Transparent photothermal hydrogels for wound visualization and accelerated healing

Abstract

Utilizing photothermal hydrogels as a wound dressing is a promising strategy to accelerate wound healing. Usually, a photothermal hydrogel has a strong light-absorbing capability, and hence its transparency can be largely sacrificed, which is unbeneficial for the visual monitoring of wound states. It remains challenging to balance the trade-off between the photothermal conversion and wound visualization for the photothermal hydrogel dressing. Herein, a composite photothermal hydrogel film with high transparency is presented for the visual monitor of the wound, which is constructed by incorporating Cs_xWO₃ nanorods into the networks of polyacrylamide hydrogels. The composite photothermal hydrogel film exhibits high light absorption in the near-infrared region and high transmittance in the visible light region. Under 980 nm laser irradiation, the composite hydrogel can be heated up to 45 °C. In vivo animal experiment on mouse skin wound model shows that the composite hydrogel film can locally heat the skin wound to accelerate healing while maintaining more than 70% transparency to realize real-time observation of the wound. This study provides the first attempt to solve the problem of opacity in photothermal hydrogel dressings, promoting the possibility of its clinical applications.

Graphic abstract

1. Introduction

Skin wounds are commonly found in accidental injuries or surgical procedures and their treatment plays a critical role in improving the patients’ life quality, and especially, acceleration of wound healing is highly desired [1], [2], [3]. Recently, hydrogels are attracting increasing attention as a wound dressing by virtues of suitable softness, humid environment, and excellent permeability, which is similar to the extracellular matrix (ECM) and can provide a suitable environment for wound healing [4], [5], [6]. In addition to these inherent superiorities, hydrogel dressings are also convenient to compound with a variety of treatment methods (e.g., medication, antibiotics, physical stimulation, etc.) to deal with complex wound conditions [7], [8], [9], [10], [11]. Among these functionalized hydrogels, photothermal hydrogel dressings that can produce heat under light have attracted widespread attention [12], [13], [14]. Under light irradiation, the generated warming of the wound with non-invasive, high spatial and temporal precision can effectively increase the blood circulation and oxygen tension in the microenvironment, thus benefiting the formation of new blood vessels and collagen deposition and accelerating healing [15], [16], [17], [18].

In general, photothermal hydrogels can be readily obtained by incorporating photothermal agents (e.g., carbon-based materials, metal materials, organic dyes, and semiconductor materials) into hydrogels [19], [20], [21], [22], [23], [24]. For example, Liu et al. used antimonene nanosheets to construct photothermal hydrogel dressing for antibacterial and accelerated healing [25]. Recently, our group has prepared photothermal hydrogel dressings by immobilizing reduced graphene oxide and carbon nanoparticles (NPs) into polyacrylamide (PAAm) and Pluronic F127-based hydrogels, which can be applied for wound treatment through photothermal warming [26, 27]. Despite the extraordinary performances of photothermal hydrogels for accelerating wound treatments, a major concern of their poor transparency poses great inconveniences that hinder their clinical applications because of the high absorption of incident light by the photothermal components [19], [20], [21], [22], [23], [24], [25], [26], [27]. Actually, due to the high incidence of complications (e.g., infection, maceration, and bleeding during wound healing), it is essential to real-time observe the wounds covered with transparent dressings to avoid serious consequences from untimely treatment [28], [29], [30], [31]. However, the simultaneous acquisition of high transparency and photothermal capability for photothermal hydrogel dressings to realize visualization as well as photothermal therapy, which are mutually exclusive properties, is a long-standing challenge and largely unexplored.

Thanks to the photothermal agent of Cs_xWO₃ nanorods (NRs), which have excellent absorption in the near-infrared (NIR) region and high transmittance in the visible light region, hydrogel dressings with Cs_xWO₃ NRs as the photothermal agent might realize the visualization of the wound during photothermal treatment [32], [33], [34], [35], [36], [37], [38]. In this article, we, for the first time, construct a composite hydrogel by immobilizing Cs_xWO₃ NRs into PAAm hydrogel and employ them as a wound dressing to realize the visualization of the wound as well as the photothermal treatment to accelerate the healing. Under NIR light irradiation, the composite hydrogel can effectively absorb light and convert it into heat, warming up the wound locally. Furthermore, it still maintains excellent transparency (higher than 70%) after multiple heating cycles, enabling real-time observation of the wound healing state. In vivo animal experiments on mice skin wound models show that under NIR light irradiation, the hydrogel film can remarkably accelerate wound healing and can allow for effective and direct observation for identifying various wound complications. This study is conducive to promoting the application of photothermal hydrogel dressings in the clinical treatment of wounds.

2. Material and methods

2.1. Materials

Tungsten hexachloride (WCl₆, 99.9%), cesium hydroxide monohydrate (CsOH∙H₂O, 95%), acrylamide (AM, purity > 99%), N, N’-Methylenebis-(acrylamide) (BIS, purity > 99%), and ammonium persulfate (APS, 99.99%) were all purchased from Aladdin. All chemicals were used as received without further purification. Deionized (DI) water was used in all experiments.

2.2. Synthesis of Cs_xWO₃ NRs

Cs_xWO₃ NRs were prepared by the solvothermal synthesis route [39]. In a typical experiment, WCl₆ (0.25 g) and CsOH·H₂O (0.08 g) were weighed and then dissolved in ethanol (40 mL), followed by the addition of 10 mL of acetic acid. The resulting solution was stirred for several minutes and subsequently transferred into a 100 mL Teflon-lined stainless-steel autoclave, and heated to 180 ˚C for 24 h. The product was collected by centrifugation, washed three times with water, and finally dispersed in DI water.

2.3. Preparation of the composite hydrogels

The PAAm/Cs_xWO₃ NRs composite hydrogels were prepared by thermally initiated crosslinking of AAm and BIS in the presence of the Cs_xWO₃ NRs. Typically, 108 mg AM, 1.08 mg BIS, 10.8 mg APS were added into 300 μL water to obtain the hydrogel precursor. Subsequently, a certain amount of Cs_xWO₃ NR suspension (concentration: 30 mg mL⁻¹) was introduced in the precursor, with its mass fraction being 0.0, 0.5, 1.0, and 2.0 mg mL⁻¹, respectively. Then, the mixture was sonicated for 2 min and was transferred into the glass splint mold with a spacing of 1.0 mm. The crosslinking was conducted at 80 ˚C for 5 min. After washing with a copious amount of water, transparent photothermal hydrogel films were obtained.

2.4. Characterizations

The morphology of the materials was characterized by using a scanning electron microscope (SEM, Hitachi SU4800) and transmission electron microscope (TEM, Titan G260-300). Dynamic light scattering (DLS, Zetasizer Nano-ZS90 Malvern) measurements were taken with a He-Ne laser (λ₀ = 633 nm) at 25 ˚C. X-ray diffraction (XRD, RKC, Smartlab-SE) analysis was carried out using Cu Kα radiation (1.5418 Å) with a scanning rate of 5° min⁻¹ from 10° to 80°. X-ray photoelectron spectroscopy (XPS) analysis was performed on a Thermo Scientific K-Alpha⁺ (ThermoFisher). The UV-Vis spectrum was obtained by a Shimadzu UV-1800 spectrophotometer. The rheological measurement of the hydrogel film was carried out on the MCR 302 rheometer (Anton Paar, Austria) at room temperature with a frequency ranging from 1 to 10 Hz at 0.5% strain amplitude (linear region).

2.5. Measurement of photothermal effect

A volume of 1.0 mL of Cs_xWO₃ NR suspension (0.0, 0.5, 1.0, and 2.0 mg mL⁻¹) contained in a 1.5 mL centrifugal tube was irradiated with a NIR laser (980 nm, 2.5 W cm⁻² Changchun New Industries Optoelectronics Tech Co., Ltd. MDL-III-2W). The temperature and thermal images were recorded every minute with a MAG32 infrared camera (Shanghai Mega Electronics Co., Ltd.). The photothermal conversion efficiency (PTCE) was calculated according to a similar method described in Yang's report [40]. In addition, the photothermal stability of Cs_xWO₃ NRs was investigated by irradiating Cs_xWO₃ NRs dispersion (2.0 mg mL⁻¹) with a 980 nm laser (2.5 W cm⁻¹) for 5 heating-cooling cycles.

2.6. Determination of the swelling ratio of hydrogel film

The swelling ratio of the composite hydrogels was measured with a reported gravimetric method [41]. To be specific, the composite hydrogel films were freeze-dried and their weights were measured by an electronic balance (W_d). Subsequently, the dried samples were immersed in phosphate-buffered saline (PBS) solution and kept for 24 h at 37 ˚C to sufficiently absorb water. After being taken out and wiping off the surface water, the hydrogels were weighted (W_w). The swelling ratio was calculated using the following Eq. 1:

$$\text{Swelling}\text{ratio}=\frac{W_w-W_d}{W_d}$$ (1)

2.7. Determination of the porosity of hydrogel film

The porosity (P_r) of the PAAm/Cs_xWO₃ NRs hydrogel film was calculated by the following Eqs. 2-4:

$${\rho }_g=\frac{W_d}{V-\left(W_w-W_d\right)/{\rho }_w}$$ (2)

$$V_p=\frac{\mathrm{V}-{\mathrm{W}}_d/{\rho }_g}{W_d}$$ (3)

$$P_r=\frac{{\mathrm{V}}_p}{V_p+1/{\rho }_g}$$ (4)

where W_w is the mass of wet hydrogel film, W_d is the mass of dry hydrogel film, and the two data were obtained by weighing the swelling hydrogel and the fully dried hydrogel. ρ_g is the density of the hydrogel backbone, ρ_w is the water density, and V_p is the volume of the pore, and these data are derived from the formula.

2.8. Animals and wound healing assay

Twenty-four female BALB/c mice aged 8-10 weeks (Changsheng Biological Technology Co., Ltd, Liaoning, China) were used for in vivo experiments. All of the animal studies were conducted according to The Guide for the Care and Use of Laboratory Animals of Huazhong University of Science and Technology, approved by the Institutional Animal Care and Use Committee, Tongji Medical College, Huazhong University of Science and Technology. The mice were randomized into four groups (n = 6 mice per group): the control group, the hydrogel group, the NIR group, and the hydrogel + NIR group. Before the experiment, the dorsal areas of the mice were depilated. A round skin wound with a diameter of 5 mm was created on the back of each mouse by a biopsy punch after intraperitoneal anesthesia. The wound in the control group and the NIR group was covered by Tegaderm (3M Health Care, USA, 20 × 20 mm). The wound in the hydrogel group and the hydrogel + NIR group was covered with a round hydrogel patch (diameter: 12 mm, thickness: 1 mm) then fixed with Tegaderm. The dressed wound in the NIR group and the hydrogel + NIR group was irradiated by the 980 nm laser (2.5 W cm⁻²) for 15 minutes each day for 3 days. On day 3^rd, dressings in all groups were removed, and then photographs of the wounds were taken every day until one group was completely healed. Three mice in each group were sacrificed randomly on day 6^th for wound histological examination. Wound areas were measured with ImageJ software, and the values were calculated as follows: wound area (%) = (wound area on a particular day/wound area on day 0) × 100%.

2.9. Wound histology

The mice in the four groups were euthanized on day 6^th and day 10^th. Individual wound tissues were fixed by immersion in 4% paraformaldehyde for 24 h, dehydrated using a series of graded ethanol, embedded in paraffin, then sectioned into 4 μm thick sections. The sections were stained with hematoxylin and eosin (H&E) and Masson according to the manufacturer's instructions and observed under an optical microscope (Olympus, Japan).

2.10. Immunohistochemistry of CD31 on endothelial cells

The sections were blocked with 10% goat serum in PBS for 1 h at room temperature to reduce nonspecific antibody binding. Then, the slides were incubated with rabbit anti-mouse CD31 primary antibody (Servicebio, China) overnight at 4 °C. After that, the slides were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody (Servicebio, China), and washed in PBS. The slides then reacted with DAB enzyme substrate, followed by hematoxylin counterstaining. Images were photographed by an optical microscope (Olympus, Japan). Three randomly selected areas were counted per slide to determine the number of blood vessels.

2.11. Different types of wound models

After depilation and anesthesia, a round skin wound with a diameter of 5 mm was created on the back of the BALB/c mouse. The bleeding wounds originate from wound modeling without hemostasis. The burns wounds were made by scalding the skin with hot water at 100 ˚C. The maceration wounds were caused by sealing the wound with plastic wrap for 24 h. To create the infected wound, 50 μL of S.aureus (1 × 10⁸ CFU mL⁻¹) were added to the normal wound. Purulent secretion could be seen in the wound after 2 days. Then, round PAAm/Cs_xWO₃ NRs hydrogel patches (diameters: 12 mm, thicknesses: 1 mm) were placed over different types of wounds to evaluate the visualization of hydrogels for wound changes.

2.12. In vivo biosafety studies

Body weights of the mice in the wound healing experiments were monitored every day. When the mice were sacrificed on day 10^th, their tissues (heart, liver, spleen, lung, and kidneys) were collected, washed with saline solution, and fixed with 4% paraformaldehyde solution. The H&E staining sections of different organs were observed under an optical microscope (Olympus, Japan). Blood samples collected from the medial canthus veins on day 10^th were used for blood routine test (BC-2800vet, Mindray, China), and blood biochemistry analysis of the serum was determined by a biochemical analyzer (Chemray, Rayto, China).

2.13. Cytotoxicity studies of composite hydrogel

NIH-3T3 fibroblasts were cultured in DMEM (Gibco, USA) with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin-streptomycin solution (Beyotime, China) at an initial density of 5 × 10³ cells/well in 96-well plates in an incubator (37 °C, 5% CO₂). The circular PAAm hydrogel and PAAm/Cs_xWO₃ NRs hydrogel patches (diameters: 3 mm, thicknesses: 1 mm) were immersed in sterile DMEM for 12 h beforehand to reach a steady state. The cells in plates were divided into three groups: Control, PAAm hydrogel, and composite hydrogel. The cells in the PAAm hydrogel group and the composite hydrogel group were respectively incubated with PAAm and composite hydrogel films, while the control group with no treatment. After 24 and 48 h of incubation, CCK-8 (Dojindo, Japan) assay was used to evaluate the cell viability in all groups. To visually observe the viability of NIH-3T3 cells cultured with hydrogels, cells were stained with Calcein-AM/PI Double Stain Kit (Yeason, China) and observed using a laser scanning confocal microscope (Olympus, FV1200).

2.14. Statistical analysis

All the data were expressed as mean value ± standard deviation (SD). All of the results were obtained in triplicate. Statistically significant differences between groups were analyzed by Student's t-test with one-way analysis of variance (*p < 0.05, **p < 0.01 and ***p < 0.001).

3. Results and discussion

3.1. Preparation and characterization of Cs_xWO₃ NRs

The composite hydrogel with high transparency and photothermal capability was generated based on the ingeniously selected photothermal materials of Cs_xWO₃ NRs in the PAAm hydrogel, as illustrated in Fig. 1. In general, Cs_xWO₃ with a rod-like structure can effectively improve the photothermal conversion performance [33]. Therefore, Cs_xWO₃ NRs were selected in this study, which were prepared by a modified solvothermal process (Fig. 1a) [39]. The Cs_xWO₃ NRs exhibited homogeneous morphology with a length and diameter of ∼50 nm and ∼15 nm, respectively (Fig. 1b) and (Fig. S1), as confirmed by the dynamic light scattering (DLS) determination (Fig. S2). Further characterization proved that the chemical composition and crystal structure of NRs are consistent with that of Cs_0.32WO₃ (JCPDS No.83-1334) (Figs. 1c, S3-5), proving the successful preparation of Cs_xWO₃ NRs.

Fig. 1.

Synthesis and characterization of Cs_xWO₃ NRs. (a) Schematic illustration of the synthesis processes of Cs_xWO₃ NRs by the solvothermal method. (b) TEM image of the Cs_xWO₃ NRs. (c) W_4f core-level XPS spectra of the Cs_xWO₃ NRs. d) UV-vis-NIR absorption spectra and photograph (the inset) of the Cs_xWO₃ NRs aqueous dispersion with different concentrations.

Unique light absorption characteristics of Cs_xWO₃ NRs lead to simultaneous visualization and considerable light-to-heat conversion. Clearly, the absorption spectrum of Cs_xWO₃ NRs aqueous suspension displayed that Cs_xWO₃ NRs possess negligible absorption in the visible region and high absorption in the NIR region with a peak at 980 nm (Fig. 1d). Cs_xWO₃ NRs demonstrated significant photothermal performance, where, upon the NIR light irradiation, the temperature of the Cs_xWO₃ NRs suspension increased immediately, and the highest temperature was reached after 15 min irradiation. When the concentrations of Cs_xWO₃ NRs were 0.0, 0.5, 1.0, and 2.0 mg mL⁻¹, the highest temperature reached after 15 min of NIR light irradiation was 33.3, 45.6, 47.2, and 50 ˚C, respectively (Fig. 2a, b). The photothermal conversion efficiency (PTCE) of the Cs_xWO₃ NRs was calculated to be 13.3% under the irradiation of NIR light of 980 nm (Fig. 2c, d) and at the same time, the suspension of the Cs_xWO₃ NRs exhibited high transparency in the visible region. Moreover, the Cs_xWO₃ NRs show good stability of photothermal conversion as evidenced by the unaltered heating capability in four cycles of on-off NIR irradiation (Fig. 2e). These results demonstrated that Cs_xWO₃ NRs can be a good candidate for the generation of viable photothermal hydrogel patches with high transparency.

Fig. 2.

Photothermal performance of the Cs_xWO₃ NRs. (a) Thermal images and (b) plots showing the time-coursed temperature of the aqueous suspension with different Cs_xWO₃ NRs concentrations under NIR laser irradiation. (c) Heating/cooling curve of the Cs_xWO₃ NRs aqueous suspension (2.0 mg mL⁻¹) under NIR laser irradiation. (d) The relationship between the cooling time and −ln(θ) obtained from the results in (c). (e) The temperature variation of Cs_xWO₃ NRs aqueous suspension (2.0 mg mL⁻¹) with four on-off cycles of laser irradiation. The NIR laser used in all the above experiments was a 980 nm laser with a power intensity of 2.5 W cm⁻².

3.2. Preparation and characterization of the PAAm/Cs_xWO₃ NRs composite hydrogels

To prepare composite hydrogel for wound dressing, NRs with four different contents were dispersed in the mixture of hydrogel precursor of AAm and N, N’-Methylenebis-(acrylamide) (BIS), followed by thermally initiated crosslinking. The as-prepared hydrogel has a porous structure with a pore size ranging from ∼40 to ∼120 µm in the dry state, and the elemental mapping images clearly demonstrate that the Cs_xWO₃ NRs were uniformly distributed in the hydrogel as evidenced by the well-distributed W signals (Fig. 3a). The rheological analysis showed that the prepared composite hydrogel film was dominated by obvious elastic solid behavior and elastic recovery property (Fig. 3b). Moreover, we show that the incorporation of NRs has a negligible effect on maintaining the excellent water swelling performance of the hydrogels. For example, average swelling ratios of the hydrogels were 1128%, 1122%, 1099%, and 1032% when the Cs_xWO₃ NRs contents were 0, 0.5, 1.0, and 2.0 mg mL⁻¹, respectively (Fig. 3c). Yet, the porosity of the hydrogels decreased with the incorporation of NRs, presumably because of the increase in crosslinking density. For instance, the average porosity of the composite hydrogels with different Cs_xWO₃ NRs loadings were 91.9%, 84.4%, 82.2%, and 77.4%, respectively (Fig. 3d). The above results demonstrated that the composite hydrogels possess excellent water absorption capacity and permeability, which is conducive to absorbing wound exudate and gas exchange, maintaining proper humidity of the wound site, thereby promising for accelerating wound healing.

Fig. 3.

Structural and mechanical properties of the composite hydrogels with different Cs_xWO₃ NRs loading fractions. (a) SEM and elemental mapping images of C, O, and W of the composite hydrogel with Cs_xWO₃ NRs content of 2 mg mL⁻¹. (b) The rheological properties of the hydrogel films. (c) Swelling ratio and (d) porosity of the hydrogel films.

Importantly, incorporation of the Cs_xWO₃ NRs into the composite hydrogel film endows it with both high transparency and photothermal conversion ability. We firstly evaluated the photothermal performance of the composite hydrogel film using a thermal camera when monitoring the temperature variation. Under NIR laser irradiation for 15 min (wavelength: 980 nm; intensity of 2.5 W cm⁻²), the equilibrium temperature of the hydrogel increased from 30.5, 34.8, 39.3, and to 42.5 ˚C with the increase of Cs_xWO₃ NRs content ranging from 0.0, 0.5, 1.0, and to 2.0 mg mL⁻¹, respectively (Fig. 4a, b). Clearly, neat hydrogel did not show any significant increase in temperature. Generally, effective hyperthermia can be achieved while avoiding burns, when the wound temperature is between 40 and 45 °C [42]. We thus selected the hydrogel film with a Cs_xWO₃ NRs content of 2.0 mg mL⁻¹ as the dressing for further in vivo experiments. Meanwhile, as the Cs_xWO₃ NRs contents in the composite hydrogel increased to 2 mg mL⁻¹, the background texts underlying the hydrogel were still visible, demonstrating the high transparency of the composite hydrogel (inset in Fig. 4c). The transmittance of the composite hydrogels was ∼ 96.4%, 86.2%, 81.9%, and 71.5% when the content of Cs_xWO₃ NRs was 0, 0.5, 1.0, and 2.0 mg mL⁻¹, respectively (Fig. 4c), indicating that the composite hydrogel film could be used as a transparent dressing to observe the wound due to the high transparency. To achieve long-term, real-time observation of the wound, transparency of the hydrogel film with 2.0 mg mL⁻¹ Cs_xWO₃ NRs under multiple photothermal cycles was explored. Our results show that the transparency of the hydrogel film did not change after three cycles of NIR light irradiation for 15 min (Fig. 4d), suggesting the good stability of the composite hydrogel film for potential applications in the wound treatment.

Fig. 4.

Photothermal effect and transparency of the composite hydrogel. (a) Thermal images and (b) plots showing the time-coursed temperature of the composite hydrogel films with different Cs_xWO₃ NRs contents under NIR laser irradiation. (c) UV-vis transmittance spectra and photograph (the inset) of hydrogels with different Cs_xWO₃ NRs contents. (d) UV-vis transmittance spectra of hydrogels after different heating cycles.

To prove the biocompatibility of the composite hydrogels, one of the most important prerequisites for the application [43, 44]. we investigated the cytocompatibility by culturing NIH-3T3 fibroblasts with the PAAm and the composite hydrogel film. The cell viability in both the PAAm hydrogel and composite hydrogel was almost ∼100% after 48h of culture (Fig. S6a), demonstrating the good biocompatibility of the hydrogels for biological applications. Furthermore, the results of LIVE/DEAD staining experiments confirmed this conclusion (Fig. S6b).

3.3. Assessment of wound visualization and healing

The complexity and long duration of wound healing have led to the possibility of various complications and timely observation with targeted treatment can effectively prevent the occurrence of serious consequences, which puts forward requirements for the real-time monitor of wound dressings [28], [29], [30], [31]. In fact, for clinical treatment, most of the complications of wounds have significant symptoms, thus direct naked-eyes observation is the most convenient and effective method of diagnosing wound disease. Therefore, to intuitively reflect the visual characteristics of the prepared composite photothermal hydrogel film on the wound, we created several models of common complications to evaluate the observation ability after covering the hydrogel film. As illustrated in Fig. 5, after covering the wound with a hydrogel film, several wound state models (normal, bleeding, burn, maceration, and infection) can still be clearly distinguished: (1) bloody secretions can be observed continuously in bleeding wounds compared with normal wounds; (2) the obvious purulent secretions suggested infection in the wounds; (3) skin tissue around immersion wounds turned white in maceration wound and turn red or inflammatory reaction appeared in burn wound. Clearly, these details in the wounds as observed through the transparent dressing demonstrated that the composite hydrogel can fully realize the observation of the wound site when being used as a photothermal dressing and improves the possibility of its clinical application.

Fig. 5.

Schematic diagram of the composite hydrogel film used for wound visualization to observe different wound conditions: normal, bleeding, burns, maceration, and infection.

After realizing the wound visualization, the BALB/c mouse model with circular dorsal full-thickness cutaneous defects was used to evaluate the performance of photothermal hydrogel for accelerating wound healing. Firstly, we irradiated the hydrogel film covering the wound on the back of the mouse with a NIR light to assess its in vivo photothermal performance. Upon the NIR light irradiation, the temperature at the wound site of the mice covered with the composite hydrogel film raised rapidly to 43.7 °C after 15 min irradiation (Fig. 6a, b). While as a control, a trivial rise of only 3 °C was observed in the mice without the composite hydrogel film. In addition, in vitro experiments on the pigskin show that no significant temperature increase was observed on both the skin and the neat hydrogel under the NIR light irradiation (Fig. S7). This also suggests that photothermal composite hydrogels are effective in photothermal skin heating. This result proves that the composite hydrogel film also has excellent photothermal conversion ability in vivo. Furthermore, these mice were randomly divided into four groups according to different treatment methods, denoted as control (without treatment), hydrogel (covered with composite hydrogel film), NIR (irradiation without composite hydrogel film), and hydrogel + NIR (irradiation with composite hydrogel film) treatment groups. The wound areas in the hydrogel group, NIR group, and hydrogel + NIR group were smaller than those of the control group on day 3 (Fig. 6c, d). In particular, the wounds in the hydrogel group and hydrogel + NIR group were smaller than the other two groups, and no obvious effusion was observed, indicating that the hydrogel could absorb wound exudate and is beneficial for the fast wounds healing in the early stage. As expected, the wounds treated with the hydrogels + NIR group healed fastest among all the four groups and closed on day 10^th. In quantitative terms, the wound area on day 3^rd in the hydrogel + NIR group (39.1 ± 3.3%) was significantly lower than those of the hydrogel group (72.3 ± 5.4%) and the NIR group (87.2 ± 2.7%). Similarly, the wound on day 10^th in the hydrogel + NIR group (4.4 ± 2.3%) was smaller than the hydrogel group (19.0 ± 0.7%) and the NIR group (14.1 ± 2.1%).

Fig. 6.

In vivo evaluation of the PAAm/Cs_xWO₃ NRs hydrogel on wound healing. (a) Thermal images and (b) temperature change on the mice treated with and without the composite NRs hydrogel film under NIR irradiation. (c) Quantification and (d) representative photographs of the wound area treated with different experiment groups. Error bars represent the standard deviation (n = 3, *p < 0.05 and ***p < 0.001). The scale bar in the last image applies to the others in (d).

Furthermore, to further verify the above results, the histological evaluation on wound regeneration in different phases was conducted by H&E and Masson's trichrome staining. The hydrogel group and hydrogel + NIR group showed more new epidermis (black arrows) on day 6^th, and the most collagen deposition could be seen in the hydrogel + NIR group (Fig. S8). This corresponds to the result of faster healing in the early stage of the two groups. On day 10^th, the hydrogel + NIR group formed an intact epidermal layer, while skin defects and scabs still existed in the other groups. Few inflammatory cells existed in the hydrogel + NIR group compared to the obvious inflammatory signs in the control group (Fig. 7a). Besides, granulation tissue formation plays an important role in the wound healing process. The granulation tissue (red double arrows) in the hydrogel + NIR group was nearly 250 μm thicker than that of the control group and hydrogel group (Fig. 7c). Masson staining in Fig. 7b shows hair follicles (blue circle) and well-arranged collagen fibers in the hydrogel + NIR group, and the quantitative analysis of the collagen area proportion confirmed more collagen deposition in the hydrogel + NIR group (18.2 ± 1.1%) than in the NIR group (13.1 ± 0.7%), hydrogel group (8.4 ± 0.3%) and control group (7.9 ± 0.1%) (Fig. 7d). These results suggest that the hydrogel with NIR irradiation can promote re-epithelization, granulation tissue formation, and collagen deposition.

Fig. 7.

Histological analysis of the tissues at the wound sites in different groups. (a) H&E and (b) Masson's staining images of the wound site tissues on day 10^th. Statistics of (c) granulation tissue thickness and (d) collagen proportion in different experimental groups on day 10^th. Error bars represent the standard deviation (n = 3, *p < 0.05 and ***p < 0.001).

Angiogenesis is critical for tissue regeneration, and neovascularization in the inflammatory stage and proliferation stage affects the wound repair process to a large extent [45], [46], [47]. To verify new blood vessel formation in different treatment groups, immunohistochemical staining of CD31, a marker of vascular endothelial cells, was conducted on day 6^th and day 10^th (Fig. 8a). Significantly more new vessels formed in the hydrogel + NIR group (101.3 ± 3.5%) than in the NIR group (68.0 ± 6.4%), hydrogel group (34.7 ± 1.5%), and control group (22.0 ± 3.1%) on day 6^th (Fig. 8b), suggesting the contribution of hydrogel and heat stimulation to neovascularization in the early healing stage. However, on day 10^th, the number of blood vessels became the least in the hydrogel + NIR group (Fig. 8c), indicating more vascular tissues had been replaced by fibroblasts and extracellular matrix (ECM), which was closer to normal skin structure compared to the other groups (Fig. S9). These results suggest that the composite hydrogel enhances new blood vessel formation in the early stage and accelerates the biological process of wound healing. In short, the photothermal composite NRs hydrogel can effectively promote wound healing under NIR irradiation while fully observing the wound site.

Fig. 8.

Promoted angiogenesis by composite photothermal hydrogel films during the wound healing process. (a) CD31 immunohistochemical staining of different experimental groups on day 6^th and day 10^th. (b) Statistics of the number of new blood vessels shown in immunohistochemical staining. (c) Statistics of the diameter of newly formed blood vessels in each group on day 6^th. Error bars represent the standard deviation (n = 3, *p < 0.05 and ^⁎⁎⁎p < 0.001).

To evaluate the in vivo biocompatibility of the composite hydrogel, weights of all the mice were recorded every day, and no significant difference existed in the four groups (Fig. S10). Liver and kidney function tests and hematology analysis from blood samples collected on day 10^th were within normal limits for different treatment groups (Fig. S11). The H&E staining of the heart, liver, spleen, lung, and kidney revealed no visible damage in the major organs of the mice (Fig. S12). These findings suggest no acute toxicity for the composite hydrogel as a wound dressing.

4. Conclusion

In summary, by taking advantage of the unique optical properties of the Cs_xWO₃ NPs and their recombination with acrylamide hydrogel, we successfully constructed a therapeutic platform for wound visualization and accelerated healing. Distinct from the reported photothermal hydrogels, the prepared hydrogels have excellent photothermal conversion ability and high transparency. The wound and its complications can be easily observed by the naked eyes through the hydrogel films, and the transmittance does not decrease after the heating-cooling cycles. The results of in vivo animal experiments show that under NIR irradiation, the hydrogel films can effectively accelerate wound healing. In general, our findings provide an illuminating insight for solving the contradiction between the photothermal conversion effect and low transparency, demonstrating potential applications of photothermal hydrogel dressings in clinical treatment considering the facile preparation and easy availability of the materials.

Declaration of Competing Interest

The authors declare that they have no conflicts of interests in this work.

Biographies

Gie Xie is a Ph.D. candidate under the supervision of Prof. Lianbin Zhang and Jintao Zhu, at the School of Chemistry and Chemical Engineering, HUST. His research interests focus on hydrogels for wound healing.

Lianbin Zhang received his B.Sc. degree in polymer material and engineering in 2005 and Ph.D. degree in polymer chemistry and physics in 2010, both from Jilin University, China. Then, he worked as a postdoctoral researcher and research scientist from 2010 to 2016 in Hongkong University of Science and Technology and King Abdullah University of Science and Technology, respectively. In Oct. 2016, Dr. Zhang joined the School of Chemistry and Chemical Engineering at HUST as a full professor. His research interests focus on responsive photonic materials and wound healing materials.