Fabrication, Properties, and Stability of Frankincense Oil and Fatty Chitosan/Polyvinyl Alcohol-Based Wound-Healing Hydrogels

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Introduction
Wound healing is a well-known physiological process that the skin goes through in response to injury and is distinguished by the interaction of numerous interconnected stages, including

Thrombogenicity tests
Thrombogenicity experiments were done using a gravimetric method to estimate how much thrombus was present on the surface of the synthetic sponges.
ACD blood was created as previously demonstrated.For 48 hours, membranes were submerged in PBS at 37 °C.
Following the incubation period, the PBS was removed, and the ACD blood was applied over the components that had undergone inspection.A positive control was also created at the same time by introducing the same quantity of ACD blood to an empty Petri dish.To encourage the blood clotting response, 20 ml of a 10 M calcium chloride solution was sprayed onto the sponges.5 ml of water was added to cease the reactions after 45 minutes.Following this, the clots were joined with an additional 5 ml of a 36% formaldehyde solution, dried, and weighed.Five further thrombogenicity tests were reported.

Characterization and Functional Group Analysis
In the current study, novel

Swelling behavior
The hydrophilic functional groups However, when compared to the control combination, the addition of Frank led to a noticeable improvement in water capacity.
The study's findings support the proactive use of pre-made sponge hydrogels for the purpose of accelerating wound healing.

In Vitro Degradation
By immersing PVA composite sponge hydrogels in phosphate-buffered saline (PBS) at a temperature of 37 °C for set periods of time, the in vitro degradation of the hydrogels was studied.
As shown in

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Berthet et al., 2017; Tamer et al., 2018).The body starts vascular contraction and blood coagulation procedures after a skin wound, which stops continuous bleeding and prevent the invasion of dangerous germs.Additionally, blood clots act as a framework that supports dermal cell migration to the site of injury, promoting wound healing and subsequent tissue remodeling (Pereira et al., 2016; Gurtner et al., 2008).However, over an extended period, hemorrhaging has repeatedly emerged as the major cause of fatality among both non-combatants and military people.Trauma victims are more likely to die during the first hour of an event (King, 2019; Alarhayem et al., Ma et al., 2020), both of which slow down the healing process.Membranes, electro-spun nanofibers, and hydrogel are wide used as wound dressings, each with their own unique compositions (Guo et al., 2021; Wang et al., 2017).Hydrogel-based wound dressings, however, provide a few unique benefits over other types of dressings, such as the ability to absorb excessive wound exudates, create a cool environment on the wound surface to reduce discomfort, and maintain the optimal moisture level in the wound bed.These features facilitate the migration and multiplication of dermal cells (Wang et al., 2019; Zhao et al., 2020).Hydrogels are also frequently used as hemostatic dressings in cases of severe bleeding because of the noteworthy adhesive qualities they display (Peng et al., and fibrinogen in the blood (Kim et al., 2021).Although injectable hydrogels have the ability to operate as hemostatic agents (Zhao et al., 2018), their application is constrained by the poor mechanical qualities of these materials.The suffering associated with the replacement or removal of injectable hydrogels is the potential downside for patients, especially those who have sustained major wounds (Sultana et al., 2021).Because of their unique architecture, which provide them a certain level of mechanical stability, 3D sponges with antibacterial, hemostatic, and antioxidant capabilities are therefore useful.Furthermore, it is essential to stress that structural dimensionality plays a critical function in aiding the adhesion and proliferation of cells that are recruited to aid in the process of wound healing (Shefe et al., 2017).Various formulations for sponge hydrogel wound dressings with antibacterial and antioxidant properties have been developed which augmented with either natural extracts or antibacterial and anti-inflammatory drugs (Haidari et al., 2020; Haidari, et al., 2021).They showed how effective they were at slowing down the growth of several dangerous bacteria and accelerating the healing of infected wounds.Cheng et al have prepared a wound dressing with potent hemostatic and antimicrobial characteristics by developing a hydrogel based tannic acid and polyvinyl alcohol (PVA) (Cheng et al., 2022).Recent studies have shown that PVA and chitosan hydrogels are excellent at accelerating the healing of damaged skin due to their antibacterial, antioxidant, and hemostatic mixed before being subjected to sonication and stirred for an hour.The mixture was then put into Petri plates and put through five cycles of -20 °C freezing for 18 hours and -25 °C thawing for 6 hours.Along with a PVA control sponge, samples of sponges with varied amounts of Frank and CS derivatives were identified as PVA, PVA/Frank, PVA/CS, PVA/Frank/CS, PVA/MNHD-CS, PVA/Frank/MNHD-CS, PVA/DNHD-CS, and PVA/Frank/DNHD-CS, respectively.The produced sponges were lyophilized after being flash-frozen in liquid nitrogen for ten minutes.Characterization of the Sponges FT-IR (KBr) apparatus (Shimadzu 8400S, Kyoto, Japan) was set up to do a total of forty scans on each sponge between the wavelengths of 400 and 4000 cm -1 Each sample was coated with a thin layer of gold under vacuum before being examined using SEM (Joel Jsm 6360LA, Tokyo, Japan) to look at the morphological alterations of the produced sponges.Thermal gravimetric analyzer (TGA) (Shimadzu 50/50H, Kyoto, Japan) with a heating rate of 10 °C/min and a nitrogen flow of 30 mL/min was used to measure 5 mg of each film in the temperature range of 20-600 °C.Sponge dried for 24 hours at 50 °C in a vacuum oven before being weighed to determine the gel fraction.After that, the sponges were re-swollen in distilled water for 24 hours until the equilibrium swelling threshold for eliminating soluble PVA was attained.The sponges were then weighed and dried in a vacuum oven at 50 °C.Each experiment was conducted five times, and the gel fractions were calculated using the following Equation: Gel fraction (%) = (1) Where W e and W i refer to the weights of the dried sponge and swollen sponge, respectively.The sponges' capacity to swell was determined by weighing them after being immersed in water for a while.500 ml of distilled water were added after 1 g of dry sponge was dipped into them.Dynamic Swelling was done at 25 °C until equilibrium was reached.Each swelled sample was removed at regular intervals and weighed after any water that had gathered on the surface had been carefully wiped away with filter sheets.The swelling ratios were calculated using the following equation five times for each experiment: Swelling ratio (%) = (2) Where Ws denotes the weight of the swollen sponge, while W d refers to the weight of the sponge at the initial time.The measurements were performed in accordance with the procedure outlined by Yin et al to estimate the porosity of the sponges (Yin et al., 2007).The dried weights of the sponges were calculated following a two-hour drying process at 50 °C in a vacuum oven.The samples were then submerged in 100% ethanol for four hours.The expanded sponges were wiped with filter paper to remove extra ethanol before being weighed.Following that, five times through the porosity analyses, Equation was used to and W 2 represent the weight of the sponge before and after immersion in absolute ethanol, V represents the volume of the sponge, and ρ represents the density of absolute ethanol.Dry sponges were weighed, dipped in 3 mL PBS (0.1 M, pH 7.4), kept at 37 °C, and removed at intervals to be gently cleaned with soft papers to remove excess water from their surfaces.The samples were then gently dried under vacuum conditions before being weighed.All experiments were carried out with five independent replications.Antibacterial Assay The antibacterial assessment of the sponges was determined by measuring optical densities and colony-forming units (CFU) per ml.First, overnight bacteria strains cultures were diluted in LB medium before and their turbidities were adapted in accordance with the McFarland 0.5 standard at 625 nm with 2x10 8 CFU/ml (Hasan et al., 2019; Hassan et al., 2022).Next, 100 mL of the diluted bacterial cultures were then added to 10 mL LB medium containing 50 mg of tested sponges, followed by 18 hours of incubation at 37°C with shaking conditions at 150 rpm.As a comparison, bacterial cultures without sponges were used as controls.After incubation, the antibacterial ability was tested by measuring of bacterial growth inhibition using a spectrophotometer at 600 nm, and then the ratio of bacterial growth inhibition was calculated using Equation (4): Bacterial growth inhibition (%) = Where OD c and OD i are the optical densities of untreated and treated bacterial cultures, respectively.Bioactive evaluation of the Sponges Phenolic contents determination Phenolic contents of the sponges were determined by reducing the yellow Folin-Ciocalteu reagent to a blue compound.First, 50 mg of each membrane was immersed in 5 ml of ethanol in order to extract the frank oil content.0.5 mL of sponge supernatant was then added to 2.0 ml of Folin-Ciocalteu reagent (10%, v/v), followed by the addition of 2 ml of sodium carbonate solution (7.5%, w/v).The mixture was kept at 50 o C for 5 minutes, after which the absorbance was measured at 760 nm using a spectrophotometer.The measurements were repeated five times and compared to the stranded curve for gallic acid (0-100 g) solutions.Antioxidant activity evaluation ABTS assay was carried out for this study through generation of radical cations by the reaction of an aqueous K 2 S 2 O 8 (3.30 mg) in 5 ml water solution with 17.2 mg ABTS.The resulting bluishgreen radical cation solution was then stored overnight below 0 degrees Celsius and in the dark.Afterward, 1 ml of the solution was diluted to a final volume of 60 mL with deionized water and labelled as the ABTS •+ solution.For the measurement of total phenolic content, the samples were extracted as described above.Then, 0.1 mL of leachate from each sponge was added to 2.0 ml of ABTS •+ solution.The ABTS •+ examination was conducted five times, and the absorbance at 730 nm was evaluated at several time periods.Hemolysis experiments Hemolysis evaluation of the sponges was carried out with a few minor modifications as previously reported by Yuan (Yuan et al., 2024).To do this assessment, anticoagulated blood 9 ml of blood was mixed with 1 ml of anticoagulant acid citrate dextrose solution (ACD).About 1 cm 2 of each film was submerged in phosphate buffer solution (PBS, pH 7.0) for 72 hours at 37 °C prior to the tested membranes being exposed to blood.The sponges were then submerged in 1 ml of ACD blood for three hours at 37 °C after the PBS had been removed.Comparable amounts of ACD blood were mixed with 7 ml of PBS and water to get the negative and positive control tubes.For proper blood-film contact, the tubes were carefully inverted three times every 30 minutes.The liquids were then transferred to new tubes and centrifuged for 15 minutes at 200 rpm to remove any remaining material.The wavelength represents the absorbance of a tested sponge, OD n represents the absorbance of the negative control, and OD p represents the absorbance of the positive control.

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crosslinked three-dimensional sponges were developed by freezing and thawing PVA and drawing inspiration from Frank, CS, MNHD-CS, and DNHD-CS.Fig. (1) showed FT-IR spectra of the composite sponge hydrogels.The predominant reason of the PVA sponge's hydrophilic characteristics is the presence of hydroxyl groups in the PVA chains, which are characterized by stretching vibration bands between 3200 and 3400 cm −1 Mansur et al., 2004; Mansur et al., 1643 cm −1 correspond to the vibration of the -CH 2 group and the acetyl carbonyl groups, respectively.Moreover, at 1444 cm −1 , the asymmetrical and symmetrical CH bending vibrations of the methyl group were seen.The band at around 1460 cm −1 corresponds to NH bending vibration is present in the sponges containing CS and MNHDCS.Peaks at 1315 and 1076 cm −1 are assigned to CN and COH groups stretching, respectively.In addition to the pre-existing bands assigned in PVA sponges, the addition of Frank leads to the appearance of a separate peak around 1445 cm −1 .A broad band between 3650 cm −1 and 3100 cm −1 , associated with the stretch of the O-H bonds, was clearly observed.As shown in the gravimetric analysis, the hydrogels were hygroscopic and had a great tendency to absorb water.This signal includes both Frank and chitosan O-H structure and water in its different strengths due to the hydrogen bonds formed.Because most important Frank signals overlap with important bands in the hydrogels, it is difficult to appreciate significant changes, although variations in signal intensities indicate the presence of Frank in the hydrogels.

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Fig. (3): TGA graphs of different of composite sponge hydrogels

Figure 4
Figure 4 shows the differential scanning calorimetry (DSC) results for composite PVA hydrogels.Wide endothermic peaks can be seen in the DSC curves in the 70-80 °C temperature range.The release of moisture that was held inside the hydrogel molecules is responsible for these peaks.The findings of this analysis are completely congruent with the authors' earlier work (Mohamed et al., 2018).The relaxation phenomena linked to the hydrogel's crystalline regions is thought to be the cause of the exothermic peaks seen between 105 and 180 °C (Liu et al., 2001).The presence of endothermic peaks in PVA at 217 °C implies the melting of PVA and the subsequent distortion of its crystal structure, which is consistent with the results of a prior study (Gupta et al., 2017).Additionally, the observed decrease in the transition temperature (T m )

Fig. ( 4
Fig. (4): DSC analysis of different formulated PVA sponge hydrogels of sponges with three-dimensional structures have long been recognized for their capacity to improve the swelling characteristics of the sponges (He et al., 2020).The PVA composite hydrogels' in vitro swelling capabilities were the subject of the experiment, as shown in Table 2 and Figure 5b.When CS was mixed, the swelling ratios showed noticeably decreased water capacity, and this decline was made even worse by N-hexadecanyl CS's increased hydrophobicity.According to these results, the PVA sponge's ability to absorb water was evaluated in /CS > PVA/CS > PVA > PVA/MNHD-CS > PVA/DNHD-CS.
Fig. (6), after 72 hours of incubation, the sponge hydrogels that had been treated with Frank and CS derivatives showed weight losses of up to 35%.When contrasting the two groups, it was shown that the PVA group's body weight decreased by 28.08 1.40%.The findings of this study show that Frank oil has a considerable impact on the rate of degradation seen in sponges.According to this study, the hydrolytic breakdown in vitro may have an effect on the drug release found in this investigation, particularly with regard to Frank oil (Liu et al., 2022; Omer et al., 2021).

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Fig. (7): Antibacterial evaluation of different sponge hydrogels against Gram positive and Gram negative bacterial strains.

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Figure 8b shows the time evolution of the PVA and other sponge hydrogels' ethanol extracts' ability to decolorize the ABTS •+ cationic radical.When it came to the ABTS •+ radical, the PVA hydrogel and other hydrogels without Frank showed a modest level of scavenging effect.The presence of hydroxyl groups in the PVA sponge's backbone is most likely the cause of this action.However, the ABTS •+ radical scavenging activity was significantly increased when Frank oil was used in sponges.Insofar as they relate to the quantification of total phenolic content, the findings of this study are consistent with the recommendations made in the preceding section.The phenolic chemicals included in Frank oil have the potential to donate electrons, which causes the reduction of ABTS •+ and subsequent decolourization, which