Optical microscopy observations of the skin biopsies after hematoxylin eosin staining show the main differences between the four samples. Figure 4a
is burnt skin after surgical cleaning before silver application. Figure 4b
is healed skin after seven days of treatment. Figure 4d
is unhealed skin after seven days of treatment, and Figure 4f
shows the same area healed after 17 days of treatment.
The burnt skin prior to Ag NP dressing application shows epidermal necrosis, diffuse perivascular infiltrate and important collagen degeneration at the level of the papillary dermis, characteristic of a deep dermal burn (Figure 4a
After seven days of treatment with Acticoat™ Flex 3, the structure of the biological tissue has been completely restored in the healed area (Figure 4b
). Under staining with toluidine blue, it can be seen that the healed skin is composed of a well-stratified epidermis, complete with basal, spinous, granular and cornified layers (Figure 4c
). Macroscopic evaluation shows more over the presence of a well vascularization at the dermal–subcutaneous interface. In the unhealed skin samples (Figure 4d,e
), the tissue organization has not been re-established. The epidermis has not yet re-formed (Figure 4d
) and the dermis still has a disorganized and irregular structure (Figure 4e
). The wound was treated with Acticoat™ Flex 3 for 10 more days. When the dressing was removed, the wound appeared healed, and a new biopsy was taken to verify the re-establishment of the tissue architecture. Optical microscopy observations of the sample (Figure 4f
) confirm the re-growth of the tissue structure, and in particular, it is possible to observe the restoration of the epidermis. This result of complete patient healing by the 17th day shows that, despite the presence of Ag NPs in the tissue and inside the cells, the healing process does not seem to be impeded. Our results agree with and support the results recently published by Gravante et al.
]. They found that Acticoat™ Flex 3 was the dressing with the shortest healing times for deep partial thickness burns (16 days average; our result, 17 days). The average healing times for sodium carboxymethyl cellulose were longer than those of nanocrystalline silver (21 days), but were shorter than paraffin gauzes (26.5 days) and collagenase cream (29 days). This data confirms that the healing process is not impeded during the treatment with Ag NPs.
TEM images of the healed skin sample after seven days are shown in (Figure 5a–g
). The sample was observed from the epidermis to a point at which Ag NPs were no longer visible, which corresponded to a depth of ~3 mm. In Figure 5a
, the presence of a great number of agglomerates of nanoparticles can be seen surrounding the fibroblasts in the upper part of the dermis. No Ag NPs or aggregates were visible in epidermis. A higher magnification of the same area shows that the aggregates are located in the extracellular matrix, close to the cell membrane. In Figure 5b
, as described by Xu [47
], a slight broadening of the intercellular space can occur and is probably due to a previous inflammatory phase. In Figure 5c
, it can be seen that agglomerates of Ag NPs enter into the cells via endocytic vesicles in the form of agglomerates, as recently observed separately by Kim [48
] and Greulich [49
] in different cell lines. The individual nanoparticles that are visible were measured to have diameters of <10 nm (Figure 5d
). After the agglomerates were released into the cytoplasm (Figure 5e
), they typically had a round shape and were located close to the mitochondria (labeled M). No Ag NPs were detected inside the nucleus (labeled NM), and no fragmented nuclei were observed. Despite the presence of Ag NPs inside the cytoplasm, the nuclear membrane is intact and is round in shape. The nucleolus visible in Figure 5b
(labeled N) confirms that the chromatin is not condensed, but that it has a structure that allows transcription to proceed. In Figure 5e
, it was observed that the mitochondria are generally located in the proximity of the nuclear membrane.
It was observed that the fibroblasts in the upper part of the dermis seem to possess a particular spatial distribution of mitochondria. We speculate that these organelles have been replicated and then moved by the microtubules [50
] to surround the nucleus to protect the DNA from possible damage caused by Ag NPs. It has been already demonstrated in different cell lines that Ag NPs are capable of generating ROS inside the cells [29
] that could damage the genetic material [32
]. We hypothesize that once the Ag NPs have been released into the cytoplasm, they generate ROS. This could result in an increase in the number of mitochondria as a response to oxidative stress, as demonstrated by Lee et al.
], to try and compensate for the reduced activity, as seen in Figure 3
. We speculate that mitochondria are moved by the microtubules around the nucleus to act as a physical, as well as “chemical”, barrier to prevent ROS and Ag NPs from reaching the nuclear membrane. If the mitochondrial membrane breaks down due to the action of ROS, antioxidative enzymes, such as mitochondrial superoxide dismutase (mtSOD), catalase, glutathione peroxidase and thioredoxin peroxidase [53
], are released from the mitochondria in the cytoplasm to quench the ROS. It has been already demonstrated that Ag NPs and Ag ions cause increased levels of SOD in human [54
] and yeast [55
] cells and that in vitro
, the administration of ROS scavengers (such as SOD, catalase, mannitol and sodium selenite) can partially block the genotoxic effects of Ag NPs in human bronchial epithelial cells [54
In the lower part of the dermis (Figure 5f,g
), the Ag NP agglomerates have a different shape and location inside the fibroblast, compared to what has been found in the upper stratus of the dermis. The agglomerates appear elongated and are located very close to the nuclear membrane. The mitochondria (labeled M) are generally distant from the agglomerates, indicated with an arrow, and appear to be undamaged and healthy (Figure 5g
As Acticoat™ Flex 3 was applied to the surface of the burn, the cells in the wound bed (lower part of the dermis) were the first to receive the Ag NPs released from the dressing. These cells were exposed to Ag NP aggregates for a longer time with respect to fibroblasts of the upper part of the dermis. During this time period, the Ag NPs could have undergone chemical changes that passivated them or quenched their ROS generating ability. In light of this hypothesis, Ag NPs could remain inside the cells, without provoking toxic effects. This could explain the different spatial distribution of the Ag NP agglomerates in the fibroblasts of the upper and lower dermis, but further investigations are necessary.
In the unhealed skin, the tissue structure is highly disorganized and the epidermis had not reformed. The TEM images in Figure 6a,b
show vesicles containing electron-dense matter, but it was not possible to verify if this material contained Ag. Unlike in the healed skin, in the unhealed skin sample, it was not possible to identify nanoparticles. It is possible that the electron-dense matter is composed of Ag salts or Ag bound to proteins via thiol or selenide groups and then precipitated [56
The SEM analyses showed the presence of particles in the unhealed skin samples. As shown in Figure 7a
, such particles have a diameter <10 μm. The EDS spectrum (Figure 7b
) shows that the particles are composed of virtually pure Ag metal. In the healed skin sample, it was not possible to identify Ag in any form. This was probably due to the low sensitivity of the technique and the absence of Ag microparticles on the surface of the sample. However, Ag microparticles were identified by SEM-EDS in the skin sample collected after 17 days of treatment, when the previously unhealed part of the wound had returned to a healthy condition.
2.2.2. Ag Release and Depth Profiles
The depth profiles of Ag concentrations in the healed and unhealed biopsies taken after seven days of treatment are shown in Figure 8
. This shows that the level of Ag in the healed sample decreases rapidly from 50.8 ± 0.8 ng mg−1
to 6.0 ± 0.2 ng mg−1
between the first and second slices. This trend continues between the third and deepest slices, to reach a range between 0.04 and 0.1 ng mg−1
. The superficial concentration of Ag in the unhealed sample after seven days of treatment was lower (37.5 ± 0.6 ng mg−1
), but the drop in the concentration profile was more gradual, as the second and third slices still had high concentrations of the metal (29.8 ± 0.5 and 9.6 ± 0.2 ng mg−1
, respectively) compared to the healed sample. The cumulative amount of Ag in the unhealed tissue was 223 ng, which is slightly higher than the value in the healed tissue of 171 ng. The sample that was collected after an additional 10 days of treatment had a Ag profile (not shown) similar to that of the healed tissue after seven-days of treatment, but with approximately double the concentrations: 110 ± 2, 31.0 ± 0.7 and 13.2 ± 0.1 ng mg−1
in the first three slices, respectively.
The comparison between the Ag profiles and the corresponding OM images of the biopsies is also shown in Figure 8
. This allows us to correlate the penetration of the metal into the tissue with its structural organization. The healed skin sample has the organized structure of a well-reconstructed tissue. The Ag NP aggregates released from the dressing were not able to penetrate deeply into the tissue, so they remained immobilized, mainly in the upper stratum of dermis, where they were covered by newly grown keratinocytes. The unhealed skin, instead, has a messy and disorderly structure and the normal connective tissue organization has not yet been re-established. Fibroblasts are low in number and the connective tissue poorly formed. This means that the Ag NPs released by Acticoat™ Flex 3 are able to pass through the connective tissue to reach the lower strata of the dermis. As observed in the quantification of Ag in the in vitro
MTT and iDMSO fractions (see above, Section 2.1.3), the Ag concentration in the skin samples increases linearly with the number of dressings applied on the wound. The same is seen in vivo
; a skin sample treated with two pieces of Acticoat™ Flex 3 has double the Ag concentration of skin samples after only one application. The slightly higher cumulative Ag concentration measured in the unhealed skin sample after seven days of treatment can be explained by considering that the presence of body fluids and exudates can increase Ag release from dressings [56
2.2.3. Clinical Observations
Our results are reinforced by the clinical observations that were collected between January 2011 to September 2012 in the Plastic Reconstructive Surgery Division. In this time interval, 98 patients were treated with Acticoat™ Flex 3 for coverage of burn wounds. A total of 102 applications, corresponding to 58,400 cm2
of dressing containing a total of nearly 48 g of high purity silver, were used. The annual use in 2011, which was the year of introduction of Acticoat™ Flex 3, was around 33,000 cm2
. Data from average monthly use in 2012 are comparable to 2011, and we foresee a stable annual use. The patients were 63 males and 45 females, with an average age of 49 years. Ninety-five patients received a single silver dressing application; three patients, two. Burning causes comprised of fire (55 patients), hot liquids (23, of which three were boiling oil) and others (six contact burns, four chemical burns). The average total body surface area (TBSA) burn percent was 23.6% ± 17.1%. Out of 98 cases, 100% reached re-epithelialization. The average amount of dressing used per patient was 602.4 ± 163.5 cm2
(minimum 200 cm2
; maximum 1100 cm2
). Re-epithelialization was spontaneous in 80 cases, and lesions were recorded as healed within 32 ± 18 days from the last silver dressing application. None of the patients in this study complained of pain or had any symptoms or signs suggesting argyria. None of the 98 patients treated with Acticoat™ Flex 3 between January 2011 to September 2012 in the Plastic Reconstructive Surgery Division was recorded to have staining of skin due to silver accumulation. Eighteen patients reached healing after autologous skin grafting. Autologous grafting was performed at 23 ± 9 days after the last silver dressing application. Healing times recorded for burn wounds are consistent with the data previously reported by other authors [34
]. Khundkar et al.
] demonstrated that Ag NP-based dressings are effective in reducing the time for re-epithelialization and the requirement for grafting in comparison to other treatment widely used in burn centers, such as silver sulfadiazine and 0.01% neomycin and polymyxin solution. Strand et al.
] compared the length of stay for hospitalized patients treated with Acticoat™ Flex 3 with those treated with Mepitel, a flexible polyamide net coated with soft silicone. The authors found the mean in-patient stay was 12.5 days when treated with Mepitel, whilst the mean stay was 4.5 days when the patients were treated with nanocrystalline silver dressing. In our study, the percentage of patients (18%) that required skin grafting is limited and comparable to the results (8%) obtained by Strand et al.
]. Literature data, clinical observations collected by the Plastic Reconstructive Surgery Division of Padua and the results obtained in this work on a limited number of samples support each other in confirming the safety of Acticoat™ Flex 3.