Enhancing pressure ulcer healing and tissue regeneration by using N-acetyl-cysteine loaded carboxymethyl cellulose/gelatin/sodium alginate hydrogel

Abstract

Prolonged pressure on the skin can result in pressure ulcers, which may lead to serious complications, such as infection and tissue damage. In this study, we evaluated the effect of a carboxymethyl cellulose/gelatin/sodium alginate (CMC/Gel/Alg) hydrogel containing N-acetyl-cysteine (NAC) on the healing of pressure ulcers. Pressure ulcers were induced by applying a magnet to the dorsum of rat skin. The wounds were then treated with sterile gauze, ChitoHeal Gel^®, and CMC/Gel/Alg hydrogel dressings with or without NAC for the other groups. We evaluated the morphology, weight loss, swelling, rheology, blood compatibility, cytocompatibility, antioxidant capacity, and wound scratch of the prepared hydrogel. MTT assay revealed that the optimum concentration of NAC was 5 mg/ml, which induced higher cell proliferation and viability. Results of the histopathological evaluation showed increased wound closure, and complete re-epithelialization in the hydrogel-containing NAC group compared to the other groups. The CMC/Gel/Alg/5 mg/ml NAC hydrogel dressing showed 84% wound closure at 14 days after treatment. Immunohistochemical results showed a decrease in the level of TNF-α on day 14 compared day 7. Results of the qPCR assay revealed that NAC hydrogel increased the expression of Collagen type I and TGF-β1 and decreased MMP2 and MMP9 mRNA on the 14th day. The results suggest that the CMC/Gel/Alg/5 mg/ml NAC hydrogel with antioxidant properties is an appropriate dressing for wound healing.

Introduction

The skin is considered part of the integumentary system, which has various functions, including a protective barrier and temperature regulation. Any disruption or break in the skin is defined as a wound. Some causes of skin wounds include surgery, burns, diabetes, and trauma [1]. Pressure ulcers are localized and chronic wounds in an area of skin or soft tissue that undergo ischemia under the influence of long-term pressure in combination with friction and shear stress, which can result in cell death and necrosis. Depending on the amount, duration, and location of pressure, different degrees of pressure ulcers are created [2]. The re-establishment of blood flow in ischemic tissues is defined as ischemic reperfusion injury. Ischemic reperfusion injury in pressure ulcers contributes to inflammation, thrombosis, tissue necrosis, and the release of harmful reactive oxygen species (ROS) [3]. Excessive ROS levels can induce programmed cell death, leading to elevated oxidative stress. The wound healing process can be delayed by many factors such as inflammation, infection, poor angiogenesis, and oxidative stress [4]. N-acetyl-cysteine (NAC) acts as an antioxidant and anti-inflammatory agent that can clear free radicals and promotes angiogenesis [5]. NAC is a precursor of glutathione production, which protects cellular components against ROS damage. NAC also promotes MMP-1 mRNA expression via the PKC/Stat3 signaling pathway and facilitates the maturation of the epidermis [4, 6]. Using tissue engineering approaches with common treatments to control and decrease pressure ulcers can help these wounds to heal faster. Tissue engineering scaffolds can be employed as carriers for different substances for tissue regeneration [7]. Many types of wound dressings such as gauze, transparent films, hydrocolloids, nanofibers, and hydrogels are used for wound healing [8]. Hydrogels are composed of hydrophilic polymer chains and are widely used for wound healing. These gels can be synthesized from natural or synthetic polymers, as well as hybrid combinations of the two [9]. Sodium alginate (Alg) is a natural polysaccharide polymer obtained from brown seaweed, which is readily available, non-toxic, and widely used for hydrogel fabrication. Alg is biocompatible and biodegradable, and can reduce bacterial infections [10]. Gelatin (Gel), a natural protein-based polymer synthesized from collagen offers several advantages as a wound dressing material. It is biocompatible, biodegradable, non-immunogenic, and promotes cell migration, adhesion, and proliferation [11]. Carboxymethyl cellulose (CMC) is a polysaccharide created from chemically modified cellulose. Carboxymethyl cellulose (CMC) is a polysaccharide that is produced from chemically modified cellulose. The use of CMC as a wound dressing helps absorb exudates, promote angiogenesis, and aid in autolytic debridement [12]. In the current study, the method of preparing scaffolds and wound healing type were highly different from those used in previous research. In this regard, multiple scaffolds based on NAC have been prepared in the form of electrospun co-lyophilization, incorporation of pH-sensitive biopolymers, oral administration, and injection. Additionally, NAC has been investigated in various types of wound healing, including corneal, full-thickness diabetic wounds, and burns. Hence, no similar study has been conducted to evaluate the effect of NAC-incorporated hydrogels on the pressure ulcer healing process [4, 13, 14]. In this study, we combined Carboxymethyl cellulose/gelatin/sodium alginate hydrogel with N-acetyl-cysteine (NAC) to accelerate the healing process of the pressure ulcer.

Materials and methods

Materials

The materials were purchased from Sigma-Aldrich (USA). NAC from BioBasic (Canada), total antioxidant capacity assay kit from Zell Bio (Germany), total RNA Extraction kit, and easy cDNA synthesis kits were obtained from the Parstous (Iran). ChitoHeal Gel^® was obtained from Chitotech (Iran). 3T3 cell line and adult Wistar rats were provided by Pasteur Institute (Iran).

Hydrogel fabrication

CMC (4% w/v), Gel (3% w/v), and Alg (6% w/v) were dissolved in autoclaved deionized water and then gently stirred to dissolve completely. Then To prepare the final hydrogel, CMC, Gel, and Alg solutions were mixed in a ratio of 8:0.5:0.5 (v/v). Next, NAC (0.1, 0.5, 5, and 10 mg/ml) was added to the CMC/Gel/Alg solution. The resulting mixture was mixed thoroughly to create a homogenous solution. CaCl₂ 75 mM was used to cross-link the final hydrogel. To prepare porous hydrogels, the final hydrogels were frozen for 24 h at −80 °C and then the hydrogel was exposed to lyophilization via a freeze drier (Zirbus, Germany) for 72 h.

Hydrogel characterization

Morphological properties

The hydrogels were freeze-dried to observe surface and pore morphology, and pore size distribution. The resulting dried scaffolds were cut into 7 mm diameter pieces, sputter-coated with gold for 250 s, and then analyzed using a scanning electron microscope (Zeiss, LS15).

Fourier transform infrared spectroscopy (FTIR)

The chemical stability of NAC and the homogeneity of the mixture of CMC, Gel, Alg, and NAC were investigated by FTIR. To assess the potential interaction between the constituents of the CMC/Gel/Alg hydrogel and the effect of adding NAC, FTIR analysis was done on the freeze-dried CMC/Gel/Alg and CMC/Gel/Alg/NAC hydrogels. Finally, FTIR spectra were collected using FTIR spectrometers (PerkinElmer, USA) in the range of 500–4400 cm⁻¹.

Weight loss and swelling analysis

Their mass loss was used to determine the degradation rate of fabricated hydrogels. Briefly, the dried hydrogel disks were weighted (W0) and each dried disk was immersed in equal-weight samples of PBS at 37 °C. On defined hours (4, 8, and 12), the hydrogels were removed from PBS and dried, and the weight loss was determined (W1). The percentage of weight loss was determined using Eq. 1:

$$Weightloss\left(\%\right)=\frac{W0-W1}{W0}\times 100$$ 1

The swelling behavior study was performed by immersing the freeze-dried hydrogels in PBS (pH 7.4) at 37 °C. Dry samples were weighed (W0) and immersed in PBS for 1, 2, 4, 6, 8, 10, 12, and 14 h. After removing the excess PBS, the swollen samples were weighed (W1). The swelling percentage was calculated using the following Equation:

$$Swellingratio\left(\%\right)=\frac{W1-W0}{W0}\times 100$$ 2

Rheological characterization

The rheological properties of the hydrogels were conducted by using Anton Paar Rheometer (MCR-302, Austria) fitted with parallel plate geometry (25 mm diameter). The shear viscosity of the hydrogels was measured in the shear rate range from 0.01–200 1/s at 25 °C.

Release study

The release of NAC from the hydrogels was specified by UV–visible spectroscopy. The CMC/Gel/Alg hydrogel with 5 mg/ml NAC was incubated in simulated body fluid (SBF) on the shaker incubator at 37 °C. On defined hours (3, 6, 8, 10, 12 and 16 h), the supernatants were removed and replaced by a new SBF solution. To quantify the amount of released NAC, the absorption of supernatants was recorded at 412 nm. Equation 3 was used to determine the NAC amount and release:

$$Drugrelease\left(\%\right)=\frac{amountofreleasedN-acetyl-cysteineinthesupernatant}{amountofloadedN-acetyl-cysteineinhydrogel}\times 100$$ 3

Blood compatibility

The percentage of blood hemolysis caused by hydrogels was evaluated with the blood compatibility test. Therefore, 200 μl of diluted blood (2 ml of fresh anticoagulated human blood in combination with 2.5 ml of normal saline) was added to each sample and incubated at 37 °C for 60 min. Next, the combinations were centrifuged at 1500 rpm for 10 min. To evaluate the absorbance of each sample at 545 nm, the supernatant was added to a 96-well plate and the absorbance of each well was measured. 200 μl diluted blood in 10 ml of normal saline was considered a negative control sample. The positive control sample was prepared by adding 200 μl diluted blood in 10 ml of deionized water. The hemolysis percentage was evaluated via Eq. 4:

$$Hemolysis(\%)=\frac{sampleabsorbance-negativecontro{l}^{\prime }sabsorbance}{positivecontro{l}^{\prime }sabsorbance-negativecontro{l}^{\prime }sabsorbance}\times 100$$ 4

Cell viability study

The MTT assay was selected to evaluate the cytotoxic effects of fabricated hydrogels against 3T3 fibroblast cell line indirectly. For hydrogel extract preparation, the hydrogel samples were immersed in 10 ml of DMEM containing 10% (v/v) FBS and 1% v/v Pen-Strep and incubated at 37 °C. At 24 and 72 h after the incubation, the media was taken out and collected under sterile conditions. The 3T3 cells were seeded in a 96-well culture plate and cultured in DMEM with 10% (v/v) FBS and 100 μg/ml of Pen-Strep in an incubator at 37 °C with 5% CO₂. After 24 h incubation, the culture medium was replaced with hydrogel extracts and subsequently incubated at 37 °C and 5% CO₂ for 24 h. Then the culture media was taken out, followed by the addition of 0.1 ml of MTT solution (0.5 mg/ml) to each well. Afterward, the plate was transferred to a dark incubator for 4 h. After removing the MTT solution, 100 μl of DMSO was added to each well plate to dissolve purple formazan crystals. After 20 min, the absorption amount was measured with a plate reader spectrophotometer at a wavelength of 570 nm. Finally, the percentage of cell viability was expressed via Eq. 5:

$$Cellviability\left(\%\right)=\frac{\left(a,b,s,o,r,b,a,n,c,e,,o,f,,t,r,e,a,t,e,d,,w,e,l,l\right)}{\left(a,b,s,o,r,b,a,n,c,e,,o,f,,c,o,n,t,r,o,l,,w,e,l,l\right)}\times 100$$ 5

In vitro wound healing scratch assay

The 3T3 fibroblast cell migration in vitro was examined using the scratch assay [15]. The 3T3 fibroblast cells were seeded in 12-well plates and cultivated until confluence was reached. The media was then removed, and the cells were scratched with a sterile 200 μl pipette tip to create a scratch wound. Each well was washed twice with PBS to remove any suspended cells. The cells were incubated with or without hydrogel extracts for 24 h, and the cell migration from the leading edge to cover the wound closure space was determined and analyzed with Image J software at 0, 24, and 48 h.

Determination of antioxidant activity

The antioxidant capacity (TAC) of NAC was assayed by the TAC assay kit, which employs an oxidation–reduction colorimetric assay. The manufacturer's protocol was followed for the measurement of TAC. In this kit, ascorbic acid was considered the standard and criterion for determining the level of TAC as the antioxidant amount, and it can determine TAC with 0.1 mM sensitivity [16]. The TAC level was calculated using the standard curves provided by the manufacturer’s instructions.

In vivo wound healing study

Wound model

In the present study, pressure ulcer was adopted on the dorsum of rats' skin according to the Stadler et al., method [17]. This model was utilized to evaluate the wound-healing efficacy of the fabricated hydrogels. All experimentation was performed according to the Shahroud University of Medical Sciences ethical committee. Thirty healthy adult male Wistar rats (200–220 g) were maintained with controlled humidity and temperature for 14 days. The rats were randomly divided into four groups: negative control, CMC/Gel/Alg hydrogel, CMC/Gel/Alg/ NAC hydrogel, and ChitoHeal Gel^®. They were then anesthetized through an intraperitoneal injection of 100 mg/kg of Ketamine and 10 mg/kg of Xylazine. The hairs of the dorsum were removed, and the skin was cleaned with 70% alcohol. Afterward, the dorsum skin was gently pulled up and loaded between two permanent magnets (5 mm in thickness and 10 mm in diameter) with a 4000 G magnetic force. The magnet was applied for 12 h with compression, and 12 h recovery (no loading pressure). In each animal, this pressure was applied for 4 days to nearly the same areas (Fig. 1). Then, the wounds were treated with sterile gauze for the control group and hydrogel wound dressings for the other groups. Finally, at 3, 7, and 14 days after treatment, the rate of wound closure and reduction of the wound size was recorded and the images were analyzed using an image analysis program (Digimizer, Belgium). Then, the calculation of wound closure was performed via Eq. 6:

$$Woundclosure\left(\%\right)=1-\frac{woundarea}{initialarea}\times 100$$ 6

Fig. 1.

The pressure ulcer induced in the rat model with the magnet

Histopathological study

Animals of all groups were sacrificed at 7 and 14 days after treatment, and the dorsum skin tissues were harvested. Then the skin tissues were randomly selected for RNA extraction, qPCR, and histopathological study. For the histopathological study, the skin tissue was immediately fixed in 10% neutral buffered formalin. The samples were processed, paraffin-embedded, and then sections with 5 μm thickness of the fixed tissue were performed. The sections were stained with Hematoxylin–eosin (H&E) and Masson's trichrome (MT) stains. The expression of tumor necrosis factor-α (TNF-α), a pro-inflammatory cytokine, was examined using immunohistochemistry (IHC) [18]. A light microscope (Olympus, Japan) was used to examine the histology slides. Comparative evaluation of dermis and epidermis formation, re-epithelialization, and angiogenesis were obtained from various groups.

Quantification of Collagen I, TGF-β1, MMP2, MMP9 by quantitative real-time PCR (qPCR)

Total RNA was extracted from the tissue samples on the 7th and 14th days of post-treatment using the Parstous Total RNA Extraction kit according to the manufacturer’s instructions. The level of GAPDH expression was used to normalize the precise mRNA expression and evaluated with the 2^−ΔΔCt technique. The primers were designed by the Allele ID version 6 software (Premier Biosoft).

Statistical analysis

The results were analyzed and plotted using Graph Pad Prism 9 software and Origin Pro software. Paired student t tests and ANOVA with the Tukey post hoc test were used to evaluate statistical significance, assuming a significance level of P < 0.05.

Results

Characterization of hydrogel

The SEM images were used to observe morphology, and evaluate the pore size distribution of the CMC/Gel/Alg and CMC/Gel/Alg/5 mg/ml NAC hydrogels (Fig. 2a, b). According to the SEM image, the pore size was statistically measured by using the Image J software. The porosity of CMC/Gel/Alg and CMC/Gel/Alg/5 mg/ml NAC hydrogels were 43.49 ± 17.52 and 14.17 ± 12.30 μm, respectively. The analytical results showed samples have interconnected porous structures, which are suitable for keratinocytes and fibroblasts migration into the hydrogel and tissue engineering applications [8].

Fig. 2.

Hydrogel characterization. The SEM micrograph was used to perform morphological characterization of a CMC/Gel/Alg hydrogel and b CMC/Gel/Alg/5 mg/ml NAC hydrogel. c FTIR spectra were used to analyze the spectra of CMC/Gel/Alg and CMC/Gel/Alg/5 mg/ml NAC hydrogels

Fourier transform infrared spectroscopy

To investigate the interaction of the NAC functional group with CMC/Gel/Alg hydrogel, the FTIR spectra of CMC/Gel/Alg hydrogel and CMC/Gel/Alg/5 mg/ml NAC hydrogel were obtained (Fig. 2c). In the CMC/Gel/Alg hydrogel spectra, a characteristic band related to the O–H bond appears clearly at 3323.92 cm⁻¹. When NAC was added to the CMC/Gel/Alg hydrogel, the bond shifted to 3291.89 cm⁻¹, which is indicative of a stretching vibration of the amide (N–H) group in the NAC and the O–H bond. The weak stretching vibration bonds at 2917.21 and 2917.55 cm⁻¹ refer to the asymmetric C–H groups in the CMC and NAC. The peaks at 1413.59 and 1416.90 cm⁻¹ refer to the asymmetric stretching vibration of the COO⁻ group in Gel and Alg. The peaks at 1323.84 and 1054.18 cm⁻¹ in CMC/Gel/Alg hydrogel and 1323.27 and 1022.56 cm⁻¹ in CMC/Gel/Alg/5 mg/ml NAC hydrogel appeared to belong to the C–O and C–O–C groups, respectively.

Weight loss and swelling analysis

The weight loss percentages and swelling ratio were shown in (Fig. 3a, b). The results indicated that in the CMC/Gel/Alg/5 mg/ml NAC hydrogels group, the weight loss increased to around 86.98% after 12 h. The swelling ratio showed that the CMC/Gel/Alg/5 mg/ml NAC hydrogels had 2174.97% swelling after 1 h. Thereafter, it gradually decreased and settled at 226.96% after 12 h. The CMC/Gel/Alg composite exhibited the highest swelling after 4 h.

Fig. 3.

Hydrogel characterization. a, b The weight loss and swelling behavior of the CMC/Gel/Alg and CMC/Gel/Alg/5 mg/ml NAC hydrogels at various intervals. c The rheology of hydrogels. d Release profile of NAC from the hydrogel. Data are shown as mean ± SD, **p < 0.01, and ****p < 0.0001

Hydrogel rheology

The spreadability (viscosity) of the hydrogels was evaluated under shear stress. As shown in Fig. 3c, the CMC/Gel/Alg/5 mg/ml NAC and CMC/Gel/Alg hydrogels showed shear thinning behavior [19]. These hydrogels demonstrated a decrease in viscosity with an increase in the shear rate, which exhibited the flow properties of these hydrogels.

Release study

Figure 3d illustrates the cumulative release profile of NAC from the CMC/Alg/Gel/5 mg/ml NAC hydrogel at different times. The amounts of NAC released during the first 3 and 6 h were 10.47 ± 1.22% and 18.12 ± 1.76%, respectively. Subsequently, a sustained release of 97.55 ± 2.45% was observed over the next 16 h.

Blood compatibility

To ensure that the hydrogels with and without NAC do not damage the blood cells, the in vitro blood compatibility test was performed. According to the previous study, a hemolysis index of below 5% is considered a critical safe hemolytic [20]. Our results demonstrated that the fabricated hydrogels were blood compatible. The hemolysis index of hydrogels was significantly lower than the positive control (P < 0.0001). The results showed our hydrogels had no significant effect on blood compatibility (Fig. 4a).

Fig. 4.

a Percentage of the blood compatibility in different groups. b The viability of 3T3 fibroblast cells on the hydrogels containing various concentrations of NAC (0.1, 0.5, 5, 10 mg/ml) was shown by the MTT assay chart. In the control group, cells were treated with pure DMEM. At each time point (24 and 48 h), all groups were compared to the control group. c Microscopic images indicate cell migration in an in vitro scratch assay with 3T3 fibroblast cells at 0, 24, and 48 h post-treatment. d Percentage of cell migration in different groups. Data are shown as mean ± SD, ****p < 0.0001

Cell viability

MTT assay was used to determine 3T3 fibroblast cell line viability on CMC/Gel/Alg hydrogel with various NAC concentrations. NAC with various concentrations (0.1, 0.5, 5, 10 mg/ml) were loaded in CMC/Gel/Alg hydrogel. Cells were treated with extracts collected from the hydrogels at 24 and 72 h in sterile conditions. According to the results, the prepared hydrogels exhibit cytocompatibility on the 3T3 fibroblast cell line in a manner that is dependent on the time and dose of treatment. Also, the propagation rate and cells proliferation of 3T3 fibroblast cells on CMC/Gel/Alg with 5 mg/ml concentration of NAC hydrogels were statistically higher than other groups at 24 and 48 h post-cell seeding (P < 0.0001) (Fig. 4b). In general, the incorporation of NAC at a concentration of 5 mg/ml, into the CMC/Gel/Alg hydrogel was more suitable for cell viability and proliferation compared to other concentrations.

In vitro wound healing scratch assay

Key components of wound healing processes, which also involve other cell types and micro-environmental factors, include fibroblast activation, proliferation, and migration. To understand the wound healing capabilities of substances, cell migration, and layer regeneration can be studied by in vitro method of mechanical scratching of cell monolayer. The migration of fibroblast cells in the wound bed is a primary process that encourages prompt wound closure [15]. A scratch assay was carried out to examine the effect of hydrogel extract treatment on the migration of the fibroblast cells (Fig. 4c). Cell migration was recorded at various time points and Image J software was used to determine the wound closure distance. The percentage of wound closure at different time points and the microscopic images are shown in (Fig. 4d). The results showed that hydrogel containing 5 mg/ml NAC filled the scratch gap by 97.18% in 48 h, which is a significant increase compared to the control and CMC/Gel/Alg groups (P < 0.0001).

Determination of antioxidant activity

The total antioxidant capacity of NAC was shown in (Fig. 5a). According to the results, the mean values of antioxidant capacity of the CMC/Gel/Alg/5 mg/ml NAC hydrogel group was 291 mM, which was a significant difference compared to CMC/Gel/Alg hydrogel group (p < 0.01).

Fig. 5.

a The TAC was evaluated in the CMC/Gel/Alg and CMC/Gel/Alg/5 mg/ml NAC hydrogel groups. The wound closure results of the different groups. b Macroscopic views of all groups with different treatments. c Percentage of wound closure at 3, 7, and 14 days of post-treatment. d H&E and MT stained microscopic sections of the pressure ulcer model. Arrows show intact areas and arrowheads show injured areas in the 1 day. Values represent the mean ± SD; ns: no significant, ns: no significant, **p < 0.01, ***p < 0.001, and ****p < 0.0001

In vivo wound healing study

The macroscopic wound healing results of the different groups were shown in (Fig. 5b). The images showed that the wound site in the CMC/Gel/Alg group was infected and inflamed, and the wound was not treated. The wound closure in the CMC/Gel/Alg/5 mg/ml NAC group was significantly higher than in the other groups (P < 0.0001, Fig. 5c). The reduction of wound size in the control, CMC/Gel/Alg, and ChitoHeal Gel^® was 37.64 ± 7.11%, 44.34 ± 9.14%, 66.35 ± 6.27%, 48.56 ± 4.61%, 59.79 ± 5.05%, 75.12 ± 3.91%, and 31.2 ± 5.02%, 49.8 ± 4.07%, 71.8 ± 4.10% after 3, 7 and 14 days of post-treatment, respectively. After adding NAC, wound closure was greater than in other groups, and the percentage of wound size in CMC/Gel/Alg/5 mg/ml NAC hydrogels was 38.52 ± 7.34, 62.68 ± 6.83, 84.32 ± 4.44% after 3, 7, and 14 days after treatment, respectively.

Histopathological study

The histological sections from the defect sites as a pressure ulcer model in 1 day were shown in Fig. 5d. The prepared hydrogels were assessed for efficient wound healing by histopathological evaluation of the animal model. The histopathological results of all groups were shown in Fig. 6a, b. In the defect sites, the layers of the skin were compressed and the epidermis and papillary dermis were destroyed. Also, as a result of the applied pressure, hair follicles, and sebaceous glands have changed in shape. Results of the histopathological evaluation showed increased wound closure, and complete re-epithelialization without fibroplasia in the hydrogel with the NAC group compared to the other groups. The CMC/Gel/Alg/5 mg/ml NAC hydrogel group showed the regeneration and formation of the epidermis, dermis, sebaceous glands, hair follicles, and angiogenesis on days 14. Collagen fibers synthesis, deposition, and maturation in wounds site be observed in MT stain. In the control group, granulation formation was observed and the epidermis was not formed after 14 days. Collagen synthesis and deposition occurred in the CMC/Gel/Alg and ChitoHeal Gel^® groups during the wound treatment period, while the epidermis has not fully formed yet. Immunohistochemical labeling with TNF-α was performed to detect changes in inflammation during pressure ulcer healing. TNF-α is a pro-inflammatory marker that is involved in the early stages of the healing process [18]. The tissue sections of the group treated with CMC/Gel/Alg/5 mg/ml NAC showed lower levels of TNF-α than the CMC/Gel/Alg group on days 7 and 14 (Fig. 6c). These results are consistent with H&E and TM staining (Fig. 6a, b).

Fig. 6.

Histological sections from the defect sites. The skin wound sections were stained with a H&E and b MT staining at 14 days. Thick black arrows: epidermal layer, thick red arrows: hair follicles, arrowhead: sebaceous glands, thin red arrows: angiogenesis, and thin black arrows: crusty scab. c IHC staining an days 7 and 14. Thick arrows: inflammatory cell. (Color figure online)

Quantitative real-time PCR

In Fig. 7, gene expression analysis of TGF-β1, Collagen type I, MMP2, and MMP9 genes in the 7th and 14th days of post-treatment were shown. Results showed that in CMC/Gel/Alg/5 mg/ml NAC hydrogel group, TGF-β1, MMP9, and MMP2 mRNA expressions were significantly increased on the 7th day in comparison with other groups (P < 0.0001). Also, on the 14th day the TGF-β1, MMP9, and MMP2 mRNA expressions were decreased (P < 0.0001, P < 0.0001, and P < 0.05, respectively). The Collagen type I mRNA expression in the NAC hydrogel group significantly increased compared to the other groups (P < 0.0001).

Fig. 7.

qPCR was used to examine Collagen type I, TGF-β1, MMP2, and MMP9 gene expressions. a Collagen type I, b TGF-β1, c MMP2, and d MMP9 expressions on the 7th and 14th days of post-treatment in the different groups. Each group was compared to the control group; values represent the mean ± SD. A considerable statistical difference from the control exists. (ns: no significant, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001).

Discussion

Pressure ulcers as non-healing chronic wounds significantly affect health and quality of life. Improved management of the inflammatory phase and faster wound healing can be achieved through the use of drugs and advanced wound dressing [21]. Hydrogel-based biomaterials provide three-dimensional networks and have many advantages in healing skin wounds [22]. We investigated the effect of the hydrogel fabricated from CMC/Gel/Alg containing NAC on enhancing pressure ulcer healing in a rat model. The blood compatibility test is performed to determine any negative effects on hemolysis, thrombosis, coagulation, and platelets of blood-contacting materials [23]. We have found that the hydrogels with and without NAC are safe and not harmful to the blood cells. This wound dressing raised the mice's fibroblast proliferation and intensified absorbance values in the MTT assay. Also, CMC/Gel/Alg/5 mg/ml NAC hydrogel filled the scratch gaps by 97.18% in 48 h, which increased significantly compared to the control group. The degradation rate of hydrogels should be tailored to tissue regeneration. The degradation rate and swelling behavior of the fabricated hydrogel demonstrated the release of NAC at an appropriate rate, suggesting its suitability for wound healing. With their shear-thinning behavior, hydrogels possess the ability to adapt to the local environment, effectively fill irregular defects, and establish optimal interfacial contact with the tissue. This unique property significantly enhances the application process by minimizing discomfort and allowing for effortless spread of the gel over the injured surface [19]. The effectiveness of the CMC/Gel/Alg/5 mg/ml NAC hydrogel in skin wound healing was assessed by histopathological analysis. The NAC hydrogel dressing showed 84% of wound closure 14 days after treatment. These results are attributed to the antioxidant, anti-inflammatory, and antibacterial efficacy of NAC [24, 25]. High levels of ROS in the pressure ulcer induction of oxidative stress can lead to cell damage and a pro-inflammatory state [26]. Prolong inflammatory phase is the main factor that can prolong the wound healing process in chronic wounds [27]. Various studies have shown that NAC has antioxidant, anti-inflammatory, and anti-infection activity [24]. NAC acts as a ROS scavenger and protects cells from oxidative damage; as a result, it may help to improve wound healing and cell proliferation [28]. Previous research has indicated that NAC can impact and suppress the activity of nuclear factor kappa B (NF-κB), which results in decreased levels of inflammatory cytokines (TNF-α) [29, 30]. Our IHC results in the NAC-treated group showed a decrease in TNF-α levels on day 14, which is indicative of decreased inflammation. Reduced TNF-α levels may have a substantial impact on MMP9 expression [31]. In the previous study, an antioxidant wound dressing containing Curcumin and NAC was employed and demonstrated this wound dressing can manage the inflammatory phase and improve wound healing [25]. Also, according to Li et al. research, NAC can help in the destruction of biofilm and decrease Pseudomonas aeruginosa, which is a bacteriological cell death agent [32]. In chronic wounds, an ongoing inflammatory response results in the proteases release and a variety of pro-inflammatory cytokines, which raise the levels of MMPs (like MMP-2 and -9) and continuously break down ECM components, causing tissue disruption [33]. During the inflammation phase of wound healing, macrophages activated, and neutrophils infiltrated around the wound site. Also, TGF-β released by platelets resulted in stimulated matrix metalloproteinase (MMP) production [34]. TGF-β1 is an important multifunctional cytokine that affects the inflammatory response, migration, and proliferation of fibroblasts and ECM molecules (such as collagen), angiogenesis, and extra-cellular matrix deposition in the wound healing and proliferation phase [35]. In the wound-healing process, increasing the TGF-β1 mRNA expressions was showed in the CMC/Gel/Alg/5 mg/ml NAC hydrogel group on the 7th day compared to other groups. In accordance with the Pakyari et al. study [36], our results demonstrated that increased TGF-β1 mRNA expression on the 14th day of the experiment resulted in increased migration and proliferation of fibroblasts, and skin re-epithelialization during the proliferative phase. We observed the low levels of TGF-β1 mRNA expression in control and CMC/Gel/Alg hydrogel groups, which was explained by previous studies that found low levels of TGF-β1 to be a pattern in chronic wounds [37]. However, TGF-β1 promotes collagen synthesis and deposition by fibroblasts, which are crucial components of ECM replacement. As a result, increased collagen type 1 mRNA expression is also observed in the CMC/Gel/Alg/5 mg/ml NAC hydrogel group on the 7th and 14th days [37, 38]. MMPs play a crucial function in controlling the degradation and deposition of the ECM and are quickly expressed and upregulated by several cells, including epidermal cells, dermal cells, and fibroblasts, during the healing of wounds [33]. Based on these findings, the MMP-2 and MMP-9 mRNA expression levels in the CMC/Gel/Alg/5 mg/ml NAC hydrogel group increased on the 7th day. The MMP-2 gene expression is important for quick wound closure [39]. The expression of MMP-9 at the borders of the wound during the wound closure resulted in keratinocytes migration to the wound site [40]. Based on the in vitro results, the keratinocytes express MMP-2 and MMP-9, whereas fibroblasts only express MMP-2. This difference in expression may explain the higher levels of MMP-2 relative to MMP-9 [41]. Angiogenesis is another crucial stage in the healing of wounds. MMP-2 and MMP-9 have played a vital role in promoting wound healing [42]. According to the Jansen study, MMP-2 mRNA expression decreased over time as wounds healed, which is consistent with our results on the 14th day [43]. Additionally, TGF-b1 inhibits the production of MMPs during the remodeling phase, which increases the accumulation of collagen fibers [36]. Another study has demonstrated that hypoxia in pressure ulcers causes’ keratinocyte migration via increasing MMP-9 mRNA expression [44]. A decrease in the mRNA expressions of MMP-2 and MMP-9 on the 14th day can be attributed to the antioxidant activity of NAC. Previously demonstrated NAC reduced the MMP-2 and MMP-9 mRNA expression during corneal healing [45, 46]. NAC inhibited the MMP-2 and MMP-9 mRNA expressions in the rats exposed to Cadmium [47].

Conclusions

Hydrogels are one of the best wound dressings that can provide the appropriate environment for wound healing. The current study evaluated the effect of CMC/Gel/Alg hydrogels containing NAC on skin wound healing in a rat model. The NAC-loaded hydrogel resulted in re-epithelialization, increased wound closure, and cell proliferation significantly more than control and CMC/Gel/Alg hydrogel without NAC groups. This study suggested that the CMC/Gel/Alg/5 mg/ml NAC hydrogel is an appropriate dressing for wound healing.

Author contributions

All authors contributed to the study conception and design. All authors read and approved the final manuscript.

Funding

This study was supported by the Shahroud University of Medical Sciences (grant No. 14010016).

Declarations

Conflict of interest

The authors have no relevant financial or non-financial interests to disclose.

Ethics approval

Animal experiments were approved by the Shahroud University of medical sciences ethical committee (ethical code: IR. SHMU. REC.1401.079).