Preclinical assessment of chitosan–polyvinyl alcohol–graphene oxide nanocomposite scaffolds as a wound dressing

Abstract

The present research aimed to examine the biological properties of chitosan (CS)–polyvinyl alcohol (PVA) scaffolds reinforced with graphene oxide (GO) nanosheets, as wound dressings. The scaffolds were characterized by various techniques. The scanning electron microscopy (SEM) and thermogravimetry analyses (TGAs) were used to investigate distribution of the GO within the polymer. The viscoelastic properties were evaluated by dynamic mechanical thermal analysis (DMTA) to examine the quality of a wound dressing. In vitro and in vivo studies were conducted to assess the biocompatibility of the scaffolds as wound dressing. The cell viability and proliferation results indicated that mouse fibroblast cells (L929) could adhere on the 50CS–50PVA/3 wt% GO scaffold. Herewith, the fabricated CS–PVA–GO nanocomposite scaffolds are suggested as promising biomaterials for skin tissue engineering and wound dressing.

Introduction

Tissue engineering was introduced as a new treatment to replace the traditional transplant method, which involves the use of polymeric or nanocomposite with or without cells.^1-3 The main role of tissue engineering is to produce artificial tissues to replace biological functions in tissue regeneration and wound healing. To use a membrane in tissue engineering, the first step is to select the right combination. Natural polymers such as CS are widely used in biomedical applications due to their suitable biological properties such as biocompatibility.^4,5 Due to the presence of the amine groups in the CS chain, this polymer has unique properties including biocompatibility, antibacterial, antiviral, non-toxic, and non-allergenic features as well as film-forming ability and high tensile expansion properties.^6-8 The CS has some disadvantages such as low mechanical properties, poor solubility in conventional solvents, and physiological environments. The PVA is known as a biocompatible and hydrophilic polymer with acceptable chemical and thermal stability properties and high-tensile strength and flexibility. Therefore, a combination of the CS and PVA should be used to increase its physical and chemical properties. Given its biocompatibility/biodegradability, this polymer hybrid can be used in various medical fields. The acceptable biological activities of CS, CS–PVA scaffold may have some beneficial effects on the biological characteristics of the compound film. ⁹

Recently, graphene oxide (GO) has received a lot of attention owing to its exceptional properties, especially mechanical properties and biocompatibility. ¹⁰ The previous researches have revealed that GO can be used as a biocompatible material. The functional groups in GO particles play a critical role in its distribution in organic solvents and polymers. In addition, the functional groups on the surface of this material improve its adhesion to polymers.^11,12 Graphene oxide helps in the proliferation of cells in a dose-dependent manner. The antifungal and antibacterial properties of GO offer a route for wound healing application. ¹³

In our previous work, we elucidated the viscoelastic properties of the fabricated CS–PVA/GO nanocomposites using TGA and dynamic mechanical thermal analysis (DMTA) tests and modeling the corresponding results with Cole–Cole plots which indeed showed enhanced properties as compared to the CS–PVA blend. ¹⁴ In addition, their antibacterial properties were investigated to be exploited as wound dressing. Herein, final evaluation on the quality of the prepared wound dressings was performed using in vitro and in vivo studies. Additionally, the morphology, structure, and hydrophilic property of scaffolds were measured by using SEM, Raman spectroscopy analysis, and contact angle measurement. The obtained results are promising for the future application of the nanocomposites as scaffolds for wound healing and dressing.

Materials and methods

Materials

The CS with a molecular weight of 161,000 g/mol, a deacetylation degree of 75.6%, and a viscosity of 1406 mPa.s. Moreover, PVA with hydrolysis degree between 98% and 99% and a molecular weight of 31,000–50,000 g/mol were purchased from Sigma–Aldrich Chemical (Milwaukee, Wisconsin, USA). The GO with a concentration of 4 g/L was also prepared through Hummer’s method. Acetic acid was produced from SD Fine Chemical. Ethanol, methanol, xylol, hematoxylin, eosin, Masson’s trichrome, fixative, paraffin, ketamine, and xylene are purchased from Merck Company. Thirty female rats with the mean weight of 180–200 g were included in this investigation. The Animal Laboratory of Guilan University of Medical Sciences prepared these animals. The Animal Ethics Committee of the same university approved all the methods used in this study.

Methods

Scaffolds Preparation

The 50CS–50PVA and the nanocomposite scaffolds were fabricated as described in our previous work. ¹⁴ In brief, aqueous solutions of CS in acetic acid (1% vol.) and PVA in water were separately prepared. The scaffolds were obtained by mixing 1:1 stoichiometric ratio (50PVA–50CS) of the two polymer solutions, followed by casting onto a transparent Petri glass dish. After drying, the films were detached from the mold and were thermally treated in vacuum oven. An ultrasonic device (Bandline Company, Germany) with a maximum power of 75 W and a frequency of 20 kHz, HD 3200 series, was used to distribute the GO in the base polymer mixture. The solutions of the composite scaffolds with the different GO contents (0.5, 2, and 3 wt%) were then prepared and sonicated for 1 h at a power of 60 W. Then, the composite scaffolds were fabricated by the same process of the 50CS-50PVA scaffold preparation.

Characterization

The nanocomposites were firstly investigated by FT-IR measurements, performed on the BRUKER Germany Tensor 27 Infrared Spectrometer used in the waveguide range of 400–4000 cm⁻¹. Furthermore, the Raman shifts were recorded over 500–2500 cm⁻¹ using a DPSS Nd:YAG laser beam with an excitation wavelength of 532 nm and a laser power of 100 mW (Teksan, TakRam N1-541) to confirm the distribution of the GO flakes in the polymer matrix. The phase and crystallinity of the structures were characterized by an X-ray diffractometer (Bruker XRD machine D8 Advance) and Cu-Kα radiation over a two theta range of 5–80°, step size of 0.02, and scan speed of 4°/min at 30 kV and 40 mA. The morphology of the scaffolds was investigated on the SEM equipped model (EM 3200 at 30 kV). The TGA curves were obtained by Shimadzu TGA 209 F1thermogravimetric instrument. The temperature range was applied from 20 to 700^oC with a ramp rate of 10^oC min⁻¹.

The viscoelastic behavior was evaluated by performing using a DMA Netzsch 242 machine. For these measurements, rectangular specimens of 4 mm width, 25 mm length, and 0.1 mm thickness were prepared. The tests were performed in three-point bending mode at a frequency of 1 Hz in the temperature range of 25–140^oC with a heating rate of 3^oC/min (ASTM: D5023-01).

The contact angle experiment was conducted to measure hydrophilicity with a precise goniometer (DSA 100, KRÜSS GmbH Co., Hamburg, Germany). A syringe was placed vertically above the scaffold surface, and a 4 μL drop of water was applied on a 1 cm² of each scaffold. Each measurement was repeated for three times at three different sites, and the mean value was reported as the contact angle.

To study biocompatibility, the quantitative toxicity and mouse fibroblast cell culture tests were performed on the samples of 50CS–50PVA blends and nanocomposite scaffolds for 48 h.

Quantitative toxicity testing is carried out in accordance with ISO10993-5 standard, using MTT cell toxicity measurement. In the fibroblast cell culture test, mouse fibroblast cells (L929) were purchased from the Pasteur Institute of Iran. The cells were maintained in an RPMI culture medium containing 10% FBS serum in an incubator with CO₂ injection capability at rates of 5% and 95% moisture at 37^oC. The cell culture with a density of 2.5 × 10⁴ cells/cm² was conducted on the polymer composition and nanocomposite, containing 3 wt% GO scaffold.

To study the biological effects of films on living organisms, polymeric bend and nanocomposite scaffolds containing 3 wt% GO were cut into a diameter of 5 mm and sterilized by ultraviolet (UV) systems. To investigate these effects, 30 mice were used in the three treatments as control, zero, and 3 wt% under two repetitions. Skin sampling was performed under five repetitions 4, 8, 12, 16, and 20 days after surgery. For pathological studies, the samples were obtained from the different solutions such as formalin 10%, the different percentages of alcohol (70, 80, 90, and 96%), methanol 100%, xylol, and paraffin were fixed, and then 5 micron thick samples were cut by a microtome device. The samples were processed for the analysis using hematoxylin and eosin and Masson’s trichrome techniques to examine the inflammation in the concerned tissues and the healing process of the wound. Then Leica’s Wild M8 microscope was used to analyze the images.

Results and discussion

Scaffold macroscopic images

Generally, 50CS–50PVA scaffolds are transparent due to the natural properties of PVA and the nature of amorphous CS. ¹⁵ With increasing GO content, the intensity of the brown tone increased and consequently the opalescence of the scaffold increased (Figure 1). ¹⁶

Figure 1.

Sample macroscopic images: (a) CS–PVA, (b) 50CS–50PVA/0.5% wt GO, (c) 50CS–50PVA/1 wt % GO, (d) 50CS–50PVA/2 wt % GO, and (e) 50CS–50PVA/3 wt % GO scaffolds.

Structural and morphological characterization

FT-IR spectra of GO, 50CS–50PVA, and 50CS–50PVA/3 wt% GO nanocomposites are indicated in Figure 2(a). The FT-IR peak at 3300 cm⁻¹ belongs to the OH bond. The C–O functionalities such as C = O (1717 cm⁻¹), C–O (1378 cm⁻¹), and C–O–C (1015 cm⁻¹) are clearly visible, corresponding to the GO nanosheets. In addition, the spectrum also shows a C = C peak at 1624 cm⁻¹ corresponding to the remaining sp² character. ¹⁷ The 50CS–50PVA/3 wt% GO nanocomposite showed a new peak at 1706 cm⁻¹ assigned to carboxyl groups from GO surface which indicates the presence of GO within the 50CS–50PVA nanocomposite. The peaks of the characteristic absorption in the curve of 50CS–50PVA/3 wt % GO are approximately similar to the curve of 50CS–50PVA.

Figure 2.

(a) The FT-IR spectra of GO, 50CS–50PVA, and 50CS–50PVA/3 wt% GO; (b) the Raman spectra of GO and 50CS–50PVA/3 wt% GO; and (c) the XRD patterns of 50CS–50PVA, 50CS–50PVA/0.5 wt% GO, and 50CS–50PVA/3 wt% GO nanocomposite scaffolds.

Figure 2(b) shows the corresponding Raman curves for GO and CS/PVA/GO nanocomposite. The GO shows two main peaks at 1343 and 1573 cm⁻¹ contributed by D and G bands, respectively. It is well-known that D band is originated from defects and disorders in the GO nanosheets, while G band is created by bond stretching of sp²-hybridized carbon atoms. ¹⁵ The Raman spectrum of 50CS–50PVA/3 wt% GO reveals both D and G bands with a slight red shift for G band at 1588 cm⁻¹. The ratio of D band to G band (I_D/I_G) is an acceptable indicator of the size of sp² domains. ¹⁸ The I_D/I_G ratio for 50CS–50PVA/3 wt% GO shows a higher value (0.91), as compared to GO nanosheets (0.80). The intensity increase of the D to G ratio denotes the higher number of small in-plane sp² domains, suggesting that GO effectively interacts with the polymer blend and is spatially arranged within the polymeric matrix. ¹⁹

The XRD patterns of the 50CS–50PVA polymer blend together with 50CS–50PVA/0.5 wt % GO and 50CS–50PVA/3 wt% GO nanocomposites are presented in Figure 2(c). In the case of 50CS–50PVA, the sharp peak at 2θ = 11.6° is assigned to the CS polymer while the peak centered at 19.7° is attributed to PVA crystals, and it is accompanied by a shoulder at ∼23°, a typical characteristics of the PVA polymer. ¹⁴ By addition of GO to the polymeric mixture, it is obvious that the first peak vanishes while the crystallinity of the major peak is enhanced. In fact, in the presence of 0.5% wt GO, the CS peak and the corresponding PVA shoulder are effectively suppressed which by further increase in the GO content to 3% wt gives rise to their complete elimination into the amorphous state.

The microstructures of the 50CS–50PVA and nanocomposite scaffolds were probed by SEM experiments. The corresponding images are presented in Figure 3. The relatively smooth surface of the 50CS–50PVA scaffold generally indicates the uniform dispersion of the CS and PVA molecules in the composition (Figure 3a). Previous studies have indicated that the surface of pure CS and PVA films separately is smooth. ¹⁶ Consequently, it is expected that the blend between them will also present a surface with the same conditions.^15,16,20 The morphology of the nanocomposite is rough and wrinkled, which is attributed to the GO (Figure 3(b) and (c)).This indicates the proper distribution of GO in the 50CS–50PVA and the placement of GO within the polymer. As the amount of GO increases by 3 wt% GO, the entire 50CS–50PVA surface is covered with the GO, and the polymer surface is less visible (Figure 3(d)).^15,21,22 Moreover, with the increase of the GO, the surface tends to become more rough, and this can be ascribed to the greater interaction between GO and polymer.^23,24

Figure 3.

Scanning electron microscopy images of (a) 50CS–50PVA, (b and c) 50CS–50PVA/1 wt% GO, and (d) 50CS–50PVA/3 wt% GO.

Thermal behavior of scaffolds

TGA is well-known as the most significant method to investigate the thermal stability of polymers. ²⁵ As presented in Figure 4, when GO is loaded into the 50CS–50PVA scaffold, the degradation stages are shifted toward higher temperatures. The major decomposition initiation temperatures (T_i) for the 50CS–50PVA and nanocomposites are around 240°C and 257°C, respectively. In the case of 50CS–50PVA/GO, the major weight loss occurs over temperature ranges of 256–465°C. A residue mass of 18% is obtained for the 50CS–50PVA as compared to 21.5 and 25% when 0.5 and 3 wt% O are added to the polymer, respectively, at similar temperatures. Clearly, the nanocomposite shows enhanced thermal stability which is mainly attributed to the following reasons ¹⁴ :

1. The effect of the physical barrier of the GO, which leads to the decomposition delay of scaffolds with increasing temperature.

2. The acceptable dispersion of GO in the 50CS–50PVA.

Figure 4.

The thermogravimetry analysis curves of 50CS–50PVA and nanocomposite scaffolds along with the degradation stages.

Viscoelastic behavior

A wound dressing should possess a certain toughness to withstand handling during application and any subsequent injury in situ. The dressing should be pliant (that is easily deformed and elongated), allowing it to conform to the body surface and hence protect the wound. ²⁶

The viscoelastic properties is essential for wound dressing, so it can tolerate the pressure of more than 40 mm Hg from the bandage tension, and thus pressure will be relaxed with time. As a result, long term conformability can be significantly increased above the immediate values based on the elastic behavior. ²⁶

Dynamic mechanical thermal analysis is simply a dynamic method to characterize the viscoelasticity of materials. A loss is often referred to as damping, and since it is appropriate to view a material damping performance in relation to its stiffness, a good expression for damping performance is the loss tangent (tan δ). The tan δ determined at 1 Hz frequency is plotted in Figure 5 as a function of temperature for both 50CS–50PVA and the nanocomposites. Figure 5 indicates that the height of tan δ of nanocomposites decreased constantly with the increasing of the content of GO. This could be related to the addition of GO that enhanced the stiffness of nanocomposites. The mobility of the polymer is reduced due to the presence of GO. Besides, the strong hindrance to chain mobility resulted from the strong interaction between functional groups on GO, CS chains, and PVA molecules, which might contribute to the decreased tan δ values.

Figure 5.

The tan delta curves for 50CS–50PVA, 50CS–50PVA/0.5 wt% GO, and 50CS–50PVA/3 wt% GO scaffolds at 1 Hz frequency.

Additionally, the tan δ peak of 50CS–50PVA/3 wt% GO at 40–140^oC is broader than the other scaffolds. The broadening of tan δ peak could be the result of good dispersion and sufficient interaction of 50CS–50PVA with the GO. Therefore, 50CS–50PVA/3 wt% GO could have appropriate viscoelastic properties.

Contact angle

In tissue engineering, scaffolds must be ideally hydrophilic as the wettability of the scaffold increases cell adherence and attachment. ²⁷ The hydrophilicity of CS is dependent on many factors such as the degree of deacetylation; however, the hydrophilicity of PVA is attributed to functional groups. The research findings indicated that the hydrophilicity of the CS–PVA blend film decreased with increasing CS content. ²⁸ Figure 6 shows the contact angle measurements of the 50CS–50PVA and 50CS–50PVA/GO. According to Figure 6, the contact angle of the 50CS–50PVA and 50CS–50PVA/1 wt% GO was observed 88.8° and 66.8°, respectively. Moreover, the contact angle of the nanocomposite significantly decreased. In this regard, numerous hydroxyl groups in the GO possibly increase the overall hydrophilicity of the scaffold. As presented in the SEM images section, the roughness of its surface could also affect the contact angle because of the increased regions of solid–liquid interface and the effect of sharp edges of rough surfaces. However, when the content of the GO reaches to 2 wt%, the contact angle increases by 81°.There are different data about increasing and decreasing the contact angle of polymers by adding GO.

Figure 6.

The contact angle diagram for 50CS–50PVA and nanocomposite scaffolds.

The hydrophilicity of the electrospun CS–PVA nanofibrous mats with the different GO contents was investigated by Yang et al. ²⁸ They documented that, with increasing the GO content, the contact angle of nanofibrous mat increased due to the inhomogeneity of the mat surface. Nevertheless, when the GO content reached 2.5 wt%, the contact angle decreased by 39.5°.

In vitro biocompatibility

Results of MTT assay 50CS–50PVA and the 50CS–50PVA/GO scaffolds are summarized in Figure 7. Although all scaffolds displayed low cytotoxicity, the 50CS–50PVA/3 wt% GO nanocomposite showed significantly lower cytotoxic potential than scaffolds. When the GO is added to 50CS–50PVA, the roughness and surface cavities of the nanocomposites increase, enhancing the survival and adhesion of the cells compared to 50CS–50PVA and playing a critical role in increasing metabolic activity. As shown in Figure 7, a significant absorbance at 570 nm was observed in the cells with addition of GO compared to polymer and a slight difference was observed even at higher concentrations of GO treated against the control sample. In general, the structure of interconnected pores ensures a suitable culture medium to guide cell adhesion, tissue proliferation and growth, and sufficient nutrient flow into the cell.^15,29 According to the researchers’ results, providing a suitable platform for cell growth is the most important and effective reason for increasing cell growth in the presence of scaffolds. ³⁰

Figure 7.

The quantification of cell proliferation rate on 50CS–50PVA and nanocomposite scaffolds, as revealed by MTT test after 48 h mouse fibroblast cell culture on the surface.

For cell–matrix adhesion, the SEM micrographs of fibroblasts L929 cells cultured onto the substrates of 50CS–50PVA, and CS–PVA/GO scaffolds, were investigated after cell seeding for 48h (Figure 8). It is clear that the morphology of nanocomposite was preserved after being soaked in the culture medium for 48h and the cells spread well on the substrates. On both 50CS–50PVA and CS–PVA/GO scaffolds, morphological changes were detected after 48 h of incubation. Round-shaped cells in 50CS–50PVA scaffold (Figure 8(a)) gradually changed into a spindle (polygonal and stretchy) (Figure 8(b)(d)). The difference of the cell morphology should be closely related to the characteristics of the scaffolds surface. The GO can be reduced oxidative stress in different doses in the cell and ultimately reduce cell weakness. ³¹ . The improved cellular response due to the presence of GO related to protein adsorption from culture media onto the GO rough surfaces (according to SEM morphology observation), significantly promoting the cell growth and proliferation. It should be noted that for the composite containing 3 wt% GO, the spindle numbers were higher than that of 0.5 wt% GO. The exfoliation and GO content in the polymer matrix can improve adhesion and growth of polygonal fibroblasts cells on the nanocomposite. At last, GO is highly biocompatible regarding the presence of epoxy, hydroxyl, and hydrophilic groups. ³²

Figure 8.

The scanning electron microscopy of cells 48 h after cell culture; (a) cells exposed to 50CS–50PVA scaffold; (b) cells exposed to 50CS–50PVA/0.5 wt% GO, and (c, d) cells exposed to 50CS–50PVA/3 wt% GO scaffold.

In vivo biocompatibility

The incision site was macro and microscopically examined to investigate the effect of 50CS–50PVA and 50CS–50PVA/3 wt% GO scaffolds in wound healing. To this end, the scaffolds were placed under the skin tissue. This has caused more damage to the tissues. On the other hand, in the first days after surgery, the inflammatory reactions of the body are completely normal because the foreign object has entered the body. Moreover, the number of fibroblast cells in the damaged tissue is higher, the wound becomes more inflamed, and these cells decrease as the wound heals. Furthermore, if less water is absorbed at the wound site, the inflammatory response increases, and the wound heals more slowly. In this regard, cell uniformity indicates completed wound healing.

Figures 9 and 10 show the macroscopic and microscopic results of tissues, respectively. During the treatment, no infection was observed in any of the animals. Wound healing in mice with 50CS–50PVA was similar to that of the control samples. In the sample, inflammatory cells are located around the wound. In addition, granulated tissue along with young and active fibroblasts is also observed. In Masson’s trichrome techniques, blue collagen strands are visible. Inside the membrane, new collagen fibers are forming and proliferating, which is due to the activity of inflammatory cells. In this regard, the farther they are from the membrane, the older the collagen fibers are.

Figure 9.

Macroscopic examination of the back area undergoing scaffolds: (a) control, (b) 50CS–50PVA, and (c) 50CS–50PVA/3 wt% GO; the subscripts 1, 2, and 3 correspond to the study after 4, 12, and 20 days after surgery, respectively.

Figure 10.

Histological analysis of back area undergoing scaffolds: (a) control, (b) 50CS–50PVA scaffold, and (c) 50CS–50PVA/3 wt% GO; the subscripts 1, 2, and 3 correspond to the examination after 4, 12, and 20 days after surgery, respectively.

The wound was examined during 20 days. In the sample with 50CS–50PVA and 50CS–50PVA/3 wt% GO, the wound was completely healed, and the incision site was not observed. In the control sample, the incision site was clearly defined while the skin tissue was fully formed. In the microscopic examination of the skin tissue sample after 20 days, it was noticed that the scar tissue was almost healed in the control sample (a). The epidermis of the skin, which is made up of a stratified squamous horny epithelium, has been completely restored; however, in Masson’s trichrome techniques, the dermis was observed in the form of a mixture of pale blue and purple, indicating that the collagen fibers were young. In sample (b), which contains 50CS–50PVA, the skin texture is perfectly normal, the wound heals and is not visible, and the skin structure is fully formed. In sample (c), the wound heals, the incision is not observed, and there is no inflammation. At the membrane site, the skin tissue was formed naturally and its structure was completed.

According to the findings, 50CS–50PVA increases the regeneration rate, which is accomplished by increasing the proliferation rate of fibroblasts, cell proliferation, and further adhesion inside the living organism. As a result, the effect of chitin accelerated the healing process of wounds CS, monomers, and oligomers with increasing the activity of inflammatory cells such as macrophages and fibroblasts. Examining the effect of CS on the early stages of wound healing revealed that CS plays an effective role in rapid wound healing by increasing the presence of white blood cells and enhancing the proliferation of fibroblasts and young capillaries to the wound surface. Accordingly, after the course of treatment, the epidermis is formed naturally, and the natural tissue appears in its original form; however, the incision is quite obvious, and the healing and growth process of cells continues in the control group. The acceleration of wound healing and the performance of the CS polymeric blends have been reported by other researchers.^33,34

Regarding the effect of GO, as it can be observed, the inflammation of the wound is less during the first days and the scaffold containing GO has higher strength under body conditions and also accelerates the healing process of the wound. Compared to rapidly absorbed polymer scaffold, GO nanocomposites can be used for wounds taking more time to be healed. Moreover, due to the function of these membranes, there is no need for cross linker to increase the strength of the scaffold. During the first days of wound healing, the extent of inflammation and wound area were less-expanded in the scaffold containing GO than in the sample containing pure polymer scaffold.^35-37

Conclusion

In summary, we successfully prepared nanocomposite scaffolds of 50CS–50PVA reinforced with GO at different contents. The SEM observations confirmed that GO has been uniformly dispersed in the matrix and well-bonded with the polymers. The TGA and tan δ curves indicated that when GO is loaded into polymer, the thermal stability and viscoelastic properties were improved. The 50CS–50PVA/3 wt% GO scaffold led to a significant increase in the cell proliferation rate. Besides, the in vivo experiment proved the ability to promote wound healing of the 50CS–50PVA/3 wt% GO scaffold. The improved thermal stability, viscoelastic and contact angle properties together with good in vivo experiment of 50CS–50PVA/3 wt% GO scaffold are promising for future bio applications such as wound dressing materials and skin tissue engineering. The authors are currently working on other types of nanocomposites scaffolds such as polycaprolactone–chitosan/GO and polyvinylidene difluoride–chitosan/GO as wound dressing.