2.2. Efficient Release Properties of Mg- and Ag-Containing Dressings

The influence of Mg in cell physiology has been well-demonstrated. Given the evidence that an ideal wound dressing should provide efficient release of the components to promote wound healing, reduce bacteria counts, and minimize tissue toxicity [12], a thorough understanding of Mg and Ag release kinetics at different time points was studied using an immersion test from 2 to 72 h. At 6 h, over 50% of the Mg ion was observed in the medium and ascended to its highest value at 72 h (2.19 mM, Figure S1, left). In addition, two different types of Mg dressings (Mg100 and Ag200 + Mg100) exhibited a similar release property (Figure S1, right). Furthermore, the release of the Ag ion in the medium was 0.5 ppm at 2 h, 0.89 ppm at 6 h, 0.88 ppm at 24 h, and 0.96 ppm at 72 h. The Ag ion was observed to rapidly release into the medium at 6 h, exhibiting good antibacterial activity, reaching approximately 92% of its concentration at 72 h (Figure S1, right).

2.3. Incorporation of Mg and Ag in Wound Dressings

Polyethylene materials are widely used in wound dressings. The mesh coated with Ag and Mg ions was prepared through sputtered technology. In the Ag200 + Mg100 dressing, the Mg ions were sputtered on top of the Ag ions to keep the Mg ions on the outermost layer of the dressing. The surface morphology of the sputtered metal ions was observed by scanning electron microscopy (SEM) at 250× and 500× magnification. Either Ag or Mg were welded steadily to maintain the integrity of the material in use (Figure S2A). In addition, surface elements such as Ag and Mg ions were studied with energy-dispersive X-ray spectroscopy (EDS) analysis. The data showed that the average proportion of Mg atoms was 4.7% in Mg100 dressing and 8.7% in Ag200 + Mg100 dressing, with Ag atoms at a low proportion of 0.15% in the latter (Figure S2B). The results indicated that Mg and Ag ions can be incorporated into our material by a sputtered process.

2.4. Good Surface Wettability of Mg100 and Ag200 + Mg100 Dressings

Wettability is one of the important abilities of sputtered technology. Traditionally, the contact angle was utilized to measure the wettability between the test liquid and the solid surface [13]. A contact angle greater than 90 degrees is needed to maintain its hydrophobic property to avoid stickiness of the dressing to the tissue wound, reduce secondary damage during dressing change, and also help to release ions. To study the surface wettability of our materials, the contact angles of different thicknesses of our dressing, such as 10, 25, 50, and 100 nm, were measured. The results showed that the contact angles of all materials were about 120 degrees. Additionally, there were no differences in the contact angle between uncoated dressing and all types of Mg dressings (Figure S3), suggesting the Mg coating did not significantly alter the hydrophobic properties of the dressing material.

2.5. Absence of Harmful Extractables from Mg100 and Ag200 + Mg100 Dressings

To study the toxic effects of Ag and Mg in our material, a standard cell toxicity test was used. According to the policy of ISO 10993-5 [14], we picked HUVEC and NIH-3T3 cell lines for cytotoxicity testing of our dressings. A maximal Mg release property of 2 mM was determined to prevent cell toxicity in our design. The extracts of Mg100 and Ag200 + Mg100 dressings were diluted into four different conditions for the following experiment. Solutions with 10% DMSO and 10% FBS were indicated as positive and negative controls, respectively. The HUVECs displayed a higher survival rate (>70%) when cultured at different levels of Mg concentrations, ranging from 0.1 to 2 mM (Figure 2A). By contrast, cultured NIH-3T3s still displayed a higher survival rate in media with 0.5 and 1 mM Mg concentrations, which were 94.63% and 78.75%, respectively. However, the survival rate of the NIH-3T3s decreased to 64.92% in the 2 mM Mg medium (Figure 2B). Although decreasing viability of fibroblasts was observed, cell viability could be reversed to higher than 70% in the Ag-Mg combination, indicating a potentially synergistic effect of Ag and Mg in the dressing. In addition, HUVECs and NIH-3T3s exhibited a high survival rate in the medium of different concentrations of Ag and Mg in the Ag200 + Mg100 dressing (Figure 2C,D). The present study showed HUVECs were able to maintain a high viability rate at this level. Even in the presence of 0.8 ppm of Ag with Mg, cell viability still reached 82.39%, indicating the absence of harmful extractables from our Ag- and Mg-based materials.

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Figure 2

The toxic effects of Mg100 and Ag200 + Mg100 dressings. Human umbilical vein endothelial cells (HUVECs) and fibroblasts (NIH-3T3s) were used for cytotoxicity testing of dressings. The effects of different levels of Mg and Ag concentrations extracted from Mg100 and Ag200 + Mg100 dressings on HUVECs and NIH-3T3s were investigated. Cell viability of HUVECs and NIH-3T3s is shown in (A–D). PC—positive control; NC—negative control.

2.6. Magnesium-Enhanced Antibacterial Ability of Silver in Wound Dressing

Testing for the antimicrobial ability of wound dressings was carried out using Staphylococcus aureus, Klebsiella pneumonia, Escherichia coli, and Pseudomonas aeruginosa [15]. According to the laboratory’s Biosafety Level (BSL) guidelines, a BSL-1 E. coli strain was chosen to test our products. The data showed that both the Mg100 and Ag200 + Mg100 dressings had lower OD600 values, with the Ag200 + Mg100 dressing showing a significantly lower value (p < 0.05, n = 3) compared to the control (Figure 3A). The obtained value was also converted into the efficiency of the bacteriostatic activity. The data indicated that the Ag200 + Mg100 dressing exhibited 40% of bacteriostatic activity against E. coli (Figure 3B), suggesting that Mg amplifies the antibacterial properties of Ag.

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Figure 3

Antibacterial ability of the Mg100 and Ag200 + Mg100 dressings. (A) A BSL-1 E. coli strain was chosen to test the antibacterial ability of the dressings. Both the Mg100 and Ag200 + Mg100 dressings had lower OD600 values, with the Ag200 + Mg100 dressing showing a significantly lower value (* p < 0.05, n = 3). (B) The percentage of bacteriostatic activity in each group is shown. OD—optical absorbance values.

2.7. Increased Migration of Fibroblasts by Mg100 and Ag200 + Mg100 Dressings

Mg was found to promote fibroblast migration and accelerate wound healing in optimal conditions [16]. To study the effect of Ag and Mg on cell migration, we performed an in vitro scratch wound assay. Fibroblasts were incubated in the medium containing either Mg100 or Ag200 + Mg100 dressings for 24 h. Images of the scratch wounds at 6 and 24 h after scratching showed that fibroblast migration into the scratch wound area was accelerated in the presence of Mg (Figure 4A). At 24 h, the scratch wound was nearly closed for the medium containing Mg100 or Ag200 + Mg100, whereas the control medium had no such effect. Quantification of the percentage of scratch wound closure using image analysis confirmed a significant acceleration of fibroblast migration in the presence of Mg100 or Ag200 + Mg100 at 6 and 24 h (Figure 4B).

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Figure 4

In vitro fibroblast migration in the presence of Mg100 and Ag200 + Mg100 dressings. (A) Fibroblasts were incubated in the medium containing Mg100 and Ag200 + Mg100 dressings for 24 h for the cell migration assay. Images show an acceleration of fibroblast migration in the experimental groups. (B) Quantification of the percentage of scratch wound closure using image analysis is shown (* p < 0.05, ** p < 0.01, *** p < 0.001 for all time points, n = 3).

4.2. Mg and Ag Synthesized Materials

Magnesium-ion and silver-ion dressings were produced by KNH Enterprise Co., Ltd., Taipei, Taiwan. In brief, a non-woven polypropylene cloth was ionic sputter coated with pure Mg and/or Ag target on the sputter coater machine for 300 s at different times before scanning electron microscopy (SEM) observation. In the combined Ag and Mg dressing, the silver ions were sputtered first, and then the magnesium ions were sputtered on top of it, so that the magnesium ions are on the outermost layer of the dressing. The thickness of the Mg coating was tested at 25, 50, 100, and 200 nm. The release concentration profile of the 100 nm group was the most ideal, so the following experiment used only the 100 nm as the Mg coating thickness. Two thicknesses of Ag coating, 200 and 2000 mg/m², were fabricated in the pilot study. The groups investigated in this study were the control (polypropylene without ionic coating), Mg100 (only Mg coating at 100 nm), Ag200 + Mg100 (Ag coating at 200 mg/m² and Mg coating at 100 nm), and Ag2000 + Mg100 (Ag coating at 2000 mg/m² and Mg coating at 100 nm) groups.

4.3. Immersion Test

A part of the synthesized Mg100 or Ag200 + Mg100 dressing material, approximately 120 cm² (N = 3), was immersed in 20 mL of phosphate buffered saline (PBS) according to the ISO 10993-12:2021 standard [33] for sample preparation and reference materials. The materials were incubated at 37 °C in a 5% CO₂ incubator for 2, 6, 24, and 72 h, and the concentration of ions contained was measured by inductively coupled plasma mass spectrometry (ICP-MS, Perkin-Elmer-SCIEX, ELAN 6100 DRC II, Woodbridge, ON, Canada). The calibration, matrix matching, and instrument conditions were described as before [34]. Briefly, acetic acid was utilized as a carbon source at the optimum concentration of 10% v/v for matrix matching in the measurement of Mg and Ag. An ICP-MS multi-element standard solution served for external calibration, while rhodium was employed as an internal standard for all measurements. To prevent particle deposition in the sample introduction system, Triton X-100 (Sigma, Kawasaki-shi, Japan) was added to all solutions at a concentration of 0.1% v/v as a detergent. Calibration involved a four-point (blank and three standards) approach, executed in a straightforward linear mode.

4.4. Micrographs

The dressing underwent a sputtering process, where Ag and Mg ions were applied using a sputter coater machine for a duration of 300 s. Scanning electron microscopy (SEM) was utilized to examine the surface morphology of the dressing, while the energy dispersive X-ray spectrometer (EDS) was employed to analyze the elemental composition present on the surface of the coated dressing.

4.5. Surface Wettability

The contact angle goniometer (First Ten Angstroms, FTA-1000B, Portsmouth, VA, USA) was used to measure surface wettability. An amount of 5 µL deionized water was dropped on the surface of the dressing, and the surface wettability was measured by the contact angle meter. When the contact angle is lower, the surface is more hydrophilic. If the surface is hydrophobic, the contact angle will be greater than 90°.

4.6. Cell Culture

Human umbilical vein endothelial cells (HUVECs, P2 to P8, H-UV001, BCRC, Hsinchu, Taiwan) and mouse fibroblasts (NIH-3T3s, P3 to P5, 60008, BCRC, Taiwan) were used for the cell cytotoxicity and migration assays. HUVECs were cultured in medium containing 10% fetal bovine serum (FBS), 1.5% heparin, 2.5% endothelial cell growth supplement (ECGS), and 1% antibiotic-antimycotic (AA). NIH-3T3s were cultured in medium containing 10% FBS, 100 U/mL penicillin G, 100 µg/mL streptomycin sulfate, 1.25 g/mL amphotericin B, and 1% AA. Both types of cells were cultured at 37 °C with 5% CO₂ in a humidified atmosphere.

4.7. Cytotoxicity Assay

HUVECs and NIH-3T3s (5.0 × 10³ cells/well) were plated in 96-well plates and incubated overnight at 37 °C in an incubator with 5% CO₂. After the cells adhered to the plate, the culture medium was replaced with six different media: one containing 10% dimethyl sulfoxide (DMSO, serving as the positive control), another with 10% FBS (fetal bovine serum, serving as the negative control), and four test media with varying concentrations of Mg and Ag. These were incubated for an additional 24 h. The test media were prepared using extracts from Mg and Ag dressings. Mg100 and Ag200 + Mg100 dressings were incubated with 20 mL of culture medium for 24 h to release Mg and Ag ions. After filtering, four different concentrations of Mg (0.1, 0.5, 1, and 2 mM) and Ag (0.04, 0.2, 0.4, and 0.8 ppm) were diluted with 10% FBS for further experiments. Cell Counting Kit-8 (CCK-8) was used for the determination of the number of viable cells in cytotoxicity assays. An amount of 10 μL of CCK-8 reagent was added per well, the plates were wrapped in aluminum foil and placed in an incubator for 3 h in the dark. Cell viability was determined by measuring the absorbance at 450 nm using an ELISA reader. Cell viability can be calculated using the following formula:

4.8. Antimicrobial Assay

The antimicrobial test was carried out according to ISO 10993-12:2021 and the United States Pharmacopoeia (USP) specifications. The tested materials were immersed in PBS, incubated at 37 °C for 24 h, and then filtered to prepare a material extract. A strain of Escherichia coli (PT-FTDH5, Protech, Taipei, Taiwan) was selected for the antimicrobial assay. To prevent the risk of infection and ensure that the microbial load is within an acceptable range, the strain concentration was controlled to be below 100 CFU (colony-forming units) according to the USP (United States Pharmacopeia) specifications to ensure the safety and quality of our products. Extracts of tested materials, such as uncoated dressing, Mg100, Ag200 + Mg100, and commercial Ag dressing (Acticoat, Smith & Nephew, London, UK), were added to each tube along with the same volume of bacteria concentration and incubated for 16 h. An amount of 2 mL of tryptic soy broth (TSB) was also added for bacterial growth. The experimental group was exposed to the same bacterial concentration and the corresponding extract concentration for 16 h. The optical density (OD) at the 600 nm absorption wavelength was measured using an ELISA reader to estimate the bacterial concentration in a liquid [35]. Analytical data can be obtained using the following formula:

4.9. Cell Migration Assay

Cell migration experiments were performed using a culture-insert (ibidi GmbH Gräfelfing, Germany). NIH-3T3s (2 × 10⁴ cells/well) were seeded and cultured in a 37 °C incubator containing 5% CO₂ until the cells’ growth was complete and without gaps. After the cells were confluent, the culture-insert could be removed to leave a 500 μm wide area in the well. The Mg100 and Ag200 + Mg100 dressings were incubated with 20 mL of culture medium for 24 h, then filtered, and 10% FBS was added to create the experimental solutions. The attached cells were fed with test solutions or control medium to observe cell migration. Following saline washes, the wound was photographed by an inverted optical microscope (Olympus, Tokyo, Japan) at the time points of 0, 6, and 24 h.

4.10. Animals

Sprague–Dawley rats (male) were purchased from BioLASCO Taiwan Co., Ltd., Taipei, Taiwan and were bred at the National Cheng Kung University animal center. The animal experimental protocol was carried out under the required conditions in accordance with the NCKU animal center standards and approved by the Institutional Animal Care and Use Committee (IACUC approval no: 107266). Diabetic rats were fed a high-fat diet with a 60% fat content. After 4 weeks of feeding, the rats were fasted for 3 days, anesthetized, and subjected to blood sampling from the tail vein (Roche blood glucose meter). Afterward, the rats were injected with streptozotocin 35 mg/kg through the tail vein. Measurement of plasma blood glucose in the fasting state was carried out on the second and sixth days after induction. Diabetes was successfully induced in rats when their fasting blood glucose levels exceeded 250 mg/dL.

4.11. Wound Healing Assay

The 5-week-old male SD rats were anesthetized and depilated. Four full-thickness excisional wounds were made on the dorsum of the mice on either side of the midline using a 10 mm biopsy punch. The wound edge was affixed to the underlying tissue using 4-0 nylon for four stitches to prevent skin retraction (Figure S6). Four wounds were randomly covered with uncoated polyethylene, Mg100, Ag200 + Mg100, and Ag2000 + Mg100 dressings, respectively, with the dressings replaced every other day. Each wound was digitally photographed and quantified at days 0, 1, 3, 5, 7, and 9 using ImageJ software 1.48 (National Institute of Health, Bethesda, MD, USA), which was normalized to the original wound areas. The equation for percent wound closure is: Percent wound closure = (1 − wound area/original wound area) × 100%.

4.12. Immunohistochemistry and Histological Analysis

On day 9, the animals were sacrificed, and the wound skins were immersed in 10% formalin fixative for 3 days and then subjected to tissue fixation and waxing. The skin tissue was dehydrated and waxed, and then the tissue was embedded into a paraffin block by a paraffin tissue embedding machine. Typically, paraffin wax used in histology for embedding tissue samples has a melting point between 56 and 58 °C. After the tissue sections were cut from the paraffin block using a microtome (4–6 μm) [36], they were stained with hematoxylin and eosin (H&E), Ki67, or CD31 to observe the structural state of the epithelial layer and the dermis layer of the wound healing site. H&E stain was observed through a microscope (Olympus BX 41 and Olympus E-M5II), and the sliced images taken under the objective lens 10× were edited into an entire wound section by Image Composite Editor. A range of 8 to 15 H&E staining images of each group was taken under the microscope objective 4×, the wound panoramic slice was edited by the computer software, and the wound distance and the total length of the epithelialization were measured by the computer software (Image Pro Plus version 7.1). The percentage of re-epithelialization in each group is as follows:

The wound pathological sections were analyzed by immunohistochemistry using Ki67 (1:50) to analyze the changes in the number of newborn keratinocytes [37]. The keratinocytes (located in the basal layer) in the epithelialization site were observed under a microscope objective 40× as neonatal keratinocytes. In the quantitative analysis, a total of 3 staining images were taken from different locations in the wound healing area in each group. The position of the basal layer in each epithelial layer of each stain was observed, and the keratinocytes that showed a positive reaction within 200 μm were counted and quantitatively analyzed. Total blood vessel counts of wound skins were analyzed by CD31 (1:100). The number and size of blood vessels composed of vascular endothelial cells in the endothelial layer were observed under a microscope with a 20× objective. In the quantitative analysis, the stained image was measured by Image Pro Plus to measure the area occupied by each group of vascular endothelial cells and the area of the whole image, thereby converting the percentage of blood vessels in the whole image. The CD31 area was calculated by the following equation:

The authors gratefully acknowledge the financial support from KNH Enterprise Co., Ltd., Taiwan, and the Biomedicine Enterprise Acceleration Project by Central Taiwan Science Park, Taiwan.