the methylglyoxal component in honey. Considering the amount of hydroxyl groups available for the reaction and the amount of methylglyoxal with respect to the total amount of both components, a low degree of crosslinking is expected. The main modifications reported in the spectrum of MC–MH, which could be attributed to the crosslinking reaction, are depicted in figure 1. In the OH vibration region, higher wavenumber values are assigned to the vibrations of 'free' hydroxyl groups, centered around 3575 cm⁻¹, and this band is not detectable in the zoom view of figure 1, for the MC–MH spectrum. Moreover, it is possible to observe three components in the OH bands centered at 3430, 3340, and 3228 cm⁻¹, in which the first two bands could be ascribable to intramolecular hydrogen bonded hydroxyl groups and the latter one to the intermolecular hydrogen bonded hydroxyl groups [58]. In addition to OH bands, it is also possible to notice a shift of the bands to the area of the intermolecular H bonds of hydroxyl groups. The intensity of this band is also increased with respect to the one in MC. A similar modification is observed for the peaks centered in the range 1100–1000 cm⁻¹, mainly related to the sugar content of the honey and the glucosidic units of the MC.

In order to verify the porous structure of the produced foams, samples were observed by SEM (figure 2). All produced samples exhibited a highly porous structure, whereas foams containing (B3/B3C) BG seem to be more dense and not homogenously porous. Additionally, higher-magnification SEM images confirmed the smooth surface of MC and MC–MH foams, whereas the addition of B3/B3C BG particles led to a rougher surface. By SEM/EDX analysis, the presence of B3/B3C BG particles in the MC–MH foams could be confirmed (figures 2(E) and (F)), and the BG particles seem to be well distributed in the MC–MH foam structure. Based on the SEM images, a pore size range of 50–200 µm was found for all foams; here, the addition of B3/B3C BG particles seems not to have a significant impact on pore size. Moreover, the produced foams possess a slightly different porosity as summarized in table 1. Also, contact angle measurements showed that the addition of MH leads to a reduction of the contact angle and therefore to a higher hydrophilicity of the produced MC-based foams. The contact angle of foams containing, additionally to MH, either B3 or B3C BG was not detectable because the foams directly absorb the water drop (table 1). It should be mentioned that the contact angle measurement performed here did not take into account the porosity and roughness of the samples, which could also have a great influence.

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In general, lyophilized hydrogels based on MC and MH can be produced by dissolving the materials in distilled water. This is a great advantage compared to other polymers, where toxic solvents are needed, which could lead to possible residuals inside the obtained samples limiting their application in the biomedical field [59, 60]. During the preparation of the MC–MH sol, no phase separation occurred and, according to SEM images and EDX measurements, the two different BG particle types could be well dispersed. The produced foams provide a high porosity of more than 90%. Additionally, all samples exhibit a suitable pore size range for migration and proliferation of cells as well as vascularization. The pore size range was found to be similar to freeze-dried hydrogels containing BG based on gelatin–hyaluronic acid [61], alginate [62], collagen–hyaluronic acid [63], and MH-containing cryogels (without BG) based on silk fibroin [19, 64] and gelatin [18]. However, compared to other studies using silk-based lyophilized foams [65] and gelatin–chitosan foams [66, 67] containing BG, which show less porosity and pore size ranges, the present MC–MH based foams containing BG appear to be more suitable for tissue-engineering applications based on their pore structure. The open structure with suitable pore size is also crucial to allow nutrient, waste, and gas exchange, cell infiltration, as well as the distribution of nutrients [68].

The produced freeze-dried foams were further mechanically characterized by a compression test, and the mean compressive strength was calculated based on the measured compression stress corresponding to 40%–60% of compression strain, as shown in figure 3. Mechanical properties affect the performance of wound dressings. Independently, if the dressing should cover a dermal wound or should fill an internal wound, the dressing should be stable enough and of sufficient structural integrity to withstand different stresses. The mechanical properties also play a crucial role in the handling in clinical routine [69]. By the addition of MH, the compression strength (table 1) of the produced foams was (not significantly) reduced, as already reported in the literature for silk fibroin cryogels containing MH [18]. However, the introduction of B3/B3C BG particles leads clearly to strengthening of the foams, whereas no significant difference between the two different borate BGs could be found. The measured compressive strength values are comparable to reported data on lyophilized foams based on silk fibroin and gelatin [18]. Therefore, the incorporation of BG is a suitable method to compensate for the lack of mechanical properties of foams, according to a 'composite' concept [70], as mentioned in the introduction. Moreover, all foams remained intact after the compression strength test, and were even able to recover their shape to a certain point after a certain time (a few hours to days). The results thus demonstrate the ability of the composite MC–MH foams containing BG to withstand (relevant) loads without breaking—an ideal property for a successful wound dressing.

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In order to assess the bioactivity of the produced freeze-dried foams in terms of formation of calcium phosphate of foam surfaces, samples were immersed in SBF for up to 14 d. The samples were then analyzed by SEM. According to figures 4(A) and (B), as expected, foams without BG show no formation of calcium phosphate on their surfaces. Foams containing additionally B3/B3C BG particles showed the first signs of calcium phosphate formation after 3 d in SBF (figures 4(C) and (D)). The formation of calcium phosphate increased with increasing immersion time in SBF, which could be also proven by SEM images (figures 4(E) and (F)). The dissolution and bioactive behavior of BGs is well documented [26], and it is characterized by a rapid exchange between glass network modifiers and hydrogen from the solution (here, SBF), leading to an increase of pH and the breaking of the glass network. Further, calcium ions as well as phosphate groups migrate from the BG and solution in order to form amorphous calcium phosphate, which, in some cases, crystallizes into hydroxyapatite [25, 26].

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To further investigate and assess the formation of calcium phosphate on the different fabricated foams, FTIR and XRD measurements were conducted. As already observed in SEM images, MC and MC–MH foams show no visible peaks in the FTIR spectra after 14 d of immersion in SBF, which could be attributed to calcium phosphate (figure 5). In contrast, the FTIR spectra of MC–MH–B3 and MC–MH–B3C foams show peaks at 1000–1100 cm⁻¹, which can be attributed to P–O stretching [71, 72]. Further, the peaks at 560 and 600 cm⁻¹ correspond to P–O bending vibrations [60]. Additionally, XRD measurements were performed in order to evaluate if the detected calcium phosphate had crystallized into hydroxyapatite. XRD spectra before immersion in SBF (not shown here) showed no measurable peaks for all different fabricated foams, which was expected since the polymeric matrix, as well as the introduced BG particles, did not contain any crystalline phase. Additionally, after immersion in SBF for 14 d, no XRD peaks were found for all fabricated foams (figure 5). This result could be due to the fact that the amount of hydroxyapatite on the surface is lower than the detection limit of XRD or due to the fact that the formed calcium phosphate is poorly crystallized (or a combination of both).

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The remaining SBF solution after the different immersion times was also investigated. By pH measurements, it was possible to observe an increase of pH with dissolution of the foams. After 14 d of immersion in SBF, MC and MC–MH samples led to a minor pH increase of up to 7.7 ± 0.1, whereas MC–MH–B3 and MC–MH–B3C samples led to an increase of up to 7.8 ± 0.1 (pure SBF had a pH of 7.5 ± 0.1). Moreover, the release of MH was analysed by UV–Vis spectroscopy. Here, it is important to mention that MH consists of several different compounds, which show different UV maxima [73]. Therefore, it is possible that some compounds are faster released than others, which could not be analyzed here. Moreover, it is also possible that some of the released compounds already degraded and therefore the real amount of release is higher than the measured amount. Nevertheless, as shown in figure 5(C), it could be confirmed that MH is released from the different fabricated foams containing MH. The addition of B3/B3C BG particles seems to favor the release of MH. This could be due to the increased surface area by the release of BG particles from the foams and the presence of interfaces between the particles and the matrix. More interestingly, the released amounts seem to not (significantly) increase over immersion time, which is a sign that MH and MC are well connected in the present freeze dried foams.

Although it is commonly agreed that the ability to form hydroxyapatite on a surface is a marker of the bioactivity potential of BGs (usually in connection with bone tissue applications), in wound healing applications, the role of hydroxyapatite in the wound-healing process is still an open question [29]. The formation of hydroxyapatite can lead to soft tissue calcifications, which should be avoided [31, 74]. On the other hand, hydroxyapatite has been shown to attract macrophages in a rabbit model [75], and it is also well known that a stable bonding can occur between BG and soft tissues [28]. Therefore, the fact that the produced composite foams did not show any crystalline phase after immersion in SBF should not be considered necessarily a disadvantage. On the other hand, the formation of calcium phosphate on foam surfaces shows that ions could be released from the BG particles, proving the previously mentioned 'second bioactivity mechanism' of BGs: the release of biologically active ions. Consequently, further in vitro cell tests as well as ICP measurements were performed to verify the bioactivity of the BGs and the suitability of the composite foams to be used as wound dressings. These results confirmed the formation of a CaP layer on the composite scaffolds, demonstrating that the BG preserves its bioactivity even if embedded in the polymeric matrix. Further studies are needed to assess and quantify the crystallinity of the deposited calcium phosphate.

ICP measurements verified that ions (namely B, Ca, Mg, P, K, and Na) could be released from the BGs used in this study. Since the amount of Na and K is already high in the CCM, leading to an inaccurate measurement, figure 6 shows only the released amounts of B, Ca, Cu, Mg, and P. No significant differences between the reference (pure CCM) and CCM containing dissolution products of MC and MC–MH could be found. In the case of MC–MH–B3 and MC–MH–B3C, significantly higher amounts of B, Ca, Mg, and P could be measured. Moreover, the only measurable difference between CCM containing IDPs of MC–MH–B3 and MC–MH–B3C is the release of copper from the Cu-doped B3 glass inside the MC–MH foam.

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As a preliminary cytotoxic study, an indirect test using MEF cells was conducted (figure 7). According to figure 7(A), the morphology of MEF cells cultured in the presence of dissolution products of MC and MC–MH foams was similar to that of the positive control, showing a dense layer of cells. The addition of B3 particles seems to not negatively influence MEF cells; however, the cell morphology seems to be different. Interestingly, according to the measured cell viability, MC and MC–MH foams led to an increase of cell viability compared to the control, whereas no significant difference could be found for B3-containing foams (figure 7(B)). On the contrary, foams containing Cu-doped BG led to a reduced number of cells according to fluorescence images and the measured cell viability. Nevertheless measurements on both samples showed better results compared to the negative control. In contrast to this preliminary test using MEF cells, the direct cell test using hDF cells showed that the addition of B3 BG particles led to a significant increase of cell viability after 1 d; however, after 7 d, no significant differences could be found (figure 8(A)). Moreover, the addition of Cu-doped BG seems not to have a negative impact on the direct cell test: no differences between foams with and without B3C BG could be found after 1 and 7 d. SEM images of hDF cells cultured on the fabricated foams for 7 d are shown in figure 8(B). The morphology is typical for hDF cells, growing in a dense layer on top of the porous foams [76–78], which corresponds well to the results obtained by cell viability measurements.

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By performing in vitro scratch tests, it is assumed that it is possible to observe cells migrating along the edge of the newly created scratch in order to establish cell–cell contact again, therefore closing the wound [79]. The test has been applied recently to phosphate BG fibers [80]. In figure 9, the results obtained by calculating the migration ratio of cells cultured in contact with dissolution products of the different foams are summarized and images of HaCaT cells directly after the scratch and after 24 h, cultured with both CCM (Control+) and 6% DMSO-CCM (Control-), are shown. A clear migration of cells on the positive control sample can be observed, whereas the addition of 6% DMSO to the CCM completely stopped the migration of HaCaT cells and even seems to have a negative impact on the cell morphology. The dissolution products of the fabricated foams did not show any negative impact on cell migration compared to the control; MH and B3 BG particles even showed a faster migration than the control (figure 9(A)).

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In summary, the performed cell biology tests showed that fabricated neat MC foams do not have any cytotoxic effect; the foams even showed a slight improvement of cell viability, proliferation, and migration. Further, by adding MH into the MC–MH foams, an increase of cell viability (shown in the indirect cell test using MEFs) could be achieved. A study performed by Sell et al [81] using hDFs, macrophages, and human pulmonary microvascular endothelial cells showed that high concentrations of MH can have a cytotoxic effect. Moreover, in the mentioned study, low concentrations of MH seem not to have any effect on the viability of all three cell types, and an in vitro scratch test using hDFs did not show any impact of MH [81]. Therefore, it seems that by combining MC and MH, a more favorable release of MH could be achieved, leading to improved cell migration. However, the effect of MH needs to be further investigated, since in the mentioned study different kinds as well as different amounts of MH were used. By introducing BG particles into the MC–MH foams, a further improvement of cell viability (shown after 1 d of direct culture of hDFs) and migration could be found, showing the great potential of B3 BG to enhance the biological performance of MC–MH foams. However, the effect of the addition of copper to B3 BG needs to be more precisely investigated. A previous study [82] showed a critical biological level of Cu²⁺ higher than 10 mg l⁻¹ on the viability of 3T3 fibroblasts, which was not exceeded here. In a similar study [83], Cu released in concentrations around 10 mg l⁻¹ did not have significant effects on endothelial cells. The uncertainty of a clear trend regarding the use of Cu for angiogenic effects indicates that more research is necessary to understand the effect of different concentrations of copper in wound-healing approaches, which needs to be investigated for different cell types and conditions.

As described in the introduction, wound healing could be negatively influenced by contaminations, which can lead to non-healing, chronic wounds. In the early phase of wound healing, a race between cells and bacteria to the surface of the wound/wound dressing occurs [1]. Therefore, besides being biocompatible, an ideal wound dressing should also provide an antibacterial effect, while not disturbing the attachment of cells and therefore the tissue-regeneration process. In order to analyze the efficiency against bacteria of MH and BG incorporated in the MC foams, antibacterial tests using E. coli and S. aureus were conducted. Both bacteria are well known for wound infections, whereas S. aureus is known to affect the initial phase of wound healing and E. coli is known for being involved in chronic wounds [1, 3, 6]. According to figure 10, MC foams did not have any impact on bacteria growth, whereas MC–MH foams showed a clear reduction of S. aureus, and even a stronger effect is observed on E. coli. The more pronounced effect of MH on E.coli has been already reported [52]. This result is in accordance with the antibacterial effect shown by MH alone. Moreover, the introduction of B3 BG particles shows a minor (not significant) effect on the growth of both kinds of bacteria. Most interestingly, the presence of B3 particles seems to disturb the antibacterial effect of MH inside the MC–MH–B3 composite foam. Moreover, the MC–MH foam containing Cu-doped B3 BG shows more pronounced antibacterial effect compared to MC–MH foams. Here, the relative bacteria viability of E.coli decreased from around 60% to almost 0% (figure 10). Therefore, it seems that the incorporation of B3C BG particles adds an extra effect for killing bacteria to the MC–MH foam and therefore increases its suitability for applications on infected and chronic wounds. Copper is a well studied antibacterial agent; for instance, copper ions have been reported to penetrate bacteria, leading to DNA degradation [84]. Based on the differences in the structure of gram-positive and gram-negative bacteria, gram-negative bacteria have a higher degree of protection and therefore copper ions are more effective against gram-positive bacteria [85, 86]. Moreover, it needs to be pointed out that both MH and antibacterial ions such as Cu offer an advantage compared to antibiotics. It is well known that in recent years, fewer new antibiotics have been developed and more resistance against available antibiotics is continuously developing [3, 24]. Unlike antibiotics, herbal extracts as MH and therapeutic ions can preserve their effectiveness, as suggested by experiments where bacteria have not developed any resistance even after continuous exposure to these compounds [87, 88].

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