Abstract
Novel physically-crosslinked PVA membranes blended with Aloe vera extract were fabricated by solution-casting method. Physically-crosslinking process is depending on the rearrangement of PVA chains forming intermolecular hydrogen bonding with removal of water molecules using propanol as a stabilizing agent. The structure of crosslinked membranes was characterized by FT-IR, SEM, TGA, and XRD analyses and confirmed via gel faction and swelling ratio studies. Caffeine and vitamin C loaded-PVA/Aloe vera membranes were bio-assessed in terms of their impact on the wound healing using Wistar albino rats as an animal model. In vitro evaluation includes protein adsorption showed that the fabricated membranes improved significantly the wound healing ability via enhancing the tissue platelet aggregation. In addition, resulting adequate in vitro release behavior for the loaded ingredients in the potential application. In-vivo results displayed that rats full-thickness wounds were remarkably reduced after PVA/Aloe vera/Vitamin C membranes treatment, as shown by a reduction in the area of the wounds when compared to wounds treated with cotton gauze and PVA/Aloe vera membranes. Furthermore, the treated wounds with PVA/Aloevera/caffeine show more wound closer comparing to that incorporate vitamin C and the PVA/Aloevera incorporated both caffeine and vitamin C give the most significant healing that show reappearance of hair covered the wound area. Histological examinations of wounds covered in membranes showed a successful re-epithelialization, demonstrating caffeine's and vitamin C’s efficacy. These results demonstrated that, PVA/Aloe Vera/Caffeine and PVA/Aloe vera/vitamin C membrane have remarkable wound healing and skin regeneration properties.
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Introduction
PVA is currently one of the most popular and oldest synthetic polymer hydrogels, and because of its excellent biocompatibility, it has been used in many advanced biomedical applications [1]. PVA hydrogels could be prepared by many different crosslinking methods chemically and physically without using chemicals that increase its pharmaceutical applications. Furthermore, hydrogels of PVA could match the water content and mimic the mechanical properties of tissues enhancing their wound healing applications [2].
However, PVA hydrogels application as a standalone wound dressing polymeric material is limited by its lack of adequate elasticity, rigid membrane, and hydrophilicity. The abundance of such polymers, ease of chemical derivatization or modification, and generally good biocompatibility make hydrogels made using PVA combined with polysaccharides, some synthetic polymers and other naturally extracting products appealing among the different hydrogels described in the literature [3].
Aloe vera has been employed for numerous centuries for its restorative and therapeutic capabilities. Several therapeutic actions for extracts of aloe leaf have been ascribed to polysaccharides established in the internal parenchymatous leaf tissue [4, 5]. After topical and systemic administration, it was demonstrated in multiple studies that aloe gel might enhance wound healing. Aloe gel’s ability to promote wound healing has been attributed to a number of methods, including keeping the site moist, enhancing epithelial cell migration, accelerating collagen maturation, and reducing inflammation [6]. Glycoprotein that was isolated from Aloe vera exhibited rise in cell migration and accelerated wound healing in a human keratinocyte monolayer. In a raft culture it exhibited stimulation of epidermal tissue formation as well as marked expression of proliferation markers on the immune-histochemical level. In hairless mice, the improved effect of this glycoprotein fraction on wound healing and cell proliferation was demonstrated [7].
Aloe vera extract naturally needs to be stabilized for membrane formation, which may be accomplished by combining it with another substance that has the potential to crosslink, such as PVA which can also mimic real tissues and can be entirely absorbed by the body. Chemical crosslinking could be used to create PVA hydrogel by different methods (e.g. polymerization reaction radically, using crosslinking chemical reagent, irradiation by high energy, or crosslinked enzymatically). Additionally, there are physical reactions that can crosslink molecules, such as ionic contact, chain crystallization polymerization, hydrogen bonds between chains, interactions of proteins, and the creation of amphiphilic block and graft copolymers [8, 9]. Our newly developed physical crosslinking method also depends on hydrogen bonding between chains. This could occur if the remaining water in the hydrogel film interconnects is removed using propanol alcohol, allowing intermolecular hydrogen bonds in PVA for replacing PVA-water hydrogen bonds. Additional crystallization results when fully hydrolyzed PVA membranes are cast in Petri dishes, dried at 36 °C for 24 h, and then soaked for 8 h in propanol. The membranes formed show enhanced mechanical strength which helps to increase the crystallization degree and, in turn, cross-linking degree of PVA membranes [10].
Among other beverages, caffeine (1,3,7-trimethylxanthine), a purine alkaloid present in plants, can be found in coffee, tea, cocoa, and soft drinks [11, 12]. Alkaloids present in coffee, cola nuts, tea, mate, cacao beans, and further plants are the three distinct methyl-xanthine’s caffeine, theophylline, and theobromine. Caffeine (1,3,7-trimethylpurine -2,6-dione) is a methyl xanthine, derived from compound of purine base that found in many organism tissues which may act as anti-inflammatory and immune-depressant agent. It has anti-inflammatory, antibacterial, skin microcirculation-activating, and sweet smell properties [13]. Compared to a foam pad and a gauze soaked in saline, caffeine promotes faster wound healing and less cell separation from the wound surface [14]. Coffee’s anti-bactericidal mechanism produce hydrogen peroxide and hyperosmotic juice, which kill bacterial cells [15]. It is referred to as a topical wound wrap that improves therapeutic outcomes by promoting the creation of new cells. Coffee powder can help with all therapeutic damage stages, reduce inflammation, promote collagen synthesis, and improve epithelialization for a faster healing process [16].
One of the water-soluble vitamins, vitamin C (ascorbic acid), is commonly available in raw fruits, vegetables, and other dietary products such as juices and sports drinks. Vitamin C's goods and by-products are becoming more popular as people become more aware of its health advantages as an essential nutrient. Even goods fortified with vitamin C, such as breakfast cereals, are becoming increasingly popular and in demand. Normally, the amount of vitamin C in different goods varies. Heating and other manufacturing methods can cause vitamin C loss in products. As a result, for quality control purposes, it is necessary to quantify vitamin C levels in foods and pharmaceutical goods [17].
This work aims to fabricate a new physically-crosslinked PVA/Aloe vera hydrogel membranes, depending on the rearrangement of PVA chains forming intermolecular hydrogen bonding with the removal of water using propanol primary alcohol stabilization of membranes originated. Crosslinked membranes were characterized by FT-IR, TGA, SEM, and XRD analyses and physicochemical properties including swelling, gel fraction, release profile, and protein adsorption were studied in details. In addition to in Vitro/Vivo studies, the effect of loaded caffeine and vitamin C on Wister albino rates via assessing the wound closing (%) with their histological changes were assessed, as well.
Materials and Methods
Materials
PVA (99% hydrolyzed, Mwt. ~ 89–98 KDa, Sigma Aldrich, Saint Louis, USA). Aloe vera leaves was obtained from local garden Tanta city, Egypt; while Aloe vera gel was freshly extracted in the lab before immediately using to avoid any oxidation or gel color change. Caffeine (98.5%, Mwt. ~ 194.19 g/mol) was purchased from Across Organics, Cole-Parmer instrument Co. Ltd, UK. Ascorbic acid (vitamin C) was from Merck KGaA, Germany. Propanol (99%, Across Chemicals, Germany). Fetal Bovine serum (FBS) was attained from (GIBCO, UK). Gentamycin Sulfate, Sigma-Aldrich, Germany).
Preparation of Aloe vera Gel
Leaves of the Aloe vera plant (from Barbaensis miller species) were harvested from a home-grown garden placed in the City of Tanta, Gharbeya, Egypt, on experiment day. Freshly plucked Aloe vera leaves ranged in length from 30 to 50 cm, indicating that the plant was three years old. To get rid of any remaining soil, the leaves were rinsed in distilled water. After removing the spikes along with the edge, the leaves were split transversely. A sharp blade was used to delicately separate the dense epidermis from the parenchyma. The parenchyma was extensively cleaned with distilled water to remove surface exudate before being homogenized in a food mixer [10, 18, 19]. The homogenized gel was centrifuged at 3000 rpm for 30 min to remove filaments. The supernatant was recovered and clarified under vacuum through a filter paper to obtain the Aloe vera clear gel.
Preparation of Physically-Crosslinked PVA/Aloe vera/Caffeine and PVA/Aloe vera/Vitamin C Hydrogel Membranes
Briefly, 10 wt.% of polyvinyl alcohol was dissolved in 10 ml of dist. water at 80 °C for 3 h, then mixed with Aloe vera extract in different ratios as displayed in Table 1. The polymeric solution mixture was kept for 2 h at 50 °C under stirring. Caffeine and Vitamin C were added to the mixture to achieve complete concentrations 0.05, 1.0, and 1.5 mg/ml to 60%PVA/40% Aloevera for in vivo studies. Then, using the conventional casting procedure in 9 cm diameter petri dishes, 1.5 mg/ml of gentamicin was applied to PVA/Aloe vera solution. The membranes of PVA/Aloe vera/ caffeine and PVA/Aloe vera/Vitamin C could be obtained after soaking in propyl alcohol for 8 h that converting membranes from soluble to crosslinked insoluble form.
Physicochemical Measurements
Gel Fraction (%)
The fabricated membranes were dried in an oven for 24 h at 50 °C and weighted (W0). To remove the leachable or soluble Aloe vera portions from the membrane, dry membrane samples were soaked in distilled water for 24 h until an equilibrium swelling weight was reached (Ws). The membrane was then weighted again (We) after being dried in an oven at 50 °C. The following equation was used to compute the gel fraction (GF %) [9, 10].
Swelling Ratio (%)
To determine the degree of swelling, membrane samples were divided into (3 × 3 cm) pieces and dried at 60 °C in an oven followed by measuring their weight to be (We). The dried samples were soaked in distilled water, maintained and incubated at 37 °C, then weighted (Ws) at specific interval times. The method described below was used to determine membrane water uptake [9, 10].
Protein Adsorption (%)
Using a UV–visible spectrophotometer set at 630 nm, the amount of adsorbed bovine serum albumin (BSA) from hydrogel membranes was quantified. From standard solutions of BSA ranging from 3.1 to 60 mg/ml, a calibration curve was created.
Membranes that have been cut into (1 cm × 1 cm pieces) was dipped in 10 ml phosphate buffer solution (pH 7.4) and incubated at 37 °C for 24 h till they achieve swelling equilibrium weight. The swelling fragments were shacked for 4 h at 37 °C in a buffer solution containing 30 mg/ml BSA. At the adsorption of protein, the membrane pieces removed gently. Protein concentrations before and after membrane sections were dipped in protein/phosphate buffer solution was measured using albumin reagent kits at 578 nm absorbance range. The difference used to determine protein adsorption for each sample [9].
In-Vitro Caffeine Release Profile
A 2100B disk dissolution system model was used to assess release behavior [20]. Two slice of the fabricated PVA/Aloe vera membranes containing caffeine (each slice has a surface area of nearly 50 cm2 and a membrane thickness ranging from 0.08 to 0.16 mm) were placed in a solution of phosphate buffer (pH 7, at 37 °C), and they were continuously shaken throughout [21]. Each vessel contains 150 ml of the dissolving buffer, the rate of stirring was set to 50 rpm and the test persisted for 48 h. Every 10 min for the first hour, and every hour for the following 4 h, 3 ml of each sample was took. To maintain a sink state, 3 ml of buffer dissolution were replaced after each sample removal. A quartz cuvette of 1 cm in diameter with a UV/vis spectrometer (Ultrospec 2000, Germany) were used to measure the samples at 302 nm. Based on the calibration curve and wave length scanning, the concentration of caffeine released from the PVA/Aloe vera hydrogels into the buffer was calculated. Three replicas of each experiment were performed.
Membrane Characterization
FT-IR Analysis
To determine the chemical composition of the chemicals in samples of PVA, PVA/Aloe vera, PVA crosslinked and PVA uncrosslinked that have been vacuumed, dried and analyzed by FTIR devices (EQUINOX 55, BRUKER, Germany). The FTIR spectra were produced by recording 64 scans with a 2 cm−1 resolution between 4000 and 400 cm−1.
XRD Analysis
To study the crystalline structure of the prepared membranes XRD-SHIMADZU 7000 Kyoto, Japan, diffractometer was used to record the X-ray diffraction (XRD) patterns using a filtered Cu Kα radiation source at 40 kV and 30 mA. The 2θ degree of each in the range (0–60) on the equatorial peak and the meridian of these membranes was measured for 10 min.
SEM Investigation
Field-emission (FE) scanning electron microscopy (SEM) model (Zeiss Leo Supra 50 VP) was used for examining the morphology of the surface of dried membrane sample layers. To minimize electron charging effects during FE-SEM characterization, the samples were sputtered with a (B5 nm) thin gold coating before analysis. The samples coated by gold were then attached to metallic stubs with conductive carbon tape before being placed in the microscope’s vacuum chamber for image capture.
TGA Thermal Analysis
The effect of heating temperature on PVA/Aloe vera based stabilized hydrogel membranes formed containing Caffeine and Vitamin C as weight loss was investigated using thermogravimetric analysis (TGA) model: (Shimadzu TGA–50, Japan) in a typical measurement. TGA was performed using a thermogravimetric analyzer at temperatures of 10 °C min−1. The loss of weight was tested at a rate flow10 mL min−1 under ambient nitrogen starting from room temperature to 800 °C.
In-Vivo Experiments
All in-vivo experiments were carried out in accordance with Tanta University’s ethical institutional protocols, which were approved by the University’s Research Committee with number (IACUC-SCI-TU-0299). In the in-vivo experiments, these guidelines include the proper protocols for feeding, locating, and sacrificing rats. Each group rats number initially set to n = 5.
Wound Closure (%)
All animal studies followed the “Guide for the Care and Use of Laboratory Animals.” As models for animal wound, male Wistar Albino rats (200–250 g, 8 weeks old) were chosen. After xylazine (5 mg/kg body weight) and ketamine hydrochloride (100 mg/kg body weight) anaesthesia, the rats dorsal hair was shaved. A circle with a diameter of about 2 cm was outlined by a marker, and the circular piece of full-thickness skin was excised with a sterile surgical scalpel and a pair of scissors. Prepared membranes were then cut into circular pieces with a 2 cm diameter (the same as the wound diameter) and soaked in sterile PBS immediately before their topical application on the wound sites every three days for two weeks., The change in the wound area was monitored on days 3, 7, 10, and 14 days the Wistar rats were divided into four groups at random (each group contain eight rats, n = 8).
The first group rats were given sterilized 60% PVA/40%Aloe vera/ Caffeine (0.05, 0.1 and 0.15 mg/ml) physically crosslinked prepared membranes that were chosen after in-vitro testing with negative control of sterile gauze. The second group is 100% PVA included vitamin C (0.05, 0.1 and 0.15 mg/ml) with negative control. The third group is 60%PVA/40%Aloevera/ vitamin C (0.05, 0.1 and 0.15 mg/ml) with negative control. Finally, the fourth group 60%PVA/40% Aloevera/ caffeine /vitamin C (0.05, 0.1 and 0.15 mg/ml for each one) with a negative control, which covered using a sterile layer of gauze (NC). All treatments were fixed with sterile gauze and fixation medical tape. The scaffolds and control membranes were changed by new ones on the third, seventh, tenth and fourteenth days [22, 23], then removing the dresses to evaluate the wounds and calculate the degree of wound closure. The equation below was used to determine the percentage of wound reduction.
Histological Study
Extracted skin tissues containing wound sites were promptly fixed in 10% formalin neutral buffered (pH 7.2) for 48 h. The biopsies were partitioned to a 5 μm thickness after being embedded in paraffin. Finally, Haematoxylin and Eosin were used to stain the sections (H&E). Epithelialization, inflammation, tissue granulation development and deposition of collagen were examined in distinct groups in a histomorphometric investigation.
These characteristics were evaluated semi-quantitatively on a 5-point scale in a histomorphometric study: 0 (absence), 1 (25%), 2 (50%), 3 (75%), and 4 (100%) (100%).The outcomes for these measures were evaluated by a comparative study performed by one independent observer who was blind to the treatment groups.
Statistical Analysis
Computerized (Aperio, Leica Biosystems) CS histological analyzer bright field scanner device was used to scan stained slides at a magnification of 209 times. After that The Image Scope tool (Aperio, Leica Biosystems) was used to evaluate digital slides. Dunn's test was used to compare all means after Kruskal Wallis analysis to compare all scores. Using (GraphPad prism 7 software), a statistical analysis was carried out. The difference between the results was believed significant statistically when the P-value was less than 0.05.
Results and Discussion
Synthesis of PVA/Aloe vera Hydrogel Membranes
To avoid reactive species introduction that could reduce and compromise the biocompatibility, chemical crosslinkers were avoided here. Where, there are methods for producing physically crosslinked PVA hydrogels by crystallization partially, including freeze–thaw alternate procedure [9], which it used to make PVA solution. Yao et al. [24] describe what appears to be a unique and direct method for physically-crosslinking 100% hydrolyzed electrospun PVA fibers while preserving the mate integrity via lower alcohols using like methanol or ethanol. Herein, we prepared a new non-spun fibers hydrogel membrane using classic casting of completely hydrolyzed PVA in a lower alcohol, e.g. propanol to form an ordered morphological hydrogel film as reported also via our published work [10].
Figure 1S (supplementary data) shows a photo of fully hydrolyzed PVA hydrogel membranes composed of PVA/Aloe vera/Caffeine, PVA/Aloe vera/Vitamin C and PVA/Aloe vera/ Caffeine/Vitamin C through casting PVA solution in Petri dishes, followed by drying for 24 h at 36 °C, and then soaking in propyl alcohol for 5 h. The entire observed mechanical of formed membranes increases as the propanol treatment increases the crystallinity degree and thus the crosslinks numbers in the PVA membranes, as well. As stated by kenawy et al. [10] and Yao et al. [24], this may occur if remaining water in the film is eliminated by alcohol, allowing PVA-water hydrogen bonds to be replaced by intermolecular polymer hydrogen bonds, which causes additional crystallization. The reaction mechanism of PVA hydrogel forming via the complete hydrolyzation of PVA with propanol as displayed in Fig. 2S (Supplementary data).
Instrumental Characterization of PVA/Aloe vera Hydrogel Membranes
FT-IR Analysis
Figure 1 shows IR spectra of PVA membrane, PVA treated membrane using propanol PVA/Aloe vera membrane treated using propanol, PVA/Aloe vera/ caffeine and PVA/Aloe vera vitamin C membranes treated propanol. In case of PVA membrane, the characteristic band is detected at ν 3253 cm−1 which is ascribed to the –OH vibration stretching; while –OH vibration bending of -OH group is represented by the peak at ν 1425 cm−1. Also, the band at ν 2925 cm−1 is corresponds to -CH2 asymmetric group with vibration stretching. The peak at about ν 1633–1561 cm−1 is related to C=C of PVA stretching vibration. Although the band at ν 837 cm−1 is corresponding to the C–C of PVA vibration stretching, the peak that attributed to C–O stretching occurs at roughly at ν 1081 cm−1 [25,26,27]. Incorporation of vitamin C and Caffeine in PVA/ Aloe vera crosslinked membranes confirmed via the appearance of absorption bands around 2100 cm−1 and 1117 cm−1 and disappearance of 1081 cm−1 absorption band in addition to the shift of free –OH to be 3418 cm−1 and 3462 cm−1 instead of 3253 cm−1 non free –OH [28].
FT-IR spectra of compositions of physically-crosslinked hydrogel membranes (a); and XRD patterns of PVA/Aloevera hydrogel membranes (b)
XRD Analysis
Figure 1 displays XRD outlines of native PVA, PVA membranes stabilized with propanol and PVA/Aloe vera stabilized with propanol hydrogel membranes. All patterns presented the key diffraction patterns of PVA about 2θ in range 18–19°. This is completely consistent with the hypothesis that non crosslinked PVA was a partly crystalline substance showed two peaks at 2θ = 19.7 and 40.57. Physically-crosslinked PVA was a more crystalline than non crosslinked showing more beak area, the four beaks were in 2θ = 19.7°, 20.39°, 23.6°, and 41.24°. According to the XRD findings, PVA and Aloe vera molecules in membranes were completely compatible. The strong crystalline reflection at 2θ = 19.7° demonstrates that PVA represents reflections from a monoclinic unit cell (d101).
The four PVA/ Aloe vera hydrogels peaks, on the other hand, did not have such sharp diffraction peaks. Crosslinked PVA/ Aloe vera was a crystalline film that showing more peaks, the four peaks were in 2θ = 18.96°, 19.8°, 28.1°, and 40.5°.
After calculating the degree of crystallization, non crosslinked PVA records (12.18%) crystallinity degree although its sharp two peaks, but crosslinked PVA membranes records about (26.9%) crystallinity degree; because the d-spacing of four peaks while PVA/Aloe vera records (26.45%) crystallinity degree. this might be due to the re-orientation of hydroxyl groups of PVA after treatment with alcohol in more symmetric directions supporting their crystallization.
The occurrence of a very small degree of crystallinity combined with a significant amount of diffuse scattering in PVA hydrogels might be due to the co-existence of a small PVA nano-crystalline phase with the bulk amorphous phase, resulting in certain diffraction patterns. With an increase in PVA and a decrease in the incorporation effect of Aloe vera in the hydrogel, there is a strong increase in the average crystallite size and crystallinity degree of obtained crosslinked membranes [10].
SEM Investigation
SEM micrographs revealed that, the shape of the non-crosslinked PVA film has more destroying cross-section than PVA crosslinked membranes (Fig. 2 up), while the same structure was found with the surface morphology of hydrogel membranes (Fig. 2, down). Membranes morphology with high Aloe vera content (Fig. 2) possesses more destroying surface and more amorphous surface and cross-sectional structures. However, the high PVA content in the hydrogel membrane forms a more arranged surface and some vertical stacks-like tubes shape structure or tinny channels in the cross-sectional structures (red arrows in Fig. 2). These results could be explained by the intermolecular hydrogen bonding that caused high developed PVA crystals formation [10, 24], that might enhance the mechanical elasticity. Similarly, it is clear that the high Aloe vera content membranes have the highest porous surface (green circles-in Fig. 2) that serve to increase hydrophilicity which are further confirmed by our results of swelling ratio as described below.
SEM micrographs of cross-sectional structure (up) and surface morphology (down) of PVA crosslinked, non-crosslinked and PVA/Aloevera crosslinked hydrogel membranes (original magnification ×500, scale 50 µm, and applied voltage 25 kV)
TGA Results
Figure 3 represents TGA thermogram of various contents of PVA/Aloe vera based stabilized hydrogel membranes formed containing caffeine and vitamin C. Results reveal significant changes in thermal decomposition profile owing to the variation of formed hydrogel membranes composition based on ratio between PVA and Aloe vera. Table (2) provides an overview of the temperatures of thermal decomposition for weight losses of 25, 50, and 75% of the original film weight.
TGA thermographs results of PVA with different contents of Aloevera stabilized hydrogel films
The initial degrading stage, which is attributed to the elimination of residues of water, humidity, or solvent vapor, is noticed between 50 and 150 ℃. Due to the breakdown and volatilization of the organic constituent of polymer such as PVA, the second degradation stage, which is detected between 250 and 500, causes the greatest residual weight loss. After about 500 °C, the curves all become flat, and the majority of the organic wastes are totally volatilized. This is the third breakdown process. It is evident that as Aloe vera concentration in hydrogel membranes grew, the temperature at which the film samples lost 50% of their original weight also increased, as shown in Table 2. This confirmed that, the Aloe vera addition improve the PVA/Aloe vera incorporated caffeine and vitamin C membranes thermal stability, due to the possibility of hydrogen bonding between the hydroxyl groups in Aloe vera and propanol with those in PVA.; in addition to the high thermal stability of native Aloe vera. The current findings are in line with those by Kenawy et al. [9, 10], who found that PVA-HES hydrogel membranes' thermal characteristics were greatly improved by adding vitamin C.
Physicochemical Properties of PVA/Aloe vera Hydrogel Membranes
Gel fraction (%)
Figure 4a shows the gel fraction (%) values of formed physically-crosslinked PVA/Aloe vera incorporated caffeine and vitamin C composite hydrogel membranes. It is clear that, the composite of highest content of PVA hydrogel membranes produces the highest physically-crosslinking degrees and the highest stability. However, incorporation of Aloe vera into PVA hydrogel membranes content decreases progressively the gel fraction percentage, in the same time the highest Aloe vera content forms the least crosslinking degree, confirming PVA crosslinking. Increased fully hydrolyzed PVA content in the film allows for more hydrogen bonding to be reformed by more crystallization, causing increasing of membrane stability and increasing the gel fraction formation [10, 24].
Effect of (PVA: Aloevera) varied ratios composition on the formed physically crosslinked composite hydrogel membranes; (a) gel fraction and (b) swelling ratio
Swelling Ratio (%)
Figure 4b shows the water uptake (%) for PVA/Aloe vera incorporated caffeine and vitamin C crosslinked membranes. It is clear that, membranes containing only PVA containing caffeine and vitamin C have the least water uptake, and with incorporating Aloe vera into the hydrogel of PVA/caffeine or PVA/vitamin C membranes increases their swelling behavior, and increasing the content of Aloe vera strongly increases the swelling behavior. This may be because the Aloe vera gels' high hydrophilicity and random dispersion increasing of amorphous pours in the PVA crystallized [10, 24].
Protein Adsorption (%)
The amount of plasma protein adsorbed onto the surface of the membrane, is a necessary test for wound dressing application. It was used to evaluate the adsorption of proteins onto the surface of membranes and the compatibility of the manufactured membranes with blood serum. Where, adsorption of blood proteins does could start the clotting process. The adsorption of bovine serum albumin (BSA) on the surface of PVA/Aloe vera/caffeine/vitamin C crosslinked membranes is shown in Fig. 5. Adsorbed BSA onto membrane surfaces as a function of surface area was studied and shown after 24 h in Fig. 5a
Protein adsorption (%) (a) of physically crosslinked PVA/Aloevera/caffeine/vitamin Chydrogel membranes. The mean standard deviation (SD; n = 3) is used to express the values. Released caffeine profile graph from PVA and PVA/Aloevera stabilized films (b)
It is evident that, increasing substitutions leads to a considerable increase in protein adsorption due to increased hydrophobicity [29]. Protein hydrophobicity outside influences protein adsorption at interfaces between solid and water. Protein molecules change their conformations more on hydrophobic surfaces than on hydrophilic surfaces. This is because the protein's hydrophobic portion interacts with the substrate's hydrophobic portion. Thus, the adsorbed BSA onto PVA/Aloe vera hydrogel membranes slightly and increased progressively as the amount of Aloe vera in the crosslinked hydrogel membranes of PVA increased.
Caffeine Release Profile (%)
The calibration curve of caffeine at 300 nm was represented in Fig. 3S (supplementary data). In phosphate buffer (pH 7) at 37 °C, caffeine release profiles from stabilized PVA/Aloe vera membranes with various Aloe vera contents are shown cumulatively in Fig. 5b. Caffeine's initial rate of release was moderate from PVA/Aloe vera hydrogels, especially after the release profile firstly 20 min, and it only minimally increased over the course of 48 h. This initial rate of release may be due to the rapidly caffeine diffusing from hydrogel membrane's near the surface area by the buffer solution's de-swelling from the caffeine-loaded hydrogel.
The lowest release rate of caffeine is detected in this order of release (100PVA < 80PVA/20Aloe vera < 60PVA/40Aloe vera < 40PVA/60Aloe vera < 20PVA/80 Aloe vera) after 48 h. In conclusion, the caffeine cumulative percentage increased notably with increasing the contents of Aloe vera in PVA/Aloe vera hydrogel membranes.
Caffeine was later released from the hydrogel more slowly than it was at first, reaching 50% of its original release after 4 h. The production of porous structure shape and spongy of PVA/Aloe vera hydrogels having high contents of Aloe vera, as was proven in SEM investigations in Fig. 2, could resulting increase in caffeine release profile as Aloe vera content increases. Additionally, as indicated in Fig. 4b, high Aloe vera concentration in hydrogels enhances the diffusion of loaded caffeine due to high water uptake possibility. This suggests that diffusion during high water uptake and the porous structure of carrier hydrogels are thought to be the main factors controlling the release of caffeine from PVA/Aloe vera hydrogel membranes. As a result, the PVA hydrogel's diffusivity decreased and the rate of drug release was reduced by the polymer's increased crystallinity [20, 21].
In-Vivo Experiments
In-Vivo Wound Closure Assay
Inflammation, the formation of new tissue, angiogenesis, and tissue remodeling are all steps of wound repair, which is a crucial homeostatic function. A major issue for individuals with venous insufficiency is abnormal or insufficient wound repair, which adds to the impairment experienced by patients with diabetes mellitus [30].
Influence of Caffeine Incorporation in Propanol-Stabilized PVA/Aloe vera Hydrogel Membranes on the In-Vivo Wound Closure (%)
Figure 6 illustrates the wound healing process in various PVA/Aloe vera/caffeine groups on the first, third, seventh, tenth, and fourteenth days of the in-vivo experiment. The diameter of wound changes over time as a result of the various caffeine concentrations. Wound closure (%) through the aforementioned groups’ detailed quantitative results are presented and summarized in (Fig. 4S, supplementary data), as well. It was observed through the presented images that; caffeine works by blocking adenosine receptors. Caffeine binds competitively adenosine receptors preventing signaling from dampening, thereby sustaining activation and increasing energy production [16]. The purine nucleoside, adenosine, and adenosine-receptor agonists have been demonstrated to improve wound healing [31, 32] in healed wounds by activating angiogenesis [33, 34] and boosting extracellular matrix production [35]. An agonist of the adenosine A2A receptor accelerates wound healing [30]. In vitro, wounds were treated with an adenosine receptor agonist increased fibroblast migration, while wounds were treated with an adenosine receptor agonist showed an increase in matrix and fibroblast infiltration [31]. Adenosine affects wound healing by binding to and activating a family of G protein-coupled receptors known as A1, A2A, A2B, and A3 [36], which regulate cyclic adenosine monophosphate (cAMP) levels [32].
Photographic images examination for monitoring the wound size reduction (wound closure %) as function of post-operation days. Images showing the influence of caffeine incorporation into PVA/Aloevera hydrogel membranes on the wound closure %
From Fig. 4S (supplementary data) the healing impact was seen in wounds treated with various caffeine ratios 0.05 mg/ml, 0.1 mg/ml and0.15 mg/ml after two weeks were about 71.5%, 84.5% and 93.5%; respectively. Gauze was used as the control group, and it only demonstrated about 65.5% wound closure. On day 7, the wounds had partially healed in all of the prepared sample treated groups, with a healing percentage of about 90%.
Influence of Vitamin C Incorporation in Propanol-Stabilized PVA Hydrogel Membranes on the In-Vivo Wound Closure (%)
The outermost epidermis and the deeper dermis are the two main layers of the skin, with very different underlying structures. The epidermis is mostly made up of cells, largely keratinocytes, and performs the majority of the skin’s barrier activities [37]. Keratinocytes are distributed in layers throughout the epidermis, and they begin to differentiate as they divide and proliferate in the direction of the dermis, away from the basal layer. Keratinization is a process that involves the creation of specialized structural proteins, the secretion of lipids, and the construction of a cross-linked protein cellular envelope. Almost all subcellular organelles, including the nucleus, disappear during differentiation [38, 39].
Figure 5S (supplementary data) displays the quantitative details of the wound closure percentages for the aforementioned groups. Figure 6 shows photos of the wound healing process in various groups on the first, third, seventh, tenth, and fourteenth days of the in-vivo experiment for tracking changes in wound diameter over time in response to various concentrations of vitamin C-loaded PVA hydrogel membranes.
Vitamin C levels in normal skin are equivalent to those seen in other bodily tissues and far exceed those found in plasma, implying active circulating accumulation. The majority of vitamin C in the skin appears to be stored in intracellular compartments, with mill-molar concentrations [40,41,42]. Ascorbic acid or vitamin C is important in collagen production, because it functions as a reducing agent which catalysis procollagen conversion to collagen by substituting a hydrogen atom instead of a hydroxyl group [43]. The three stages of dermal wound healing are matrix deposition of connective tissue, contraction, and epithelization.
A period of hemostasis and inflammatory activity precedes this proteoglycans, Collagen, and proteins attachment are deposited during tissue matrix deposition to develop a new extracellular matrix. Contractions are controlled by myofibroblasts, which are dependent on AA for collagen formation [44]. Epithelialization, in turn, is dependent on AA, as this high metabolic process necessitates increased nutrition and oxygen intake. Vascular endothelial cells upregulate hypoxiainducible factor-1a (HIF-1) to enhance angiogenesis via modulating vascular endothelial cell growth factor (VEGF) [45]. Fibroblasts, cells, epithelial, cytokines, and immune cells all play a role in the healing process. The removal of bacteria and tissue debris is aided by neutrophils and specialist macrophages. To do so, they must phagocytize and break down these dangerous microorganisms, for which they are thought to require AA [46]. In fact, the concentration of AA in circulating white blood cells is 10–30 times that in plasma [47]. When you consider the effect of AA shortage on the generation of reactive oxygen species (ROS) in tissue cells, it's easy to realize how it could lead to inefficient wound healing [48]. Figure 6 showed the vital role of vitamin C loaded onto PVA membranes for accelerating the process of healing, compared to control membrane group without vitamin C.
From Fig. 5S (supplementary data), the healing effect was observed after 2 weeks in wounds treated with different ratio of vitamin C, 0.05 mg/ml is about 63.2%, 0.1 mg/ml is about 78.6% and 0.15 mg/ml is about 84%. Gauze, used as a control group, only demonstrated about 55.5% wound closure. All of the wounds in the prepared sample treated groups had around 70% of their original healing capacity by day 7.
Influence of Vitamin C Incorporation in Propanol-Stabilized PVA/Aloe vera Hydrogel Membranes on the Wound Closure (%) In vivo
The detailed quantitative results of the wound closure percentages for wounds treated with different vitamin C contents loaded PVA/Aloe vera hydrogel membranes are presented in Fig. 6S (supplementary data). Figure 7 illustrates the images of wound healing process in various groups on the first, third, seventh, tenth, and fourteenth days of the in-vivo experiment for wound diameter change tracking with time.
Photographic images examination for monitoring the wound size reduction (wound closure %) as function of post-operation days. Images showing the influence of vitamin C incorporation into PVA hydrogel membranes on the wound closure %
As seen in Fig. 7, the healing effect was observed after 2 weeks in wounds treated with different ratio of vitamin C, where 0.05 mg/ml of loaded vitamin C shows healing about 71%, 0.1 mg/ml is about 81.5% and 0.15 mg/ml is about 91.5%; respectively (Fig. 6S, supplementary data). Compared to the highest vitamin C without Aloe vera is about 84% and compered to gauze, as a control group, showed a wound closure rate of only 55.5%. On day 7, 90% or more of the prepared sample treated groups' wounds have partially healed (Fig. 6S, supplementary data) (Fig. 8).
Photographic images examination for monitoring the wound size reduction (wound closure %) as function of post-operation days. Images showing the influence of vitamin C incorporation into PVA/Aloevera hydrogel membranes on the wound closure %
Influence of Caffeine and Vitamin C Incorporation in Propanol-Stabilized PVA/Aloe vera Hydrogel Membranes on the Wound Closure (%) In vivo
As concluded from presented data in Fig. 9 and Fig. 7S (supplementary data), the healing effect was observed after only one week in wounds treated with caffeine and vitamin C (ration 1:1) and Aloe vera, wound is healed about 85%, however when the content of caffeine and vitamin C (1:1) increases, the wound closure % is significantly increased to 95% and 99%; respectively. Gauze was used as the control group and only achieved a wound closure rate of about 55.5%. All of the wounds in the prepared sample treated groups had primarily and quickly healed by day 7, with a healing percentage of about 95%.
Photographic images examination for monitoring the wound size reduction (wound closure %) as function of post-operation days. Images showing the influence of caffeine and vitamin C incorporation into PVA/Aloevera hydrogel membranes on the wound closure %
Histological /Histomorphometric Test
Control negative group A’s epidermis and dermal normal structures can be seen in detail via microscopic images of hematoxylin and eosin-stained skin sections (Figs. 10 and 11). While, epidermal necrosis was seen covered by thick scar (thick yellow arrows) accompanied with granulation tissue (*) infiltrated with intense neutrophils infiltrated with polymorphonuclear inflammatory cells (PMNs) in superficial dermis in the positive control group C + . Microscopic images of skin slices from groups treated with 0.05 and 0.1 mg/ml Caffeine (Fig. 10), demonstrate that wound gaps are filled with the developed granulation tissue (*). Deposition of mature collagen begins (*) in groups that received 0.15 mg/ml of caffeine as a treatment. In groups that received treatments of 0.1 and 0.15 mg/ml caffeine, full new epidermal layer development is complete (blue arrows). The degree of scar contraction, re-epithelization (black arrows), dermal inflammation, neo-vascularization (red arrows) and collagen fibers maturation (*) were screened in treated groups and compared to the control. Scar contraction increased gradually for groups of PVA/ Vitamin C (Fig. 10) and disappeared in groups PVA/ Alovera/ Caffeine/ Vitamin C (Fig. 11). Re-epithelization appears in groups PVA/vitamin C (Fig. 10), compared to groups of PVA/ Alovera/caffeine/vitamin C (Fig. 11). Dermal inflammation slightly decreased in groups 3, moderately decreased in groups PVA/Alovera/vitamin C (Fig. 11) and markedly decreased in groups PVA/Alovera/ caffeine/vitamin C (Fig. 11). Mature collagen fibers deposition (*) starts in groups PVA/Alovera/vitamin C and completed in groups PVA/Alovera/Caffeine/ Vitamin C (Fig. 11). New skin appendages formation appears (blue arrows) in groups 9 &10. (× 100 bar 100).
Representative histological sections of skin tissues for positive and negative control (up), skin tissue regeneration at 14 days’ post-treatment for the different content of Caffeine in PVA/Aloevera stabilized membrane (middle), and skin tissue regeneration at 14 days’ post-treatment of different content of vitamin C in PVA propanol stabilized films (down). All sections were stained with H&E staining
Representative histological sections of skin tissues for positive and negative control (up), skin tissue regeneration at 14 days’ post-treatment of different content of vitamin C in PVA/Aloevera propanol stabilized films (middle), and skin tissue regeneration at 14 days’ post-treatment of different content of Caffeine and Vitamin C in PVA/Aloevera propanol stabilized films (down). All sections were stained with H&E staining
Histomorphometric results confirmed that, membranes treated with caffeine treated have an epitheliogenesis proliferation; especially for the sample of highest caffeine content. Furthermore, growth in tissue granulation and inflammation decrease compared to control group and the increase in mature collagen deposition for caffeine treated groups (Table 3). Group that was treated with caffeine and control gauze show increase in collagen synthesis in that may be the result of an increase in fibroblastic cells.
Conclusions
Novel physically-crosslinked PVA/Aloe vera hydrogel membranes were fabricated and stabilized by propanol. Physically-crosslinking of PVA was further proved via membrane characterization by FT-IR, SEM, TGA, and XRD analyses. Moreover, swelling ratio (%), gel fraction, protein adsorption and caffeine release (%) of formed membranes were studied. The absorption peak of −OH group appeared in FT-IR is confirming the presence of inter and intra-molecular H–H bonding between PVA chains. Also, SEM results showed the blending with Aloe vera interchange the morphology of the surface whereas increasing the content of Aloe vera increased the pores in the surface. Moreover, physically crosslinked PVA membranes exhibited high gel fraction (%), compared to that with PVA incorporated Aloe vera, with less swollen hydrogel membranes. The addition of Aloe vera to PVA enhanced the thermal stability of physically-crosslinked membranes, as shown in TGA chart. In-Vitro biological evaluation showed that, the accumulative release of caffeine and Vitamin C-loaded membranes and protein adsorption increased by increasing the Aloe vera content; which enhances tissue platelet aggregation and improved healing process. However, In-Vivo studies using Albino rates showed that the addition of caffeine and vitamin C to crosslinked PVA membranes dramatically increased the closure and healing rate with an excellent ratio of the wound, compared with both PVA/Alovera and PVA membranes. Furthermore, incorporation of Aloe vera into PVA enhances wound healing, compared with PVA hydrogel membranes. Histopathological examination also revealed that wounds treated with caffeine and vitamin C loaded-PVA/Aloe vera membranes have a high wound closure%, as well as improved re-epithelialization and reduced inflammation. Accordingly, it could be concluded that the membranes derived from caffeine and vitamin C-loaded PVA/Alovera are regarded as effective biomaterials for accelerating of topical wound healing rates.
Data Availability
All data and materials are available in the paper.
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All authors acknowledge Tanta University, Egypt for funding this project.
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Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB). The authors extend their appreciation to the Scientific Research Fund at Tanta University, Egypt for funding this work through research project code: TU:03–19-02.
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MAE-M and ZSG: have conducted the experimental part and written the original and final draft. MEK: has participated the experimental part and tissue culture experiments. EAK and E-RK: study design, characterization and supervision and reviewed the original manuscript.
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Kenawy, ER., El-Meligy, M.A., Ghaly, Z.S. et al. Novel Physically-Crosslinked Caffeine and Vitamin C-Loaded PVA/Aloe Vera Hydrogel Membranes for Topical Wound Healing: Synthesis, Characterization and In-Vivo Wound Healing Tests. J Polym Environ 32, 2140–2157 (2024). https://doi.org/10.1007/s10924-023-03083-7
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DOI: https://doi.org/10.1007/s10924-023-03083-7