{"paper_id":"2d1bae88-b4b9-4797-9a0f-9092e9bf78f4","body_text":"Dual-Crosslinked Oxidized Pectin/N-Succinyl Chitosan Hydrogel Containing Graphene Oxide Nanosheets for Tissue Engineering Application | 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 Dual-Crosslinked Oxidized Pectin/N-Succinyl Chitosan Hydrogel Containing Graphene Oxide Nanosheets for Tissue Engineering Application Atefeh Afroozan Bazghaleh, Mojtaba Akbari Dogolsar, Jalal Barzin This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-922109/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Biopolymer-based hydrogels are commonly used in clinical applications. In the present study, N- succinyl chitosan (NSC), oxidized pectin (OP), and graphene oxide (GO) were used to develop a new dual-crosslinked hydrogel system. The dynamic OP/NSC/GO hydrogel showed quick gelation and great injectability due to the cooperation of hydrogen interaction between the GO nanosheets and the NSC and OP macromolecules and Schiff-based crosslinking by amino and aldehyde functional groups of polysaccharide derivatives. The performance of the above-mentioned hydrogel was improved when the GOs were embedded. When the GO content was 6 (mg/ml), the hydrogel showed the best overall performance, with a 10-minute healing time, a quick gelation time (~ 13s), acceptable swelling ability, suitable conductivity, great hemocompatibility, and strong biological compatibility. These results showed that the composite hydrogel could be used as a promising conductive injectable self-healing hydrogel for tissue engineering applications. Cellular Metabolism Cellular & Molecular Neuroscience Double cross-linked network Schiff- base reaction Hydrogen bond Self-healing hydrogel Tissue engineering Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Introduction Tissue engineering is the process of designing and creating functional substitutes for the damaged tissues and organs through growing the cells into a scaffold that mimics the target tissue's extracellular matrix (ECM) (Spicer, 2020b ). For this purpose, hydrogels, a three-dimensional cross-linked network backbone containing a large amount of water (50–90%) are promising materials for tissue engineering applications. To support cell proliferation, migration, and differentiation, hydrogels could be designed which permit oxygen and nutrient transport. Also, they supply a 3D, highly hydrated environment for cells that mimics the original soft tissues. This is mainly attributed to the unique properties of hydrogels, including their high permeability, excellent biocompatibility, non-toxicity, non-immunogenicity, biodegradability, tunable physical and chemical properties, tissue-like elasticity as well as the capability to encapsulate bioactive molecules, cells, or drugs to mimic an extracellular matrix (ECM) (Spicer, 2020a , Zhao et al., 2020 ). Recently, stimulus-responsive hydrogels have become a hot topic in biomedical applications because of their versatility. Self-healing hydrogel has appealing features such as maintaining the integrity of the structure and improving the mechanical properties. The most common preparation method for self-healing hydrogels is introducing the dynamic covalent (boronate ester bond (Ji et al., 2019 , Figueiredo et al., 2019 ), disulfide bond (Guo et al., 2017 ), imine chemistry (Schiff-base reaction) (Xu et al., 2018 , Liu et al., 2018 ) and Diels-Alder reaction (Lin et al., 2018 , Bi et al., 2019 ), etc.) or the non-covalent bonds (hydrophobic association (Deng et al., 2018 ), electrostatic interactions (Pu et al., 2017 ), hydrogen bonding (Ye et al., 2017 ), host-guest inclusion (Jin et al., 2019 ), etc.) into the hydrogel system. However, the crosslinking curing mechanism affects the stability of the formed hydrogel directly (Zhang et al., 2020 ). So, due to the moderate reaction conditions, efficient reversibility of imine bonds, tunable properties, and stimuli-responsive performance of the resulting materials, imine bonding (CH = N) has attracted significant interest in the biological applications. Also, water molecules are the only by-products which are released in the imine reaction (Yu et al., 2021 , de Lima et al., 2020 ). The appearance of the non-toxic, ecofriendly and biocompatible polymers derived from natural sources have introduced a new and interesting dimension to the development of the low-cost and scalable biomaterials for tissue engineering applications. Chitosan, a linear polysaccharide with cationic nature is more attractive than the other bio-polymers due to its intrinsic biocompatibility, biodegradability, bio-adhesivity, hydrophilicity, high viscosity, and antibacterial properties. It could be crosslinked via reaction between its amino groups along the backbone and other polymers such as hyaluronic acid (Zhang et al., 2018 ), alginate (Bagher et al., 2020 ), silk fibroin (Eivazzadeh-Keihan et al., 2021 ), and cellulose (Xu et al., 2019 ). However, its poor solubility in physiological solvents due to its strong intermolecular hydrogen bonding limits its biomedical applications (Yu et al., 2021 ). Therefore, to expand its application, N-Succinyl-chitosan (NSC), which is a water-soluble chitosan derivative could be synthesized via the introduction of succinyl groups at the N-position of the glucosamine units of chitosan. This makes it an excellent candidate to design polymeric scaffolds for tissue regeneration (Bashir et al., 2017 , Kamoun, 2016 ). Pectin (PE), which is also called polygalacturonic acid, is an anionic polysaccharide extracted from primary cell plants. Pectin has been widely used in a variety of biomedical applications, due to its important properties such as biocompatibility, biodegradability, capability to inhibiting the activity of macrophages and neutrophils, anti-inflammatory effects, and the capability to restrain cells, drugs, and genes (Li et al., 2019 , Kaushik et al., 2020 ). On the backbone chain, there are hydroxyl, carboxyl and carboxymethyl groups which makes it readily susceptible to functionalization and modification. PE could be oxidized by reacting with sodium periodate to produce OP (Tummalapalli et al., 2016 , Li et al., 2020a ). Through the oxidation reaction, aldehyde groups are introduced in the PE molecular chain, which are able to participate in Schiff mechanism. So, they could react with amine groups of NSC and form imine bonds. Since the stability of the hydrogel network composed of imine bonds is usually poor, and the mechanical properties are relatively weak, academics have paid widespread attention to the dual cross-linked (DC) network hydrogels. This is because such DC network hydrogels optimize and combine different properties of each network structure, including dual physically cross-linked hydrogels, dual chemically cross-linked hydrogels. It should be noted that the hybrid cross-linked hydrogels could potentially resolve these issues and results in materials appropriate for tissue engineering applications (Li et al., 2020b , Qin et al., 2019 ). For this purpose, graphene oxide (GO), a precursor of chemically converted graphene, is a widely investigated nanomaterial. Due to its oxygen-containing functional groups (i.e., hydroxyl, carboxyl, carbonyl, and epoxy groups), it could be easily combined with polymers for enhancing the hydrogel's mechanical properties. Moreover, it has good electrical conductivity, excellent chemical stability, easy accessibility, appropriate mechanical and antibacterial properties (Hua et al., 2017 , Zhou et al., 2018 ). Therefore, in this study, the dual-crosslinked OP/NSC/GO hydrogels were fabricated via a dynamic imine bond and a hydrogen bond. The gelling time, morphology, rheology, swelling ratio, degradation, conductivity, self-healing, and injectability were examined. Also, in vitro biocompatibility and hemolysis rates were evaluated. Experimental Section Materials Chitosan (Mw 190–310 kDa), pectin from citrus peels (galacturonic acid ≥ 74.0%), graphite powder, succinic anhydride, sodium periodate, ethylene glycol, hydroxylamine hydrochloride, methanol and sodium hydroxide were obtained from Sigma–Aldrich. Ethanol, acetone, potassium permanganate, sodium nitrate, sulfuric acid 98%, and hydrogen peroxide, were obtained from commercially available suppliers (Merck KGaA). All chemicals were of analytical grade purity and they were used as received without any further purification. The aqueous solutions were prepared in deionized water. Synthesis of N-succinyl chitosan (NSC) As previously described, NSC was synthesized with slight modifications (Bashir et al., 2017 ). In short, the purified chitosan (0.5 g) was suspended in 75 mL aqueous solution of acetic acid (5.0% v/v). This mixture was placed magnetic stirring at 50 ° C for 1 h. After that, dilution of the solution was done by adding 75 mL methanol. Subsequently, succinic anhydride (2.5 g) was dissolved in 60 mL acetone and mixed with chitosan solution. The stirring was continued at 50 ° C for 24 hours. After 24 h, the pH of the mixture was adjusted to 12 with NaOH (1.0 mol/L) solution, and the formation of a clear solution was observed. The stirring of this clear solution was continued for further 5 hours. Afterward, 150mL ethanol (96%) was added to form precipitates followed by filtration to separate the precipitates. These precipitates were dispersed in ethanol for 24 hours. Then, dispersed precipitated product was washed with several times with ethanol and acetone to remove excess reagents and dried using freeze-dryer for 4 hours. The final product was stored at room temperature. The degree of the substitution (DS) of NSC was determined to be 0.52 according to the described previously (Bashir et al., 2017 ). Synthesis of oxidized pectin (OP) Synthesis of OP was according to the previously reported procedure with slight modifications (Ahadi et al., 2019 ). Initially, 0.5 g of pectin was dispersed in 10 mL ethanol (96%). Then 10 mL of sodium periodate solution (0.5 M) was added and the temperature was controlled at 40 ° C . After the reaction was carried out for 8 h in a dark environment. The appropriate amount of ethylene glycol was added to continue the reaction for 2 h to remove the unreacted oxidant. After the reaction, the product was dialyzed in deionized water (dialysis bag, molecular weight cutoff: 8–12 kDa) for 3 days to remove the excess sodium periodate, then frozen at − 20 ℃ and lyophilized at − 80 ℃ for 24 h. The degree of oxidation (DO) was determined by potentiometric titration, and DO value was 42.284 ± 0.897 %. Synthesis of graphene oxide (GO) GO was prepared using graphite powder via the modified Hummers method (Shahriary and Athawale, 2014 ). In brief, the graphite powder (1 g) and sodium nitrate (0.5 g) were added to sulfuric acid 98% (23 mL) under stirring. While using an ice bath and keeping the temperature at 0 ° C (to prevent overheating and explosion), potassium permanganate (3g) was added gradually to the mixture under the same conditions. Subsequently, the mixture was stirred at 35 °C for 24 h. Then, the solution was diluted with distilled water, and the reaction was finally completed by adding 30% hydrogen peroxide (5mL). The resulting mixture was washed with distilled water until the filtrate showed neutral pH. The obtained solid was freeze-dried for 24 h. Preparation of the OP/NCS/GO hydrogels NSC and OP were dissolved in the phosphate-buffered saline (PBS, pH 7.4 and the room temperature for 5 h) separately to form a 3 wt. % solution. The various contents of GO (0mg/ml, 2mg/ml, 4mg/ml, and 6mg/ml) was dispersed into OP solutions (3 wt. %) with the aid of ultrasound. Then, the solutions were stored at 4℃ for further use. The preparation of OP/NSC/GO hydrogels was carried out using a system of two interconnected syringes. Equal volumes of the NSC and OP/GO solution were loaded into two separate Luer-Lock syringes, respectively, and then both syringes were connected via a connector. The solutions were thoroughly mixed by pressing alternately on each of the plungers (more than three times). Upon thorough mixing, the entire syringe contents were pushed into one of the syringes, the connector in conjunction with the empty syringe was disengaged, and the prepared hydrogel was ejected and liberally deposited in suitable mold (10 mm diameter) for further investigation. According to the difference in the contents of GO, the hydrogels were coded as OP/NSC/GO-0, OP/NSC/GO-2, OP/NSC/GO-4, and OP/NSC/GO-6 hydrogels. The procedures of synthesizing the hydrogels have been shown in Figs. 1 and 2 . Gelation time test The gelation time of hydrogels was measured by the tube inversion method. A mixture of 1 ml of OP/NSC/GO solution was poured into a glass bottle with a diameter of 20 mm, and it was placed at room temperature. The gelatin time is determined by the time it takes for the mixture to stop flowing when the glass bottle is inverted. Swelling measurements To evaluation the swelling ratio, the same shape and size of freeze-dried hydrogel samples were weighed (W d ) and soaked in PBS (pH = 7.4) at 37° C . After 48 h, samples were removed and the water remaining on the surface was absorbed by filter paper. Then, the swollen hydrogel was immediately weighed (W s ) and the swelling ratio was calculated from the formula: In vitro degradation analysis The degradation of the OP/NSC/GO hydrogels was examined for the weight loss under the aqueous condition (PBS at 37 o C) for 4 weeks. Firstly, the initial weight of lyophilized OP/NSC/GO hydrogels was accurately weighed (W 0 ). After predetermined time intervals, hydrogels were removed from the medium and, they were freeze-dried. Then, the sample was accurately weighed and recorded as (W 1 ). The PBS was replaced by fresh PBS every 3 days. All measurements were performed in duplicate. The following formula calculated the degradation ratios of the OP/NSC/GO hydrogels: Characterizations Fourier transform infrared spectroscopy–attenuated total reflectance (FTIR–ATR) spectroscopy was carried out with a JASCO-4700 FTIR spectrometer, and the wavelength range was set at 500–4000 cm − 1 at room temperature. Scanning electron microscopy (SEM) and transmission electron spectroscopy (TEM) images were taken with a JEOL JSM-IT300S microscope (Tokyo, Japan), which works at a 5kV accelerating voltage and a JEM-100CX electron microscope, respectively. X-ray diffraction (XRD) analysis was performed using Philips PW1730 X-ray diffractometer with Cu Kα radiation (λ = 0.15405 nm) which operates at 40 kV and 40 mA. Data were collected from 5 to 50° 2θ at room temperature. The hydrogel (600µL) was formed in a cubic container, and an avometer was used to measure the conductivity using the electric circuit. Rheological Analysis Rheological properties of hydrogels were carried out on an Anton Paar MCR-302 rheometer at room temperature using a 25 mm diameter parallel plate. The frequency sweep test was analyzed over the range of 1–100 rad/s at a fixed strain rate of 1%, and in the strain sweep test, the strain ranged from 0 to 1000%. Considering the strain sweep results, the self-healing properties were quantitatively evaluated by the damage-healing cycles which were continuous step switches from 1% (120 s for each interval) to 300% (60 s for each interval), with a constant frequency of 1 Hz at room temperature. The viscosity of hydrogels was monitored at the different shear rates to characterize the injectability quantitatively. The viscosity-shear rate curves were obtained at room temperature, and the viscosity was recorded when the corresponding shear rate was in the range of 1-100 s − 1 . Hemolysis rate Hemolysis testing of the prepared hydrogels was performed according to the previously reported procedure with slight modifications (Iqbal et al., 2017 ). Fresh human blood from a healthy donor was collected in 5ml EDTA Vacutainer. The freeze-dried hydrogel samples of OP/NSC/GO0, OP/NSC/GO2, OP/NSC/GO4, and OP/NSC/GO6 were ground into powder. 25 mg of the powdered sample was weighed and poured into the test tube, then 10 mL of normal saline was slowly added. All tubes were incubated at 37 ° C ) for 30 min. Subsequently, 0.1 mL EDTA blood was added to the test tube with a micropipette. After shocking and mixing, well, it was kept at a constant temperature at 37°C for 60 min. Then, the tubes were removed, and they were centrifuged at 1500 rpm for 10 min. The absorbance values of the supernatants at 545 nm were measured with an ultraviolet spectrophotometer using Lambda 25 UV/Vis spectrophotometer. The following formula calculated the hemolysis rate of hydrogel samples: Where A 1 , A 2 , A 3 are the absorbance of the hydrogel sample group, the positive control group (10 mL distilled water, 0.1 mL human EDTA blood, without hydrogel sample material), and the negative control group (10 mL normal saline, 0.1 mL human EDTA blood, without hydrogel sample material), respectively. Cytotoxicity assay Evaluation of cytotoxicity of the hydrogels was conducted using MTT (3-[4, [5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay and in terms of ISO 10993-5:2009 (Biological evaluation of medical devices: Tests for in vitro cytotoxicity) using mouse fibroblast L-929 cells. Firstly, the prepared hydrogels were sterilized by UV irradiation for 2 h, then they were added to a 24-well culture plate. Also, L-929 cells with a density of 1.0 × 10 6 cells were seeded on each hydrogel to be assessed using the standard MTT test. After culturing for 24 h, MTT assays were conducted to test the cell growth. The wells without hydrogels were set as the controls. Results And Discussion Characterization of GO The FTIR-ATR spectra of the graphite powder and the GO have been shown in Fig. 3 . As shown in Fig. 3 a, in the graphite powder spectrum, no significant peak was observed. While, the FTIR-ATR spectrum of GO showed characteristic bands at 3179 cm − 1 , 1726cm − 1 , 1617 cm − 1 , 1232 cm − 1 , and 1041 cm − 1 which are attributed to the hydroxyl group (-OH), carboxyl group (C = O), aromatic ring (C = C), epoxy group (C-O-C) and alkoxy group (C-O), respectively. These hydrophilic oxygen-containing functional groups supply the GO sheets with high water dispersibility (Chen et al., 2013 ). According to Sandhya and et al., the characteristic peak of graphite was reported to be at 2θ = 26.25° (Sandhya et al., 2018 ). As shown in Fig. 3 b, for GO, the 2θ peak shifted to around 10.93°, which indicated that the graphite was fully oxidized into GO. The morphological structure of GO was evaluated by TEM and SEM (Fig. 3 ). The SEM images of GO layers have been indicated in Fig. 3 c, representing a porous shape. Figure 3 d showed the GO TEM image with the paper-like morphology that has wrinkles on its surface. Characterization of hydrogels FTIR-ATR Evidence for the modification of the chitosan (CS) and pectin (PE) was observed in the infrared spectrum (Fig. 4 ). Comparison of the CS infrared spectra with NSC spectra demonstrated the vanishing of the peak at 1590 cm − 1 (the primary amine bending vibration) in NSC. The emergence of a new absorption band at 1550 cm − 1 provides direct evidence of the chitosan modification. Moreover, the absorption band at 1401 cm − 1 could be attributed to the symmetric stretching of the COO-, and it confirms the formation of N-succinyl chitosan (Bashir et al., 2017 ). Furthermore, FTIR-ATR results confirmed the formation of aldehyde groups in the oxidized pectins (OP). Pristine pectin showed the broad peak at 3347 cm − 1 that was assigned to the hydroxyl group's stretching band. The two bands located at 1719 and 1606 cm − 1 were assigned to the ester carbonyl stretching vibrations and asymmetric carboxylate stretching vibrations, respectively. The aldehyde peak of OP, which emerges at 1726 cm − 1 and 883 cm − 1 , belonged to the aldehyde symmetric vibrational band and the hemiacetal formation of free aldehyde groups, respectively. FT-IR-ATR was also used to verify the chemical structure of the hydrogels (Fig. 4 ). For the spectrum of the OP/NSC/GO-0 hydrogel, the hemiacetal structure's characteristic peaks at 871 cm − 1 , along with the appearance of stretching vibrations of the C = N bond at 1631 cm − 1 , demonstrated that the Schiff-base reaction occurred between the –CHO groups of OP and the NH 2 groups of NSC. Some peaks are increased for the other hydrogels, but there is no obvious change in the spectra' peak position. This demonstrates that the hydrogen bonds are formed between the oxygen-containing GO's functional groups and the hydroxyl groups on the NSC and OP molecular chains. Gelation time Gelation times of various amounts of GO are shown in Fig. 5 . In the Gels, the OP acts as a macromolecular chemical cross-linker making a spontaneous and fast chemical crosslinking reaction between the aldehyde of OP and the amino of NSC (Fig. 5 .a). Moreover, with the content of GO increasing in the hydrogels, a shorter gelation time was observed. This might be due to the hydrophilic nature of the GO. The GO nanosheets contain numerous –COOH and –OH groups, forming hydrogen bonds with the NSC and OP macromolecules. Meanwhile, the higher content of GO will produce a higher amount of hydrogen bonds that facilitate network structure formation, accelerate the gelation process, and shorten the gelation time. So the formation of the cross-linked network structure is fast (< 30s). Swelling and degradation Figure 5 illustrates the swelling and degradation behavior of the composite hydrogels in the PBS at 37 \\(^\\circ C\\) . According to Fig. 5 b, OP/NSC/GO-0 showed the highest swelling ratio among the four GO-containing hydrogels. This is because of the lowest crosslinking density and mechanical properties of the OP/NSC/GO-0 hydrogels, that water molecules could enter the hydrogel network. Also, when the GO content increased from 0mg/ml to 6mg/ml, the swelling ratio of the hydrogels was reduced. This might have been due to the hydrogen bonds between GO nanosheets and the NSC and OP macromolecules, which formed denser crosslinking points, thus the capacity of hydrogels would be decreased to form the hydrogen bonds with water molecules. Having biodegradability and preserving minimal resistance are the common requirements for porous hydrogels used in tissue engineering. The presence of GO in the composite gel scaffold could maintain their weight for up to 21days (Fig. 5 c). After incubation for 21 days, the degradation ratio of OP/NSC/GO-6 hydrogel (67.78 ± 2.65%) was lower than those of OP/NSC/GO-4, OP/NSC/GO-2, and OP/NSC/GO-0 (70.50 ± 3.83, 73.52 ± 5.10, and 94.90 ± 1.98%, respectively). It was due to the addition of GO, which enhanced the hydrogel's crosslinking density; and therefore improved its stability. Such findings showed that during in vitro culture, the composite hydrogel's structure was stable. SEM The cross-sectional morphology of composite hydrogels with different GO content was observed by SEM (see Fig. 6 a-d). As shown in Fig. 6 , OP/NSC/GO owned the continuous three-dimensional porous structure. This would lead to the structural and compositional similarity of hydrogels with the natural extracellular matrix, as well as high oxygen permeability. There were no significant variations in the micro-morphology of the four hydrogels prepared with various GO loadings. The pores were morphologically homogeneous with smooth walls and there were dense voids. The network created by the Schiff base cross-linking between NSC and OP and the hydrogen bonds between the polymer chains and GO nanosheets caused the pore size of hydrogels to decrease slightly as the GO content increased (from 286.69 to 213.10, 184.91, and 127.573µm, respectively). Therefore, being orderly and compact, the hydrogels with GO exhibited better mechanical properties than hydrogels without GO. Conductivity of the OP/NSC/GO hydrogels Besides self-healing and injectability, conductivity is also a desired property for the hydrogels used in tissue engineering, mainly when it is used for tissues under electrophysiological conditions (Zheng et al., 2020 ). The tissue conductivity (ventricular muscle, nerve, lung, cardiac, and skeletal muscle) ranges orderly between 0.03 and 0.6 S/m. Thus, researchers must consider this issue when engineering various tissues in the body, so that their designed electroconductive nano-biomaterials scaffolds satisfy these electrical characteristics. Therefore, we then explored the conductivity of the OP/NSC/GO hydrogels. The conductivity of OP/NSC/GO hydrogels with different GO contents has been shown in Fig. 7 . All hydrogels showed a conductivity of the order 0.0035–0.112 S/m. It is worth mentioning that the suitable conductivity for native cardiac tissue is in the range from 0.005 to 0.16 S/m (Mostafavi et al., 2020 ), which is in the range of the OP/NSC/GO conductivity hydrogels that we prepared. Rheological property The rheological analysis at both strain and frequency sweep modes (Fig. 8 a, b) was done to get the hydrogels' mechanical property. Throughout the test, the storage modulus (G') was consistently higher than (G\") in the frequency range (1-100 rad/s) at a fixed strain of 1%. Figure 8 a shows that the storage modulus G′ and loss modulus G′′ of the hydrogels did not change significantly, suggesting that the hydrogel is stable and elastic. However, the elastic modulus does not change apparently as the strain increases, indicating the stability of the OP/NSC/GO imine bond and homogeneity of all these hydrogels (Fig. 8 b). The best mechanical properties were for samples with the largest G′ and G′′ that contained 6 mg/ml GO. This may be due to the increased cross-linking density between NSC and OP, as well as the hydrogen bonding between GO nanosheets and polymer chains. As a result, the OP/NSC/GO-6 were selected for more investigations. Self-healing and injectable properties of OP/NSC/GO hydrogels To evaluate the hydrogels' self-healing ability, the hydrogels' strain amplitude sweep test was first performed to determine the critical point of the hydrogel between liquid and solid-state (Fig. 9 a). The results showed that the intersection point between the storage modulus (G') and loss modulus (G'') was at the strain of 83.52%. When the strain is larger than this value, G' would be lower than G'', indicating the collapse of the hydrogel, and change in its physical form from solid to fluid. Then, a series of rheological recovery tests were carried out with the G' and G'' of the hydrogels versus time under higher strain to evaluate the self-healing ability of the hydrogel (Fig. 9 b). A large strain of 300% was applied to break the network structure. It decreased the G' from ~ 759 Pa to ~ 63 Pa and induced gel-to-sol transition. When switched into the low strain, the G' of the hydrogel returned quickly to the original value with the recovery of the hydrogel structure, thus indicating the rapid self-healing property of the OP/NSC/GO-6 hydrogel. According to the results, the broken structure recovered rapidly, and after periods of breaking and reforming, there was still the nearly normal hydrogel. A macroscopic self-healing test was conducted to further assess the self-healing behavior of the OP/NSC/GO-6 hydrogel. As shown in Fig. 10 , the square shape hydrogel was cut into two halves, and then they were reconnected to allow them to be self-healed at 37℃. After 10 min of incubation at 37 ℃ without any external stimulus, the ruptured hydrogel could entirely integrate, and the blended integral hydrogels could be lifted with laboratory tweezers. It was held up under the force of gravity, indicating the self-healing performance of the OP/NSC/GO-6 hydrogel. Such self-healing ability attributes to the Schiff-based bonds and hydrogen interactions of the OP/NSC-GO-6 hydrogel. We used both observable and qualitative approaches to confirm the injectability of the OP/NSC-GO-6 hydrogel. The viscosity of the OP/NSC-GO-6 hydrogel reduced significantly as the shear rate increased from 1 to 100 s-1, indicating the shear-thinning behavior (see Fig. 11 ). Furthermore, given the shear-thinning property of the OP/NSC-GO-6 hydrogel, it could also be rapidly injected via a syringe (3mL, G23) without clogging. This indicated that OP/NSC-GO-6 hydrogel has suitable injectability. The Schiff base linkages and hydrogen bonds were separated under pressure when the hydrogel was squeezed into the syringe, leading to the transfiguration of the hydrogel, so that it could flow like a liquid into the needle. The extruded hydrogel was combined to make a single component because of its self-healing capacity. This led to showing its ability to homogeneously encapsulate the cells or drugs and transplant them into the tissue in a less invasive manner. Hemolysis rate The material's hemolysis rate is a crucial indicator of biological materials' blood compatibility. The hemolysis phenomenon occurs when a foreign body is in contact with the blood which damages or ruptures the red blood cells. The lower the calculated hemolysis rate is, the better would be, the material's blood compatibility. The apparent hemolysis phenomenon was observed in the positive control tube, as shown in Fig. 12 . The positive control group's blood cells have ruptured, and the solution was uniform and red. Furthermore, the blood cells were deposited at the tube in the negative control group, and the solution was transparent. The hydrogel samples' hemolysis activity resembled somehow the negative control sample. That is, some RBCs were deposited in the tube and a large number of them were adsorbed in the hydrogel. The supernatant was mainly colorless and transparent. Biomaterials would be suitable for biomedical applications if the hemolysis rate is less than 5%, according to international guidelines. The hemolysis rate values obtained for all hydrogels range between 1.38% and 0.34% (as it could be seen in Table 1 ), indicating that all materials have excellent blood compatibility properties. Table 1 In vitro hemocompatibility analysis of OP/NSC/GO hydrogels Sample Hemolysis rate(%) OP/NSC/GO-0 1.38 OP/NSC/GO-2 0.92 OP/NSC/GO-4 0.57 OP/NSC/GO-6 0.34 Cytotoxicity test using MTT assay To evaluate the suitability of using OP/NSC/GO hydrogels as biomedical materials, an MTT assay was used to determine the toxicity of synthesized hydrogels. As shown in Fig. 13 , during the whole incubation period, the cell viabilities of the fibroblast (L929) were higher than 80 %, suggesting that all hydrogels had no cytotoxicity to fibroblast cells (L929). Notably, the results also found that the cell viability was higher with the addition of GO. Furthermore, compared to OP/NSC/GO-2, OP/NSC/GO-4, OP/NSC/GO-6, the cell viabilities significantly decrease with the rise of GO content. This could be explained by the fact that as the GO content in the hydrogels increases, the GO content in the extracts increases, and the GO shows a strong cytotoxic effect at high concentrations. Conclusion This study introduced a new dual-crosslinked hydrogel system with suitable injectability, excellent self-healing, and adequate conductivity. Through embedding the GO into the doubly Schiff-base crosslinking by amino and aldehyde groups of the NSC and OP, respectively and hydrogen bonding between the GO sheets and the polymer chains, the OP/NSC/GO composite hydrogels were successfully prepared. For the cmposite hydrogel's adjustable performance, it is critical to integrate the GO. Composite hydrogels had a faster gelation time, greater stability, and better mechanical properties than pure hydrogels. Overall, In our analysis, the composite hydrogel with a GOs content of 6 mg/mL displayed the best performance, with an adequate swelling ratio and stability. The hydrogels had excellent anti-hemolytic properties when they were examined in terms of hemolytic potential. Furthermore, the cytotoxicity testing revealed that the hydrogel had no biological toxicity when tested on mouse embryonic fibroblasts, and the cell viability of the various samples exceeded 80 %. These findings suggest that the constructed hydrogel could be a suitable choice for tissue engineering, especially in electroactive tissues. Declarations Acknowledgements The authors would like to thank Dr. Mohammad Taghi Razavi Tousi for kind assistance on Hemolytic assays. References Ahadi F, Khorshidi S, Karkhaneh A (2019) A hydrogel/fiber scaffold based on silk fibroin/oxidized pectin with sustainable release of vancomycin hydrochloride. Eur Polym J 118:265–274 Bagher Z, Ehterami A, Safdel MH, Khastar H, Semiari H, Asefnejad A, Davachi SM, Mirzaii M, Salehi M (2020) Wound healing with alginate/chitosan hydrogel containing hesperidin in rat model. Drug Deliv Sci Technol 55:101379 Bashir S, Teo Y. Y, Naeem S, Ramesh S, Ramesh K (2017) pH responsive N-succinyl chitosan/Poly (acrylamide-co-acrylic acid) hydrogels and in vitro release of 5-fluorouracil. 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Polymer 140:150–157 Liu J, Zhang X, Chen X, Qu L, Zhang L, Li W, Zhang A (2018) Stimuli-responsive dendronized polymeric hydrogels through Schiff-base chemistry showing remarkable topological effects. Polym Chem 9:378–387 Mostafavi E, Medina-Cruz D, Kalantari K, Taymoori A, Soltantabar P, Webster TJ (2020) Electroconductive nanobiomaterials for tissue engineering and regenerative medicine. J BIOELECTR 2:120–149 Pu W, Jiang F, Chen P, Wei B (2017) A POSS based hydrogel with mechanical robustness, cohesiveness and a rapid self-healing ability by electrostatic interaction. Soft Matter 13:5645–5648 Qin Y, Wang J, Qiu C, Xu X, Jin Z (2019) A dual cross-linked strategy to construct moldable hydrogels with high stretchability, good self-recovery, and self-healing capability. J Agric Food Chem 67:3966–3980 Sandhya P, Jose J, Sreekala M, Padmanabhan M, Kalarikkal N, Thomas S (2018) Reduced graphene oxide and ZnO decorated graphene for biomedical applications. Ceram Int 44:15092–15098 Shahriary L, Athawale AA (2014) Graphene oxide synthesized by using modified hummers approach. Int J Renew Energy Environ Eng 2:58–63 Spicer CD (2020a) Hydrogel scaffolds for tissue engineering: the importance of polymer choice. Polym Chem 11:184–219 Spicer CD (2020b) Hydrogel scaffolds for tissue engineering: the importance of polymer choice. Polym Chem 2:184–219 Tummalapalli M, Berthet M, Verrier B, Deopura B, Alam M, Gupta B (2016) Drug loaded composite oxidized pectin and gelatin networks for accelerated wound healing. Int J Pharm 505:234–245 Xu C, Zhan W, Tang X, Mo F, Fu L, Lin B (2018) Self-healing chitosan/vanillin hydrogels based on Schiff-base bond/hydrogen bond hybrid linkages. Polym Test 66:155–163 Xu Q, Ji Y, Sun Q, Fu Y, Xu Y, Jin L (2019) Fabrication of cellulose nanocrystal/chitosan hydrogel for controlled drug release. Nanomaterials 9:253 Ye X, Li X, Shen Y, Chang G, Yang J, Gu Z (2017) Self-healing pH-sensitive cytosine-and guanosine-modified hyaluronic acid hydrogels via hydrogen bonding. Polymer 108:348–360 Yu R, De Saint-Cyr L, C, Soussan L, Barboiu M, Li S (2021) Anti-bacterial dynamic hydrogels prepared from O-carboxymethyl chitosan by dual imine bond crosslinking for biomedical applications. Int J Biol Macromol 167:1146–1155 Zhang W, Jin X, Li H, Zhang R-R, Wu C-W (2018) Injectable and body temperature sensitive hydrogels based on chitosan and hyaluronic acid for pH sensitive drug release. Carbohydr Polym 186:82–90 Zhang X, Pan Y, Li S, Xing L, Du S, Yuan G, Li J, Zhou T, Xiong D, Tan H (2020) Doubly crosslinked biodegradable hydrogels based on gellan gum and chitosan for drug delivery and wound dressing. Int J Biol Macromol 164:2204–2214 Zhao H, Liu M, Zhang Y, Yin J, Pei R (2020) Nanocomposite hydrogels for tissue engineering applications. Nanoscale 12:14976–14995 Zheng F, Li R, He Q, Koral K, Tao J, Fan L, Xiang R, Ma J, Wang N, Yin Y (2020) The electrostimulation and scar inhibition effect of chitosan/oxidized hydroxyethyl cellulose/reduced graphene oxide/asiaticoside liposome based hydrogel on peripheral nerve regeneration in vitro. Mater Sci Eng C 109:110560 Zhou J, Yang X, Liu W, Wang C, Shen Y, Zhang F, Zhu H, Sun H, Chen J, Lam J (2018) Injectable OPF/graphene oxide hydrogels provide mechanical support and enhance cell electrical signaling after implantation into myocardial infarct. Theranostics 8:3317 Supplementary Files graphical2300.tiff Cite Share Download PDF Status: Posted Version 1 posted 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. 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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-922109\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":55001046,\"identity\":\"942da0d2-7557-4bec-8d0e-8fa7df4b0155\",\"order_by\":0,\"name\":\"Atefeh Afroozan Bazghaleh\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of Guilan\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Atefeh\",\"middleName\":\"Afroozan\",\"lastName\":\"Bazghaleh\",\"suffix\":\"\"},{\"id\":55001047,\"identity\":\"45533644-3ace-4624-98f0-db8e15f7dada\",\"order_by\":1,\"name\":\"Mojtaba Akbari Dogolsar\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCklEQVRIiWNgGAWjYHCCBAbGBhDNxgYkJORAzAMP8GtJbEDWYgzWkoBfCyOyFqABEJtxA932hucPfu6wAzKOpT34uccifX7Y4YdAW+zkdBuwazE7cyCxsfdMMpCRdtyw55lE7sbbaQZALcnGZgdwaLmRkNjA28bMYHYgvU2C5wBQy+wEkJYDidvwaGn821bPYHb+eZvknwMS6Yaz0z8Q1NLM23YYyEg7Jg20JUFeOoeALUC/zJY9c5zH7MazdGOZAxKGG6RzCg4kGODxy/GehI9vd1TLmZ1PM3v45kCdvPzs9M0fPlTYyeHSwsDAkwAm4XwDsEoDXMpBgB3NMPkGfKpHwSgYBaNgJAIAHCxpxDG0k0oAAAAASUVORK5CYII=\",\"orcid\":\"https://orcid.org/0000-0002-7576-676X\",\"institution\":\"University of Guilan\",\"correspondingAuthor\":true,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Mojtaba\",\"middleName\":\"Akbari\",\"lastName\":\"Dogolsar\",\"suffix\":\"\"},{\"id\":55001048,\"identity\":\"36e180ae-5806-47b7-a2f3-cb960c870f74\",\"order_by\":2,\"name\":\"Jalal Barzin\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Iran Polymer Research Center: Iran Polymer and Petrochemical Research Institute\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Jalal\",\"middleName\":\"\",\"lastName\":\"Barzin\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2021-09-19 21:42:55\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-922109/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-922109/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":14190870,\"identity\":\"95e43482-4ca1-49af-a52b-2b58637d1531\",\"added_by\":\"auto\",\"created_at\":\"2021-10-01 15:08:12\",\"extension\":\"jpg\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":49803,\"visible\":true,\"origin\":\"\",\"legend\":\"Schematic of the synthesis of OP/NSC/GO hydrogel.\",\"description\":\"\",\"filename\":\"fig1.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-922109/v1/27dc7ebd5d4a093599cbf913.jpg\"},{\"id\":14190967,\"identity\":\"bebac8b3-bf14-4aa2-bc4e-f9cb6cf5fe91\",\"added_by\":\"auto\",\"created_at\":\"2021-10-01 15:11:13\",\"extension\":\"jpg\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":100349,\"visible\":true,\"origin\":\"\",\"legend\":\"Scheme of double cross-linked processes.\",\"description\":\"\",\"filename\":\"fig2.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-922109/v1/f03ae2dce63057fe269319eb.jpg\"},{\"id\":14190872,\"identity\":\"7d8fd853-432f-4f9b-b13c-c322f2e31279\",\"added_by\":\"auto\",\"created_at\":\"2021-10-01 15:08:13\",\"extension\":\"jpg\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":117943,\"visible\":true,\"origin\":\"\",\"legend\":\"Characterization of GO; a) FTIR-ATR, b) X-ray diffraction pattern, c), and d) SEM and TEM image, respectively.\",\"description\":\"\",\"filename\":\"fig3.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-922109/v1/afdec4e7dd4c75e255e84a33.jpg\"},{\"id\":14190965,\"identity\":\"2488374d-8add-446d-9d3f-d175feb1a9d9\",\"added_by\":\"auto\",\"created_at\":\"2021-10-01 15:11:13\",\"extension\":\"jpg\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":91083,\"visible\":true,\"origin\":\"\",\"legend\":\"FTIR-ATR spectra of various sample.\",\"description\":\"\",\"filename\":\"fig4.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-922109/v1/4d627fc55d0a048a3d8d2c26.jpg\"},{\"id\":14191462,\"identity\":\"93ff3e8f-cc68-4239-b00f-620285c49c4b\",\"added_by\":\"auto\",\"created_at\":\"2021-10-01 15:17:13\",\"extension\":\"jpg\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":79735,\"visible\":true,\"origin\":\"\",\"legend\":\"Composite hydrogels with different amounts of GO; a) Gelation time, b) Swelling ratio, and c) degradation behavior.\",\"description\":\"\",\"filename\":\"fig5.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-922109/v1/6e89be410d6c7e4e97e178c7.jpg\"},{\"id\":14190871,\"identity\":\"3f925b5f-de37-4c2b-bc2f-5ec6e472bbe8\",\"added_by\":\"auto\",\"created_at\":\"2021-10-01 15:08:13\",\"extension\":\"jpg\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":132015,\"visible\":true,\"origin\":\"\",\"legend\":\"Morphological characterization of hydrogels: (a) OP/NSC/GO-0, (b) OP/NSC/GO-2, (c) OP/NSC/GO-4 and (d) OP/NSC/GO-6. Scale bar: 500μm\",\"description\":\"\",\"filename\":\"fig6.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-922109/v1/c6fbbeb1e7e2e164bf6443e8.jpg\"},{\"id\":14191461,\"identity\":\"43eb19ab-8532-46bc-bec9-a6766837d56c\",\"added_by\":\"auto\",\"created_at\":\"2021-10-01 15:17:13\",\"extension\":\"jpg\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":63973,\"visible\":true,\"origin\":\"\",\"legend\":\"Conductivity results of the hydrogels.\",\"description\":\"\",\"filename\":\"fig7.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-922109/v1/d942467de7f941e8d70c01e1.jpg\"},{\"id\":14190971,\"identity\":\"1cf72f2d-b530-44f6-98f2-d84f79f12be6\",\"added_by\":\"auto\",\"created_at\":\"2021-10-01 15:11:13\",\"extension\":\"jpg\",\"order_by\":8,\"title\":\"Figure 8\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":90384,\"visible\":true,\"origin\":\"\",\"legend\":\"a) Rheology measurement of the hydrogels under the frequency-sweep, b) The storage modulus G′ and loss modulus G″ were plotted logarithmically against strains of the corresponding hydrogel samples.\",\"description\":\"\",\"filename\":\"fig8.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-922109/v1/f2887c83e8596f068f92a1ee.jpg\"},{\"id\":14191162,\"identity\":\"fb8a40d4-13ab-45dd-a8cd-add29e916180\",\"added_by\":\"auto\",\"created_at\":\"2021-10-01 15:14:13\",\"extension\":\"jpg\",\"order_by\":9,\"title\":\"Figure 9\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":67738,\"visible\":true,\"origin\":\"\",\"legend\":\"a) Strain amplitude sweep of OP/NSCGO-6 hydrogel, b) Amplitude oscillatory sweep when alternate step strain switched from 1% to 300%.\",\"description\":\"\",\"filename\":\"fig9.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-922109/v1/baa55592813a5076ce457a72.jpg\"},{\"id\":14191164,\"identity\":\"c1d59c3c-666c-4a10-a17d-2c070c404b70\",\"added_by\":\"auto\",\"created_at\":\"2021-10-01 15:14:13\",\"extension\":\"jpg\",\"order_by\":10,\"title\":\"Figure 10\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":62395,\"visible\":true,\"origin\":\"\",\"legend\":\"Macroscopic self-healing process of OPNSC/GO-6 hydrogels. The scale bar is 1.5 cm.\",\"description\":\"\",\"filename\":\"fig10.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-922109/v1/da76a2153c2db0cd6368d812.jpg\"},{\"id\":14190876,\"identity\":\"61c06fa5-39bd-4c0a-ae28-5573368ef426\",\"added_by\":\"auto\",\"created_at\":\"2021-10-01 15:08:13\",\"extension\":\"jpg\",\"order_by\":11,\"title\":\"Figure 11\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":59944,\"visible\":true,\"origin\":\"\",\"legend\":\"Viscosity measurements of OP/NSC/GO-6 hydrogel; extrusion of hydrogel through a 23-G needle and writing by using OP/NSC/GO-6 as ink.\",\"description\":\"\",\"filename\":\"fig11.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-922109/v1/2d2153dee5c1424f89e6a0d2.jpg\"},{\"id\":14190882,\"identity\":\"077d9c56-9c79-493e-bfbb-d6832f786913\",\"added_by\":\"auto\",\"created_at\":\"2021-10-01 15:08:13\",\"extension\":\"jpg\",\"order_by\":12,\"title\":\"Figure 12\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":106231,\"visible\":true,\"origin\":\"\",\"legend\":\"Photographs of corresponding RBCs solutions after centrifugation. The picture shows the Hemolysis of the negative control sample, positive control sample, and (A) OP/NSC/GO-0, (B) OP/NSC/GO-2, (C) OP/NSC/GO-4, and (D) OP/NSC/GO-6.\",\"description\":\"\",\"filename\":\"fig12.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-922109/v1/f993c414b5b802edb362b0d3.jpg\"},{\"id\":14190881,\"identity\":\"b068c773-f564-4841-8b22-7bd907913630\",\"added_by\":\"auto\",\"created_at\":\"2021-10-01 15:08:13\",\"extension\":\"jpg\",\"order_by\":13,\"title\":\"Figure 13\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":82943,\"visible\":true,\"origin\":\"\",\"legend\":\"MTT assay results. a) Histogram comparing percentage of the live cells on 24 h, b) Mouse fibroblast cells cytotoxicity assessment of hydrogels; A1) OP/NSC/GOs, A2) OP/NSC/GO-2, A3) OP/NSC/GO-4, A4) OP/NSC/GO-6 Images were acquired on an inverted microscope from cells (after 24 h).\",\"description\":\"\",\"filename\":\"fig13.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-922109/v1/353622b19c55b952dd00117f.jpg\"},{\"id\":15401174,\"identity\":\"a3059291-cc85-4432-8a06-80a01829ba45\",\"added_by\":\"auto\",\"created_at\":\"2021-11-10 15:52:43\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":1294880,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-922109/v1/1ab0a420-98dc-4e27-8a4a-9f77c92144b7.pdf\"},{\"id\":14190883,\"identity\":\"5011e852-5190-420c-af27-b1fae127028b\",\"added_by\":\"auto\",\"created_at\":\"2021-10-01 15:08:13\",\"extension\":\"tiff\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":17628216,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"graphical2300.tiff\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-922109/v1/fba1c209be514bc47e08ce7b.tiff\"}],\"financialInterests\":\"\",\"formattedTitle\":\"\\u003cp\\u003eDual-Crosslinked Oxidized Pectin/N-Succinyl Chitosan Hydrogel Containing Graphene Oxide Nanosheets for Tissue Engineering Application\\u003c/p\\u003e\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eTissue engineering is the process of designing and creating functional substitutes for the damaged tissues and organs through growing the cells into a scaffold that mimics the target tissue's extracellular matrix (ECM) (Spicer, \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e2020b\\u003c/span\\u003e). For this purpose, hydrogels, a three-dimensional cross-linked network backbone containing a large amount of water (50\\u0026ndash;90%) are promising materials for tissue engineering applications. To support cell proliferation, migration, and differentiation, hydrogels could be designed which permit oxygen and nutrient transport. Also, they supply a 3D, highly hydrated environment for cells that mimics the original soft tissues. This is mainly attributed to the unique properties of hydrogels, including their high permeability, excellent biocompatibility, non-toxicity, non-immunogenicity, biodegradability, tunable physical and chemical properties, tissue-like elasticity as well as the capability to encapsulate bioactive molecules, cells, or drugs to mimic an extracellular matrix (ECM) (Spicer, \\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e2020a\\u003c/span\\u003e, Zhao et al., \\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e). Recently, stimulus-responsive hydrogels have become a hot topic in biomedical applications because of their versatility. Self-healing hydrogel has appealing features such as maintaining the integrity of the structure and improving the mechanical properties. The most common preparation method for self-healing hydrogels is introducing the dynamic covalent (boronate ester bond (Ji et al., \\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e, Figueiredo et al., \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e), disulfide bond (Guo et al., \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e), imine chemistry (Schiff-base reaction) (Xu et al., \\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e, Liu et al., \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e) and Diels-Alder reaction (Lin et al., \\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e, Bi et al., \\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e), etc.) or the non-covalent bonds (hydrophobic association (Deng et al., \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e), electrostatic interactions (Pu et al., \\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e), hydrogen bonding (Ye et al., \\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e), host-guest inclusion (Jin et al., \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e), etc.) into the hydrogel system. However, the crosslinking curing mechanism affects the stability of the formed hydrogel directly (Zhang et al., \\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e). So, due to the moderate reaction conditions, efficient reversibility of imine bonds, tunable properties, and stimuli-responsive performance of the resulting materials, imine bonding (CH\\u0026thinsp;=\\u0026thinsp;N) has attracted significant interest in the biological applications. Also, water molecules are the only by-products which are released in the imine reaction (Yu et al., \\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e, de Lima et al., \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eThe appearance of the non-toxic, ecofriendly and biocompatible polymers derived from natural sources have introduced a new and interesting dimension to the development of the low-cost and scalable biomaterials for tissue engineering applications. Chitosan, a linear polysaccharide with cationic nature is more attractive than the other bio-polymers due to its intrinsic biocompatibility, biodegradability, bio-adhesivity, hydrophilicity, high viscosity, and antibacterial properties. It could be crosslinked via reaction between its amino groups along the backbone and other polymers such as hyaluronic acid (Zhang et al., \\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e), alginate (Bagher et al., \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e), silk fibroin (Eivazzadeh-Keihan et al., \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e), and cellulose (Xu et al., \\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). However, its poor solubility in physiological solvents due to its strong intermolecular hydrogen bonding limits its biomedical applications (Yu et al., \\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). Therefore, to expand its application, N-Succinyl-chitosan (NSC), which is a water-soluble chitosan derivative could be synthesized via the introduction of succinyl groups at the N-position of the glucosamine units of chitosan. This makes it an excellent candidate to design polymeric scaffolds for tissue regeneration (Bashir et al., \\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e, Kamoun, \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003ePectin (PE), which is also called polygalacturonic acid, is an anionic polysaccharide extracted from primary cell plants. Pectin has been widely used in a variety of biomedical applications, due to its important properties such as biocompatibility, biodegradability, capability to inhibiting the activity of macrophages and neutrophils, anti-inflammatory effects, and the capability to restrain cells, drugs, and genes (Li et al., \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e, Kaushik et al., \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e). On the backbone chain, there are hydroxyl, carboxyl and carboxymethyl groups which makes it readily susceptible to functionalization and modification. PE could be oxidized by reacting with sodium periodate to produce OP (Tummalapalli et al., \\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e, Li et al., \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e2020a\\u003c/span\\u003e). Through the oxidation reaction, aldehyde groups are introduced in the PE molecular chain, which are able to participate in Schiff mechanism. So, they could react with amine groups of NSC and form imine bonds.\\u003c/p\\u003e \\u003cp\\u003eSince the stability of the hydrogel network composed of imine bonds is usually poor, and the mechanical properties are relatively weak, academics have paid widespread attention to the dual cross-linked (DC) network hydrogels. This is because such DC network hydrogels optimize and combine different properties of each network structure, including dual physically cross-linked hydrogels, dual chemically cross-linked hydrogels. It should be noted that the hybrid cross-linked hydrogels could potentially resolve these issues and results in materials appropriate for tissue engineering applications (Li et al., \\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e2020b\\u003c/span\\u003e, Qin et al., \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). For this purpose, graphene oxide (GO), a precursor of chemically converted graphene, is a widely investigated nanomaterial. Due to its oxygen-containing functional groups (i.e., hydroxyl, carboxyl, carbonyl, and epoxy groups), it could be easily combined with polymers for enhancing the hydrogel's mechanical properties. Moreover, it has good electrical conductivity, excellent chemical stability, easy accessibility, appropriate mechanical and antibacterial properties (Hua et al., \\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e, Zhou et al., \\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e). Therefore, in this study, the dual-crosslinked OP/NSC/GO hydrogels were fabricated via a dynamic imine bond and a hydrogen bond. The gelling time, morphology, rheology, swelling ratio, degradation, conductivity, self-healing, and injectability were examined. Also, in vitro biocompatibility and hemolysis rates were evaluated.\\u003c/p\\u003e\"},{\"header\":\"Experimental Section\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eMaterials\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eChitosan (Mw 190\\u0026ndash;310 kDa), pectin from citrus peels (galacturonic acid\\u0026thinsp;\\u0026ge;\\u0026thinsp;74.0%), graphite powder, succinic anhydride, sodium periodate, ethylene glycol, hydroxylamine hydrochloride, methanol and sodium hydroxide were obtained from Sigma\\u0026ndash;Aldrich. Ethanol, acetone, potassium permanganate, sodium nitrate, sulfuric acid 98%, and hydrogen peroxide, were obtained from commercially available suppliers (Merck KGaA). All chemicals were of analytical grade purity and they were used as received without any further purification. The aqueous solutions were prepared in deionized water.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eSynthesis of N-succinyl chitosan (NSC)\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAs previously described, NSC was synthesized with slight modifications (Bashir et al., \\u003cspan class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e). In short, the purified chitosan (0.5 g) was suspended in 75 mL aqueous solution of acetic acid (5.0% v/v). This mixture was placed magnetic stirring at 50 \\u0026deg;\\u003cem\\u003eC\\u003c/em\\u003e for 1 h. After that, dilution of the solution was done by adding 75 mL methanol. Subsequently, succinic anhydride (2.5 g) was dissolved in 60 mL acetone and mixed with chitosan solution. The stirring was continued at 50 \\u0026deg;\\u003cem\\u003eC\\u003c/em\\u003e for 24 hours. After 24 h, the pH of the mixture was adjusted to 12 with NaOH (1.0 mol/L) solution, and the formation of a clear solution was observed. The stirring of this clear solution was continued for further 5 hours. Afterward, 150mL ethanol (96%) was added to form precipitates followed by filtration to separate the precipitates. These precipitates were dispersed in ethanol for 24 hours. Then, dispersed precipitated product was washed with several times with ethanol and acetone to remove excess reagents and dried using freeze-dryer for 4 hours. The final product was stored at room temperature. The degree of the substitution (DS) of NSC was determined to be 0.52 according to the described previously (Bashir et al., \\u003cspan class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eSynthesis of oxidized pectin (OP)\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eSynthesis of OP was according to the previously reported procedure with slight modifications (Ahadi et al., \\u003cspan class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). Initially, 0.5 g of pectin was dispersed in 10 mL ethanol (96%). Then 10 mL of sodium periodate solution (0.5 M) was added and the temperature was controlled at 40 \\u0026deg;\\u003cem\\u003eC\\u003c/em\\u003e. After the reaction was carried out for 8 h in a dark environment. The appropriate amount of ethylene glycol was added to continue the reaction for 2 h to remove the unreacted oxidant. After the reaction, the product was dialyzed in deionized water (dialysis bag, molecular weight cutoff: 8\\u0026ndash;12 kDa) for 3 days to remove the excess sodium periodate, then frozen at \\u0026minus;\\u0026thinsp;20 \\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e℃\\u003c/span\\u003e\\u003c/span\\u003e and lyophilized at \\u0026minus;\\u0026thinsp;80 \\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e℃\\u0026nbsp;\\u003c/span\\u003e\\u003c/span\\u003efor 24 h. The degree of oxidation (DO) was determined by potentiometric titration, and DO value was 42.284\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.897 %.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eSynthesis of graphene oxide (GO)\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eGO was prepared using graphite powder via the modified Hummers method (Shahriary and Athawale, \\u003cspan class=\\\"CitationRef\\\"\\u003e2014\\u003c/span\\u003e). In brief, the graphite powder (1 g) and sodium nitrate (0.5 g) were added to sulfuric acid 98% (23 mL) under stirring. While using an ice bath and keeping the temperature at 0 \\u0026deg;\\u003cem\\u003eC\\u003c/em\\u003e (to prevent overheating and explosion), potassium permanganate (3g) was added gradually to the mixture under the same conditions. Subsequently, the mixture was stirred at 35 \\u0026deg;C for 24 h. Then, the solution was diluted with distilled water, and the reaction was finally completed by adding 30% hydrogen peroxide (5mL). The resulting mixture was washed with distilled water until the filtrate showed neutral pH. The obtained solid was freeze-dried for 24 h.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003ePreparation of the OP/NCS/GO hydrogels\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNSC and OP were dissolved in the phosphate-buffered saline (PBS, pH 7.4 and the room temperature for 5 h) separately to form a 3 wt. % solution. The various contents of GO (0mg/ml, 2mg/ml, 4mg/ml, and 6mg/ml) was dispersed into OP solutions (3 wt. %) with the aid of ultrasound. Then, the solutions were stored at 4℃ for further use. The preparation of OP/NSC/GO hydrogels was carried out using a system of two interconnected syringes. Equal volumes of the NSC and OP/GO solution were loaded into two separate Luer-Lock syringes, respectively, and then both syringes were connected via a connector. The solutions were thoroughly mixed by pressing alternately on each of the plungers (more than three times). Upon thorough mixing, the entire syringe contents were pushed into one of the syringes, the connector in conjunction with the empty syringe was disengaged, and the prepared hydrogel was ejected and liberally deposited in suitable mold (10 mm diameter) for further investigation. According to the difference in the contents of GO, the hydrogels were coded as OP/NSC/GO-0, OP/NSC/GO-2, OP/NSC/GO-4, and OP/NSC/GO-6 hydrogels. The procedures of synthesizing the hydrogels have been shown in Figs. \\u003cspan class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e and \\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eGelation time test\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe gelation time of hydrogels was measured by the tube inversion method. A mixture of 1 ml of OP/NSC/GO solution was poured into a glass bottle with a diameter of 20 mm, and it was placed at room temperature. The gelatin time is determined by the time it takes for the mixture to stop flowing when the glass bottle is inverted.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eSwelling measurements\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTo evaluation the swelling ratio, the same shape and size of freeze-dried hydrogel samples were weighed (W\\u003csub\\u003ed\\u003c/sub\\u003e) and soaked in PBS (pH\\u0026thinsp;=\\u0026thinsp;7.4) at 37\\u0026deg;\\u003cem\\u003eC\\u003c/em\\u003e. After 48 h, samples were removed and the water remaining on the surface was absorbed by filter paper. Then, the swollen hydrogel was immediately weighed (W\\u003csub\\u003es\\u003c/sub\\u003e) and the swelling ratio was calculated from the formula:\\u003c/p\\u003e\\n\\u003cdiv class=\\\"Equation\\\" id=\\\"Equa\\\"\\u003e\\n \\u003cdiv class=\\\"mathdisplay\\\" id=\\\"FileID_Equa\\\" name=\\\"EquationSource\\\"\\u003e\\u003cimg 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vitro degradation analysis\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe degradation of the OP/NSC/GO hydrogels was examined for the weight loss under the aqueous condition (PBS at 37\\u003csup\\u003eo\\u003c/sup\\u003eC) for 4 weeks. Firstly, the initial weight of lyophilized OP/NSC/GO hydrogels was accurately weighed (W\\u003csub\\u003e0\\u003c/sub\\u003e). After predetermined time intervals, hydrogels were removed from the medium and, they were freeze-dried. Then, the sample was accurately weighed and recorded as (W\\u003csub\\u003e1\\u003c/sub\\u003e). The PBS was replaced by fresh PBS every 3 days. All measurements were performed in duplicate. The following formula calculated the degradation ratios of the OP/NSC/GO hydrogels:\\u003c/p\\u003e\\n\\u003cdiv class=\\\"Equation\\\" id=\\\"Equb\\\"\\u003e\\n \\u003cdiv class=\\\"mathdisplay\\\" id=\\\"FileID_Equb\\\" name=\\\"EquationSource\\\"\\u003e\\u003cimg 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\\\"\\u003e\\u003c/div\\u003e\\n\\u003c/div\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCharacterizations\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eFourier transform infrared spectroscopy\\u0026ndash;attenuated total reflectance (FTIR\\u0026ndash;ATR) spectroscopy was carried out with a JASCO-4700 FTIR spectrometer, and the wavelength range was set at 500\\u0026ndash;4000 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e at room temperature. Scanning electron microscopy (SEM) and transmission electron spectroscopy (TEM) images were taken with a JEOL JSM-IT300S microscope (Tokyo, Japan), which works at a 5kV accelerating voltage and a JEM-100CX electron microscope, respectively. X-ray diffraction (XRD) analysis was performed using Philips PW1730 X-ray diffractometer with Cu K\\u0026alpha; radiation (\\u0026lambda;\\u0026thinsp;=\\u0026thinsp;0.15405 nm) which operates at 40 kV and 40 mA. Data were collected from 5 to 50\\u0026deg; 2\\u0026theta; at room temperature. The hydrogel (600\\u0026micro;L) was formed in a cubic container, and an avometer was used to measure the conductivity using the electric circuit.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eRheological Analysis\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eRheological properties of hydrogels were carried out on an Anton Paar MCR-302 rheometer at room temperature using a 25 mm diameter parallel plate. The frequency sweep test was analyzed over the range of 1\\u0026ndash;100 rad/s at a fixed strain rate of 1%, and in the strain sweep test, the strain ranged from 0 to 1000%.\\u003c/p\\u003e\\n\\u003cp\\u003eConsidering the strain sweep results, the self-healing properties were quantitatively evaluated by the damage-healing cycles which were continuous step switches from 1% (120 s for each interval) to 300% (60 s for each interval), with a constant frequency of 1 Hz at room temperature.\\u003c/p\\u003e\\n\\u003cp\\u003eThe viscosity of hydrogels was monitored at the different shear rates to characterize the injectability quantitatively. The viscosity-shear rate curves were obtained at room temperature, and the viscosity was recorded when the corresponding shear rate was in the range of 1-100 s\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eHemolysis rate\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eHemolysis testing of the prepared hydrogels was performed according to the previously reported procedure with slight modifications (Iqbal et al., \\u003cspan class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e). Fresh human blood from a healthy donor was collected in 5ml EDTA Vacutainer. The freeze-dried hydrogel samples of OP/NSC/GO0, OP/NSC/GO2, OP/NSC/GO4, and OP/NSC/GO6 were ground into powder. 25 mg of the powdered sample was weighed and poured into the test tube, then 10 mL of normal saline was slowly added. All tubes were incubated at 37 \\u003cspan style='color: rgb(0, 0, 0); font-family: \\\"Times New Roman\\\"; font-size: medium; font-style: normal; font-variant-ligatures: normal; font-variant-caps: normal; font-weight: 400; letter-spacing: normal; orphans: 2; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration-style: initial; text-decoration-color: initial; display: inline !important; float: none;'\\u003e\\u0026deg;\\u003cem\\u003eC\\u003c/em\\u003e\\u003c/span\\u003e\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e)\\u003c/span\\u003e\\u003c/span\\u003e for 30 min. Subsequently, 0.1 mL EDTA blood was added to the test tube with a micropipette. After shocking and mixing, well, it was kept at a constant temperature at 37\\u0026deg;C for 60 min. Then, the tubes were removed, and they were centrifuged at 1500 rpm for 10 min. The absorbance values of the supernatants at 545 nm were measured with an ultraviolet spectrophotometer using Lambda 25 UV/Vis spectrophotometer. The following formula calculated the hemolysis rate of hydrogel samples:\\u003c/p\\u003e\\n\\u003cdiv class=\\\"Equation\\\" id=\\\"Equc\\\"\\u003e\\n \\u003cdiv class=\\\"mathdisplay\\\" id=\\\"FileID_Equc\\\" name=\\\"EquationSource\\\"\\u003e\\u003cimg 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\\\"\\u003e\\u003c/div\\u003e\\n\\u003c/div\\u003e\\n\\u003cp\\u003eWhere A\\u003csub\\u003e1\\u003c/sub\\u003e, A\\u003csub\\u003e2\\u003c/sub\\u003e, A\\u003csub\\u003e3\\u003c/sub\\u003e are the absorbance of the hydrogel sample group, the positive control group (10 mL distilled water, 0.1 mL human EDTA blood, without hydrogel sample material), and the negative control group (10 mL normal saline, 0.1 mL human EDTA blood, without hydrogel sample material), respectively.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCytotoxicity assay\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eEvaluation of cytotoxicity of the hydrogels was conducted using MTT (3-[4, [5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay and in terms of ISO 10993-5:2009 (Biological evaluation of medical devices: Tests for in vitro cytotoxicity) using mouse fibroblast L-929 cells. Firstly, the prepared hydrogels were sterilized by UV irradiation for 2 h, then they were added to a 24-well culture plate. Also, L-929 cells with a density of 1.0 \\u0026times; 10\\u003csup\\u003e6\\u003c/sup\\u003e cells were seeded on each hydrogel to be assessed using the standard MTT test. After culturing for 24 h, MTT assays were conducted to test the cell growth. The wells without hydrogels were set as the controls.\\u003c/p\\u003e\"},{\"header\":\"Results And Discussion\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eCharacterization of GO\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe FTIR-ATR spectra of the graphite powder and the GO have been shown in Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e. As shown in Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ea, in the graphite powder spectrum, no significant peak was observed. While, the FTIR-ATR spectrum of GO showed characteristic bands at 3179 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e, 1726cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e, 1617 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e, 1232 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e, and 1041 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e which are attributed to the hydroxyl group (-OH), carboxyl group (C\\u0026thinsp;=\\u0026thinsp;O), aromatic ring (C\\u0026thinsp;=\\u0026thinsp;C), epoxy group (C-O-C) and alkoxy group (C-O), respectively. These hydrophilic oxygen-containing functional groups supply the GO sheets with high water dispersibility (Chen et al., \\u003cspan class=\\\"CitationRef\\\"\\u003e2013\\u003c/span\\u003e).\\u003c/p\\u003e\\n\\u003cp\\u003eAccording to Sandhya and et al., the characteristic peak of graphite was reported to be at 2\\u0026theta;\\u0026thinsp;=\\u0026thinsp;26.25\\u0026deg; (Sandhya et al., \\u003cspan class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e). As shown in Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eb, for GO, the 2\\u0026theta; peak shifted to around 10.93\\u0026deg;, which indicated that the graphite was fully oxidized into GO.\\u003c/p\\u003e\\n\\u003cp\\u003eThe morphological structure of GO was evaluated by TEM and SEM (Fig.\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e). The SEM images of GO layers have been indicated in Fig.\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ec, representing a porous shape. Figure\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ed showed the GO TEM image with the paper-like morphology that has wrinkles on its surface.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCharacterization of hydrogels\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cdiv class=\\\"Section2\\\" id=\\\"Sec4\\\"\\u003e\\n \\u003ch2\\u003eFTIR-ATR\\u003c/h2\\u003e\\n \\u003cp\\u003eEvidence for the modification of the chitosan (CS) and pectin (PE) was observed in the infrared spectrum (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e). Comparison of the CS infrared spectra with NSC spectra demonstrated the vanishing of the peak at 1590 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e (the primary amine bending vibration) in NSC. The emergence of a new absorption band at 1550 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e provides direct evidence of the chitosan modification. Moreover, the absorption band at 1401 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e could be attributed to the symmetric stretching of the COO-, and it confirms the formation of N-succinyl chitosan (Bashir et al., \\u003cspan class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e). Furthermore, FTIR-ATR results confirmed the formation of aldehyde groups in the oxidized pectins (OP). Pristine pectin showed the broad peak at 3347 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e that was assigned to the hydroxyl group\\u0026apos;s stretching band. The two bands located at 1719 and 1606 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e were assigned to the ester carbonyl stretching vibrations and asymmetric carboxylate stretching vibrations, respectively. The aldehyde peak of OP, which emerges at 1726 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e and 883 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e, belonged to the aldehyde symmetric vibrational band and the hemiacetal formation of free aldehyde groups, respectively.\\u003c/p\\u003e\\n \\u003cp\\u003eFT-IR-ATR was also used to verify the chemical structure of the hydrogels (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e). For the spectrum of the OP/NSC/GO-0 hydrogel, the hemiacetal structure\\u0026apos;s characteristic peaks at 871 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e, along with the appearance of stretching vibrations of the C\\u0026thinsp;=\\u0026thinsp;N bond at 1631 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e, demonstrated that the Schiff-base reaction occurred between the \\u0026ndash;CHO groups of OP and the NH\\u003csub\\u003e2\\u003c/sub\\u003e groups of NSC. Some peaks are increased for the other hydrogels, but there is no obvious change in the spectra\\u0026apos; peak position. This demonstrates that the hydrogen bonds are formed between the oxygen-containing GO\\u0026apos;s functional groups and the hydroxyl groups on the NSC and OP molecular chains.\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eGelation time\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003eGelation times of various amounts of GO are shown in Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e. In the Gels, the OP acts as a macromolecular chemical cross-linker making a spontaneous and fast chemical crosslinking reaction between the aldehyde of OP and the amino of NSC (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e.a). Moreover, with the content of GO increasing in the hydrogels, a shorter gelation time was observed. This might be due to the hydrophilic nature of the GO. The GO nanosheets contain numerous \\u0026ndash;COOH and \\u0026ndash;OH groups, forming hydrogen bonds with the NSC and OP macromolecules. Meanwhile, the higher content of GO will produce a higher amount of hydrogen bonds that facilitate network structure formation, accelerate the gelation process, and shorten the gelation time. So the formation of the cross-linked network structure is fast (\\u0026lt;\\u0026thinsp;30s).\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eSwelling and degradation\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003eFigure\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e illustrates the swelling and degradation behavior of the composite hydrogels in the PBS at 37\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(^\\\\circ C\\\\)\\u003c/span\\u003e\\u003c/span\\u003e. According to Fig.\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eb, OP/NSC/GO-0 showed the highest swelling ratio among the four GO-containing hydrogels. This is because of the lowest crosslinking density and mechanical properties of the OP/NSC/GO-0 hydrogels, that water molecules could enter the hydrogel network. Also, when the GO content increased from 0mg/ml to 6mg/ml, the swelling ratio of the hydrogels was reduced. This might have been due to the hydrogen bonds between GO nanosheets and the NSC and OP macromolecules, which formed denser crosslinking points, thus the capacity of hydrogels would be decreased to form the hydrogen bonds with water molecules.\\u003c/p\\u003e\\n \\u003cp\\u003eHaving biodegradability and preserving minimal resistance are the common requirements for porous hydrogels used in tissue engineering. The presence of GO in the composite gel scaffold could maintain their weight for up to 21days (Fig.\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ec). After incubation for 21 days, the degradation ratio of OP/NSC/GO-6 hydrogel (67.78\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2.65%) was lower than those of OP/NSC/GO-4, OP/NSC/GO-2, and OP/NSC/GO-0 (70.50\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.83, 73.52\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;5.10, and 94.90\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.98%, respectively). It was due to the addition of GO, which enhanced the hydrogel\\u0026apos;s crosslinking density; and therefore improved its stability. Such findings showed that during in vitro culture, the composite hydrogel\\u0026apos;s structure was stable.\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv class=\\\"Section2\\\" id=\\\"Sec5\\\"\\u003e\\n \\u003ch2\\u003eSEM\\u003c/h2\\u003e\\n \\u003cp\\u003eThe cross-sectional morphology of composite hydrogels with different GO content was observed by SEM (see Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003ea-d). As shown in Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003e, OP/NSC/GO owned the continuous three-dimensional porous structure. This would lead to the structural and compositional similarity of hydrogels with the natural extracellular matrix, as well as high oxygen permeability. There were no significant variations in the micro-morphology of the four hydrogels prepared with various GO loadings. The pores were morphologically homogeneous with smooth walls and there were dense voids. The network created by the Schiff base cross-linking between NSC and OP and the hydrogen bonds between the polymer chains and GO nanosheets caused the pore size of hydrogels to decrease slightly as the GO content increased (from 286.69 to 213.10, 184.91, and 127.573\\u0026micro;m, respectively). Therefore, being orderly and compact, the hydrogels with GO exhibited better mechanical properties than hydrogels without GO.\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eConductivity of the OP/NSC/GO hydrogels\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003eBesides self-healing and injectability, conductivity is also a desired property for the hydrogels used in tissue engineering, mainly when it is used for tissues under electrophysiological conditions (Zheng et al., \\u003cspan class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e). The tissue conductivity (ventricular muscle, nerve, lung, cardiac, and skeletal muscle) ranges orderly between 0.03 and 0.6 S/m. Thus, researchers must consider this issue when engineering various tissues in the body, so that their designed electroconductive nano-biomaterials scaffolds satisfy these electrical characteristics. Therefore, we then explored the conductivity of the OP/NSC/GO hydrogels. The conductivity of OP/NSC/GO hydrogels with different GO contents has been shown in Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003e. All hydrogels showed a conductivity of the order 0.0035\\u0026ndash;0.112 S/m. It is worth mentioning that the suitable conductivity for native cardiac tissue is in the range from 0.005 to 0.16 S/m (Mostafavi et al., \\u003cspan class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e), which is in the range of the OP/NSC/GO conductivity hydrogels that we prepared.\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eRheological property\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003eThe rheological analysis at both strain and frequency sweep modes (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003ea, b) was done to get the hydrogels\\u0026apos; mechanical property. Throughout the test, the storage modulus (G\\u0026apos;) was consistently higher than (G\\u0026quot;) in the frequency range (1-100 rad/s) at a fixed strain of 1%. Figure \\u003cspan class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003ea shows that the storage modulus G\\u0026prime; and loss modulus G\\u0026prime;\\u0026prime; of the hydrogels did not change significantly, suggesting that the hydrogel is stable and elastic. However, the elastic modulus does not change apparently as the strain increases, indicating the stability of the OP/NSC/GO imine bond and homogeneity of all these hydrogels (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003eb). The best mechanical properties were for samples with the largest G\\u0026prime; and G\\u0026prime;\\u0026prime; that contained 6 mg/ml GO. This may be due to the increased cross-linking density between NSC and OP, as well as the hydrogen bonding between GO nanosheets and polymer chains. As a result, the OP/NSC/GO-6 were selected for more investigations.\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eSelf-healing and injectable properties of OP/NSC/GO hydrogels\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003eTo evaluate the hydrogels\\u0026apos; self-healing ability, the hydrogels\\u0026apos; strain amplitude sweep test was first performed to determine the critical point of the hydrogel between liquid and solid-state (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003ea). The results showed that the intersection point between the storage modulus (G\\u0026apos;) and loss modulus (G\\u0026apos;\\u0026apos;) was at the strain of 83.52%. When the strain is larger than this value, G\\u0026apos; would be lower than G\\u0026apos;\\u0026apos;, indicating the collapse of the hydrogel, and change in its physical form from solid to fluid. Then, a series of rheological recovery tests were carried out with the G\\u0026apos; and G\\u0026apos;\\u0026apos; of the hydrogels versus time under higher strain to evaluate the self-healing ability of the hydrogel (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003eb). A large strain of 300% was applied to break the network structure. It decreased the G\\u0026apos; from ~\\u0026thinsp;759 Pa to ~\\u0026thinsp;63 Pa and induced gel-to-sol transition. When switched into the low strain, the G\\u0026apos; of the hydrogel returned quickly to the original value with the recovery of the hydrogel structure, thus indicating the rapid self-healing property of the OP/NSC/GO-6 hydrogel. According to the results, the broken structure recovered rapidly, and after periods of breaking and reforming, there was still the nearly normal hydrogel.\\u003c/p\\u003e\\n \\u003cp\\u003eA macroscopic self-healing test was conducted to further assess the self-healing behavior of the OP/NSC/GO-6 hydrogel. As shown in Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e10\\u003c/span\\u003e, the square shape hydrogel was cut into two halves, and then they were reconnected to allow them to be self-healed at 37℃. After 10 min of incubation at 37 ℃ without any external stimulus, the ruptured hydrogel could entirely integrate, and the blended integral hydrogels could be lifted with laboratory tweezers. It was held up under the force of gravity, indicating the self-healing performance of the OP/NSC/GO-6 hydrogel. Such self-healing ability attributes to the Schiff-based bonds and hydrogen interactions of the OP/NSC-GO-6 hydrogel.\\u003c/p\\u003e\\n \\u003cp\\u003eWe used both observable and qualitative approaches to confirm the injectability of the OP/NSC-GO-6 hydrogel. The viscosity of the OP/NSC-GO-6 hydrogel reduced significantly as the shear rate increased from 1 to 100 s-1, indicating the shear-thinning behavior (see Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e11\\u003c/span\\u003e). Furthermore, given the shear-thinning property of the OP/NSC-GO-6 hydrogel, it could also be rapidly injected via a syringe (3mL, G23) without clogging. This indicated that OP/NSC-GO-6 hydrogel has suitable injectability. The Schiff base linkages and hydrogen bonds were separated under pressure when the hydrogel was squeezed into the syringe, leading to the transfiguration of the hydrogel, so that it could flow like a liquid into the needle. The extruded hydrogel was combined to make a single component because of its self-healing capacity. This led to showing its ability to homogeneously encapsulate the cells or drugs and transplant them into the tissue in a less invasive manner.\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eHemolysis rate\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003eThe material\\u0026apos;s hemolysis rate is a crucial indicator of biological materials\\u0026apos; blood compatibility. The hemolysis phenomenon occurs when a foreign body is in contact with the blood which damages or ruptures the red blood cells. The lower the calculated hemolysis rate is, the better would be, the material\\u0026apos;s blood compatibility. The apparent hemolysis phenomenon was observed in the positive control tube, as shown in Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e12\\u003c/span\\u003e. The positive control group\\u0026apos;s blood cells have ruptured, and the solution was uniform and red. Furthermore, the blood cells were deposited at the tube in the negative control group, and the solution was transparent. The hydrogel samples\\u0026apos; hemolysis activity resembled somehow the negative control sample. That is, some RBCs were deposited in the tube and a large number of them were adsorbed in the hydrogel. The supernatant was mainly colorless and transparent. Biomaterials would be suitable for biomedical applications if the hemolysis rate is less than 5%, according to international guidelines. The hemolysis rate values obtained for all hydrogels range between 1.38% and 0.34% (as it could be seen in Table \\u003cspan class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e), indicating that all materials have excellent blood compatibility properties. \\u0026nbsp;\\u003c/p\\u003e\\n \\u003ctable border=\\\"1\\\" id=\\\"Tab1\\\"\\u003e\\n \\u003ccaption language=\\\"En\\\"\\u003e\\n \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 1\\u003c/div\\u003e\\n \\u003cdiv class=\\\"CaptionContent\\\"\\u003e\\n \\u003cp\\u003eIn vitro hemocompatibility analysis of OP/NSC/GO hydrogels\\u003c/p\\u003e\\n \\u003c/div\\u003e\\n \\u003c/caption\\u003e\\n \\u003cthead\\u003e\\n \\u003ctr\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eSample\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eHemolysis rate(%)\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/thead\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eOP/NSC/GO-0\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e1.38\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eOP/NSC/GO-2\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e0.92\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eOP/NSC/GO-4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e0.57\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eOP/NSC/GO-6\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e0.34\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n \\u003c/table\\u003e\\n \\u003cp\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cbr\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eCytotoxicity test using MTT assay\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003cp\\u003eTo evaluate the suitability of using OP/NSC/GO hydrogels as biomedical materials, an MTT assay was used to determine the toxicity of synthesized hydrogels. As shown in Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e13\\u003c/span\\u003e, during the whole incubation period, the cell viabilities of the fibroblast (L929) were higher than 80 %, suggesting that all hydrogels had no cytotoxicity to fibroblast cells (L929). Notably, the results also found that the cell viability was higher with the addition of GO. Furthermore, compared to OP/NSC/GO-2, OP/NSC/GO-4, OP/NSC/GO-6, the cell viabilities significantly decrease with the rise of GO content. This could be explained by the fact that as the GO content in the hydrogels increases, the GO content in the extracts increases, and the GO shows a strong cytotoxic effect at high concentrations.\\u003c/p\\u003e\\n\\u003c/div\\u003e\"},{\"header\":\"Conclusion\",\"content\":\"\\u003cp\\u003eThis study introduced a new dual-crosslinked hydrogel system with suitable injectability, excellent self-healing, and adequate conductivity. Through embedding the GO into the doubly Schiff-base crosslinking by amino and aldehyde groups of the NSC and OP, respectively and hydrogen bonding between the GO sheets and the polymer chains, the OP/NSC/GO composite hydrogels were successfully prepared. For the cmposite hydrogel's adjustable performance, it is critical to integrate the GO. Composite hydrogels had a faster gelation time, greater stability, and better mechanical properties than pure hydrogels. Overall, In our analysis, the composite hydrogel with a GOs content of 6 mg/mL displayed the best performance, with an adequate swelling ratio and stability. The hydrogels had excellent anti-hemolytic properties when they were examined in terms of hemolytic potential. Furthermore, the cytotoxicity testing revealed that the hydrogel had no biological toxicity when tested on mouse embryonic fibroblasts, and the cell viability of the various samples exceeded 80 %. These findings suggest that the constructed hydrogel could be a suitable choice for tissue engineering, especially in electroactive tissues.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003ch2\\u003eAcknowledgements\\u003c/h2\\u003e \\u003cp\\u003eThe authors would like to thank Dr. Mohammad Taghi Razavi Tousi for kind assistance on Hemolytic assays.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eAhadi F, Khorshidi S, Karkhaneh A (2019) A hydrogel/fiber scaffold based on silk fibroin/oxidized pectin with sustainable release of vancomycin hydrochloride. Eur Polym J 118:265\\u0026ndash;274\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBagher Z, Ehterami A, Safdel MH, Khastar H, Semiari H, Asefnejad A, Davachi SM, Mirzaii M, Salehi M (2020) Wound healing with alginate/chitosan hydrogel containing hesperidin in rat model. Drug Deliv Sci Technol 55:101379\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBashir S, Teo Y. Y, Naeem S, Ramesh S, Ramesh K (2017) pH responsive N-succinyl chitosan/Poly (acrylamide-co-acrylic acid) hydrogels and in vitro release of 5-fluorouracil. PLoS One 12:e0179250\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBi B, Ma M, Lv S, Zhuo R, Jiang X (2019) In-situ forming thermosensitive hydroxypropyl chitin-based hydrogel crosslinked by Diels-Alder reaction for three dimensional cell culture. Carbohydr Polym 212:368\\u0026ndash;377\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eChen J, Yao B, Li C, Shi G (2013) An improved Hummers method for eco-friendly synthesis of graphene oxide. Carbon 64:225\\u0026ndash;229\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eDe Lima EL, Vasconcelos NF, Da Silva Maciel J, Andrade FK, Vieira RS, Feitosa JP A (2020) Injectable hydrogel based on dialdehyde galactomannan and N-succinyl chitosan: a suitable platform for cell culture. J Mater Sci Mater Med. 31: 1\\u0026ndash;13\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eDeng Y, Hussain I, Kang M, Li K, Yao F, Liu S, Fu G (2018) Self-recoverable and mechanical-reinforced hydrogel based on hydrophobic interaction with self-healable and conductive properties. Chem Eng J 353:900\\u0026ndash;910\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eEivazzadeh-Keihan R, Radinekiyan F, Aliabadi HA, M, Sukhtezari S, Tahmasebi B, Maleki A, Madanchi H (2021) Chitosan hydrogel/silk fibroin/Mg(OH)2 nanobiocomposite as a novel scaffold with antimicrobial activity and improved mechanical properties. 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Theranostics 8:3317\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Double cross-linked network, Schiff- base reaction, Hydrogen bond, Self-healing hydrogel, Tissue engineering\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-922109/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-922109/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eBiopolymer-based hydrogels are commonly used in clinical applications. In the present study, N- succinyl chitosan (NSC), oxidized pectin (OP), and graphene oxide (GO) were used to develop a new dual-crosslinked hydrogel system. The dynamic OP/NSC/GO hydrogel showed quick gelation and great injectability due to the cooperation of hydrogen interaction between the GO nanosheets and the NSC and OP macromolecules and Schiff-based crosslinking by amino and aldehyde functional groups of polysaccharide derivatives. The performance of the above-mentioned hydrogel was improved when the GOs were embedded. When the GO content was 6 (mg/ml), the hydrogel showed the best overall performance, with a 10-minute healing time, a quick gelation time (~\\u0026thinsp;13s), acceptable swelling ability, suitable conductivity, great hemocompatibility, and strong biological compatibility. These results showed that the composite hydrogel could be used as a promising conductive injectable self-healing hydrogel for tissue engineering applications.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Dual-Crosslinked Oxidized Pectin/N-Succinyl Chitosan Hydrogel Containing Graphene Oxide Nanosheets for Tissue Engineering Application\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2021-10-01 15:08:10\",\"doi\":\"10.21203/rs.3.rs-922109/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"9c30c4c2-f9e7-4dea-9220-c375ac68dfaf\",\"owner\":[],\"postedDate\":\"October 1st, 2021\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[{\"id\":7581207,\"name\":\"Cellular Metabolism\"},{\"id\":7581208,\"name\":\"Cellular \\u0026 Molecular Neuroscience\"}],\"tags\":[],\"updatedAt\":\"2021-11-10T15:52:38+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2021-10-01 15:08:10\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-922109\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-922109\",\"identity\":\"rs-922109\",\"version\":[\"v1\"]},\"buildId\":\"cBFmMYwuxLRRLfASyISRj\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}