{"paper_id":"0641a5a2-a094-4675-91c9-11aff3e8b3af","body_text":"Fabrication of composite hydrogels from Carrageenan-cl-carboxymethyl chitosan-PVA incorporated ZiF-8 for wound healing applications | 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 Research Article Fabrication of composite hydrogels from Carrageenan-cl-carboxymethyl chitosan-PVA incorporated ZiF-8 for wound healing applications Tooba Yasin, Muhammad Shahzad Zafar, Muhammad Umar Aslam Khan, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6345624/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Jun, 2025 Read the published version in Polymer Bulletin → Version 1 posted 10 You are reading this latest preprint version Abstract The skin is the largest organ of the human body, protecting it from the external environment and pathogens and minimizing the risk of injury. Hydrogels have attracted a lot of interest lately because of their biomimetic structure and inherent extracellular matrix characteristics. Hydrogels hold great promise for application in wound healing, mainly because they can very conveniently provide bioactive ingredients. In this work, composite hydrogels were developed for wound healing from carrageenan, polyvinyl alcohol, and carboxymethyl chitosan by blending method. These hydrogels were characterized by advanced techniques such as FTIR, XRD, DSC, and SEM-EDX, which were used to study their structural, thermal, surface morphology, and elemental behavior. Their physiochemical analyses were performed using swelling degradation, water contact angle, and gel fraction. The increasing ZiF-8 causes more surface roughness, with decreased swelling in different media (Aqueous>PBS>NaCl). The increasing ZiF-8 amount causes less hydrophilic behavior and biodegradation with increasing gel fraction. The cytocompatibility of ZiF-8-based composite hydrogels and their potential antibacterial activities were performed against Gram-positive and Gram-negative, which were enhanced with increasing ZiF-8. The increasing ZiF-8 caused more cell viability and proliferation with proper cell morphology and enhanced in vivo wound healing. Hence, the results show that synthesized composite hydrogels may be a potential candidate for numerous wound repair applications. Biodegradable Natural polymers ZiF-8@Hydrogels Polymeric composite Wound healing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Highlights • Fabrication of the composite hydrogels by incorporating ZiF-8 as a bioactive dressing material for wound healing. • The development of composite hydrogels using a simple blending method from natural polymers for wound healing applications. • Hydrogels with enhanced physicochemical properties with pH-responsiveness. • Cytocompatible composite hydrogels via cell viability and proliferation with mature cell morphology. • Enhance wound healing by increasing the ZiF-8 amount in the composite hydrogels. : The graphical abstract illustrates the fabrication of composite hydrogels incorporated with Zif-8 into the polymeric matrix of carrageenans, carboxymethyl chitosan and polyvinyl alcohol and their potential antibacterial and wound healing applications. 1. Introduction The skin is an essential organ in the human body that functions as a barrier of protection against various external environmental bacterial attacks and external environmental damages. Numerous forms of trauma, burns, ulcers, and other harmful events frequently impair the skin’s otherwise resolute barrier and interfere with its essential role in sensory perception [ 1 ]. Even in the absence of external threats, the skin is vulnerable to damage. When its function at any level is compromised, it can lead to distress on many levels for the individual and society. Therefore, one of the most important subjects in biomedicine is skin healing. The mainstays of effective wound closure are dressings that promote rapid healing [ 2 , 3 ]. Among the available dressings, hydrogel-based wound dressings are particularly distinguished for their ability to facilitate this process. They provide a barrier against contaminants and maintain a wound site hydrated, two crucial elements in promoting efficient tissue repair. At present, bioactive hydrogels are gaining recognition as advanced wound dressings [ 3 ]. These excellent dressing materials exhibit several unique properties that make them suitable for this application. They can be tailored to adapt to the shapes of irregularly and deeply contoured wounds. They can also be designed to incorporate drugs and other bioactive agents. The past decade has seen an explosive growth of interest in using hydrogels for wound dressings [ 4 ]. Nonetheless, there is a significant deficit in understanding their performance as dressing materials. Bioactive hydrogels that gel quickly and that have wound-healing properties that are accelerated beyond what other materials can do are particularly interesting for use in wound care. One reliable way to develop such materials is to use multiple kinds of reversible interactions as crosslinkers in the gel structure. It increases the hydrogel’s antibacterial properties and enhances its wound-healing capabilities [ 5 ]. Bacterial contamination is still a significant problem in wound healing, especially in chronic wounds. These wounds show a healing delay because they display a never-ending inflammation phase that leads to chronic inflammation [ 6 , 7 ]. An increased microbial bioburden and oxidative stress will surely suffice. We are making significant efforts, which include the administration of antibiotics, to circumvent this issue and make progress toward the healing of wounds. The application of bioactive compounds and the incorporation of novel nanomaterials into wound dressings reduce inflammation in some way [ 8 – 10 ]. The emergence of drug-resistant pathogens now represents a major threat to public health and constitutes a huge socio-economic burden. Synthetic antibiotic drugs, when used excessively, bring about this emergence. One potential solution to this issue is to employ wound dressings that contain non-antibiotic treatments that are effective against bacterial infections. Natural antimicrobial compounds are biocompatible and environmentally friendly, and they provide excellent infection treatments, unlike antibiotics [ 11 , 12 ]. Metal-organic frameworks are highly porous nanomaterials that have gained popularity due to their uniqueness in the biomedical field. ZIF-8 has a strong antimicrobial effect that makes it a powerful agent against any dangerous bacteria that could infect a wound [ 13 ]. ZiF-8 protects the wound from bacterial attack due to its potential antibacterial activities that accelerate the healing of infected wounds [ 14 ]. Chitosan (CS) has also gained considerable attention for medical applications because it has inherent antibacterial properties. At the same time, the many amino groups along the backbone allow you to modify chitosan with moderate covalent interactions. Chitosan demonstrates biocompatibility, promotes cell growth and attachment, and has controllable degradation properties [ 15 , 16 ]. It makes chitosan a highly suitable candidate for many biomedical applications. Because of its innate antibacterial properties, chitosan has attracted great interest in the wound healing application arena. Additionally, carboxymethyl chitosan (CMCs), a derivative of chitosan, is widely applied in drug delivery and is even more often used as a material in tissue engineering. CMCs are also being extensively studied in the area of wound healing, and their antibacterial activities accelerate infected wound healing [ 17 ]. Carrageenans are naturally occurring polymers taken from red algae that belong to the Rhodophyceae group of plants. These materials are widely used in the food and biomedical industries as emulsifiers, thickeners, gel-forming agents, and stabilizers [ 18 ]. They are polysaccharides, and their various types are categorized by the amount and accessibility of sulfate groups on the disaccharide monomer units. Kappa (κ-) carrageenan has one sulfate group; iota (ι-) carrageenan has two; and lambda (λ-) carrageenan has three sulfate groups per disaccharide monomer unit. These have several potential applications in wound healing and drug delivery due to their hydrogen bonding or ionic interactions [ 19 ]. Polyvinyl alcohol (PVA) is the synthetic polymer of choice, winning out for a variety of very good reasons: its non-cytotoxicity, hydrophilicity, biocompatibility, transparency, and ease of incorporation into swelled-state forms [ 20 , 21 ]. PVA, a promising biomaterial, is particularly notable for its easy processing and nearly ideal mechanical and chemical properties that can be varied over a wide range. This hydrophilic material can form hydrogen bonds with other hydrophilic substances, making it suitable for numerous applications—everything from contact lenses to cartilage, drug delivery systems to wound dressings, and skin replacement components [ 22 ]. Herein, we have developed composite hydrogel with enhanced antibacterial activities and accelerated wound closure for wound healing applications. Different formulations of the hydrogel were produced from carrageenan (CG), polyvinyl alcohol (PVA), and carboxymethyl chitosan (CMCs) and were incorporated with varying amounts of ZiF-8. To the best of our knowledge, these formulations of novel hydrogel systems have never been reported before using a simple blending method. We have employed several techniques to study the structural, morphological, thermal, and mechanical properties of composite hydrogels. The physicochemical analysis was also performed to determine the swelling and degradation behavior of the composite hydrogels, and their in vitro assay was performed to evaluate cell viability, proliferation, and morphology assays using fibroblast cell lines. Finally, we used a standard in vivo model to assess the wound healing potential of the composite hydrogel. 2. Materials and methods 2.1. Materials Carrageenan (CAS number: 9000-07-1, light brown powder), carboxymethyl chitosan (CAS number: 83512-85-0, light yellow powder with carboxymethylation =/> 80%), polyvinyl alcohol (CAS number: 9002-89-5, Mw 31,000–50,000), and Tetraethoxysilane (CAS number: 78-10-4, Mw 208,33 g/mol) were obtained from Merck, Germany. Sodium chloride (NaCl), acetic acid, absolute ethanol, and potassium persulfate (K 2 S 2 O 8 ) were purchased from Sigma Aldrich. All the chemicals are analytical grade chemicals used as received. 2.2. Biological materials and reagents Gram (positive and negative) bacterial pathogens were (Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli)), and Fibroblast (3T3) cell lines were purchased from ATCC (Manassa, VA, USA), and preserved as per ATCC recommendations. α-MEM medium, Fetal Bovine Serum (FBS), Penicillin, and Streptomycin were obtained from Hyclone Laboratories Inc. and ThermoFisher Scientific, respectively. The live/dead assay was performed using the kit obtained from Invitrogen (R37601). BOBO-3 Iodide (red) and Calcein-AM (green) were used to assess the cell morphology, and the assay was performed according to the supplier’s instructions. Male Sprague-Dawley (SD) was supplied by the National Institutes of Health (NIH) in Islamabad, Pakistan. 2.3. Methods 2.3.1. Synthesis of ZIF-8 We have synthesized the ZIF-8 according to already reported by Attwa et al . [ 23 ]. In brief, we mixed zinc hexahydrate, an organic linker, and 2-methylimidazole in deionized water in a ratio of 1:35:1238. Then, 2-methylimidazole (H-MIM) (12 g) and zinc nitrate (1.25 g) were dissolved in deionized water. The solution containing Zn-ions was added dropwise into H-MIM while stirring vigorously at room temperature for 2 h. The polymeric solution formed a milky color, which indicates the formation of ZiF-8 and was collected by centrifuging at 4500 rpm for 30 minutes. It was washed thrice with deionized water, followed by oven-drying at 60 ºC for 24 h to remove moisture or water contents. The ZIF-8 nanoparticles were stored in glass vials to incorporate into the polymeric composite to develop a composite hydrogel system. 2.3.2. Development of composite polymeric systems The composite hydrogels based on polymers were developed by mixing carrageenan, carboxymethyl chitosan, polyvinyl alcohol, and ZIF-8 particles. Briefly, carrageenan (0.5 g) and carboxymethyl chitosan (0.3 g) were dissolved in distilled water separately. Then, polyvinyl alcohol (0.2 g) was dissolved in additional distilled water (15 mL) at 80 o C and combined with the other components. All the polymeric solutions were mixed and stirred at 60°C for 2 h to obtain homogeneous polymeric solutions. We have added different amounts of ZIF-8 particles (3, 6, and 10%) into the polymeric solution. A fixed concentration of TEOS (180 µL) was dissolved in 5ml ethanol, poured dropwise into the whorl of the polymeric media, and stirred at 60°C for 1 h. The initiator KPS (0.05) was then added to the polymeric media for effective crosslinks and allowed to be stirred at 60°C for 3 h. After 3 h, the composite polymeric solution was poured into the Petri plate. We have summarized the components of the composite in Table 1 . Table 1 The table summarizes the composition of the fabricated composite hydrogels. Sr. No. Carrageenan (g) Carboxymethyl chitosan (g) Polyvinyl alcohol (g) TEOS (µL) ZiF-8 (%) 1. 0.5 0.3 0.2 180 3 2. 0.5 0.3 0.2 180 6 3. 0.5 0.3 0.2 180 10 2.3.3. Fabrication of composite hydrogels The Petri plates were placed in the freezer and subsequently freeze-dried to obtain various formulations of the porous composite hydrogel system. The composite hydrogels were coded according to the varying quantities of ZiF-8 (CPC-ZiF-3%, CPC-ZiF-6%, and CPC-ZiF-10%). The polymeric solution was stored in glass vials for biological testing, while the produced composite hydrogel films were packed in an airtight plastic bag for further characterization and analysis. The chemical reaction of the composite hydrogels that we have proposed is illustrated in Fig. 1 . 3. Characterizations The composite hydrogels were structurally analyzed with the help of the PerkinElmer Diamond 1000 spectrophotometer, performing Fourier transform infrared spectroscopy over the 4000 − 400 cm − 1 wavelength range. An X-ray diffractometer (Bruker AXS D8 Advance XRD) was used to carry out the phase analysis over a range of 20°-80° to determine amorphous or crystalline behavior. The surface morphology was determined using a scanning electron microscope (SEM Jeol, Japan), and the hydrogel samples were gold-sputtered before analysis. The analysis of wetting was also conducted by measuring the angle of contact with water using equipment JY-82, Dingsheng, Chengde, China, to determine the nature of hydrogels—whether they are hydrophobic or hydrophilic. 3.1. Swelling analysis The swelling analysis of composite hydrogel was performed in different media (electrolyte (2M NaCl), phosphate buffer solution (PBS), and water) and different pH ranges at 37 °C for 24 h. The hydrogel samples were cut into square shapes weighing 50mg ( W i ) and placed in the abovementioned media. The samples were removed from the media after 24 h and surface water with tissue paper and weighed the swallow weight (W f ). The swelling percentage of the composite hydrogels was calculated by Eq. 1 . $$\\:\\varvec{S}\\varvec{w}\\varvec{e}\\varvec{l}\\varvec{l}\\varvec{i}\\varvec{n}\\varvec{g}\\:\\left(\\varvec{\\%}\\right)=\\frac{{\\varvec{W}}_{\\varvec{f}}-{\\varvec{W}}_{\\varvec{i}}}{{\\varvec{W}}_{\\varvec{i}}}\\:\\times\\:100$$ 1 In the above equation, W i refers to the initial weight, and W f shows the final weight of the hydrogel sample. 3.2. Degradation analysis The degradation of composite hydrogels was determined in PBS media having pH 7.4 at 37 °C after different time intervals (1–7 days). The initial weight of dried hydrogels was recorded ( W i ) and then placed in PBS media. The composite hydrogel samples were taken out from PBS media after at time ( t ). The composite hydrogels were dried in an oven to measure the weight at a time “ t ” ( W t ), and the degradation was determined by Eq. 2 . $$\\:\\varvec{D}\\varvec{e}\\varvec{g}\\varvec{r}\\varvec{a}\\varvec{d}\\varvec{a}\\varvec{t}\\varvec{i}\\varvec{o}\\varvec{n}\\:\\left(\\varvec{\\%}\\right)=\\frac{{\\varvec{W}}_{\\varvec{o}}-{\\varvec{W}}_{\\varvec{t}}}{{\\varvec{W}}_{\\varvec{o}}}\\:\\times\\:100$$ 2 3.3. Gel fraction The gel fraction is one of the essential properties of the composite hydrogels. The personage gel fraction of the composite hydrogels was calculated the gel fraction by Eq. 3 . $$\\:\\varvec{G}\\varvec{e}\\varvec{l}\\:\\varvec{f}\\varvec{r}\\varvec{a}\\varvec{c}\\varvec{t}\\varvec{i}\\varvec{o}\\varvec{n}\\:\\left(\\varvec{\\%}\\right)=\\frac{{\\varvec{W}}_{\\varvec{o}}-{\\varvec{W}}_{1}}{{\\varvec{W}}_{1}}\\:\\times\\:100$$ 3 3.4. In vitro studies 3.4.1. Cell morphology We studied the fibroblast cell lines’ behavior in association with hydrogels by performing Live/Dead assays. We determined the cells’ morphology after different time intervals (24, 48, and 72 h) against the composite hydrogels. The well plates were incubated for variable time intervals, whereas we carried out the cell culturing in DMEM media against the synthesized hydrogels. We used the Live/Dead assay kit (Invitrogen R37601) to perform the cellular morphology assessments, and we observed the cells using the Olympus fluorescent microscope (Olympus, FV300). 3.4.2. Cell viability and proliferation We analyzed the cytocompatibility of composite hydrogels by performing cell viability and proliferation assays on 3t3 cells. We used different dilutions (1.5–2.5 mg/mL) of the composite hydrogels to perform the assays, and we took the measurements at various time points (24–72 hours) to determine how well the cells were doing. The cytotoxicity of the composite hydrogels was performed using Neutral Red assays, as reported by Repetto et al . [ 24 ]. Briefly, the cell culture plates were treated with different hydrogel dilutions and incubated at 37 °C in a CO 2 (5%) and humidity (95%) environment. The 40 µg/mL neutral red medium was applied to cells and incubated. The leftover NR medium from the cells was removed by rinsing with a phosphate buffer solution. Additionally, a destaining solution composed of deionized water (50%), absolute ethanol (49%), distilled water (50%), and acetic acid (1%) was also used to remove the neutral red medium at 37 °C for ten minutes. The cell density was measured by a microplate reader (Bio-Tek, ELx-800, USA) at wavelength 570 nm. The percentage of cell viability was determined by Eq. 3 . $$\\:Cell\\:viability\\:\\%=\\frac{\\text{O}\\text{D}s-\\text{O}\\text{D}c}{\\text{O}\\text{D}c}\\times\\:100$$ 3 On the other hand, OD c = optical density of control and OD s = optical density of sample. 3.4.3. Antibacterial activities Composite hydrogels’ antibacterial properties were evaluated in relation to Gram-positive and Gram-negative bacteria, including Escherichia coli and Staphylococcus aureus. We have investigated antibacterial activities using the disc diffusion approach. Petri dishes containing the hot molten agar were set to solidify; a sterile cotton swab was used to apply the bacterial culture to the solid agar. Then, 65 µL of hydrogel was placed into wells and incubated at 37°C 24 h. After 24 h, the antibacterial activities were determined by zone inhibition, which was measured in millimeters by the regular ruler. 3.5. In vivo assay A full-thickness wound model has been developed to assess the efficacy of composite hydrogels in vivo in wound healing. In this study, SD rats were used for in vivo testing. After administering anesthesia, the dorsal area was shaved. A 20-mm diameter full-thickness wound was created in the back of each rat. A volume of 50 µL of composite hydrogel was applied to the wounds following the random assignment of the rats into four experimental groups (n = 5 per group). Digital images of the scars were captured on days 0, 3, 7, 10, and 14 using a Nikon camera to monitor the progression of wound healing over time. All experimental procedures involving animals were conducted in strict adherence to ethical standards and were reviewed and approved by the NIH, Islamabad. 3.6. Statistical analysis The software for statistics (IBM, SPSS Statistics 21) was used for the statistical analysis of the data. Means with standard errors (mean ± SE) were calculated and presented in the figures as Y-error bars. Significance levels used were: *(p < 0.05), **(p < 0.01), and ***(p < 0.001) with sample sizes of n = 3. 4. Results and discussions 4.1. FTIR analysis The structural and functional groups of the composite hydrogels were studied using the FTIR spectrum (Fig. 1 (a and b)). The broadband peak at 3600 − 3200 cm − 1 is due to O − H and − NH 2 functional groups [ 25 , 26 ]. The vibration peaks at 2923 cm − 1 show the presence of − CH 3 functional groups in CMCs, CG, and PVA [ 25 ]. The appearance of broadband at 3600 − 3200 cm − 1 indicates inter and intramolecular hydrogen bonding between PVA, CMCs, CG, and TEOS. The characteristic IR peaks at 1500–1600 cm − 1 are attributed to the N-H bending of the amide part in the acetyl group of CMCs [ 25 ]. The C − O stretch in CMCs and PVA appears at 1050–1100 cm − 1 . The vibration peaks at 1250 and 1290 cm − 1 are attributed to − Si − O−C and − Si − O−Si, which are happening to the crosslinking between the polymeric chains [ 27 ]. The peaks at 1152 and 846 cm − 1 are the characteristics of polysaccharides, and these peaks are due to pyranose rings and saccharines, respectively. The functional peaks at 1073 cm –1 are due to C–O stretching vibrations of pyranose, which is the characteristic peak of GG and CMCs [ 28 ]. The FTIR vibrational band around 1262 cm − 1 corresponds to the sulfate ester group of carrageenan [ 29 ]. The expected range of the Zn–N stretch vibration can be found at 421 cm –1 and could be due to the limitation of the FTIR instrument. The functional peaks at 1401 cm –1 and 1631 cm –1 are assigned to –CH 2 COOH and –C = O, respectively, in CMCs [ 30 , 31 ]. Thus, all the peaks and functional groups confirm the successful fabrication of composite hydrogels. 4.2. XRD analysis The crystalline behavior of the composite hydrogels was determined by X-ray diffraction pattern, as shown in Fig. 1 (c). The polymers are usually amorphous and do not show sharp peaks in the XRD pattern but broad peaks from 2θ 0-20 o . The XRD pattern of ZiF-8-based composite hydrogels demonstrated that adding ZiF-8 particles to a polymeric matrix may significantly affect the intensity of the XRD signal [ 32 ]. As the ZiF-8 particles are highly crystalline, incorporating ZiF-8 into the polymers and well dispersion enhances the crystalline features of the composite system. The XRD peaks of a composition containing lower ZiF-8 concentration (3%) are comparatively less sharp. In contrast, the higher ZiF-8 concentrations (6% and 10%) increase the crystallinity of polymeric material, resulting in sharper and more intense peaks. When the ZiF-8 particles are adequately dispersed in the polymer matrix, individual particles contribute to the diffraction signal, increasing the intensity of XRD peaks. The non-uniform dispersion of ZiF-8 particles causes aggregate formation and produces XRD signals that are less intense and broader than the signals produced by well-dispersed samples. Thus, the XRD analysis assures that the incorporated ZiF-8 composite hydrogels are successfully fabricated. 4.3. Thermal behavior The thermal resistance of composite hydrogels was evaluated using differential scanning calorimetry, as shown in Fig. 1 (d). The composite material exhibited thermal stability up to 220 o C, after which the material absorbed heat and underwent melting. The appearance of the shoulder peak indicates some secondary thermal processes that are closely associated yet discrete from primary events [ 33 ]. The complex network and structure of composite hydrogels involving several types of intramolecular or intermolecular interactions are responsible for the appearance of shoulder peaks [ 34 ]. The concentration of ZiF-8 affects the melting behavior of polymeric composite material. At the lower ZiF-8 concentration (3%), the particles of ZiF-8 may undergo proper dispersion within the polymer solution, bringing minor modifications in the polymer structure and leading to a less intense peak. As the concentration of ZiF-8 particles increases to 6% and 10%, the interaction of ZiF-8 particles and polymer chains becomes more robust, leading to a more prominent alteration in polymer melting behavior, potentially enhancing the intensity of the shoulder peak. Table 2 shows the melting temperatures of synthesized films. Increasing ZiF-8 concentration from 3–10% increases the melting temperature from 226 to 230 °C. Higher ZiF-8 concentrations enhance polymers’ crosslinking and stability, increasing melting points. Table 2 Thermal behavior of the composite hydrogels. Sr. No. Codes T onset (°C) T m (°C) T offset (°C) 1 CPC-ZiF-3% 220 226 231 2 CPC-ZiF-6% 220 228 233 3 CPC-ZiF-10% 220 230 235 4.4. Surface morphology and elemental analysis The surface structure of synthesized material was analyzed by recording SEM micrographs, as shown in Fig. 2 , at resolutions of 100µm and 200 µm. The SEM images show different pore sizes and shapes of fabricated composite hydrogels, as shown in Fig. 2 (a-f). Introducing highly porous ZiF-8 particles in the polymer blend induces variable pore distribution, leading to numerous biological processes, including cell proliferation and migration [ 35 ]. In this work, the concentration of ZiF-8 was increased from 3–10% to obtain a more uniform pore size distribution. The wound dressing with 3% ZiF-8 particles shows lesser porosity and irregular pore sizes. As the concentration increased to 6% and 10%, the porosity increased, and pore shapes and sizes became more spherical and uniform. The increasing ZiF-8 concentration causes more porosity, absorbing additional wound exudate and other biological fluids to keep the wound environment moist, supporting wound healing and repairing [ 36 ]. It can be observed that increasing the amount of ZiF-8 regulates the pore size distribution, which increases surface area. The elemental analysis of the composite hydrogels has been presented in tabular form for the quantitative analysis as in Fig. 2 (g-i), and the graphical analysis to show the peak intensity in Fig. 2 (j-l). It can be seen that increasing the amount of ZiF-8 in the polymeric matrix increases the peak intensity of the zinc and the elemental analysis also confirm that there is not foreign element, which may cause the toxicity. Hence, the different morphology and the elemental analysis of the composite hydrogels confirmed the successful fabrication of the composite hydrogels. 4.5. Swelling in aqueous, PBS, and NaCl media The hydrophilic character of the polymers that make up the hydrogels accounts for their swelling tendency. Their water uptake capacity is severalfold greater than their weight, and the increasing amount of ZiF-8 added to the polymeric matrix of the composite hydrogels has different swelling trends at various pH levels. The swelling characteristics of fabricated composite hydrogels were studied in aqueous media to varying pHs (Fig. 3 (a)). It was observed that the fabricated hydrogels exhibited more swelling in acidic medium at pH = 3 and less swelling in basic media at pH = 11. Maximum swelling was found in neutral media (pH = 7). However, the composite hydrogels have shown excellent swelling characteristics. In the acidic media, H + is excessively available, and these protonate the amino groups, which are available at the backbone of the chitosan to produce ammonium ions [ 37 ]. Increasing protonation at acidic pH causes more charge, which causes repulsion and osmosis, increasing the media penetration into the polymeric. With the increase in pH, the swelling increased and reached a maximum at neutral pH due to deprotonation. The swelling behavior follows the trend of Aqueous > PBS > NaCl media. Further increased pH caused the decrease in swelling because of the formation of ammonium ions, which decreases interactions between polymer chains. The maximum swelling at pH-7 would be because of the maximum mobility of the polymeric chain, which results in increased swelling. Similarly, the swelling behavior was found in PBS (Fig. 3 (b)) and NaCl (Fig. 3 (c)) media. Increasing ZiF-8 concentration reduces the swelling ratio because more ZiF-8 particles impart additional crosslinking and restrict the mobility of chains within the polymer network, making it difficult to absorb more water. Figure 3 (a-c) displays how composites containing 3% ZiF-8 particles exhibit significant swelling. Increasing the concentration of ZiF-8 to 6% and 10% reduced the swelling response. Since swelling acts as a barrier against subsequent infections and keeps the area surrounding the lesion wet, it is crucial to the healing process of wounds. Because of this property, composite hydrogel is a more effective wound dressing that promotes wound healing. 4.5.1. Biodegradation analysis Figure 3 (d) demonstrates how the biodegradation of composite material was tracked in a PBS medium at 37 °C and 7.4 pH. All the compositions consisting of varying amounts of ZiF-8 show different degradation behavior. The formulation with 3% ZiF-8 degraded rapidly while the degradation process was slow, raising ZiF-8 concentration to 6%. The composite hydrogels containing 10% ZiF-8 degraded the least. The extent of crosslinking governs the rate of degradation. The higher concentration of ZiF-8 increases the mechanical strength and thermal stability, reducing the biodegradation rate. The polymer chains are strongly bound and less vulnerable to decomposition. Increasing ZiF-8 may also alter the degradation pathways by stimulating heterogeneous degradation procedures. The highly porous structure of ZiF-8 particles retards the penetration of oxygen, moisture, and several other degrading agents into the polymer solution slowing down the degradation mechanism. The fabricated composite system is significant for absorbing excessive wound exudate and maintaining a moist environment of the wound along with controlled biodegradation. 4.5.2. Wettability Wettability study is one crucial surface phenomenon for determining if biomaterials are hydrophilic or hydrophobic. A critical feature that characterizes how the hydrogels interact with the biofluids is their hydrophilic nature. The hydrophilic hydrogel can potentially absorb the wound exudate, which helps with wound care and treatment [ 38 ]. Therefore, wettability determines the hydrophilicity by measuring the water contact angle, which helps to understand the cellular interaction with the hydrogel during wound healing [ 39 ]. The wettability of the produced composite hydrogels was determined at different time intervals (Fig. 4 (a)), and it was shown that raising ZiF-8 causes the water contact angle to rise and changes the pattern from more hydrophilic to less hydrophilic morphology. Because the tightly packed polymeric chains occupy the hydrophilicity-causing functional groups, it may result from reduced mobility. The increasing ZiF-8 amount has hydrophilicity trends such as CPC-ZiF-3% > CPC-ZiF-6% > CPC-ZiF-10%. The optimized amount of ZiF-8 causes the required wettability for specific applications. Several factors influence the cellular interaction with the hydrogels, including wettability, surface morphology, roughness, stiffness, surface charge, and chemistry [ 40 ]. Optimized wettability promotes cellular interaction via cell adherence proliferation and influences the cell-hydrogel interaction supporting wound healing [ 41 ]. Hence, the different wettability confirms the fabrication of different composite hydrogels. 4.5.3. Gel fraction The degree of crosslinking determines the gel fraction of composite hydrogels, so a higher degree of crosslinking results in a higher gel fraction. Based on our observation of the gel fraction percentage (Fig. 4 (b)), we have found that the percentage of gel fraction increases as the amount of ZiF-8 increases. The CPC-ZiF-3% has the least gel fraction, and CPC-ZiF-10% has the maximum value of the gel fraction, while CPC-ZiF-6% has a value between both the composition of the composite hydrogels. Since the increasing amount of Zif-8 increases its interaction with the polymeric chains, it has been via different interactions, including hydrogen bonding and other weaker forces (ionic-liquid and van der Waal forces, etc.). Hence, the increasing amount of ZiF-8 increases the interaction between the chains via hydrogen bonding with different functional groups of the polymers, as illustrated in Scheme-I, and it acts as a crosslinker to reduce the mobility of the polymeric chains. 4.6. In vitro assays 4.6.1. Cell morphology The morphology of fibroblast cells was analyzed by biological assays after the time intervals of 24, 48, and 72h, as depicted in Fig. 5 (A). The morphological studies of fibroblast cells help understand the cell response to numerous stimuli, including growth factors and components of the extracellular matrix and cytokines. The structural modifications also demonstrate wound healing by disclosing cell migration, proliferation, and extracellular matrix synthesis. The appropriate shape of fibroblast cells is crucial when engaging with various substrates, such as hydrogels, for wound repair or tissue regeneration applications. Figure 5 (A) shows the elongated, cylindrical, spindle-like fibroblast cells with proper cell adherence [ 34 ]. The spindle-shaped cells navigate via the extracellular matrix and reach the wound region to stimulate wound repair. By increasing the concentration of ZiF-8 nanoparticles in composite hydrogels and the contact time of composite hydrogels to fibroblast cells, cells attain more cylindrical morphology for better tissue repair. The shape of these cells facilitates the synthesis and alignment of extracellular matrix fibers, such as collagen and fibronectin, to provide structural integrity for tissue repair. An increase in ZiF-8 concentration has also boosted the stimulation of cell re-epithelialization by the cell population. Zinc is a well-known metal and essential co-enzyme to several enzymes with potential and promising biological properties [ 42 ]. The zinc from ZiF-8 particles controls the enzymatic activity during the cell division and synthesis of DNA, which are crucial for cell migration and proliferation [ 43 ]. Zinc ion lowers oxidative stress, reduces cell damage, and promotes healthy cellular morphology and function, as zinc is a trace element that acts as an antioxidant. Zinc ions also act as a cofactor for collagen synthesis, so higher zinc ions enhance the structural support needed for wound repair [ 44 ]. Hence, the increasing ZiF-8 causes an increase in cell density with mature cell morphology and is helpful for wound healing. 4.6.2. Cell viability and proliferation Cell viability and proliferation have been studied against multiple contact times (dilutions) via 3T3 cell lines of bioactive composite hydrogels, as demonstrated in Fig. 5 (B). Cell viability and proliferation are critical to be studied as these directly contribute to repairing damaged tissue and wound healing. Figure 5 B (a–c) illustrates the correlation between improved cell viability and increases in ZiF-8 concentration and contact times. Figure 5 B (d-f) shows nearly identical behavior for cell proliferation. ZiF-8 releases zinc ions continuously to ensure their optimum level, which controls numerous cellular mechanisms involved in wound repair [ 34 ]. These ions maintain the number of viable cells and increase cell proliferation, leading to the most effective wound repair. Polymeric hydrogels contain several functional groups, including hydroxyl, amine, and sulfonate ester groups, which interact with many receptors or binding sites to promote fibroblast cell adhesion [ 45 ]. The cells get adhered via integrin, and various cell adhesion molecules and proteoglycans are adhered to via electrostatic interactions, hydrogen bonding, or chemical crosslinking reactions [ 46 ]. These interactions promote cell adherence, viable cells, and proliferation, making hydrogels suitable for wound repair applications. 4.7. Antibacterial activities The antibacterial mechanism is depicted in Fig. 5 (C), while the antibacterial efficacy of the composite hydrogels using Gram (positive and negative) bacteria strains. These include E. coli and S. aureus, presented in Fig. 5 (D). The hydrogels demonstrate varied antibacterial properties due to their distinct formulations and structural characteristics. It can be seen that the hydrogels with the least ZiF-8 have the least antibacterial activities, while the hydrogel with the maximum amount of ZiF-8 has enhanced antibacterial activities. The antibacterial efficacy of the hydrogels follows the sequence CPC-ZiF-3% < CPC-ZiF-6% < CPC-ZiF-10%, potentially due to the sustained release of ZiF-8 from the polymeric matrix of the hydrogels. The hydrogels have a synergistic effect of ZIF-8 and polymeric matrix as the polymeric part in hydrogel may interact with phospholipids and lipopolysaccharides of the bacterial membrane. The polymers may penetrate and rupture the bacterial membrane to hinder bacterial growth. The other possibility could be that the polymers may bind with the DNA and inhibit the replication of bacteria, causing antibacterial activities. Various bacteria may colonize the injured region and cause inflammation, pain, and infections, and excessive skin tissue exudate may damage the healthy tissues [ 47 ]. The antibacterial properties of the hydrogels suggest that integrating ZIF-8 into the polymeric matrix with varying crosslinking may yield distinct antibacterial effects. The antibacterial mechanism is attributed to Zn 2+ , which may produce reactive oxygen species (ROS) that compromise bacterial integrity, resulting in extensive bacterial eradication. The Zn ions have a positive charge, and the bacterial membrane has a negative charge that can penetrate the bacterial membrane. It interacts with the replication process of DNA to hinder bacterial growth. The Zn 2+ may accumulate over bacterial membranes and cause antibacterial activities by rupturing bacterial membranes to cause antibacterial activities [ 48 ]. Since zinc ions are released to the wound site under control and have the potential to break down bacterial membranes, ZIF-8 has outstanding antibacterial properties. It also works as a co-enzyme and potential material for generating reactive oxygen species to kill bacteria [ 49 ]. Hydrogels acquire the ability to thwart bacterial invasion by safeguarding the wound from pathogenic bacteria while also absorbing wound exudate and maintaining a microenvironment suitable for wound healing and regeneration. Incorporating ZiF-8 into the polymeric matrix may facilitate the controlled release of ZiF-8 on demand at the wound site. The sustained release of antibacterial agents is crucial for wound healing to protect bacterial pathogens. The crosslinker possesses multiple hydroxyl functional groups that confer structural properties, such as surface roughness and hydrophilic characteristics [ 50 ]. Hence, the fabricated hydrogels have different potential antibacterial activities that increase with increasing amounts of ZiF-8. 4.8. Wound healing In this work, we evaluated the effectiveness of different hydrogel formulations containing ZIF-8 through in vivo wound healing experiments, as shown in Fig. 6 (a-c). As part of the treatment plan, the composite hydrogels were applied to wounds at predetermined intervals of 0, 3, 7, 10, and 14 days in order to evaluate how well they promoted tissue repair and regeneration and wound closure gradually. We have illustrated different wound phases, as shown in Fig. 6 (a). The hydrogel without ZiF-8 has been used as a control, and it also exhibits a desirable wound-healing capacity. It has been demonstrated that the addition of zinc to the composite ZIF-8@hydrogels greatly accelerates the healing of wounds. The hydrogels containing different amounts of ZiF-8 have different wound-healing behaviors. The group exhibited exceptional healing in comparison to the scars seen in the blank control group. The CPC-ZIF-10% formulation was noteworthy for its ability to promote efficient and regenerative tissue repair by dramatically speeding up the wound-healing process while preventing scarring. The majority of hydrogel formulations were found to have a wound healing rate of about 40% after 7 days of treatment, suggesting that they were moderately effective in promoting tissue repair during this period. The CPC-ZIF-10% formulation, on the other hand, performed better, showing over 50% wound healing in the same time frame. The special qualities of the CPC-ZIF-10% hydrogel, which are probably caused by the combined effects of ZiF-8 and the polymeric matrix, are responsible for this increased efficacy. Because CPC-ZiF-10% contains the highest concentration of ZiF-8 that releases more zinc and therefore CPC-ZIF-10% demonstrates greater therapeutic potential than CPC-ZiF-3%, CPC-ZiF-6%, and control, as shown in Fig. 6 (b and c). The maximum amount of ZiF-8 in CPC-ZiF-6% makes it a viable option for advanced wound care applications. Zinc is a vital trace element that is crucial to many physiological and biochemical functions that are required to preserve the health of cells and systems. Zinc plays an important role in catalysis, structure, and regulation as a vital component of many enzymes and is necessary for many metabolic processes [ 51 ]. Because of its involvement in cellular functions like DNA synthesis, protein metabolism, and tissue repair, it is essential for efficient cellular operation. Zinc has a major impact on cellular behavior in the context of wound healing by improving cell viability, encouraging differentiation, and facilitating cell migration and proliferation, especially for fibroblasts and keratinocytes, which are essential for tissue regeneration [ 52 , 53 ]. Zinc also helps to modulate inflammatory responses, promotes angiogenesis, and facilitates the synthesis of collagen—all of which are essential for the remodeling and repair of damaged tissues. Zinc reduces complications and promotes functional tissue restoration by taking part in these complex biological processes, which not only encourages wound closure but also enhances healing quality [ 52 ]. 5. Conclusions Herein, we produced composite hydrogels for wound healing applications, combining synthetic polymers (PVA) with biopolymers (CMCs and CG). ZiF-8 is incorporated into the polymeric matrix of hydrogels. The FTIR confirmed the multifunctionality of the hydrogels, while XRD revealed their amorphous and crystalline behavior. These hydrogels are porous, and as the amount of ZiF-8 increases, the uniform pore distribution causes the pore size to decrease as the surface area increases. Increased ZiF-8 content increases thermal stability in these composite hydrogels, and hydrogel CPC-ZiF-10% pore size-controlled maximum stability. The composite hydrogels with the least amount of ZiF-8 cause less cell density, viability, and proliferation. The composite hydrogel CPC-ZiF-3% possessed the least cellular behavior, and CPC-ZiF-10% exhibited maximum cytocompatibility with mature cell morphology. Similarly, CPC-ZiF-10% exhibited excellent wound healing without causing any inflammation. Hence, the fabricated composite hydrogels are potential wound dressing materials for wound healing applications. Declarations Conflicts of Interest: All authors declared no conflict of interest. Author Contribution Credit Authorship:Conceptualization, Muhammad Umar Aslam Khan, and Humaira Masood Siddiqui; Data curation, Tooba Yasin, Hina Khan, and Thamir M Eid; Formal analysis, Hayat M. Thamir M Eid, and Muhamad Umar Aslam Khan; Funding acquisition, Muhammad Umar Aslam Khan, Thamir M Eid, and Aneela Javed; Investigation, Muhammad Umar Aslam Khan, and Muhammad Shahzad Zafar; Methodology, Muhammad Umar Aslam Khan, Humaira Masood Siddiqui, and Muhammad Shahzad Zafar; Project administration, Muhammad Umar Aslam Khan, Hayat M. Albishi, and Humaira Masood Siddiqui; Resources, Muhammad Umar Aslam Khan, Hayat M. Albishi, and Thamir M Eid; Software, Muhammad Umar Aslam Khan, Hina Khan, and Tooba Yasin; Supervision, Muhammad Umar Aslam Khan, and Humaira Masood Siddiqui; Validation, Hayat M. Albishi, Thamir M Eid, and Anela Javed; Visualization, Tooba Yasin, Hina Khan, and Muhammad; Writing–original draft, Tooba Yasin, Muhammad Umar Aslam Khan; Writing – review & editing, Muhammad Umar Aslam Khan, and Humaira Masood Siddiqi. Data availability: The data contained within the manuscript. References Zhang, H., et al., Developing natural polymers for skin wound healing. 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Xiang, G., et al., A Zn-MOF-GOx-based cascade nanoreactor promotes diabetic infected wound healing by NO release and microenvironment regulation. Acta Biomaterialia, 2024. Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Graphicalabstract.jpg Graphical abstract: The graphical abstract illustrates the fabrication of composite hydrogels incorporated with Zif-8 into the polymeric matrix of carrageenans, carboxymethyl chitosan and polyvinyl alcohol and their potential antibacterial and wound healing applications. scheme1.jpg Scheme-I. Chemical illustrations have been proposed for fabricating composite hydrogel from carrageenan, carboxymethyl chitosan, and polyvinyl alcohol incorporated with ZiF-8 via crosslinking by TEOS. Cite Share Download PDF Status: Published Journal Publication published 11 Jun, 2025 Read the published version in Polymer Bulletin → Version 1 posted Editorial decision: Revision requested 23 Apr, 2025 Reviews received at journal 22 Apr, 2025 Reviews received at journal 16 Apr, 2025 Reviewers agreed at journal 07 Apr, 2025 Reviewers agreed at journal 04 Apr, 2025 Reviewers agreed at journal 03 Apr, 2025 Reviewers invited by journal 02 Apr, 2025 Editor assigned by journal 02 Apr, 2025 Submission checks completed at journal 31 Mar, 2025 First submitted to journal 31 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-6345624\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":446812669,\"identity\":\"2f0bb05f-e251-4281-9b6f-34d77ac35932\",\"order_by\":0,\"name\":\"Tooba Yasin\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Quaid-i-Azam University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Tooba\",\"middleName\":\"\",\"lastName\":\"Yasin\",\"suffix\":\"\"},{\"id\":446812671,\"identity\":\"47583c6d-b99b-4101-b4e2-7db7ccfee241\",\"order_by\":1,\"name\":\"Muhammad Shahzad Zafar\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"SKKU Advanced Institute of Nanotechnology (SAINT), Sungkyunkwan University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Muhammad\",\"middleName\":\"Shahzad\",\"lastName\":\"Zafar\",\"suffix\":\"\"},{\"id\":446812672,\"identity\":\"64ba821f-7483-4597-b2fd-daab74d73c45\",\"order_by\":2,\"name\":\"Muhammad Umar Aslam Khan\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6klEQVRIiWNgGAWjYLCCBIP/BgYMDIwPgGwePqK0fKhgBmlhNgBpYSNGB+OMM2AtbBIgHkEtBtcOP/vM28ZmbM5+9ljl1xw7GTYG5oePbuDTcjvNeDZvG4+ZZU9e2m3ZbclAh7EZG+fg1ZJgzMzbJmFjcCDH7LbkNmagFh42afxa0j8DtRjYGJx/Y1Ysua2eGC05xkDvJ5gZ3MgxY/y47TBhLZK3c4qBgXzA2ODGG2Npxm3HediYCfiF73b6ZmBUHjDccD7H8OPPbdX2/OzNDx/j04ICmHnAJLHKQYDxBymqR8EoGAWjYMQAAHsIRhpMGb3GAAAAAElFTkSuQmCC\",\"orcid\":\"\",\"institution\":\"Qatar University\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Muhammad\",\"middleName\":\"Umar Aslam\",\"lastName\":\"Khan\",\"suffix\":\"\"},{\"id\":446812673,\"identity\":\"3b24198c-2376-42a5-9188-20553c5a2967\",\"order_by\":3,\"name\":\"Hina Khan\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"National University of Sciences and Technology\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Hina\",\"middleName\":\"\",\"lastName\":\"Khan\",\"suffix\":\"\"},{\"id\":446812675,\"identity\":\"0c957300-c1c0-424a-a707-399370bb8c4b\",\"order_by\":4,\"name\":\"Hayat M. 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composite hydrogels, (a) FTIR spectrum ranging 3600-500 cm\\u003csup\\u003e‒1\\u003c/sup\\u003e and (b) FTIR spectrum ranging 3600-500 cm\\u003csup\\u003e‒1\\u003c/sup\\u003e, (c) XRD of the composite hydrogels and (d) thermal behavior of the composite hydrogels with temperature ranging from 0-800 °C.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6345624/v1/1d075822d52f3fa1d31ffe57.jpg\"},{\"id\":81253670,\"identity\":\"f4087bf7-7eba-4b3c-a34b-cb9a22048439\",\"added_by\":\"auto\",\"created_at\":\"2025-04-24 04:00:15\",\"extension\":\"jpg\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":125297,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThe surface morphology at different magnifications (200 and 100 µm) and elemental analysis of the composite hydrogel SEM coupled with EDX.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6345624/v1/7448f5c12e470c893ce23b3a.jpg\"},{\"id\":81253674,\"identity\":\"d0e7b000-675b-4778-bb43-255ead109a68\",\"added_by\":\"auto\",\"created_at\":\"2025-04-24 04:00:15\",\"extension\":\"jpg\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":84431,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThe swelling of composite hydrogels in (a) aqueous, (b) phosphate buffer saline media, (c) electrolyte (NaCl) media, and d) degradation in phosphate buffer saline media at 37 \\u003csup\\u003eo\\u003c/sup\\u003eC.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6345624/v1/0e98b27815963f69242bf362.jpg\"},{\"id\":81253677,\"identity\":\"b2591f15-1324-4d6e-a960-0f19afc35b15\",\"added_by\":\"auto\",\"created_at\":\"2025-04-24 04:00:15\",\"extension\":\"jpg\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":43663,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e(a) wettability at different time intervals (b) and gel fraction of the composite hydrogels.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6345624/v1/e7dfc26f890690d035fc70ec.jpg\"},{\"id\":81254183,\"identity\":\"f395d082-f7da-4115-bd11-5b78d7c4e17e\",\"added_by\":\"auto\",\"created_at\":\"2025-04-24 04:08:15\",\"extension\":\"jpg\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":129670,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThe in vitro biological activities, A) cell morphology of the fibroblast cell lines after different time intervals (24-72 h), B) cell viability and proliferation of the fibroblast cell lines after different time intervals (24-72 h), C) antibacterial mechanism and D) antibacterial activities (Gram-positive and Gram-negative) against the composite hydrogels.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"5.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6345624/v1/5ad7653a6305b28b6735f6f5.jpg\"},{\"id\":81255054,\"identity\":\"ed8cde03-05c9-43b8-8cd3-a859b6ea7f30\",\"added_by\":\"auto\",\"created_at\":\"2025-04-24 04:16:15\",\"extension\":\"jpg\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":92797,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThe wound healing using composite hydrogels using in vivo SD rat model, (a) different wound healing phases, developing full-thickness wound model, wound treatment using composite hydrogels, and complete wound healing. (b) digital photographs of wound healing and (c) wound contraction percentage.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"6.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6345624/v1/9d03ebb36398dbdb91ff4671.jpg\"},{\"id\":84726860,\"identity\":\"730f70f1-444a-476a-8965-c7deb65e3213\",\"added_by\":\"auto\",\"created_at\":\"2025-06-16 16:08:37\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":1625061,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6345624/v1/6a0cf6f8-b338-424a-9219-ea97d8d59981.pdf\"},{\"id\":81254180,\"identity\":\"8fc9ee02-357f-407e-adf2-145bc8213cfb\",\"added_by\":\"auto\",\"created_at\":\"2025-04-24 04:08:15\",\"extension\":\"jpg\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":64697,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eGraphical abstract:\\u003c/strong\\u003e The graphical abstract illustrates the fabrication of composite hydrogels incorporated with Zif-8 into the polymeric matrix of carrageenans, carboxymethyl chitosan and polyvinyl alcohol and their potential antibacterial and wound healing applications.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Graphicalabstract.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6345624/v1/37027e05fdc3c5cb81b21403.jpg\"},{\"id\":81254181,\"identity\":\"1c5afefe-fff6-41cd-a42f-62a5077201be\",\"added_by\":\"auto\",\"created_at\":\"2025-04-24 04:08:15\",\"extension\":\"jpg\",\"order_by\":2,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":88930,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eScheme-I.\\u003c/strong\\u003e Chemical illustrations have been proposed for fabricating composite hydrogel from carrageenan, carboxymethyl chitosan, and polyvinyl alcohol incorporated with ZiF-8 via crosslinking by TEOS.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"scheme1.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6345624/v1/055b5b0448b9322ab09b815c.jpg\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Fabrication of composite hydrogels from Carrageenan-cl-carboxymethyl chitosan-PVA incorporated ZiF-8 for wound healing applications\",\"fulltext\":[{\"header\":\"Highlights\",\"content\":\"\\u003cp\\u003e\\u0026bull; Fabrication of the composite hydrogels by incorporating ZiF-8 as a bioactive dressing material for wound healing.\\u003c/p\\u003e\\u003cp\\u003e\\u0026bull; The development of composite hydrogels using a simple blending method from natural polymers for wound healing applications.\\u003c/p\\u003e\\u003cp\\u003e\\u0026bull; Hydrogels with enhanced physicochemical properties with pH-responsiveness.\\u003c/p\\u003e\\u003cp\\u003e\\u0026bull; Cytocompatible composite hydrogels via cell viability and proliferation with mature cell morphology.\\u003c/p\\u003e\\u003cp\\u003e\\u0026bull; Enhance wound healing by increasing the ZiF-8 amount in the composite hydrogels.\\u003c/p\\u003e\\u003cp\\u003e : The graphical abstract illustrates the fabrication of composite hydrogels incorporated with Zif-8 into the polymeric matrix of carrageenans, carboxymethyl chitosan and polyvinyl alcohol and their potential antibacterial and wound healing applications.\\u003c/p\\u003e\"},{\"header\":\"1. Introduction\",\"content\":\"\\u003cp\\u003eThe skin is an essential organ in the human body that functions as a barrier of protection against various external environmental bacterial attacks and external environmental damages. Numerous forms of trauma, burns, ulcers, and other harmful events frequently impair the skin\\u0026rsquo;s otherwise resolute barrier and interfere with its essential role in sensory perception [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e]. Even in the absence of external threats, the skin is vulnerable to damage. When its function at any level is compromised, it can lead to distress on many levels for the individual and society. Therefore, one of the most important subjects in biomedicine is skin healing. The mainstays of effective wound closure are dressings that promote rapid healing [\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e]. Among the available dressings, hydrogel-based wound dressings are particularly distinguished for their ability to facilitate this process. They provide a barrier against contaminants and maintain a wound site hydrated, two crucial elements in promoting efficient tissue repair. At present, bioactive hydrogels are gaining recognition as advanced wound dressings [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e]. These excellent dressing materials exhibit several unique properties that make them suitable for this application. They can be tailored to adapt to the shapes of irregularly and deeply contoured wounds. They can also be designed to incorporate drugs and other bioactive agents. The past decade has seen an explosive growth of interest in using hydrogels for wound dressings [\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e]. Nonetheless, there is a significant deficit in understanding their performance as dressing materials. Bioactive hydrogels that gel quickly and that have wound-healing properties that are accelerated beyond what other materials can do are particularly interesting for use in wound care. One reliable way to develop such materials is to use multiple kinds of reversible interactions as crosslinkers in the gel structure. It increases the hydrogel\\u0026rsquo;s antibacterial properties and enhances its wound-healing capabilities [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eBacterial contamination is still a significant problem in wound healing, especially in chronic wounds. These wounds show a healing delay because they display a never-ending inflammation phase that leads to chronic inflammation [\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e]. An increased microbial bioburden and oxidative stress will surely suffice. We are making significant efforts, which include the administration of antibiotics, to circumvent this issue and make progress toward the healing of wounds. The application of bioactive compounds and the incorporation of novel nanomaterials into wound dressings reduce inflammation in some way [\\u003cspan additionalcitationids=\\\"CR9\\\" citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e]. The emergence of drug-resistant pathogens now represents a major threat to public health and constitutes a huge socio-economic burden. Synthetic antibiotic drugs, when used excessively, bring about this emergence. One potential solution to this issue is to employ wound dressings that contain non-antibiotic treatments that are effective against bacterial infections. Natural antimicrobial compounds are biocompatible and environmentally friendly, and they provide excellent infection treatments, unlike antibiotics [\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eMetal-organic frameworks are highly porous nanomaterials that have gained popularity due to their uniqueness in the biomedical field. ZIF-8 has a strong antimicrobial effect that makes it a powerful agent against any dangerous bacteria that could infect a wound [\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e]. ZiF-8 protects the wound from bacterial attack due to its potential antibacterial activities that accelerate the healing of infected wounds [\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e]. Chitosan (CS) has also gained considerable attention for medical applications because it has inherent antibacterial properties. At the same time, the many amino groups along the backbone allow you to modify chitosan with moderate covalent interactions. Chitosan demonstrates biocompatibility, promotes cell growth and attachment, and has controllable degradation properties [\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e]. It makes chitosan a highly suitable candidate for many biomedical applications. Because of its innate antibacterial properties, chitosan has attracted great interest in the wound healing application arena. Additionally, carboxymethyl chitosan (CMCs), a derivative of chitosan, is widely applied in drug delivery and is even more often used as a material in tissue engineering. CMCs are also being extensively studied in the area of wound healing, and their antibacterial activities accelerate infected wound healing [\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eCarrageenans are naturally occurring polymers taken from red algae that belong to the Rhodophyceae group of plants. These materials are widely used in the food and biomedical industries as emulsifiers, thickeners, gel-forming agents, and stabilizers [\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e]. They are polysaccharides, and their various types are categorized by the amount and accessibility of sulfate groups on the disaccharide monomer units. Kappa (κ-) carrageenan has one sulfate group; iota (ι-) carrageenan has two; and lambda (λ-) carrageenan has three sulfate groups per disaccharide monomer unit. These have several potential applications in wound healing and drug delivery due to their hydrogen bonding or ionic interactions [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e]. Polyvinyl alcohol (PVA) is the synthetic polymer of choice, winning out for a variety of very good reasons: its non-cytotoxicity, hydrophilicity, biocompatibility, transparency, and ease of incorporation into swelled-state forms [\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e]. PVA, a promising biomaterial, is particularly notable for its easy processing and nearly ideal mechanical and chemical properties that can be varied over a wide range. This hydrophilic material can form hydrogen bonds with other hydrophilic substances, making it suitable for numerous applications\\u0026mdash;everything from contact lenses to cartilage, drug delivery systems to wound dressings, and skin replacement components [\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eHerein, we have developed composite hydrogel with enhanced antibacterial activities and accelerated wound closure for wound healing applications. Different formulations of the hydrogel were produced from carrageenan (CG), polyvinyl alcohol (PVA), and carboxymethyl chitosan (CMCs) and were incorporated with varying amounts of ZiF-8. To the best of our knowledge, these formulations of novel hydrogel systems have never been reported before using a simple blending method. We have employed several techniques to study the structural, morphological, thermal, and mechanical properties of composite hydrogels. The physicochemical analysis was also performed to determine the swelling and degradation behavior of the composite hydrogels, and their in vitro assay was performed to evaluate cell viability, proliferation, and morphology assays using fibroblast cell lines. Finally, we used a standard in vivo model to assess the wound healing potential of the composite hydrogel.\\u003c/p\\u003e\"},{\"header\":\"2. Materials and methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.1. Materials\\u003c/h2\\u003e \\u003cp\\u003eCarrageenan (CAS number: 9000-07-1, light brown powder), carboxymethyl chitosan (CAS number: 83512-85-0, light yellow powder with carboxymethylation =/\\u0026gt; 80%), polyvinyl alcohol (CAS number: 9002-89-5, Mw 31,000\\u0026ndash;50,000), and Tetraethoxysilane (CAS number: 78-10-4, Mw 208,33 g/mol) were obtained from Merck, Germany. Sodium chloride (NaCl), acetic acid, absolute ethanol, and potassium persulfate (K\\u003csub\\u003e2\\u003c/sub\\u003eS\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e8\\u003c/sub\\u003e) were purchased from Sigma Aldrich. All the chemicals are analytical grade chemicals used as received.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.2. Biological materials and reagents\\u003c/h2\\u003e \\u003cp\\u003eGram (positive and negative) bacterial pathogens were (Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli)), and Fibroblast (3T3) cell lines were purchased from ATCC (Manassa, VA, USA), and preserved as per ATCC recommendations. α-MEM medium, Fetal Bovine Serum (FBS), Penicillin, and Streptomycin were obtained from Hyclone Laboratories Inc. and ThermoFisher Scientific, respectively. The live/dead assay was performed using the kit obtained from Invitrogen (R37601). BOBO-3 Iodide (red) and Calcein-AM (green) were used to assess the cell morphology, and the assay was performed according to the supplier\\u0026rsquo;s instructions. Male Sprague-Dawley (SD) was supplied by the National Institutes of Health (NIH) in Islamabad, Pakistan.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.3. Methods\\u003c/h2\\u003e \\u003cdiv id=\\\"Sec6\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.3.1. Synthesis of ZIF-8\\u003c/h2\\u003e \\u003cp\\u003eWe have synthesized the ZIF-8 according to already reported by Attwa \\u003cem\\u003eet al\\u003c/em\\u003e. [\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e]. In brief, we mixed zinc hexahydrate, an organic linker, and 2-methylimidazole in deionized water in a ratio of 1:35:1238. Then, 2-methylimidazole (H-MIM) (12 g) and zinc nitrate (1.25 g) were dissolved in deionized water. The solution containing Zn-ions was added dropwise into H-MIM while stirring vigorously at room temperature for 2 h. The polymeric solution formed a milky color, which indicates the formation of ZiF-8 and was collected by centrifuging at 4500 rpm for 30 minutes. It was washed thrice with deionized water, followed by oven-drying at 60 \\u0026ordm;C for 24 h to remove moisture or water contents. The ZIF-8 nanoparticles were stored in glass vials to incorporate into the polymeric composite to develop a composite hydrogel system.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec7\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.3.2. Development of composite polymeric systems\\u003c/h2\\u003e \\u003cp\\u003eThe composite hydrogels based on polymers were developed by mixing carrageenan, carboxymethyl chitosan, polyvinyl alcohol, and ZIF-8 particles. Briefly, carrageenan (0.5 g) and carboxymethyl chitosan (0.3 g) were dissolved in distilled water separately. Then, polyvinyl alcohol (0.2 g) was dissolved in additional distilled water (15 mL) at 80 \\u003csup\\u003eo\\u003c/sup\\u003eC and combined with the other components. All the polymeric solutions were mixed and stirred at 60\\u0026deg;C for 2 h to obtain homogeneous polymeric solutions. We have added different amounts of ZIF-8 particles (3, 6, and 10%) into the polymeric solution. A fixed concentration of TEOS (180 \\u0026micro;L) was dissolved in 5ml ethanol, poured dropwise into the whorl of the polymeric media, and stirred at 60\\u0026deg;C for 1 h. The initiator KPS (0.05) was then added to the polymeric media for effective crosslinks and allowed to be stirred at 60\\u0026deg;C for 3 h. After 3 h, the composite polymeric solution was poured into the Petri plate. We have summarized the components of the composite in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e.\\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\\u003eThe table summarizes the composition of the fabricated composite hydrogels.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"6\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c6\\\" colnum=\\\"6\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eSr. No.\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eCarrageenan (g)\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eCarboxymethyl chitosan\\u003c/p\\u003e \\u003cp\\u003e(g)\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003ePolyvinyl alcohol\\u003c/p\\u003e \\u003cp\\u003e(g)\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003eTEOS\\u003c/p\\u003e \\u003cp\\u003e(\\u0026micro;L)\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003eZiF-8\\u003c/p\\u003e \\u003cp\\u003e(%)\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e1.\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0.5\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0.3\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0.2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e180\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e3\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e2.\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0.5\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0.3\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0.2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e180\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e6\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e3.\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0.5\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0.3\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0.2\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e180\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e10\\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 \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.3.3. Fabrication of composite hydrogels\\u003c/h2\\u003e \\u003cp\\u003eThe Petri plates were placed in the freezer and subsequently freeze-dried to obtain various formulations of the porous composite hydrogel system. The composite hydrogels were coded according to the varying quantities of ZiF-8 (CPC-ZiF-3%, CPC-ZiF-6%, and CPC-ZiF-10%). The polymeric solution was stored in glass vials for biological testing, while the produced composite hydrogel films were packed in an airtight plastic bag for further characterization and analysis. The chemical reaction of the composite hydrogels that we have proposed is illustrated in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e.\\u003c/p\\u003e\"},{\"header\":\"3. Characterizations\",\"content\":\"\\u003cp\\u003eThe composite hydrogels were structurally analyzed with the help of the PerkinElmer Diamond 1000 spectrophotometer, performing Fourier transform infrared spectroscopy over the 4000\\u0026thinsp;\\u0026minus;\\u0026thinsp;400 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e wavelength range. An X-ray diffractometer (Bruker AXS D8 Advance XRD) was used to carry out the phase analysis over a range of 20\\u0026deg;-80\\u0026deg; to determine amorphous or crystalline behavior. The surface morphology was determined using a scanning electron microscope (SEM Jeol, Japan), and the hydrogel samples were gold-sputtered before analysis. The analysis of wetting was also conducted by measuring the angle of contact with water using equipment JY-82, Dingsheng, Chengde, China, to determine the nature of hydrogels\\u0026mdash;whether they are hydrophobic or hydrophilic.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec10\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.1. Swelling analysis\\u003c/h2\\u003e \\u003cp\\u003eThe swelling analysis of composite hydrogel was performed in different media (electrolyte (2M NaCl), phosphate buffer solution (PBS), and water) and different pH ranges at 37 \\u0026deg;C for 24 h. The hydrogel samples were cut into square shapes weighing 50mg (\\u003cem\\u003eW\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003ei\\u003c/em\\u003e\\u003c/sub\\u003e) and placed in the abovementioned media. The samples were removed from the media after 24 h and surface water with tissue paper and weighed the swallow weight (W\\u003csub\\u003e\\u003cem\\u003ef\\u003c/em\\u003e\\u003c/sub\\u003e). The swelling percentage of the composite hydrogels was calculated by Eq.\\u0026nbsp;\\u003cspan refid=\\\"Equ1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e.\\u003cdiv id=\\\"Equ1\\\" class=\\\"Equation\\\"\\u003e\\u003cdiv format=\\\"TEX\\\" class=\\\"mathdisplay\\\" id=\\\"FileID_Equ1\\\" name=\\\"EquationSource\\\"\\u003e\\n$$\\\\:\\\\varvec{S}\\\\varvec{w}\\\\varvec{e}\\\\varvec{l}\\\\varvec{l}\\\\varvec{i}\\\\varvec{n}\\\\varvec{g}\\\\:\\\\left(\\\\varvec{\\\\%}\\\\right)=\\\\frac{{\\\\varvec{W}}_{\\\\varvec{f}}-{\\\\varvec{W}}_{\\\\varvec{i}}}{{\\\\varvec{W}}_{\\\\varvec{i}}}\\\\:\\\\times\\\\:100$$\\u003c/div\\u003e\\u003cdiv class=\\\"EquationNumber\\\"\\u003e1\\u003c/div\\u003e\\u003c/div\\u003e\\u003c/p\\u003e \\u003cp\\u003eIn the above equation, \\u003cem\\u003eW\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003ei\\u003c/em\\u003e\\u003c/sub\\u003e refers to the initial weight, and \\u003cem\\u003eW\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003ef\\u003c/em\\u003e\\u003c/sub\\u003e shows the final weight of the hydrogel sample.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.2. Degradation analysis\\u003c/h2\\u003e \\u003cp\\u003eThe degradation of composite hydrogels was determined in PBS media having pH 7.4 at 37 \\u0026deg;C after different time intervals (1\\u0026ndash;7 days). The initial weight of dried hydrogels was recorded (\\u003cem\\u003eW\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003ei\\u003c/em\\u003e\\u003c/sub\\u003e) and then placed in PBS media. The composite hydrogel samples were taken out from PBS media after at time (\\u003cem\\u003et\\u003c/em\\u003e). The composite hydrogels were dried in an oven to measure the weight at a time \\u0026ldquo;\\u003cem\\u003et\\u003c/em\\u003e\\u0026rdquo; (\\u003cem\\u003eW\\u003c/em\\u003e\\u003csub\\u003e\\u003cem\\u003et\\u003c/em\\u003e\\u003c/sub\\u003e), and the degradation was determined by Eq.\\u0026nbsp;\\u003cspan refid=\\\"Equ2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e.\\u003cdiv id=\\\"Equ2\\\" class=\\\"Equation\\\"\\u003e\\u003cdiv format=\\\"TEX\\\" class=\\\"mathdisplay\\\" id=\\\"FileID_Equ2\\\" name=\\\"EquationSource\\\"\\u003e\\n$$\\\\:\\\\varvec{D}\\\\varvec{e}\\\\varvec{g}\\\\varvec{r}\\\\varvec{a}\\\\varvec{d}\\\\varvec{a}\\\\varvec{t}\\\\varvec{i}\\\\varvec{o}\\\\varvec{n}\\\\:\\\\left(\\\\varvec{\\\\%}\\\\right)=\\\\frac{{\\\\varvec{W}}_{\\\\varvec{o}}-{\\\\varvec{W}}_{\\\\varvec{t}}}{{\\\\varvec{W}}_{\\\\varvec{o}}}\\\\:\\\\times\\\\:100$$\\u003c/div\\u003e\\u003cdiv class=\\\"EquationNumber\\\"\\u003e2\\u003c/div\\u003e\\u003c/div\\u003e\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.3. Gel fraction\\u003c/h2\\u003e \\u003cp\\u003eThe gel fraction is one of the essential properties of the composite hydrogels. The personage gel fraction of the composite hydrogels was calculated the gel fraction by Eq.\\u0026nbsp;\\u003cspan refid=\\\"Equ4\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e.\\u003cdiv id=\\\"Equ3\\\" class=\\\"Equation\\\"\\u003e\\u003cdiv format=\\\"TEX\\\" class=\\\"mathdisplay\\\" id=\\\"FileID_Equ3\\\" name=\\\"EquationSource\\\"\\u003e\\n$$\\\\:\\\\varvec{G}\\\\varvec{e}\\\\varvec{l}\\\\:\\\\varvec{f}\\\\varvec{r}\\\\varvec{a}\\\\varvec{c}\\\\varvec{t}\\\\varvec{i}\\\\varvec{o}\\\\varvec{n}\\\\:\\\\left(\\\\varvec{\\\\%}\\\\right)=\\\\frac{{\\\\varvec{W}}_{\\\\varvec{o}}-{\\\\varvec{W}}_{1}}{{\\\\varvec{W}}_{1}}\\\\:\\\\times\\\\:100$$\\u003c/div\\u003e\\u003cdiv class=\\\"EquationNumber\\\"\\u003e3\\u003c/div\\u003e\\u003c/div\\u003e\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.4. In vitro studies\\u003c/h2\\u003e \\u003cdiv id=\\\"Sec14\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e3.4.1. Cell morphology\\u003c/h2\\u003e \\u003cp\\u003eWe studied the fibroblast cell lines\\u0026rsquo; behavior in association with hydrogels by performing Live/Dead assays. We determined the cells\\u0026rsquo; morphology after different time intervals (24, 48, and 72 h) against the composite hydrogels. The well plates were incubated for variable time intervals, whereas we carried out the cell culturing in DMEM media against the synthesized hydrogels. We used the Live/Dead assay kit (Invitrogen R37601) to perform the cellular morphology assessments, and we observed the cells using the Olympus fluorescent microscope (Olympus, FV300).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec15\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e3.4.2. Cell viability and proliferation\\u003c/h2\\u003e \\u003cp\\u003eWe analyzed the cytocompatibility of composite hydrogels by performing cell viability and proliferation assays on 3t3 cells. We used different dilutions (1.5\\u0026ndash;2.5 mg/mL) of the composite hydrogels to perform the assays, and we took the measurements at various time points (24\\u0026ndash;72 hours) to determine how well the cells were doing. The cytotoxicity of the composite hydrogels was performed using Neutral Red assays, as reported by Repetto \\u003cem\\u003eet al\\u003c/em\\u003e. [\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e]. Briefly, the cell culture plates were treated with different hydrogel dilutions and incubated at 37 \\u0026deg;C in a CO\\u003csub\\u003e2\\u003c/sub\\u003e (5%) and humidity (95%) environment. The 40 \\u0026micro;g/mL neutral red medium was applied to cells and incubated. The leftover NR medium from the cells was removed by rinsing with a phosphate buffer solution. Additionally, a destaining solution composed of deionized water (50%), absolute ethanol (49%), distilled water (50%), and acetic acid (1%) was also used to remove the neutral red medium at 37 \\u0026deg;C for ten minutes. The cell density was measured by a microplate reader (Bio-Tek, ELx-800, USA) at wavelength 570 nm. The percentage of cell viability was determined by Eq.\\u0026nbsp;\\u003cspan refid=\\\"Equ4\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e.\\u003cdiv id=\\\"Equ4\\\" class=\\\"Equation\\\"\\u003e\\u003cdiv format=\\\"TEX\\\" class=\\\"mathdisplay\\\" id=\\\"FileID_Equ4\\\" name=\\\"EquationSource\\\"\\u003e\\n$$\\\\:Cell\\\\:viability\\\\:\\\\%=\\\\frac{\\\\text{O}\\\\text{D}s-\\\\text{O}\\\\text{D}c}{\\\\text{O}\\\\text{D}c}\\\\times\\\\:100$$\\u003c/div\\u003e\\u003cdiv class=\\\"EquationNumber\\\"\\u003e3\\u003c/div\\u003e\\u003c/div\\u003e\\u003c/p\\u003e \\u003cp\\u003eOn the other hand, OD\\u003csub\\u003ec\\u003c/sub\\u003e = optical density of control and OD\\u003csub\\u003es\\u003c/sub\\u003e = optical density of sample.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec16\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e3.4.3. Antibacterial activities\\u003c/h2\\u003e \\u003cp\\u003eComposite hydrogels\\u0026rsquo; antibacterial properties were evaluated in relation to Gram-positive and Gram-negative bacteria, including Escherichia coli and Staphylococcus aureus. We have investigated antibacterial activities using the disc diffusion approach. Petri dishes containing the hot molten agar were set to solidify; a sterile cotton swab was used to apply the bacterial culture to the solid agar. Then, 65 \\u0026micro;L of hydrogel was placed into wells and incubated at 37\\u0026deg;C 24 h. After 24 h, the antibacterial activities were determined by zone inhibition, which was measured in millimeters by the regular ruler.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec17\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.5. In vivo assay\\u003c/h2\\u003e \\u003cp\\u003eA full-thickness wound model has been developed to assess the efficacy of composite hydrogels in vivo in wound healing. In this study, SD rats were used for in vivo testing. After administering anesthesia, the dorsal area was shaved. A 20-mm diameter full-thickness wound was created in the back of each rat. A volume of 50 \\u0026micro;L of composite hydrogel was applied to the wounds following the random assignment of the rats into four experimental groups (n\\u0026thinsp;=\\u0026thinsp;5 per group). Digital images of the scars were captured on days 0, 3, 7, 10, and 14 using a Nikon camera to monitor the progression of wound healing over time. All experimental procedures involving animals were conducted in strict adherence to ethical standards and were reviewed and approved by the NIH, Islamabad.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec18\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.6. Statistical analysis\\u003c/h2\\u003e \\u003cp\\u003eThe software for statistics (IBM, SPSS Statistics 21) was used for the statistical analysis of the data. Means with standard errors (mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;SE) were calculated and presented in the figures as Y-error bars. Significance levels used were: *(p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05), **(p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01), and ***(p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001) with sample sizes of n\\u0026thinsp;=\\u0026thinsp;3.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"4. Results and discussions\",\"content\":\"\\u003cdiv id=\\\"Sec20\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e4.1. FTIR analysis\\u003c/h2\\u003e \\u003cp\\u003eThe structural and functional groups of the composite hydrogels were studied using the FTIR spectrum (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e (a and b)). The broadband peak at 3600\\u0026thinsp;\\u0026minus;\\u0026thinsp;3200 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e is due to O\\u0026thinsp;\\u0026minus;\\u0026thinsp;H and \\u0026minus;\\u0026thinsp;NH\\u003csub\\u003e2\\u003c/sub\\u003e functional groups [\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e]. The vibration peaks at 2923 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e show the presence of \\u0026minus;\\u0026thinsp;CH\\u003csub\\u003e3\\u003c/sub\\u003e functional groups in CMCs, CG, and PVA [\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e]. The appearance of broadband at 3600\\u0026thinsp;\\u0026minus;\\u0026thinsp;3200 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e indicates inter and intramolecular hydrogen bonding between PVA, CMCs, CG, and TEOS. The characteristic IR peaks at 1500\\u0026ndash;1600 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e are attributed to the N-H bending of the amide part in the acetyl group of CMCs [\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e]. The C\\u0026thinsp;\\u0026minus;\\u0026thinsp;O stretch in CMCs and PVA appears at 1050\\u0026ndash;1100 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e. The vibration peaks at 1250 and 1290 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e are attributed to \\u0026minus;\\u0026thinsp;Si\\u0026thinsp;\\u0026minus;\\u0026thinsp;O\\u0026minus;C and \\u0026minus;\\u0026thinsp;Si\\u0026thinsp;\\u0026minus;\\u0026thinsp;O\\u0026minus;Si, which are happening to the crosslinking between the polymeric chains [\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e]. The peaks at 1152 and 846 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e are the characteristics of polysaccharides, and these peaks are due to pyranose rings and saccharines, respectively. The functional peaks at 1073 cm\\u003csup\\u003e\\u0026ndash;1\\u003c/sup\\u003e are due to C\\u0026ndash;O stretching vibrations of pyranose, which is the characteristic peak of GG and CMCs [\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e]. The FTIR vibrational band around 1262 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e corresponds to the sulfate ester group of carrageenan [\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e]. The expected range of the Zn\\u0026ndash;N stretch vibration can be found at 421 cm\\u003csup\\u003e\\u0026ndash;1\\u003c/sup\\u003e and could be due to the limitation of the FTIR instrument. The functional peaks at 1401 cm\\u003csup\\u003e\\u0026ndash;1\\u003c/sup\\u003e and 1631 cm\\u003csup\\u003e\\u0026ndash;1\\u003c/sup\\u003e are assigned to \\u0026ndash;CH\\u003csub\\u003e2\\u003c/sub\\u003eCOOH and \\u0026ndash;C\\u0026thinsp;=\\u0026thinsp;O, respectively, in CMCs [\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e]. Thus, all the peaks and functional groups confirm the successful fabrication of composite hydrogels.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec21\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e4.2. XRD analysis\\u003c/h2\\u003e \\u003cp\\u003eThe crystalline behavior of the composite hydrogels was determined by X-ray diffraction pattern, as shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e (c). The polymers are usually amorphous and do not show sharp peaks in the XRD pattern but broad peaks from 2θ 0-20\\u003csup\\u003eo\\u003c/sup\\u003e. The XRD pattern of ZiF-8-based composite hydrogels demonstrated that adding ZiF-8 particles to a polymeric matrix may significantly affect the intensity of the XRD signal [\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e]. As the ZiF-8 particles are highly crystalline, incorporating ZiF-8 into the polymers and well dispersion enhances the crystalline features of the composite system. The XRD peaks of a composition containing lower ZiF-8 concentration (3%) are comparatively less sharp. In contrast, the higher ZiF-8 concentrations (6% and 10%) increase the crystallinity of polymeric material, resulting in sharper and more intense peaks. When the ZiF-8 particles are adequately dispersed in the polymer matrix, individual particles contribute to the diffraction signal, increasing the intensity of XRD peaks. The non-uniform dispersion of ZiF-8 particles causes aggregate formation and produces XRD signals that are less intense and broader than the signals produced by well-dispersed samples. Thus, the XRD analysis assures that the incorporated ZiF-8 composite hydrogels are successfully fabricated.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec22\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e4.3. Thermal behavior\\u003c/h2\\u003e \\u003cp\\u003eThe thermal resistance of composite hydrogels was evaluated using differential scanning calorimetry, as shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e (d). The composite material exhibited thermal stability up to 220 \\u003csup\\u003eo\\u003c/sup\\u003eC, after which the material absorbed heat and underwent melting. The appearance of the shoulder peak indicates some secondary thermal processes that are closely associated yet discrete from primary events [\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e]. The complex network and structure of composite hydrogels involving several types of intramolecular or intermolecular interactions are responsible for the appearance of shoulder peaks [\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e]. The concentration of ZiF-8 affects the melting behavior of polymeric composite material. At the lower ZiF-8 concentration (3%), the particles of ZiF-8 may undergo proper dispersion within the polymer solution, bringing minor modifications in the polymer structure and leading to a less intense peak. As the concentration of ZiF-8 particles increases to 6% and 10%, the interaction of ZiF-8 particles and polymer chains becomes more robust, leading to a more prominent alteration in polymer melting behavior, potentially enhancing the intensity of the shoulder peak. Table\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e shows the melting temperatures of synthesized films. Increasing ZiF-8 concentration from 3\\u0026ndash;10% increases the melting temperature from 226 to 230 \\u0026deg;C. Higher ZiF-8 concentrations enhance polymers\\u0026rsquo; crosslinking and stability, increasing melting points.\\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab2\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 2\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eThermal behavior of the composite hydrogels.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"5\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eSr. No.\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eCodes\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eT\\u003csub\\u003eonset\\u003c/sub\\u003e (\\u0026deg;C)\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eT\\u003csub\\u003em\\u003c/sub\\u003e (\\u0026deg;C)\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003eT\\u003csub\\u003eoffset\\u003c/sub\\u003e (\\u0026deg;C)\\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\\u003e1\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eCPC-ZiF-3%\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e220\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e226\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e231\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003e2\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eCPC-ZiF-6%\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e220\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e228\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e233\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003e3\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eCPC-ZiF-10%\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e220\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e230\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e235\\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 \\u003cdiv id=\\\"Sec23\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e4.4. Surface morphology and elemental analysis\\u003c/h2\\u003e \\u003cp\\u003eThe surface structure of synthesized material was analyzed by recording SEM micrographs, as shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e, at resolutions of 100\\u0026micro;m and 200 \\u0026micro;m. The SEM images show different pore sizes and shapes of fabricated composite hydrogels, as shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e (a-f). Introducing highly porous ZiF-8 particles in the polymer blend induces variable pore distribution, leading to numerous biological processes, including cell proliferation and migration [\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e]. In this work, the concentration of ZiF-8 was increased from 3\\u0026ndash;10% to obtain a more uniform pore size distribution. The wound dressing with 3% ZiF-8 particles shows lesser porosity and irregular pore sizes. As the concentration increased to 6% and 10%, the porosity increased, and pore shapes and sizes became more spherical and uniform. The increasing ZiF-8 concentration causes more porosity, absorbing additional wound exudate and other biological fluids to keep the wound environment moist, supporting wound healing and repairing [\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e]. It can be observed that increasing the amount of ZiF-8 regulates the pore size distribution, which increases surface area. The elemental analysis of the composite hydrogels has been presented in tabular form for the quantitative analysis as in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e (g-i), and the graphical analysis to show the peak intensity in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e (j-l). It can be seen that increasing the amount of ZiF-8 in the polymeric matrix increases the peak intensity of the zinc and the elemental analysis also confirm that there is not foreign element, which may cause the toxicity. Hence, the different morphology and the elemental analysis of the composite hydrogels confirmed the successful fabrication of the composite hydrogels.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec24\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e4.5. Swelling in aqueous, PBS, and NaCl media\\u003c/h2\\u003e \\u003cp\\u003eThe hydrophilic character of the polymers that make up the hydrogels accounts for their swelling tendency. Their water uptake capacity is severalfold greater than their weight, and the increasing amount of ZiF-8 added to the polymeric matrix of the composite hydrogels has different swelling trends at various pH levels. The swelling characteristics of fabricated composite hydrogels were studied in aqueous media to varying pHs (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e (a)). It was observed that the fabricated hydrogels exhibited more swelling in acidic medium at pH\\u0026thinsp;=\\u0026thinsp;3 and less swelling in basic media at pH\\u0026thinsp;=\\u0026thinsp;11. Maximum swelling was found in neutral media (pH\\u0026thinsp;=\\u0026thinsp;7). However, the composite hydrogels have shown excellent swelling characteristics. In the acidic media, H\\u003csup\\u003e+\\u003c/sup\\u003e is excessively available, and these protonate the amino groups, which are available at the backbone of the chitosan to produce ammonium ions [\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e]. Increasing protonation at acidic pH causes more charge, which causes repulsion and osmosis, increasing the media penetration into the polymeric. With the increase in pH, the swelling increased and reached a maximum at neutral pH due to deprotonation. The swelling behavior follows the trend of Aqueous\\u0026thinsp;\\u0026gt;\\u0026thinsp;PBS\\u0026thinsp;\\u0026gt;\\u0026thinsp;NaCl media. Further increased pH caused the decrease in swelling because of the formation of ammonium ions, which decreases interactions between polymer chains. The maximum swelling at pH-7 would be because of the maximum mobility of the polymeric chain, which results in increased swelling. Similarly, the swelling behavior was found in PBS (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e (b)) and NaCl (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e (c)) media. Increasing ZiF-8 concentration reduces the swelling ratio because more ZiF-8 particles impart additional crosslinking and restrict the mobility of chains within the polymer network, making it difficult to absorb more water. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e (a-c) displays how composites containing 3% ZiF-8 particles exhibit significant swelling. Increasing the concentration of ZiF-8 to 6% and 10% reduced the swelling response. Since swelling acts as a barrier against subsequent infections and keeps the area surrounding the lesion wet, it is crucial to the healing process of wounds. Because of this property, composite hydrogel is a more effective wound dressing that promotes wound healing.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cdiv id=\\\"Sec25\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e4.5.1. Biodegradation analysis\\u003c/h2\\u003e \\u003cp\\u003eFigure \\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e (d) demonstrates how the biodegradation of composite material was tracked in a PBS medium at 37 \\u0026deg;C and 7.4 pH. All the compositions consisting of varying amounts of ZiF-8 show different degradation behavior. The formulation with 3% ZiF-8 degraded rapidly while the degradation process was slow, raising ZiF-8 concentration to 6%. The composite hydrogels containing 10% ZiF-8 degraded the least. The extent of crosslinking governs the rate of degradation. The higher concentration of ZiF-8 increases the mechanical strength and thermal stability, reducing the biodegradation rate. The polymer chains are strongly bound and less vulnerable to decomposition. Increasing ZiF-8 may also alter the degradation pathways by stimulating heterogeneous degradation procedures. The highly porous structure of ZiF-8 particles retards the penetration of oxygen, moisture, and several other degrading agents into the polymer solution slowing down the degradation mechanism. The fabricated composite system is significant for absorbing excessive wound exudate and maintaining a moist environment of the wound along with controlled biodegradation.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec26\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e4.5.2. Wettability\\u003c/h2\\u003e \\u003cp\\u003eWettability study is one crucial surface phenomenon for determining if biomaterials are hydrophilic or hydrophobic. A critical feature that characterizes how the hydrogels interact with the biofluids is their hydrophilic nature. The hydrophilic hydrogel can potentially absorb the wound exudate, which helps with wound care and treatment [\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e]. Therefore, wettability determines the hydrophilicity by measuring the water contact angle, which helps to understand the cellular interaction with the hydrogel during wound healing [\\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e]. The wettability of the produced composite hydrogels was determined at different time intervals (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e (a)), and it was shown that raising ZiF-8 causes the water contact angle to rise and changes the pattern from more hydrophilic to less hydrophilic morphology. Because the tightly packed polymeric chains occupy the hydrophilicity-causing functional groups, it may result from reduced mobility. The increasing ZiF-8 amount has hydrophilicity trends such as CPC-ZiF-3% \\u0026gt; CPC-ZiF-6% \\u0026gt; CPC-ZiF-10%. The optimized amount of ZiF-8 causes the required wettability for specific applications. Several factors influence the cellular interaction with the hydrogels, including wettability, surface morphology, roughness, stiffness, surface charge, and chemistry [\\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e]. Optimized wettability promotes cellular interaction via cell adherence proliferation and influences the cell-hydrogel interaction supporting wound healing [\\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e]. Hence, the different wettability confirms the fabrication of different composite hydrogels.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec27\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e4.5.3. Gel fraction\\u003c/h2\\u003e \\u003cp\\u003eThe degree of crosslinking determines the gel fraction of composite hydrogels, so a higher degree of crosslinking results in a higher gel fraction. Based on our observation of the gel fraction percentage (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e (b)), we have found that the percentage of gel fraction increases as the amount of ZiF-8 increases. The CPC-ZiF-3% has the least gel fraction, and CPC-ZiF-10% has the maximum value of the gel fraction, while CPC-ZiF-6% has a value between both the composition of the composite hydrogels. Since the increasing amount of Zif-8 increases its interaction with the polymeric chains, it has been via different interactions, including hydrogen bonding and other weaker forces (ionic-liquid and van der Waal forces, etc.). Hence, the increasing amount of ZiF-8 increases the interaction between the chains via hydrogen bonding with different functional groups of the polymers, as illustrated in Scheme-I, and it acts as a crosslinker to reduce the mobility of the polymeric chains.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec28\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e4.6. In vitro assays\\u003c/h2\\u003e \\u003cdiv id=\\\"Sec29\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e4.6.1. Cell morphology\\u003c/h2\\u003e \\u003cp\\u003eThe morphology of fibroblast cells was analyzed by biological assays after the time intervals of 24, 48, and 72h, as depicted in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e (A). The morphological studies of fibroblast cells help understand the cell response to numerous stimuli, including growth factors and components of the extracellular matrix and cytokines. The structural modifications also demonstrate wound healing by disclosing cell migration, proliferation, and extracellular matrix synthesis. The appropriate shape of fibroblast cells is crucial when engaging with various substrates, such as hydrogels, for wound repair or tissue regeneration applications. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e (A) shows the elongated, cylindrical, spindle-like fibroblast cells with proper cell adherence [\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e]. The spindle-shaped cells navigate via the extracellular matrix and reach the wound region to stimulate wound repair. By increasing the concentration of ZiF-8 nanoparticles in composite hydrogels and the contact time of composite hydrogels to fibroblast cells, cells attain more cylindrical morphology for better tissue repair. The shape of these cells facilitates the synthesis and alignment of extracellular matrix fibers, such as collagen and fibronectin, to provide structural integrity for tissue repair. An increase in ZiF-8 concentration has also boosted the stimulation of cell re-epithelialization by the cell population. Zinc is a well-known metal and essential co-enzyme to several enzymes with potential and promising biological properties [\\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e]. The zinc from ZiF-8 particles controls the enzymatic activity during the cell division and synthesis of DNA, which are crucial for cell migration and proliferation [\\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e43\\u003c/span\\u003e]. Zinc ion lowers oxidative stress, reduces cell damage, and promotes healthy cellular morphology and function, as zinc is a trace element that acts as an antioxidant. Zinc ions also act as a cofactor for collagen synthesis, so higher zinc ions enhance the structural support needed for wound repair [\\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e]. Hence, the increasing ZiF-8 causes an increase in cell density with mature cell morphology and is helpful for wound healing.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec30\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e4.6.2. Cell viability and proliferation\\u003c/h2\\u003e \\u003cp\\u003eCell viability and proliferation have been studied against multiple contact times (dilutions) via 3T3 cell lines of bioactive composite hydrogels, as demonstrated in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e (B). Cell viability and proliferation are critical to be studied as these directly contribute to repairing damaged tissue and wound healing. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eB (a\\u0026ndash;c) illustrates the correlation between improved cell viability and increases in ZiF-8 concentration and contact times. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eB (d-f) shows nearly identical behavior for cell proliferation. ZiF-8 releases zinc ions continuously to ensure their optimum level, which controls numerous cellular mechanisms involved in wound repair [\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e]. These ions maintain the number of viable cells and increase cell proliferation, leading to the most effective wound repair. Polymeric hydrogels contain several functional groups, including hydroxyl, amine, and sulfonate ester groups, which interact with many receptors or binding sites to promote fibroblast cell adhesion [\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e]. The cells get adhered via integrin, and various cell adhesion molecules and proteoglycans are adhered to via electrostatic interactions, hydrogen bonding, or chemical crosslinking reactions [\\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e46\\u003c/span\\u003e]. These interactions promote cell adherence, viable cells, and proliferation, making hydrogels suitable for wound repair applications.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec31\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e4.7. Antibacterial activities\\u003c/h2\\u003e \\u003cp\\u003eThe antibacterial mechanism is depicted in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e (C), while the antibacterial efficacy of the composite hydrogels using Gram (positive and negative) bacteria strains. These include E. coli and S. aureus, presented in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e (D). The hydrogels demonstrate varied antibacterial properties due to their distinct formulations and structural characteristics. It can be seen that the hydrogels with the least ZiF-8 have the least antibacterial activities, while the hydrogel with the maximum amount of ZiF-8 has enhanced antibacterial activities. The antibacterial efficacy of the hydrogels follows the sequence CPC-ZiF-3% \\u0026lt; CPC-ZiF-6% \\u0026lt; CPC-ZiF-10%, potentially due to the sustained release of ZiF-8 from the polymeric matrix of the hydrogels. The hydrogels have a synergistic effect of ZIF-8 and polymeric matrix as the polymeric part in hydrogel may interact with phospholipids and lipopolysaccharides of the bacterial membrane. The polymers may penetrate and rupture the bacterial membrane to hinder bacterial growth. The other possibility could be that the polymers may bind with the DNA and inhibit the replication of bacteria, causing antibacterial activities. Various bacteria may colonize the injured region and cause inflammation, pain, and infections, and excessive skin tissue exudate may damage the healthy tissues [\\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e47\\u003c/span\\u003e]. The antibacterial properties of the hydrogels suggest that integrating ZIF-8 into the polymeric matrix with varying crosslinking may yield distinct antibacterial effects. The antibacterial mechanism is attributed to Zn\\u003csup\\u003e2+\\u003c/sup\\u003e, which may produce reactive oxygen species (ROS) that compromise bacterial integrity, resulting in extensive bacterial eradication. The Zn ions have a positive charge, and the bacterial membrane has a negative charge that can penetrate the bacterial membrane. It interacts with the replication process of DNA to hinder bacterial growth. The Zn\\u003csup\\u003e2+\\u003c/sup\\u003e may accumulate over bacterial membranes and cause antibacterial activities by rupturing bacterial membranes to cause antibacterial activities [\\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e48\\u003c/span\\u003e]. Since zinc ions are released to the wound site under control and have the potential to break down bacterial membranes, ZIF-8 has outstanding antibacterial properties. It also works as a co-enzyme and potential material for generating reactive oxygen species to kill bacteria [\\u003cspan citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e49\\u003c/span\\u003e]. Hydrogels acquire the ability to thwart bacterial invasion by safeguarding the wound from pathogenic bacteria while also absorbing wound exudate and maintaining a microenvironment suitable for wound healing and regeneration. Incorporating ZiF-8 into the polymeric matrix may facilitate the controlled release of ZiF-8 on demand at the wound site. The sustained release of antibacterial agents is crucial for wound healing to protect bacterial pathogens. The crosslinker possesses multiple hydroxyl functional groups that confer structural properties, such as surface roughness and hydrophilic characteristics [\\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e50\\u003c/span\\u003e]. Hence, the fabricated hydrogels have different potential antibacterial activities that increase with increasing amounts of ZiF-8.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec32\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e4.8. Wound healing\\u003c/h2\\u003e \\u003cp\\u003eIn this work, we evaluated the effectiveness of different hydrogel formulations containing ZIF-8 through in vivo wound healing experiments, as shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003e (a-c). As part of the treatment plan, the composite hydrogels were applied to wounds at predetermined intervals of 0, 3, 7, 10, and 14 days in order to evaluate how well they promoted tissue repair and regeneration and wound closure gradually. We have illustrated different wound phases, as shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003e (a). The hydrogel without ZiF-8 has been used as a control, and it also exhibits a desirable wound-healing capacity. It has been demonstrated that the addition of zinc to the composite ZIF-8@hydrogels greatly accelerates the healing of wounds. The hydrogels containing different amounts of ZiF-8 have different wound-healing behaviors. The group exhibited exceptional healing in comparison to the scars seen in the blank control group. The CPC-ZIF-10% formulation was noteworthy for its ability to promote efficient and regenerative tissue repair by dramatically speeding up the wound-healing process while preventing scarring.\\u003c/p\\u003e \\u003cp\\u003eThe majority of hydrogel formulations were found to have a wound healing rate of about 40% after 7 days of treatment, suggesting that they were moderately effective in promoting tissue repair during this period. The CPC-ZIF-10% formulation, on the other hand, performed better, showing over 50% wound healing in the same time frame. The special qualities of the CPC-ZIF-10% hydrogel, which are probably caused by the combined effects of ZiF-8 and the polymeric matrix, are responsible for this increased efficacy. Because CPC-ZiF-10% contains the highest concentration of ZiF-8 that releases more zinc and therefore CPC-ZIF-10% demonstrates greater therapeutic potential than CPC-ZiF-3%, CPC-ZiF-6%, and control, as shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003e (b and c). The maximum amount of ZiF-8 in CPC-ZiF-6% makes it a viable option for advanced wound care applications. Zinc is a vital trace element that is crucial to many physiological and biochemical functions that are required to preserve the health of cells and systems. Zinc plays an important role in catalysis, structure, and regulation as a vital component of many enzymes and is necessary for many metabolic processes [\\u003cspan citationid=\\\"CR51\\\" class=\\\"CitationRef\\\"\\u003e51\\u003c/span\\u003e]. Because of its involvement in cellular functions like DNA synthesis, protein metabolism, and tissue repair, it is essential for efficient cellular operation. Zinc has a major impact on cellular behavior in the context of wound healing by improving cell viability, encouraging differentiation, and facilitating cell migration and proliferation, especially for fibroblasts and keratinocytes, which are essential for tissue regeneration [\\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e52\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e53\\u003c/span\\u003e]. Zinc also helps to modulate inflammatory responses, promotes angiogenesis, and facilitates the synthesis of collagen\\u0026mdash;all of which are essential for the remodeling and repair of damaged tissues. Zinc reduces complications and promotes functional tissue restoration by taking part in these complex biological processes, which not only encourages wound closure but also enhances healing quality [\\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e52\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"5. Conclusions\",\"content\":\"\\u003cp\\u003eHerein, we produced composite hydrogels for wound healing applications, combining synthetic polymers (PVA) with biopolymers (CMCs and CG). ZiF-8 is incorporated into the polymeric matrix of hydrogels. The FTIR confirmed the multifunctionality of the hydrogels, while XRD revealed their amorphous and crystalline behavior. These hydrogels are porous, and as the amount of ZiF-8 increases, the uniform pore distribution causes the pore size to decrease as the surface area increases. Increased ZiF-8 content increases thermal stability in these composite hydrogels, and hydrogel CPC-ZiF-10% pore size-controlled maximum stability. The composite hydrogels with the least amount of ZiF-8 cause less cell density, viability, and proliferation. The composite hydrogel CPC-ZiF-3% possessed the least cellular behavior, and CPC-ZiF-10% exhibited maximum cytocompatibility with mature cell morphology. Similarly, CPC-ZiF-10% exhibited excellent wound healing without causing any inflammation. Hence, the fabricated composite hydrogels are potential wound dressing materials for wound healing applications.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e \\u003ch2\\u003eConflicts of Interest:\\u003c/h2\\u003e \\u003cp\\u003eAll authors declared no conflict of interest.\\u003c/p\\u003e \\u003c/p\\u003e\\u003ch2\\u003eAuthor Contribution\\u003c/h2\\u003e\\u003cp\\u003eCredit Authorship:Conceptualization, Muhammad Umar Aslam Khan, and Humaira Masood Siddiqui; Data curation, Tooba Yasin, Hina Khan, and Thamir M Eid; Formal analysis, Hayat M. Thamir M Eid, and Muhamad Umar Aslam Khan; Funding acquisition, Muhammad Umar Aslam Khan, Thamir M Eid, and Aneela Javed; Investigation, Muhammad Umar Aslam Khan, and Muhammad Shahzad Zafar; Methodology, Muhammad Umar Aslam Khan, Humaira Masood Siddiqui, and Muhammad Shahzad Zafar; Project administration, Muhammad Umar Aslam Khan, Hayat M. Albishi, and Humaira Masood Siddiqui; Resources, Muhammad Umar Aslam Khan, Hayat M. Albishi, and Thamir M Eid; Software, Muhammad Umar Aslam Khan, Hina Khan, and Tooba Yasin; Supervision, Muhammad Umar Aslam Khan, and Humaira Masood Siddiqui; Validation, Hayat M. Albishi, Thamir M Eid, and Anela Javed; Visualization, Tooba Yasin, Hina Khan, and Muhammad; Writing\\u0026ndash;original draft, Tooba Yasin, Muhammad Umar Aslam Khan; Writing \\u0026ndash; review \\u0026amp; editing, Muhammad Umar Aslam Khan, and Humaira Masood Siddiqi.\\u003c/p\\u003e\\u003ch2\\u003eData availability:\\u003c/h2\\u003e \\u003cp\\u003eThe data contained within the manuscript.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eZhang, H., et al., \\u003cem\\u003eDeveloping natural polymers for skin wound healing.\\u003c/em\\u003e Bioactive Materials, 2024. \\u003cstrong\\u003e33\\u003c/strong\\u003e: p. 355-376.\\u003c/li\\u003e\\n\\u003cli\\u003eFarahani, M. and A. 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Zhuang, \\u003cem\\u003eAntibacterial activity of chitosan and its derivatives and their interaction mechanism with bacteria: Current state and perspectives.\\u003c/em\\u003e European Polymer Journal, 2020. \\u003cstrong\\u003e138\\u003c/strong\\u003e: p. 109984.\\u003c/li\\u003e\\n\\u003cli\\u003eLiu, Y., et al., \\u003cem\\u003eZIF-8 modified multifunctional injectable photopolymerizable GelMA hydrogel for the treatment of periodontitis.\\u003c/em\\u003e Acta Biomaterialia, 2022. \\u003cstrong\\u003e146\\u003c/strong\\u003e: p. 37-48.\\u003c/li\\u003e\\n\\u003cli\\u003eDeng, L., et al., \\u003cem\\u003eBacterial cellulose-based hydrogel with antibacterial activity and vascularization for wound healing.\\u003c/em\\u003e Carbohydrate polymers, 2023. \\u003cstrong\\u003e308\\u003c/strong\\u003e: p. 120647.\\u003c/li\\u003e\\n\\u003cli\\u003eLiu, Y., et al., \\u003cem\\u003eZIF-8-modified multifunctional bone-adhesive hydrogels promoting angiogenesis and osteogenesis for bone regeneration.\\u003c/em\\u003e ACS applied materials \\u0026amp; interfaces, 2020. \\u003cstrong\\u003e12\\u003c/strong\\u003e(33): p. 36978-36995.\\u003c/li\\u003e\\n\\u003cli\\u003eWang, M., et al., \\u003cem\\u003eConstruction of enzyme-MOFs composite with carbon dots: A strategy to enhance the activity and increase the growth rate of the enzyme-ZIF-8 composite.\\u003c/em\\u003e International Journal of Biological Macromolecules, 2025: p. 139985.\\u003c/li\\u003e\\n\\u003cli\\u003eCai, J., et al., \\u003cem\\u003eMultifunctional PDA/ZIF8 based hydrogel dressing modulates the microenvironment to accelerate chronic wound healing by ROS scavenging and macrophage polarization.\\u003c/em\\u003e Chemical Engineering Journal, 2024. \\u003cstrong\\u003e487\\u003c/strong\\u003e: p. 150632.\\u003c/li\\u003e\\n\\u003cli\\u003eXiang, G., et al., \\u003cem\\u003eA Zn-MOF-GOx-based cascade nanoreactor promotes diabetic infected wound healing by NO release and microenvironment regulation.\\u003c/em\\u003e Acta Biomaterialia, 2024.\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"},{\"header\":\"Scheme 1\",\"content\":\"\\u003cp\\u003eScheme 1 is available in the Supplementary Files section.\\u003c/p\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"polymer-bulletin\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"pobu\",\"sideBox\":\"Learn more about [Polymer Bulletin](http://link.springer.com/journal/289)\",\"snPcode\":\"289\",\"submissionUrl\":\"https://submission.nature.com/new-submission/289/3\",\"title\":\"Polymer Bulletin\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false},\"keywords\":\"Biodegradable, Natural polymers, ZiF-8@Hydrogels, Polymeric composite, Wound healing\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-6345624/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-6345624/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eThe skin is the largest organ of the human body, protecting it from the external environment and pathogens and minimizing the risk of injury. Hydrogels have attracted a lot of interest lately because of their biomimetic structure and inherent extracellular matrix characteristics. Hydrogels hold great promise for application in wound healing, mainly because they can very conveniently provide bioactive ingredients. In this work, composite hydrogels were developed for wound healing from carrageenan, polyvinyl alcohol, and carboxymethyl chitosan by blending method. These hydrogels were characterized by advanced techniques such as FTIR, XRD, DSC, and SEM-EDX, which were used to study their structural, thermal, surface morphology, and elemental behavior. Their physiochemical analyses were performed using swelling degradation, water contact angle, and gel fraction. The increasing ZiF-8 causes more surface roughness, with decreased swelling in different media (Aqueous\\u0026gt;PBS\\u0026gt;NaCl). The increasing ZiF-8 amount causes less hydrophilic behavior and biodegradation with increasing gel fraction. The cytocompatibility of ZiF-8-based composite hydrogels and their potential antibacterial activities were performed against Gram-positive and Gram-negative, which were enhanced with increasing ZiF-8. The increasing ZiF-8 caused more cell viability and proliferation with proper cell morphology and enhanced in vivo wound healing. Hence, the results show that synthesized composite hydrogels may be a potential candidate for numerous wound repair applications.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Fabrication of composite hydrogels from Carrageenan-cl-carboxymethyl chitosan-PVA incorporated ZiF-8 for wound healing applications\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-04-24 04:00:10\",\"doi\":\"10.21203/rs.3.rs-6345624/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Revision requested\",\"date\":\"2025-04-23T09:09:20+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2025-04-22T14:30:53+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2025-04-16T15:16:28+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"112393766415019210682550470842588968607\",\"date\":\"2025-04-07T12:23:07+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"59632842529832298671930682532492088560\",\"date\":\"2025-04-04T12:14:36+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"254841470717020736897108548108991513164\",\"date\":\"2025-04-03T09:25:36+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2025-04-02T11:43:15+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2025-04-02T11:41:41+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2025-04-01T03:19:34+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Polymer Bulletin\",\"date\":\"2025-03-31T14:09:53+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"polymer-bulletin\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"pobu\",\"sideBox\":\"Learn more about [Polymer Bulletin](http://link.springer.com/journal/289)\",\"snPcode\":\"289\",\"submissionUrl\":\"https://submission.nature.com/new-submission/289/3\",\"title\":\"Polymer Bulletin\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false}}],\"origin\":\"\",\"ownerIdentity\":\"07e9a67b-db3e-48c0-91a5-7cb8c4c379ce\",\"owner\":[],\"postedDate\":\"April 24th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2025-06-16T16:06:58+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-6345624\",\"link\":\"https://doi.org/10.1007/s00289-025-05827-y\",\"journal\":{\"identity\":\"polymer-bulletin\",\"isVorOnly\":false,\"title\":\"Polymer Bulletin\"},\"publishedOn\":\"2025-06-11 15:57:18\",\"publishedOnDateReadable\":\"June 11th, 2025\"},\"versionCreatedAt\":\"2025-04-24 04:00:10\",\"video\":\"\",\"vorDoi\":\"10.1007/s00289-025-05827-y\",\"vorDoiUrl\":\"https://doi.org/10.1007/s00289-025-05827-y\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-6345624\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-6345624\",\"identity\":\"rs-6345624\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}