Evaluation of Anti-Inflammatory Activity of Hydrogel Containing New Hybrid Nano catalyst of Ibuprofen-Loaded on Chitosan Nanoparticles for Full Thickness Burn Repair

Evaluation of Anti-Inflammatory Activity of Hydrogel Containing New Hybrid Nano catalyst of Ibuprofen-Loaded on Chitosan Nanoparticles for Full Thickness Burn Repair | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Evaluation of Anti-Inflammatory Activity of Hydrogel Containing New Hybrid Nano catalyst of Ibuprofen-Loaded on Chitosan Nanoparticles for Full Thickness Burn Repair Seyedeh-Sara Hashemi, Ali-Akbar Mohammadi, Ali-Mohammad Fallah Tafti, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4741694/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective Tissue engineering represents a promising approach for restoring or improving the functionality of damaged or missing tissues. This study investigates the fabrication and characterization of a novel hydrogel scaffold incorporating chitosan nanoparticles and New Hybrid Nano catalyst of Ibuprofen for wound healing applications. Methods The hydrogel was synthesized using a solution casting method and cross-linked with calcium chloride. A new hybrid nano catalyst derivative of ibuprofen exhibiting superior analgesic effects compared to ibuprofen was synthesized and incorporated into the hydrogel. Extensive characterization using FTIR, XRD, SEM, mechanical testing, swelling studies, degradation analysis, and cell viability assays was performed to evaluate the structural, physical, and biological properties of the scaffolds. In addition to, hydrogels containing new hybrid nano catalyst derivative of ibuprofen (compound "a") assessed as wound dressing for full-thickness wound. Results In vitro results demonstrated that the 3% chitosan nanoparticle-loaded hydrogel possessed optimal physico-chemical characteristics, porosity, biocompatibility, and supported human fibroblast cell proliferation. In vivo studies using a full-thickness wound model in rats revealed accelerated wound closure, reduced inflammation, and enhanced angiogenesis for wounds treated with the ibuprofen derivative-loaded hydrogel compared to controls. Discussion Overall, this novel alginate/collagen/chitosan nanoparticle hydrogel incorporating an ibuprofen prodrug represents a promising biomaterial for facilitating wound healing through its analgesic, anti-inflammatory, and pro-angiogenic effects. This represents a pioneering effort in developing ibuprofen-supplemented scaffolds for enhanced wound healing. Anti-Inflammatory Activity New Hybrid Nano catalyst of Ibuprofen Chitosan Nanoparticle Burn Repair Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Tissue regeneration (TE) is a multidisciplinary domain that integrates engineering and natural sciences in the progression of biological materials to substitute, mend, and enhance the functionality of impaired or missing tissues[ 1 ]. Conventional medical and surgical interventions result in adverse effects on patients as a consequence of organ necrosis and tissue depletion. Nevertheless, engineered tissues and organs present a novel approach to address specific ailments. Scaffold production represents a crucial phase in the tissue regeneration procedure[ 2 , 3 ]. The utilization of tissue regeneration scaffolds can alleviate the injury of different tissues. Tissues or synthetic organs are implanted into the patients' bodies, and the scaffolds must correspond to the shape of the impaired tissue or organ architecture. Furthermore, scaffolds ought to furnish suitable mechanical characteristics and steadiness to endure pressures and sustain the soundness of the devised configuration [ 4 , 5 ]. Presently, the benchmark for medical treatment for permanent wound closure is the application of an autologous split-thickness skin graft (STSG) comprised of epidermis and a small segment of papillary dermis. Despite effectively sealing the wound, this procedure commonly triggers the contraction of the wound site owing to the absence of dermis. Augmenting the thickness of the autograft leads to heightened trauma in the donor region and is linked to delayed recuperation of the donor site, formation of scars, alterations in pigmentation, escalated discomfort, and the susceptibility to donor site infections. Moreover, individuals enduring severe burns or trauma confront restricted availability of skin, hence, the thickness of the STSG and the volume of grafted skin tissue must be minimized to enable repetitive harvesting and prompt wound closure [ 6 , 7 ]. Devising a straightforward and efficient treatment approach is imperative to tackle this issue. In recent years, tissue regeneration has achieved notable advancements in formulating biological substitutes to reinstate or enhance tissue functionality [ 8 , 9 ]. Within tissue regeneration, a supportive framework like bioscaffolds can be fabricated by encapsulating the distinct biological and physical attributes of the target tissue to simulate the composition and microenvironment of the tissue for cellular proliferation [ 10 , 11 ]. Production of scaffolds to establish a conducive microenvironment for cellular proliferation serves as a fundamental groundwork for tissue regeneration to mend wounds, encompassing burn injuries [ 12 , 13 ]. Consequently, we opted for a scaffold incorporating alginate/collagen/chitosan nanoparticles in conjunction with a derivative of an ibuprofen medication for wound recovery, alleviation of inflammatory and pain. Sodium Alginate (ALG), a linear anionic polysaccharide sourced from brown algae or bacteria, and hydrophilic nature, exceptional biocompatibility, and remarkable liquid absorption capacity render it’s a compelling choice for wound dressings. Alginate-based wound dressings are as an approved polymeric material in the biomedical and engineering that particularly noteworthy is its ability to activate macrophages and induce monocytes to generate interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) to expedite the healing of chronic wounds [ 14 – 16 ]. Chitosan, a natural polysaccharide extensively employed in medical applications, is derived from chitin and has garnered more attention than its base polymer chitin due to its pH responsiveness, biocompatibility, and bioactive attributes. Chitosan nanoparticles have emerged as promising polymeric and biological nanoparticles that have captured significant interest in recent years. These nanoparticles demonstrate considerable potential as nanocarriers for encapsulating substances like drugs or active compounds, facilitating targeted delivery to specific sites and enabling controlled release [ 17 – 20 ]. Ibuprofen has garnered significant attention in recent years due to its analgesic and anti-inflammatory attributes. Derivatives of Ibuprofen function by impeding the prostaglandin synthesis, leading to its classification as a non-steroidal anti-inflammatory drug as it does not interfere with the pituitary gland and adrenal gland interactions. Despite the advantageous aspects of Ibuprofen, it is accompanied by adverse effects, particularly on the gastrointestinal system. Consequently, there is a demand for the conversion of Ibuprofen into safer prodrugs. Through a sequence of modifications, the aim is to enhance the analgesic properties of the drug while minimizing side effects [ 21 – 23 ]. Methods Materials Sodium Alginate (BDH Co., UK), low molecular weight Chitosan (Sigma–Aldrich Co., Germany), and Calcium Chloride (Merck KGaA Co., Germany), Dulbecco's Modified Eagle Medium (DMEM), 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO) were purchased from were obtained from Sigma-Aldrich (USA). Chi–NPs were provided from US-Nano (USA). Fetal Bovine Serum (FBS) was purchased from Shellmax (Iran). Deionized water was used for all the experiments. Diethyl ether was purchased from Merck (USA). The human skin fibroblast cell line (HNFF-P18) was purchased from the National Cell Bank of Iran (NCBI, Pasteur Institute, Tehran, Iran). Fabrication of hydrogel films Sodium alginate and Collagen (3%w/v) with a ratio of 2/1 were dissolved in distilled water and mixed under magnetically stirring for 24 h. Then, 200 µl, 1%w/v, 3% w/v and 5% w/v of chitosan nanoparticles (Chi-NP) were added into the 10 ml ALG/Col solutions, respectively, and stirred overnight at 400 rpm. Hydrogel crosslinking method The solution containing calcium chloride (CaCl2) 0.25 M as a cross-linker is prepared by combining 254 mg of CaCl2 in 8 ml of deionized water. Then 100 ml of this cross-linker solution is added to the deionized water to crosslink the alginate and preserve the material's structure in the following steps. After the hydrogels were covered entirely with calcium chloride solution for 25 min, washed several times with PBS. Fourier transform infrared (FTIR) spectroscopy The FTIR spectra of Gel, PVA, and TiO2-NPs powders, as well as Gel/PVA and Gel/PVA/TiO2-NPs hydrogel films, were characterized by a spectrometer (Tensor II, Bruker, Germany) to identify their chemical bonds and functional groups in the wavenumber range of 4000–400 cm-1. Mechanical properties of Hydrogel The measurement of the radius and height of cylindrical hydrogel denoted as r and h respectively is essential. The determination of the elastic modulus of the hydrogel is carried out utilizing an electronic universal testing machine operating at a velocity of 1 mm/min until the specimen's height is diminished by 80%. The parameters S and h represent the cross-sectional area and height of the scaffolds, while L1 and L2 signify the pressure levels at the initial and final points of the linear section, and D1 and D2 indicate the displacement of the scaffolds. The calculation of the scaffolds' elastic modulus is performed through the subsequent formula: Ε = ε⁄σ= [(L_1-L_2) ⁄S] ⁄ [(D_1-D_2) ⁄h] Determine the degradation ratio The assessment of the weight loss of the hydrogel scaffolds is conducted over intervals of 3, 6, 9, 12, and 15 days while the scaffolds are immersed in a lysozyme solution at 37℃. The scaffolds are extracted every 3 days, rinsed twice with deionized water, dried, and then weighed. The degradation ratio is computed using the subsequent formula where m0 denotes the initial weight and mt signifies the ultimate weight of the scaffold post-drying: Degradation ratio (%) = ((mt-m0)) ⁄m0 ×100 Water vapor transmission rate (WVTR) The Water vapor transmission rate (WVTR) of samples was determined by fixing 2 cm diameter circular samples using instant glue on the opening of plastic tubes that had been one-third filled with water (approx. 7 ml). A glass desiccator was used to set the environmental humidity at 5075%. After 3 days, the tubes were weighed and VP measured by: $$\:\text{W}\text{V}\text{T}\text{R}=\frac{{\text{w}}_{0}-{\text{w}}_{1}}{\text{A}\times\:24}$$ Where initially weighed ( \(\:{\text{w}}_{0}\) ), weight after 24 hours \(\:\:{(\text{w}}_{1})\) , A is the area (cm 2 ). Three repeats were considered for each sample. Measure the contact angle and conduct a swelling examination The determination of the contact angle of the hydrogel scaffolding is executed utilizing a contact protractor. The assessment of swelling behavior is achieved through gravimetric analysis, where the weight of the dried scaffold (m0) is quantified on an analytical balance, followed by immersion in PBS at 37℃ for 4 hours. After the specified duration, excess moisture on the sample surface is eliminated using filter paper, and the sample is precisely reweighed (mt). The swelling behavior is quantified by employing the ensuing formula: Swelling ratio (%) = ((mt-m0)) ⁄m0 ×100 Evaluate the morphology and size distribution of the pores in Hydrogel The visual examination of the surface morphology of the scaffolds necessitates the use of a scanning electron microscope (SEM) equipped with a voltage of 10 kV. Before imaging, the specimens were sputtered with a thin layer of gold using a sputter coater (Q150R-ES, Quorum Technologies, UK). The average porosity of each sample was determined by the image processing method. Analyze the porosity of hydrogel scaffolds The determination of scaffold porosity entails the utilization of ethanol as a non-solvent liquid for porosity measurement purposes. The methodology involves immersing the scaffold in a graduated cylinder filled with ethanol, proceeding to place it in a vacuum oven for 5 minutes to eliminate air bubbles completely. The initial volume of ethanol denoted as V1 and the combined volume of ethanol and scaffold regarded as V2 are measured. Subsequently, the scaffold is cautiously withdrawn, and the residual ethanol volume is quantified, with this remaining ethanol volume designated as V3. The calculation of scaffold porosity is performed using the ensuing formula: Porosity (%) = [((v1-v3)) ⁄ ((v1-v2)] ×100 Biocompatibility Biocompatibility of the support will be assessed using the MTT technique. In this manner, the supports were immersed in 75% ethanol and exposed to ultraviolet radiation for 12 hours, subsequently the supports were rinsed twice with PBS for 1 hour each time to eliminate the residual ethanol and ultraviolet rays. Next, HDF fibroblasts are seeded on the supports with a concentration of 5x104 cells/mL and cultivated in a complete medium comprising α-DMEM, 10% FBS, penicillin-streptomycin, and 5% CO2 in an incubation chamber with a temperature of 37°C. Following a cultivation period of 3 days, the MTT assay will be measured utilizing an ELISA reader. Procedure for synthesis of new hybrid nanocatalyst of Ibuprofen (IBP) According to our previous study in obtaining the best product of new hybrid nanocatalyst of IBP, first we [BMIm]SS catalyzed the reaction of IBP with epoxides to afford the new IBP 1,2-diol mono esters in good to excellent yields [ 23 ]. The products were tested in vivo for the analgesic properties on female mice using formalin test. Formalin test Experiments were performed on male mice (25–30g, n = 15) purchased and kept in Comparative and Experimental Medicine Center of Shiraz University of Medical Sciences, Shiraz, Iran. Animal selection, care and sacrifice, protocols, were adhered to the Animal Care Committee of Iran Veterinary Organization guidelines. The mice were kept under a standard 12 h light/dark cycle at 21 ± 2ºC and have with ad libitum access to food and water. Animals were randomly divided into 3 equal groups of control, positive control and treatment. Before the inception of the experiment, mice were moved to a testing lab for at least 60 minutes and put in the formalin testing boxes for habituation for at least 30 minutes. A mirror was placed underneath at a 45° angle to allow a clear view of the paws. Formalin (20 µL; SC) was injected into right foot hind. These behaviors were scored and recording in several time (5.10,15,20,25, and 30 min) after formalin injection. In vivo evaluation Adult Sprague-Dawley male rats (200 g, 7 weeks old) were purchased from the Comparative and Experimental Medicine Center of Shiraz University of Medical Sciences, Shiraz, Iran. They were kept under sterilized conditions and light/dark cycle of 12 hours and an ambient temperature of 22.0 ± 2.0˚C. The animals had free access to food and water. During interventions, animals were anesthetized using Ketamine (5 mg/kg; Woerden, Netherlands) and xylazine (20 mg/kg; Alfazyne, Woerden, Netherlands) intraperitoneally (IP). All experimental procedures were according to Iran Veterinary Organization rules and principles of working with laboratory animals (IR.SUMS.AEC.1402.073). A total number of 36 adult healthy rats were enrolled and randomly divided into control and three experimental groups. A 1 cm2 full-thickness (third-degree) burn was induced to all animals in all groups using a plastic ring exposed to boiling water for 10 seconds These groups were observed over 3 time periods of 3, 7, and 14 days to assess parameters such as repair progression, bleeding, angiogenesis, and inflammation. The experimental groups comprised the following: - The first group served as the control, receiving no interventions (3 rat specimens per time point). - The second group was the control group treated solely with hydrogel base, excluding nanoparticles and ibuprofen (3 rat specimens per time point). - The third group underwent therapeutic procedures involving hydrogel with chitosan nanoparticles (3 rat specimens per time point). - The fourth group constituted the second experimental group, subjected to therapeutic measures utilizing hydrogel with chitosan nanoparticles and ibuprofen (3 rat specimens per time point). Subsequent to the interventions, the rats were humanely euthanized on days 3, 7, and 14, and wound samples were collected for histological analysis, preserved in 10% formalin. Subsequently, the samples will be processed into slides and stained with hematoxylin-eosin for pathological examination under a light microscope. Results In order to achieve the desired hydrogel scaffold, several tests were performed in terms of physical and mechanical properties. The primary goal of this research was to form a hydrogel scaffold using natural polymers and to create an analgesic property to it using a new hybrid nanocatalyst of IBP, and then to examine the morphology, physical properties, mechanical properties, cytotoxicity, cell growth and proliferation. Finally, in-vivo test of the scaffolds done. FTIR Characterization of the Samples FTIR spectroscopy was used to investigate functional groups and bonds formed from raw materials as well as bonds formed in nanocomposite hydrogel. For this purpose, the FTIR spectrum was taken from the pure material, hydrogel and synthesized nano-composite hydrogel, and the results are shown in Fig. 1 . The mechanical properties of scaffold: The physical characteristics of all nanocomposite films were assessed in order to gauge the performance of the film in line with its physical capacity. Young's modulus or the gradient of the graph is derived from the linear segment of the stress-strain graph. The linear segment signifies the elastic zone while the non-linear segment signifies the plastic zone. The ultimate tensile strength is the threshold at which the specimen withstands the highest stress and experiences rupture. The outcomes illustrated in Fig. 2 indicate that the ALG/Col membrane exhibits the minimum yield strength and elastic modulus. In light of the outcomes, escalating the loading of nanoparticles in the polymer matrix leads to the enhancement of physical characteristics such as Young's modulus and yield stress. In vitro degradation Biodegradability Fig. 3 delineates the mass reduction of scaffolds over a 21-day period in saline phosphate buffer solution. Alginate/collagen scaffolds alone and with varying proportions of chitosan nanoparticles were assessed for degradability. The findings from the inquiries reveal that hydrophilic ALG/Col frameworks exhibit a relatively rapid degradability rate for the liberation of any medications and antibacterial substances, such as chitosan nanoparticles. Alginate, collagen, and chitosan natural polymers all possess significant water absorption characteristics and thus can be utilized in the production of biocompatible hydrogels. Examination of the swelling behavior of hydrogels and Water contact angle The findings indicate that the proportion of expansion rises steadily with the incorporation of chitosan nanomaterial proportion and remains nearly steady in the 3% and 5% nanomaterial specimen, which is attributed to the interplay of the existing connections and the linking of the compounds. Concerning the inclusion of nanomaterials, various hypotheses were deliberated concerning the extent of hydrogel expansion in the existence of nanomaterials, such that it can be deemed as a highly absorbent material. Also, the increase in the contact angle in the ALG/Col/CS-NPs sample is caused by the irregularity of the surface and the tendency of the sample to water due to the hydrophilic nature of the nanoparticles. Modifying alginate-based films by incorporating nanoparticles in significant concentrations increases its aqueous affinity and, conversely, decreases mechanical properties (Fig. 4 ). These observations are attributed to the stable formation of hydrogen bonds between oxygen in the nanoparticle and hydrogen in alginate, which are potential reaction sites to retain water molecules. Examining the moisture vapor transmission rate : The outcomes of the moisture vapor transmission rate (MVTR) of the specimens are in agreement with the scanning electron microscope (SEM) findings, such that the MVTR of the specimens with lower density surpasses that of the specimens with higher density. It appears that the process of vapor transfer of moisture is reliant on the hydrophilicity of the constituent components of the specimen and the cross-linking (Fig. 4 ). Scanning electron microscope : Examining the hydrogel structure with an electron microscope shows that with the addition of chitosan nanoparticles, the amount of fibers and the size of the pores are gradually reduced and the structure of the layer becomes uniform, which indicates the formation of interaction between polymers and nanoparticles. The pores in the nanocomposite should be such that the fibroblast cells can migrate into the scaffold. Considering that the size of a fibroblast cell is approximately 30–50 microns, the pores should be large enough and interconnected to facilitate the transfer of cells and nutrients to the scaffold. Observing the size of the pores indicates the appropriate concentration of the scaffold containing chitosan nanoparticles (Fig. 5 ). Pain test results of new hybrid nanocatalyst of IBP: As illustrated in Table 1 , the analgesic effect of the new hybrid nanocatalyst IBP; a, b and c, which were the best samples in our previous study, was evaluated by formalin test and the best sample, which was sample a, was selected and added to the hydrogel. This sample consistently outperformed ibuprofen even in the initial and subsequent 5 minutes, showing a significant difference with ibuprofen and a superior analgesic effect. Hence, it is inferred that this compound has a significant analgesic effect compared to ibuprofen. In addition, it can be seen that ibuprofen and the synthesized compound performed better than the control group in both cases. Table 1 Effects of subcutaneous formalin injection (20 µl) into the hairy skin of the right hind leg. The score of formalin-induced nociceptive behaviors was measured every 5 minutes for 30 minutes (n = 15). Compound H5 H10 H15 H20 H25 H30 a4h (Mean ± SD) 0.67 ± 0.06 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 b4k (Mean ± SD) 1.13 ± 0.03 0.03 ± 0.03 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 c4o (Mean ± SD) 1.03 ± 0.03 0.02 ± 0.03 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 Control (Mean ± SD) 1.87 ± 0.03 1.03 ± 0.03 0.72 ± 0.03 0.60 ± 0.05 0.45 ± 0.05 0.45 ± 0.05 IBP (Mean ± SD) 1.58 ± 0.03 0.53 ± 0.03 0.22 ± 0.03 0.12 ± 0.03 0.00 ± 0.00 0.00 ± 0.00 Biocompatibility As the injury dressing typically acts as a transient substitute for the epidermis, it must possess outstanding biocompatibility. Cytotoxicity stands as a crucial criterion for assessing the biocompatibility of substances. The cell survival of murine fibroblasts within the permeable framework configuration was scrutinized for 24 hours and 48 hours utilizing the MTT technique (Fig. 6 ). The elevated viability signifies commendable biocompatibility with negligible impact on cellular growth for the frameworks devised in this study. In Fig. 6 , the cell survival in the control group is inferior to the groups of nanoparticles and IBP, suggesting that nanoparticles in a specific quantity potentially enhance cellular propagation and the proliferation mechanism endures. The acquired outcomes revealed that ALG/Col/3%CS-NPs /IBP specimen exhibits the highest biocompatibility in comparison to other formulated hydrogels. It appears that the incorporation of nanoparticles into the hydrogel can notably enhance cellular propagation as well as cell survival by encouraging cells to elevate growth factors. Nonetheless, the cell survival of the reference and ALG/Col specimens was akin, and the biocompatibility of the ALG/Col/1%CS-NPs specimen was substandard compared to that of the ALG/Col/5%CS-NPs specimen due to the reduced porosity, which could adversely impact cellular adhesion. Also, the presence of IBP not only did not decrease cell viability, but also increased cell viability. In vivo wound healing evaluation Derived from the outcomes obtained from laboratory examinations, the finest Samples were chosen and contemplated for in vivo investigations. Amid the diverse proportions of chitosan nanoparticles, 3% was regarded as optimum, and amid the variations of ibuprofen, derivative was designated as prime, and hydrogels encompassing 3% of chitosan nanoparticles and 10 mg of ibuprofen derivative a were taken into account. Executing in vivo investigations to cure the full thickness wound of male rats in the control and treatment groups were assessed. Wound assessment was carried out on days 0, 3, 7 and 14, and the morphological exploration exhibited a remarkable decline in the dimensions of the wound on day 7, and visual juxtaposition of the wound on day 14 indicated entire closure of the wound in the group containing ibuprofen derivative as opposed to the group lacking ibuprofen derivative and control. This issue denotes that all wounds treated with scaffolds with ibuprofen derivatives close earlier due to the mitigation of inflammation (Fig. 7 ). The pathological assessment on the 7th day subsequent to wound establishment and treatment with alginate-collagen-chitosan nanoparticle and derivative a scaffold in contrast to the control group and positive control reveals the notable impact of this scaffold in the recuperation process, as discerned in the control group, coagulation necrosis and Epidermis is entirely obliterated and not regenerated, there is intense vascular expansion and absence of tissue bud formation. Nevertheless, in the treatment cluster, budding tissue is evolving in the dermis and regeneration of the epidermis has commenced. These outcomes are evidently superior in the group receiving the ibuprofen derivative, signifying that pain management quickens the recuperation process. These outcomes are in harmony with the investigations of other scholars. On the fourteenth day, in the positive control group, the tissue bud is expanding, the inflammatory cells and vascular expansion have dwindled, and we can observe the regularization of the tissue. However, in the treatment groups, we observe an absence of inflammation and no hemorrhage in the tissue, and no infection is observed, and we have incomplete tissue mending with a mature tissue bud, total repair with a mature tissue bud, angiogenesis and regularization of collagen fibers can be observed, inflammatory cells and hemorrhage is not observed. In the group of alginate-collagen-chitosan nanoparticle and ibuprofen derivative, the restoration is entire and the keratin tissue is completely developed. We also observe hair growth in the affected region (Fig. 8 ). Discussion Currently, wound dressings are typically composed of natural materials like collagen, hyaluronic acid or chitosan [ 5 , 24 , 25 ]. These materials exhibit good biocompatibility and promote tissue regeneration, however, the physical characteristics of hydrogels manufactured from these materials are indicated to be relatively subpar. Consequently, they are vulnerable to damage from contact or friction. The findings in our investigation surpassed the outcomes of numerous hydrogels that have been documented [ 26 , 27 ]. Collagen–Alginate hydrogel with a chitosan nanoparticle is a biodegradable polymer with strong mechanical properties, good biocompatibility, low cost, and an easily tunable degradation profile. In the context of a wound dressing, it is imperative for hydrogels to possess the capacity to absorb exudates from the wound area and retain moisture to safeguard the newly regenerated tissue. Alginate is a material with a propensity for attracting moisture that is anticipated to soak up moisture vapor and augment MVTR[ 16 ]. Off-the-shelf dressings exhibit MVTR figures ranging from 76 grams per square meter per day to 9360 grams per square meter per day. Quinn and colleagues demonstrated that an acceptable MVTR of 2500 − 2000 grams per square meter per day can maintain the wound site damp to avert desiccation or excessive fluid discharge[ 28 ]. As depicted in Fig. 4 , all hydrogels exhibit elevated MVTR. The impact of CS-NPs content is delineated substantially augmented the MVTR of the nanocomposite membrane[ 29 ]. This is because the integration of nanomaterials can establish an extensive convoluted route and the interplay between alginate and nanomaterials can considerably enhance the wetness affinity of the nanocomposite membrane[ 29 ]. As well as Integration of CS-NPs into the polymer matrix hinders the mobility of the ALG polymer chain due to the robust interaction between the nanoparticles and the polymer matrix [ 30 ]. Additionally, the elastic modulus surged with the rise in nanoparticle concentration as documented in the literature [ 31 ], despite some authors presenting the contrary trend [ 32 ]. In certain investigations, it has been documented that the particle loading exceeding 1% of nanoparticles results in a reduction in the resilience of the polymer network against fracture and a decline in the robustness of the polymer network, which is an outcome of the polymer distortion around the nanoparticles [ 33 ]. SEM images validated the homogeneous structure and porosity of the scaffolds. The scaffolds exhibited favorable biocompatibility and supported human fibroblast cell proliferation, with the 3% chitosan nanoparticle concentration showing optimal results. Incorporation of chitosan nanoparticles enhanced the scaffolds' properties. In this investigation, we achieved our objective, IBP was successfully incorporated into alginate/ Collagen /chitosan nanoparticle hydrogel to create analgesic, anti-inflammatory, degradable scaffolds for wound healing application. Results showed that proliferation of human dermal fibroblasts to the freshly prepared Ibuprofen loaded scaffolds was not reduced at the new hybrid nanocatalyst of IBP. Also, cell attachment increased in compare in the control unloaded scaffolds. This showed that loading IBP into the scaffolds did not change their basic ability to act as fibers for skin cell attachment and migration which is encouraging for developing these fibers as tissue guides for wound healing applications[ 34 ]. A new hybrid nanocatalyst of IBP exhibited greater pain-relieving impacts than ibuprofen ten minutes following the formalin experiment, substance a demonstrated a superior pain-relieving effect than ibuprofen. Concerning pain-relieving characteristics, the synthesized substances possess a swifter absorption than ibuprofen due to their lipophilicity, hence they can be advantageous for acute pains that are acute, such as burns, cuts, and similar discomforts. We utilized it in hydrogel to manage full-thickness wounds. It is imperative to note that by safeguarding the acidic faction of ibuprofen, we endeavored to diminish its side effects. Regarding wound healing, a certain level of inflammation accelerates wound healing because the release of pro-inflammatory cytokines attracts cells into the wound area the converse is also true and too high a level of inflammation can retard wound healing [ 34 , 35 ]. We observed that our natural hydrogels are able to support the attachment, migration and proliferation of normal skin cells, helping to accelerate wound healing by providing an immediate replacement substrate for undamaged skin cells at the wound margin. The simultaneous release of an anti-inflammatory from this natural scaffold will calm down the aggressive inflammatory beds of the wound and accelerate the healing process. Conclusion We present a conceptually simple approach to a wound healing dressing which provides a temporary substrate for the cells in the wound bed to migrate along while delivering Ibuprofen as an anti-inflammatory agent and pain reliever. This scaffold exhibiting robust mechanical properties, biodegradability, and controlled drug release for mitigating inflammation, and the need to remove this dressing. The hydrogel containing ibuprofen derivatives accelerated wound closure and promoted angiogenesis by supporting cell attachment and migration compared to the control group. This represents a pioneering effort in the development of ibuprofen-containing scaffolds for wound healing. Given that the number of chronic non-healing wounds is high, such dressings could be useful in the treatment of burns and chronic wounds, where approaches to simultaneously manage pain and accelerate wound re-epithelialization are needed. Declarations Acknowledgments Author Contributions Conceptualization, S-S.H., A-A.M., A-M FT., and M.N.SR.; methodology, S-S.H., A-A.M., A-M FT., and M.N.SR.; validation, S.S.H., A.A.M., and A-M FT; formal analysis, S-S.H., and A-M FT; investigation, S-S.H., and A-M FT; resources, S-S.H. and A-M FT.; data curation, S-S.H.; writing original draft preparation, S-S.H., and A-M FT.; writing—review and editing, S-S.H. and A-M FT; visualization, S-S.H., and A-M FT.; supervision, S-S.H., and A-M FT.; project administration, S-S.H., A-A.M., A-M FT., and M.KR.; funding acquisition, M.KR. All authors have read and agreed to the published version of the manuscript. Funding Not applicable. Data Availability Statement The data that support the findings of this study are available from the corresponding author upon reasonable request. Ethics approval and consent to participate The study was approved by the Institutional Ethics Committee of Shiraz University of Medical Sciences (IR.SUMS.AEC.1402.073). Consent for publication Not applicable. Competing interests The authors declare no competing interests. References Hashemi SS, Jowkar S, Mahmoodi M, Rafati AR, Mehrabani D, Zarei M, Keshavarzi A: Biochemical Methods in Production of Three-Dimensional Scaffolds from Human Skin: A Window in Aesthetic Surgery . World J Plast Surg 2018, 7 (2):204-211. Hashemi SS, Mohammadi AA, Kabiri H, Hashempoor MR, Mahmoodi M, Amini M, Mehrabani D: The healing effect of Wharton's jelly stem cells seeded on biological scaffold in chronic skin ulcers: A randomized clinical trial . Journal of cosmetic dermatology 2019, 18 (6):1961-1967. Hashemi SS, Mohammadi AA, Moshirabadi K, Zardosht M: Effect of dermal fibroblasts and mesenchymal stem cells seeded on an amniotic membrane scaffold in skin regeneration: A case series . 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Amirsadeghi A, Jafari A, Hashemi S-S, Kazemi A, Ghasemi Y, Derakhshanfar A, Shahbazi M-A, Niknezhad SV: Sprayable antibacterial Persian gum-silver nanoparticle dressing for wound healing acceleration . Materials Today Communications 2021, 27 :102225. Wainwright DJ, Bury SB: Acellular Dermal Matrix in the Management of the Burn Patient . Aesthetic Surgery Journal 2011, 31 (7 Supplement):13S-23S. Amirsadeghi A, Khorram M, Hashemi SS: Preparation of multilayer electrospun nanofibrous scaffolds containing soluble eggshell membrane as potential dermal substitute . Journal of Biomedical Materials Research Part A 2021. Mehrabani D, Nazempour M, Mehdinavaz-Aghdam R, Hashemi S-S, Jalli R, Moghadam MS, Zare S, Jamhiri I, Moayedi J, Karimi-Busheri F: MRI tracking of human Wharton’s jelly stem cells seeded onto acellular dermal matrix labeled with superparamagnetic iron oxide nanoparticles in burn wounds . Burns & Trauma 2022, 10 :tkac018. Nazempour M, Mehrabani D, Mehdinavaz‐Aghdam R, Hashemi SS, Derakhshanfar A, Zare S, Zardosht M, Moayedi J, Vahedi M: The effect of allogenic human Wharton's jelly stem cells seeded onto acellular dermal matrix in healing of rat burn wounds . Journal of cosmetic dermatology 2020, 19 (4):995-1001. Yanat M, Schroën K: Preparation methods and applications of chitosan nanoparticles; with an outlook toward reinforcement of biodegradable packaging . Reactive and Functional Polymers 2021, 161 :104849. Zhang M, Zhao X: Alginate hydrogel dressings for advanced wound management . International Journal of Biological Macromolecules 2020, 162 :1414-1428. Sobhanian P, Khorram M, Hashemi S-S, Mohammadi A: Development of nanofibrous collagen-grafted poly (vinyl alcohol)/gelatin/alginate scaffolds as potential skin substitute . International journal of biological macromolecules 2019, 130 :977-987. Cai D, Chen S, Wu B, Chen J, Tao D, Li Z, Dong Q, Zou Y, Chen Y, Bi C et al : Construction of multifunctional porcine acellular dermal matrix hydrogel blended with vancomycin for hemorrhage control, antibacterial action, and tissue repair in infected trauma wounds . Mater Today Bio 2021, 12 :100127-100127. Wang Y, Zhao Y, Qiao L, Zou F, Xie Y, Zheng Y, Chao Y, Yang Y, He W, Yang S: Cellulose fibers-reinforced self-expanding porous composite with multiple hemostatic efficacy and shape adaptability for uncontrollable massive hemorrhage treatment . Bioact Mater 2021, 6 (7):2089-2104. Xu J, Fang H, Zheng S, Li L, Jiao Z, Wang H, Nie Y, Liu T, Song K: A biological functional hybrid scaffold based on decellularized extracellular matrix/gelatin/chitosan with high biocompatibility and antibacterial activity for skin tissue engineering . International Journal of Biological Macromolecules 2021, 187 :840-849. Hashemi S-S, Saadatjo Z, Mahmoudi R, Delaviz H, Bardania H, Rajabi S-S, Rafati A, Zarshenas MM, Barmak MJ: Preparation and evaluation of polycaprolactone/chitosan/Jaft biocompatible nanofibers as a burn wound dressing . Burns 2022, 48 (7):1690-1705. Katritzky AR, Jishkariani D, Narindoshvili T: Convenient Synthesis of Ibuprofen and Naproxen Aminoacyl, Dipeptidoyl and Ester Derivatives . Chemical Biology & Drug Design 2009, 73 (6):618-626. Redasani VK, Bari SB: Synthesis and evaluation of mutual prodrugs of ibuprofen with menthol, thymol and eugenol . European Journal of Medicinal Chemistry 2012, 56 :134-138. Rad MNS, Behrouz S, Atashbasteh E, Hashemi S-S: Butyl methyl imidazolium silica sulfate (BMIm) SS: A novel hybrid nano‐catalyst for highly efficient synthesis of new 1, 2-diol monoesters of ibuprofen as the novel prodrugs of ibuprofen having potent analgesic property . Bioorganic Chemistry 2021, 107 :104570. Asvar Z, Pirbonyeh N, Emami A, Hashemi S-S, Fadaie M, Ebrahiminezhad A, Mirzaei E: Enhancing antibacterial activity against multi-drug resistant wound bacteria: Incorporating multiple nanoparticles into chitosan-based nanofibrous dressings for effective wound regeneration . Journal of Drug Delivery Science and Technology 2024, 95 :105542. Hashemi S-S, Pirmoradi M, Rafati A, Kian M, Mohammadi AA, Ali M: A human acellular dermal matrix coated with zinc oxide nanoparticles accelerates tendon repair in patients with hand flexor tendon injuries in zone 5 of the hand . 2024. Chen K, Liu J, Yang X, Zhang D: Preparation, optimization and property of PVA-HA/PAA composite hydrogel . Materials Science and Engineering: C 2017, 78 :520-529. Priya SG, Gupta A, Jain E, Sarkar J, Damania A, Jagdale PR, Chaudhari BP, Gupta KC, Kumar A: Bilayer Cryogel Wound Dressing and Skin Regeneration Grafts for the Treatment of Acute Skin Wounds . ACS Applied Materials & Interfaces 2016, 8 (24):15145-15159. Wu P, Fisher A, Foo P, Queen D, Gaylor J: In vitro assessment of water vapour transmission of synthetic wound dressings . Biomaterials 1995, 16 (3):171-175. Salehi E, Khajavian M, Sahebjamee N, Mahmoudi M, Drioli E, Matsuura T: Advances in nanocomposite and nanostructured chitosan membrane adsorbents for environmental remediation: A review . Desalination 2022, 527 :115565. Cao X, Chen Y, Chang PR, Stumborg M, Huneault MA: Green composites reinforced with hemp nanocrystals in plasticized starch . Journal of Applied Polymer Science 2008, 109 (6):3804-3810. Islam MS, Masoodi R, Rostami H: The Effect of Nanoparticles Percentage on Mechanical Behavior of Silica-Epoxy Nanocomposites . Journal of Nanoscience 2013, 2013 :1-10. Wu S, Chen X, Yi M, Ge J, Yin G, Li X, He M: Improving Thermal, Mechanical, and Barrier Properties of Feather Keratin/Polyvinyl Alcohol/Tris(hydroxymethyl)aminomethane Nanocomposite Films by Incorporating Sodium Montmorillonite and TiO₂ . Nanomaterials (Basel) 2019, 9 (2):298. Zolfi M, Khodaiyan F, Mousavi M, Hashemi M: Development and characterization of the kefiran-whey protein isolate-TiO2 nanocomposite films . International Journal of Biological Macromolecules 2014, 65 :340-345. Cantón I, Mckean R, Charnley M, Blackwood KA, Fiorica C, Ryan AJ, MacNeil S: Development of an Ibuprofen‐releasing biodegradable PLA/PGA electrospun scaffold for tissue regeneration . Biotechnology and bioengineering 2010, 105 (2):396-408. Batool F, Morand D-N, Thomas L, Bugueno IM, Aragon J, Irusta S, Keller L, Benkirane-Jessel N, Tenenbaum H, Huck O: Synthesis of a novel electrospun polycaprolactone scaffold functionalized with ibuprofen for periodontal regeneration: An in vitro andin vivo study . Materials 2018, 11 (4):580. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4741694","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":330818375,"identity":"8567a518-8843-4ed8-a12b-d52e72505103","order_by":0,"name":"Seyedeh-Sara Hashemi","email":"","orcid":"","institution":"Shiraz University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Seyedeh-Sara","middleName":"","lastName":"Hashemi","suffix":""},{"id":330818378,"identity":"a9dbed65-9d9e-4ffa-953d-8d3b3a32a244","order_by":1,"name":"Ali-Akbar Mohammadi","email":"","orcid":"","institution":"Shiraz University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ali-Akbar","middleName":"","lastName":"Mohammadi","suffix":""},{"id":330818381,"identity":"3c8b544a-8c2a-48a1-92d6-9b8e3c30a5af","order_by":2,"name":"Ali-Mohammad Fallah Tafti","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIie2RvwrCMBCHLwTbJdq1DuIzOApKnyW46jucCHHxz6qTDyGIY0PByQdQMvUBhLqpKHhtXW11E8wHB78L95ELAbBYfhAX+TBkCFDj1CVUjluiCJrPFIcUtoA8fKhQw0V6VKq4GsPrFpq0T2XduW2atCFLzv0CRUjU0z20FAfHDGYmDby+3LxXApAYVhWwXJmYLPBqgSK8GPVDQZAp7YkJyhVfYkS3yEyBi5EfKDFGDeX3FGfqOEWThlHhW4TX0/FJdbpzL9odLnfTXY1HOjkXKC98KvodpiAPX3D/ZthisVj+hSfRKU4O1+JZ1AAAAABJRU5ErkJggg==","orcid":"","institution":"Shiraz University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Ali-Mohammad","middleName":"Fallah","lastName":"Tafti","suffix":""},{"id":330818382,"identity":"616b6baa-e026-49d2-8820-81d82fbb63c3","order_by":3,"name":"Marzieh Karami Rad","email":"","orcid":"","institution":"Shiraz University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Marzieh","middleName":"Karami","lastName":"Rad","suffix":""},{"id":330818383,"identity":"2674518f-8621-4064-8244-1f4002fcdb22","order_by":4,"name":"Mohammad Navid Soltani Rad","email":"","orcid":"","institution":"Shiraz University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"Navid Soltani","lastName":"Rad","suffix":""}],"badges":[],"createdAt":"2024-07-15 08:45:44","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4741694/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4741694/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":62270924,"identity":"11b932d7-ecca-41b2-9a5a-3987b331693e","added_by":"auto","created_at":"2024-08-12 10:13:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":61071,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectroscopy diagrams for Sodium Alginate (ALG)/ Collagen (Col), Chitosan nanoparticle (CS-NPs) and new hybrid nanocatalyst of Ibuprofen (IBP)\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4741694/v1/653ef3721f0c528d022e6484.png"},{"id":62270096,"identity":"12a1c9cb-1397-420b-97a6-5069740920a1","added_by":"auto","created_at":"2024-08-12 10:05:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":51012,"visible":true,"origin":"","legend":"\u003cp\u003eMechanical properties of hydrogels, (A) ultimate tensile strength; and (B) young's modulus.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4741694/v1/4e5ca45be93ba1d4bbe74dd4.png"},{"id":62270095,"identity":"6450d914-bebf-4ac5-b5cb-c46a4fd759d9","added_by":"auto","created_at":"2024-08-12 10:05:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":22886,"visible":true,"origin":"","legend":"\u003cp\u003eDegradation behavior of ALG/Col hydrogel and Gel/PVA/CS-NPs nanocomposites (1, 3, and 5%) during 21 days.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4741694/v1/6bd49280be82718ea3e275fb.png"},{"id":62270091,"identity":"7e11ebfa-3703-4e27-8ab4-ecc9c63bee08","added_by":"auto","created_at":"2024-08-12 10:05:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":71875,"visible":true,"origin":"","legend":"\u003cp\u003eThe graph of the Swelling ratio, WVTR, and WCA of ALG/Col hydrogel and ALG/Col/CS-NPs nanocomposites.\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4741694/v1/93f694c05ad6ce11da22bfd6.png"},{"id":62270097,"identity":"dd151505-ca3b-4e9f-9973-47c2bcee4ad7","added_by":"auto","created_at":"2024-08-12 10:05:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":240262,"visible":true,"origin":"","legend":"\u003cp\u003eSEM micrographs of the cross section of various porous scaffolds (ALG/Col (A)and ALG/Col /3%CS-NPs (B)); Pore size distribution histograms of the hydrogels (ALG/Col (C, E.G) and ALG/Col /3%CS-NPs (D, F, H))\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4741694/v1/759e9260b7b3428d66358127.png"},{"id":62270092,"identity":"02ddf4c5-7004-4f17-b53c-a36d2e28a082","added_by":"auto","created_at":"2024-08-12 10:05:38","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":19618,"visible":true,"origin":"","legend":"\u003cp\u003eThe cell viability assessment using MTT assay for different nanocomposites.\u003c/p\u003e","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4741694/v1/3417050615a9a198e09f56a7.png"},{"id":62270094,"identity":"58a7651b-2d7a-45bd-b6fa-e4436fcb1bf1","added_by":"auto","created_at":"2024-08-12 10:05:38","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":366372,"visible":true,"origin":"","legend":"\u003cp\u003eOptical images expressive the wound site at different time points (scale bar = 10 mm), and (b) obtained wound closure percentages for control, Sham (ALG/Col), Treatment 1(ALG/Col /3%CS-NPs), and treatment2 (ALG/Col /3%CS-NPs/IBP) groups (***p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4741694/v1/79aae4122e0148a381e56e8b.png"},{"id":62270098,"identity":"c05e339e-812f-49a8-9082-633fd89a4e5c","added_by":"auto","created_at":"2024-08-12 10:05:39","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":854470,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathological analyses of wound healing. H\u0026amp; E-stained sections of wounds within 3,7,14 days in control, Sham (ALG/Col), Treatment 1(ALG/Col /3%CS-NPs), and treatment2 (ALG/Col /3%CS-NPs/IBP) groups.\u003c/p\u003e","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-4741694/v1/25db7960a254046c725d8319.png"},{"id":70502484,"identity":"ebb4b0a6-3eaa-4fc4-8ee1-ee9342d169f9","added_by":"auto","created_at":"2024-12-03 21:46:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3361562,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4741694/v1/a363c629-1e8b-4e4c-83cd-ed8d57a71a5f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluation of Anti-Inflammatory Activity of Hydrogel Containing New Hybrid Nano catalyst of Ibuprofen-Loaded on Chitosan Nanoparticles for Full Thickness Burn Repair","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTissue regeneration (TE) is a multidisciplinary domain that integrates engineering and natural sciences in the progression of biological materials to substitute, mend, and enhance the functionality of impaired or missing tissues[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Conventional medical and surgical interventions result in adverse effects on patients as a consequence of organ necrosis and tissue depletion. Nevertheless, engineered tissues and organs present a novel approach to address specific ailments. Scaffold production represents a crucial phase in the tissue regeneration procedure[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The utilization of tissue regeneration scaffolds can alleviate the injury of different tissues. Tissues or synthetic organs are implanted into the patients' bodies, and the scaffolds must correspond to the shape of the impaired tissue or organ architecture. Furthermore, scaffolds ought to furnish suitable mechanical characteristics and steadiness to endure pressures and sustain the soundness of the devised configuration [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Presently, the benchmark for medical treatment for permanent wound closure is the application of an autologous split-thickness skin graft (STSG) comprised of epidermis and a small segment of papillary dermis. Despite effectively sealing the wound, this procedure commonly triggers the contraction of the wound site owing to the absence of dermis. Augmenting the thickness of the autograft leads to heightened trauma in the donor region and is linked to delayed recuperation of the donor site, formation of scars, alterations in pigmentation, escalated discomfort, and the susceptibility to donor site infections. Moreover, individuals enduring severe burns or trauma confront restricted availability of skin, hence, the thickness of the STSG and the volume of grafted skin tissue must be minimized to enable repetitive harvesting and prompt wound closure [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Devising a straightforward and efficient treatment approach is imperative to tackle this issue. In recent years, tissue regeneration has achieved notable advancements in formulating biological substitutes to reinstate or enhance tissue functionality [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Within tissue regeneration, a supportive framework like bioscaffolds can be fabricated by encapsulating the distinct biological and physical attributes of the target tissue to simulate the composition and microenvironment of the tissue for cellular proliferation [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Production of scaffolds to establish a conducive microenvironment for cellular proliferation serves as a fundamental groundwork for tissue regeneration to mend wounds, encompassing burn injuries [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Consequently, we opted for a scaffold incorporating alginate/collagen/chitosan nanoparticles in conjunction with a derivative of an ibuprofen medication for wound recovery, alleviation of inflammatory and pain.\u003c/p\u003e \u003cp\u003eSodium Alginate (ALG), a linear anionic polysaccharide sourced from brown algae or bacteria, and hydrophilic nature, exceptional biocompatibility, and remarkable liquid absorption capacity render it\u0026rsquo;s a compelling choice for wound dressings. Alginate-based wound dressings are as an approved polymeric material in the biomedical and engineering that particularly noteworthy is its ability to activate macrophages and induce monocytes to generate interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) to expedite the healing of chronic wounds [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eChitosan, a natural polysaccharide extensively employed in medical applications, is derived from chitin and has garnered more attention than its base polymer chitin due to its pH responsiveness, biocompatibility, and bioactive attributes. Chitosan nanoparticles have emerged as promising polymeric and biological nanoparticles that have captured significant interest in recent years. These nanoparticles demonstrate considerable potential as nanocarriers for encapsulating substances like drugs or active compounds, facilitating targeted delivery to specific sites and enabling controlled release [\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIbuprofen has garnered significant attention in recent years due to its analgesic and anti-inflammatory attributes. Derivatives of Ibuprofen function by impeding the prostaglandin synthesis, leading to its classification as a non-steroidal anti-inflammatory drug as it does not interfere with the pituitary gland and adrenal gland interactions. Despite the advantageous aspects of Ibuprofen, it is accompanied by adverse effects, particularly on the gastrointestinal system. Consequently, there is a demand for the conversion of Ibuprofen into safer prodrugs. Through a sequence of modifications, the aim is to enhance the analgesic properties of the drug while minimizing side effects [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eSodium Alginate (BDH Co., UK), low molecular weight Chitosan (Sigma\u0026ndash;Aldrich Co., Germany), and Calcium Chloride (Merck KGaA Co., Germany), Dulbecco's Modified Eagle Medium (DMEM), 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO) were purchased from were obtained from Sigma-Aldrich (USA). Chi\u0026ndash;NPs were provided from US-Nano (USA). Fetal Bovine Serum (FBS) was purchased from Shellmax (Iran). Deionized water was used for all the experiments. Diethyl ether was purchased from Merck (USA). The human skin fibroblast cell line (HNFF-P18) was purchased from the National Cell Bank of Iran (NCBI, Pasteur Institute, Tehran, Iran).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eFabrication of hydrogel films\u003c/h2\u003e \u003cp\u003eSodium alginate and Collagen (3%w/v) with a ratio of 2/1 were dissolved in distilled water and mixed under magnetically stirring for 24 h. Then, 200 \u0026micro;l, 1%w/v, 3% w/v and 5% w/v of chitosan nanoparticles (Chi-NP) were added into the 10 ml ALG/Col solutions, respectively, and stirred overnight at 400 rpm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eHydrogel crosslinking method\u003c/h2\u003e \u003cp\u003eThe solution containing calcium chloride (CaCl2) 0.25 M as a cross-linker is prepared by combining 254 mg of CaCl2 in 8 ml of deionized water. Then 100 ml of this cross-linker solution is added to the deionized water to crosslink the alginate and preserve the material's structure in the following steps. After the hydrogels were covered entirely with calcium chloride solution for 25 min, washed several times with PBS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eFourier transform infrared (FTIR) spectroscopy\u003c/h2\u003e \u003cp\u003eThe FTIR spectra of Gel, PVA, and TiO2-NPs powders, as well as Gel/PVA and Gel/PVA/TiO2-NPs hydrogel films, were characterized by a spectrometer (Tensor II, Bruker, Germany) to identify their chemical bonds and functional groups in the wavenumber range of 4000\u0026ndash;400 cm-1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMechanical properties of Hydrogel\u003c/h2\u003e \u003cp\u003eThe measurement of the radius and height of cylindrical hydrogel denoted as r and h respectively is essential. The determination of the elastic modulus of the hydrogel is carried out utilizing an electronic universal testing machine operating at a velocity of 1 mm/min until the specimen's height is diminished by 80%. The parameters S and h represent the cross-sectional area and height of the scaffolds, while L1 and L2 signify the pressure levels at the initial and final points of the linear section, and D1 and D2 indicate the displacement of the scaffolds. The calculation of the scaffolds' elastic modulus is performed through the subsequent formula:\u003c/p\u003e \u003cp\u003eΕ\u0026thinsp;=\u0026thinsp;ε\u0026frasl;σ= [(L_1-L_2) \u0026frasl;S] \u0026frasl; [(D_1-D_2) \u0026frasl;h]\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDetermine the degradation ratio\u003c/h2\u003e \u003cp\u003eThe assessment of the weight loss of the hydrogel scaffolds is conducted over intervals of 3, 6, 9, 12, and 15 days while the scaffolds are immersed in a lysozyme solution at 37℃. The scaffolds are extracted every 3 days, rinsed twice with deionized water, dried, and then weighed. The degradation ratio is computed using the subsequent formula where m0 denotes the initial weight and mt signifies the ultimate weight of the scaffold post-drying:\u003c/p\u003e \u003cp\u003eDegradation ratio (%) = ((mt-m0)) \u0026frasl;m0 \u0026times;100\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eWater vapor transmission rate (WVTR)\u003c/h2\u003e \u003cp\u003eThe Water vapor transmission rate (WVTR) of samples was determined by fixing 2 cm diameter circular samples using instant glue on the opening of plastic tubes that had been one-third filled with water (approx. 7 ml). A glass desiccator was used to set the environmental humidity at 5075%. After 3 days, the tubes were weighed and VP measured by:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\text{W}\\text{V}\\text{T}\\text{R}=\\frac{{\\text{w}}_{0}-{\\text{w}}_{1}}{\\text{A}\\times\\:24}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eWhere initially weighed (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\text{w}}_{0}\\)\u003c/span\u003e\u003c/span\u003e), weight after 24 hours\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:{(\\text{w}}_{1})\\)\u003c/span\u003e\u003c/span\u003e, A is the area (cm\u003csup\u003e2\u003c/sup\u003e). Three repeats were considered for each sample.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eMeasure the contact angle and conduct a swelling examination\u003c/h2\u003e \u003cp\u003eThe determination of the contact angle of the hydrogel scaffolding is executed utilizing a contact protractor. The assessment of swelling behavior is achieved through gravimetric analysis, where the weight of the dried scaffold (m0) is quantified on an analytical balance, followed by immersion in PBS at 37℃ for 4 hours. After the specified duration, excess moisture on the sample surface is eliminated using filter paper, and the sample is precisely reweighed (mt). The swelling behavior is quantified by employing the ensuing formula:\u003c/p\u003e \u003cp\u003eSwelling ratio (%) = ((mt-m0)) \u0026frasl;m0 \u0026times;100\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEvaluate the morphology and size distribution of the pores in Hydrogel\u003c/h2\u003e \u003cp\u003eThe visual examination of the surface morphology of the scaffolds necessitates the use of a scanning electron microscope (SEM) equipped with a voltage of 10 kV. Before imaging, the specimens were sputtered with a thin layer of gold using a sputter coater (Q150R-ES, Quorum Technologies, UK). The average porosity of each sample was determined by the image processing method.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalyze the porosity of hydrogel scaffolds\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe determination of scaffold porosity entails the utilization of ethanol as a non-solvent liquid for porosity measurement purposes. The methodology involves immersing the scaffold in a graduated cylinder filled with ethanol, proceeding to place it in a vacuum oven for 5 minutes to eliminate air bubbles completely. The initial volume of ethanol denoted as V1 and the combined volume of ethanol and scaffold regarded as V2 are measured. Subsequently, the scaffold is cautiously withdrawn, and the residual ethanol volume is quantified, with this remaining ethanol volume designated as V3. The calculation of scaffold porosity is performed using the ensuing formula:\u003c/p\u003e \u003cp\u003ePorosity (%) = [((v1-v3)) \u0026frasl; ((v1-v2)] \u0026times;100\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eBiocompatibility\u003c/h2\u003e \u003cp\u003eBiocompatibility of the support will be assessed using the MTT technique. In this manner, the supports were immersed in 75% ethanol and exposed to ultraviolet radiation for 12 hours, subsequently the supports were rinsed twice with PBS for 1 hour each time to eliminate the residual ethanol and ultraviolet rays. Next, HDF fibroblasts are seeded on the supports with a concentration of 5x104 cells/mL and cultivated in a complete medium comprising α-DMEM, 10% FBS, penicillin-streptomycin, and 5% CO2 in an incubation chamber with a temperature of 37\u0026deg;C. Following a cultivation period of 3 days, the MTT assay will be measured utilizing an ELISA reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eProcedure for synthesis of new hybrid nanocatalyst of Ibuprofen (IBP)\u003c/h2\u003e \u003cp\u003eAccording to our previous study in obtaining the best product of new hybrid nanocatalyst of IBP, first we [BMIm]SS catalyzed the reaction of IBP with epoxides to afford the new IBP 1,2-diol mono esters in good to excellent yields [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The products were tested in vivo for the analgesic properties on female mice using formalin test.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eFormalin test\u003c/h2\u003e \u003cp\u003eExperiments were performed on male mice (25\u0026ndash;30g, n\u0026thinsp;=\u0026thinsp;15) purchased and kept in Comparative and Experimental Medicine Center of Shiraz University of Medical Sciences, Shiraz, Iran. Animal selection, care and sacrifice, protocols, were adhered to the Animal Care Committee of Iran Veterinary Organization guidelines. The mice were kept under a standard 12 h light/dark cycle at 21\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026ordm;C and have with ad libitum access to food and water. Animals were randomly divided into 3 equal groups of control, positive control and treatment. Before the inception of the experiment, mice were moved to a testing lab for at least 60 minutes and put in the formalin testing boxes for habituation for at least 30 minutes. A mirror was placed underneath at a 45\u0026deg; angle to allow a clear view of the paws. Formalin (20 \u0026micro;L; SC) was injected into right foot hind. These behaviors were scored and recording in several time (5.10,15,20,25, and 30 min) after formalin injection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eIn vivo evaluation\u003c/h2\u003e \u003cp\u003eAdult Sprague-Dawley male rats (200 g, 7 weeks old) were purchased from the Comparative and Experimental Medicine Center of Shiraz University of Medical Sciences, Shiraz, Iran. They were kept under sterilized conditions and light/dark cycle of 12 hours and an ambient temperature of 22.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0˚C. The animals had free access to food and water. During interventions, animals were anesthetized using Ketamine (5 mg/kg; Woerden, Netherlands) and xylazine (20 mg/kg; Alfazyne, Woerden, Netherlands) intraperitoneally (IP). All experimental procedures were according to Iran Veterinary Organization rules and principles of working with laboratory animals (IR.SUMS.AEC.1402.073).\u003c/p\u003e \u003cp\u003eA total number of 36 adult healthy rats were enrolled and randomly divided into control and three experimental groups. A 1 cm2 full-thickness (third-degree) burn was induced to all animals in all groups using a plastic ring exposed to boiling water for 10 seconds\u003c/p\u003e \u003cp\u003eThese groups were observed over 3 time periods of 3, 7, and 14 days to assess parameters such as repair progression, bleeding, angiogenesis, and inflammation. The experimental groups comprised the following: - The first group served as the control, receiving no interventions (3 rat specimens per time point). - The second group was the control group treated solely with hydrogel base, excluding nanoparticles and ibuprofen (3 rat specimens per time point). - The third group underwent therapeutic procedures involving hydrogel with chitosan nanoparticles (3 rat specimens per time point). - The fourth group constituted the second experimental group, subjected to therapeutic measures utilizing hydrogel with chitosan nanoparticles and ibuprofen (3 rat specimens per time point). Subsequent to the interventions, the rats were humanely euthanized on days 3, 7, and 14, and wound samples were collected for histological analysis, preserved in 10% formalin. Subsequently, the samples will be processed into slides and stained with hematoxylin-eosin for pathological examination under a light microscope.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eIn order to achieve the desired hydrogel scaffold, several tests were performed in terms of physical and mechanical properties. The primary goal of this research was to form a hydrogel scaffold using natural polymers and to create an analgesic property to it using a new hybrid nanocatalyst of IBP, and then to examine the morphology, physical properties, mechanical properties, cytotoxicity, cell growth and proliferation. Finally, in-vivo test of the scaffolds done.\u003c/p\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eFTIR Characterization of the Samples\u003c/h2\u003e \u003cp\u003eFTIR spectroscopy was used to investigate functional groups and bonds formed from raw materials as well as bonds formed in nanocomposite hydrogel. For this purpose, the FTIR spectrum was taken from the pure material, hydrogel and synthesized nano-composite hydrogel, and the results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eThe mechanical properties of scaffold:\u003c/h2\u003e \u003cp\u003eThe physical characteristics of all nanocomposite films were assessed in order to gauge the performance of the film in line with its physical capacity. Young's modulus or the gradient of the graph is derived from the linear segment of the stress-strain graph. The linear segment signifies the elastic zone while the non-linear segment signifies the plastic zone. The ultimate tensile strength is the threshold at which the specimen withstands the highest stress and experiences rupture. The outcomes illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e indicate that the ALG/Col membrane exhibits the minimum yield strength and elastic modulus. In light of the outcomes, escalating the loading of nanoparticles in the polymer matrix leads to the enhancement of physical characteristics such as Young's modulus and yield stress.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro degradation\u003c/h2\u003e \u003cp\u003eBiodegradability Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e delineates the mass reduction of scaffolds over a 21-day period in saline phosphate buffer solution. Alginate/collagen scaffolds alone and with varying proportions of chitosan nanoparticles were assessed for degradability.\u003c/p\u003e \u003cp\u003eThe findings from the inquiries reveal that hydrophilic ALG/Col frameworks exhibit a relatively rapid degradability rate for the liberation of any medications and antibacterial substances, such as chitosan nanoparticles. Alginate, collagen, and chitosan natural polymers all possess significant water absorption characteristics and thus can be utilized in the production of biocompatible hydrogels.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eExamination of the swelling behavior of hydrogels and Water contact angle\u003c/h2\u003e \u003cp\u003eThe findings indicate that the proportion of expansion rises steadily with the incorporation of chitosan nanomaterial proportion and remains nearly steady in the 3% and 5% nanomaterial specimen, which is attributed to the interplay of the existing connections and the linking of the compounds. Concerning the inclusion of nanomaterials, various hypotheses were deliberated concerning the extent of hydrogel expansion in the existence of nanomaterials, such that it can be deemed as a highly absorbent material.\u003c/p\u003e \u003cp\u003eAlso, the increase in the contact angle in the ALG/Col/CS-NPs sample is caused by the irregularity of the surface and the tendency of the sample to water due to the hydrophilic nature of the nanoparticles. Modifying alginate-based films by incorporating nanoparticles in significant concentrations increases its aqueous affinity and, conversely, decreases mechanical properties (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These observations are attributed to the stable formation of hydrogen bonds between oxygen in the nanoparticle and hydrogen in alginate, which are potential reaction sites to retain water molecules.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eExamining the moisture vapor transmission rate\u003c/b\u003e:\u003c/h2\u003e \u003cp\u003eThe outcomes of the moisture vapor transmission rate (MVTR) of the specimens are in agreement with the scanning electron microscope (SEM) findings, such that the MVTR of the specimens with lower density surpasses that of the specimens with higher density. It appears that the process of vapor transfer of moisture is reliant on the hydrophilicity of the constituent components of the specimen and the cross-linking (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eScanning electron microscope\u003c/b\u003e:\u003c/h2\u003e \u003cp\u003eExamining the hydrogel structure with an electron microscope shows that with the addition of chitosan nanoparticles, the amount of fibers and the size of the pores are gradually reduced and the structure of the layer becomes uniform, which indicates the formation of interaction between polymers and nanoparticles. The pores in the nanocomposite should be such that the fibroblast cells can migrate into the scaffold. Considering that the size of a fibroblast cell is approximately 30\u0026ndash;50 microns, the pores should be large enough and interconnected to facilitate the transfer of cells and nutrients to the scaffold. Observing the size of the pores indicates the appropriate concentration of the scaffold containing chitosan nanoparticles (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003ePain test results of new hybrid nanocatalyst of IBP:\u003c/h2\u003e \u003cp\u003eAs illustrated in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the analgesic effect of the new hybrid nanocatalyst IBP; a, b and c, which were the best samples in our previous study, was evaluated by formalin test and the best sample, which was sample a, was selected and added to the hydrogel. This sample consistently outperformed ibuprofen even in the initial and subsequent 5 minutes, showing a significant difference with ibuprofen and a superior analgesic effect. Hence, it is inferred that this compound has a significant analgesic effect compared to ibuprofen. In addition, it can be seen that ibuprofen and the synthesized compound performed better than the control group in both cases.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of subcutaneous formalin injection (20 \u0026micro;l) into the hairy skin of the right hind leg. The score of formalin-induced nociceptive behaviors was measured every 5 minutes for 30 minutes (n\u0026thinsp;=\u0026thinsp;15).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eH10\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eH15\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eH20\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eH25\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eH30\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ea4h\u003c/b\u003e (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eb4k\u003c/b\u003e (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ec4o\u003c/b\u003e (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eControl\u003c/b\u003e (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIBP\u003c/b\u003e (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eBiocompatibility\u003c/h2\u003e \u003cp\u003eAs the injury dressing typically acts as a transient substitute for the epidermis, it must possess outstanding biocompatibility. Cytotoxicity stands as a crucial criterion for assessing the biocompatibility of substances. The cell survival of murine fibroblasts within the permeable framework configuration was scrutinized for 24 hours and 48 hours utilizing the MTT technique (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The elevated viability signifies commendable biocompatibility with negligible impact on cellular growth for the frameworks devised in this study. In Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, the cell survival in the control group is inferior to the groups of nanoparticles and IBP, suggesting that nanoparticles in a specific quantity potentially enhance cellular propagation and the proliferation mechanism endures. The acquired outcomes revealed that ALG/Col/3%CS-NPs /IBP specimen exhibits the highest biocompatibility in comparison to other formulated hydrogels. It appears that the incorporation of nanoparticles into the hydrogel can notably enhance cellular propagation as well as cell survival by encouraging cells to elevate growth factors. Nonetheless, the cell survival of the reference and ALG/Col specimens was akin, and the biocompatibility of the ALG/Col/1%CS-NPs specimen was substandard compared to that of the ALG/Col/5%CS-NPs specimen due to the reduced porosity, which could adversely impact cellular adhesion. Also, the presence of IBP not only did not decrease cell viability, but also increased cell viability.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eIn vivo wound healing evaluation\u003c/h2\u003e \u003cp\u003eDerived from the outcomes obtained from laboratory examinations, the finest Samples were chosen and contemplated for in vivo investigations. Amid the diverse proportions of chitosan nanoparticles, 3% was regarded as optimum, and amid the variations of ibuprofen, derivative was designated as prime, and hydrogels encompassing 3% of chitosan nanoparticles and 10 mg of ibuprofen derivative a were taken into account. Executing in vivo investigations to cure the full thickness wound of male rats in the control and treatment groups were assessed. Wound assessment was carried out on days 0, 3, 7 and 14, and the morphological exploration exhibited a remarkable decline in the dimensions of the wound on day 7, and visual juxtaposition of the wound on day 14 indicated entire closure of the wound in the group containing ibuprofen derivative as opposed to the group lacking ibuprofen derivative and control. This issue denotes that all wounds treated with scaffolds with ibuprofen derivatives close earlier due to the mitigation of inflammation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe pathological assessment on the 7th day subsequent to wound establishment and treatment with alginate-collagen-chitosan nanoparticle and derivative a scaffold in contrast to the control group and positive control reveals the notable impact of this scaffold in the recuperation process, as discerned in the control group, coagulation necrosis and Epidermis is entirely obliterated and not regenerated, there is intense vascular expansion and absence of tissue bud formation. Nevertheless, in the treatment cluster, budding tissue is evolving in the dermis and regeneration of the epidermis has commenced. These outcomes are evidently superior in the group receiving the ibuprofen derivative, signifying that pain management quickens the recuperation process. These outcomes are in harmony with the investigations of other scholars. On the fourteenth day, in the positive control group, the tissue bud is expanding, the inflammatory cells and vascular expansion have dwindled, and we can observe the regularization of the tissue. However, in the treatment groups, we observe an absence of inflammation and no hemorrhage in the tissue, and no infection is observed, and we have incomplete tissue mending with a mature tissue bud, total repair with a mature tissue bud, angiogenesis and regularization of collagen fibers can be observed, inflammatory cells and hemorrhage is not observed. In the group of alginate-collagen-chitosan nanoparticle and ibuprofen derivative, the restoration is entire and the keratin tissue is completely developed. We also observe hair growth in the affected region (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eCurrently, wound dressings are typically composed of natural materials like collagen, hyaluronic acid or chitosan [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. These materials exhibit good biocompatibility and promote tissue regeneration, however, the physical characteristics of hydrogels manufactured from these materials are indicated to be relatively subpar. Consequently, they are vulnerable to damage from contact or friction. The findings in our investigation surpassed the outcomes of numerous hydrogels that have been documented [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Collagen\u0026ndash;Alginate hydrogel with a chitosan nanoparticle is a biodegradable polymer with strong mechanical properties, good biocompatibility, low cost, and an easily tunable degradation profile.\u003c/p\u003e \u003cp\u003eIn the context of a wound dressing, it is imperative for hydrogels to possess the capacity to absorb exudates from the wound area and retain moisture to safeguard the newly regenerated tissue. Alginate is a material with a propensity for attracting moisture that is anticipated to soak up moisture vapor and augment MVTR[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Off-the-shelf dressings exhibit MVTR figures ranging from 76 grams per square meter per day to 9360 grams per square meter per day. Quinn and colleagues demonstrated that an acceptable MVTR of 2500\u0026thinsp;\u0026minus;\u0026thinsp;2000 grams per square meter per day can maintain the wound site damp to avert desiccation or excessive fluid discharge[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, all hydrogels exhibit elevated MVTR. The impact of CS-NPs content is delineated substantially augmented the MVTR of the nanocomposite membrane[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This is because the integration of nanomaterials can establish an extensive convoluted route and the interplay between alginate and nanomaterials can considerably enhance the wetness affinity of the nanocomposite membrane[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. As well as Integration of CS-NPs into the polymer matrix hinders the mobility of the ALG polymer chain due to the robust interaction between the nanoparticles and the polymer matrix [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Additionally, the elastic modulus surged with the rise in nanoparticle concentration as documented in the literature [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], despite some authors presenting the contrary trend [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In certain investigations, it has been documented that the particle loading exceeding 1% of nanoparticles results in a reduction in the resilience of the polymer network against fracture and a decline in the robustness of the polymer network, which is an outcome of the polymer distortion around the nanoparticles [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSEM images validated the homogeneous structure and porosity of the scaffolds. The scaffolds exhibited favorable biocompatibility and supported human fibroblast cell proliferation, with the 3% chitosan nanoparticle concentration showing optimal results. Incorporation of chitosan nanoparticles enhanced the scaffolds' properties.\u003c/p\u003e \u003cp\u003eIn this investigation, we achieved our objective, IBP was successfully incorporated into alginate/ Collagen /chitosan nanoparticle hydrogel to create analgesic, anti-inflammatory, degradable scaffolds for wound healing application. Results showed that proliferation of human dermal fibroblasts to the freshly prepared Ibuprofen loaded scaffolds was not reduced at the new hybrid nanocatalyst of IBP. Also, cell attachment increased in compare in the control unloaded scaffolds. This showed that loading IBP into the scaffolds did not change their basic ability to act as fibers for skin cell attachment and migration which is encouraging for developing these fibers as tissue guides for wound healing applications[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. A new hybrid nanocatalyst of IBP exhibited greater pain-relieving impacts than ibuprofen ten minutes following the formalin experiment, substance a demonstrated a superior pain-relieving effect than ibuprofen. Concerning pain-relieving characteristics, the synthesized substances possess a swifter absorption than ibuprofen due to their lipophilicity, hence they can be advantageous for acute pains that are acute, such as burns, cuts, and similar discomforts. We utilized it in hydrogel to manage full-thickness wounds. It is imperative to note that by safeguarding the acidic faction of ibuprofen, we endeavored to diminish its side effects.\u003c/p\u003e \u003cp\u003eRegarding wound healing, a certain level of inflammation accelerates wound healing because the release of pro-inflammatory cytokines attracts cells into the wound area the converse is also true and too high a level of inflammation can retard wound healing [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. We observed that our natural hydrogels are able to support the attachment, migration and proliferation of normal skin cells, helping to accelerate wound healing by providing an immediate replacement substrate for undamaged skin cells at the wound margin. The simultaneous release of an anti-inflammatory from this natural scaffold will calm down the aggressive inflammatory beds of the wound and accelerate the healing process.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWe present a conceptually simple approach to a wound healing dressing which provides a temporary substrate for the cells in the wound bed to migrate along while delivering Ibuprofen as an anti-inflammatory agent and pain reliever. This scaffold exhibiting robust mechanical properties, biodegradability, and controlled drug release for mitigating inflammation, and the need to remove this dressing.\u003c/p\u003e \u003cp\u003eThe hydrogel containing ibuprofen derivatives accelerated wound closure and promoted angiogenesis by supporting cell attachment and migration compared to the control group. This represents a pioneering effort in the development of ibuprofen-containing scaffolds for wound healing. Given that the number of chronic non-healing wounds is high, such dressings could be useful in the treatment of burns and chronic wounds, where approaches to simultaneously manage pain and accelerate wound re-epithelialization are needed.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, S-S.H., A-A.M., A-M FT., and M.N.SR.; methodology, S-S.H., A-A.M., A-M FT., and M.N.SR.; validation, S.S.H., A.A.M., and \u0026nbsp;A-M FT; formal analysis, S-S.H., and A-M FT; investigation, S-S.H., and A-M FT; resources, S-S.H. and A-M FT.; data curation, S-S.H.; writing original draft preparation, S-S.H., and A-M FT.; writing\u0026mdash;review and editing, S-S.H. and A-M FT; visualization, S-S.H., and A-M FT.; supervision, S-S.H., and A-M FT.; project administration, \u0026nbsp;S-S.H., A-A.M., A-M FT., and M.KR.; funding acquisition, M.KR. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the Institutional Ethics Committee of Shiraz University of Medical Sciences (IR.SUMS.AEC.1402.073).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHashemi SS, Jowkar S, Mahmoodi M, Rafati AR, Mehrabani D, Zarei M, Keshavarzi A: \u003cstrong\u003eBiochemical Methods in Production of Three-Dimensional Scaffolds from Human Skin: A Window in Aesthetic Surgery\u003c/strong\u003e. \u003cem\u003eWorld J Plast Surg \u003c/em\u003e2018, \u003cstrong\u003e7\u003c/strong\u003e(2):204-211.\u003c/li\u003e\n\u003cli\u003eHashemi SS, Mohammadi AA, Kabiri H, Hashempoor MR, Mahmoodi M, Amini M, Mehrabani D: \u003cstrong\u003eThe healing effect of Wharton\u0026apos;s jelly stem cells seeded on biological scaffold in chronic skin ulcers: A randomized clinical trial\u003c/strong\u003e. \u003cem\u003eJournal of cosmetic dermatology \u003c/em\u003e2019, \u003cstrong\u003e18\u003c/strong\u003e(6):1961-1967.\u003c/li\u003e\n\u003cli\u003eHashemi SS, Mohammadi AA, Moshirabadi K, Zardosht M: \u003cstrong\u003eEffect of dermal fibroblasts and mesenchymal stem cells seeded on an amniotic membrane scaffold in skin regeneration: A case series\u003c/strong\u003e. \u003cem\u003eJournal of Cosmetic Dermatology \u003c/em\u003e2021.\u003c/li\u003e\n\u003cli\u003eZhao P, Shen M, Gu Y, Zhu S, Wang F: \u003cstrong\u003eOxidation behavior of NiCrAlY coatings prepared by arc ion plating using various substrate biases: Effects of chemical composition and thickness of the coatings\u003c/strong\u003e. \u003cem\u003eCorrosion Science \u003c/em\u003e2017, \u003cstrong\u003e126\u003c/strong\u003e:317-323.\u003c/li\u003e\n\u003cli\u003eHashemi S-S, Rajabi S-S, Mahmoudi R, Ghanbari A, Zibara K, Barmak MJ: \u003cstrong\u003ePolyurethane/chitosan/hyaluronic acid scaffolds: providing an optimum environment for fibroblast growth\u003c/strong\u003e. \u003cem\u003eJournal of Wound Care \u003c/em\u003e2020, \u003cstrong\u003e29\u003c/strong\u003e(10):586-596.\u003c/li\u003e\n\u003cli\u003eBush K, Gertzman AA: \u003cstrong\u003eProcess Development and Manufacturing of Human and Animal Acellular Dermal Matrices\u003c/strong\u003e. 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This study investigates the fabrication and characterization of a novel hydrogel scaffold incorporating chitosan nanoparticles and New Hybrid Nano catalyst of Ibuprofen for wound healing applications.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe hydrogel was synthesized using a solution casting method and cross-linked with calcium chloride. A new hybrid nano catalyst derivative of ibuprofen exhibiting superior analgesic effects compared to ibuprofen was synthesized and incorporated into the hydrogel. Extensive characterization using FTIR, XRD, SEM, mechanical testing, swelling studies, degradation analysis, and cell viability assays was performed to evaluate the structural, physical, and biological properties of the scaffolds. In addition to, hydrogels containing new hybrid nano catalyst derivative of ibuprofen (compound \"a\") assessed as wound dressing for full-thickness wound.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn vitro results demonstrated that the 3% chitosan nanoparticle-loaded hydrogel possessed optimal physico-chemical characteristics, porosity, biocompatibility, and supported human fibroblast cell proliferation. In vivo studies using a full-thickness wound model in rats revealed accelerated wound closure, reduced inflammation, and enhanced angiogenesis for wounds treated with the ibuprofen derivative-loaded hydrogel compared to controls.\u003c/p\u003e\u003ch2\u003eDiscussion\u003c/h2\u003e \u003cp\u003eOverall, this novel alginate/collagen/chitosan nanoparticle hydrogel incorporating an ibuprofen prodrug represents a promising biomaterial for facilitating wound healing through its analgesic, anti-inflammatory, and pro-angiogenic effects. This represents a pioneering effort in developing ibuprofen-supplemented scaffolds for enhanced wound healing.\u003c/p\u003e","manuscriptTitle":"Evaluation of Anti-Inflammatory Activity of Hydrogel Containing New Hybrid Nano catalyst of Ibuprofen-Loaded on Chitosan Nanoparticles for Full Thickness Burn Repair","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-12 10:05:34","doi":"10.21203/rs.3.rs-4741694/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2b685aeb-d0bb-4c0b-9ff5-ee13f5c2d117","owner":[],"postedDate":"August 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-12-03T21:38:21+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-12 10:05:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4741694","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4741694","identity":"rs-4741694","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}