Ca-Zn-Ag Alginate Aerogels for Wound Healing Applications: Swelling Behavior in Simulated Human Body Fluids and Effect on Macrophages

Chronic non-healing wounds represent a substantial economic burden to healthcare systems and cause a considerable reduction in quality of life for those affected. Approximately 0.5–2% of the population in developed countries are projected to experience a chronic wound in their lifetime, necessitating further developments in the area of wound care materials. The use of aerogels for wound healing applications has increased due to their high exudate absorbency and ability to incorporate therapeutic substances, amongst them trace metals, to promote wound-healing. This study evaluates the swelling behavior of Ca-Zn-Ag-loaded alginate aerogels and their metal release upon incubation in human sweat or wound fluid substitutes. All aerogels show excellent liquid uptake from any of the formulas and high liquid holding capacities. Calcium is only marginally released into the swelling solvents, thus remaining as alginate bridging component aiding the absorption and fast transfer of liquids into the aerogel network. The zinc transfer quota is similar to those observed for common wound dressings in human and animal injury models. With respect to the immune regulatory function of zinc, cell culture studies show a high availability and anti-inflammatory activity of aerogel released Zn-species in RAW 264.7 macrophages. For silver, the balance between antibacterial effectiveness versus cytotoxicity remains a significant challenge for which the alginate aerogels need to be improved in the future. An increased knowledge of the transformations that alginate aerogels undergo in the course of the fabrication as well as during wound fluid exposure is necessary when aiming to create advanced, tissue-compatible aerogel products.


Introduction
Chronic wounds are a health issue with major impact on patients' living quality and generate considerable costs for healthcare systems and societies [1,2]. It has been estimated that around 0.5 to 2% of the population in developed countries will experience a chronic wound during their lifetime [3,4].

Aerogel Preparation
Synthesis and characterization of the aerogel particles used within this study (Table 1) is described in a previous paper [30]. Briefly 1 wt% sodium alginate solutions were mixed with cross linkers in a defined cation to alginate ratio to achieve cross-linking degrees of either Q1 (1.8 mmol Cation /g dry alginate ) or Q0.5 (0.9 mmol Cation /g dry alginate ). In case of Ca-Zn-Ag aerogels AgNO 3 was admixed into the gelling solution in a total amount of 0.91 mmol AgNO3 /g dry alginate . Subsequently aerogels were prepared via stepwise CO 2 -induced internal setting gelation, solvent exchange and supercritical carbon dioxide-assisted drying [12]. The aerogels were subsequently stored in the dark at 4 • C in closed thermoplastic containers. Data previously published in [30]. n.d. = not detected.

Serum Albumin Binding Assay
To evaluate albumin binding capacity, the aerogel swelling procedure was performed in SBF-albumin fluid. The remaining levels of protein in the swelling supernatant were measured by using the bicinchoninic acid (BCA) assay [34].

Antibacterial Test
Antimicrobial effectiveness was studied by measuring the effect of SBF-albumin swelling supernatants on growth (OD 600 cell density) and viability (MTT assay; reduction of the yellow tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) of Escherichia coli strain BL21(D3) and Staphylococcus warneri (strain dsm-20316) [35]. Briefly, overnight cultures of bacteria were diluted 1:250 in their respective culture media and grown in 96-well plate cavities in the presence of aerogel swelling supernatans or metal salt solutions for 4 h. OD600 bacterial density was measured on an Infinite M200 microplate reader (Tecan, Crailsheim, Germany). Subsequently, bacteria were treated with 0.1 mg/l MTT in culture medium following 30 min lysis in isopropanol and determination of the formazan absorption at 570 nm with a reference wavelength of 630 nm (Tecan, Crailsheim, Germany). Incubations with aerogel-untreated SBF-albumin were included to define the maximum possible bacterial growth and metabolic activity (100% control).

Cell Culture
RAW 264.7 macrophages [36] were used as cell model to examine the immune modulatory properties of aerogel swelling supernatants. Cells were grown in DMEM containing 10% FCS (heat inactivated for 30 min at 56 • C), 2 mM l-glutamine, 100 µg/mL potassium penicillin, and 100 µg/mL streptomycin.

Griess Assay
Stimulation of Raw 264.7 cells with bacterial lipopolysaccharides (LPS) triggers the production of the antimicrobial effector nitric monoxide (NO • ). Its stable metabolite nitrite can be detected following reaction with the Griess reagent [37]. Briefly, cells were grown in 96-well plate cavities up to 70-75% confluence before 24 h stimulation with 100 ng/mL Escherichia coli LPS or LPS together with either 50 µl aerogel supernatants or metal salt solutions in a total volume of 200 µl growth medium. Afterwards cell supernatants were collected, and cell layers used for viability measurements applying 0.25 mg/mL MTT. Cell supernatants from viable cells were analyzed for NO • content by the Griess method [39]. In brief, 50 µl of cell supernatant was mixed with 50 µl of 1% sulfanilamide (dissolved in 5% H 3 PO 4 ) for 10 min. 50 µl 0.1% N-(1-naphtyl) ethylenediamine dihydrochloride solution was added and reactions incubated for another 10 min in the dark. Nitrite production was determined photometrically at 520 nm in a Tecan Infinite M200 reader and quantified based on a standard curve from 0.78 to 50 µM sodium nitrite.

Statistical Analyses
The data shown are based on three independently performed experiments. The statistical significance of the experimental results was calculated with GraphPad prism software version 8.02 (GraphPad Software Inc., San Diego, CA, USA) using the tests indicated in the respective figure legends.

Aerogel Swelling Behavior and Metal Release
Liquid uptake and metal release from the Ca-Zn-Ag aerogels was previously studied using bidistilled water and a HEPES-based buffer for swelling [30]. However, for medical applications a pre-clinical assessment under conditions closer to an intact or wounded human skin milieu is required. Therefore, we re-assessed these parameters applying (1) ISO-standardized sweat formulations [42] mimicking the composition and pH of human perspirations, (2) a simulated body fluid (SBF) with ion concentrations equal to those of human blood plasma [33], and (3) a SBF with added albumin, as this is the most abundant blood plasma protein (accounting for~60% of human blood proteins,~0.53-0.75 mM) [43]. Moreover, albumin is released into wound fluids, in particular during inflammation where increased capillary permeability allows leakage of this protein into the extravascular space [44]. As shown in Figure 1A, the aerogels are superabsorbers from any of the selected formulations. The open-porous structure and high surface area of these alginate aerogels [30] promotes rapid liquid uptake from any of the investigated human body fluid substitutes within a previously established time frame sufficient for achieving maximum swelling of the aerogels [30]. We anticipate the water vapor permeability of the alginate-based gels to be in the range of common calcium alginate wound dressings (0.00001-0.00002 kg/m.Pa.s) since the chemical structure of the calcium alginate aerogel does not vary significantly [45].

Aerogel Swelling Behavior and Metal Release
Liquid uptake and metal release from the Ca-Zn-Ag aerogels was previously studied using bidistilled water and a HEPES-based buffer for swelling [30]. However, for medical applications a pre-clinical assessment under conditions closer to an intact or wounded human skin milieu is required. Therefore, we re-assessed these parameters applying (1) ISO-standardized sweat formulations [42] mimicking the composition and pH of human perspirations, (2) a simulated body fluid (SBF) with ion concentrations equal to those of human blood plasma [33], and (3) a SBF with added albumin, as this is the most abundant blood plasma protein (accounting for ~60% of human blood proteins, ~0.53-0.75 mM) [43]. Moreover, albumin is released into wound fluids, in particular during inflammation where increased capillary permeability allows leakage of this protein into the extravascular space [44]. As shown in Figure 1A, the aerogels are superabsorbers from any of the selected formulations. The open-porous structure and high surface area of these alginate aerogels [30] promotes rapid liquid uptake from any of the investigated human body fluid substitutes within a previously established time frame sufficient for achieving maximum swelling of the aerogels [30]. We anticipate the water vapor permeability of the alginate-based gels to be in the range of common calcium alginate wound dressings (0.00001-0.00002 kg/m.Pa.s) since the chemical structure of the calcium alginate aerogel does not vary significantly [45]. Khattab et al. [46] recently reported a cellulose-based liquid-stretchable aerogel sensor suitable for assessment of skin sweat status. This study also identified alginate aerogels as promising ingredients in wound dressings or cosmetic products to trap human sebum and perspirations. From  Khattab et al. [46] recently reported a cellulose-based liquid-stretchable aerogel sensor suitable for assessment of skin sweat status. This study also identified alginate aerogels as promising ingredients in wound dressings or cosmetic products to trap human sebum and perspirations. From a medical perspective the perspiration-soaking ability of Ca-Zn-Ag aerogels is advantageous when aiming to restrict moisture lesions in skin and associated periwound skin damage. In this respect, the multication-loaded alginate aerogels seem to be particularly well suited for uptake of sweat-mimicking fluids ( Figure 1A), but more comprehensive work is required to understand the underlying aspect of swelling and liquid uptake from perspirations in detail.
The liquid uptake of synthetic sweat formulas and SBFs was distinctly lower than the quota determined for bidistilled water as swelling agent (~4000-7000%, [30]). Presumably, a disintegration of the alginate aerogel occurs following treatment with human body fluid substitutes. These contain a considerable amount of monovalent ions (Na + , K + ) and phosphates, displacing the bivalent bridging cations from the egg-box cavity. Such disintegration was previously reported for several other alginate aerogels [24,[47][48][49]. Cation release varied substantially depending on the swelling solution as well as the investigated cation ( Figure 1C). Upon wetting with SBF, zinc was more efficiently released from the alginate aerogels than calcium. This was also observed after swelling of these aerogels in Na + -enriched HEPES, confirming the weaker attachment of zinc to the alginate gel [30,50].
In physiological environments such as human plasma or wound fluids, the aerogels will be surrounded by a protein corona that may alter their surface and porosity. The results of the protein binding assay revealed the highest albumin adsorption onto CaZnQ1 aerogels ( Figure 1B), probably because of their higher surface area (Table 1). Approximately 60% of the BSA contained in the SBF-albumin was bound by the CaZnQ1 aerogel. In blood, albumin is crucial for binding, storage and transport of zinc. Almost 80% of total plasma zinc (~14 µM) is thought to be bound to human albumin, constituting the bulk of the potentially exchangeable plasma zinc pool [43,51]. However, under normal conditions only 2% of the circulating albumin molecules carry a zinc ion [52], leaving a vast capacity for accepting zinc from topically applied wound dressings (such as Ca-Zn-Ag aerogels). Here the presence of albumin enhanced zinc release from alginate aerogels over SBF solutions with no protein ( Figure 1C). Thus, it is quite likely that systemic albumin will also act as a zinc-carrier in the wounded tissue. The calcium affinity of human albumin is rather low (K d = 0.67 mM [43]). Hence, BSA only marginally contributes to calcium release from alginate aerogels into SBF-albumin ( Figure 1C). Regarding clinical applications the total calcium and zinc concentrations in any of the aerogel SBF-albumin swelling concentrations ( Figure S1) were close to those determined in sera from skin-injured humans and animals treated with Ca/Zn-ion impregnated wound dressings [29,53,54]. The considerable silver release from the CaZnQ1Ag aerogels into the SBF-albumin solution ( Figure 1C, Supplementary Figure S1) is a matter of concern. Silver levels in the swelling supernatants were orders of magnitude higher than those measured in wound fluids or sera from patients topically treated with Ag-coated foam dressing Acticoat or nitrate silver sulfadiazine (~2-10 µM Ag in these patients' sera) [55,56]. Local toxicity or argyrosis upon topic treatment with CaZnQ1Ag aerogels have to be considered, necessitating fine tuning of the silver quantity released by the alginate aerogels.

Antibacterial Effectiveness of Aerogel Swelling Supernatants
Escherichia coli and Staphylococcus warneri were selected as representatives for gram-positive and gram-negative bacteria. These species represent relevant opportunistic or nosocomial pathogens in wounded tissues [57,58]. The turbidity measurement of microbial cultures revealed very limited bactericidal effectiveness of serial dilutions of SBF-albumin swelling supernatants from Ca and/or Zn loaded aerogels. Likewise, application of inorganic zinc and calcium salts showed almost no toxicity (Figure 2A,B). For CaZnQ1Ag a concentration-dependent effect was observed with a greater impact on Escherichia coli. Based on the estimated amount of silver in the SBF-albumin swelling supernatants (~1700 µM; Supplementary Figure S1) the actual effectiveness was lower compared to AgNO 3 , suggesting a different speciation of silver released from the aerogel, altering its effect, e.g., by affecting its availability for bacteria (Figure 2A,B). Still the total quantity of silver within the undiluted CaZnQ1Ag swelling concentrate delivers silver far in excess of bactericidal concentrations [35,59]. In principle, determination of bacterial metabolic activity with the MTT assay confirms the results of the OD600 assay. AgNO3 and the CaZnQ1Ag swelling supernatant were particularly effective in inhibiting bacterial metabolism ( Figure 3A,B). For the CaZnQ1Ag aerogel-soluble mediators a maximum bacterial metabolic inhibitory effect of ~ 50% was observed, where the least diluted 1:20 and 1:40 samples were comparable in antibacterial efficacy within the experimental fluctuation margins. ZnO showed no statistically relevant antibacterial efficacy against Escherichia coli and Staphylococcus warneri over the entire concentration range when evaluating the MTT-reducing activity ( Figure 3A,B). We attribute the slight increase in E. coli OD600 density following high ZnO treatment (1000 µM; Figure 2A) to the assembly of ZnO particles in bacteria enriched growth media. ZnO-related increase in turbidity was not observed in media in the absence of bacteria, which we included as negative controls. In principle, determination of bacterial metabolic activity with the MTT assay confirms the results of the OD 600 assay. AgNO 3 and the CaZnQ1Ag swelling supernatant were particularly effective in inhibiting bacterial metabolism ( Figure 3A,B). For the CaZnQ1Ag aerogel-soluble mediators a maximum bacterial metabolic inhibitory effect of~50% was observed, where the least diluted 1:20 and 1:40 samples were comparable in antibacterial efficacy within the experimental fluctuation margins. ZnO showed no statistically relevant antibacterial efficacy against Escherichia coli and Staphylococcus warneri over the entire concentration range when evaluating the MTT-reducing activity ( Figure 3A,B). We attribute the slight increase in E. coli OD600 density following high ZnO treatment (1000 µM; Figure 2A) to the assembly of ZnO particles in bacteria enriched growth media. ZnO-related increase in turbidity was not observed in media in the absence of bacteria, which we included as negative controls. Bacterial burden is believed to play an extensive role in impaired healing of chronic wounds and the development of infection-related complications. Yet, it has to be noted that the present study aimed to evaluate, in particular, the antibacterial effectiveness of the aerogel soluble metal fractions. Still, functionality of the aerogels in real wound situations is also affected by intermolecular interactions between bacteria and gel surfaces [60,61] in the local human skin milieu. These interactions would be identifiable in an ex-vivo human skin model [62], followed by application on animals and finally human volunteers in clinical trials. Bacterial burden is believed to play an extensive role in impaired healing of chronic wounds and the development of infection-related complications. Yet, it has to be noted that the present study aimed to evaluate, in particular, the antibacterial effectiveness of the aerogel soluble metal fractions. Still, functionality of the aerogels in real wound situations is also affected by intermolecular interactions between bacteria and gel surfaces [60,61] in the local human skin milieu. These interactions would be identifiable in an ex-vivo human skin model [62], followed by application on animals and finally human volunteers in clinical trials.

Immune Modulatory Potency of Aerogel Swelling Supernatants
Macrophages as part of the innate immune system are crucial for wound healing. They initiate an inflammatory response immediately upon wounding, which can trigger infiltration of neutrophils and thus accelerate bacterial clearance. However, prolonged inflammation is detrimental and can contribute to chronification of wounds and healing difficulties [63]. The sensing of infection and strengthening of innate immunity by macrophages are mediated by pattern recognition receptors, including Toll-like receptor TLR4 [64]. TLR4 complexes, localized on the cell surface of macrophages, can register intact bacteria as well as isolated bacterial LPS. Sensing of pathogens by this receptor drives intracellular signaling cascades mediating the production of inflammatory factors. Here the activation status of the MAPK-pathway (MAPK= mitogen-activated protein kinase) is decisive with regard to the quantities of inflammatory mediators produced. As part of the MAPK-pathway, the protein kinase ERK1/2 is regulated in a phosphorylation-dependent manner. Thus, covalent phosphate attachment to ERK1/2 protein leads to increased kinase activity while dephosphorylation restores baseline activity [37,65]. Previous studies revealed a regulatory role of intracellular free zinc ions on TLR4-signaling with zinc augmenting ERK1/2 phosphorylation in macrophages [37,40,66]. These observations led us to evaluate the modulatory potency of zinc-containing aerogel swelling supernatants in cultivated RAW 264.7 macrophages, focusing on TLR4 signaling. Western blot experiments were performed to analyze RAW 264.7 ERK1/2 phosphorylation upon stimulation with E. coli suspensions in the absence or presence of aerogel swelling supernatants (Figure 4). Applying two different antibodies specific for either total ERK1/2 or phosphorylated ERK1/2 (Thr202/Tyr204) allowed monitoring the overall amount of cellular ERK1/2 and also its phosphorylation status [37,40]. In addition, Zinpyr-1, a membrane-permeant fluorescent sensor with a high affinity for Zn (K d = 0.7 ± 0.1 nM; [38]), but insensitive to calcium and silver [67], was used for the investigation of intracellular free zinc ( Figure 5).

Immune Modulatory Potency of Aerogel Swelling Supernatants
Macrophages as part of the innate immune system are crucial for wound healing. They initiate an inflammatory response immediately upon wounding, which can trigger infiltration of neutrophils and thus accelerate bacterial clearance. However, prolonged inflammation is detrimental and can contribute to chronification of wounds and healing difficulties [63]. The sensing of infection and strengthening of innate immunity by macrophages are mediated by pattern recognition receptors, including Toll-like receptor TLR4 [64]. TLR4 complexes, localized on the cell surface of macrophages, can register intact bacteria as well as isolated bacterial LPS. Sensing of pathogens by this receptor drives intracellular signaling cascades mediating the production of inflammatory factors. Here the activation status of the MAPK-pathway (MAPK= mitogen-activated protein kinase) is decisive with regard to the quantities of inflammatory mediators produced. As part of the MAPK-pathway, the protein kinase ERK1/2 is regulated in a phosphorylation-dependent manner. Thus, covalent phosphate attachment to ERK1/2 protein leads to increased kinase activity while dephosphorylation restores baseline activity [37,65]. Previous studies revealed a regulatory role of intracellular free zinc ions on TLR4-signaling with zinc augmenting ERK1/2 phosphorylation in macrophages [37,40,66]. These observations led us to evaluate the modulatory potency of zinc-containing aerogel swelling supernatants in cultivated RAW 264.7 macrophages, focusing on TLR4 signaling. Western blot experiments were performed to analyze RAW 264.7 ERK1/2 phosphorylation upon stimulation with E. coli suspensions in the absence or presence of aerogel swelling supernatants (Figure 4). Applying two different antibodies specific for either total ERK1/2 or phosphorylated ERK1/2 (Thr202/Tyr204) allowed monitoring the overall amount of cellular ERK1/2 and also its phosphorylation status [37,40]. In addition, Zinpyr-1, a membrane-permeant fluorescent sensor with a high affinity for Zn (Kd = 0.7 ± 0.1 nM; [38]), but insensitive to calcium and silver [67], was used for the investigation of intracellular free zinc ( Figure 5).   TLR4 stimulation with E. coli dose-dependently increased phosphorylation of ERK1/2 ( Figure  4A,B). In case of co-incubation with bacteria and aerogel CaZnQ1 or CaZnQ1Ag swelling supernatants diluted to a final zinc concentration of 100 µM, ERK1/2 phosphorylation was increased. A CaCl2 bonus up to 200 µM added to the DMEM medium (basal calcium concentration ~1.3 mM) had no impact on this TLR4-dependent signaling pathway ( Figure 4D). The calcium input from the SBF-albumin aerogel supernatants was ~150 µM (calculated from the metal concentration of undiluted supernatant shown in Figure S1). For AgNO3 a dose-dependent inhibitory effect on ERK1/2 phosphorylation was observed ( Figure 4D), yet silver delivered from the CaZnQ1Ag swelling supernatant (final concentration of ~17 µM, based on data in Figure S1) was below the effectual dose ( Figure 4D). Thus, effects of the aerogel SBF-albumin mobile fraction on ERK1/2 phosphorylation are predominantly attributable to zinc species that affect the TLR4 cascade either via extracellular activation of the ZnR/GPR39 G-protein coupled receptor [68] or via intracellular zincergic signaling. An intracellular regulatory mechanism is supported by the estimation of the cellular free zinc levels ( Figure 5A-C), showing almost comparable increase in Zinpyr-1 fluorescence upon incubation of RAW 264.7 with either Zn-aerogel swelling supernatant solutions or ZnSO4/ZnO, suggesting comparable zinc availability.
In direct comparison to inorganic zinc sources (ZnSO4/ZnO), RAW 264.7 macrophages responded with stronger ERK1/2 phosphorylation to aerogel swelling supernatants containing the same amount of zinc ions ( Figure 4B,C). This leads to the assumption that aerogel alginate backbone TLR4 stimulation with E. coli dose-dependently increased phosphorylation of ERK1/2 ( Figure 4A,B). In case of co-incubation with bacteria and aerogel CaZnQ1 or CaZnQ1Ag swelling supernatants diluted to a final zinc concentration of 100 µM, ERK1/2 phosphorylation was increased. A CaCl 2 bonus up to 200 µM added to the DMEM medium (basal calcium concentration~1.3 mM) had no impact on this TLR4-dependent signaling pathway ( Figure 4D). The calcium input from the SBF-albumin aerogel supernatants was~150 µM (calculated from the metal concentration of undiluted supernatant shown in Figure S1). For AgNO 3 a dose-dependent inhibitory effect on ERK1/2 phosphorylation was observed ( Figure 4D), yet silver delivered from the CaZnQ1Ag swelling supernatant (final concentration of 17 µM, based on data in Figure S1) was below the effectual dose ( Figure 4D). Thus, effects of the aerogel SBF-albumin mobile fraction on ERK1/2 phosphorylation are predominantly attributable to zinc species that affect the TLR4 cascade either via extracellular activation of the ZnR/GPR39 G-protein coupled receptor [68] or via intracellular zincergic signaling. An intracellular regulatory mechanism is supported by the estimation of the cellular free zinc levels ( Figure 5A-C), showing almost comparable increase in Zinpyr-1 fluorescence upon incubation of RAW 264.7 with either Zn-aerogel swelling supernatant solutions or ZnSO 4 /ZnO, suggesting comparable zinc availability.
In direct comparison to inorganic zinc sources (ZnSO 4 /ZnO), RAW 264.7 macrophages responded with stronger ERK1/2 phosphorylation to aerogel swelling supernatants containing the same amount of zinc ions ( Figure 4B,C). This leads to the assumption that aerogel alginate backbone components induce TLR4 or other ERK-dependent signaling pathways, either by acting as a ligand for plasma membrane receptors or affecting their downstream signaling pathways. This hypothesis is consistent with the observation of higher ERK1/2 baseline phosphorylation for aerogel swelling supernatant treatment in the absence of E. coli ( Figure 4C). In accordance, Fang et al. [69] showed the induction of TLR4-signaling by alginate-derived guluronate oligosaccharide in RAW 264.7 macrophages.
TLR4 signaling in macrophages induces the production of many pro-inflammatory molecules, amongst them the effector nitric monoxide. NO • plays a critical role in the wound-healing process. It is important as a noxious defense molecule against infectious organisms, mediates vasodilation in blood vessels and also coordinates host immune cells, thereby locally controlling the immune response [70]. RAW 264.7 cells activated with E. coli LPS for 24 h generated around 28 µM of nitrite, almost the same amount as in previous studies [71,72]. Zinc orchestrates and regulates the inflammatory balance within the local tissue environment [73]. Swelling supernatants obtained from ZnQ0.5 aerogels dose-dependently decreased LPS-induced nitrite concentrations in cell supernatants. This was not due to cytotoxicity, because viability was not affected by the ZnQ0.5 swelling supernatant under these conditions ( Figure 6A,B). The influence on TLR4-induced NO • release was comparable in efficiency to inorganic zinc ( Figure 6B). The anti-inflammatory potency of the Zn alginate aerogels provided by the wound fluid-accessible zinc fraction could be advantageous for the treatment of wounds showing signs of chronic inflammation and delayed healing. On the other hand, the application of the CaZnQ1Ag aerogels has to be viewed much more carefully, as indications of silver cytotoxicity were found in the cell culture studies upon prolonged incubation ( Figure 6B). Similarly, in a recently published in vitro screening of clinically applied wound dressings, the silver alginate Biatain ® scored rather poorly in cytocompatibility [74]. Very likely the amount of silver released by the alginate wound pad overstrained the cellular buffering capacity, thus damaging the cells irreversibly.

Conclusions
Alginate aerogels augmented with multiple cations can provide the next generation of superabsorbent medical devices for advanced wound care. This work shows that exposure of Ca-Zn-Ag alginate aerogels to body fluid formulations designed to match the chemical composition of human sweat or wound fluids led to substantial metal transfer into the supernatants. Calcium was only moderately released into any of the swelling solvents, thus remaining as alginate bridging component aiding the absorbency and fast transfer of liquids into the aerogel network. Zinc release Figure 6. Impact of aerogel swelling supernatants on LPS-induced nitric monoxide production in RAW 264.7 cells. Supernatants from aerogels or metal salt solutions generated in SBF-albumin were applied on RAW 264.7 macrophage cells to study their impact on bacterial LPS-induced NO • production, measured by its stable degradation product nitrite (A), and the corresponding viability (B) of RAW 264.7 cells. The designations on the X axes state the final amounts of aerogel-derived zinc and the final concentration of metal salts within the incubations. n.d. = not detected. Data are presented as means ± SEM of three independent experiments. Statistically significant differences from LPS-treatment (A) or control (B) are marked by asterisks (** p < 0.01, *** p < 0.001; One-Way ANOVA with Tukey's Multiple Comparison Test).

Conclusions
Alginate aerogels augmented with multiple cations can provide the next generation of superabsorbent medical devices for advanced wound care. This work shows that exposure of Ca-Zn-Ag alginate aerogels to body fluid formulations designed to match the chemical composition of human sweat or wound fluids led to substantial metal transfer into the supernatants. Calcium was only moderately released into any of the swelling solvents, thus remaining as alginate bridging component aiding the absorbency and fast transfer of liquids into the aerogel network. Zinc release into wound fluid-like liquids was close to those observed for common zinc ion-impregnated wound care products in human and animal injury models. The zinc-species within these swelling supernatants were able to modulate inflammatory signaling pathways in macrophages, with an anti-inflammatory outcome similar to inorganic zinc sources (ZnSO 4 /ZnO). For silver, the balance between antibacterial effectiveness versus cytotoxicity remains a significant challenge for which the alginate aerogels need to be improved in the future. Based on the results found for macrophages, an in-depth analysis of the multi-cation-loaded alginate aerogels on other cell types involved in the wound healing process is required prior to preclinical testing on animals or human volunteers. Additionally, the aspect of wound material sterilization needs to be considered. Physical treatments, such as plasma processing, may generate reactive oxygen species leading to the depolymerization of the alginate polysaccharide chains, thereby impacting swelling properties and release of bioactive soluble metal mediators from the aerogels. Furthermore, the long-term stability of alginate-based aerogels under conditions recommended by ICH (International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use) has to be checked as there are no data available in the literature up to now. Increased knowledge of the transformations that alginates undergo in the course of the wound material fabrication, storage as well as during wound fluid exposure is necessary when aiming to create advanced, tissue compatible aerogel products.

Conflicts of Interest:
The authors declare no conflict of interest.