CMC-Na | Dna Repair Signal

Preparation of self-regulating/anti-adhesive hydrogels and their ability to promote healing in burn wounds

Min Liang, Zhongmin Chen, Fuping Wang, Lan Liu, Runan Wei, Mei Zhang

Abstract:

Few burn dressings can self-regulate the optimal humidity levels that are required for wound healing, while also providing good anti-adhesive properties to prevent damage that can occur when wound dressings are changed. Consequently, a water-soluble carboxymethylcellulose sodium/ sodium alginate/chitosan (CMC-Na/SA/CS) composite hydrogel has been developed as a potential burn wound dressing, with orthogonal testing revealing an optimal ratio of CMC-Na, SA, and CS as 2, 3, and 1 wt % for hydrogel preparation, respectively. The resultant hydrogel has been formulated into composite wound dressings that were then used for the treatment of deep second degree burn wounds in Sprague–Dawley (SD) rats. Analysis of the physical properties of this dressing revealed that it exhibits good water vapor permeability properties that promote the healing of deep second-degree burn wounds. The pro-healing mechanism of the dressing has been investigated Vascular endothelial growth factor (VEGF) expression was upregulated and basic fibroblast growth factor (bFGF) expression was downregulated in the early periods of wound healing, with upregulation of bFGF then occurring at a later stage of wound healing. At the same time, the wound dressing decreased the levels of tumor necrosis factor-α and interleukin6, thus validating its beneficial effect on the wound healing process at a biomolecular level. In conclusion, this new hydrogel dressing was shown to exhibit excellent self-regulatory and anti-adhesive properties that synergistically promote the healing of burn wounds in rats, thus providing promising results that may have clinical applications. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res B Part B: 00B: 000–000, 2018.

Key Words: burn dressing, self-regulation, anti-adhesion, promotion of wound healing

INTRODUCTION

Burn wounds occur widely, causing serious damage or loss of skin tissue. These burn wounds often leading to serious medical complications, such as nonhealing wounds, pressure ulcers, extensive scarring, and secondary infections.1 Despite impressive developments made in burn management procedures, there are still significant risks of complications associated with burn wounds that can result in lengthy medical treatments and an increase in patient mortality.2 Therefore, it is still necessary to identify suitable burn dressings to control wound infection and promote healing.1,3 An ideal burn dressings should exhibit the following characteristics4: (1) absorb superfluous exudate to keep the wound moist, while enabling the effective dissolution of necrotic tissue and fibrin; (2) not increase the risk of wound infection; (3) readily attach to healthy skin, but avoid conglutination with neo-formative granulation tissue that can result in secondary injures during dressing replacement; (4) maintain and promote the release of multiple bioactive factors, or drugs that are helpful to the healing process; (5) accelerate the formation of granulation tissue and the rate of reepithelialization at the wound site. Although there are many different types of dressings under development and on the market,5–10 none of these dressings currently fulfills all the characteristics required of an ideal burn dressing.
Dressings for different types of wounds face different challenges at different stages of the wound healing process.11 Plasma-like fluid exudate caused by increased capillary permeability is normally produced in the early stages of a burn injury, so dressings in the early phase of wound care need to exhibit strong hygroscopicity to absorb exudate.12 Water evaporation during the later periods of wound healing results in the wound becoming dry and scabbing, with neoformative granulation tissue capable of growing into the large meshes that are present in traditional gauze dressings,13 which can result in secondary injuries being caused when dressings are changed. Therefore, an ideal burn wound dressing should ideally maintain a moist environment with the correct humidity levels, while also preventing adhesion from the wound. Therefore, an ideal scenario would be to develop a wound dressing that could adjust and adapt to the varying moisture levels present in the wound environment during the healing process.
Hydrogel is a water-swollen three-dimensional network based on hydrophilic polymer chains.14 Due to their high water absorption capacity and the ability to maintain threedimensional structure, hydrogels have been widely used as wound dressings.7 Hydrogel dressings are semi-occlusive, with hydrogels are designed to hydrate wounds, rehydrate eschar, and aid in autolytic debridement. Autolytic debridement without harm to granulation or epithelial cells is important advantage of hydrogel dressings. Hydrogels have a marked cooling and soothing effect on the skin, which is valuable in burns and painful wounds. Moreover, hydrogels can absorb some exudate and provide a moist environment for cell migration. Generally speaking, hydrogels are recommended for wounds that range from dry to mildly exudating and can be used to degrade slough on the wound surface.15
This study has involved the preparation of a watersoluble hydrogel as a wound dressing that is derived from a water-soluble matrix CMC-Na/SA that has been incorporated into a chitosan matrix using a high-speed blending process. The optimum ratio of the primary reagents used to prepare this hydrogel has been determined using an orthogonal testing strategy using self-regulatory and anti-adhesive properties as evaluation indexes. The resultant hydrogel has been applied topically to deep second-degree burn wounds in SD rats to enable their anti-adhesion and wound healing performance to be evaluated.

MATERIALS AND METHODS

Materials

Carboxymethylcellulose sodium, sodium alginate, propanediol, NaCl, and CaCl2 were purchased from Chengdu Kelong Chemical Reagent Co., Ltd, China. Chitosan [viscosity of (400 20) mPa S] was purchased from Shandong Xiya Chemical Reagent Co., Ltd, China. Medical polypropylene nonwoven fabric (aperture of 1.5 mm) was purchased from Zhejiang Xinhai nonwoven manufacturing Co., Ltd, China. Gel (type A from porcine skin) was purchased from Tianjing DaMao Chemical Reagent Co., Ltd, China. Fibrinogen (Fg) (from ox blood, purity >85%) and bovine serum albumin (BSA) (purity ≥98%) were purchased from Beijing Haiwei gene technology Co., Ltd, China. Thrombin (Tb) was purchased from Hefei Bomei biotechnology Co., Ltd, China. Sterile dressing and DuoDERM™ dressing were purchased from Qingdao HaiRuo Co., Ltd, China and ConvaTec Co., Ltd, China, respectively. ELISA kits testing for TNF-α and IL-6 were purchased from Xiamen Huijia biotechnology Co., Ltd, China. Primary antibodies for VEGF (Rabbit anti rat, PB0084), antibody diluent (AR1016), secondary antibody (anti rabbit) (SV0002), and 3,30-diaminobenzidine tetrahydrochloride (DAB) kit (AR1022) were purchased from Wuhan BOSTER technology Co., Ltd, China. Primary antibody for bFGF (Rabbit anti rat, K001614) were purchased from Beijing Solarbio technology Co., Ltd, China. All chemicals and solvents were used without further purification.

Animals

Sixty healthy male SD rats (200 10 g) were obtained from the Animal Experiment Center of DaPing Hospital (Chongqing, China). All the animals were kept at constant temperature (25C) and lighting (12 h light/dark cycle). Principles of laboratory animal care were followed and all animal experiments were carried out following the Guidelines for the Care and Use of Laboratory Animals at the institute.

Preparation of self-regulating/anti-adhesive (SR-AA) hydrogel

Water-soluble colloidal matrices were prepared by dissolving CMC-Na and SA in propanediol aqueous solution (1 wt %) using mechanical stirrer [speed(s) = 500 r/min]. Defined amounts of CS were then added to the stirred CMCNa/SA colloidal solution (s = 700 r/min). The medical polypropylene nonwoven fabric was combined with colloidal solution, with time being allowed to ensure that any bubbles formed in the addition process were eliminated. The composite system was ultimately heated at 60C for 2 h to allow thermoforming to prepared double-layer structured hydrogel for burn dressings [thickness(T)dressing = 1 mm].

Optimization of the composition of hydrogel materials using orthogonal testing

Based on factors and levels of experiment (Table I), an L9(3)4 orthogonal testing designed to optimize the selfregulatory and anti-adhesive properties were used to optimize the ratio of components used to prepare the hydrogel materials (Table II).
Determination of subindexes. Simulated exudation of burn wound and simulated burn skin were prepared by mixing solution (25 mmol/L CaCl22H2O and 142 mmol/L NaCl16) and gelatin solution (35 wt %).17 A gravimetric method18 was used to determine the water absorption rates (WA) and water retention rates (WR) of dressings [length (L) × width (W) × T = 2 × 2 × 0.1 cm]. An alginate medical dressing test19 was used to determinate the moisture absorption rate (MA) and moisture donation rate (MD) of dressings.
Results were calculated according to the following formulas: where m0 is the dry weight of the sample; m1 is the wet weight of the sample after complete immersion in simulated wound exudation at 37C for 2 h; m2 is the weight of sample after it had been wrapped in filter paper and centrifuged for 10 min (s = 10,000 r/min); w0 is the weight of the sample after it had been exposed to a specific environment [T = 20C, relative humidity (RH) = 65%] for 24 h to regain moisture; w1 is the wet weight of a sample exposed to simulated wound exudation for 2 h at 37C that had been suspended it in air for 30 s; w2 is the weight of simulated skin; w3 is the weight of a sample after it had been attached to the surface of simulated skin for 24 h at 37C.
A fibrin matrix (Fg/BSA/Tb) was used to prepare simulated burn wounds20 to test the adhesion force and water solubility of dressings in vitro.
Samples were exposed to the surface of the simulated wound at 37C for 4 h, before then being installed to a Microcomputer controlled mechanical testing machine (DNS20, CIMACH China) using a self-made hook that penetrated the center of the samples. Tensile tests were carried out (s = 50 mm/min) until the dressing was completely stripped from the simulated wound, with the maximum stripping force measured as the adhesion force between the dressing and simulated wound.
A bespoke constant pressure output liquid device was used to maintain a flow rate of physiological buffer solutions (PBS, 0.01 M) at 2.55 cm/s when changing dressings. PBS flow was adjusted to be perpendicular to the ground at a horizontal angle of 45 to the dressing, with a distance between the outlet of PBS and the top of the sample of 10 cm. Samples were exposed to the surface of the simulated wound at 37C for 4 h, then the water solubility of the dressings in vitro was evaluated indirectly by measuring the period of time taken from PBS first contacted the dressing to when the dressing was completely separated from the simulated wound.
Comprehensive score of multi-indices. The self-regulatory property was evaluated by WA, WR, MA, and MD. The antiadhesive property was evaluated by adhesion force and water solubility. A multi-indices test formula method21 was used to calculated the comprehensive scores by combining subindexes as follows: where Xij and Dij represents the measured value and standardized value of the i index for a j test, respectively. The value of the same indices was standardized using the maximum value of each index as a reference. Ei was the weight coefficient based on the importance of each index on an evaluated performance. EMA = 0.2, EMD = 0.2, and Eadhesion force = Ewater solubility = 0.5. Optimal performance levels for the dressing corresponded to high scores for self-regulatory performance and low scores for anti-adhesive performance.

Physical characterization

Determination of water vapor permeability (WVP). Fixed circular samples [diameter (D) = 5 cm] were positioned at the apertures of glass bottles [D = 4 cm, height (H) = 5 cm] that contained simulated wound exudation (20 mL). No material, single layer, and double layer nonwoven fabrics were used as a blank control group, negative control group, and positive control group, respectively. Evaporation of liquid through the sample at 37C was monitored by measuring loss of weight from the system (bottle and liquid) over time. The WVP of the samples was determined by the water vapor transmission rate (WVTR) (g m−2 d−1) (n = 5): where m3 is the weight of the initial system, m4 is the weight of the system at time t, t is the measuring time, and A is the area of the aperture of the glass bottle.
Scanning electron microscopy (SEM). The microstructure and surface morphology of freeze-dried hydrogels were observed using SEM (LEO-supra35, ZEISS Germany), with samples being sputtered with an ultrathin layer of gold prior to analysis.
Treatment on rats with deep second-degree burn injury Animal studies were carried out to evaluate the effect of the SR-AA dressing on wound healing. SD rats were intraperitoneally anesthetized using sodium pentobarbital (35 mg/kg) and the hair on the rats’ back shaved using an electrical clipper. A beaker fixed test tube clamp was placed in 80C water bath until the temperature of cup bottom was constant, and the temperature was measured by the hand-held infrared radiation thermometer (SM380, JingDa China). After adjusting the rats position to ensure the flat back skin, the beaker with hot water in water bath (20 mL) was vertically contacted with the back skin of the rats for 20 s to scald a circular deep second degree burn wound (D = 2.4 cm). No external force was applied during the contact process, but depending on the weight of beaker, test tube clamp, and water (35 g). The degree of burn was confirmed after 24 h by staining wound skin slides with hematoxylin and eosin (H&E). Sixty burnt SD male rats were randomly divided into four groups (n = 15 in each group): a blank control group (without any treatment), a sterile dressing group (dressing made using medical tape and viscose fiber), a commercial dressing group (DuoDERM™), and an SR-AA dressing group. The dressings of each group were replaced once daily, and three rats were sacrificed on 4, 8, 12, 16, and 20 days after burn injury.
Morphological observation and healing rate of wound. Wounds were observed at 0, 1, 4, 8, 12, 16, and 20 days after burn injury, with wound areas being analyzed using Image Pro Plus 6.0. The wound healing rate (WHR) was defined using the following equation: where An is the wound area on a given day after burn injury and A0 is the initial wound area.
Histological analysis. Skin from full-thickness wounds was taken from rats of each group at 4, 8, 12, 16, and 21 days after injury, respectively. These tissue samples were fixed in 10% formaldehyde solutions, then embedded in paraffin, and cut into 5-mm-thick sections. The slides were then stained with H&E and Masson’s trichrome, before being visualized using an inverted optical microscope (IX71, Olympus, Japan).
Detection of inflammatory factor. Arterial blood was taken from anesthetized rats of each group at 4, 8, 12, 16, and 21 days after injury, respectively. These blood samples were spontaneous coagulated at 26C, before being centrifuged for 20 min (s = 2000 r/min, T = 4 C) (D3024R, SCILOGEX). Supernatant was collected for ELISA determination of TNF-α and IL-6 as described in the commercial kit instructions.
Immunohistochemical investigation. Expression levels of VEGF and bFGF were determined using immunohistochemical staining techniques. Paraffin sections of wound sites were deparaffinized, rehydrated, and microwave-heated for 15 min in 0.01 M citrate buffer solution (pH 6.0) to enable antigen recovery. Samples were then treated with 3% hydrogen peroxide (H2O2)–methanol solution for 10 min, to eliminate any endogenous peroxidase activity. Nonspecific adsorption of tissues was prevented via treatment with 5% BSA for 30 min at 37C. Diluted primary antibodies for VEGF (or bFGF) were added to tissues and incubated overnight at 4C, washed three times with PBS (for 5 min) and the sections were then incubated with a secondary antibody for 30 min at 25C, before being immersed in DAB chromogenic agent for 6 min. Sections were then rinsed with water, counterstained with hematoxylin, and cover-slipped using neutral balsam, with each sample then being observed using an inverted microscope. Each section was randomly visualized from 5 orientations at high magnification (400×), with integrated optical densities (IOD) expressed in the same mode being measured using Image Pro Plus 6.0 to enable semi-quantitative analysis (n = 5).

Statistical analysis

Statistical analysis of data was performed by one-way analysis of variance (ANOVA) using IBM SPSS 19.0 software. Results were expressed as mean standard deviation, with p values <0.05 indicating significance.

RESULTS

Optimization of the ratio of reagents used for the preparation of hydrogels by orthogonal test

The measured subindex values and comprehensive scores obtained for each group from orthogonal testing using selfregulatory and anti-adhesive properties of the hydrogel as major indicators are shown in Table II. These results (Table III) revealed that the impact factors (p < 0.05 or p < 0.01) for the self-regulatory property of the hydrogel (Table IV) were in the following order: concentration of CMC-Na > concentration of CS > concentration of SA. Similarly, it was found that the impact factors (p < 0.01) of the anti-adhesive properties of the hydrogel were in the order: concentration of SA > concentration of CS > concentration of CMC-Na (Tables V and VI). The optimal ratio of components was then determined via simultaneous screening for selfregulatory and anti-adhesive properties, which gave optimal concentrations for CMC-Na, SA, and CS of 2, 3, and 1 wt % in propanediol aqueous solution (1 wt %), respectively.

Physical characterization

WVTR analysis of the SR-AA dressing gave a value of 1810.777 131.679 g m−2 over 24 h (Table VII), which was similar to the value obtained for double-layer nonwoven fabric group. SEM analysis of the microstructure of the composite hydrogel revealed that it displayed a uniform pore structure with a controlled pore size distribution of 30–80 μm (Figure 1), that was consistent with the good WVP observed for the SR-AA dressing.

Treatment of rats with deep second-degree burn injuries

Morphological observation and wound healing rates. No significant differences were observed between the rat groups, with an obvious inflammatory response and significant edema being present during the first day after injury. After the fourth day, the wounds from each group began to scab, however, there was less edema from the wounds of group C and D and their scabs were softer than those for groups A and B. On the eighth day, the scabs of groups A and B were rough and hard, with some of the rats from group C showing signs of shedding on the edges of their scabs. However, there were signs of shedding at the edges of scabs and regrowth of hair around the wound sites of all the rats from group D. Some rats from group D exhibited narrowing of wounds after 12 days, whose scabs had been totally shed. At the 16th day, the scabs of rats from group A remained intact, with the scabs of some rats from groups B and C having been completely shedded. The scabs of rats from group D were completely shedded with newly granulated tissue; however, some of the wounds have healed completely. New epithelial tissue had grown to completely cover the wounds of the majority of the rats from group D at the 20th day, with only a few rats from this cohort retaining small wounds (Figure 2). Wound healing rates at the 20th day for the blank control group, sterile dressing group, commercial dressing group, and SR-AA dressing group were found to be (79.82 12.32)%, (86.30 10.01)%, (94.46 4.03)%, and (99.48 1.03)%, respectively (Figure 3). Therefore, these wound healing studies demonstrate that the SR-AA dressing facilitates good healing of deep second-degree burn wounds in SD rats.
Histological analysis. Our new SR-AA dressing and commercial dressing groups demonstrated more extensive reepithelialization of desquamated epithelial regions and different infiltration of inflammatory cells that the other two groups. On the fourth and eighth day, in SR-AA dressing treated wounds, neutrophils and macrophages were observed. On the 12th day, the infiltrate contained numerous histiocytes and multinucleated giant cells in SR-AA dressing group. Furthermore, in SR-AA dressing treated wounds, lymphocytes were predominated at 16th and 20th day with occasional histiocytes. The wounds of groups C and D were completely healed at the 20th day; however, there was no skin present covering accessory organs in group C, although the epidermal layer was thickened. In contrast, skin covering accessory organs and connective tissues in rats from group D were well formed after 20 days, with a consistency that was similar to that of normal skin (Figure 4). Severe collagen degeneration and necrosis was found in all groups in the immediate stages of after burn injury; however, greater collagen proliferation and a more orderly arrangement of collagenous fibers was seen to develop in the rats from group D (Figure 5). Therefore, it was concluded that the SR-AA dressing was operating to promote histopathological changes in burn wounds that resulted in more rapid wound healing and the growth of normal skin.
Inflammatory factor detection. The standard curves for expression levels of TNF-α and IL-6 were found to be y = 1957.70357 + (−1957.96047)/(1 + (x/5.7489E6)0.65856) (R2 = 0.99895) and y = 4.30141 + (−4.37376)/(1 + (x/93. 00444)1.36475) (R2 = 0.99954), respectively. The expression levels of TNF-α and IL-6 in the serum of normal rats were found to be (32.46 2.06) ng L−1 and (14.78 1.19) ng L−1, respectively. Both these expression levels were significantly higher in rats with burn wounds than in normal rats. The levels of both factors were highest immediately after the burn wound was formed, with a downward trend in levels as time passed, corresponding to an overall decrease in inflammation. Additionally, it was seen that the relative levels of these two factors in the SR-AA dressing group were lowest at each time point, with expression levels of TNF-α at 16 d and IL-6 at 20 days close to the levels measured in normal rats. Therefore, it appears that the SR-AA dressing facilitates more rapid downregulation of TNF-α and IL-6 [Figure 6(A,B)] to normal levels as the wound healing process proceeds.
Immunohistochemical investigation. The expression levels of VEGF and bFGF in each group were detected by semiquantitative analysis. These results showed that the SR-AA dressing upregulated the expression levels of VEGF expression directly after burn injury, thus indicating that it was playing a key role in the wound healing process [Figure 7 (A)]. The SR-AA dressing was also shown downregulate expression of bFGF during the early phase of wound healing;

DISCUSSION

The SR-AA hydrogel prepared in this study was designed to be soluble in physiological buffer solutions and to avoid physical injuries to burn wounds that can occur when dressings are changed. Therefore, water-soluble polymers (carboxymethylcellulose sodium and sodium alginate) were chosen to prepare a water-soluble matrix that would then be attached to medical polypropylene non-woven fabric to afford a composite dressing for testing. Dissolution of the hydrogel in physiological buffer solutions occur when hydrophilic groups of the matrix interact with water molecules, resulting in swelling that further exposes internal hydrophilic groups until the material becomes fully dissolved.22 Chitosan is a well-known component that has been widely used an anti-tissue adhesion agent,23,24that exhibits its antiadhesion effects via the following modes of action. (1) It can form a viscoelastic barrier,25 where the chitosan-based material becomes deposited between the gaps in wound tissues to separate and protect their inner surfaces. (2) A hemostatic effect26 can occur where positively charged ammonium groups attract negatively charged platelets and erythrocytes that accelerates platelet adhesion and stimulates vascular contraction.27 (3) Tissue healing can occur through the promotion of epithelial cell growth that inhibits excess proliferation of fibroblasts, thus reducing the synthesis of collagen fibers and weakening the degree of fibrous adhesion.28 A low dose of propanediol was also added as a supplementary moisturizer to increase the moisture retention properties of the hydrogel.29 The mechanical properties of the dressing were improved by thermally binding the hydrogel to the polypropylene non-woven fabric using a thermo-forming process. This gave a composite hydrogel dressing that was designed to provide burn wounds with the correct humidity levels throughout the healing process. It was also designed to function as a nonadhering surface that would prevent damage when the dressing was changed.
A dressing used to treat a burn is exposed to a large amount of exudate generated by the wound. The components of burn wound exudate are complex, containing many biomolecules and electrolytes that are as part of the wound healing process. The composition of exudate is also dependent on the wound type and the stage of healing,30 which can make it difficult to completely simulate the exact composition of exudate produced by burn wounds. In this study, an aqueous solution of 25 mmol/L CaCl2ċH2O and 142 mmol/L NaCl16 was used to simulate exudate for in vitro studies. The anti-adhesion properties of the dressing were explored using a biological matrix designed to simulate a burn wound, based on the use of a coagulation cascade reaction that produces fibrin.31 Therefore, fibrinogen in its soluble state was converted into gel state fibrin via thrombin catalytic cleavage, affording a fibrin polymer that was then combined with albumin to form a fibrin clot.32 The presence of burn wounds is often accompanied by the onset of other physiological effects, such as local congestion, edema, cell necrosis, and pathological changes of blood vessels.33 Therefore, the use of artificial biological matrices based on the blood coagulation process can simplify experiments designed to assess the potential of new dressings for the treatment of burn wounds.
The composition of a hydrogel clearly affects the selfregulatory and anti-adhesive properties of any wound dressing, with optimal levels of chitosan known to play an important role in controlling their anti-adhesive properties. Other factors that influence these properties include the concentration of propanediol used in hydrogel preparation, the thickness of the hydrogel, and the temperature and time used in the thermo-forming process. However, this study focused on determining the best ratio of reagents used for hydrogel production, with all these other factors remaining fixed at the start of the optimization process.
Four subindices were considered as factors to influence the overall score assigned for the self-regulatory performance of the dressings that were produced, with optimal ratios of reagents being determined by varying the weight distribution of the four subindices (EWA = EWR = EMA = EMD = 0.25; EWA = EWR = 0.2; and EMA = EMD = 0.3). The primary role of the dressing is to control the moistness of the wound site, which means that the WA and WR values have a significant influence on its self-regulatory performance, with the final weight allocation used for each subindex determined as EWA = EWR = 0.3 and EMA = EMD = 0.2. Similarly, orthogonal testing results were also used to optimize the weight distribution of the two subindices controlling the dressings antiadhesion properties (Eadhesion force = 0.4 and Ewater solubility = 0.6; Eadhesion force = 0.6 and Ewater solubility = 0.4). Finally, the adhesion force and water solubility of the hydrogel can be seen as having the same influence on the dressings anti-adhesion properties (Eadhesion force = Ewater solubility = 0.5). These studies revealed that the optimal ratio of components for preparing the hydrogel for the dressing, based on both its self-regulatory and antiadhesive properties, was carboxymethylcellulose sodium (2 wt %), sodium alginate (3 wt %), and chitosan (1 wt %) in a 1 wt % propanediol aqueous solution.
An ideal burn dressing should maintain effective gas exchange between the wound and the external environment, while preventing excess gas exchange that can result in drying of the wound that can make it more difficult to heal. Alternatively, too little gas exchange can result in accumulation of CO2 and other exhaust gases produced by necrotic cells within the wound region. An increase in CO2 pressure will also increase the acidity of wound and create a hypoxic environment that reduces the ability of tissue cells to regenerate, while providing an environment for anaerobic bacteria to grow, both of which can lead to slow healing.34 Therefore, it is generally accepted that the WVTR of burn dressings needs to be maintained within an optimal range to promote wound healing. The WVTR of normal skin is around 204 g m−2 per day, while the WVTR of injured skin in first degree burn wounds is 279 g m−2 per day and around 5238 g m−2 per day in granulation wounds.35 The WVTR value obtained in our SR-AA dressing was (1810.777 131.679) g m−2 per day, which is slightly lower than the recommended value for medical dressings (2500 g m−2 per day35). However, this dressing has been designed for the treatment of deep second degree burn wounds, so it is reasonable to expect that the ideal WVTR of an ideal dressing for this type of application may be lower.36,37 Two major absorption and separation processes are involved when water vapor passes through a wound dressing. Therefore, an increase in the number of pores in the dressing material can increase the rate of adsorption of water vapor, while a decrease in the thickness of adsorbent material in the dressing can increase the rate of loss of water molecules from the wound.35 The microstructure of the SR-AA hydrogel composite used in this study exhibited moderate pore size and a dense layer thickness, affording it good water vapor permeability properties for the treatment of deep second-degree burn wounds.
Wound healing is a complex biological event which requires the cooperation of multiple cells and different biological pathways, including hemostasis, inflammatory response, structural repair, and wound contraction.38 Once a wound is formed, it enters a rapid repair phase based on formation of a platelet plug, vasoconstriction, and formation of a fibrin clot. Release of chemokines occur as part of the blood clotting process that increase the permeability of peripheral capillaries to enable neutrophils, monocytes, and T lymphocytes to be attracted to the injured site. Structural repair at the wound site then occurs, involving re-epithelialization and formation and remodeling of granulation tissue. Granulation tissue is connective tissue that is composed of new capillaries, fibroblasts, and inflammatory cells that serve to promote the growth of fibrous tissues that enable the regeneration of blood vessels and nerves. Finally, skin cells around the wound site regrow, forming a uniform layer that eventually covers the wound site to form an effective barrier to the external environment.39
The main raw materials used in the SR-AA dressing are natural polymers, which have excellent biocompatibility.40–42 Based on the principle that a moist environment is desirable for the promotion of wound healing,43 a hydrogel matrix was developed in this study to prepare dressings for the treatment of dry burn wounds. Chitosan was included in the hydrogel to promote epithelial cell growth,44 which was important for healing of deep second-degree burn wound in rats. The wound healing rates for rats using our SR-AA dressings to treat burn wounds reached around 95% (standard for wound healing in clinical)45 at 20 days after injury, which was significantly better than for the control group and other dressings trialed in this study. These results showed that the SR-AA dressing could promote the healing of deep second-degree burn wounds in SD rats. Furthermore, predominant macrophages-based response creates more favorable environment for healing,46 thus the promote healing performance of SR-AA dressing is expected to further study from the perspective of inflammatory cells.
This study also compared the levels of inflammatory response and cytokine expression between each group to try and determine the healing mechanism of the SR-AA dressing. Complex inflammatory reactions occur during the process of wound healing, which involve the interaction of a variety of inflammatory cytokines. Bacteria present in burn wounds are strong stimulators of monocytes, macrophages, and lymphocytes, which can induce the synthesis and release of inflammatory mediators such as TNF-α, IL, γ interferon, NO, and so forth,33 that enhance levels of inflammation. In this study, two proinflammatory cytokines (TNF-α and IL-647) that are closely related to wound healing were selected as indicators to determine the level of inflammatory response in rats. These results showed that the SR-AA dressing could significantly inhibit the expression of TNF-α and IL-6 in rats, thereby reducing the inflammatory response to help accelerate the healing process.
Growth factor is a class of polypeptide that has a significant regulatory effect on cell growth and differentiation in vivo.48 In this study, two kinds of growth factors that are closely related to wound healing were selected for analysis. VEGF is a strong and effective proangiogenic growth factor which can promote the formation of capillaries and accelerate neovascularization.49,50 VEGF promotes the fission and proliferation of vascular endothelial cells and increases the permeability of venules, which result in improved vascular endothelial cell migration and capillary angiogenesis.51 The abundant blood supply present after angiogenesis provides sufficient oxygen and nutrients for wound healing to occur, which is important for the formation of granulation tissue and the survival of keratinocytes. The upregulated expression of VEGF that is facilitated by the SR-AA dressing during wound healing can accelerate the proliferation of capillaries to synergistically promote the wound healing process. bFGF is produced by fibroblasts, endothelial cells, smooth muscle cells, and chondrocytes, which has a significant effect on the wound healing process. The effects of bFGF include the following52,53: (1) Induced formation of microvascular vessels.
(2) Stimulation of cell proliferation at wound sites at an early stage of wound repair, caused by macrophages and endothelial cells releasing bFGF that cause inflammatory cells and pericytes to migrate to the wound that stimulate the further proliferation and migration of fibroblasts and keratinocytes. (3) Promotion of collagen synthesis, with newly formed extracellular matrix stimulated by fibroblasts to increase collagen content. Low expression levels of bFGF at an early stage of healing can promote angiogenesis, with high expression levels promoting epithelial regeneration, while angiogenic effects were inhibited in the stage of scar healing.54 Therefore, the SR-AA dressing downregulates the expression of bFGF at an early stage of healing to promote angiogenesis. Conversely, it causes bFGF expression to be upregulated at a later stage of healing to promote regeneration of epithelial tissue, thus synergistically promoting the rapid healing of wound tissues.

CONCLUSION

A water-soluble CMC-Na/SA/CS hydrogel composite has been developed as a wound dressing which has been applied as a potential treatment for burn wounds in rats. A solution of CMC-Na (2 wt %), SA (3 wt %), and CS (1 wt %) in1 wt % propanediol aqueous solution was used to prepare a hydrogel with optimal self-regulating/anti-adhesive properties. A composite wound dressing prepared from this optimal hydrogel exhibited good water vapor permeability that effectively promoted the healing of deep second-degree burn wounds in SD rats. Use of this hydrogel dressing resulted in upregulation of VEGF during the wound healing process, downregulation of bFGF in the early phases of wound healing, upregulation of bFGF in the late period of wound healing, and decreased levels of TNF-α and IL-6. These biological effects all combine to synergistically promote the rapid healing of burn wound tissue when the new composite hydrogel was used as a dressing. Further research is currently underway to explore the prohealing mechanism of these hydrogel composite dressings, while large animal experiments will be carried out to determine the potential of using these dressings for the treatment of burn injuries in humans.

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