Bioinspired Laponite-Reinforced Gelatin/Alginate Nanocomposite Hydrogels with Tunable Properties for Enhanced Cartilage Tissue Engineering

Bioinspired Laponite-Reinforced Gelatin/Alginate Nanocomposite Hydrogels with Tunable Properties for Enhanced Cartilage Tissue Engineering | 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 Article Bioinspired Laponite-Reinforced Gelatin/Alginate Nanocomposite Hydrogels with Tunable Properties for Enhanced Cartilage Tissue Engineering Faezeh Shahedi Aliabad, Elnaz Tamjid, Mitra Tavakoli, Parvin Najafi, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8037436/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 Cartilage tissue engineering offers a promising strategy for the regeneration of damaged cartilage, a tissue with limited self-repair capacity. The development of bioengineered scaffolds that closely mimic the mechanical and biological properties of native cartilage is critical to restoring its function. In this study, we fabricated and characterized gelatin/alginate hybrid hydrogels incorporating varying concentrations of laponite nanoclay particles (0, 1, and 2 wt.%) for potential application in cartilage tissue engineering. The microstructural, mechanical, physicochemical, and biological properties of the hydrogel nanocomposites were systematically evaluated using FTIR, XRD, SEM, swelling and degradation analyses, compression testing, rheological measurements, MTT assay, and cell adhesion studies. The incorporation of laponite nanoclay significantly influenced the hydrogel properties, reducing pore size (from 46 ± 9 µm to 26 ± 7 µm), decreasing the swelling ratio (by ~70% and ~50%), and lowering the degradation rate (from 50% to 40% and 2.5% after 6 hours and 21 days, respectively). Additionally, mechanical strength more than doubled, and the hydrogels exhibited shear-thinning behavior. All formulations maintained a high hydration level (>90%), providing a favorable environment for cell viability and proliferation. These findings indicate that laponite nanoclay-reinforced gelatin/alginate hydrogels possess a biomimetic structure, tunable mechanics, controlled biodegradability, and excellent biocompatibility, making them promising candidates for cartilage tissue engineering applications. Biological sciences/Biotechnology Physical sciences/Engineering Physical sciences/Materials science Cartilage tissue engineering Hydrogel Characterization Shear-thinning Gelatin Alginate Laponite Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 1-Introduction Cartilage is an avascular tissue that has inferior intrinsic repair capability. Until now, different approaches including autologous chondrocyte implantation, mosaicplasty, osteochondral allograft, and microfracture, have been employed to repair damaged cartilage 1 . However, in many cases, these approaches cannot generate adequate tissue for damaged cartilage. Having emerged in early 1991, tissue engineering has been a promising applied method in cartilage hurts with a concentration on stem cells, scaffolds, and growth factors. Full reconstruction of damaged tissue requires scaffolds that mimic the properties of the given tissue and support the newly formed tissue until complete growth 2 . Since the emergence of tissue engineering, a wide range of biomaterials has been investigated for cartilage repair and regeneration. Hydrogels, nonetheless, have been receiving the highest interest, especially in being used as scaffolds in cartilage, as they have highly comparable features to extracellular matrix (ECM). Hydrogels, by their most common definition, are water-swollen and cross-linked networks confirmed by the reaction of monomers. Various natural and synthetic polymers have been exploited to prepare cartilage scaffolds. The most used natural biomaterials for this purpose are collagen 3 , gelatin 4 , 5 , alginate 6–89 , and chitosan 10 – 12 . Alginates, a natural biomaterial, are widely utilized copolymers in various biomedical applications, including drug delivery 13 , tissue engineering 14 , and wound healing 9 . Alginates are nontoxic at controlled concentrations, and they have high biocompatibility, relatively low price, and good gelation in the presence of cations such as Ca 2 + 15,16 . They are blocks of linear unbranched copolymers with different patterns of blocks of acids that are linked with covalent bonds 17 . These patterns are mainly MM, GG, GM, M, and G. When two G-blocks of alginate are positioned adjacently, they tend to form cross-links through interactions with multivalent cations such as Ca²⁺, Ba²⁺, Fe²⁺, Sr²⁺, and Al³⁺. 15 . The involvement of these cations in ionic binding zones between G-blocks leads to a gelation mechanism, resulting in the formation of a three-dimensional network commonly referred to as an "egg-box" structure. 1819 . However, a limitation of alginate is the lack of cell attachment motifs, leading to weak interactions between cells and material. Moreover, it degrades very slowly and in an uncontrolled manner 20 , 21 . Obtained from hydrolyzed collagens, gelatin is one of the most favorable biomaterials in tissue engineering. This natural polymer has excellent biocompatibility and contains Arginylglycylaspartic acid (RGD) peptide, promoting cell proliferation and migration 22 . However, it suffers from a high degradation rate and poor mechanical properties 202324 . Natural biomaterials-based hydrogels are usually more biocompatible, while synthetic ones possess better mechanical properties 25 . Some of the hydrogels, therefore, are produced by the combination of natural and synthetic biomaterials, to simultaneously provide biocompatibility, bio-functionality, and suitable mechanical properties and degradation rate. As another approach to enhance mechanical properties, the hybrid natural hydrogels can be reinforced by various types of nanomaterials, for instance, carbon nanotubes 26 , metal oxide nanoparticles 27 , and nanoclays 25 . Among them, laponite is a nano clay with the formula Na + 0.7 [(Mg 5.5 Li 0.3 ) Si 8 O 20 (OH) 4 ] −0.7 , is a synthetic nano silicate obtained from salts of lithium, sodium, magnesium, and sodium silicate. Laponite has a disk-like structure, with diameters around 25 nm and thicknesses of approximately 0.9 nm 20 17 , and has negative charges distributed on the surfaces of the platelet and positive charges on the surface of the edges. The electrostatic interaction between laponite nanoclays brings about the formation of specific microstructures. The mechanism of formation of these is still a topic of discussion. Dispersion microstructure is highly related to the concentration of the laponite. At a concentration lower than 2 wt.% of laponite, a “house of card” microstructure is proposed, while at a concentration above this amount, two suggestions are reported: (i) a Wigner repulsive glass or (ii) a house of card microstructure 28 . Laponite can be an ideal additive to form hydrogel nanocomposites with greater features, particularly mechanical and rheological properties, and enhance cell-hydrogel interaction for many applications 2930, 31 . Taking laponite nanoclays' biological activities into consideration, non-toxicity 32 , improving cell proliferation 33 and cell viability, and enhancement of cell adhesion 34 have been reported in the literature. For example, Rajabi et al. achieved the highest toughness of gelatin-based nanocomposite hydrogels at 1 wt.% laponite concentration. The addition of laponite caused an enhancement in both the elongation and tensile strength of the samples by inducing chemical cross-linking 23 . Davila et al., following rheological studies on alginate and laponite/alginate solutions, reported a pronounced shear-thinning behavior upon the addition of laponite. It has been reported that a "house of cards" structure forms due to electrostatic interactions between charged laponite platelets at very low shear rates, leading to an increase in viscosity. 17 . In this study, for the first time, three hydrogel samples based on gelatin and alginate with 0,1 and 2 wt.% laponite were comprehensively characterized and the effect of laponite content on the microstructure, mechanical and rheological properties was studied. 2-Experimental procedure 2.1 Materials Sodium alginate extracted from brown algae with a viscosity lower than 2000 cP was purchased from Sigma Aldrich Inc., USA. Gelatin (from Bovine gelatin with 260–280 g bloom, type B,) was purchased from Gelatin Halal Inc., Iran. The crosslinker, calcium chloride anhydrous obtained from Biobasic, Canada. Extra pure XLG laponite was obtained from BYK Company, Germany. 2.2 Hydrogel preparation Three hydrogel samples with different contents of laponite nanoparticles were prepared to investigate the effect of the laponite nanoparticles on Gelatin-Alginate composite hydrogel. Initially, gelatin, alginate, and laponite powders were accurately mixed with given proportions, 4:2:0,1,2 respectively. After that, the mixtures were gradually added to a certain amount of deionized water at 55°C, using a magnetic stirrer. Then, the beaker was sealed using a cap to prevent evaporation and maintain the exact proportion of precursors. The solution was stirred for 3 h and complete homogeneity was achieved. In the second stage, for gelation of the solutions, samples were put in a 0.1 M CaCl 2 in an oven at 37°C overnight. Finally, the three hydrogel samples were washed with deionized water. 2.3 Characterization of samples 2-3-1- Morphological characterization The morphological characterization of the prepared hydrogels was carried out using scanning electron microscopy (SEM), FEI, Quantum 2000, USA. For SEM analysis, the samples first were put in a freezer at -80°C. After being completely frozen, the samples were lyophilized for 24 h, to dry out with the least influence on the microstructures. The samples were then coated with a gold nanolayer to enhance contrast. After that, the images were analyzed using Image J software. 2.3.2 Structural analyses The structural characterizations of prepared hydrogels were performed using the Fourier Transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) analyses. The chemical composition of precursors (gelatin, alginate, and laponite) and prepared hydrogel nanocomposites were examined by the FTIR. Before the FTIR analysis, the hydrogel samples were lyophilized. Tests were conducted using an FTIR spectrometer Bruker, equinox55 in the attuned total reflectance mode. The transmittance evaluation wavelength was in the range of 4000 − 400 cm − 1 . The crystal structures and phase analyses of the initial and lyophilized hydrogel samples were studied using XRD. All samples for this analysis were in powder form. The diffraction angle for scanning the samples was set within the range of 2θ = 5–80° with the scanning step size and speed of 0.02 and 0.4 s, respectively. 2-3-3- Physical properties of samples The physical properties of samples were investigated by studying their swelling and degradation behavior. To study the swelling behavior of the hydrogel nanocomposites, the cylindrical samples with height and diameter of 1.4 cm and 1 cm, respectively, were fabricated. For data reliability, three identical samples of each hydrogel were tested. These hydrogel samples were immersed into a certain volume of Phosphate-buffered saline (PBS) for different times (20, 40, 60, 120, 240, and, 360 min) at 37°C. After immersion, the samples were removed from the PBS solution. Next, the surface water was removed and the samples were weighed (wet weight (w w )). Then, the samples were frozen at -80 and lyophilized and weighted, (dry weight (w d )). Finally, swelling present (SW%) and hydration degree (HD%) was calculated from Equations 1 and 2 respectively. To investigate the degradation behavior of hydrogels, first, the samples were lyophilized and weighed. This was considered as the initial weight, w 1 . Then, three identical samples of each hydrogel were incubated in PBS buffer for certain periods,1, 3, 7, 14, and 21 days. After incubation for specific periods, each sample was freeze-dried and weighed, W 2 . The degradation ratio (Deg%) was achieved using Eq. 3. 2-3-4- Mechanical and rheological properties The compression and shear rheology tests were conducted to evaluate the mechanical properties of the prepared hydrogel nanocomposites. To examine the compressive behavior of the hydrogels, three samples of each hydrogel, measuring 14 mm in height and 9 mm in diameter, were prepared. Before the tests, samples were immersed in PBS solution for 30 min to be fully hydrated. The compressive properties of the hydrogels were examined using a Hounsfield-H10Ks machine equipped with a 100 N load cell. The compression tests were carried out under the strain rate of 6 \(\:\times\:\) 10 −2 s − 1 and up to the strain of 0.5. Moreover, after 10 days of preparation of samples, the rheological measurements were conducted on the cross-linked hydrogel samples with a diameter of 14 mm and a thickness of 4 mm. The frequency sweep test and amplitude sweep test were carried out using a modular compact rheometer (Anton par, MCR 502) fitted with a 25 nm parallel-plate geometry and gap size of 1mm. Amplitude sweep tests were made at a constant angular frequency of 10 rad s − 1 and shear strains ranging from 0.1 to 100%. The frequency sweep test was carried out at a constant shear strain of 0.1% with an angular frequency range of 0.1 to 628 rad s − 1 . The viscosity of non-cross-linked solutions was evaluated using (Anton par, MCR 502). Before the tests, samples were kept in the fridge, 4°C, for 10 days. Tests were carried out at 25°C and viscosity variations against shear rate in the range of 0.01–1000 s − 1 were measured. The curves were fitted with the Carreau model using Eq. 4 using OriginPro software and all curves represented curve fitting with plausible Adjusted R-squared. 2-3-5- Biological properties of samples The viability of cultured cells and cell adhesion assessments were performed to evaluate the biological properties of prepared hydrogel nanocomposites. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was performed based on an international standard (ISO)10993-5. For this purpose, based on ISO 10993-12, extracts of 1-day, 3-day, and 7-days were obtained from incubated hydrogels in complete medium culture. Mouse fibroblast cells (L929, Iranian Biological Resources Center, Iran) at a density of 5000 cells were seeded in a 96-well plate along with 200 µL of Dalbecco’s Modified Eagle Medium (DMEM) culture medium incubated for 24h (5% CO 2 , 37 ֯ C). The culture medium was supplemented with 10% of inactivated fetal bovine serum (FBS), and 1% of penicillin-streptomycin. After 24 h of incubation, the old medium was discarded and 200 ul of prepared extract was added to each well and the cells were incubated for another 24 h. Then, the extraction medium was removed, wells were washed with sterilized PBS solution and 200 ul of MTT solution (5 mg/ml in PBS) was added to each well, and the plate was wrapped in aluminum foil and then incubated for 4 h at 37 ◦ c. Formazan was dissolved by adding 200 µL of DMSO to each well. Finally, spectrophotometric quantifications were applied at 570 nm by an ELIZA microplate reader, BioTek, ELX800, USA. Cell adhesion of the hydrogels was evaluated by SEM observations. Preparing the sample is carried out in three main stages cell culture, fixation of the cells, and imaging. Human bone marrow mesenchymal stem cells (hBMSCs, Royan Institute, Iran) were seeded on a thin layer of the cross-linked hydrogels spin-coated on the microscope slides. Cells were cultivated in Minimum Essential Medium α (alpha-MEM) culture medium, supplemented with 16% FBS, 1% L-glutamine, and 1% pen-strep. About 3500 cells were seeded on each sample, located in 6-well plates, and incubated in a Co 2 incubator. After 15 hours of culture, samples were immersed in glutaraldehyde solution for 1 hour to be fixed. Then samples were washed with PBS and dehydrated by dipping them in a graded ethanol bath. Finally, after being sputter-coated with a nanolayer of gold, samples were imaged by SEM. 2-4- Statistical analysis OriginPro 2016 (Origin Lab Corporation, USA) was utilized for statistical evaluation. All data were analyzed by an analysis of variance (ANOVA) and demonstrated as the mean ± standard deviation (SD). The level of significance of all data was considered as *p ≤ 0.05. 3- Results and discussion 3-1- Morphology of hydrogel nanocomposite SEM micrographs of three groups of hydrogel samples with different contents of laponite are shown in Fig. 1 . Obviously, all hydrogels are highly porous with interconnected pores. Samples with 1 wt.% and 2 wt.% of laponite (named Lap 1% and Lap 2%) demonstrate more pores in smaller size ranges, meaning adjacency of macro (> 50) and micropores in their microstructure. The average size of the pores (Fig. 1 .d) and percentage of porosity (Fig. 1 .e) in the hydrogels are as follows: Lap 0%: 45.7 ± 9.4 and 51.4 ± 0.6%, Lap 1%: 41.8 ± 7.8 and 61.6 ± 3.3%, and Lap 2%:25.7 ± 6.6 and 60.4 ± 1%. In the hydrogel without laponite (named as Lap 0%), however, macro pores dominantly exist in the microstructure and there is an insignificant amount of very fine pores, 2–4 µm, in the walls. Furthermore, as the laponite content increases, the distribution of pore sizes becomes closer to the normal distribution. It seems that additional laponite to the structure results in the formation of new pores and thicker pore walls through electrostatic interactions and hydrogen binding between laponite and the matrix 23 . When laponite is incorporated into the gelatin/alginate hydrogel matrix, the laponite hydrogel creates a more complex and interconnected network structure. This resulted in a smaller average pore size for the samples containing laponite. In cartilage tissue engineering, the reported scaffold pore size ranges from 10–500 µm. 35 By adding 2 wt.% of Lap to the hydrogel, the pore's size decreases by half, and the percentage of porosity increases significantly (about 10%). The porosity of the hydrogel plays a significant role in resembling the structure of the native cartilage ECM. It helps with water uptake, oxygen and nutrient exchange, cell adhesion, and migration in the hydrogel. For in vitro cell biology tests, a porosity range of 48–90% is reported, which provides a favorable environment for cells. 36 , 37 In a study by Cidonio, Gianluca, et al. it was found that LAP-gellan gum (GG) constructs exhibited more porous networks 38 . The study results also show that incorporating Lap increases the hydrogel’s porosity, which aligns with these findings. 3-2- Chemical analyses of hydrogel nanocomposite The chemical functional groups of sodium alginate, gelatin, laponite, Lap 0%, Lap 2% samples, and the crosslinking within the hydrogel were analyzed using FTIR. As illustrated in Fig. 2 , the peaks observed for sodium alginate within the range of 3200–3600 cm⁻¹ correspond to O-H stretching 39 . Additionally, absorptions in the range of 1410–1600 cm⁻¹ are attributed to C = O bonds 40 . The peaks at 1030 cm − 1 and 1101 cm − 1 are attributed to the C-C and COC groups, respectively. In the Lap 0% spectrum, sodium alginate peaks have been shifted from 1600 cm − 1 to 1634 cm − 1 . This can be due to ionic cross-linking of the structure by CaCl 2 and the formation of an egg-box structure 41 . In the gelatin spectrum, the appeared peaks at 3284 cm − 1 and 3066 cm − 1 are ascribed to N-H stretching vibration of primary amine groups of gelatin 42 and 1523 cm − 1 , 1241 cm − 1 are related to N-H of amid II and III bonds respectively 4344 . The peaks at 2940 cm − 1 and 1631 are attributed to the alkyl group and C = O group of amid bond, respectively 44 . For laponite, H-O-H bond absorption was revealed at 1633 cm − 1 . Moreover, the Si-O and Si-O-Si peaks were observed at 962 cm − 1 45, 46 . Clearly, in the Lap 0% spectrum, there are no signs of laponite peaks, However, in samples with 2 wt.% laponite (Lap 2%), the related peaks to laponite can be traced, due to laponite interactions with the matrix, Si-O and Si-O-Si bonds wavenumber shifted from 962 cm − 1 to 996 cm − 1 and the Mg-O wave number shifted from 650 cm − 1 to 642 cm − 1 . After the incorporation of Lap, the characteristic peaks of gelatin and alginate still existed which shows the successful loading of laponite nanoparticles inside the hydrogel. Compared to the gelatin, Lap 0% and Lap 2% exhibit absorption peaks around peak shifts from 1445 to 1438 and from 1526 to 1543, which is presumably due to interactions and crosslinking between the amine groups of gelatin and the carboxyl group in alginate. Figure 3 depicts the XRD patterns of the alginate, laponite, gelatin, Lap 0%, and Lap 2% samples. As can be observed, the characteristic peaks at 6.5º, 19.97º, 35.07º, and 60.9º of the laponite can be detected in the XRD pattern of the Lap 2%, demonstrating the presence of laponite nanoparticles in this sample. Gelatin and alginate are amorphous materials and so, there are no sharp peaks in their XRD patterns 47 , 48 . However, a sharp peak at 32.16º was observed in the XRD patterns of alginate, Lap 0%, and Lap 2% which can be related to NaCl or CaCo 3 impurities in the alginate 15 , 49 . Notably, the peaks of 19.97 and 35.07 shifted to lower angle peaks of 19.93 and 34.05 respectively. This could be owing to the intercalation of polymer chains into laponite platelets and increasing the distance between platelets 21 . Polymer compounds can be used to cover the surface charge of platelets, which reduces the electrostatic repulsion between them. The possible mechanisms for physical cross-linking in these hydrogels are supported by FTIR and XRD studies, as well as previous research. During hydrogel crosslinking, Lap nanoparticles interact with polymer chains through hydroxyl groups that can form hydrogen bonds with functional groups in gelatin and alginate 42 , 46 . When laponite is mixed with water, it releases sodium ions which create a negative charge on the surface of the platelets. However, the edges of the platelets have a positive charge. This property allows laponite to be easily dispersed in water and establish an electrostatic interaction with gelatin and alginate polymer 42 . 3-3- Physical properties Assessing the swelling ratio of hydrogels is crucial, as swelling or water absorption reflects the hydrogel's capacity to transmit and absorb physiological fluids 21 . This characteristic, which determines the hydrogel's ability to transport nutrients and remove waste, is essential for maintaining cell viability within the hydrogel structure due to its impact on hydration and nutrient exchange 22 . However, swelling should be up to a certain amount, otherwise, it causes rapid degradation of the scaffold structure. As mentioned earlier, to investigate the swelling behavior of the hydrogels, they were immersed in PBS for certain durations, and their weight losses were measured. Figure 4 represents the swelling percentage of the samples in different durations. The highest swelling percentage was achieved for the Lap 0% sample. It can be observed that the addition of laponite to the hydrogels significantly reduced the swelling percentage. The involvement of hydrogen and electrostatic interactions between laponite and matrix provides a network with a higher density of cross-linking which has been explained in the results of FTIR analysis, leading to the creation of finer pores with a lower ability to absorb the solution. Also, the results are in good agreement with previous studies 50 . For instance, Akhtar et al. reported that adding 0.3% w/v Cu-Ag doped mesoporous bioactive glass nanoparticles to a hydrogel prepared from oxidized alginate, gelatin, and silk fibroin reduced the hydrogel's water absorption capacity by decreasing the size of its pores 51 . SEM imaging results also showed a significant reduction in pore size within the hydrogels upon the addition of Lap nanoparticles. The hydration degree determines the appropriateness of hydrogels for the proliferation and growth of the cells 22 . It has been reported that hydrogels with hydration degrees higher than 90% are suitable for cell proliferation and growth 22 . In this study, after immersion of three numbers of each sample in PBS for 6 h, the average hydration degree was calculated. Although the hydration degrees decreased with increasing laponite concentration, they were more than 90% for all three hydrogels, Fig. 5 . It has been reported that a concentration of 3% laponite in gelatin/alginate hydrogel makes the hydrogel unsuitable for cell proliferation and growth 22 . For ECM formation and cell proliferation, the used biomaterial scaffolds should be biodegradable. However, the degradation rate of scaffolds should be proportionate to the rate of regeneration of cells. To evaluate the degradation behavior of the hydrogels, their weight loss was measured after immersed in PBS and the degradation rate was calculated for the given durations (Fig. 6 ). As can be observed, for Lap 0% and Lap 1%, the degradation rate is very high at the initial time of immersion (1 day) in PBS solution while in the case of the Lap 2% sample is much lower. Following the initial time of immersion period, the degradation rate of samples generally slowed down. However, the addition of the degradation rate of the samples decreased as a function of the laponite contents. The severely high degradation rate on day 1 in the sample without laponite is due to weak interaction between gelatin and alginate and consequently release of gelatin in the PBS as it is water soluble 20 . Also, Bider et al demonstrated that gelatin could be released from alginate dialdehyde-gelatin hydrogel 52 . The addition of laponite, however, reduced the degradation rate of the samples because of the combination effect of strengthening interactions between components, and reduction of water penetration in the structure, resulting from smaller pore sizes 23 , 24 , 53 . It has been reported that hydrogels with lower porosity or larger pore sizes tend to degrade faster than those with higher porosity or smaller pore sizes. The structure of the hydrogel, specifically the mesh size of its cross-linked network, also affects the degradation profile. Highly cross-linked hydrogels with smaller mesh sizes demonstrate a longer degradation period. Zhu et al., reported that the degradation rate of hydrogel based on alginate dialdehyde (oxidized alginate) and gelatin after 24 hours was around 2.5%, consistent with the current study's results 54 . In this study, the addition of nanoparticles has increased the amount of cross-linking, leading to long-term stability and decreased degradation rate of the construct. 3-4- Mechanical properties Compressive stress-strain curves of hydrogel nanocomposites containing various contents of laponite are presented in Fig. 7 a. All curves experienced the same trend in two main regions. The first region, in which the stress slope was low, was due to the initial deformation of samples. The second region started with an abrupt increase in the slope. This was a result of the resistance of the network against external stresses 55 . The strength of samples at 50% of strain has been presented in Fig. 7 c. Results show the highest strength value has been achieved for the Lap 1% sample. The contribution of 1 wt.% of the laponite leads to a significant increase in the strength from almost 10.61 ± 1.54 kPa for sample Lap 0% to 26.81 ± 1.92 kPa. This enhancement in the compressive strength could be due to additional ionic, van der Waals, and hydrogen bonds created by the addition of the laponite nanosheets leading to increasing the density of cross-linking of the hydrogel networks 23 . This pressure modulus is comparable to values reported in other studies; for instance, a gelatin and silk-based hydrogel developed for cartilage tissue engineering demonstrated a maximum modulus of 30 kPa 56 . By further addition of the laponite content to 2 wt.%, the compressive strength dropped to 17.61 ± 0.68 kPa. This reduction in compressive strength is due to agglomeration of the laponites at higher concentrations that voids some areas of the microstructure of laponite and causes inhomogeneity 14 . The maximum compressive modulus of hydrogels, which are used for cartilage tissue engineering, is usually lower than that of native articular cartilage. However, this value is still suitable for supporting cells to secrete cartilage tissue ECM, as it falls within the range for soft tissue. The mechanical properties of the hydrogel are also influenced by architectural parameters, such as porosity, pore size, and pore shape 56 . The viscoelastic behavior of the samples was analyzed by oscillatory shear tests. In the diagram of the strain sweep test shown in Fig. 8 a, all the hydrogel samples represent viscoelastic behavior, since there are both storage modulus (Gʹ) and loss modulus (Gʺ) in their diagrams. Samples with a storage modulus higher than the loss modulus tend to have solid-like behavior 40 . Herein, higher storage modules of the samples indicate their solid-like behavior. Based on the strain sweep test results, all the samples demonstrated linear viscoelastic (LVE) behavior in the strain range of 10 − 2 to 1%. Therefore, the frequency sweep test was carried out at a constant strain of 0.1%. Storage modules for all samples are higher than loss modules, indicating their solid-like behavior (Fig. 8 b). In the Lap 1% and Lap 2% samples, cross-linking is intensified by physical gelation between polymer chains and laponite sheets. 24 It is shown that the Laponite operates as a physical crosslinking agent through reversible non-covalent interactions between nanoplatelets and the biopolymer matrix, thus improving the storage modulus of the bioink from 45 to 277 Pa at 1wt.% concentration. Amplitude sweep test can be considered to assess of mechanical strength of cross-linked hydrogels 45 . The samples with higher storage modules and loss modules show better mechanical stability. Based on this, as can be seen in the diagram samples containing laponite demonstrated better mechanical stability. This is improved more by increasing the laponite concentration to 2 wt.%. The injectability and printability of hydrogels are highly dependent on the shear-thinning viscosity behavior 41 . Although alginate and gelatin, solely, have Newton fluid behavior, their compound solution demonstrates shear-thinning behavior because of physical interaction between their polymer chains 40 . This behavior is also observed when the laponite nanoparticles are added to their solution and form the house of card structure 2144, 57 . Represented in Fig. 9 . is the viscosity behavior of the samples. As observed in Fig. 9 a. with increasing shear rate viscosity of all samples significantly decreases, meaning they show shear-thinning behavior. Curve fitting results also demonstrate the shear-thinning behavior of the samples as well-fitted curves with the Carreau equation with a parameter of 0 < n < 1. The addition of laponite to the structure increases the viscosity of the samples. The viscosity for Lap 1% is noticeably higher than others. This could be a result of keeping the samples at 4°C which leads to a conversion in the conformation of the gelatin chains from “random coil” to “triple helix”, making the structure more complex and more viscous 58 . It seems that due to the contribution of gelatin in physical interactions with the complex structure components, all helixes could not convert to random coils after reaching room temperature. As a result, the sample Lap 0% represented a higher viscosity compared to previous similar works 22 . Nevertheless, increasing laponite content to 2 wt.% decreased viscosity values even lower than that observed for the sample without laponite. This could be owing to the agglomeration of the laponite particles in some parts of the structure 49 which might increase the possibility of transition of the triple helix to the random coil in the other parts. Liu et.al investigated the viscosity behavior of the hydrogels with a combination of gelatin 5 wt.%, alginate 1 wt.%, and laponite (0–3 wt.%). Our results represented higher viscosity with the addition of lower amounts of laponite 22 which could be related to the effect of 4°C. 3-5- Biological properties The MTT assay on L929 fibroblast cells was conducted to assess the cell viability behavior of the hydrogels. In this study, hydrogel extracts were used as substrates in an indirect method to evaluate the effect of the prepared hydrogels on cell viability. In this study, the effect of prepared hydrogels on cell viability, evaluated indirectly utilizing MTT assay. As shown in Fig. 9 , all samples noticeably improved the viability of the cells. Results are in a good correlation with the degradation test results. On day 1, the sample Lap 2% showed lower cell viability than the samples Lap 0% and Lap 1%. This is caused by higher amounts of gelatin released, by degradation, in the latter samples. It is widely reported that gelatin improves the proliferation and viability of cells. Additionally, gelatin contains intrinsic cell adhesive peptide sequences including RGD (arginine-glycine-aspartic) which might have contributed to cell attachment and proliferation. Additionally, increased DNA content in cells provided evidence of enhanced cell viability 56 . In day 3 samples, a significant enhancement in viability was observed. This phenomenon is due to the release of a greater amount of gelatin compared to that observed on day 1. However, in day 7 samples, the viability of cells decreased. This might be attributable to the gradual degradation of gelatin by proteases in the FBS as a part of the complete culture medium during the extraction stage. Based on previous works, gelatin, alginate, and laponite in the concentration range used in this study not only are nontoxic and biocompatible but also increase cell viability 22 , 24 . For example, Ghadiri et al. reported that the cells in 1-day extracts were 4 times as viable as those in control samples 21 . Previous studies showed that Laponite-based nanocomposites promote cell proliferation, differentiation, and attachment. Therefore, the higher cell viability observed in the extracts of hydrogels could be due to the release of silica from the Lap into the extracts which improves cell proliferation. Silica has been reported to upregulate the expression of genes involved in cell growth and differentiation 21 . A critically important feature of scaffolds in tissue engineering is supporting cell adhesion, and cell spreading, playing a vital role in the enhancement of cell proliferation and formation of the ECM 50 . For assessment of the cell adhesion behavior of the hydrogels, hBMSC cells were cultured on a thin layer of the hydrogels. The cells, after 15 h, were fixed and dehydrated using glutaraldehyde and ethanol to be prepared for imaging. Figure 11 shows the morphology of the attached cell on the control sample and the three hydrogels. As can be seen, hBMSCs have well attached to the surface of all samples and produced filopodia. It has been reported that, due to the lack of necessary adhesion motifs in the alginate structure, cells tend to adopt a completely rounded shape and aggregate on its surface 50 . In this research, effective cell adhesion to the surfaces is primarily attributed to the abundance of cell-adhesive motifs within the gelatin structure 20 , 22 . Laponite can also improve cell adhesion because some adhesion proteins are inclined to be absorbed by silicate nanoparticles and silicate nanoparticles could provide cell absorption focal points in the structure 21 . In a study by Kafili et al., it was demonstrated that Laponite-containing hydrogels release magnesium (Mg), silicon (Si), and lithium (Li) ions, as confirmed. This release led to improved bioactivity, promoted cell proliferation, and created a more favorable microenvironment for the adhesion of fibroblast cells. 59 4- Conclusion In this study, the gelatin/ alginate hydrogels with different laponite concentrations (0,1 and 2 wt.%) were successfully synthesized for cartilage applications. The effects of laponite concentrations on microstructure evolutions and physiochemical, biological, and mechanical properties of the hydrogels were investigated. The following items are the main findings of this research: The incorporation of laponite to the gelatin/ alginate hydrogel increased pore size variability, enhancing the material's suitability for tissue regeneration applications. The increased cross-linking density of the hydrogels along with smaller pore size by increasing laponite concentrations also decreased the swelling ratio and degradation rate of the samples. Mechanical testing indicated an optimal laponite concentration for significantly enhancing Young's modulus and strength at 50% deformation, with the ideal concentration of laponite nanoparticles determined to be 1 wt.%. It was determined that cellular viability and adherence were suitably in line with expectations. Overall, it appears that gelatin/alginate/ laponite hydrogels can be suitable for regenerating cartilage tissue. Declarations Data availability Available by request from the authors. Declaration of competing interest Authors have no conflicts to declare. Funding Declaration This study was not supported by any sponsor or funder. CRediT authorship contribution statement Faezeh Shahedi Aliabad: Data curation, Formal analysis, Investigation, Project administration, Visualization, Writing- original draft, Writing-review & editing. Elnaz Tamjid: Conceptualization, Data curation, Formal analysis, Methodology, Resources, Supervision, Validation, Writing-review & editing. Mitra Tavakoli: Formal analysis, Methodology, Resources, Validation, Writing-review & editing. Parvin Najafi: Investigation, Project administration, Visualization, Writing-review & editing. Hossein Alipour: Investigation, Project administration, Visualization, Writing-review & editing. Acknowledgements The authors would like to acknowledge the Department of Research Affairs of Tarbiat Modares University References Li J, Chen G, Xu X, et al. Advances of injectable hydrogel-based scaffolds for cartilage regeneration. 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09:58:45","extension":"jpeg","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":565170,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8037436/v1/60cf3b4d58ff7d52bad93956.jpeg"},{"id":96981145,"identity":"a7a5ccbe-7df8-4b4c-ae2e-2313a1089475","added_by":"auto","created_at":"2025-11-28 09:14:44","extension":"jpeg","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":160758,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8037436/v1/0b636c1807dc0015935b8778.jpeg"},{"id":96981149,"identity":"5a2dd211-adf3-4802-b12c-2200c925d63e","added_by":"auto","created_at":"2025-11-28 09:14:45","extension":"jpeg","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":418636,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8037436/v1/7ba90ae30db1c5d55e23c1a5.jpeg"},{"id":96981131,"identity":"1ff13edc-90d1-4ccb-ad06-d5a90b2f65a6","added_by":"auto","created_at":"2025-11-28 09:14:44","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":194693,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8037436/v1/fbf3506e1c19c1083cd61c6f.png"},{"id":96981140,"identity":"7edf6193-e7cb-4a33-90dc-bb4216aa7858","added_by":"auto","created_at":"2025-11-28 09:14:44","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":7319,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-8037436/v1/96c88199bc19aa6b5d7a9fbc.png"},{"id":96981137,"identity":"1d835033-d305-4d83-a5ce-7e3c3ea03178","added_by":"auto","created_at":"2025-11-28 09:14:44","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":31322,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-8037436/v1/7e8ba51b8ba6f937d2b59633.png"},{"id":96981144,"identity":"0e972d70-4422-4820-9035-1f215bc7934e","added_by":"auto","created_at":"2025-11-28 09:14:44","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":27245,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8037436/v1/bcd5928c5dff3092ffbe01c2.png"},{"id":97138577,"identity":"5cf54759-010c-44fe-a65c-5eb0e6b5b4a0","added_by":"auto","created_at":"2025-12-01 09:59:06","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":12065,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8037436/v1/d2c08aa3667b83f88c5c6824.png"},{"id":96981138,"identity":"4048cb1f-9bad-425f-b118-8b3c0ef6ecc1","added_by":"auto","created_at":"2025-11-28 09:14:44","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":17765,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8037436/v1/2caa11c70d380526b0ea8fca.png"},{"id":97138215,"identity":"2c3f238f-260b-4e88-ac05-dfe28f514e8c","added_by":"auto","created_at":"2025-12-01 09:58:38","extension":"png","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6090,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8037436/v1/987d16921da7b29ce1a3beae.png"},{"id":97139346,"identity":"8e951528-d76c-496c-a53f-7ba965216559","added_by":"auto","created_at":"2025-12-01 10:00:06","extension":"png","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":16876,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8037436/v1/e74a1dd1bad167bef3cf6e3b.png"},{"id":96981151,"identity":"dce6914c-dcdd-48c8-a6d6-715cc6bbedeb","added_by":"auto","created_at":"2025-11-28 09:14:45","extension":"png","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":40266,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8037436/v1/1aed375a490caea0dbc0eeca.png"},{"id":97137707,"identity":"a50ee85f-1a6d-402f-8104-fc90146a5c40","added_by":"auto","created_at":"2025-12-01 09:58:05","extension":"png","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":19989,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8037436/v1/c71e9fa14e53dd9b4dac6005.png"},{"id":96981139,"identity":"06e8b029-2321-4004-ba29-d789ee41623b","added_by":"auto","created_at":"2025-11-28 09:14:44","extension":"png","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":39654,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8037436/v1/f04c00293f550ca06587da88.png"},{"id":97137169,"identity":"1717f612-1edc-4f35-826f-d2eaf1c95eb7","added_by":"auto","created_at":"2025-12-01 09:57:24","extension":"xml","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":140926,"visible":true,"origin":"","legend":"","description":"","filename":"9552fee774724117a99ab9129688621a1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8037436/v1/b2910aac86b63f97fa61a0c0.xml"},{"id":96981142,"identity":"f38ceeef-075d-4199-86d0-c6572afc984d","added_by":"auto","created_at":"2025-11-28 09:14:44","extension":"html","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":153988,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8037436/v1/09ffb89ef72eb792dc2909ea.html"},{"id":96981115,"identity":"0be75c45-bc5f-4a46-ae4f-6966941eb8e5","added_by":"auto","created_at":"2025-11-28 09:14:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":479213,"visible":true,"origin":"","legend":"\u003cp\u003eSEM micrograph of the horizontal cross-section of the freeze-dried hydrogel samples gelatin/alginate hydrogels with different contents of laponite (a) 0, (b) 1 wt.%, and (c) 2 wt.% . The average size of the pores and the porosity (%) of hydrogels were analyzed by different SEM images (n=3) using ImageJ software 1.52v. The porosity (%) was processed by calculating the percentage area occupied by pores compared to the total visible cross-sectional area of the SEM images.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8037436/v1/482dacd998222e303563a141.png"},{"id":97138885,"identity":"90dbb8d9-ac78-43da-b7a4-a2dc568388e4","added_by":"auto","created_at":"2025-12-01 09:59:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":132271,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectra of pure gelatin, laponite, alginate, and the gelatin/ alginate hydrogels without and with 2 wt.% of laponite.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8037436/v1/f28db0ccf41c3f8da0f77527.png"},{"id":97136991,"identity":"7d8b7484-e6d2-4f78-b31a-698a2b92e7d4","added_by":"auto","created_at":"2025-12-01 09:57:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":122385,"visible":true,"origin":"","legend":"\u003cp\u003eThe XRD patterns of the alginate, laponite, gelatin, Lap 0%, and Lap 2% samples.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8037436/v1/e8c4f8700605c176aae91291.png"},{"id":96981117,"identity":"30f89f54-b4de-4403-abac-b00e8cda575b","added_by":"auto","created_at":"2025-11-28 09:14:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":78342,"visible":true,"origin":"","legend":"\u003cp\u003eSwelling percentage of gelatin /alginate and gelatin /alginate/ laponite hydrogels after immersion in PBS.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8037436/v1/f18e2edae132fd61077d0a6a.png"},{"id":96981120,"identity":"13f059ea-a5e4-4ced-822d-825d0200ba7c","added_by":"auto","created_at":"2025-11-28 09:14:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":53751,"visible":true,"origin":"","legend":"\u003cp\u003ehydration degree of the hydrogels after 6 h of immersion in PBS.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8037436/v1/eb034ddc8a06ad40c18e0f8b.png"},{"id":96981122,"identity":"50fee2c3-504e-46fe-9f4d-a6a7794ee0fd","added_by":"auto","created_at":"2025-11-28 09:14:44","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":75993,"visible":true,"origin":"","legend":"\u003cp\u003eDegradation rate of the gelatin/ alginate hydrogel and gelatin/ alginate/laponite nanocomposites after immersion in PBS.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8037436/v1/e10576e38e4a06c087dd6ce2.png"},{"id":96981119,"identity":"497f4106-1045-4502-9ae4-8820d1f39f01","added_by":"auto","created_at":"2025-11-28 09:14:44","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":92923,"visible":true,"origin":"","legend":"\u003cp\u003ecompressive deformation characteristics of the hydrogels: a) compressive flow stress, b) elastic modules, and c) stresses at 50% of compression.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8037436/v1/57dcf87995c315747def7f79.png"},{"id":96981135,"identity":"7a70ec64-95fe-4074-a47a-f5a161a7862f","added_by":"auto","created_at":"2025-11-28 09:14:44","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":165929,"visible":true,"origin":"","legend":"\u003cp\u003eStorage modulus (solid symbols) and loss modulus (open symbols) as a function of the (a) shear strain and (b) angular frequency.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8037436/v1/25b9b75aa7a7f011d6611524.png"},{"id":97137110,"identity":"aefd5901-4faf-4f84-b56e-96d14f958f9b","added_by":"auto","created_at":"2025-12-01 09:57:23","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":91551,"visible":true,"origin":"","legend":"\u003cp\u003eViscosity behavior of the samples: a) viscosity as a function of shear rate. b) Viscosity-shear rate curves fitted using the Carreau equation.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-8037436/v1/faa19d31d7ca6f3ba632f99a.png"},{"id":97139151,"identity":"02b8ab86-77ce-4e97-ac2c-f15269891a12","added_by":"auto","created_at":"2025-12-01 09:59:41","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":60354,"visible":true,"origin":"","legend":"\u003cp\u003e\u0026nbsp;Cell viability of the L929 fibroblast cells cultured on extracts of three different hydrogels at different times.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-8037436/v1/4e16ed111711b1a34d7fbc1b.png"},{"id":96981128,"identity":"30a0a536-4f0e-4f76-9683-ca1a081077a9","added_by":"auto","created_at":"2025-11-28 09:14:44","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":222947,"visible":true,"origin":"","legend":"\u003cp\u003eSEM image of hBMS on (a) control sample substrate (b) gelatin/ alginate hydrogel substrate c) gelatin/ alginate with 1wt. % laponite hydrogel substrate d) gelatin/ alginate with 2wt. % laponite hydrogel substrate.\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-8037436/v1/6f7a7edc8757a6247f2a3c29.png"},{"id":106223110,"identity":"9c4ed349-1ca3-496c-8fd5-005956084c3d","added_by":"auto","created_at":"2026-04-06 10:12:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2316240,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8037436/v1/7c086684-c89e-4b6b-b8a2-8040fba3909c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bioinspired Laponite-Reinforced Gelatin/Alginate Nanocomposite Hydrogels with Tunable Properties for Enhanced Cartilage Tissue Engineering","fulltext":[{"header":"1-Introduction","content":"\u003cp\u003eCartilage is an avascular tissue that has inferior intrinsic repair capability. Until now, different approaches including autologous chondrocyte implantation, mosaicplasty, osteochondral allograft, and microfracture, have been employed to repair damaged cartilage\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. However, in many cases, these approaches cannot generate adequate tissue for damaged cartilage. Having emerged in early 1991, tissue engineering has been a promising applied method in cartilage hurts with a concentration on stem cells, scaffolds, and growth factors. Full reconstruction of damaged tissue requires scaffolds that mimic the properties of the given tissue and support the newly formed tissue until complete growth \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eSince the emergence of tissue engineering, a wide range of biomaterials has been investigated for cartilage repair and regeneration. Hydrogels, nonetheless, have been receiving the highest interest, especially in being used as scaffolds in cartilage, as they have highly comparable features to extracellular matrix (ECM). Hydrogels, by their most common definition, are water-swollen and cross-linked networks confirmed by the reaction of monomers. Various natural and synthetic polymers have been exploited to prepare cartilage scaffolds. The most used natural biomaterials for this purpose are collagen\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, gelatin\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, alginate \u003csup\u003e6\u0026ndash;89\u003c/sup\u003e, and chitosan\u003csup\u003e\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAlginates, a natural biomaterial, are widely utilized copolymers in various biomedical applications, including drug delivery\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, tissue engineering\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, and wound healing\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Alginates are nontoxic at controlled concentrations, and they have high biocompatibility, relatively low price, and good gelation in the presence of cations such as Ca\u003csup\u003e2\u0026thinsp;+\u0026thinsp;15,16\u003c/sup\u003e. They are blocks of linear unbranched copolymers with different patterns of blocks of acids that are linked with covalent bonds \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. These patterns are mainly MM, GG, GM, M, and G. When two G-blocks of alginate are positioned adjacently, they tend to form cross-links through interactions with multivalent cations such as Ca\u0026sup2;⁺, Ba\u0026sup2;⁺, Fe\u0026sup2;⁺, Sr\u0026sup2;⁺, and Al\u0026sup3;⁺.\u003csup\u003e15\u003c/sup\u003e. The involvement of these cations in ionic binding zones between G-blocks leads to a gelation mechanism, resulting in the formation of a three-dimensional network commonly referred to as an \"egg-box\" structure.\u003csup\u003e1819\u003c/sup\u003e. However, a limitation of alginate is the lack of cell attachment motifs, leading to weak interactions between cells and material. Moreover, it degrades very slowly and in an uncontrolled manner \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eObtained from hydrolyzed collagens, gelatin is one of the most favorable biomaterials in tissue engineering. This natural polymer has excellent biocompatibility and contains Arginylglycylaspartic acid (RGD) peptide, promoting cell proliferation and migration \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. However, it suffers from a high degradation rate and poor mechanical properties \u003csup\u003e202324\u003c/sup\u003e. Natural biomaterials-based hydrogels are usually more biocompatible, while synthetic ones possess better mechanical properties\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Some of the hydrogels, therefore, are produced by the combination of natural and synthetic biomaterials, to simultaneously provide biocompatibility, bio-functionality, and suitable mechanical properties and degradation rate.\u003c/p\u003e\u003cp\u003eAs another approach to enhance mechanical properties, the hybrid natural hydrogels can be reinforced by various types of nanomaterials, for instance, carbon nanotubes \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, metal oxide nanoparticles \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, and nanoclays \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Among them, laponite is a nano clay with the formula Na\u003csub\u003e+\u0026thinsp;0.7\u003c/sub\u003e [(Mg\u003csub\u003e5.5\u003c/sub\u003eLi\u003csub\u003e0.3\u003c/sub\u003e) Si\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e20\u003c/sub\u003e(OH)\u003csub\u003e4\u003c/sub\u003e] \u003csup\u003e\u0026minus;0.7\u003c/sup\u003e, is a synthetic nano silicate obtained from salts of lithium, sodium, magnesium, and sodium silicate. Laponite has a disk-like structure, with diameters around 25 nm and thicknesses of approximately 0.9 nm \u003csup\u003e20 17\u003c/sup\u003e, and has negative charges distributed on the surfaces of the platelet and positive charges on the surface of the edges. The electrostatic interaction between laponite nanoclays brings about the formation of specific microstructures. The mechanism of formation of these is still a topic of discussion. Dispersion microstructure is highly related to the concentration of the laponite. At a concentration lower than 2 wt.% of laponite, a \u0026ldquo;house of card\u0026rdquo; microstructure is proposed, while at a concentration above this amount, two suggestions are reported: (i) a Wigner repulsive glass or (ii) a house of card microstructure \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Laponite can be an ideal additive to form hydrogel nanocomposites with greater features, particularly mechanical and rheological properties, and enhance cell-hydrogel interaction for many applications \u003csup\u003e2930,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eTaking laponite nanoclays' biological activities into consideration, non-toxicity \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, improving cell proliferation \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e and cell viability, and enhancement of cell adhesion\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e have been reported in the literature. For example, Rajabi et al. achieved the highest toughness of gelatin-based nanocomposite hydrogels at 1 wt.% laponite concentration. The addition of laponite caused an enhancement in both the elongation and tensile strength of the samples by inducing chemical cross-linking \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Davila et al., following rheological studies on alginate and laponite/alginate solutions, reported a pronounced shear-thinning behavior upon the addition of laponite. It has been reported that a \"house of cards\" structure forms due to electrostatic interactions between charged laponite platelets at very low shear rates, leading to an increase in viscosity. \u003csup\u003e17\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn this study, for the first time, three hydrogel samples based on gelatin and alginate with 0,1 and 2 wt.% laponite were comprehensively characterized and the effect of laponite content on the microstructure, mechanical and rheological properties was studied.\u003c/p\u003e"},{"header":"2-Experimental procedure","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1 Materials\u003c/h2\u003e\n \u003cp\u003eSodium alginate extracted from brown algae with a viscosity lower than 2000 cP was purchased from Sigma Aldrich Inc., USA. Gelatin (from Bovine gelatin with 260\u0026ndash;280 g bloom, type B,) was purchased from Gelatin Halal Inc., Iran. The crosslinker, calcium chloride anhydrous obtained from Biobasic, Canada. Extra pure XLG laponite was obtained from BYK Company, Germany.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2 Hydrogel preparation\u003c/h2\u003e\n \u003cp\u003eThree hydrogel samples with different contents of laponite nanoparticles were prepared to investigate the effect of the laponite nanoparticles on Gelatin-Alginate composite hydrogel. Initially, gelatin, alginate, and laponite powders were accurately mixed with given proportions, 4:2:0,1,2 respectively. After that, the mixtures were gradually added to a certain amount of deionized water at 55\u0026deg;C, using a magnetic stirrer. Then, the beaker was sealed using a cap to prevent evaporation and maintain the exact proportion of precursors. The solution was stirred for 3 h and complete homogeneity was achieved. In the second stage, for gelation of the solutions, samples were put in a 0.1 M CaCl\u003csub\u003e2\u003c/sub\u003e in an oven at 37\u0026deg;C overnight. Finally, the three hydrogel samples were washed with deionized water.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3 Characterization of samples\u003c/h2\u003e\n\u003c/div\u003e\n\u003ch3\u003e2-3-1- Morphological characterization\u003c/h3\u003e\n\u003cp\u003eThe morphological characterization of the prepared hydrogels was carried out using scanning electron microscopy (SEM), FEI, Quantum 2000, USA. For SEM analysis, the samples first were put in a freezer at -80\u0026deg;C. After being completely frozen, the samples were lyophilized for 24 h, to dry out with the least influence on the microstructures. The samples were then coated with a gold nanolayer to enhance contrast. After that, the images were analyzed using Image J software.\u003c/p\u003e\n\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\n \u003cdiv class=\"Heading\"\u003e\u003cstrong\u003e2.3.2 Structural analyses\u003c/strong\u003e\u003c/div\u003e\n \u003cp\u003eThe structural characterizations of prepared hydrogels were performed using the Fourier Transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) analyses. The chemical composition of precursors (gelatin, alginate, and laponite) and prepared hydrogel nanocomposites were examined by the FTIR. Before the FTIR analysis, the hydrogel samples were lyophilized. Tests were conducted using an FTIR spectrometer Bruker, equinox55 in the attuned total reflectance mode. The transmittance evaluation wavelength was in the range of 4000\u0026thinsp;\u0026minus;\u0026thinsp;400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003eThe crystal structures and phase analyses of the initial and lyophilized hydrogel samples were studied using XRD. All samples for this analysis were in powder form. The diffraction angle for scanning the samples was set within the range of 2\u0026theta;\u0026thinsp;=\u0026thinsp;5\u0026ndash;80\u0026deg; with the scanning step size and speed of 0.02 and 0.4 s, respectively.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003e2-3-3- Physical properties of samples\u003c/h3\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThe physical properties of samples were investigated by studying their swelling and degradation behavior. To study the swelling behavior of the hydrogel nanocomposites, the cylindrical samples with height and diameter of 1.4 cm and 1 cm, respectively, were fabricated. For data reliability, three identical samples of each hydrogel were tested. These hydrogel samples were immersed into a certain volume of Phosphate-buffered saline (PBS) for different times (20, 40, 60, 120, 240, and, 360 min) at 37\u0026deg;C. After immersion, the samples were removed from the PBS solution. Next, the surface water was removed and the samples were weighed (wet weight (w\u003csub\u003ew\u003c/sub\u003e)). Then, the samples were frozen at -80 and lyophilized and weighted, (dry weight (w\u003csub\u003ed\u003c/sub\u003e)). Finally, swelling present (SW%) and hydration degree (HD%) was calculated from Equations \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e respectively.\u003c/p\u003e\n \u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003eTo investigate the degradation behavior of hydrogels, first, the samples were lyophilized and weighed. This was considered as the initial weight, w\u003csub\u003e1\u003c/sub\u003e. Then, three identical samples of each hydrogel were incubated in PBS buffer for certain periods,1, 3, 7, 14, and 21 days. After incubation for specific periods, each sample was freeze-dried and weighed, W\u003csub\u003e2\u003c/sub\u003e. The degradation ratio (Deg%) was achieved using Eq. 3.\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAogAAABLCAYAAADzqnaJAAAAAXNSR0IArs4c6QAAAARnQU1BAACxjwv8YQUAAAAJcEhZcwAADsMAAA7DAcdvqGQAAAzVSURBVHhe7d1faFtlA8fxX973euppvVFkjJ7uQjYpaFpka28GNqUDEWbbVIYUWlYTxZu5FVpEof5Zs12JdG1ZQYratN1AdIn9AxNMFLR1Nij2Yj0BRbxK1tUr78578fYcc06yNW2zreu+HziIz/PkJG3OOL8+/07Atm1bAAAAwIb/+AsAAADwcCMgAgAAwIOACAAAAA8CIgAAADwIiAAAAPAgIAIAAMCDgAgAAAAPAiIAAAA8CIgAAADwICACAADAg4AIAAAADwIiAAAAPAiIAAAA8CAgAgAAwIOAuEXZbFbRaFTxeNxftWfFYjFFo1Fls1l/FQAA2IMCtm3b/kKUlslkdOLECV25ckV1dXX+6j0tnU6rq6vrofzZAQB42BAQy5TP53Xw4EF9+eWXamxs9Fc/FJyQ+MMPP6i6utpfDQAA9giGmMs0Pj6u5ubmhzYcSlJjY6MMw9D4+Li/CgAA7CEExDKNjY3ppZde8pRFo1EFAgFVVVUpk8lIG8PQgUDA/f94PK6qqipPm7stHo8rEAgoEAh45kpWVVVpdHRU2victbW1RW0209PTo7GxMX8xAADYQwiIZchms7IsS0899ZSnfHh4WMFgUOFw2J2XNz097flvOBxWQ0ODPv3003s2dy8cDqu/v1+maSocDkuSksmk1tbWdOnSJUlSXV2dTp06pf7+frdNOQ4dOiTLspTP5/1VAABgjyAgluGvv/6SNoZY/Xp6ejw9cPPz8xoZGdH8/Ly0MXdxdXVVra2tBa8qj9MLeKcjFov5XyZJam9vl2VZbq/lxMSEEomElpaW3HA3MzOj7u5u3yvLs7Ky4i8CAAB7BAFxh1544QWtra0pmUwqmUyqublZzz//vJaWlpTNZnX58mWdOXPG85p4PK7a2lpPWSm2bW96nD171v8yaaOH0DRNTU9PK5/Py7Istba2yjAMLSwsKJPJyDRN1dTUSBtBNhaLqaqq6o7BEwAA7H0ExC0oNaxaU1OjUCikq1ev6urVq+ru7nbD2cLCgi5duqSXX37Zbe8EL8uyCs5yd5w6dUpTU1NaWFjQ6dOnpY3h5y+++EKjo6N644033LYrKyu6fv26bt68qUQiob6+voIzAQCAhwnb3JQhm83KNE2lUqmSw8zxeFzRaFSmaWpxcVGSNDAwoKmpKTU3N2t4eNj/EgUCAd3tX73zuQ3D0I0bN1RdXa1kMqmTJ096Pqufsxn47Oysv0rpdFpNTU3K5XJsdQMAwB5FD2IZampqZJqm/vzzT3+VVDDM7PTSqWAOYG9vr6ftvVRTU+MuonHCnDMXsqenx9f6XxcuXCgZaiXp+++/l2mahEMAAPYwehDLFIvFdO3atZK9attxL3oQt2NgYEDt7e23XXFdX1+vtra22859BAAADz56EMvU3d2tH3/8Uel02l+1Zc7K4lJzGu+nwnA4MDDgr3a3ytnuymcAALA5Z+FoqXtxKbFYTNFoVNls1l+1ffYWmaZpS/IcoVDInpyc9DfdkVwuZ4dCIff8uVyuqN4wDHt5edlTfjctLy/bpmnu6D2dn8k5hoaG/E3ui8nJyaLvtVAlfvZy5XI5u6Ojw04kEv4q27ZtO5FIuNehaZolr72hoSHbMAxbkh0MBu/J5wYAYKdyuZwdDAaL7m2Tk5N2MBh079EdHR2ebJRKpSp6n95yQEylUrYkO5VK2bZt25Zl2UNDQ7YkOxKJ+JtvWyQSsfv7+23btu2Ojo6ic0cikfsSrizLsiORSNEXt5eNjIzYkUjEtizLX1XEsqySgd4uCP13Ok8ikXCDnXONFXKuP+f371x7hW2dcLi8vOyGTcMwSn4mAAB2k1AoVJRvhoaG7GAw6N4/nXuhPxs5IbES97sdB0SHc6OuVHAyTdN9j1QqZYdCIbeukr8AVF4kErGDwaDn+3H+IvJfzIUmJyfdfxSlrjG74NyFDMPwnNc0TfePC3uj97OS1yYAAHdDKpWyDcPwF5ckyR4ZGfEX28FgsChgbkfF5iA689K+/fZbtyyZTKq+vl6BQEC1tbVFz/wt3Ji58Ein07Isq+SWMvl8Xl1dXfrkk0/KXknb0tJS9B7+o6Wlxf8ybNPw8LDq6+vV0tKifD6vfD6vlpYW1dfX33Z1tDb2aNxs8cv8/Lyam5s9ZQ0NDe68C+exiEePHnXrnQU3f/zxh1sGAMBu8/nnn2/6+Nt0Oq1oNKr+/v6SO6X09PRobGzMX7xlFQuITlgrvFEfP35cp0+flm3b+uijj9TZ2enWx+NxnTt3Tt98841s29bQ0JAMw3CDoWmaJReEjI+Pq7m5Wfv27VNtba0CgcCmv8zZ2dmiJ5D4j0qtTsb/FYbEcsJhuSzL0qOPPuov1tzcnFTwWMRHHnnE10K6du2avwgAgF1jfn5eBw4c8Be7AoGAmpqaND8/r2eeecZfLUk6dOiQLMva8ULYigVEv/HxcXcPPm3sv+c8XUQbvTkNDQ1u787Zs2e1trbm3uCDwaC+/vprSdLHH3+sY8eOKZPJ6Ny5cxocHFRfX5/ee+895XI5WZZV1DtZKf6eRo5/j80MDg7KsixZlqXBwUF/dUUZhuEvKvLYY4/5iwAA2DUsy9KRI0f8xS7btpXL5XTmzBl1dnbe8bG4Kysr/qItqVhAdJLqsWPHJEk//fSTlpaWPIHCsiytr69Lkvbv36+5uTm3l3B0dFSS9OSTT0qSPvjgA01NTSkQCOjWrVvq7u5WX1+fhoeHVV1drbm5OXcD6La2Ns/Qtt9Ohpj9PY0c/x6bef3112WapkzT1Ntvv+2v3hbDMNxrqFBDQ4Mkad++fZKkv//+29dCevbZZ/1FAAA8UKqrq9Xb26tgMHhXR8YqFhAvX74sSQqFQlLBM4r9ocKZYxYOh9XR0aGmpiYFAgGdP39eiURCNTU17utXV1dlbwz/Oue/3XDynfb+YYj53otGo7IsS7Ozs5qdndXi4qKi0ai/2ZY1Nzdrfn7eU7a6uqrnnntO2phvaBiGvvvuO7feuTYOHz7slgEAsBuV6uAopdx1GNu144CYzWYVi8X02muvqb+/3x0yfuWVVzQ3N+f2DDrP93Vu1slkUrdu3XID2urqqvsYOL9sNqvz58/fcQ6bExBw/0WjUS0uLmp2dlbV1dWqrq7eUkh0eqNLPdrw1Vdf1dLSkjulYHR0VDdv3vRs3h2JRHTx4kVlMhnl83lduHBBwWDwttcXAAC7gWma+vXXX/3FamlpUTgcdu+P6XRac3Nz6urq8jd1Pf300/6irfEva97M7TbKLrWp8dDQkGdD48I2hfvdOYdhGCWXZnd0dBSVm6Zpj4yMuHvrsYXJ7rCTfRCXl5ftUCjkuS5Kfbf+TbD92+Hkcjk7Eom45+jo6LjtewIAsFtEIpGS28EtLy/bkUjEvfc5GagUJ3vt1H17FnM0GlVdXZ1niXY8HldnZ6cKP1IymdQ777yjxcVFt0wbj6s7ceKELMtSf3+/3n//fU89AADAgySdTuvFF1/UjRs3tj2EXF9fr7a2tk23jdvMjoeYt+vixYtaX193u0vz+bx++eUXmabpadfa2loUDrUx18yZo0g4BAAAD7rGxkY1NDRofHzcX1WWZDKptbU1z7Sr7bpvATGRSGhmZkaPP/64AoGADh48KMuyihYgAAAAPCw+++wzzczMbHn7vkwmozfffFNXrlzZdu9jofs2xAwAAIBi+Xxe4+PjWl9fL2uUdHR0VJlMRm+99Za7G8xOERB3CWf+pSRNTk662/lUVVXpww8/VG9vr2feZWEbAACASiIg7iIDAwOamprS6uqqtDGX4Pjx4woGg+48zFgsVvZfFAAAANtx3+Ygolh7e7ssy1Imk5EkTUxMKJFIaGlpyV3MMzMzU5HJpwAAALdDQNxF6urqZJqmpqenlc/nZVmWWltbZRiGFhYWlMlkZJqmZ36BswH5nZ7HCAAAsBX/fffdd9/1F+L++eeffzQxMaEDBw7oyJEjOnz4sH7//Xf9/PPP+u2339Td3a39+/dLGyuWvvrqK2WzWT3xxBM6evSo/3QAAABbxhzEXSabzco0TRmG4W6UmUwmdfLkSZmmWXJPSKf3cKebYgIAAIgh5t2npqZGwWBQ4XDY3cfIeYZwT0+PrzUAAEDl0YO4B9CDCAAAKokexD3g+vXrWl9f9xcDAABsCz2ID7B0Oq2mpiZPGV8nAADYKQIiAAAAPBhiBgAAgAcBEQAAAB4ERAAAAHgQEAEAAOBBQAQAAIAHAREAAAAeBEQAAAB4EBABAADgQUAEAACABwERAAAAHgREAAAAeBAQAQAA4EFABAAAgAcBEQAAAB4ERAAAAHgQEAEAAOBBQAQAAIAHAREAAAAeBEQAAAB4EBABAADgQUAEAACABwERAAAAHv8DU3A12w4C67UAAAAASUVORK5CYII=\"\u003e\u003c/p\u003e\n\u003ch3\u003e2-3-4- Mechanical and rheological properties\u003c/h3\u003e\n\u003cp\u003eThe compression and shear rheology tests were conducted to evaluate the mechanical properties of the prepared hydrogel nanocomposites. To examine the compressive behavior of the hydrogels, three samples of each hydrogel, measuring 14 mm in height and 9 mm in diameter, were prepared. Before the tests, samples were immersed in PBS solution for 30 min to be fully hydrated. The compressive properties of the hydrogels were examined using a Hounsfield-H10Ks machine equipped with a 100 N load cell. The compression tests were carried out under the strain rate of 6\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\times\\:\\)\u003c/span\u003e\u003c/span\u003e10\u003csup\u003e\u0026minus;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and up to the strain of 0.5.\u003c/p\u003e\n\u003cp\u003eMoreover, after 10 days of preparation of samples, the rheological measurements were conducted on the cross-linked hydrogel samples with a diameter of 14 mm and a thickness of 4 mm. The frequency sweep test and amplitude sweep test were carried out using a modular compact rheometer (Anton par, MCR 502) fitted with a 25 nm parallel-plate geometry and gap size of 1mm. Amplitude sweep tests were made at a constant angular frequency of 10 rad s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and shear strains ranging from 0.1 to 100%. The frequency sweep test was carried out at a constant shear strain of 0.1% with an angular frequency range of 0.1 to 628 rad s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe viscosity of non-cross-linked solutions was evaluated using (Anton par, MCR 502). Before the tests, samples were kept in the fridge, 4\u0026deg;C, for 10 days. Tests were carried out at 25\u0026deg;C and viscosity variations against shear rate in the range of 0.01\u0026ndash;1000 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were measured. The curves were fitted with the Carreau model using Eq. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e using OriginPro software and all curves represented curve fitting with plausible Adjusted R-squared.\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/p\u003e\n\u003ch3\u003e2-3-5- Biological properties of samples\u003c/h3\u003e\n\u003cp\u003eThe viability of cultured cells and cell adhesion assessments were performed to evaluate the biological properties of prepared hydrogel nanocomposites. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was performed based on an international standard (ISO)10993-5. For this purpose, based on ISO 10993-12, extracts of 1-day, 3-day, and 7-days were obtained from incubated hydrogels in complete medium culture. Mouse fibroblast cells (L929, Iranian Biological Resources Center, Iran) at a density of 5000 cells were seeded in a 96-well plate along with 200 \u0026micro;L of Dalbecco\u0026rsquo;s Modified Eagle Medium (DMEM) culture medium incubated for 24h (5% CO\u003csub\u003e2\u003c/sub\u003e, 37 ֯ C). The culture medium was supplemented with 10% of inactivated fetal bovine serum (FBS), and 1% of penicillin-streptomycin. After 24 h of incubation, the old medium was discarded and 200 ul of prepared extract was added to each well and the cells were incubated for another 24 h. Then, the extraction medium was removed, wells were washed with sterilized PBS solution and 200 ul of MTT solution (5 mg/ml in PBS) was added to each well, and the plate was wrapped in aluminum foil and then incubated for 4 h at 37\u003csup\u003e◦\u003c/sup\u003e c. Formazan was dissolved by adding 200 \u0026micro;L of DMSO to each well. Finally, spectrophotometric quantifications were applied at 570 nm by an ELIZA microplate reader, BioTek, ELX800, USA.\u003c/p\u003e\n\u003cp\u003eCell adhesion of the hydrogels was evaluated by SEM observations. Preparing the sample is carried out in three main stages cell culture, fixation of the cells, and imaging. Human bone marrow mesenchymal stem cells (hBMSCs, Royan Institute, Iran) were seeded on a thin layer of the cross-linked hydrogels spin-coated on the microscope slides. Cells were cultivated in Minimum Essential Medium \u003cstrong\u003e\u0026alpha;\u003c/strong\u003e (alpha-MEM) culture medium, supplemented with 16% FBS, 1% L-glutamine, and 1% pen-strep. About 3500 cells were seeded on each sample, located in 6-well plates, and incubated in a Co\u003csub\u003e2\u003c/sub\u003e incubator. After 15 hours of culture, samples were immersed in glutaraldehyde solution for 1 hour to be fixed. Then samples were washed with PBS and dehydrated by dipping them in a graded ethanol bath. Finally, after being sputter-coated with a nanolayer of gold, samples were imaged by SEM.\u003c/p\u003e\n\u003ch3\u003e2-4- Statistical analysis\u003c/h3\u003e\n\u003cp\u003eOriginPro 2016 (Origin Lab Corporation, USA) was utilized for statistical evaluation. All data were analyzed by an analysis of variance (ANOVA) and demonstrated as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). The level of significance of all data was considered as *p\u0026thinsp;\u0026le;\u0026thinsp;0.05.\u003c/p\u003e"},{"header":"3- Results and discussion","content":"\u003cp\u003e\u003cb\u003e3-1- Morphology of hydrogel nanocomposite\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSEM micrographs of three groups of hydrogel samples with different contents of laponite are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Obviously, all hydrogels are highly porous with interconnected pores. Samples with 1 wt.% and 2 wt.% of laponite (named Lap 1% and Lap 2%) demonstrate more pores in smaller size ranges, meaning adjacency of macro (\u0026gt;\u0026thinsp;50) and micropores in their microstructure. The average size of the pores (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.d) and percentage of porosity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.e) in the hydrogels are as follows: Lap 0%: 45.7\u0026thinsp;\u0026plusmn;\u0026thinsp;9.4 and 51.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6%, Lap 1%: 41.8\u0026thinsp;\u0026plusmn;\u0026thinsp;7.8 and 61.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3%, and Lap 2%:25.7\u0026thinsp;\u0026plusmn;\u0026thinsp;6.6 and 60.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1%. In the hydrogel without laponite (named as Lap 0%), however, macro pores dominantly exist in the microstructure and there is an insignificant amount of very fine pores, 2\u0026ndash;4 \u0026micro;m, in the walls. Furthermore, as the laponite content increases, the distribution of pore sizes becomes closer to the normal distribution. It seems that additional laponite to the structure results in the formation of new pores and thicker pore walls through electrostatic interactions and hydrogen binding between laponite and the matrix\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. When laponite is incorporated into the gelatin/alginate hydrogel matrix, the laponite hydrogel creates a more complex and interconnected network structure. This resulted in a smaller average pore size for the samples containing laponite. In cartilage tissue engineering, the reported scaffold pore size ranges from 10\u0026ndash;500 \u0026micro;m.\u003csup\u003e35\u003c/sup\u003e By adding 2 wt.% of Lap to the hydrogel, the pore's size decreases by half, and the percentage of porosity increases significantly (about 10%). The porosity of the hydrogel plays a significant role in resembling the structure of the native cartilage ECM. It helps with water uptake, oxygen and nutrient exchange, cell adhesion, and migration in the hydrogel. For in vitro cell biology tests, a porosity range of 48\u0026ndash;90% is reported, which provides a favorable environment for cells.\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eIn a study by Cidonio, Gianluca, et al. it was found that LAP-gellan gum (GG) constructs exhibited more porous networks\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. The study results also show that incorporating Lap increases the hydrogel\u0026rsquo;s porosity, which aligns with these findings.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003e3-2- Chemical analyses of hydrogel nanocomposite\u003c/h3\u003e\n\u003cp\u003eThe chemical functional groups of sodium alginate, gelatin, laponite, Lap 0%, Lap 2% samples, and the crosslinking within the hydrogel were analyzed using FTIR. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the peaks observed for sodium alginate within the range of 3200\u0026ndash;3600 cm⁻\u0026sup1; correspond to O-H stretching \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Additionally, absorptions in the range of 1410\u0026ndash;1600 cm⁻\u0026sup1; are attributed to C\u0026thinsp;=\u0026thinsp;O bonds \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. The peaks at 1030 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1101 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are attributed to the C-C and COC groups, respectively. In the Lap 0% spectrum, sodium alginate peaks have been shifted from 1600 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 1634 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. This can be due to ionic cross-linking of the structure by CaCl\u003csub\u003e2\u003c/sub\u003e and the formation of an \u003cem\u003eegg-box\u003c/em\u003e structure\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. In the gelatin spectrum, the appeared peaks at 3284 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 3066 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are ascribed to N-H stretching vibration of primary amine groups of gelatin\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e and 1523 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 1241 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are related to N-H of amid II and III bonds respectively\u003csup\u003e4344\u003c/sup\u003e. The peaks at 2940 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1631 are attributed to the alkyl group and C\u0026thinsp;=\u0026thinsp;O group of amid bond, respectively \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. For laponite, H-O-H bond absorption was revealed at 1633 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Moreover, the Si-O and Si-O-Si peaks were observed at 962 cm\u003csup\u003e\u0026minus;\u0026thinsp;1 45,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Clearly, in the Lap 0% spectrum, there are no signs of laponite peaks, However, in samples with 2 wt.% laponite (Lap 2%), the related peaks to laponite can be traced, due to laponite interactions with the matrix, Si-O and Si-O-Si bonds wavenumber shifted from 962 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 996 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and the Mg-O wave number shifted from 650 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 642 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. After the incorporation of Lap, the characteristic peaks of gelatin and alginate still existed which shows the successful loading of laponite nanoparticles inside the hydrogel. Compared to the gelatin, Lap 0% and Lap 2% exhibit absorption peaks around peak shifts from 1445 to 1438 and from 1526 to 1543, which is presumably due to interactions and crosslinking between the amine groups of gelatin and the carboxyl group in alginate.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e depicts the XRD patterns of the alginate, laponite, gelatin, Lap 0%, and Lap 2% samples. As can be observed, the characteristic peaks at 6.5\u0026ordm;, 19.97\u0026ordm;, 35.07\u0026ordm;, and 60.9\u0026ordm; of the laponite can be detected in the XRD pattern of the Lap 2%, demonstrating the presence of laponite nanoparticles in this sample. Gelatin and alginate are amorphous materials and so, there are no sharp peaks in their XRD patterns\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. However, a sharp peak at 32.16\u0026ordm; was observed in the XRD patterns of alginate, Lap 0%, and Lap 2% which can be related to NaCl or CaCo\u003csub\u003e3\u003c/sub\u003e impurities in the alginate\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Notably, the peaks of 19.97 and 35.07 shifted to lower angle peaks of 19.93 and 34.05 respectively. This could be owing to the intercalation of polymer chains into laponite platelets and increasing the distance between platelets\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Polymer compounds can be used to cover the surface charge of platelets, which reduces the electrostatic repulsion between them. The possible mechanisms for physical cross-linking in these hydrogels are supported by FTIR and XRD studies, as well as previous research. During hydrogel crosslinking, Lap nanoparticles interact with polymer chains through hydroxyl groups that can form hydrogen bonds with functional groups in gelatin and alginate\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. When laponite is mixed with water, it releases sodium ions which create a negative charge on the surface of the platelets. However, the edges of the platelets have a positive charge. This property allows laponite to be easily dispersed in water and establish an electrostatic interaction with gelatin and alginate polymer\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003e3-3- Physical properties\u003c/h3\u003e\n\u003cp\u003eAssessing the swelling ratio of hydrogels is crucial, as swelling or water absorption reflects the hydrogel's capacity to transmit and absorb physiological fluids\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. This characteristic, which determines the hydrogel's ability to transport nutrients and remove waste, is essential for maintaining cell viability within the hydrogel structure due to its impact on hydration and nutrient exchange\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. However, swelling should be up to a certain amount, otherwise, it causes rapid degradation of the scaffold structure. As mentioned earlier, to investigate the swelling behavior of the hydrogels, they were immersed in PBS for certain durations, and their weight losses were measured. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e represents the swelling percentage of the samples in different durations. The highest swelling percentage was achieved for the Lap 0% sample. It can be observed that the addition of laponite to the hydrogels significantly reduced the swelling percentage. The involvement of hydrogen and electrostatic interactions between laponite and matrix provides a network with a higher density of cross-linking which has been explained in the results of FTIR analysis, leading to the creation of finer pores with a lower ability to absorb the solution. Also, the results are in good agreement with previous studies\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. For instance, Akhtar et al. reported that adding 0.3% w/v Cu-Ag doped mesoporous bioactive glass nanoparticles to a hydrogel prepared from oxidized alginate, gelatin, and silk fibroin reduced the hydrogel's water absorption capacity by decreasing the size of its pores\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. SEM imaging results also showed a significant reduction in pore size within the hydrogels upon the addition of Lap nanoparticles.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe hydration degree determines the appropriateness of hydrogels for the proliferation and growth of the cells\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. It has been reported that hydrogels with hydration degrees higher than 90% are suitable for cell proliferation and growth \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. In this study, after immersion of three numbers of each sample in PBS for 6 h, the average hydration degree was calculated. Although the hydration degrees decreased with increasing laponite concentration, they were more than 90% for all three hydrogels, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. It has been reported that a concentration of 3% laponite in gelatin/alginate hydrogel makes the hydrogel unsuitable for cell proliferation and growth\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFor ECM formation and cell proliferation, the used biomaterial scaffolds should be biodegradable. However, the degradation rate of scaffolds should be proportionate to the rate of regeneration of cells. To evaluate the degradation behavior of the hydrogels, their weight loss was measured after immersed in PBS and the degradation rate was calculated for the given durations (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). As can be observed, for Lap 0% and Lap 1%, the degradation rate is very high at the initial time of immersion (1 day) in PBS solution while in the case of the Lap 2% sample is much lower. Following the initial time of immersion period, the degradation rate of samples generally slowed down. However, the addition of the degradation rate of the samples decreased as a function of the laponite contents. The severely high degradation rate on day 1 in the sample without laponite is due to weak interaction between gelatin and alginate and consequently release of gelatin in the PBS as it is water soluble\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Also, Bider et al demonstrated that gelatin could be released from alginate dialdehyde-gelatin hydrogel\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. The addition of laponite, however, reduced the degradation rate of the samples because of the combination effect of strengthening interactions between components, and reduction of water penetration in the structure, resulting from smaller pore sizes\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. It has been reported that hydrogels with lower porosity or larger pore sizes tend to degrade faster than those with higher porosity or smaller pore sizes. The structure of the hydrogel, specifically the mesh size of its cross-linked network, also affects the degradation profile. Highly cross-linked hydrogels with smaller mesh sizes demonstrate a longer degradation period. Zhu et al., reported that the degradation rate of hydrogel based on alginate dialdehyde (oxidized alginate) and gelatin after 24 hours was around 2.5%, consistent with the current study's results\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. In this study, the addition of nanoparticles has increased the amount of cross-linking, leading to long-term stability and decreased degradation rate of the construct.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003e3-4- Mechanical properties\u003c/h3\u003e\n\u003cp\u003eCompressive stress-strain curves of hydrogel nanocomposites containing various contents of laponite are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea. All curves experienced the same trend in two main regions. The first region, in which the stress slope was low, was due to the initial deformation of samples. The second region started with an abrupt increase in the slope. This was a result of the resistance of the network against external stresses\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. The strength of samples at 50% of strain has been presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec. Results show the highest strength value has been achieved for the Lap 1% sample. The contribution of 1 wt.% of the laponite leads to a significant increase in the strength from almost 10.61\u0026thinsp;\u0026plusmn;\u0026thinsp;1.54 kPa for sample Lap 0% to 26.81\u0026thinsp;\u0026plusmn;\u0026thinsp;1.92 kPa. This enhancement in the compressive strength could be due to additional ionic, van der Waals, and hydrogen bonds created by the addition of the laponite nanosheets leading to increasing the density of cross-linking of the hydrogel networks\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. This pressure modulus is comparable to values reported in other studies; for instance, a gelatin and silk-based hydrogel developed for cartilage tissue engineering demonstrated a maximum modulus of 30 kPa\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. By further addition of the laponite content to 2 wt.%, the compressive strength dropped to 17.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68 kPa. This reduction in compressive strength is due to agglomeration of the laponites at higher concentrations that voids some areas of the microstructure of laponite and causes inhomogeneity \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The maximum compressive modulus of hydrogels, which are used for cartilage tissue engineering, is usually lower than that of native articular cartilage. However, this value is still suitable for supporting cells to secrete cartilage tissue ECM, as it falls within the range for soft tissue. The mechanical properties of the hydrogel are also influenced by architectural parameters, such as porosity, pore size, and pore shape\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe viscoelastic behavior of the samples was analyzed by oscillatory shear tests. In the diagram of the strain sweep test shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea, all the hydrogel samples represent viscoelastic behavior, since there are both storage modulus (Gʹ) and loss modulus (Gʺ) in their diagrams. Samples with a storage modulus higher than the loss modulus tend to have solid-like behavior \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Herein, higher storage modules of the samples indicate their solid-like behavior. Based on the strain sweep test results, all the samples demonstrated linear viscoelastic (LVE) behavior in the strain range of 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e to 1%. Therefore, the frequency sweep test was carried out at a constant strain of 0.1%. Storage modules for all samples are higher than loss modules, indicating their solid-like behavior (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb). In the Lap 1% and Lap 2% samples, cross-linking is intensified by physical gelation between polymer chains and laponite sheets.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e It is shown that the Laponite operates as a physical crosslinking agent through reversible non-covalent interactions between nanoplatelets and the biopolymer matrix, thus improving the storage modulus of the bioink from 45 to 277 Pa at 1wt.% concentration. Amplitude sweep test can be considered to assess of mechanical strength of cross-linked hydrogels\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. The samples with higher storage modules and loss modules show better mechanical stability. Based on this, as can be seen in the diagram samples containing laponite demonstrated better mechanical stability. This is improved more by increasing the laponite concentration to 2 wt.%.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe injectability and printability of hydrogels are highly dependent on the shear-thinning viscosity behavior\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Although alginate and gelatin, solely, have Newton fluid behavior, their compound solution demonstrates shear-thinning behavior because of physical interaction between their polymer chains\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. This behavior is also observed when the laponite nanoparticles are added to their solution and form the house of card structure\u003csup\u003e2144,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Represented in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e. is the viscosity behavior of the samples. As observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea. with increasing shear rate viscosity of all samples significantly decreases, meaning they show shear-thinning behavior. Curve fitting results also demonstrate the shear-thinning behavior of the samples as well-fitted curves with the Carreau equation with a parameter of 0\u0026thinsp;\u0026lt;\u0026thinsp;n \u0026lt;\u0026thinsp;1. The addition of laponite to the structure increases the viscosity of the samples. The viscosity for Lap 1% is noticeably higher than others. This could be a result of keeping the samples at 4\u0026deg;C which leads to a conversion in the conformation of the gelatin chains from \u0026ldquo;random coil\u0026rdquo; to \u0026ldquo;triple helix\u0026rdquo;, making the structure more complex and more viscous\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. It seems that due to the contribution of gelatin in physical interactions with the complex structure components, all helixes could not convert to random coils after reaching room temperature. As a result, the sample Lap 0% represented a higher viscosity compared to previous similar works\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Nevertheless, increasing laponite content to 2 wt.% decreased viscosity values even lower than that observed for the sample without laponite. This could be owing to the agglomeration of the laponite particles in some parts of the structure\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e which might increase the possibility of transition of the triple helix to the random coil in the other parts. Liu et.al investigated the viscosity behavior of the hydrogels with a combination of gelatin 5 wt.%, alginate 1 wt.%, and laponite (0\u0026ndash;3 wt.%). Our results represented higher viscosity with the addition of lower amounts of laponite\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e which could be related to the effect of 4\u0026deg;C.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003e3-5- Biological properties\u003c/h3\u003e\n\u003cp\u003eThe MTT assay on L929 fibroblast cells was conducted to assess the cell viability behavior of the hydrogels. In this study, hydrogel extracts were used as substrates in an indirect method to evaluate the effect of the prepared hydrogels on cell viability.\u003c/p\u003e\u003cp\u003eIn this study, the effect of prepared hydrogels on cell viability, evaluated indirectly utilizing MTT assay. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e, all samples noticeably improved the viability of the cells. Results are in a good correlation with the degradation test results. On day 1, the sample Lap 2% showed lower cell viability than the samples Lap 0% and Lap 1%. This is caused by higher amounts of gelatin released, by degradation, in the latter samples. It is widely reported that gelatin improves the proliferation and viability of cells. Additionally, gelatin contains intrinsic cell adhesive peptide sequences including RGD (arginine-glycine-aspartic) which might have contributed to cell attachment and proliferation. Additionally, increased DNA content in cells provided evidence of enhanced cell viability\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. In day 3 samples, a significant enhancement in viability was observed. This phenomenon is due to the release of a greater amount of gelatin compared to that observed on day 1. However, in day 7 samples, the viability of cells decreased. This might be attributable to the gradual degradation of gelatin by proteases in the FBS as a part of the complete culture medium during the extraction stage. Based on previous works, gelatin, alginate, and laponite in the concentration range used in this study not only are nontoxic and biocompatible but also increase cell viability\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. For example, Ghadiri et al. reported that the cells in 1-day extracts were 4 times as viable as those in control samples\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Previous studies showed that Laponite-based nanocomposites promote cell proliferation, differentiation, and attachment. Therefore, the higher cell viability observed in the extracts of hydrogels could be due to the release of silica from the Lap into the extracts which improves cell proliferation. Silica has been reported to upregulate the expression of genes involved in cell growth and differentiation\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eA critically important feature of scaffolds in tissue engineering is supporting cell adhesion, and cell spreading, playing a vital role in the enhancement of cell proliferation and formation of the ECM\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. For assessment of the cell adhesion behavior of the hydrogels, hBMSC cells were cultured on a thin layer of the hydrogels. The cells, after 15 h, were fixed and dehydrated using glutaraldehyde and ethanol to be prepared for imaging. Figure\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e shows the morphology of the attached cell on the control sample and the three hydrogels. As can be seen, hBMSCs have well attached to the surface of all samples and produced filopodia. It has been reported that, due to the lack of necessary adhesion motifs in the alginate structure, cells tend to adopt a completely rounded shape and aggregate on its surface \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. In this research, effective cell adhesion to the surfaces is primarily attributed to the abundance of cell-adhesive motifs within the gelatin structure\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Laponite can also improve cell adhesion because some adhesion proteins are inclined to be absorbed by silicate nanoparticles and silicate nanoparticles could provide cell absorption focal points in the structure \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. In a study by Kafili et al., it was demonstrated that Laponite-containing hydrogels release magnesium (Mg), silicon (Si), and lithium (Li) ions, as confirmed. This release led to improved bioactivity, promoted cell proliferation, and created a more favorable microenvironment for the adhesion of fibroblast cells.\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"4- Conclusion","content":"\u003cp\u003eIn this study, the gelatin/ alginate hydrogels with different laponite concentrations (0,1 and 2 wt.%) were successfully synthesized for cartilage applications. The effects of laponite concentrations on microstructure evolutions and physiochemical, biological, and mechanical properties of the hydrogels were investigated. The following items are the main findings of this research:\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eThe incorporation of laponite to the gelatin/ alginate hydrogel increased pore size variability, enhancing the material's suitability for tissue regeneration applications.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eThe increased cross-linking density of the hydrogels along with smaller pore size by increasing laponite concentrations also decreased the swelling ratio and degradation rate of the samples.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eMechanical testing indicated an optimal laponite concentration for significantly enhancing Young's modulus and strength at 50% deformation, with the ideal concentration of laponite nanoparticles determined to be 1 wt.%.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eIt was determined that cellular viability and adherence were suitably in line with expectations. Overall, it appears that gelatin/alginate/ laponite hydrogels can be suitable for regenerating cartilage tissue.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAvailable by request from the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors have no conflicts to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was not supported by any sponsor or funder.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFaezeh Shahedi Aliabad:\u0026nbsp;\u003c/strong\u003eData curation, Formal analysis, Investigation, Project administration, Visualization, Writing- original draft, Writing-review \u0026amp; editing. \u003cstrong\u003eElnaz Tamjid:\u0026nbsp;\u003c/strong\u003eConceptualization, Data curation, Formal analysis, Methodology, Resources, Supervision, Validation, Writing-review \u0026amp; editing.\u0026nbsp;\u003cstrong\u003eMitra Tavakoli:\u003c/strong\u003e Formal analysis, Methodology, Resources, Validation, Writing-review \u0026amp; editing.\u003cstrong\u003e\u0026nbsp;Parvin Najafi:\u0026nbsp;\u003c/strong\u003eInvestigation, Project administration, Visualization, Writing-review \u0026amp; editing. \u003cstrong\u003eHossein Alipour:\u0026nbsp;\u003c/strong\u003eInvestigation, Project administration, Visualization, Writing-review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to acknowledge the Department of Research Affairs of Tarbiat Modares\u003c/p\u003e\n\u003cp\u003eUniversity\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eLi J, Chen G, Xu X, et al. 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Development of bioinspired nanocomposite bioinks based on decellularized amniotic membrane and hydroxyethyl cellulose for skin tissue engineering. \u003cem\u003eCellulose\u003c/em\u003e. 2024; 31:1-25. doi:10.1007/s10570-024-05797-w\u003c/li\u003e\n\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":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cartilage tissue engineering, Hydrogel, Characterization, Shear-thinning, Gelatin, Alginate, Laponite","lastPublishedDoi":"10.21203/rs.3.rs-8037436/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8037436/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCartilage tissue engineering offers a promising strategy for the regeneration of damaged cartilage, a tissue with limited self-repair capacity. The development of bioengineered scaffolds that closely mimic the mechanical and biological properties of native cartilage is critical to restoring its function. In this study, we fabricated and characterized gelatin/alginate hybrid hydrogels incorporating varying concentrations of laponite nanoclay particles (0, 1, and 2 wt.%) for potential application in cartilage tissue engineering. The microstructural, mechanical, physicochemical, and biological properties of the hydrogel nanocomposites were systematically evaluated using FTIR, XRD, SEM, swelling and degradation analyses, compression testing, rheological measurements, MTT assay, and cell adhesion studies. The incorporation of laponite nanoclay significantly influenced the hydrogel properties, reducing pore size (from 46 ± 9 µm to 26 ± 7 µm), decreasing the swelling ratio (by ~70% and ~50%), and lowering the degradation rate (from 50% to 40% and 2.5% after 6 hours and 21 days, respectively). Additionally, mechanical strength more than doubled, and the hydrogels exhibited shear-thinning behavior. All formulations maintained a high hydration level (\u0026gt;90%), providing a favorable environment for cell viability and proliferation. These findings indicate that laponite nanoclay-reinforced gelatin/alginate hydrogels possess a biomimetic structure, tunable mechanics, controlled biodegradability, and excellent biocompatibility, making them promising candidates for cartilage tissue engineering applications.\u003c/p\u003e","manuscriptTitle":"Bioinspired Laponite-Reinforced Gelatin/Alginate Nanocomposite Hydrogels with Tunable Properties for Enhanced Cartilage Tissue Engineering","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-28 09:14:39","doi":"10.21203/rs.3.rs-8037436/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fe0553ed-b648-43ee-a95f-62cd22cfc988","owner":[],"postedDate":"November 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":58561853,"name":"Biological sciences/Biotechnology"},{"id":58561854,"name":"Physical sciences/Engineering"},{"id":58561855,"name":"Physical sciences/Materials science"}],"tags":[],"updatedAt":"2026-04-06T10:11:39+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-28 09:14:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8037436","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8037436","identity":"rs-8037436","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}