Alginate-Gelatin Composite Hydrogels for Next-Generation 3D Bio-Printing in Tissue Engineering

Alginate-Gelatin Composite Hydrogels for Next-Generation 3D Bio-Printing in 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 Research Article Alginate-Gelatin Composite Hydrogels for Next-Generation 3D Bio-Printing in Tissue Engineering Mohaimenul Khan, Md Alamgir Hossain This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7163757/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 The development of hydrogel bioinks faces several obstacles, including optimizing the printing parameters of bioinks, maintaining tissue vascularization, and ensuring good mechanical strength, among others. In this work, alginate-gelatin hydrogel bioinks are developed assessing the physical properties, including swelling properties, thermal properties, stiffness, and rheological properties and cell survivability. CaCl 2 was used as a cross-linker to enhance the bio-inks’ mechanical stability. FTIR analysis of Ca 2+ crosslinked with sodium alginate-gelatin (SA-G) reports a slight shift in symmetric stretching carboxyl groups. Morphological structure of optimized SA-G bio-ink showed well porous interconnected net like structure. The swelling results show an inverse relationship with increasing the proportion of sodium alginate. Stiffness indicates the resistance of the hydrogel bioink's surface to deformation under applied load. Higher stiffness indicates solid behaviour, while lower stiffness indicates a viscous structure. The storage modulus (G'), loss modulus (G"), and phase angle, as measured by a rotational rheometer, which indicates the solid point, viscous point, and viscoelastic point. Cells (Schwann cells, Cancer cells and the co culture cells) survivability in 2D or monolayer system confirms the non-toxicity of the developed hydrogels for 3D/4D bioprinting. The 3D bio-printing was carried by extrusion bio printing process. 3D bio printed structure's stability and well size porous structure were analyzed by pore size and the life dead assay showed the live and dead cells after the bio-printing at day 10 using fluorescence microscopy. Thus the developed hydrogel can play a crucial roles for tissue engineering. Hydrogels Cancer and normal cells 3D Bio-printing Tissue Engineering Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 1. Introduction Bioink plays a crucial role in 3D bioprinting. Groll and colleagues (Groll et al., 2018) describe bioinks as a mixture of cells that can be processed using automated biofabrication technologies, which may also include biologically active components and biomaterials (Li et al., 2020 ). Bioink materials enable scientists to create complex biological structures by manipulating living cells and their environments (Ozkerim et al., 2018). Contemporary bioinks are being engineered to enhance significantly both printability and biocompatibility (Kyle et al., 2004). This is accomplished by meticulously regulating a variety of physical, chemical, and biological characteristics. An ideal ink must meet the biological requirements concerning cell compatibility, as well as the physical and mechanical demands of the printing process (Chung et al.,2013). It should exhibit excellent printability, robust mechanical strength, and stability (Mobaraki et al., 2020 ). The ink must demonstrate gel-like properties or possess adequate viscosity to be dispensed as a self-supporting filament (Barrulas et al.,2023). Nonetheless, if the gel exhibits excessive strength, the substantial shear forces required to expel the ink may lead to cell mortality and the gel's fracturing (Kong et al.,2003). Bioinks must be consistent with suitable bioprinting methods to create functional living structures that exhibit the necessary biological and mechanical properties. Bioinks must provide a non-toxic extracellular matrix environment that facilitates cell adhesion, communication, growth, and differentiation, while also ensuring high cell viability after printing (Zhang et al.,2018). In certain instances, it will be suitable for the scaffold to degrade in a controlled manner over time (Chung et al.,2013). The degradation rate of bioinks, assessed through time or remaining mass, is influenced by external conditions such as temperature, pH, enzyme presence, and vibration, as well as internal factors including the scaffold’s chemical composition, the incorporation of nanoparticles or nanofibers, polymer chain length, and any surface modifications applied to the scaffold (Shokrani et al.,2022). Hydrogels are polymeric materials frequently used in tissue engineering due to their low cytotoxicity and structural resemblance to the extracellular matrix (ECM) (Frampton et al.,2011). Hydrogels are recognised as optimal materials for biomedical applications due to their physical properties closely resembling those of the physiological tissue environment. Hydrogel is a substance composed of a three-dimensional polymer network that retains a significant amount of water, typically exceeding 90% (Chen et al.,2023). The extensively hydrated network structure facilitates the exchange of gases and nutrients, making it as a compelling choice for developing inks used in bioprinting. The combination of hydrogels presents an opportunity to merge the unique properties of each hydrogel component, allowing for the customization of the overall hydrogel to meet specific needs (Rosellini et al.,2009;Dong et al.,2006;Chung et al.,2013). Cross-linking serves as a stabilization mechanism in polymer chemistry, facilitating the extension of polymer chains in various directions to create a network structure (Maitra et al.,2014). A cross-link refers to a bond, which can be either ionic or covalent, that connects one polymer chain to another. The effectiveness of cross-linking is influenced by various factors, including the pH of the reaction, the type of starch used, the quantity and nature of the cross-linking agent, the reaction temperature and duration, and the level of substitution present (Dhull et al.,2023). Due to the diverse array of potential structures in cross-linked networks, it is crucial to employ analytical techniques to evaluate important structural parameters, such as cross-link density, molecular weight between cross-links, variations in chain length between cross-links, network uniformity, and the fraction of polymer that remains unbound to the network (Nielsen et al.,1969). The process of crosslinking plays a crucial role in influencing the behavior of loaded cells at the cellular level, as well as affecting the mechanical and physicochemical characteristics of the bioprinted constructs. It is essential to achieve a balance between the level of crosslinking and the ability to print, as reduced crosslinking can improve the flow of bioink, whereas increased crosslinking can lead to greater stiffness and potentially obstruct printability (GhavamiNejad et al.,2020). Essential biomechanical properties that can be assessed encompass rheology (including viscosity, shear-thinning, viscoelasticity, and thixotropy), gelation kinetics, crosslinking, and network architecture (Murphy et al., 2013 ; Jia et al., 2014; Blaeser et al., 2016 ; Tarassoli et al., 2021 ). Despite the considerable advancements in these research areas, existing bioprinting systems encounter various limitations. Addressing these challenges is crucial to fully leverage bioprinting's capabilities in tissue engineering and regenerative medicine (Vyas et al., 2019). The limitations associated with the production of bioinks include the absence of specialized extracellular matrix components (ECM) proteins tailored for different cell types, inadequate cell interaction, variability in the degradation of tissue compared to tissue formation, toxicity of degradation byproducts, instability, and the deterioration of structural integrity of the bioprinting scaffold throughout various phases of the bioprinting process (Hospodiuk et al.,2017). The development of printable biomaterials and 3D printing techniques that simulate tissue functions is essential; however, analyzing the advantages and disadvantages of existing fabrication methods can inform future investigations (Askari et al., 2021 ). Selecting the appropriate materials is crucial for producing hydrogels. These materials significantly impact biocompatibility, cellular viability, and the mechanical properties of bioprinted structures, all of which are crucial for achieving successful bioprinting outcomes (Sánchez et al., 2020). The printability of bioinks is influenced by their viscosity, surface tension, and cross-linking capabilities, as well as the surface characteristics of the printer nozzle Kyle et al.,2018). Stem cells from the periodontal ligament were incorporated into GelMA hydrogel at concentrations of 3%, 5%, and 10% (Zhu et al., 2023). A 10% GelMA concentration showed reduced cell viability and survival; however, the addition of periodontal ligament stem cells facilitated new cell generation (Almeida et al., 2024 ). The composition of alginate and gelatin methacryloyl (GelMA) has been demonstrated recently using a coaxial dispensing system (Colosi et al.,2016). GelMA, when used at concentrations below 5% w/v, provides beneficial conditions for cellular activity; however, it lacks adequate printability independently. The combination with alginate yields a bioink that exhibits mechanical stabilization due to the presence of physically cross-linked fibers. The coaxial needle system enables precise regulation of the gelation kinetics of this bioink by modifying the concentrations of alginate and CaCl₂. After bioprinting, the hydrogel construct undergoes additional reinforcement via UV cross-linking of GelMA (Hölzl et al., 2016 ). Gelatin–alginate bioinks were used to encapsulate myoblasts and investigate the mechanical properties of the printed constructs (Zhang et al., 2018). Employing a dual-nozzle system, they developed 3D filaments featuring diverse structural configurations. The methodology encompassed a two-step cross-linking approach: initially, physical cross-linking of gelatin was conducted at low temperatures during the printing phase, followed by ionic cross-linking of alginate using Ca 2+ ions after printing. Although there was a reduction in mechanical strength over time, the constructs maintained their mechanical durability due to their low porosity and angled geometry (Ozkerim et al., 2018). In this research, we utilize alginate and gelatin hydrogels to prepare the bioink, with CaCl 2 serving as the crosslinker. Alginate is a linear polysaccharide composed of monomers known as mannuronic and guluronic acids. The initiation of alginate gelation occurs through the release of CaCl 2 ions, leading to the formation of egg-box structures between alginate chains (Draget et al.,1990; Stokke et al., 2000 ; Siew et al., 2005 ). Gelatin is a protein derived from the denaturation of the triple helix structure of collagen ( Panouillé et al.,2005). Alginate exhibits several benefits, including bioinertness, affordability, accessibility, tunability, biocompatibility, biodegradability, and tissue-specific mechanical properties. Nonetheless, it also has limitations, such as insufficient degradation, the absence of cell-binding motifs, restricted cell-material interactions, high hydrophilicity, rapid gelation that can cause nozzle clogging, inconsistencies in printing, and inadequate dimensional stability (Datta et al.,2023). Gelatin exhibits numerous significant advantages, including biocompatibility, non-immunogenicity, and hydrophilicity (Kuijpers et al.,2000). This material functions as a thermoreversible gel, transitioning to a solid state at reduced temperatures while exhibiting a loss of mechanical stability in physiological environments. To preserve its structural integrity at temperatures below 37°C, chemical modification is necessary (Datta et al.,2023). In this research, we analyzed the formulation of alginate-gelatin hydrogels bioink through Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), swelling properties, and thermal properties. In addition, we investigated the rheological properties such that storage modulus, loss modulus, and cell viability of the bioink composition. We then printed the 3D structure through the bioinks layer by layer and analysed the bioprinted structure’s stability using optical microscopic imaging. Rationally, the developed alginate-gelatin bioink is a promising candidate for 3D/4D bioprinting in various potential applications. 2. Experimental Procedure 2.1 Materials The materials used Sodium Alginate (sodium polymannuronate) bought from Research-Lab Fine Chem Industries, Gelatin from Merck Specialities Private Limited, calcium chloride (CaCl 2 , anhydrous, granular, ≤ 7.0 mm, ≥ 93.0%) obtained from Sigma Aldrich, and phosphate-buffered saline (PBS, 10× concentrate, powder, pH 7.2) acquired from HiMedia Laboratories Pvt. Ltd. 2.2 Bioink Preparation Various proportions of sodium alginate and gelatin solutions were mixed to prepare the bioink solution. The mass of the alginate powder and gelatin powder is measured in the following proportion: 1% Sodium Alginate (w/v) + 9% Gelatin (w/v);2% Sodium Alginate (w/v) + 8% Gelatin (w/v);3% Sodium Alginate (w/v) + 7% Gelatin (w/v);4% Sodium Alginate (w/v) + 6% Gelatin (w/v);5% Sodium Alginate (w/v) + 5% Gelatin (w/v). A schematic diagram of the bioink preparation method is shown in Figure 1.Initially, we combined various desired concentrations of Sodium alginate powder with 25 mL of water and mixed different concentrations of gelatin powder with 25 mL of water in a beaker. Subsequently, we combined 25 mL of sodium alginate solution with 25 mL of gelatin solution. We subjected the mixture to a magnetic stirrer at 80°C for 1.5 hours to ensure complete dissolution of the solution. Subsequently, we added 50 mL of 0.5 M CaCl 2 to the solution as a cross-linking agent. Subsequently, we positioned the solutions within a test tube and placed them in the freezer for freeze-drying. After removing the sample from the freeze dryer, we sliced the solution into small pieces, creating a 3D-shaped structure. Then, the solution was placed under the table lamp for evaporation. After 4 days, it was removed from the lamp and placed in sunlight for 4 days to accelerate the evaporation process. After completing the evaporation process in sunlight, the solution was moved to a magnetic stirrer at 50 °C. Thus, water was removed from the sodium alginate-gelatin solution. 2.3 Characterization Process 2.3.1 Fourier Transform Infrared Spectroscopy ( FTIR) for bond formation analysis The molecular structure and chemical composition of pure sodium alginate, gelatin, and five compositions of sodium alginate-gelatin bio-ink solution with CaCl 2 crosslinkers were analyzed by Fourier transform infrared spectroscopy (FTIR). The Shimadzu IRTracer-100 model device interpreted this property. 2.3.2 Scanning Electron Microscopy (SEM) for surface morphology analysis The surface morphology of the five developed sodium alginate hydrogel bio-ink compositions, with CaCl 2 crosslinkers, pure sodium alginate, and gelatin, was analyzed using scanning electron microscopy (SEM) at an accelerating voltage of 5 kV. All samples were carefully coated with a thin layer of gold under a vacuum before starting the SEM analysis. 2.3.3 Thermal Stability Analysis Thermogravimetric analysis (TGA), differential thermal analysis (DTA), and derivative thermogravimetry (DTG) were conducted carefully under a nitrogen gas flow of 10 mL/min to assess the thermal stability properties of five compositions of sodium alginate-gelatin with CaCl 2 as a crosslinker, pure sodium alginate, and gelatin. All the samples weighed between 5 and 14 mg and were heated to 250 °C. 2.3.4 Swelling Behavior The entire composition of Sodium Alginate (SA)-Gelatin (G) hydrogel bioink, along with the CaCl 2 crosslinker, was immersed in a 40 mL Phosphate-Buffered Saline (PBS) solution. The weight of the compositions was recorded at intervals of 1 hour, 3 hours, 7 hours, 12 hours, 18 hours, and 24 hours to assess swelling. The determination of the swelling percentage of the compositions is conducted through the application of the formula: Where w t represents the swollen weight of the composition at different time points, and w o represents the initial (dry) weight of the composition. 2.3.5 Statistical Analysis A one-sample t-test was performed using GraphPad Prism 10.2.0 (392) to analyse variables that significantly affected (p-value < 0.05) the swelling rate composition. The quantitative data are shown as the mean ± standard deviation.Legend **** on the figure indicates p<0.0001,*** indicates p = 0.0003 and 0.0005 ,** indicates p = 0.0017. 2.3.6 Assessment of Rheological Parameter The evaluation of the rheological behaviour of the hydrogel bioinks involved measuring shear modulus, loss modulus, and phase angle through the use of a rotational rheometer configured with a parallel plate geometry. The hydrogel samples were meticulously positioned between the plates, ensuring a consistent gap of about 1 mm, and the edges were sealed with mineral oil to avert dehydration throughout the testing process. A strain sweep test was initially performed at a constant frequency (usually 1 Hz) to determine the linear viscoelastic region (LVR), where the storage modulus (G′), loss modulus (G″), and phase angle (δ) exhibited independence from strain. This facilitated the selection of an appropriate strain value for the following frequency and time sweeps. A frequency sweep was conducted within the LVR, typically ranging from 0.1 to 100 rad/s, to examine the relationship of G′, G″, and δ with oscillatory frequency, highlighting the viscoelastic characteristics and structural stability of the bioinks across varying deformation rates. Time sweep tests were performed at a constant strain and frequency to assess the bioink's stability over time, mimicking conditions like crosslinking or gelation behaviour. Throughout these experiments, variations in G′ and G″ were observed to evaluate structural development. The shear modulus was derived from the storage modulus, indicating the stiffness of the hydrogel matrix. The phase angle, derived from the ratio of G″ to G′, reflects the equilibrium between elastic and viscous characteristics. All tests were performed at either room temperature or 37°C, based on the specific application requirements. Measurements were conducted a minimum of three times to ensure consistency, and the resulting data were analysed with the rheometer software. Graphs illustrating modulus and phase angle about strain, frequency, and time were created to analyse the rheological characteristics of various bioink formulations. This thorough evaluation offered valuable information regarding the printability, mechanical stability, and viscoelastic characteristics of the hydrogels in contexts pertinent to bioprinting. 2.3.7 3D/4D Printing Process A scaffold is a 3D structure for culturing cells. The sustainability of the printed cells depends on the printed structures. For instance, the stress distribution is significantly better in dome structures compared to cubic structures (Pati et al.,2015). Till now, many authors have reported different types of scaffold design (Wang et.,2016;Reddy et al.,2015) architectures for 3D/4D printing for engineering applications. Figure 2 represents the schematic diagam of the 3D bio-printing process of Schwann cells, cancer cells and the co-cultured cells with the mixing of algiante-gelatin bioinks at temperature 29.5 degree celcius. We used an extrusion-based bioprinting process on the bioink. The bioink was loaded into the cartridge or syringe and then applied to the bioprinter. The bioink was extruded layer by layer with applied pressure to form a 3D structure. The extrusion parameters, such as printing speed, pressure, and nozzle height,porosity etc were optimized to ensure good printability. Basic Parameters for 3D/4D Bio-printing Scaffolds Design Parameters Porosity The maximum porosity will be used keeping the mechanical properties in constant Size of the pore 400 µm to 2000 µm Structure of the pore Various structures (circular, rectangular, hexagonal etc.) maximum connected Needle diameters 100-400 µm Gas pressures 0.08 to 0.26 MPa Number of Layers 1-10 Velocity 5 to 30 mm/s Height of the scaffolds 1 to10 mm Dimension 5×5×5 mm Temperature 25-33 Degree Celsius Cross-linker is used to bind the molecules internally as well as improve the mechanical properties and provide more stability (Discher et al.,2005). Based on the biopolymers, the cross-linker is used to modify the compositions. To crosslink alginate-gelatin 3D printed hydrogels, CaCl 2 was used as a crosslinking agent in the current work. Ca 2+ helps to crosslink the printed scaffolds. These crosslinked hydrogels are biodegradable and very useful for materials in the regeneration of medicine and tissue engineering applications. Ca 2+ provides mechanical stability and reduces the swelling of the hydrogels. 2.3.8 Cell Viability of the printed samples Cancer Cell line (H1975) and Schwann cell line (hTERT NF1 ipnNF95.11c, #CRL-3391) were purchased from ATCC The medium used for the cells cultured were DMEM, 10% FBS and 1.0% antibiotic following the general protocol of cell culture at the incubator of 37°C with humidified atmosphere in the culture flask/petri dish of 5% CO 2 within the cell. The cells were transferred into cryovials using DMSO and stored at -80°C for short-term use. For long-term storage, they were kept in liquid nitrogen vapour phase at -196 °C. 70% ethanol was used to sterilize all the other items used for these purposes. Trypan blue was used as a reagent to count the cells with a hemocytometer. The sub-cultures of the cells were performed using a standard protocol. The adherent cells were suspended in trypsin-EDTA solution. Schwann cells (hTERT NF1 ipnNF95.11c, #CRL-3391) and cancer cells (H1975) were first cultured in a cell culture flask/petri dish in a monolayer system. Following the same protocol, co-culture of H1975 and hTERT NF1 ipnNF95.11c, #CRL-3391 was executed in the monolayer system. After culturing and sub-culturing the cells in the subsequent phase three, the cells were cultured into the bulk hydrogels. Schwann cells, as well as cancer cells and the co-culture cells, were added to the cartridges before printing cells with the hybrid hydrogel. All the cartridges will be sterilized with 70% ethanol. After printing all the designed scaffolds, CaCl 2 , CaCl 2 was added to crosslink the models for 10 minutes. The printed hydrogel with cells was then kept in DMEM with 10% FBS and 1% antibiotic inside the petri dish. Finally, the Petri dish with printed hydrogels was kept at 37°C to maintain a physiological environment. 2.3.9 Optical Microscopic Image Analysis The evaluation of optical image of lyophilized hybrid samples was performed in order to figure out the tomography of the 3D printed hydrogel scaffolds. The OMI images of the prepared compositions of 1% Alginate (w/v) + 9% Gelatin (w/v), 3% Alginate (w/v) + 7% Gelatin (w/v), 4% Alginate (w/v) + 6% Gelatin (w/v) and 5% Alginate (w/v) + 5% Gelatin (w/v) with added 0.5M CaCl 2 were observed using optical microscope. The OMI was implemented the structures features of the scaffolds architectures. The printed scaffolds were lyophilization at -80 degree Celsius. The water was completely removed and then scaffolds was kept under the microscope to observe the topographical characteristics of the samples. The optical image of the hybrid hydrogels was taken to analysis the pore structures of the hydrogel. The actual strand size is the diameter of the printer nozzle diameter. The diameter of the used nozzle in the current experiment was 410 μm. The achievable strand size was 0.80 times of the nozzle diameter. 3. Experimental Result and Discussion 3.1 Chemical and molecular structure analysis by Fourier Transform Infrared Spectroscopy (FTIR) The development of hydrogel structures is influenced by multiple factors related to their three-dimensional network configuration. Gelatin forms complexes with ionic polysaccharides primarily through electrostatic attractions and hydrogen bonding. The ionic interactions among compatible functional groups facilitate the attachment of gelatin chain segments to sodium alginate chains. Nevertheless, the hydroxyl and carboxyl groups found in both gelatin and sodium alginate are not expected to participate in reactions with epoxy groups. The main organizing principle is based on the interaction between the structuring agent and the amino group present in the lysine residues of the gelatin chains (Maikovych et al.,2025). The mechanism that enhances the toughness of the SA-G gel was investigated using Fourier Transform Infrared Spectroscopy (FT-IR). Figures 4 (A) and 4(B) illustrate the FT-IR spectra for pure sodium alginate and gelatin, respectively. The spectrum of sodium alginate displays absorption peaks at 1600 cm − 1 and 1414 cm − 1 , corresponding to the symmetric and asymmetric stretching vibrations of the − COO − group in alginates. Similarly, the spectrum for gelatin displays peaks at 1632 cm − 1 and 1539 cm − 1 , which correspond to the C = O and C − N stretching vibrations found in the amide I band, along with the bending vibrations of the − NH group in the amide II band (Sartori et al.,1997) . Figures 4 (C–H) illustrate samples exhibiting different SA-G ratios that underwent crosslinking with CaCl 2 . The samples exhibit variations including shifts in peaks, modifications in peak shapes, and the emergence of new bands, all of which are affected by the hydrogel composition. For example, the absorption band around 3278 cm − 1 , associated with O–H stretching, exhibits broadening, a shift to higher wave numbers, and a reduction in intensity as the concentration of SA decreases. A decrease in intramolecular bonding can explain this phenomenon. The asymmetric stretching peak of the − COO − group at 1628 cm − 1 in the SA/G 5/5 sample, linked to the interaction between − COO − groups in SA and CaCl 2 , shifts to a broadband at 1631 cm − 1 (amide I, C = O, and C − N stretching) in the SA/G 1/9 sample, arising from the − NH 2 group in gelatin. Similarly, the symmetric stretching peak of − COO − at 1432 cm − 1 exhibits a shift to lower wave numbers in all samples, indicating the presence of ionic interactions between CaCl 2 and − COO − groups in SA. The results align with previous research conducted by Sartori and colleagues (Sartori et al., 1997 ) and Cathell and colleagues (Cathell et al., 2007) on CaCl 2 -crosslinked alginate thin films. Furthermore, the SA/G 1/9 sample exhibits the emergence of two new absorption bands at 1532 cm − 1 and 1244 cm − 1 , corresponding to amide II and III, respectively. The observed bands are indicative of C–N stretching coupled with N–H bending, a phenomenon that intensifies with an increase in gelatin content within the hydrogel. Moreover, with a decrease in SA concentration, the relative intensities of the C–C and C–O stretching bands at 1160 cm − 1 and 1028 cm − 1 progressively diminish, although their positions stay unchanged. The decrease in intensity is probably due to the weakening of these bonds as they adjust to the coordination structure around CaCl 2 ions (Sartori et al.,1997;Cathell et al.,2007;Saarai et al.,2013). From the above discussion, we conclude that the presence of an asymmetric COO- group indicates that alginate is crosslinked with Ca 2+ and confirms ionic bonding. In contrast, the presence of a symmetric COO- group confirms the formation of a Ca 2+ -alginate bioink, which plays a crucial role in the gelation process. The O-H stretching group indicates the presence of a hydrophilic group, which enhances cell viability. The amide II band confirms the protein secondary structure (gelatin), while the amide III band signifies the interaction between gelatin and the alginate network without chemical degradation. 3.2 Surface Structure Analysis By SEM The surface morphology of 3D hydrogels was examined through scanning electron microscopy (SEM). Since the SEM process requires sample dehydration, all specimens were subjected to lyophilisation as outlined in the Materials and Methods section (Mirek et al.,2022). Figure 5 displays SEM images at a magnification scale of 20 µm, emphasising the cross-section of the grid structure. The scaffold exhibited a remarkably porous and interconnected structure (Hashimi et al.,2021;Meligy et al.,2022), resulting from the drying process and the use of a high vacuum during sputter coating. The porous structure demonstrated benefits for cell adhesion, proliferation, and tissue development, as it improved nutrient diffusion and metabolic exchange within the scaffold (Abert et al.,2023). The analysis of the surface structure revealed that the scaffold's components are covalently bonded, forming a strong and interconnected network. The distinct separation between the scaffold's particles or structural units indicates a precisely defined and cohesive architecture (Khademi et al.,2025). From the images, a 3% alginate + 7% gelatin + 0.5 M CaCl 2 solution exhibits well-defined porous structures with interconnected pores, which are suitable for cell culture. The distribution of pores is more homogeneous than that of others, allowing for consistent crosslinking and preventing the formation of weak or necrotic regions in 3D culture. The porous structure of 3% alginate, 7% gelatin, and 0.5 M CaCl 2 permits cell migration, nutrient diffusion, and waste removal. The interconnected pores of this composition facilitate cell communication and promote the uniform distribution of cells throughout the composition. The presence of a smooth inner surface and uniform structure indicates more efficient mixing and crosslinking, rendering the composition advantageous for cell adhesion and proliferation. The structure of this composition of hydrogels exhibits a greater resemblance to the native extracellular matrix (ECM) due to its highly well-defined porous structure and interconnected pores, which promote significant cell viability, migration, and tissue formation. 3.3 Thermal Stability Analysis In the TG curve, it is observed that 10.91% of the total mass decayed at 192°C, 5.24% of the mass was lost between 192°C and 250°C, and 83.85% of the mass remained at 250°C. In gelatin, 13.73% of the total mass decayed at 161°C, 4.29% of the mass was lost at 161°C, and 82.98% of the mass remained at 250°C. 12.49% mass lost at 138.2°C and 2.32% mass degraded from 138.2°C and 84.88% mass remained in 1% SA + 9% G + 0.5 M CaCl 2 .11.22% mass lost at 139.02°C and 2.88% mass degraded from 139.02°C and 84.86% mass remained in 2% SA + 8% G + 0.5 M CaCl 2 .13.25% mass lost at 124.67°C and 2.73% mass degraded from 124.67°C and 84.02% mass remained in 3% SA + 7% G + 0.5 M CaCl 2 .13.47% mass lost at 155.15°C and 4.82% mass degraded from 155.15°C and 81.71% mass remained in 4% SA + 6% G + 0.5 M CaCl 2 .12.56% mass lost at 124°C and 3.92% mass degraded from 124°C and 83.52% mass remained in 5% SA + 5% G + 0.5 M CaCl 2 (Thakur et al.,2024). The DTA curve indicates the recorded temperatures for the endothermic and exothermic peaks as follows: Sodium alginate shows distinct peaks at 51.97°C and 159.09°C, respectively. The observed peaks for gelatin occur at 73.09°C and 211.78°C. The observed temperatures for the combination of 1% SA + 9% G + 0.5 M CaCl 2 are 79.18°C, indicating an endothermic reaction, and 194.54°C, which signifies an exothermic reaction. Similarly, the endothermic and exothermic peaks for the composition of 2% SA + 8% G + 0.5 M CaCl 2 are observed at 62.31°C and 194.17°C, respectively. In contrast, for the mixture of 3% SA + 7% G + 0.5 M CaCl 2 , these peaks are found at 57.70°C and 200.27°C. The peaks for the mixture consisting of 4% SA, 6% G, and 0.5 M CaCl 2 are observed at 57.29°C and 191.34°C. Finally, for the combination of 5% SA + 5% G + 0.5 M CaCl 2 , the observed temperatures are 60.90°C (endothermic) and 190.60°C (exothermic) (Thakur et al.,2024) Figure 8 presents the weight loss rates for different combinations: 1% Sodium Alginate (SA) + 9% Gelatin (G) + 0.5 M CaCl 2 , 2% Sodium Alginate (SA) + 8% Gelatin (G) + 0.5 M CaCl 2 , 3% Sodium Alginate (SA) + 7% Gelatin (G) + 0.5 M CaCl 2 , 4% Sodium Alginate (SA) + 6% Gelatin (G) + 0.5 M CaCl 2 , and 5% Sodium Alginate (SA) + 5% Gelatin (G) + 0.5 M CaCl 2 , Sodium Alginate (SA), Gelatin (G) with corresponding rates of 79, 65, 60.34, 60.65, 59.31, 51.7, and 75°C, respectively (Wang et al.,2023). From the above discussion, a 3% alginate + 7% gelatin + 0.5 M CaCl 2 composition is more suitable for cell culture, as it exhibits greater thermal stability and displays the highest exothermic peak (200.27°C). The highest exothermic peaks indicate a good crosslink structure. The mass loss (13.25%) of 3% alginate + 7% gelatin + 0.5 M CaCl 2 is moderate compared to other compositions, which signifies good hydration and importance for supporting a moist, cell-friendly environment for cell culture. The structural resilience of this composition is higher than that of other compositions due to the presence of a higher rate of remaining mass (84.02%), which indicates strong resistance to the breakdown of the composition. The DTG curve of this composition exhibits a smoother profile, indicating a uniform polymer blend that is suitable for consistent 3D bioprinting and reliable gel performance. Figure 9 illustrates the stages of swelling of scaffolds at various periods for all compositions. Figure 10 represents the results of the swelling test for all compositions. The recorded average swelling percentages at the 1-hour mark for the different concentrations were as follows: 1% SA − 9% G yielded 1237.24%; 2% SA − 8% G produced 627.84%; 3% SA − 7% G showed 506.364%; 4% SA − 6% G reached 440.183%; and 5% SA − 5% G resulted in 269.607%. The compositions demonstrated a progressive increase, reaching the peak mean swelling percentage at the 18-hour mark. The recorded values are 1776.94%, 1232.17%, 1164.27%, 1105.01%, and 908.48%, which correspond to 1% SA -9% G, 2% SA -8% G, 3% SA -7% G, 4% SA -6% G, and 5% SA -5% G respectively. The average swelling percentage started to decline at the 24-hour mark. The recorded values are 1545.18%, 1029.96%, 996.14%, 919.52%, and 800.89% for the respective categories of 1% SA -9% G, 2% SA -8% G, 3% SA -7% G, 4% SA -6% G, and 5% SA -5%. The p-values for 1% SA-9% G and 2% SA-8% are both p < 0.0001, demonstrating a highly significant level. 3% SA-7% G (p = 0.0003), 4% SA-6% G (p = 0.0005), and 5% SA-5% G (p = 0.0017) demonstrate reduced significance relative to the earlier findings. The findings indicate that increased Sodium alginate (SA) concentration diminishes the average swelling percentage. According to Fig. 10 ,2% alginate + 8% gelatin + 0.5M CaCl 2 and 3% alginate + 7% gelatin + 0.5M CaCl 2 are good for cell culture and bioprinting. 2% alginate + 8% gelatin + 0.5M CaCl 2 composition shows strong statistical significance but 3% alginate + 7% gelatin + 0.5M CaCl 2 exhibits less strong statistical significance. Both compositions exhibit moderate discrepancies; however, the 3% alginate + 7% gelatin + 0.5 M CaCl 2 swelling rate is notably more moderate and mechanically suitable than those of the others. A higher gelatin amount and a lower alginate amount indicate the printed structure is more viscous. On the other hand, lower gelatin amount and higher alginate amount indicate that the printed structure is more solid. For this reason, 3% alginate + 7% gelatin + 0.5 M CaCl 2 is not so viscous and not so solid. Hence,3% alginate + 7% gelatin + 0.5 M CaCl 2 printed structure is more suitable for cell culture and bioprinting than 2% alginate + 8% gelatin + 0.5M CaCl 2 and others. 3.5 Optimization for 3D/4D printed scaffolds 3.5.1 Analysis of the elastic, viscous and the damping characteristics of the samples Keeping the 3D printed hybrid scaffolds under the oscillatory force and measuring the response, the elastic, viscous and the damping characteristics of the samples have been measured. The time scale of this test is frequency of oscillation. Strain, phase angles are measured from a range of frequencies by the applied sinusoidal stress. The storage modulus, G ′ = \(\:{\sigma\:}_{0}\frac{\text{cos}\delta\:}{{\gamma\:}_{0}}\) ; (3.5.1.1) where δ is the phase angle, \(\:{\sigma\:}_{0}\) is the component of stress to stress ratio in phase and \(\:{{\gamma\:}}_{0}\) is the strain to strain ratio in phase. Similarly, G ″ = \(\:{\sigma\:}_{0}\frac{\text{Sin}\delta\:}{{\gamma\:}_{0}}\) ; (3.5.1.2) It is noted that for perfect elastic materials G″=0, where δ = 0 i.e. the wave forms are in phase. For perfect viscous material, the stress and the strain waveforms are completely out of phase. i.e., δ = 0, G′=0 and hence, G″ is finite. The loss factor, tan δ = G″/G′ (3.5.1.3) Figure 11 (a) illustrates the relation of storage modulus(G′) and loss modulus (G″) of 3D printed hydrogel scaffold of 1% alginate and 9% gelatin with crosslinker 0.5MCaCl 2 over strain amplitudes. The storage modulus(G’) gradually decreases conversely, the loss modulus gradually increases. The storage and loss modulus of the hydrogel measured at constant temperature materials of the hydrogel show solid fluid like behavior until it reaches to the critical strain. The critical strain for polymer materials is near to 1 in log scale. The critical strain point is also known as the gel point. In the gel point maximum crosslinking is happened of the polymers. After the gel point the storage modulus is less the loss modulus. In this stage, the 3D alginate gelatin hydrogel breaks up the 3D structure and starts to behave as fluid materials. The tangent of the materials is increasing slowly but it is almost strain independent. Figure 11 (b) shows the frequency sweep graph of the 3D printed hydrogel scaffold. Only up to 100 rads − 1 the elastic modulus was measured. Within this short range it is found that the storage modulus is greater than the loss modulus and the storage modulus is almost frequency independent. The hydrogel scaffolds show this property below the critical strain or gel point. This property indicates that the printed hydrogel scaffolds is solid gel like. Figure 11 (c) shows the time sweep of the hydrogel. Within very short time the there is no change of the storage and loss modulus. The tangent is also time independent within this short time. Figure 11 (d-f) illustrates the rheological characteristics of 2% Alginate (w/v) + 8% Gelatin (w/v) + 0.5M CaCl 2 of the 3D printed hydrogel scaffolds. According to Fig. 11 (d) the storage modulus is greater than the loss modulus and phase angle is increasing gradually. After the gel point the phase angle is increased as the loss modulus is greater than the storage modulus at this stage. Gel point is greater than 1 in log scale. The incorporation of alginate 2% alginate and 8% gelatin, the rheological properties has increased. Figure 11 (e) represents G′, G″ and phase angle over frequency. The storage and loss modulus are measured up to 10KHz in log scale. Within this frequency range G′ is always greater than G″. The time sweep curve shows that within the short period there is no change of storage and loss modulus. Figure 11 (g-i) demonstrate the rheological characteristics of 3% Alginate (w/v) + 7% Gelatin (w/v) + 0.5M CaCl 2 of the 3D printed hydrogel scaffolds. From Fig. 11 (g), it is seen that the gel point of the 3D printed hydrogel is slightly greater than 1 in log scale. Up to the gel points the hybrid hydrogel shows the linear elastic behavior and it is in jelly like solid materials. After the gel points with increasing the strain the 3D structures of the materials will collapse and G’’ will be larger than the G’ and the 3D printed hydrogel will behave like fluid materials. Figure 11 (h) shows the frequency response storage and loss modulus within the gel point. The storage modulus is always higher than the loss modulus. Both of G′ and G″ is independent of frequency within this frequency range. Figure 11 (i) represents the time sweep graph and it is clearly seen that there is no change of G′ and G″ within this small time. Figure 11 (j-l) demonstrate the rheological characteristics of 4% Alginate (w/v) + 6% Gelatin (w/v) + 0.5M CaCl 2 of the 3D printed hydrogel scaffolds. According to the Fig. 11 (j), the gel point of the hydrogel is greater than 1 in log scale. The 3% alginate and 7% gelatin has increased both the storage and loss modulus of the binary hybrid polymers hydrogels scaffolds. Up to the gel point the materials is solid jelly like and very much suitable for used as a bio-ink for DIW bio-printing. After the gel points the materials start to behave like fluid like behavior. Figure 9 (k) represent the frequency response G′, G″ and phase angle graph. Within 10KHz log scale frequency range, G′ is always greater than G″ and it is almost frequency independent. Figure 11 (l) is time sweep curve and it is seen that within small time there is no change of G′ and G″. Figure 11 (m-o) elucidate the rheological characteristics of 5% Alginate (w/v) + 5% Gelatin (w/v) + 0.5M CaCl 2 of the 3D printed hydrogel scaffolds. According to the Fig. 11 (m), it is observed that up to the critical strain, the G’ and G’’ both are straight line, i.e. the alginate-gelatin binary composites shows elastic behavior and it is jelly-like solid materials. This property indicates that this composite can be used as a bio-ink. With increasing the proportion of the alginate and decreasing the gelatin the stiffness has increased than the previous proportions. Figure 11 (n) is the frequency sweep curve and it is observed that G′ is greater than the G″ and independent of frequency i.e. the materials jelly like solid materials and can be used as a bio-inks. Figure 11 (o) indicates the time sweep and it is found that there is no change of G′ and G″ within this time period. Although this time is very small with compare to the experimental time of the 3D printed hydrogel scaffolds. Table 1 Storage modulus, loss modulus and loss tangent of alginate-gelatin hydrogels with 0.5M CaCl 2 cross-linker Composites Cross-linker G′ (KPa) G″ (KPa) tanδ 5% Alginate + 5% Gelatin Calcium Chloride 10 minutes 16.2 5.4 0.34 4% Alginate + 6% Gelatin 13.1 6.0 0.46 3% Alginate + 7% Gelatin 10.02 3.0 0.29 2% Alginate + 8% Gelatin 3.0 0.6 0.21 1% Alginate + 9% Gelatin 2.0 0.9 0.45 The loss tangent of the developed bio-ink hydrogels are an important parameters for developing the desired bio-inks for 3D bio-printing. The loss tangent mentioned in the table-2 is from 0.21 to 0.46. The loss tangent value is 0.20 to 0.45 is suitable for meaningful unique bio-printing (Liu et al.,2019). Hence 4% alginate and 6% gelatin is not significant for 3D bioprinting. The composition 3% alginate + 7% gelatin + 0.5M CaCl 2 shows excellent viscoelastic balance among them. Balance viscoelasticity mimics native extracellular matrix (ECM), which plays a crucial role in stem cell fate decision and cell-matrix interactions. Optimal viscoelasticity supports long cell viability and proliferation. Thus, we conclude that balanced viscoelasticity improves the relevance of in vitro models and implanted cells. 3.5.2 Cells Viability of 2D/Monolayer System The MTT is used to identify the toxicity of the materials. The cells viability of H 1975, #CRL 3991 and the co-culture of both of them in 3% alginate and 7% gelatin bio-inks and 0.5M Calcium chloride have been determined by MTT without counting cells in details. In this process, indissoluble formazan is made from water soluble MTT. The insoluble formazan turns into the solubilized one and then then the absorption of the concentration is measured at the different wavelengths using molecular devices the Fluorescence Microplate Readers. The cells viability of cancer cells (H1975), Schwann cells (hTERT NF1 ipnNF95.11c, #CRL-3391) and the co culture of two cells are conducted to figure out the behavior of cells in 2D monolayer of hydrogels (3% alginate and 7% gelatin ). The cells density used in the current experiment is listed in table-2. Table 2 Cells density used for 2D/monolayer cultures Sl. Cells name Cells per ml Cells per ul Cells used per well of 96 plates 1 H1975 1.32×10 6 1320 8ul 2 #CRL-3391 2.1×10 5 210 48ul 3 H1975 & #CRL-3391 2.12×10 6 2120 5ul For executing the MTT experiment, the medium was removed and washed the wells with PBS. 100ul new fresh medium was added with the adherent cells and then 10ul of mM MTT stock solution injected into the medium. The treated cells were kept warm for four hours at 37°C. After incubating, 100ul of SDS solution was added in each of the plates and pipetting for mixing thoroughly for further incubating in humidified chamber for four hours. The samples were mixed again after incubating with the pipettes. The absorbance was read at 570nm using the Fluorescence Microplate Readers. Table 3 Cells survival rate (in percentage) of H1975, #CRL 3991 and the culture of H1975 and #CRL 3991 Sl Cells Name Live Cells Dead Cells Day 1 Day 3 Day 7 Day 1 Day 3 Day 7 1 H1975 (Cancer) 82.26 93.88 91.68 17.74 6.12 8.32 2 #CRL 3991 84.18 87.69 82.84 15.82 12.31 17.16 3 of H1975 & #CRL 3991 79.76 85.69 85.30 20.24 14.31 14.70 The measured absorbance values using wavelength 570 nm cells viability as shown in the Table 3 . Cells viability measurement: Cell viability = \(\:\frac{{A}_{t}-{A}_{Blank}}{{A}_{c\:}-{A}_{Blank}}\) ×100%; Where, A t =Absorbance of the treated samples A c = Absorbance of the control samples A Blank = without cells only medium presents The multi-label micro reader measured the intensity of the emitting light of the treated, control cells and the blank. Figure 12 presents the cells viability of cancer cells, Schwann cell and the co-cultures of both of the cells at different days.The number of live H1975, (Cancer) #CRL 3991(Schwann), the co-culture cells at the day 1 are 82.26, 84.18 and 79.76 at day 3 93.88, 87.69 and 85.69 and 91.68 82.84, 85.30 at day 7. The maximum densities of the cells show at day 3. For H1975, the cells grow rapidly and the optimal densities at 48 to 72 hours. At day 7, the cells proliferation reduced and onward. For # CRL 3991cells the maximum survival rate is at day 3 to day 4 and the proliferation rate increased up to day 7. 3.5.3 Cells Proliferation of 3D System by Live-dead Assay by Fluorescence Microscopy Figure 13 (A) shows the schematic diagram of 3D bio printing of alginate-gelatin hydrogel. 3D cubic structures of layer 1 to 3 were printed by setting the fundamental parameters of nozzle diameters 410µm, speed 10mm/s, pressure 1.5 to 2.2 KPa and the temperature 29.5°C. The solvent of the hydrogel were DMEM, FBS (10%) and antibiotic (1%). All the scaffolds were submerged into 0.5M calcium chloride (CaCl 2 ) cross linker for 10 minutes. After completion of the gelation process, the extra cross linkers was removed by washing PBS. Before printing the cells with the alginate-gelatin hydrogel, the cells were subcultures 3 times for getting the normal level of the cells. The cells density of H 1975 and #CRL-3991 are mentioned in Table 5 . After printing the cells with hydrogels the cells laden printed scaffolds were put into the calcium chloride cross-linker for 10 minutes. Fresh medium was added after washing with PBS. At 37°C, 5% CO 2 the 3D printed cells laden hydrogels were incubated for further treatment. The cells proliferation rate is measured by following the MTT absorbance data at wavelength 570nm. H 1975 cells proliferation rate is observed maximum at day 3 and after the following days the cells proliferation rate decreased which is observed at day 7. The cells #CRL-3991 shows the highest proliferation at day 3 but the proliferation increases with increasing times. From Fig. 13 (B) it is clearly seen that the proliferation of H 1975 and #CRL-3991 cells in the alginate hydrogels is very much significant at the cells survivability is around 90%. Table 4 Cells density used for 3D Bio-printing Sl. Cells name Cells per ml Cells per µl Cells used in 5ml hydrogels 1 H1975 1.32×10 6 1320 250 µl 2 #CRL-3391 2.1×10 5 210 1ml The live dead image of the H 1975 cells of 3D printed scaffold observed using fluorescence optical microscopy. The kits of two colors of wavelength for live represents green and the absorbance- emission spectral wave lengths were 488nm and 515nm and for dead cells the corresponding image was red the emission excitation wavelength was 570nm and emission wavelength was 602nm. The kit is purchased from Thermo Fisher SCIENTIFIC. Following the general protocol, the cells laden 3D bio printed scaffolds were stained and live dead image images were taken. Figure 13 (C) shows the live cells image which is represented by green colors. The live cells show the clear fluorescence indication. Figure 13 (D) represents the Live-Dead cells. The green colors is representing the live H-1975 cells and the red color represents the dead H 1975 cells. Figure 13 (E) represents solely the dead cells indicated by red color. The live dead images were taken at day 10 using fluorescence microscopy. 3.5.4 Optical Image Analysis The table below shows the average strand size as well as the average pore size of the printed hydrogels. Table 5 Optical characteristics of the 3d printed hybrid scaffolds Composites Strands size(um) Pore size (um) 1% Alginate (w/v) + 9% Gelatin (w/v) + 0.5M CaCl 2 414.14 880.577 2% Alginate (w/v) + 8% Gelatin (w/v) + 0.5M CaCl 2 301.25 923.23 3% Alginate (w/v) + 7% Gelatin (w/v) + 0.5M CaCl 2 294.62 854.66 5% Alginate (w/v) + 5% Gelatin (w/v) + 0.5M CaCl 2 317.37 883.48 The morphological structure of the 3D bio-printed scaffolds is porous and no significant difference of the same composite. But for different composites the porosity varies. Computer Aided Design (CAD) model of stl file have different porosities were used to fabricate hydrogel structures. 1 to 13 layers successfully. The porosity of the highest layer was higher than the successive printed lower one. With increasing the printing layers the swelling of the lower increased due to the gravitational force. It was found that for layer 1 to 7 the porosity was no significant change. The printed structures have proper channel to pass oxygen and nutrition within the microenvironment of the individual tissue to nourish it for surviving inside the scaffolds. Porosity is the most important parameters for increasing the cell numbers and for the viability of the cells. The printed scaffolds need sufficient space for growing the cells successfully. The structure of the scaffolds need to protect from collapse, hence to choice the scaffolds in the experiment is good for biomedical applications. 4% sodium alginate + 6% gelatin + 0.5M CaCl 2 sample was not visible in the optical microscope due tan𝛅 value exceeding 0.45. 4. Conclusion In conclusion, the assessment of alginate gelatin hydrogel bioinks with CaCl 2 crosslinker revealed their enormous potential for 3D/4D bioprinting applications, as indicated by various physical, chemical, mechanical, and biological inspections. In this context, CaCl 2 is an effective crosslinking agent that improves the mechanical strength of the solutions. The FTIR analysis aligns with the intended bond formation. FTIR results validated the efficient mixture of alginate and gelatin through distinct peaks that correspond to hydroxyl, carboxyl, and amide groups, which indicate intermolecular interactions. The SEM results of all compositions demonstrated a well-structured porous microstructure network that facilitates effective nutrient diffusion and cell propagation. The TGA, DTA, and DTG analyses indicate that this material exhibits excellent the rmoresponsive properties and strong thermal stability, which directly influence nutrient diffusion and cellular viability. The findings from the swelling test suggest that the SA-G composition possesses excellent swelling characteristics. Swelling characteristics provided an optimal and hydrated microenvironment for cell survival and growth. The evaluation of rheological properties, including storage modulus, loss modulus, and phase angle, revealed shear thinning behavior across strain, frequency, and time sweeps, indicating structural and printed stability. Phase angle maintained below 45 degrees, indicating solid-like properties of the gels, which is essential for preserving shape fidelity post-printing. The cell viability of developed bioink’s compositon indicate the non-toxicity of the bioink composition. Cell viability assays demonstrated significant biocompatibility with Schwann cells, cancer cells, and co-cultures, indicating robust cellular adhesion, proliferation, and metabolic activity within the printed constructs. Co-culture systems exhibited improved cell-cell interactions and diverse tissue modeling. The 3D bioprinted structures maintained their geometrical integrity with minimal deformation, demonstrating outstanding shape retention and structural stability. Optical microscopy validated the consistent formation of strands, the presence of interconnected pores, and the distribution of cells within the hydrogel matrix. Live-dead staining effectively demonstrated the presence of viable cell populations throughout the layers, confirming the supportive microenvironment established by the bioink. The co-culture system demonstrated synergistic effects, indicating its potential application in intricate tissue modeling. The collective findings confirm that alginate-gelatin hydrogels satisfy essential criteria for bio-fabrication, providing a stable, cell-supportive, and customizable platform for advanced 3D/4D bioprinting. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7163757","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":488782115,"identity":"83ae9bf3-c9c1-4116-9dd1-be160de073ce","order_by":0,"name":"Mohaimenul Khan","email":"","orcid":"","institution":"Khulna University of Engineering \u0026 Technology","correspondingAuthor":false,"prefix":"","firstName":"Mohaimenul","middleName":"","lastName":"Khan","suffix":""},{"id":488782116,"identity":"a3e430b8-c14e-4494-a63d-e4ffac60f19a","order_by":1,"name":"Md Alamgir Hossain","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYBACPmYQWcGQwMDABhPjYZDAp4WNGaTnDExLAjFaGIBaGNtI0sLOf/Bx4bw7eQYH2NIkPv5gkOdv4D14g4DDmI1nbntWDNRyTHJGAoPhjAN8yRYEtLBJ8247nLjhAHubNE8CA+MGBh4zQt4HapkD1fIngcGeSC0NIC1sx6SB3k8kRouxMc+xZ4kzD7MlW/akSSTPOEzAL/z8Bx8+5qm5k9h3vM3wxg8bG9v+9l78IQYFBxgUDoMZQCcxE6EerEW+gTiVo2AUjIJRMAIBANpBP6lL+/v8AAAAAElFTkSuQmCC","orcid":"","institution":"Khulna University of Engineering \u0026 Technology","correspondingAuthor":true,"prefix":"","firstName":"Md","middleName":"Alamgir","lastName":"Hossain","suffix":""}],"badges":[],"createdAt":"2025-07-19 10:23:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7163757/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7163757/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87288758,"identity":"af82af10-be99-422b-a278-8ef07aa5567d","added_by":"auto","created_at":"2025-07-22 11:07:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":566980,"visible":true,"origin":"","legend":"\u003cp\u003eSodium alginate-gelatin bio-ink preparation method\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7163757/v1/2b19844fdbf382afb50ad121.png"},{"id":87288759,"identity":"0c038b1a-7878-477a-aad4-9b5b1c6e01d0","added_by":"auto","created_at":"2025-07-22 11:07:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":552281,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of 3D Bio-printed process with Schwann cells, Cancer cells and the co culture cells with nozzle diameter 410μm, speed 10mm/s, pressure 1.5-2.2 KPa at near the sol-gel temperature of 29.50°C.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7163757/v1/2108e17a33c421ea6bb1eeb3.png"},{"id":87289926,"identity":"e8550e32-b5d9-49f4-930f-d35963288500","added_by":"auto","created_at":"2025-07-22 11:23:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":303524,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of combination of alginate-gelatin hydrogel synthesis (Maikovych et al.,2025).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7163757/v1/439d412d607a541a5d4f8eef.png"},{"id":87288762,"identity":"d0dfbf19-a3c6-48bd-8c98-6a3670a975c6","added_by":"auto","created_at":"2025-07-22 11:07:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":349391,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectra of sodium alginate hydrogel bioinks (A) Sodium Alginate (SA) (B) Gelatin (G), (C) 1% Sodium Alginate (SA) + 9% Gelatin (G) + 0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e, (D) 2% Sodium Alginate (SA) + 8% Gelatin (G) + 0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e, (E) 3% Sodium Alginate (SA) + 7% Gelatin (G) + 0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e, (F) 4% Sodium Alginate (SA) + 6% Gelatin (G) + 0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e, (G) 5% Sodium Alginate (SA) + 5% Gelatin (G) + 0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e (H) region of chemical bond of all compositions\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7163757/v1/eede87138499dfdc685b73c2.png"},{"id":87289928,"identity":"09869831-aa93-4a48-b231-5b0813bc2f06","added_by":"auto","created_at":"2025-07-22 11:23:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":905818,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological structure of sodium alginate-gelatin hydrogel bioinks (A) Sodium Alginate (SA) (B) Gelatin (G) (C)\u0026nbsp; 1% Sodium Alginate (SA) + 9% Gelatin (G) + 0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e, (D) 2% Sodium Alginate (SA) + 8% Gelatin (G) + 0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e, (E) 3% Sodium Alginate (SA) + 7% Gelatin (G) + 0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e, (F) 4% Sodium Alginate (SA) + 6% Gelatin (G) + 0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e, (G) 5% Sodium Alginate (SA) + 5% Gelatin (G) + 0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7163757/v1/6bda3384127672ac561f7dce.png"},{"id":87288767,"identity":"11b5d568-9509-4e65-8f82-fbeace310c65","added_by":"auto","created_at":"2025-07-22 11:07:09","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":198400,"visible":true,"origin":"","legend":"\u003cp\u003eTG analysis of sodium alginate-gelatin hydrogel bioinks (i) Sodium Alginate (SA), (ii) Gelatin (G) (iii) 1% Sodium Alginate (SA) + 9% Gelatin (G) + 0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e, (iv)2% Sodium Alginate (SA) + 8% Gelatin (G) + 0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e, (v) 3% Sodium Alginate (SA) + 7% Gelatin (G) + 0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e, (vi) 4% Sodium Alginate (SA) + 6% Gelatin (G) + 0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e, (vii) 5% Sodium Alginate (SA) + 5% Gelatin (G) + 0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7163757/v1/04ddaa23a2fd05bf8a692751.png"},{"id":87289542,"identity":"46ec458c-9e70-4030-9284-04edc0641ae4","added_by":"auto","created_at":"2025-07-22 11:15:09","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":146660,"visible":true,"origin":"","legend":"\u003cp\u003eDTA analysis of sodium alginate-gelatin hydrogel bioinks (i) Sodium Alginate (SA), (ii) Gelatin (G) (iii) 1% Sodium Alginate (SA) + 9% Gelatin (G) + 0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e, (iv)2% Sodium Alginate (SA) + 8% Gelatin (G) + 0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e, (v) 3% Sodium Alginate (SA) + 7% Gelatin (G) + 0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e, (vi) 4% Sodium Alginate (SA) + 6% Gelatin (G) + 0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e, (vii) 5% Sodium Alginate (SA) + 5% Gelatin (G) + 0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7163757/v1/4b071742e5ca730bd3dc0ba5.png"},{"id":87288763,"identity":"13c4fb8d-86db-4d94-a643-6bf06555dfa8","added_by":"auto","created_at":"2025-07-22 11:07:09","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":60001,"visible":true,"origin":"","legend":"\u003cp\u003eDTG Curve of sodium alginate-gelatin hydrogel bioinks (i) Sodium Alginate (SA), (ii) Gelatin (G) (iii) 1% Sodium Alginate (SA) + 9% Gelatin (G) + 0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e, (iv)2% Sodium Alginate (SA) + 8% Gelatin (G) + 0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e, (v) 3% Sodium Alginate (SA) + 7% Gelatin (G) + 0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e, (vi) 4% Sodium Alginate (SA) + 6% Gelatin (G) + 0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e, (vii) 5% Sodium Alginate (SA) + 5% Gelatin (G) + 0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7163757/v1/7b48402dc0badbaec5aa3881.png"},{"id":87289545,"identity":"39d63f71-2ff2-4b95-8bf7-991514469b3d","added_by":"auto","created_at":"2025-07-22 11:15:09","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":885461,"visible":true,"origin":"","legend":"\u003cp\u003ePhases of the sodium alginate-gelatin bioinks at different periods (i) Sodium Alginate (SA), (ii) Gelatin (G) (iii) 1% Sodium Alginate (SA) + 9% Gelatin (G) + 0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e, (iv)2% Sodium Alginate (SA) + 8% Gelatin (G) + 0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e, (v) 3% Sodium Alginate (SA) + 7% Gelatin (G) + 0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e, (vi) 4% Sodium Alginate (SA) + 6% Gelatin (G) + 0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e, (vii) 5% Sodium Alginate (SA) + 5% Gelatin (G) + 0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-7163757/v1/7e33a11bc22005ad670c33fd.png"},{"id":87288765,"identity":"4e51441d-f531-4a2f-bb6f-8eaeae91babb","added_by":"auto","created_at":"2025-07-22 11:07:09","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":51518,"visible":true,"origin":"","legend":"\u003cp\u003eMean swelling percentage of sodium alginate-gelatin hydrogel bioinks (a) 1% Sodium Alginate (SA) + 9% Gelatin (G) + 0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e, (b) 2% Sodium Alginate (SA) + 8% Gelatin (G) + 0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e, (c) 3% Sodium Alginate (SA) + 7% Gelatin (G) + 0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e,\u0026nbsp; (d) 4% Sodium Alginate (SA) + 6% Gelatin (G) + 0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e, (e)5% Sodium Alginate (SA) + 5% Gelatin (G) + 0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e with one sample t test by using GraphPad Prism 10.2.0 (392)\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-7163757/v1/d82c564d5df244ee5d197621.png"},{"id":87288775,"identity":"f8f71aef-e4fd-429f-97ca-d5652e88a297","added_by":"auto","created_at":"2025-07-22 11:07:09","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":655116,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Storage (G′) and loss modulus (G″) and phase angle (θ) against strain (b) G′, G″ and phase angle (θ) over frequency (c) G′, G″ and phase angle (θ) against time of the 1 % Alginate (w/v) + 9% Gelatin (w/v) + 0.5M CaCl\u003csub\u003e2\u003c/sub\u003e of the 3D printed hydrogel scaffolds.\u003c/p\u003e\n\u003cp\u003e(d) Storage (G′) and loss modulus (G″) and phase angle (θ) against strain (e) G′, G″ and phase angle (θ) over frequency (f) G′, G″ and phase angle (θ) against time of the 2% Alginate (w/v) + 8% Gelatin (w/v) + 0.5M CaCl\u003csub\u003e2\u003c/sub\u003e of the 3D printed hydrogel scaffolds.\u003c/p\u003e\n\u003cp\u003e(g) Storage (G′) and loss modulus (G″) and phase angle (θ) against strain (h) G′, G″ and phase angle (θ) over frequency (i) G′, G″ and phase angle (θ) against time of the 3% Alginate (w/v) + 7% Gelatin (w/v) + 0.5M CaCl\u003csub\u003e2\u003c/sub\u003e of the 3D printed hydrogel scaffolds.\u003c/p\u003e\n\u003cp\u003e(j) Storage (G′) and loss modulus (G″) and phase angle (θ) against strain (k) G′, G″ and phase angle (θ) over frequency (l) G′, G″ and phase angle (θ) against time of the 4% Alginate (w/v) + 6% Gelatin (w/v) + 0.5M CaCl\u003csub\u003e2\u003c/sub\u003e of the 3D printed hydrogel scaffolds.\u003c/p\u003e\n\u003cp\u003e(m) Storage (G′) and loss modulus (G″) and phase angle (θ) against strain (n) G′, G″ and phase angle (θ) over frequency (o) G′, G″ and phase angle (θ) against time of the 5% Alginate (w/v) + 5% Gelatin (w/v) + 0.5M CaCl\u003csub\u003e2\u003c/sub\u003e of the 3D printed hydrogel scaffolds.\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-7163757/v1/f1e6310f19c0bfc03283d008.png"},{"id":87289547,"identity":"9f4bc3b8-fe95-4a76-843e-ddd491a5a233","added_by":"auto","created_at":"2025-07-22 11:15:09","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":449640,"visible":true,"origin":"","legend":"\u003cp\u003eCells viability of cancer cells (H1975), 4.2×105/ml Schwann cells (hTERT NF1 ipnNF95.11c, #CRL-3391) and, the co-culture of the two cells. Figure shows the cells viability measurements of H 1975, #CRL-3991 \u0026amp; co-culture of the two cells at different days. The control is present for comparing with lives and dead cells.\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-7163757/v1/3da1fae5c0f6428b6604591b.png"},{"id":87289933,"identity":"8f0c1842-f301-4c1c-8af8-50348aa84e40","added_by":"auto","created_at":"2025-07-22 11:23:09","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":1150757,"visible":true,"origin":"","legend":"\u003cp\u003e(A-E) Proliferation of 3D bio printing alginate-gelatin hydrogel with embedded H1975, #CRL-3991 and the co-cultures of them after day 10. Figure (A) shows the successive steps of the 3D bio-printed 3 layers with laden cells of H -1975, #CRL 3991 and the mixtures of the cells. Figure (B) represents cells proliferation rates at different days respectively. Figure (C) fluorescence microscopic image of H 1975 cells. Figure (D) shows the live –dead image of H 1975 and Figure (E) represents the fluorescence microscopic image of dead cancer cells.\u003c/p\u003e","description":"","filename":"13.png","url":"https://assets-eu.researchsquare.com/files/rs-7163757/v1/14c07182fa09657010dbb646.png"},{"id":87289935,"identity":"d97dbd90-bd58-4529-9279-81dd304d1916","added_by":"auto","created_at":"2025-07-22 11:23:09","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":1590414,"visible":true,"origin":"","legend":"\u003cp\u003eOptical image of lyophilized hybrid samples of sodium alginate-gelatin hydrogel bioinks (A-D): 1% Alginate (w/v) + 9% Gelatin (w/v), 2% Alginate (w/v) + 8% Gelatin (w/v), 3% Alginate (w/v) + 7% Gelatin (w/v) and 5% Alginate (w/v) + 5% Gelatin (w/v) with added 0.5M CaCl\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"14.png","url":"https://assets-eu.researchsquare.com/files/rs-7163757/v1/323b9d1eda5e5c430ac9eb5e.png"},{"id":89242065,"identity":"d8a25887-8bd5-4e6e-bf5a-0c36054c7864","added_by":"auto","created_at":"2025-08-17 20:31:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10108934,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7163757/v1/19494ebc-126c-411d-b525-93c8eb7455da.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Alginate-Gelatin Composite Hydrogels for Next-Generation 3D Bio-Printing in Tissue Engineering","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eBioink plays a crucial role in 3D bioprinting. Groll and colleagues (Groll et al., 2018) describe bioinks as a mixture of cells that can be processed using automated biofabrication technologies, which may also include biologically active components and biomaterials (Li et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Bioink materials enable scientists to create complex biological structures by manipulating living cells and their environments (Ozkerim et al., 2018). Contemporary bioinks are being engineered to enhance significantly both printability and biocompatibility (Kyle et al., 2004). This is accomplished by meticulously regulating a variety of physical, chemical, and biological characteristics.\u003c/p\u003e\u003cp\u003eAn ideal ink must meet the biological requirements concerning cell compatibility, as well as the physical and mechanical demands of the printing process (Chung et al.,2013). It should exhibit excellent printability, robust mechanical strength, and stability (Mobaraki et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The ink must demonstrate gel-like properties or possess adequate viscosity to be dispensed as a self-supporting filament (Barrulas et al.,2023). Nonetheless, if the gel exhibits excessive strength, the substantial shear forces required to expel the ink may lead to cell mortality and the gel's fracturing (Kong et al.,2003). Bioinks must be consistent with suitable bioprinting methods to create functional living structures that exhibit the necessary biological and mechanical properties. Bioinks must provide a non-toxic extracellular matrix environment that facilitates cell adhesion, communication, growth, and differentiation, while also ensuring high cell viability after printing (Zhang et al.,2018). In certain instances, it will be suitable for the scaffold to degrade in a controlled manner over time (Chung et al.,2013). The degradation rate of bioinks, assessed through time or remaining mass, is influenced by external conditions such as temperature, pH, enzyme presence, and vibration, as well as internal factors including the scaffold\u0026rsquo;s chemical composition, the incorporation of nanoparticles or nanofibers, polymer chain length, and any surface modifications applied to the scaffold (Shokrani et al.,2022).\u003c/p\u003e\u003cp\u003eHydrogels are polymeric materials frequently used in tissue engineering due to their low cytotoxicity and structural resemblance to the extracellular matrix (ECM) (Frampton et al.,2011). Hydrogels are recognised as optimal materials for biomedical applications due to their physical properties closely resembling those of the physiological tissue environment. Hydrogel is a substance composed of a three-dimensional polymer network that retains a significant amount of water, typically exceeding 90% (Chen et al.,2023). The extensively hydrated network structure facilitates the exchange of gases and nutrients, making it as a compelling choice for developing inks used in bioprinting. The combination of hydrogels presents an opportunity to merge the unique properties of each hydrogel component, allowing for the customization of the overall hydrogel to meet specific needs (Rosellini et al.,2009;Dong et al.,2006;Chung et al.,2013).\u003c/p\u003e\u003cp\u003eCross-linking serves as a stabilization mechanism in polymer chemistry, facilitating the extension of polymer chains in various directions to create a network structure (Maitra et al.,2014). A cross-link refers to a bond, which can be either ionic or covalent, that connects one polymer chain to another. The effectiveness of cross-linking is influenced by various factors, including the pH of the reaction, the type of starch used, the quantity and nature of the cross-linking agent, the reaction temperature and duration, and the level of substitution present (Dhull et al.,2023). Due to the diverse array of potential structures in cross-linked networks, it is crucial to employ analytical techniques to evaluate important structural parameters, such as cross-link density, molecular weight between cross-links, variations in chain length between cross-links, network uniformity, and the fraction of polymer that remains unbound to the network (Nielsen et al.,1969). The process of crosslinking plays a crucial role in influencing the behavior of loaded cells at the cellular level, as well as affecting the mechanical and physicochemical characteristics of the bioprinted constructs. It is essential to achieve a balance between the level of crosslinking and the ability to print, as reduced crosslinking can improve the flow of bioink, whereas increased crosslinking can lead to greater stiffness and potentially obstruct printability (GhavamiNejad et al.,2020).\u003c/p\u003e\u003cp\u003eEssential biomechanical properties that can be assessed encompass rheology (including viscosity, shear-thinning, viscoelasticity, and thixotropy), gelation kinetics, crosslinking, and network architecture (Murphy et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Jia et al., 2014; Blaeser et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Tarassoli et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Despite the considerable advancements in these research areas, existing bioprinting systems encounter various limitations. Addressing these challenges is crucial to fully leverage bioprinting's capabilities in tissue engineering and regenerative medicine (Vyas et al., 2019). The limitations associated with the production of bioinks include the absence of specialized extracellular matrix components (ECM) proteins tailored for different cell types, inadequate cell interaction, variability in the degradation of tissue compared to tissue formation, toxicity of degradation byproducts, instability, and the deterioration of structural integrity of the bioprinting scaffold throughout various phases of the bioprinting process (Hospodiuk et al.,2017).\u003c/p\u003e\u003cp\u003eThe development of printable biomaterials and 3D printing techniques that simulate tissue functions is essential; however, analyzing the advantages and disadvantages of existing fabrication methods can inform future investigations (Askari et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Selecting the appropriate materials is crucial for producing hydrogels. These materials significantly impact biocompatibility, cellular viability, and the mechanical properties of bioprinted structures, all of which are crucial for achieving successful bioprinting outcomes (S\u0026aacute;nchez et al., 2020). The printability of bioinks is influenced by their viscosity, surface tension, and cross-linking capabilities, as well as the surface characteristics of the printer nozzle Kyle et al.,2018).\u003c/p\u003e\u003cp\u003eStem cells from the periodontal ligament were incorporated into GelMA hydrogel at concentrations of 3%, 5%, and 10% (Zhu et al., 2023). A 10% GelMA concentration showed reduced cell viability and survival; however, the addition of periodontal ligament stem cells facilitated new cell generation (Almeida et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe composition of alginate and gelatin methacryloyl (GelMA) has been demonstrated recently using a coaxial dispensing system (Colosi et al.,2016). GelMA, when used at concentrations below 5% w/v, provides beneficial conditions for cellular activity; however, it lacks adequate printability independently. The combination with alginate yields a bioink that exhibits mechanical stabilization due to the presence of physically cross-linked fibers. The coaxial needle system enables precise regulation of the gelation kinetics of this bioink by modifying the concentrations of alginate and CaCl₂. After bioprinting, the hydrogel construct undergoes additional reinforcement via UV cross-linking of GelMA (H\u0026ouml;lzl et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eGelatin\u0026ndash;alginate bioinks were used to encapsulate myoblasts and investigate the mechanical properties of the printed constructs (Zhang et al., 2018). Employing a dual-nozzle system, they developed 3D filaments featuring diverse structural configurations. The methodology encompassed a two-step cross-linking approach: initially, physical cross-linking of gelatin was conducted at low temperatures during the printing phase, followed by ionic cross-linking of alginate using Ca\u003csup\u003e2+\u003c/sup\u003e ions after printing. Although there was a reduction in mechanical strength over time, the constructs maintained their mechanical durability due to their low porosity and angled geometry (Ozkerim et al., 2018).\u003c/p\u003e\u003cp\u003eIn this research, we utilize alginate and gelatin hydrogels to prepare the bioink, with CaCl\u003csub\u003e2\u003c/sub\u003e serving as the crosslinker. Alginate is a linear polysaccharide composed of monomers known as mannuronic and guluronic acids. The initiation of alginate gelation occurs through the release of CaCl\u003csub\u003e2\u003c/sub\u003e ions, leading to the formation of egg-box structures between alginate chains (Draget et al.,1990; Stokke et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Siew et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Gelatin is a protein derived from the denaturation of the triple helix structure of collagen ( Panouill\u0026eacute; et al.,2005). Alginate exhibits several benefits, including bioinertness, affordability, accessibility, tunability, biocompatibility, biodegradability, and tissue-specific mechanical properties. Nonetheless, it also has limitations, such as insufficient degradation, the absence of cell-binding motifs, restricted cell-material interactions, high hydrophilicity, rapid gelation that can cause nozzle clogging, inconsistencies in printing, and inadequate dimensional stability (Datta et al.,2023). Gelatin exhibits numerous significant advantages, including biocompatibility, non-immunogenicity, and hydrophilicity (Kuijpers et al.,2000). This material functions as a thermoreversible gel, transitioning to a solid state at reduced temperatures while exhibiting a loss of mechanical stability in physiological environments. To preserve its structural integrity at temperatures below 37\u0026deg;C, chemical modification is necessary (Datta et al.,2023). In this research, we analyzed the formulation of alginate-gelatin hydrogels bioink through Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), swelling properties, and thermal properties. In addition, we investigated the rheological properties such that storage modulus, loss modulus, and cell viability of the bioink composition. We then printed the 3D structure through the bioinks layer by layer and analysed the bioprinted structure\u0026rsquo;s stability using optical microscopic imaging. Rationally, the developed alginate-gelatin bioink is a promising candidate for 3D/4D bioprinting in various potential applications.\u003c/p\u003e"},{"header":"2. Experimental Procedure","content":"\u003cp\u003e\u003cstrong\u003e2.1 Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe materials used \u0026nbsp;Sodium Alginate (sodium polymannuronate) bought from Research-Lab Fine Chem Industries, Gelatin from Merck Specialities Private Limited, calcium chloride (CaCl\u003csub\u003e2\u003c/sub\u003e, anhydrous, granular, \u0026le; 7.0 mm, \u0026ge; 93.0%) obtained from Sigma Aldrich, and phosphate-buffered saline (PBS, 10\u0026times; concentrate, powder, pH 7.2) acquired from HiMedia Laboratories Pvt. Ltd.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Bioink Preparation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVarious proportions of sodium alginate and gelatin solutions were mixed to prepare the bioink solution. The mass of the alginate powder and gelatin powder is measured in the following proportion: 1% Sodium Alginate (w/v) + 9% Gelatin (w/v);2% Sodium Alginate (w/v) + 8% Gelatin (w/v);3% Sodium Alginate (w/v) + 7% Gelatin (w/v);4% Sodium Alginate (w/v) + 6% Gelatin (w/v);5% Sodium Alginate (w/v) + 5% Gelatin (w/v).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA schematic diagram of the bioink preparation method is shown in Figure 1.Initially, we combined various desired concentrations of Sodium alginate powder with 25 mL of water and mixed different concentrations of gelatin powder with 25 mL of water in a beaker. Subsequently, we combined 25 mL of sodium alginate solution with 25 mL of gelatin solution. We subjected the mixture to a magnetic stirrer at 80\u0026deg;C for 1.5 hours to ensure complete dissolution of the solution. Subsequently, we added 50 mL of 0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e to the solution as a cross-linking agent. Subsequently, we positioned the solutions within a test tube and placed them in the freezer for freeze-drying. After removing the sample from the freeze dryer, we sliced the solution into small pieces, creating a 3D-shaped structure.\u003c/p\u003e\n\u003cp\u003eThen, the solution was placed under the table lamp for evaporation. After 4 days, it was removed from the lamp and placed in sunlight for 4 days to accelerate the evaporation process. After completing the evaporation process in sunlight, the solution was moved to a magnetic stirrer at 50 \u0026deg;C. Thus, water was removed from the sodium alginate-gelatin solution.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003e2.3 Characterization Process\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.1 Fourier Transform Infrared Spectroscopy\u003c/strong\u003e (\u003cstrong\u003eFTIR) for bond formation analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe molecular structure and chemical composition of pure sodium alginate, gelatin, and five compositions of sodium alginate-gelatin bio-ink solution with CaCl\u003csub\u003e2\u003c/sub\u003e crosslinkers were analyzed by Fourier transform infrared spectroscopy (FTIR). The Shimadzu IRTracer-100 model device interpreted this property.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.2 Scanning Electron Microscopy (SEM) for surface morphology analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe surface morphology of the five developed sodium alginate hydrogel bio-ink compositions, with CaCl\u003csub\u003e2\u003c/sub\u003e crosslinkers, pure sodium alginate, and gelatin, was analyzed using scanning electron microscopy (SEM) at an accelerating voltage of 5 kV. All samples were carefully coated with a thin layer of gold under a vacuum before starting the SEM analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.3 Thermal Stability Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThermogravimetric analysis (TGA), differential thermal analysis (DTA), and derivative thermogravimetry (DTG) were conducted carefully under a nitrogen gas flow of 10 mL/min to assess the thermal stability properties of five compositions of sodium alginate-gelatin with CaCl\u003csub\u003e2\u003c/sub\u003e as a crosslinker, pure sodium alginate, and gelatin. All the samples weighed between 5 and 14 mg and were heated to 250 \u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.4 Swelling Behavior\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe entire composition of Sodium Alginate (SA)-Gelatin (G) hydrogel bioink, along with the CaCl\u003csub\u003e2\u003c/sub\u003e crosslinker, was immersed in a 40 mL Phosphate-Buffered Saline (PBS) solution. \u0026nbsp;The weight of the compositions was recorded at intervals of 1 hour, 3 hours, 7 hours, 12 hours, 18 hours, and 24 hours to assess swelling. The determination of the swelling percentage of the compositions is conducted through the application of the formula:\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\" width=\"561\" height=\"85\"\u003e\u003c/p\u003e\n\u003cp\u003eWhere w\u003csub\u003et\u003c/sub\u003e represents the swollen weight of the composition at different time points, and w\u003csub\u003eo\u003c/sub\u003e represents the initial (dry) weight of the composition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.5 Statistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA one-sample t-test was performed using GraphPad Prism 10.2.0 (392) to analyse variables that significantly affected (p-value \u0026lt; 0.05) the swelling rate composition. The quantitative data are shown as the mean \u0026plusmn; standard deviation.Legend **** on the figure indicates p\u0026lt;0.0001,*** indicates p = 0.0003 and 0.0005 ,** indicates p = 0.0017.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.6 Assessment of Rheological Parameter\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe evaluation of the rheological behaviour of the hydrogel bioinks involved measuring shear modulus, loss modulus, and phase angle through the use of a rotational rheometer configured with a parallel plate geometry. \u0026nbsp; The hydrogel samples were meticulously positioned between the plates, ensuring a consistent gap of about 1 mm, and the edges were sealed with mineral oil to avert dehydration throughout the testing process. \u0026nbsp;A strain sweep test was initially performed at a constant frequency (usually 1 Hz) to determine the linear viscoelastic region (LVR), where the storage modulus (G\u0026prime;), loss modulus (G\u0026Prime;), and phase angle (\u0026delta;) exhibited independence from strain. \u0026nbsp; This facilitated the selection of an appropriate strain value for the following frequency and time sweeps.\u003c/p\u003e\n\u003cp\u003eA frequency sweep was conducted within the LVR, typically ranging from 0.1 to 100 rad/s, to examine the relationship of G\u0026prime;, G\u0026Prime;, and \u0026delta; with oscillatory frequency, highlighting the viscoelastic characteristics and structural stability of the bioinks across varying deformation rates. \u0026nbsp;Time sweep tests were performed at a constant strain and frequency to assess the bioink\u0026apos;s stability over time, mimicking conditions like crosslinking or gelation behaviour. \u0026nbsp;Throughout these experiments, variations in G\u0026prime; and G\u0026Prime; were observed to evaluate structural development. \u0026nbsp;The shear modulus was derived from the storage modulus, indicating the stiffness of the hydrogel matrix. \u0026nbsp;The phase angle, derived from the ratio of G\u0026Prime; to G\u0026prime;, reflects the equilibrium between elastic and viscous characteristics.\u003c/p\u003e\n\u003cp\u003eAll tests were performed at either room temperature or 37\u0026deg;C, based on the specific application requirements. \u0026nbsp;Measurements were conducted a minimum of three times to ensure consistency, and the resulting data were analysed with the rheometer software. \u0026nbsp; Graphs illustrating modulus and phase angle about strain, frequency, and time were created to analyse the rheological characteristics of various bioink formulations. \u0026nbsp;This thorough evaluation offered valuable information regarding the printability, mechanical stability, and viscoelastic characteristics of the hydrogels in contexts pertinent to bioprinting.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.7 3D/4D Printing Process\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA scaffold is a 3D structure for culturing cells. The sustainability of the printed cells depends on the printed structures. For instance, the stress distribution is significantly better in dome structures compared to cubic structures (Pati et al.,2015). Till now, many authors have reported different types of scaffold design (Wang et.,2016;Reddy et al.,2015) architectures for 3D/4D printing for engineering applications.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFigure 2 represents \u0026nbsp;the schematic diagam of the 3D bio-printing process of Schwann cells, cancer cells and the co-cultured cells with the mixing of algiante-gelatin bioinks at temperature 29.5 degree celcius.\u003c/p\u003e\n\u003cp\u003eWe used an extrusion-based bioprinting process on the bioink. The bioink was loaded into the cartridge or syringe and then applied to the bioprinter. The bioink was extruded layer by layer with applied pressure to form a 3D structure. The extrusion parameters, such as printing speed, pressure, and nozzle height,porosity etc were optimized to ensure good printability. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBasic Parameters for 3D/4D Bio-printing\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"631\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 631px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eScaffolds Design Parameters\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 191px;\"\u003e\n \u003cp\u003ePorosity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 440px;\"\u003e\n \u003cp\u003eThe maximum porosity will be used keeping the mechanical properties in constant\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 191px;\"\u003e\n \u003cp\u003eSize of the pore\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 440px;\"\u003e\n \u003cp\u003e400 \u0026micro;m to 2000 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 191px;\"\u003e\n \u003cp\u003eStructure of the pore\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 440px;\"\u003e\n \u003cp\u003eVarious structures (circular, rectangular, hexagonal etc.) maximum connected\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 191px;\"\u003e\n \u003cp\u003eNeedle diameters\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 440px;\"\u003e\n \u003cp\u003e100-400 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 191px;\"\u003e\n \u003cp\u003eGas pressures\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 440px;\"\u003e\n \u003cp\u003e0.08 to 0.26 MPa\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 191px;\"\u003e\n \u003cp\u003eNumber of Layers\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 440px;\"\u003e\n \u003cp\u003e1-10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 191px;\"\u003e\n \u003cp\u003eVelocity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 440px;\"\u003e\n \u003cp\u003e5 to 30 mm/s\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 191px;\"\u003e\n \u003cp\u003eHeight of the scaffolds\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 440px;\"\u003e\n \u003cp\u003e1 to10 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 191px;\"\u003e\n \u003cp\u003eDimension\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 440px;\"\u003e\n \u003cp\u003e5\u0026times;5\u0026times;5 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 191px;\"\u003e\n \u003cp\u003eTemperature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 440px;\"\u003e\n \u003cp\u003e25-33 Degree Celsius\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eCross-linker is used to bind the molecules internally as well as improve the mechanical properties and provide more stability (Discher et al.,2005). Based on the biopolymers, the cross-linker is used to modify the compositions. To crosslink alginate-gelatin 3D printed hydrogels, CaCl\u003csub\u003e2\u003c/sub\u003e was used as a crosslinking agent in the current work. Ca\u003csup\u003e2+\u003c/sup\u003e helps to crosslink the printed scaffolds. These crosslinked hydrogels are biodegradable and very useful for materials in the regeneration of medicine and tissue engineering applications. Ca\u003csup\u003e2+\u003c/sup\u003e provides mechanical stability and reduces the swelling of the hydrogels.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.8 Cell Viability of the printed samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCancer Cell line (H1975) and Schwann cell line (hTERT NF1 ipnNF95.11c, #CRL-3391) were purchased from ATCC The medium used for the cells cultured were DMEM, 10% FBS and 1.0% antibiotic following the general protocol of cell culture at the incubator of 37\u0026deg;C with humidified atmosphere in the culture flask/petri dish of 5% CO\u003csub\u003e2\u003c/sub\u003e within the cell. The cells were transferred into cryovials using DMSO and stored at -80\u0026deg;C for short-term use. For long-term storage, they were kept in liquid nitrogen vapour phase at -196 \u0026deg;C. 70% ethanol was used to sterilize all the other items used for these purposes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTrypan blue was used as a reagent to count the cells with a hemocytometer. The sub-cultures of the cells were performed using a standard protocol. The adherent cells were suspended in trypsin-EDTA solution. Schwann cells (hTERT NF1 ipnNF95.11c, #CRL-3391) and cancer cells (H1975) were first cultured in a cell culture flask/petri dish in a monolayer system. Following the same protocol, co-culture of H1975 and hTERT NF1 ipnNF95.11c, #CRL-3391 was executed in the monolayer system. After culturing and sub-culturing the cells in the subsequent phase three, the cells were cultured into the bulk hydrogels. Schwann cells, as well as cancer cells and the co-culture cells, were added to the cartridges before printing cells with the hybrid hydrogel. All the cartridges will be sterilized with 70% ethanol. After printing all the designed scaffolds, CaCl\u003csub\u003e2\u003c/sub\u003e, CaCl\u003csub\u003e2\u003c/sub\u003e was added to crosslink the models for 10 minutes. The printed hydrogel with cells was then kept in DMEM with 10% FBS and 1% antibiotic inside the petri dish. Finally, the Petri dish with printed hydrogels was kept at 37\u0026deg;C to maintain a physiological environment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.9 Optical Microscopic Image Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe evaluation of optical image of lyophilized hybrid samples was performed in order to figure out the tomography of the 3D printed hydrogel scaffolds. \u0026nbsp;The OMI images of the prepared compositions of 1% Alginate (w/v) + 9% Gelatin (w/v), 3% Alginate (w/v) + 7% Gelatin (w/v), 4% Alginate (w/v) + 6% Gelatin (w/v) and 5% Alginate (w/v) + 5% Gelatin (w/v) with added 0.5M CaCl\u003csub\u003e2\u003c/sub\u003e were observed using optical microscope. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe OMI was implemented the structures features of the scaffolds architectures. The printed scaffolds were lyophilization at -80 degree Celsius. The water was completely removed and then scaffolds was kept under the microscope to observe the topographical characteristics of the samples. The optical image of the hybrid hydrogels was taken to analysis the pore structures of the hydrogel. The actual strand size is the diameter of the printer nozzle diameter. The diameter of the used nozzle in the current experiment was 410 \u0026mu;m. The achievable strand size was 0.80 times of the nozzle diameter.\u0026nbsp;\u003c/p\u003e"},{"header":"3. Experimental Result and Discussion","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Chemical and molecular structure analysis by Fourier Transform Infrared Spectroscopy (FTIR)\u003c/h2\u003e\n \u003cp\u003eThe development of hydrogel structures is influenced by multiple factors related to their three-dimensional network configuration. Gelatin forms complexes with ionic polysaccharides primarily through electrostatic attractions and hydrogen bonding. The ionic interactions among compatible functional groups facilitate the attachment of gelatin chain segments to sodium alginate chains. Nevertheless, the hydroxyl and carboxyl groups found in both gelatin and sodium alginate are not expected to participate in reactions with epoxy groups. The main organizing principle is based on the interaction between the structuring agent and the amino group present in the lysine residues of the gelatin chains (Maikovych et al.,2025).\u003c/p\u003e\n \u003cp\u003eThe mechanism that enhances the toughness of the SA-G gel was investigated using Fourier Transform Infrared Spectroscopy (FT-IR). Figures \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e(A) and 4(B) illustrate the FT-IR spectra for pure sodium alginate and gelatin, respectively. The spectrum of sodium alginate displays absorption peaks at 1600 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1414 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, corresponding to the symmetric and asymmetric stretching vibrations of the \u0026minus;\u0026thinsp;COO\u0026thinsp;\u0026minus;\u0026thinsp;group in alginates. Similarly, the spectrum for gelatin displays peaks at 1632 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1539 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which correspond to the C\u0026thinsp;=\u0026thinsp;O and C\u0026thinsp;\u0026minus;\u0026thinsp;N stretching vibrations found in the amide I band, along with the bending vibrations of the \u0026minus;\u0026thinsp;NH group in the amide II band (Sartori et al.,1997) .\u003c/p\u003e\n \u003cp\u003eFigures \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e(C\u0026ndash;H) illustrate samples exhibiting different SA-G ratios that underwent crosslinking with CaCl\u003csub\u003e2\u003c/sub\u003e. The samples exhibit variations including shifts in peaks, modifications in peak shapes, and the emergence of new bands, all of which are affected by the hydrogel composition. For example, the absorption band around 3278 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, associated with O\u0026ndash;H stretching, exhibits broadening, a shift to higher wave numbers, and a reduction in intensity as the concentration of SA decreases. A decrease in intramolecular bonding can explain this phenomenon.\u003c/p\u003e\n \u003cp\u003eThe asymmetric stretching peak of the \u0026minus;\u0026thinsp;COO\u0026thinsp;\u0026minus;\u0026thinsp;group at 1628 cm\u0026thinsp;\u0026minus;\u0026thinsp;1 in the SA/G 5/5 sample, linked to the interaction between \u0026minus;\u0026thinsp;COO\u0026thinsp;\u0026minus;\u0026thinsp;groups in SA and CaCl\u003csub\u003e2\u003c/sub\u003e, shifts to a broadband at 1631 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (amide I, C\u0026thinsp;=\u0026thinsp;O, and C\u0026thinsp;\u0026minus;\u0026thinsp;N stretching) in the SA/G 1/9 sample, arising from the \u0026minus;\u0026thinsp;NH\u003csub\u003e2\u003c/sub\u003e group in gelatin. Similarly, the symmetric stretching peak of \u0026minus;\u0026thinsp;COO\u0026thinsp;\u0026minus;\u0026thinsp;at 1432 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e exhibits a shift to lower wave numbers in all samples, indicating the presence of ionic interactions between CaCl\u003csub\u003e2\u003c/sub\u003e and \u0026minus;\u0026thinsp;COO\u0026thinsp;\u0026minus;\u0026thinsp;groups in SA. The results align with previous research conducted by Sartori and colleagues (Sartori et al., \u003cspan class=\"CitationRef\"\u003e1997\u003c/span\u003e) and Cathell and colleagues (Cathell et al., 2007) on CaCl\u003csub\u003e2\u003c/sub\u003e-crosslinked alginate thin films.\u003c/p\u003e\n \u003cp\u003eFurthermore, the SA/G 1/9 sample exhibits the emergence of two new absorption bands at 1532 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1244 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, corresponding to amide II and III, respectively. The observed bands are indicative of C\u0026ndash;N stretching coupled with N\u0026ndash;H bending, a phenomenon that intensifies with an increase in gelatin content within the hydrogel. Moreover, with a decrease in SA concentration, the relative intensities of the C\u0026ndash;C and C\u0026ndash;O stretching bands at 1160 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1028 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e progressively diminish, although their positions stay unchanged. The decrease in intensity is probably due to the weakening of these bonds as they adjust to the coordination structure around CaCl\u003csub\u003e2\u003c/sub\u003e ions (Sartori et al.,1997;Cathell et al.,2007;Saarai et al.,2013).\u003c/p\u003e\n \u003cp\u003eFrom the above discussion, we conclude that the presence of an asymmetric COO- group indicates that alginate is crosslinked with Ca\u003csup\u003e2+\u003c/sup\u003e and confirms ionic bonding. In contrast, the presence of a symmetric COO- group confirms the formation of a Ca\u003csup\u003e2+\u003c/sup\u003e-alginate bioink, which plays a crucial role in the gelation process. The O-H stretching group indicates the presence of a hydrophilic group, which enhances cell viability. The amide II band confirms the protein secondary structure (gelatin), while the amide III band signifies the interaction between gelatin and the alginate network without chemical degradation.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Surface Structure Analysis By SEM\u003c/h2\u003e\n \u003cp\u003eThe surface morphology of 3D hydrogels was examined through scanning electron microscopy (SEM). Since the SEM process requires sample dehydration, all specimens were subjected to lyophilisation as outlined in the Materials and Methods section (Mirek et al.,2022).\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e displays SEM images at a magnification scale of 20 \u0026micro;m, emphasising the cross-section of the grid structure. The scaffold exhibited a remarkably porous and interconnected structure (Hashimi et al.,2021;Meligy et al.,2022), resulting from the drying process and the use of a high vacuum during sputter coating. The porous structure demonstrated benefits for cell adhesion, proliferation, and tissue development, as it improved nutrient diffusion and metabolic exchange within the scaffold (Abert et al.,2023). The analysis of the surface structure revealed that the scaffold\u0026apos;s components are covalently bonded, forming a strong and interconnected network. The distinct separation between the scaffold\u0026apos;s particles or structural units indicates a precisely defined and cohesive architecture (Khademi et al.,2025).\u003c/p\u003e\n \u003cp\u003eFrom the images, a 3% alginate + 7% gelatin + 0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e solution exhibits well-defined porous structures with interconnected pores, which are suitable for cell culture. The distribution of pores is more homogeneous than that of others, allowing for consistent crosslinking and preventing the formation of weak or necrotic regions in 3D culture. The porous structure of 3% alginate, 7% gelatin, and 0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e permits cell migration, nutrient diffusion, and waste removal. The interconnected pores of this composition facilitate cell communication and promote the uniform distribution of cells throughout the composition. The presence of a smooth inner surface and uniform structure indicates more efficient mixing and crosslinking, rendering the composition advantageous for cell adhesion and proliferation. The structure of this composition of hydrogels exhibits a greater resemblance to the native extracellular matrix (ECM) due to its highly well-defined porous structure and interconnected pores, which promote significant cell viability, migration, and tissue formation.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e3.3 Thermal Stability Analysis\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eIn the TG curve, it is observed that 10.91% of the total mass decayed at 192\u0026deg;C, 5.24% of the mass was lost between 192\u0026deg;C and 250\u0026deg;C, and 83.85% of the mass remained at 250\u0026deg;C. In gelatin, 13.73% of the total mass decayed at 161\u0026deg;C, 4.29% of the mass was lost at 161\u0026deg;C, and 82.98% of the mass remained at 250\u0026deg;C. 12.49% mass lost at 138.2\u0026deg;C and 2.32% mass degraded from 138.2\u0026deg;C and 84.88% mass remained in 1% SA\u0026thinsp;+\u0026thinsp;9% G\u0026thinsp;+\u0026thinsp;0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e.11.22% mass lost at 139.02\u0026deg;C and 2.88% mass degraded from 139.02\u0026deg;C and 84.86% mass remained in 2% SA\u0026thinsp;+\u0026thinsp;8% G\u0026thinsp;+\u0026thinsp;0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e.13.25% mass lost at 124.67\u0026deg;C and 2.73% mass degraded from 124.67\u0026deg;C and 84.02% mass remained in 3% SA\u0026thinsp;+\u0026thinsp;7% G\u0026thinsp;+\u0026thinsp;0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e.13.47% mass lost at 155.15\u0026deg;C and 4.82% mass degraded from 155.15\u0026deg;C and 81.71% mass remained in 4% SA\u0026thinsp;+\u0026thinsp;6% G\u0026thinsp;+\u0026thinsp;0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e.12.56% mass lost at 124\u0026deg;C and 3.92% mass degraded from 124\u0026deg;C and 83.52% mass remained in 5% SA + 5% G\u0026thinsp;+\u0026thinsp;0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e (Thakur et al.,2024).\u003c/p\u003e\n \u003cp\u003eThe DTA curve indicates the recorded temperatures for the endothermic and exothermic peaks as follows: Sodium alginate shows distinct peaks at 51.97\u0026deg;C and 159.09\u0026deg;C, respectively. The observed peaks for gelatin occur at 73.09\u0026deg;C and 211.78\u0026deg;C. The observed temperatures for the combination of 1% SA\u0026thinsp;+\u0026thinsp;9% G\u0026thinsp;+\u0026thinsp;0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e are 79.18\u0026deg;C, indicating an endothermic reaction, and 194.54\u0026deg;C, which signifies an exothermic reaction. Similarly, the endothermic and exothermic peaks for the composition of 2% SA\u0026thinsp;+\u0026thinsp;8% G\u0026thinsp;+\u0026thinsp;0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e are observed at 62.31\u0026deg;C and 194.17\u0026deg;C, respectively. In contrast, for the mixture of 3% SA\u0026thinsp;+\u0026thinsp;7% G\u0026thinsp;+\u0026thinsp;0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e, these peaks are found at 57.70\u0026deg;C and 200.27\u0026deg;C. The peaks for the mixture consisting of 4% SA, 6% G, and 0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e are observed at 57.29\u0026deg;C and 191.34\u0026deg;C. Finally, for the combination of 5% SA\u0026thinsp;+\u0026thinsp;5% G\u0026thinsp;+\u0026thinsp;0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e, the observed temperatures are 60.90\u0026deg;C (endothermic) and 190.60\u0026deg;C (exothermic) (Thakur et al.,2024)\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e presents the weight loss rates for different combinations: 1% Sodium Alginate (SA)\u0026thinsp;+\u0026thinsp;9% Gelatin (G)\u0026thinsp;+\u0026thinsp;0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e, 2% Sodium Alginate (SA)\u0026thinsp;+\u0026thinsp;8% Gelatin (G)\u0026thinsp;+\u0026thinsp;0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e, 3% Sodium Alginate (SA)\u0026thinsp;+\u0026thinsp;7% Gelatin (G)\u0026thinsp;+\u0026thinsp;0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e, 4% Sodium Alginate (SA)\u0026thinsp;+\u0026thinsp;6% Gelatin (G)\u0026thinsp;+\u0026thinsp;0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e, and 5% Sodium Alginate (SA)\u0026thinsp;+\u0026thinsp;5% Gelatin (G)\u0026thinsp;+\u0026thinsp;0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e, Sodium Alginate (SA), Gelatin (G) with corresponding rates of 79, 65, 60.34, 60.65, 59.31, 51.7, and 75\u0026deg;C, respectively (Wang et al.,2023).\u003c/p\u003e\n \u003cp\u003eFrom the above discussion, a 3% alginate\u0026thinsp;+\u0026thinsp;7% gelatin\u0026thinsp;+\u0026thinsp;0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e composition is more suitable for cell culture, as it exhibits greater thermal stability and displays the highest exothermic peak (200.27\u0026deg;C). The highest exothermic peaks indicate a good crosslink structure. The mass loss (13.25%) of 3% alginate\u0026thinsp;+\u0026thinsp;7% gelatin\u0026thinsp;+\u0026thinsp;0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e is moderate compared to other compositions, which signifies good hydration and importance for supporting a moist, cell-friendly environment for cell culture. The structural resilience of this composition is higher than that of other compositions due to the presence of a higher rate of remaining mass (84.02%), which indicates strong resistance to the breakdown of the composition. The DTG curve of this composition exhibits a smoother profile, indicating a uniform polymer blend that is suitable for consistent 3D bioprinting and reliable gel performance.\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e illustrates the stages of swelling of scaffolds at various periods for all compositions. Figure \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e represents the results of the swelling test for all compositions. The recorded average swelling percentages at the 1-hour mark for the different concentrations were as follows: 1% SA \u0026minus;\u0026thinsp;9% G yielded 1237.24%; 2% SA \u0026minus;\u0026thinsp;8% G produced 627.84%; 3% SA \u0026minus;\u0026thinsp;7% G showed 506.364%; 4% SA \u0026minus;\u0026thinsp;6% G reached 440.183%; and 5% SA \u0026minus;\u0026thinsp;5% G resulted in 269.607%. The compositions demonstrated a progressive increase, reaching the peak mean swelling percentage at the 18-hour mark. The recorded values are 1776.94%, 1232.17%, 1164.27%, 1105.01%, and 908.48%, which correspond to 1% SA -9% G, 2% SA -8% G, 3% SA -7% G, 4% SA -6% G, and 5% SA -5% G respectively. The average swelling percentage started to decline at the 24-hour mark. The recorded values are 1545.18%, 1029.96%, 996.14%, 919.52%, and 800.89% for the respective categories of 1% SA -9% G, 2% SA -8% G, 3% SA -7% G, 4% SA -6% G, and 5% SA -5%. The p-values for 1% SA-9% G and 2% SA-8% are both p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, demonstrating a highly significant level. 3% SA-7% G (p\u0026thinsp;=\u0026thinsp;0.0003), 4% SA-6% G (p\u0026thinsp;=\u0026thinsp;0.0005), and 5% SA-5% G (p\u0026thinsp;=\u0026thinsp;0.0017) demonstrate reduced significance relative to the earlier findings. The findings indicate that increased Sodium alginate (SA) concentration diminishes the average swelling percentage.\u003c/p\u003e\n \u003cp\u003eAccording to Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e,2% alginate\u0026thinsp;+\u0026thinsp;8% gelatin\u0026thinsp;+\u0026thinsp;0.5M CaCl\u003csub\u003e2\u003c/sub\u003e and 3% alginate\u0026thinsp;+\u0026thinsp;7% gelatin\u0026thinsp;+\u0026thinsp;0.5M CaCl\u003csub\u003e2\u003c/sub\u003e are good for cell culture and bioprinting. 2% alginate\u0026thinsp;+\u0026thinsp;8% gelatin\u0026thinsp;+\u0026thinsp;0.5M CaCl\u003csub\u003e2\u003c/sub\u003e composition shows strong statistical significance but 3% alginate\u0026thinsp;+\u0026thinsp;7% gelatin\u0026thinsp;+\u0026thinsp;0.5M CaCl\u003csub\u003e2\u003c/sub\u003e exhibits less strong statistical significance. Both compositions exhibit moderate discrepancies; however, the 3% alginate\u0026thinsp;+\u0026thinsp;7% gelatin\u0026thinsp;+\u0026thinsp;0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e swelling rate is notably more moderate and mechanically suitable than those of the others. A higher gelatin amount and a lower alginate amount indicate the printed structure is more viscous. On the other hand, lower gelatin amount and higher alginate amount indicate that the printed structure is more solid. For this reason, 3% alginate\u0026thinsp;+\u0026thinsp;7% gelatin\u0026thinsp;+\u0026thinsp;0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e is not so viscous and not so solid. Hence,3% alginate\u0026thinsp;+\u0026thinsp;7% gelatin\u0026thinsp;+\u0026thinsp;0.5 M CaCl\u003csub\u003e2\u003c/sub\u003e printed structure is more suitable for cell culture and bioprinting than 2% alginate\u0026thinsp;+\u0026thinsp;8% gelatin\u0026thinsp;+\u0026thinsp;0.5M CaCl\u003csub\u003e2\u003c/sub\u003e and others.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5 Optimization for 3D/4D printed scaffolds\u003c/h2\u003e\n \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\n \u003ch2\u003e3.5.1 Analysis of the elastic, viscous and the damping characteristics of the samples\u003c/h2\u003e\n \u003cp\u003eKeeping the 3D printed hybrid scaffolds under the oscillatory force and measuring the response, the elastic, viscous and the damping characteristics of the samples have been measured. The time scale of this test is frequency of oscillation. Strain, phase angles are measured from a range of frequencies by the applied sinusoidal stress.\u003c/p\u003e\n \u003cp\u003eThe storage modulus,\u003c/p\u003e\n \u003cp\u003eG\u003csup\u003e\u0026prime;\u003c/sup\u003e= \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\sigma\\:}_{0}\\frac{\\text{cos}\\delta\\:}{{\\gamma\\:}_{0}}\\)\u003c/span\u003e\u003c/span\u003e; (3.5.1.1)\u003c/p\u003e\n \u003cp\u003ewhere \u0026delta; is the phase angle,\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\sigma\\:}_{0}\\)\u003c/span\u003e\u003c/span\u003e is the component of stress to stress ratio in phase and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{{\\gamma\\:}}_{0}\\)\u003c/span\u003e\u003c/span\u003e is the strain to strain ratio in phase. Similarly,\u003c/p\u003e\n \u003cp\u003eG\u003csup\u003e\u0026Prime;\u003c/sup\u003e= \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\sigma\\:}_{0}\\frac{\\text{Sin}\\delta\\:}{{\\gamma\\:}_{0}}\\)\u003c/span\u003e\u003c/span\u003e; (3.5.1.2)\u003c/p\u003e\n \u003cp\u003eIt is noted that for perfect elastic materials G\u0026Prime;=0, where \u0026delta;\u0026thinsp;=\u0026thinsp;0 i.e. the wave forms are in phase.\u003c/p\u003e\n \u003cp\u003eFor perfect viscous material, the stress and the strain waveforms are completely out of phase.\u003c/p\u003e\n \u003cp\u003ei.e., \u0026delta;\u0026thinsp;=\u0026thinsp;0, G\u0026prime;=0 and hence, G\u0026Prime; is finite.\u003c/p\u003e\n \u003cp\u003eThe loss factor,\u003c/p\u003e\n \u003cp\u003etan \u0026delta;\u0026thinsp;=\u0026thinsp;G\u0026Prime;/G\u0026prime; (3.5.1.3)\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003e(a) illustrates the relation of storage modulus(G\u0026prime;) and loss modulus (G\u0026Prime;) of 3D printed hydrogel scaffold of 1% alginate and 9% gelatin with crosslinker 0.5MCaCl\u003csub\u003e2\u003c/sub\u003e over strain amplitudes. The storage modulus(G\u0026rsquo;) gradually decreases conversely, the loss modulus gradually increases. The storage and loss modulus of the hydrogel measured at constant temperature materials of the hydrogel show solid fluid like behavior until it reaches to the critical strain. The critical strain for polymer materials is near to 1 in log scale. The critical strain point is also known as the gel point. In the gel point maximum crosslinking is happened of the polymers. After the gel point the storage modulus is less the loss modulus. In this stage, the 3D alginate gelatin hydrogel breaks up the 3D structure and starts to behave as fluid materials. The tangent of the materials is increasing slowly but it is almost strain independent. Figure \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003e(b) shows the frequency sweep graph of the 3D printed hydrogel scaffold. Only up to 100 rads\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e the elastic modulus was measured. Within this short range it is found that the storage modulus is greater than the loss modulus and the storage modulus is almost frequency independent. The hydrogel scaffolds show this property below the critical strain or gel point. This property indicates that the printed hydrogel scaffolds is solid gel like. Figure \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003e(c) shows the time sweep of the hydrogel. Within very short time the there is no change of the storage and loss modulus. The tangent is also time independent within this short time.\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003e(d-f) illustrates the rheological characteristics of 2% Alginate (w/v)\u0026thinsp;+\u0026thinsp;8% Gelatin (w/v)\u0026thinsp;+\u0026thinsp;0.5M CaCl\u003csub\u003e2\u003c/sub\u003e of the 3D printed hydrogel scaffolds. According to Fig. \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003e(d) the storage modulus is greater than the loss modulus and phase angle is increasing gradually. After the gel point the phase angle is increased as the loss modulus is greater than the storage modulus at this stage. Gel point is greater than 1 in log scale. The incorporation of alginate 2% alginate and 8% gelatin, the rheological properties has increased. Figure \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003e(e) represents G\u0026prime;, G\u0026Prime; and phase angle over frequency. The storage and loss modulus are measured up to 10KHz in log scale. Within this frequency range G\u0026prime; is always greater than G\u0026Prime;. The time sweep curve shows that within the short period there is no change of storage and loss modulus.\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003e(g-i) demonstrate the rheological characteristics of 3% Alginate (w/v)\u0026thinsp;+\u0026thinsp;7% Gelatin (w/v)\u0026thinsp;+\u0026thinsp;0.5M CaCl\u003csub\u003e2\u003c/sub\u003e of the 3D printed hydrogel scaffolds. From Fig. \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003e(g), it is seen that the gel point of the 3D printed hydrogel is slightly greater than 1 in log scale. Up to the gel points the hybrid hydrogel shows the linear elastic behavior and it is in jelly like solid materials. After the gel points with increasing the strain the 3D structures of the materials will collapse and G\u0026rsquo;\u0026rsquo; will be larger than the G\u0026rsquo; and the 3D printed hydrogel will behave like fluid materials. Figure \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003e(h) shows the frequency response storage and loss modulus within the gel point. The storage modulus is always higher than the loss modulus. Both of G\u0026prime; and G\u0026Prime; is independent of frequency within this frequency range. Figure \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003e(i) represents the time sweep graph and it is clearly seen that there is no change of G\u0026prime; and G\u0026Prime; within this small time.\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003e(j-l) demonstrate the rheological characteristics of 4% Alginate (w/v)\u0026thinsp;+\u0026thinsp;6% Gelatin (w/v)\u0026thinsp;+\u0026thinsp;0.5M CaCl\u003csub\u003e2\u003c/sub\u003e of the 3D printed hydrogel scaffolds. According to the Fig. \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003e(j), the gel point of the hydrogel is greater than 1 in log scale. The 3% alginate and 7% gelatin has increased both the storage and loss modulus of the binary hybrid polymers hydrogels scaffolds. Up to the gel point the materials is solid jelly like and very much suitable for used as a bio-ink for DIW bio-printing. After the gel points the materials start to behave like fluid like behavior. Figure \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e(k) represent the frequency response G\u0026prime;, G\u0026Prime; and phase angle graph. Within 10KHz log scale frequency range, G\u0026prime; is always greater than G\u0026Prime; and it is almost frequency independent. Figure \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003e(l) is time sweep curve and it is seen that within small time there is no change of G\u0026prime; and G\u0026Prime;.\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003e(m-o) elucidate the rheological characteristics of 5% Alginate (w/v)\u0026thinsp;+\u0026thinsp;5% Gelatin (w/v)\u0026thinsp;+\u0026thinsp;0.5M CaCl\u003csub\u003e2\u003c/sub\u003e of the 3D printed hydrogel scaffolds. According to the Fig. \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003e(m), it is observed that up to the critical strain, the G\u0026rsquo; and G\u0026rsquo;\u0026rsquo; both are straight line, i.e. the alginate-gelatin binary composites shows elastic behavior and it is jelly-like solid materials. This property indicates that this composite can be used as a bio-ink. With increasing the proportion of the alginate and decreasing the gelatin the stiffness has increased than the previous proportions. Figure \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003e(n) is the frequency sweep curve and it is observed that G\u0026prime; is greater than the G\u0026Prime; and independent of frequency i.e. the materials jelly like solid materials and can be used as a bio-inks. Figure \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003e(o) indicates the time sweep and it is found that there is no change of G\u0026prime; and G\u0026Prime; within this time period. Although this time is very small with compare to the experimental time of the 3D printed hydrogel scaffolds.\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eStorage modulus, loss modulus and loss tangent of alginate-gelatin hydrogels with 0.5M CaCl\u003csub\u003e2\u003c/sub\u003e cross-linker\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eComposites\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCross-linker\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eG\u0026prime;\u003c/p\u003e\n \u003cp\u003e(KPa)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eG\u0026Prime;\u003c/p\u003e\n \u003cp\u003e(KPa)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003etan\u0026delta;\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5% Alginate + 5% Gelatin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"5\"\u003e\n \u003cp\u003eCalcium Chloride\u003c/p\u003e\n \u003cp\u003e10 minutes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.34\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4% Alginate\u0026thinsp;+\u0026thinsp;6% Gelatin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e13.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.46\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3% Alginate\u0026thinsp;+\u0026thinsp;7% Gelatin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.29\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2% Alginate\u0026thinsp;+\u0026thinsp;8% Gelatin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1% Alginate\u0026thinsp;+\u0026thinsp;9% Gelatin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.45\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003eThe loss tangent of the developed bio-ink hydrogels are an important parameters for developing the desired bio-inks for 3D bio-printing. The loss tangent mentioned in the table-2 is from 0.21 to 0.46. The loss tangent value is 0.20 to 0.45 is suitable for meaningful unique bio-printing (Liu et al.,2019). Hence 4% alginate and 6% gelatin is not significant for 3D bioprinting.\u003c/p\u003e\n \u003cp\u003eThe composition 3% alginate\u0026thinsp;+\u0026thinsp;7% gelatin\u0026thinsp;+\u0026thinsp;0.5M CaCl\u003csub\u003e2\u003c/sub\u003e shows excellent viscoelastic balance among them. Balance viscoelasticity mimics native extracellular matrix (ECM), which plays a crucial role in stem cell fate decision and cell-matrix interactions. Optimal viscoelasticity supports long cell viability and proliferation. Thus, we conclude that balanced viscoelasticity improves the relevance of in vitro models and implanted cells.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\n \u003ch2\u003e3.5.2 Cells Viability of 2D/Monolayer System\u003c/h2\u003e\n \u003cp\u003eThe MTT is used to identify the toxicity of the materials. The cells viability of H 1975, #CRL 3991 and the co-culture of both of them in 3% alginate and 7% gelatin bio-inks and 0.5M Calcium chloride have been determined by MTT without counting cells in details. In this process, indissoluble formazan is made from water soluble MTT. The insoluble formazan turns into the solubilized one and then then the absorption of the concentration is measured at the different wavelengths using molecular devices the Fluorescence Microplate Readers.\u003c/p\u003e\n \u003cp\u003eThe cells viability of cancer cells (H1975), Schwann cells (hTERT NF1 ipnNF95.11c, #CRL-3391) and the co culture of two cells are conducted to figure out the behavior of cells in 2D monolayer of hydrogels (3% alginate and 7% gelatin ). The cells density used in the current experiment is listed in table-2.\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eCells density used for 2D/monolayer cultures\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSl.\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCells name\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCells per ml\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCells per ul\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCells used per well of 96 plates\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH1975\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.32\u0026times;10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1320\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8ul\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e#CRL-3391\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.1\u0026times;10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e210\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e48ul\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH1975 \u0026amp; #CRL-3391\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.12\u0026times;10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2120\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5ul\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003eFor executing the MTT experiment, the medium was removed and washed the wells with PBS. 100ul new fresh medium was added with the adherent cells and then 10ul of mM MTT stock solution injected into the medium. The treated cells were kept warm for four hours at 37\u0026deg;C. After incubating, 100ul of SDS solution was added in each of the plates and pipetting for mixing thoroughly for further incubating in humidified chamber for four hours. The samples were mixed again after incubating with the pipettes. The absorbance was read at 570nm using the Fluorescence Microplate Readers.\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eCells survival rate (in percentage) of H1975, #CRL 3991 and the culture of H1975 and #CRL 3991\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"2\" rowspan=\"2\"\u003e\n \u003cp\u003eSl\u003c/p\u003e\n \u003cp\u003eCells Name\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eLive Cells\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eDead Cells\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDay 1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDay 3\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDay 7\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDay 1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDay 3\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDay 7\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH1975 (Cancer)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e82.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e93.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e91.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.32\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e#CRL 3991\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e84.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e87.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e82.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e15.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e12.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17.16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eof H1975 \u0026amp; #CRL 3991\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e79.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e85.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e85.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e14.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e14.70\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003eThe measured absorbance values using wavelength 570 nm cells viability as shown in the Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003eCells viability measurement:\u003c/p\u003e\n \u003cp\u003eCell viability =\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{{A}_{t}-{A}_{Blank}}{{A}_{c\\:}-{A}_{Blank}}\\)\u003c/span\u003e\u003c/span\u003e\u0026times;100%;\u003c/p\u003e\n \u003cp\u003eWhere,\u003c/p\u003e\n \u003cp\u003eA\u003csub\u003et\u003c/sub\u003e =Absorbance of the treated samples\u003c/p\u003e\n \u003cp\u003eA\u003csub\u003ec\u003c/sub\u003e = Absorbance of the control samples\u003c/p\u003e\n \u003cp\u003eA\u003csub\u003eBlank\u003c/sub\u003e = without cells only medium presents\u003c/p\u003e\n \u003cp\u003eThe multi-label micro reader measured the intensity of the emitting light of the treated, control cells and the blank. Figure \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003e presents the cells viability of cancer cells, Schwann cell and the co-cultures of both of the cells at different days.The number of live H1975, (Cancer) #CRL 3991(Schwann), the co-culture cells at the day 1 are 82.26, 84.18 and 79.76 at day 3 93.88, 87.69 and 85.69 and 91.68 82.84, 85.30 at day 7. The maximum densities of the cells show at day 3. For H1975, the cells grow rapidly and the optimal densities at 48 to 72 hours. At day 7, the cells proliferation reduced and onward. For # CRL 3991cells the maximum survival rate is at day 3 to day 4 and the proliferation rate increased up to day 7.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e\n \u003ch2\u003e3.5.3 Cells Proliferation of 3D System by Live-dead Assay by Fluorescence Microscopy\u003c/h2\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e13\u003c/span\u003e(A) shows the schematic diagram of 3D bio printing of alginate-gelatin hydrogel. 3D cubic structures of layer 1 to 3 were printed by setting the fundamental parameters of nozzle diameters 410\u0026micro;m, speed 10mm/s, pressure 1.5 to 2.2 KPa and the temperature 29.5\u0026deg;C. The solvent of the hydrogel were DMEM, FBS (10%) and antibiotic (1%).\u003c/p\u003e\n \u003cp\u003eAll the scaffolds were submerged into 0.5M calcium chloride (CaCl\u003csub\u003e2\u003c/sub\u003e) cross linker for 10 minutes. After completion of the gelation process, the extra cross linkers was removed by washing PBS.\u003c/p\u003e\n \u003cp\u003eBefore printing the cells with the alginate-gelatin hydrogel, the cells were subcultures 3 times for getting the normal level of the cells. The cells density of H 1975 and #CRL-3991 are mentioned in Table \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e. After printing the cells with hydrogels the cells laden printed scaffolds were put into the calcium chloride cross-linker for 10 minutes. Fresh medium was added after washing with PBS.\u003c/p\u003e\n \u003cp\u003eAt 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e the 3D printed cells laden hydrogels were incubated for further treatment. The cells proliferation rate is measured by following the MTT absorbance data at wavelength 570nm. H 1975 cells proliferation rate is observed maximum at day 3 and after the following days the cells proliferation rate decreased which is observed at day 7. The cells #CRL-3991 shows the highest proliferation at day 3 but the proliferation increases with increasing times. From Fig. \u003cspan class=\"InternalRef\"\u003e13\u003c/span\u003e(B) it is clearly seen that the proliferation of H 1975 and #CRL-3991 cells in the alginate hydrogels is very much significant at the cells survivability is around 90%.\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eCells density used for 3D Bio-printing\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSl.\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCells name\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCells per ml\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCells per \u0026micro;l\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCells used in 5ml hydrogels\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH1975\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.32\u0026times;10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1320\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e250 \u0026micro;l\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e#CRL-3391\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.1\u0026times;10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e210\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1ml\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003eThe live dead image of the H 1975 cells of 3D printed scaffold observed using fluorescence optical microscopy. The kits of two colors of wavelength for live represents green and the absorbance- emission spectral wave lengths were 488nm and 515nm and for dead cells the corresponding image was red the emission excitation wavelength was 570nm and emission wavelength was 602nm. The kit is purchased from Thermo Fisher SCIENTIFIC. Following the general protocol, the cells laden 3D bio printed scaffolds were stained and live dead image images were taken. Figure \u003cspan class=\"InternalRef\"\u003e13\u003c/span\u003e(C) shows the live cells image which is represented by green colors. The live cells show the clear fluorescence indication. Figure \u003cspan class=\"InternalRef\"\u003e13\u003c/span\u003e(D) represents the Live-Dead cells. The green colors is representing the live H-1975 cells and the red color represents the dead H 1975 cells. Figure \u003cspan class=\"InternalRef\"\u003e13\u003c/span\u003e(E) represents solely the dead cells indicated by red color. The live dead images were taken at day 10 using fluorescence microscopy.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e3.5.4 Optical Image Analysis\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe table below shows the average strand size as well as the average pore size of the printed hydrogels.\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003ctable id=\"Tab5\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eOptical characteristics of the 3d printed hybrid scaffolds\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eComposites\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStrands size(um)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePore size\u003c/p\u003e\n \u003cp\u003e(um)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1% Alginate (w/v)\u0026thinsp;+\u0026thinsp;9% Gelatin (w/v)\u0026thinsp;+\u0026thinsp;0.5M CaCl\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e414.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e880.577\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2% Alginate (w/v)\u0026thinsp;+\u0026thinsp;8% Gelatin (w/v)\u0026thinsp;+\u0026thinsp;0.5M CaCl\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e301.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e923.23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3% Alginate (w/v)\u0026thinsp;+\u0026thinsp;7% Gelatin (w/v)\u0026thinsp;+\u0026thinsp;0.5M CaCl\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e294.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e854.66\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5% Alginate (w/v)\u0026thinsp;+\u0026thinsp;5% Gelatin (w/v)\u0026thinsp;+\u0026thinsp;0.5M CaCl\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e317.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e883.48\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003eThe morphological structure of the 3D bio-printed scaffolds is porous and no significant difference of the same composite. But for different composites the porosity varies. Computer Aided Design (CAD) model of stl file have different porosities were used to fabricate hydrogel structures. 1 to 13 layers successfully. The porosity of the highest layer was higher than the successive printed lower one. With increasing the printing layers the swelling of the lower increased due to the gravitational force. It was found that for layer 1 to 7 the porosity was no significant change. The printed structures have proper channel to pass oxygen and nutrition within the microenvironment of the individual tissue to nourish it for surviving inside the scaffolds. Porosity is the most important parameters for increasing the cell numbers and for the viability of the cells. The printed scaffolds need sufficient space for growing the cells successfully. The structure of the scaffolds need to protect from collapse, hence to choice the scaffolds in the experiment is good for biomedical applications. 4% sodium alginate\u0026thinsp;+\u0026thinsp;6% gelatin\u0026thinsp;+\u0026thinsp;0.5M CaCl\u003csub\u003e2\u003c/sub\u003e sample was not visible in the optical microscope due tan𝛅 value exceeding 0.45.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn conclusion, the assessment of alginate gelatin hydrogel bioinks with CaCl\u003csub\u003e2\u003c/sub\u003e crosslinker revealed their enormous potential for 3D/4D bioprinting applications, as indicated by various physical, chemical, mechanical, and biological inspections. In this context, CaCl\u003csub\u003e2\u003c/sub\u003e is an effective crosslinking agent that improves the mechanical strength of the solutions. The FTIR analysis aligns with the intended bond formation. FTIR results validated the efficient mixture of alginate and gelatin through distinct peaks that correspond to hydroxyl, carboxyl, and amide groups, which indicate intermolecular interactions. The SEM results of all compositions demonstrated a well-structured porous microstructure network that facilitates effective nutrient diffusion and cell propagation. The TGA, DTA, and DTG analyses indicate that this material exhibits excellent the rmoresponsive properties and strong thermal stability, which directly influence nutrient diffusion and cellular viability. The findings from the swelling test suggest that the SA-G composition possesses excellent swelling characteristics. Swelling characteristics provided an optimal and hydrated microenvironment for cell survival and growth. The evaluation of rheological properties, including storage modulus, loss modulus, and phase angle, revealed shear thinning behavior across strain, frequency, and time sweeps, indicating structural and printed stability. Phase angle maintained below 45 degrees, indicating solid-like properties of the gels, which is essential for preserving shape fidelity post-printing. The cell viability of developed bioink\u0026rsquo;s compositon indicate the non-toxicity of the bioink composition. Cell viability assays demonstrated significant biocompatibility with Schwann cells, cancer cells, and co-cultures, indicating robust cellular adhesion, proliferation, and metabolic activity within the printed constructs. Co-culture systems exhibited improved cell-cell interactions and diverse tissue modeling. The 3D bioprinted structures maintained their geometrical integrity with minimal deformation, demonstrating outstanding shape retention and structural stability. Optical microscopy validated the consistent formation of strands, the presence of interconnected pores, and the distribution of cells within the hydrogel matrix. Live-dead staining effectively demonstrated the presence of viable cell populations throughout the layers, confirming the supportive microenvironment established by the bioink. The co-culture system demonstrated synergistic effects, indicating its potential application in intricate tissue modeling. The collective findings confirm that alginate-gelatin hydrogels satisfy essential criteria for bio-fabrication, providing a stable, cell-supportive, and customizable platform for advanced 3D/4D bioprinting.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eMohaimenul Khan: Data curation; Methodology; Formal analysis ; Writing \u0026ndash; review . Dr Md Alamgir Hossain: Supervision; Project administration; Funding acquisition; Conceptualization ; Methodology, Validation ; Resources ; Writing \u0026ndash; editing .\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eNo\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbert AA, Akiah MA (2023) Preparation of Bioink for Hydrogel Printing in Additive Manufacturing. Malaysian J Compos Sci Manuf 12(1):43\u0026ndash;50\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAl-Hashimi N, Babenko M, Saaed M, Kargar N, ElShaer A (2021) The impact of natural and synthetic polymers in formulating micro and nanoparticles for anti-diabetic drugs. Curr Drug Deliv 18(3):271\u0026ndash;288\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlmeida ND, Carneiro CA, de Marco AC, Porto VC, Fran\u0026ccedil;a R (2024) 3D Bioprinting Techniques and Bioinks for Periodontal Tissues Regeneration-A Literature Review. 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Clinical Oral Investigations, 27(9),5153\u0026ndash;5170\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"Hydrogels, Cancer and normal cells, 3D Bio-printing, Tissue Engineering","lastPublishedDoi":"10.21203/rs.3.rs-7163757/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7163757/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe development of hydrogel bioinks faces several obstacles, including optimizing the printing parameters of bioinks, maintaining tissue vascularization, and ensuring good mechanical strength, among others. In this work, alginate-gelatin hydrogel bioinks are developed assessing the physical properties, including swelling properties, thermal properties, stiffness, and rheological properties and cell survivability. CaCl\u003csub\u003e2\u003c/sub\u003e was used as a cross-linker to enhance the bio-inks\u0026rsquo; mechanical stability. FTIR analysis of Ca\u003csup\u003e2+\u003c/sup\u003e crosslinked with sodium alginate-gelatin (SA-G) reports a slight shift in symmetric stretching carboxyl groups. Morphological structure of optimized SA-G bio-ink showed well porous interconnected net like structure. The swelling results show an inverse relationship with increasing the proportion of sodium alginate. Stiffness indicates the resistance of the hydrogel bioink's surface to deformation under applied load. Higher stiffness indicates solid behaviour, while lower stiffness indicates a viscous structure. The storage modulus (G'), loss modulus (G\"), and phase angle, as measured by a rotational rheometer, which indicates the solid point, viscous point, and viscoelastic point. Cells (Schwann cells, Cancer cells and the co culture cells) survivability in 2D or monolayer system confirms the non-toxicity of the developed hydrogels for 3D/4D bioprinting. The 3D bio-printing was carried by extrusion bio printing process. 3D bio printed structure's stability and well size porous structure were analyzed by pore size and the life dead assay showed the live and dead cells after the bio-printing at day 10 using fluorescence microscopy. Thus the developed hydrogel can play a crucial roles for tissue engineering.\u003c/p\u003e","manuscriptTitle":"Alginate-Gelatin Composite Hydrogels for Next-Generation 3D Bio-Printing in Tissue Engineering","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-22 11:07:04","doi":"10.21203/rs.3.rs-7163757/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":"f0b1ce5c-d1a8-45a6-a706-c69fc971f788","owner":[],"postedDate":"July 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-17T20:23:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-22 11:07:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7163757","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7163757","identity":"rs-7163757","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}