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Polyvinyl alcohol/gum Arabic hydrogel preparation and cytotoxicity for wound healing improvement

  • Mohamed A. Elblbesy , Taha A. Hanafy and Mamdouh M. Shawki EMAIL logo
Published/Copyright: July 2, 2022
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Abstract

The application of pure polyvinyl alcohol (PVA) hydrogel as wound dressing faces many restrictions due to its insufficient elasticity, stiff membrane, and very limited hydrophilicity. These drawbacks can be limited through cross-linking with other natural biopolymers such as gum Arabic (GA). PVA hydrogels blended with six different GA concentrations were prepared. The characterization of these hydrogels was performed by Fourier transform infrared spectrophotometer, X-ray diffraction, and scanning electron microscope. The swelling ratio (% SR) percentage has been calculated. The possible cytotoxicity was determined using a sulforhodamine B assay. Wound healing test was performed on human skin fibroblast cells. Our results indicated that by increasing GA concentration in PVA hydrogel, the % SR increases and the cytotoxicity effect decreases. The results indicate also a significant gradual decrease in the wound area with time as the GA concentration increases in the PVA hydrogel. Therefore, GA improves the biological applications of PVA hydrogel.

1 Introduction

Wound healing is a reparative process involving a sequence of continual inflammation and repair to restore normal tissue function. Many kinds of cells including epithelial cells, endothelial cells, inflammatory cells, platelets, and fibroblasts all work together to restore normal activities during this phase. Many studies have been carried out to develop a way of wound regeneration and healing that involves the use of various cover materials (dressings) to help with optimal wound management (1). The goal of wound closure research has been to improve the design of the cover material in order to speed up wound healing. A hydrogel is one form of wound closure material that is being developed. Hydrogels have the advantage of being able to work well in a moist environment (2). They swell without being dissolved due to their ability to absorb a huge amount of water (3).

Different materials are used for the preparation of hydrogels, and several more are being studied for their potential application in tissue engineering. Various synthetic polymers, such as polyvinyl alcohol (PVA), are used to make the hydrogel scaffolds (4).

Natural polymers (such as collagen, chitin derivatives and chitosan, alginic acid and sodium alginate, starch and starch derivatives, dextran, glucan, gelatin, poly-N-acetyl glucosamine, hyaluronic acid or hyaluronan, bacterial cellulose, and keratin) and synthetic polymers (such as polyurethane, methyl methacrylate, proplast, or alloplastic, poly(N-vinylpyrrolidone), polyethylene glycol, poly(N-isopropylacrylamide), nanoparticles composite-polymers, clay nanocomposite-membranes, metal oxides composite-membranes, and carbon-based materials composite-membranes) have been used separately or in combination for the preparation of dressings (5). By fostering more rapid epithelialization, polymeric wound dressings based on hydrogels aided the healing of pressure ulcers. As a result, 85% of wounds were healed with hydrogel dressings, compared to 50% for wounds repaired with standard gauze dressings (6).

Blended polymers for medical applications are designed to interact with biological systems in order to assess, address, and increase the body’s function, or to replace any tissues or organs (7). Natural or synthetic polymers in blended materials assemble the desired features of each material on its own; however, blended polymer materials are always mixed with PVA to improve mechanical and physicochemical properties of generated polymeric materials (8).

PVA is a polyhydroxy polymer and a chemically resistant emulsifier with good adhesion properties. Because of its membrane-forming abilities and hydrophilic qualities, PVA is commonly selected as a cross-linker (9). PVA is nontoxic, water-soluble, biodegradable, with favorable thermal property, and flexibility (10). On the other hand, there are some restrictions for using PVA hydrogel alone as a wound dressing polymeric material due to the insufficient elasticity, stiff membrane, and very limited hydrophilicity characteristics of PVA hydrogel. Through cross-linking and blending, these limitations can be solved by combining PVA with other biopolymers (11). These interactions can improve PVA’s absorption and mechanical characteristics in aqueous solutions and physiological fluids, hence increasing its biological activity.

PVA is cross-linked using a variety of chemical cross-linking agents such as anhydrides, epichlorohydrin, and aldehydes. Because these substances are synthetic, they are extremely harmful to the living cells and the environment. Furthermore, they have a low molecular weight and can easily pass via a variety of portals into physiological systems (12). As a result, there is a greater demand for biocompatible green and nontoxic cross-linkers in biomedical applications. That can be achieved through using natural polymers as cross-linkers instead of chemical agents (13). Water absorption capacity, water–vapor permeability, and transmission of manufactured hydrogels can all be considerably improved by combining PVA with natural polymers (5).

Acacia gum, known as gum Arabic (GA), is a plant substance that is considered safe for use in human food, cosmetics, and medicinal formulations (14). There are about 100,000 tons of GA used around the world yearly (15). GA is a natural biopolymer composed of about 97% polysaccharide and less than 3% protein (16). It is hydrophilic, brittle, and can be biodegradable easily (17). GA has branching polymeric structures providing excellent cohesion and adhesion characteristics. GA is widely utilized in the food sector (as dietary fiber) and pharmaceutical preparations (as suspending and emulsifying agents, tablet binder, demulcent, and emollient in cosmetics) due to its unique qualities (18).

The aim of the current research is the formation and characterization of PVA hydrogel conjugated with different GA concentrations. Fourier transform infrared spectrophotometer (FTIR), X-ray diffraction (XRD), and scanning electron microscope (SEM) were used to characterize the formed PVA hydrogels conjugated with different GA concentrations. Their swelling behavior, cell cytotoxicity, and their efficiency for wound healing have been studied.

2 Materials and methods

2.1 Preparation of hydrogel

PVA with a molecular weight of 85,000 was supplied by laboratory Reagent, Techno Pharmchem Haryana, India. GA was sundried and grained using a serrated disk grinder to obtain small-sized particles. Therefore, 1 g of PVA was added to 100 mL of triply distilled water at 80°C with continuous stirring to obtain the clear transparent solution. An aqueous GA solution was obtained by adding the proper weight of GA in 50 mL of triply distilled water at 60°C. The mass fraction of GA w (wt%) was calculated according to the following equation:

(1) w ( wt % ) = W GA W P + W GA × 100

where W GA and W P represent the weights of GA and PVA, respectively. By using Eq. 1, different concentrations of GA solutions (10, 15, 20, 25, and 50 wt%) were calculated. To obtain PVA/GA x blend sample, PVA and GA solutions were mixed together at 60°C for 6 h with continuous stirring. The final solution of the PVA/GA x blend was then poured into polypropylene dishes and dried at 40°C on a leveled plate for 3 days until the solvent was completely evaporated. The flexible uniform and transparent films, with a thickness of 0.1 mm, had been obtained and retained in the desiccators for further characterization (12).

2.2 Characterization of the prepared hydrogels

FTIR spectrum of the prepared hydrogels was recorded on FTIR (Bruker, Germany) in the region of 4,000 to 500 cm−1. The XRD pattern has been performed using the XPERT-PRO powder diffractometer system, with 2θ (4–80°), with a minimum step size of 2θ: 0.026, and at the wavelength (Kα) = 1.54614°. The surface morphology of the hydrogels was measured using an SEM (ZEISS, Germany) (19). Swelling characteristics of all the prepared hydrogels were studied. Swelling of the hydrogels was observed by immersing the gels in 50 mL of distilled water at pH 7 for 120 min at room temperature (19). The amount of absorbed water is commonly indicated as water uptake or percent of swelling ratio (% SR). % SR is determined using the following equation (19):

(2) %  SR =   W t W o W o × 100

where W t is the weight of the swollen hydrogel at different time intervals and W o is the weight of dried hydrogel.

2.3 Cell culture

Human skin fibroblast was obtained from Nawah Scientific Inc. (Mokatam, Cairo, Egypt). Cells were maintained in DMEM media supplemented with 100 mg‧mL−1 of streptomycin, 100 units‧mL−1 of penicillin, and 10% of heat-inactivated fetal bovine serum in humidified, 5% (v/v) CO2 atmosphere at 37°C.

2.4 Cytotoxicity assay

The sulforhodamine B (SRB) assay was used to determine cell viability. In 96-well plates, aliquots of 100 μL cell suspension (5 × 103 cells) were incubated in a complete medium for 1 day. Aliquots of 100 μL media containing PVA hydrogel or PVA-GAx hydrogels were used at different concentrations (0.001–1,000 μg‧mL−1) to treat the cells. After 3 days of the treatment, cells were fixed by changing medium with 150 μL of 10% trichloroacetic acid and incubating at 4°C for 1 h. After removing the trichloroacetic acid solution, the cells were washed five times with distilled water. Aliquots of 70 μL of SRB in 1% (v/v) acetic acid (SRB solution) were added and incubated at room temperature for 10 min in the dark. Plates were washed three times with 1% acetic acid and air-dried overnight. Then, 150 μL of Tris base solution (pH 10.5, 10 mM) was added to dissolve the protein-bound SRB stain, and the absorbance was measured at 540 nm with a BMGLABTECH®-FLUOstar Omega microplate reader (Ortenberg, Germany) (20,21).

2.5 Wound healing test

For the scratch wound experiment, cells were plated at a density of 3 × 105/well onto a coated 6-well plate and grown overnight in 5% DMEM media supplied with fetal bovine serum at 37°C and 5% CO2. Horizontal scratches were inserted into the confluent monolayer the next day; the plate was carefully washed with phosphate buffer saline, control wells were refilled with fresh medium, and the tested wells were treated with fresh media containing PVA hydrogel or PVA-GAx hydrogels. At 0, 24, 48, 72, and 96 h, images were obtained with an inverted microscope. In between these time intervals, the plates were incubated at 37°C with 5% CO2 (13,14). The captured photos were analyzed by MII Image-View software version 3.7 to determine the percent of wound area (22).

2.6 Statistical analysis

All data were calculated using three independent replicates and expressed as mean and standard deviation. The difference between different groups was assessed using ANOVA. A two-tailed Student’s t-test was used to examine the significance of differences between experimental groups (Excel 2013 Microsoft, USA). When the p-value was less than 0.05, the results were considered statistically significant.

3 Results and discussion

The PVA hydrogels are currently used in many biomedical applications such as drug delivery systems, engineering wound dressings, and scaffolds for wound care (23). Characterization, cytotoxicity, and wound healing tests have been performed.

3.1 Fourier transform infrared spectrophotometer (FTIR)

FTIR spectroscopy was performed to assess the possible interactions between GA and PVA. FTIR spectra of these materials vary according to their compositions and may be able to demonstrate the presence of a complex formation and the interaction between the various constituents. Figure 1 exhibits the characteristics of IR bands of raw GA. The strong and wide absorption band at 3,421 cm−1 is due to the stretching vibration of OH groups. The band at 2,921 cm−1 is assigned to the asymmetric stretching vibration of CH groups. The bands at 1,672 and 1,448 cm−1 are related to the bending of OH and CH groups, respectively. The band at 1,020 cm−1 is due to the stretching of the C–O groups (24,25,26). In the case of pristine PVA film (Figure 1), the spectrum exhibits a strong band at 3,642–3,068 cm−1 which is due to OH stretching vibration mode. The bands at 2,941 and 2,855 cm−1 are corresponding to the asymmetric and symmetric stretching vibration of CH groups. The band at 1,758 cm−1 is related to the stretching vibration of an acetyl carbonyl C═O group. The band at 1,568 cm−1 corresponds to OH and CH bending vibrations (27,28,29).

Figure 1 
                  FTIR spectra of GA, PVA hydrogel, and the prepared PVA/GA
                        x
                     . Concentrations (x) of GA are from 5% to 50%.
Figure 1

FTIR spectra of GA, PVA hydrogel, and the prepared PVA/GA x . Concentrations (x) of GA are from 5% to 50%.

Figure 1 also presents FTIR spectra of PVA/GA x , where x = 5, 10, 15, 20, 25, and 50 wt% of GA. From the figure, it is clear that both OH stretching and asymmetric CH bands of the blended PVA/GA x samples have been reduced in their intensity. Also, the peak position of these two bands has shifted to a lower frequency with the increase of the concentration of GA inside the PVA structure from 5 to 50 wt%. The behavior of the IR spectrum suggests a probable interaction between PVA and GA in their blend. This can be assigned to the cross-linking formation inside the blend PVA samples. This cross-linking comes from the reaction between the OH and CH groups of GA and C═O and OH groups of PVA structure (30,31). This indicates the existence of hydrogen bonding and electrostatic interactions in the blend PVA/GA x samples. This interaction can be confirmed by the presence of the shift of peak position of the C═O band of PVA to lower frequency with the increase of the concentration ratio of GA within the PVA/GA x blend samples. Where the absorption band of C═O for PVA/GA x samples was located at 1,741, 1,724, 1,706, 1,690, 1,706, and 1,689 cm−1 for PVA contains 5, 10, 15, 20, 25, and 50 wt% of GA, in the same order, respectively.

The blending of PVA with GA caused some bands to disappear or overlapped with the absorption bands of PVA such as the band of the asymmetric stretching vibration of CH groups of GA, which was obtained at 2,921 cm−1. This band may be overlapped with the vibration stretching of the OH band of PVA (32). The bands at 1,672 and 1,448 cm−1, which are related to the bending of OH and CH groups, respectively, disappeared. The disappearance of some IR bands for PVA/GA x blend samples may be attributed to the strong interactions between OH and CH groups of GA and the carbonyl groups C═O of PVA. This may be indicating the miscibility of PVA and GA in their blends and the cross-linking formation within the two polymers via their functional groups of them.

3.2 X-ray diffraction (XRD)

XRD analysis of GA, PVA, and PVA/GA x blend (x = 0, 5, 10, 15, 20, 25, or 50 wt%) is presented in Figure 2. Pure PVA exhibits strong diffraction peaks at 2θ = 20°. This demonstrates that PVA is semicrystalline, with both crystalline and amorphous structures (33,34). The occurrence of strong inter and intramolecular hydrogen bonding between distinct molecules of PVA chains is attributed to the crystalline character of PVA (35). It is noticed that PVA/GA x blends show a broad peak at 2θ = 10° and 20°, which is typical of semicrystallinity of both GA and PVA, respectively. These findings show a significant change in the degree of crystallinity of the PVA/GA x blends, as well as an increase in the amorphous regions (36). The broadness of the two peaks is increased with the increase of GA within the PVA sample. It is demonstrated that the GA molecules were successful in disrupting the PVA crystal structure. Broad peaks in XRD are normally related to amorphous material structures. GA molecules have a hydrophilic nature which causes more hydrogen bonding between the GA and water. This leads to an increase in the cross-linking ratio within the PAV/GA x blend sample. This can be explained in terms of a decrease in the crystallinity of our samples, due to bulky units of GA. In other words, the hydrogen bonding due to hydroxyl groups (OH) crystallizes PVA molecules through physical cross-linking, but the steric hindrance of the bulky properties of GA disturbs the PVA chain and decreases the crystallinity. Also, the hydroxyl groups increase the hydrogen bond interactions between OH groups of PVA and the amide groups of GA. This can form a dimer between PVA and GA molecules leading to a rise in the cross-linking between them.

Figure 2 
                  XRD of GA, PVA hydrogel, and the prepared PVA/GA
                        x
                     . Concentrations (x) of GA are from 5% to 50%.
Figure 2

XRD of GA, PVA hydrogel, and the prepared PVA/GA x . Concentrations (x) of GA are from 5% to 50%.

3.3 Microscopic study

For the topographical study of specimens, SEM is a promising technique. It gives important information regarding the size and shape of the examined material. Figure 3 shows the scanning electron micrographs of pure PVA and PVA/GA x (x = 5, 10, 15, 20, 25, or 50 wt%). A smooth and homogeneous surface for pure PVA was obtained, as expected (37). The micrographs reflect the good compatibility and the miscibility of the two polymers of PVA and GA. The membranes’ surface was dense, and no obvious pores were observed. In PVA/GA x blend composites, the cross-linking formation between the OH groups in both PVA and GA as well as the hydrogen bonding formation can be obtained during the blending process. The SEM micrographs of PVA/GA x demonstrate that GA was well dispersed in the PVA matrix. This dispersion increases as the GA concentration increases. These results imply that the incorporation of GA into the PVA matrix has led to morphological changes in PVA/GA x blend composites.

Figure 3 
                  Scanning electron micrographs (x = 10,000, and the scale bar is 1 µm) of the pure PVA and the PVA/GA
                        x
                      hydrogels. Concentrations (x) of GA are from 5% to 50%.
Figure 3

Scanning electron micrographs (x = 10,000, and the scale bar is 1 µm) of the pure PVA and the PVA/GA x hydrogels. Concentrations (x) of GA are from 5% to 50%.

3.4 Swelling characteristics

The impact of adding different GA concentrations on the swelling properties of PVA hydrogel was investigated. As shown in Figure 4, there is a significant increase in % SR as the GA concentration increases in PVA hydrogel. This increase is most likely owing to the ionization of carboxylic groups (COOH) of glucuronic acid (which presents in many gums such as GA), a more hydrophilic structure due to the OH groups, and the space-preventing effect generated by space isomerism during hybrid hydrogel formation. When compared to pure PVA hydrogel, the inclusion of GA boosted the network’s water uptake capacity. Because carboxylic group ionization accumulates negative charges inside the hydrogel network, their repulsion causes additional solvent diffusion into the network (38). Our results are in agreement with other findings in which the addition of polysaccharides improves the swelling capacity of PVA/GA blends (19,39).

Figure 4 
                  Percentage of the swelling ratio of the pure PVA and the PVA/GA
                        x
                      hydrogels. Concentrations (x) of GA are from 5% to 50%.
Figure 4

Percentage of the swelling ratio of the pure PVA and the PVA/GA x hydrogels. Concentrations (x) of GA are from 5% to 50%.

3.5 Cytotoxicity measurement

The lethal dose at which 50% of cell death occurs (LD50) is the main parameter that indicates the safety of the applied concentration. A significant gradual increase in the required concentration induces LD50 as the concentration of GA increases in the hydrogel. These results indicate that GA significantly decreases the cytotoxicity of PVA in a concentration-dependent manner. LD50 ranged from 531 ± 0.17 µg‧mL−1 in case of PVA/GA0% to 600 ± 0.56 µg‧mL−1 for PVA/GA50%. Based upon LD50 results, 100 µg‧mL−1 hydrogel concentration was selected for further application where the cell viability % is ranged between 82.27 ± 0.27 µg‧mL−1 in the case of PVA/GA0% and 89.23 ± 0.53µg‧mL−1 for PVA/GA50%. The cytotoxicity data are shown in Figure 5.

Figure 5 
                  The cytotoxicity assessment of the pure PVA and the PVA/GA
                        x
                      hydrogels. Concentrations (x) of GA are from 5% to 50%.
Figure 5

The cytotoxicity assessment of the pure PVA and the PVA/GA x hydrogels. Concentrations (x) of GA are from 5% to 50%.

Many researchers agreed that GA has significant properties that can improve the cell viability with the great biodegradability of PVA-GA (40). GA has been shown to have powerful antioxidant effects (41). GA extract is also reported to be hemostatic, nonhemolytic, and antimicrobial in nature (42).

3.6 Wound healing test

Thousands of patients suffer from various types of epidermal or skin damage each year (5). Wound healing is a complicated process in which several types of cells play essential roles at various stages. As a result, different wound healing treatments must be used at different times. Stopping bleeding and avoiding pathogen infection are critical in the early stages (43), while avoiding scar formation and speeding up wound healing time are critical in the later stages (44). Providing moist conditions during the wound healing process can accelerate the fibroblast cell proliferation and avoid the creation of scars (45), can also reduce the pain sensation, and prevent the water loss from the wound as in burn wound cases (46). All that can be achieved by the properties of PVA-GA, and as GA concentration increases, these properties increase. Full epithelialization and wound healing are achieved through fibroblast proliferation and keratinocyte migration. This process is highly improved in the lack of wound exudates, which can be absorbed and retained by hydrogels. GA is hydrophilic and so can improve wound healing by creating a moist environment that minimizes necrotic tissue buildup through apoptosis, accelerates angiogenesis, and enhances the interaction of growth factors with their target cells (47).

In this study, the wound punch for all the groups started at 5 ± 0.056 mm at 0 h. The effect of the different PVA/GA x groups on the wound healing compared to control is shown in Figure 6, indicating the concentration-dependent manner of improvement in wound healing as GA concentration increases in the hydrogel. The wound is completely healed at 96 h in the control group while it is achieved at only 48 h in the PVA/GA50% group as shown in Figure 7. An extremely high statistically gradual decrease in the percentage of original wound area with time as the GA concentration increases in the hydrogel.

Figure 6 
                  Percentage of wound area with time for control, pure PVA, and the PVA/GA
                        x
                      hydrogels. Concentrations (x) of GA are from 5% to 50%.
Figure 6

Percentage of wound area with time for control, pure PVA, and the PVA/GA x hydrogels. Concentrations (x) of GA are from 5% to 50%.

Figure 7 
                  Inverted microscope images of percentage of original wound area for control, pure PVA, and the PVA/GA
                        x
                      hydrogels. Concentrations (x) of GA are from 5% to 50%.
Figure 7

Inverted microscope images of percentage of original wound area for control, pure PVA, and the PVA/GA x hydrogels. Concentrations (x) of GA are from 5% to 50%.

Aside from GA’s moist, antioxidant, and antibacterial properties, several studies have been conducted on the structural and electrical conductivity properties of GA (48), indicating that GA can change the physical and electrical properties of a synthetic polymer like PVA by increasing its conductivity value by two to threefold, potentially speeding up wound healing (49,50,51).

4 Conclusion

We have reported the synthesis and characterization of PVA hydrogel blended with different GA concentrations. The addition of GA highly improves the swelling properties of PVA hydrogel. The cytotoxicity of the prepared hydrogel decreases as the GA concentration increases. The required concentration that induces 50% of cell death (LD50) is 531 ± 0.17 and 600 ± 0.56 μg for pure PVA and PVA/GA50%, respectively. The results indicate a significant gradual decrease in the wound area with time as the GA concentration increases in the PVA hydrogel. Complete wound healing is achieved at only 48 h in the PVA/GA50% group compared to 96 h in pure PVA. Blending GA with PVA improves the properties of PVA hydrogel for biological application.


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  1. Funding information: Authors state no funding involved.

  2. Author contributions: Mohamed A. Elblbesy: conceptualization, formal analysis, methodology, writing – original draft, funding acquisition, validation; Taha A. Hanafy: conceptualization, formal analysis, methodology, funding acquisition, writing – original draft; Mamdouh M. Shawki: conceptualization, formal analysis, methodology, writing – original draft, funding acquisition, investigation.

  3. Conflict of interest: Authors state no conflict of interest.

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Received: 2022-02-20
Revised: 2022-05-12
Accepted: 2022-05-13
Published Online: 2022-07-02

© 2022 Mohamed A. Elblbesy et al., published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

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