Digital Light Processing Printed Hydrogel Scaffolds with Adjustable Modulus

Digital Light Processing Printed Hydrogel Scaffolds with Adjustable Modulus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Digital Light Processing Printed Hydrogel Scaffolds with Adjustable Modulus Feng Xu, Hang Jin, Huiquan Wu, Acan Jiang, Bin Qiu, Lingling Liu, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4083780/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 13 You are reading this latest preprint version Abstract Hydrogels are extensively explored as biomaterials for tissue scaffolds, and their controlled fabrication has been the subject of wide investigation. However, the tedious mechanical property adjusting process through formula control hindered their application for diverse tissue scaffolds. To overcome this limitation, we proposed a two-step process to realize simple adjustment of mechanical modulus over a broad range, by combining digital light processing (DLP) and post-processing steps. UV-curable hydrogels are 3D printed via DLP, with the ability to create complex 3D patterns. Subsequent post-processing with Fe 3+ ions bath induces secondary crosslinking of hydrogel scaffolds, tuning the modulus as required through soaking in solutions with different Fe 3+ concentrations. This innovative two-step process offers high-precision (10 µm) and broad modulus adjusting capability (15.8–345 kPa), covering a broad range of tissues in the human body. As a practical demonstration, hydrogel scaffolds with tissue-mimicking patterns were printed for cultivating cardiac tissue and vascular scaffolds, which can effectively support tissue growth and induce tissue morphologies. Physical sciences/Engineering/Biomedical engineering Physical sciences/Materials science/Soft materials Biological sciences/Biotechnology/Tissue engineering Digital Light Processing Double Network Hydrogels Hydrogel Scaffolds Adjustable Modulus Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Tissue scaffold plays an important role in tissue engineering, providing structural support for the biomimetic characteristics of tissues 1 . The main motivation is to replicate the specific structures and mechanical environments of human tissues, thereby promoting effective tissue remodeling 2 . However, the morphology and modulus of soft tissues vary widely within the human body, e.g. liver (modulus < 10 kPa 3,4 ) possesses functional hepatic lobules arranged in a radial distribution, cardiac tissue (modulus ~ 10 kPa 5–10 ) exhibits an organized orientation, blood vessels (modulus ~ 10 3 kPa 11 ) feature network-like structures. Moreover, the modulus of cardiac tissue increases with development, starting at approximately 12 kPa in the embryonic stage and reaching around 30 kPa in adulthood 12 . This makes the manufacturing of diverse tissue scaffolds challenging while aiming to match the complexed structural and mechanical properties. Hydrogels find extensive applications in tissue engineering, due to their excellent properties including flexibility, stretchability, and biocompatibility 13–16 . UV-curable hydrogels, in particular, offer a versatile solution for creating high-resolution 3D structures through techniques like Stereolithography or Digital Light Processing (DLP) 17–21 . It is feasible to efficiently and rapidly manufacture bionic tissue scaffolds with macro-microscopic structures. For example, tissue scaffolds with structures like networked blood vessel scaffolds 22,23 , tendon-like scaffolds 24 , and irregular-shaped bone and cartilage engineering scaffolds 25,26 can be manufactured using UV-curable hydrogel in DLP fabrication 27 . And DLP-printed hydrogel scaffolds can easily replicate the specific structures of diverse tissue. Currently, the mechanical properties of hydrogel scaffolds can be generally modulated through UV exposure control 28,29 , structural design 30 , and formula adjustment 23,31 . UV energy intensity and exposure time have a pronounced effect on the mechanical behavior of hydrogels 28,29 . However, unavoidable scattering, transmission, and refraction of light occur during printing, resulting in varying crosslinking densities across different parts of the scaffold (where monomers are not fully crosslinked). This becomes more pronounced in scaffolds with complex structures 27,29 . Structural design is another important way to modify the modulus of a scaffold. Incorporating hollow structures into the scaffold reduces the overall stiffness of a sample 28,30 . The cells sense the local modulus of scaffolds, which indicates a structural resolution of micrometer scale is required. Such methods would greatly reduce the efficiency and raise the cost of printing 32,33 . By adjusting the components or ratios in the formula, the polymer network and the modulus of the DLP-printed sample can be regulated remarkably 23,31 . An increase in the monomer content or the number of unsaturated bonds in the formula can raise the crosslinking density of the polymer network rapidly 29,34 . On the contrary, if components prone to hydrolysis are added to the polymer network, the crosslinking density of the polymer network decrease, which results in a reduction of the modulus 35 . The presence of additives in solid form into the polymer network will also enhance the sample modulus. The addictive and hydrogels form composite materials, and the cured sample exhibited higher modulus with increasing the additive contents 36 . However, scaffolds produced with the same formula have a limited modulus adjusting range, making it challenging to match the mechanical environment required by a variety of tissues. On the other hand, altering the formula reduces versability, raises printing costs, and lowers its maneuverability. Herein, we report a two-step process to realize simple adjustment of mechanical modulus over a broad range, by combining DLP and post-processing steps. Through this two-step process, hydrogel scaffolds with exceptional spatial resolution, biomimetic microstructural, and adjustable modulus were fabricated. These attributes represent a distinctive advantage not attainable through other bioprinting processes. The fabricated scaffold, mirroring the morphology and modulus of native tissue, effectively induced the cultivation of well-organized, synchronized beating cardiac tissue or branching coronary artery. Importantly, this manufacturing process is user-friendly and broadly applicable to ion-crosslinked double network hydrogel systems, and is expected to provide an important method for biomimetic tissue engineering. Experimental Materials Acrylamide (AAm) (Macklin, Shanghai, China) as the monomer; poly(ethylene glycol) diacrylate (PEGDA, Mw = 1000) (Macklin, Shanghai, China) as crosslinker. Sodium alginate (Alg) (Rhawn, Shanghai, China) was introduced as a second crosslinking network, Lithiumpheny-2,4,6-trimethyl-benzoylphosphinate (LAP) (Energy Chemical, Shanghai, China) photoinitiator, Tartrazine (Aladdin, Shanghai, China) as a UV absorber. FeCl 3 ·6H 2 O (Acmec, Shanghai, China) was used as ionic crosslinks to adjust the modulus of the sample. Deionized water (DI water) was produced for laboratory pure water systems (Master-Q30UT, HHitech, Shanghai, China). Preparation of UV-curable Hydrogel Solutions The proposed UV-curable hydrogel formula was a mixture of AAm, PEGDA, and Alg precursors. The basic printing solution was developed with a composition of AAm: PEGDA: LAP: Tartrazine: DI water = 1: 0.03: 0.03: 0.015: 4. The hydrogel solution was prepared at five concentrations of Alg (0, 1, 2, 4, and 6% Alg/AAm ratios). Alg solution was prepared by dissolving quantitative powder in 100 g of DI water under magnetic stirring for 12 hours at 35°C. 0.75 g of LAP and 0.375 g of Tartrazine were dissolved in the Alg solution by stirring for 2 hours at 25°C. 25 g AAm and 0.75 g PEGDA were added to the above mixture solution and stirred for 5 hours to obtain the desired UV-curable hydrogel solution. It is worth noting that stirring should be conducted in the dark, and the resulting hydrogel solution should be stored in a refrigerator. Hydrogel Samples Manufacturing The samples were fabricated using a 3D printer (S240, BMF Precision Tech Inc., Chong Qin, China) based on digital light processing technology, which provides a resolution of 10 μm (Fig. 1a). A 405 nm light source was used, and the light energy density was adjusted to 43.1 mW/cm 2 for our experiments. The 3D digital model was sliced using CHITUBOX V1.9.4, with 10-40 μm for each layer, corresponding to an exposure time of 4-6 s. The printed samples were immersed in a 40 wt% ethanol solution for 15 min to dissolve any hydrogel solution on the surface. All sample surface was dried using a high-pressure air gun. Then the samples were post-cured under UV light (1000 mW, 15 min). Subsequently, the samples were soaked in a corresponding concentration of Fe 3+ solution for 24 h to guarantee a complete ion exchange and crosslinking with alginate in the ion bath (Fig. 1b) 37 . To ensure uniform crosslinking, a sufficient amount of Fe 3+ ion bath solution is provided so as to maintain the stability of the ion concentration during the soaking (Fig. 1c). Characterization of Hydrogels The rheological properties of hydrogel solutions were measured using a rotational viscometer (MCR302, Anton Paar, Graz, Austria). All measurements were performed at 25°C (except for temperature scans) and low viscosity solutions were tested using a cone-and-plate geometry of CP50-1 (cone angle: 1°, diameter: 50 mm). The swelling behavior of hydrogel samples was evaluated using rectangular specimens measuring 16 mm ´ 10 mm ´ 2 mm. The lengthwise deformation of the samples was recorded for 7 days after crosslinking with Fe 3+ at concentrations of 0, 0.005 M, 0.01 M, 0.02 M, and 1 M, for alginate content of 0 wt%, 1 wt%, 2 wt%, 4 wt%, and 6 wt% (Alg/AAm). At room temperature, the mechanical properties of hydrogel samples were measured using an electromechanical universal testing machine (E43, Meters Industrial Systems, USA) @2 mm/min with a 50 N load cell. Each sample was tested and recorded with a dial caliper with a precision of 0.01 mm. Unless otherwise specified, the hydrogel samples were subjected to ion bath treatment for 1 day and soaked in DI water for 7 days. According to the diffusion formula of ions in an aqueous solution, prolonged soaking allows for the uniform diffusion of Fe 3+ into the hydrogel samples 37 . The structures of testing models and hydrogel scaffolds were characterized and measured using a digital microscope (DSX1000, Olympus Corporation, Japan). After ion bath treatment, PAAm-Alg hydrogels were cut into equal-sized pieces (~6 mm ´ 6 mm ´ 3 mm) for biocompatibility testing. After rinsing thoroughly with phosphate-buffered saline (PBS), the hydrogel samples were incubated in quantitative DMEM medium (the ratio of surface area to medium volume is 3 cm 2 /mL) for 72 hours @37°C. The biocompatibility was tested on fibroblast cells cultured in the extract of hydrogels using Enzyme Markers (K3 Touch, Thermo Fisher, USA), and a CCK-8 kit (Shanghai Beyoncé Bio, C0043) after 72 hours. Generation of HiPSC Derived Cardiomyocytes and Endothelial Cells HiPSC (Beijing Cel-lapy Biological Technology Co. Ltd., Beijing, China) were cultured on matrigel-coated (0.15%, Corning, 356234) six-well plates in StemFlexTM medium (Gibco, A3349401) at 37℃ in 5% CO 2 . The hiPSCs were labeled with green fluorescent by introducing an EGFP expressional element in an AAVS1 locus. HiPSC-CMs were cultured according to previous publications 38,39 . Human Coronary Artery Endothelial Cells (HCAEC) (FengHuiShengWu, CL0117) were cultivated as adherent cells in Endothelial Cell Medium (ECM, Sciencell, 1001). When the cell confluence reached 80%, a 0.05% trypsin solution was employed for a digestion period of 8-10 minutes. To halt the enzymatic digestion, a serum-containing culture medium was used. Subsequently, the cells were gently detached using a pipette, transferred to a centrifuge tube, and subjected to centrifugation at 200 g for 5 minutes. After discarding the supernatant, the cell pellet was resuspended in ECM to achieve a concentration of 5×10 6 /mL and then seeded onto the hydrogel substrates. Seeding and Culture of the Cardiomyocytes and Endothelial Cells All samples underwent sterilization through γ-ray irradiation. The hydrogel scaffolds were immersed in the culture medium three consecutive times, with each immersion lasting no less than 3 hours. Subsequently, the samples were immersed in the culture medium for more than 72 hours and stored in a refrigerator. Before cell seeding, all hydrogel scaffolds were coated with porcine gelatin (Sigma, V900863) at 2% w/v in phosphate-buffered saline (PBS) for 2 days @37°C. Cardiac tissue scaffolds: a cell suspension was prepared by mixing cardiomyocytes and fibroblasts with a ratio of 2:1. 500 μl cell suspension (3 ´10 5 /well) was seeded on a hydrogel scaffold, and all samples were incubated for 12 h @37°C to allow cell attachment. Subsequently, the devices were cultured in the RPMI 1640 Medium with 3% KnockOut Serum Replacement @37℃, and 5% CO 2 every 2-3 days changes of media for the duration of the experiment. Vasculature-like hydrogel scaffolds: 15 μL of endothelial cell suspension (7.5 ´10 4 /well) were pipetted onto the central groove of the samples. Culture medium was added 4 hours later. For immunofluorescence staining, cells were fixed with 4% paraformaldehyde for 20 min, followed by permeabilization with 0.25% Triton-X 100 (Sigma-Aldrich) in PBS for 20 min at room temperature. The morphology of tissues cultured on the scaffold was recorded using a fluorescence microscope (MF52-N, Mshot, China). Furthermore, rhythmic contractions of tissue were induced using a custom-made electrical stimulation device, employing a pair of electrodes to generate a uniform electric field. Unless otherwise specified, the electrical field strength applied to the cardiac tissue by the stimulation device was set at 8 V/cm. The cardiac tissue contractions were recorded through video documentation. Statistical analysis The data were indicated by the mean ± standard deviation (SD). We conducted swelling, surface morphology, and mechanical testing calculations for each experimental group at least three times. We used OriginPro2017 software for statistical analysis was applied to compare the mean values within each group. The data of Stress-strain curves in the paper has undergone smoothing processing. Ethical approval The cardiomyocytes and endothelial cells used in the study were commercially cells. All cells experiment in the manuscript were approved by the Declaration of Helsinki guidelines. Results and discussions Printability and Swelling Behaviors of the Hydrogel Formula To meet the desired printability and swelling behaviors, the AAm-Alg hydrogel formula was carefully designed, as shown in Fig. 1. AAm has been extensively studied as a UV-curable hydrogel solution. To minimize the toxicity of printed samples, the hydrogel formula was improved by using LAP and Tartrazine, which have superior biocompatibility. And PEGDA (Mw = 1000) was added as a crosslinker to form rigid chains connecting the flexible chains of polyacrylamide (PAAm), enhancing the mechanical properties of the polymer network 40,41 . Among the variety of choices, a formable formula (AAm: PEGDA: LAP: Tartrazine: DI water =1: 0.03: 0.03: 0.015: 4) was selected. A sufficient fluidity of the UV-curable formula is crucial to reduce the difficulty of the recoating process in DLP printing, thereby improving both the printing efficiency and quality. A hydrogel solution with low viscosity enhances printability, including continuity and thickness control 42,43 . However, to create a dual-network hydrogel with an adjustable modulus, Alg was added into the formula. The addition of alginate increased the viscosity and brought distinct non-Newtonian fluid behavior to the hydrogel solution, as shown in Fig. 2a. At a low shear rate of 10 s -1 , the viscosity of the hydrogel solution (0-6 wt% Alg/AAm) increased from 3.58 mPa·s to 168 mPa·s (Fig. 2a),. Besides, the rheological properties of this formula are temperature-sensitive. In a temperature scan ranging from 25 to 60℃, the 4 wt% solution viscosity decreased from 61.4 mPa·s to 26.4 mPa·s @50 s -1 (Fig. S1). In addition, the swelling behaviors of hydrogels are closely related to Alg. Cured PAAm-Alg hydrogels exhibit hydrophilicity due to the abundant hydrophilic functional groups in the polymer chains. Therefore, those cured samples swelled at immersion in solutions, resulting in undesirable shape changes 44 . Excessive deformation is not conducive to printing scaffolds with high resolution. Advanced dual-network hydrogel formula design helps the swelling control. The deformation extent of 0-6 wt% (Alg/AAm) UV-curable hydrogel samples in DI water, 0.005 M, 0.01 M, 0.02 M, and 1 M Fe 3+ environments was recorded for 7 days. At the same Alg content, the samples treated in different ion baths exhibit similar deformations (Fig. S2). However, the swelling deformations ratio of the samples decreased with a higher content of alginate in the same ion bath (Fig. 2b). Notably, the addition of alginate created a double network hydrogel that limited swelling even in DI water without ionic crosslinking. After 1 M Fe 3+ bath treatment, the swelling deformation of 4wt% and 6wt% samples (~130%, and ~120%, respectively) were comparable and acceptable. Ultimately, balancing the viscosity and swelling requirements of the formula, a UV-curable hydrogel solution containing 4wt% alginate was selected in this study. To visualize the swelling deformation of hydrogel samples, a 10 mm hydrogel ruler (4wt% Alg/AAm) was printed. In Figure 2c, the state of the hydrogel ruler was shown directly after printing, either with 1 M Fe 3+ bath treatment, or after immersion in DI water. The ratio of their length to width was nearly equal (2.67, 2.53, 2.67 respectively). It could be confirmed that, after swelling, the manufactured hydrogel samples undergo consistent deformation on a macroscopic scale. It is worth mentioning that the cost-effective UV-curable hydrogel (priced at approximately $10 for 100 g solution) exclusively incorporates readily accessible commercial-grade raw materials, thereby diminishing the entry threshold for 3D bioprinting. Optimization of DLP Parameters To achieve high-resolution printing, the optimal printing parameters for the UV-curable hydrogel solution were investigated. Based on the Beer-Lambert law, the curing behavior of photocurable inks can be described by Jacobs equation 43,45 : where C d is the depth of cure at a given exposure E , D p is the transmission depth of UV-curable solutions, and E c is the critical exposure intensity. Printing hollow structures with a transverse channel using different energy densities inputted on the top layer was performed (Fig. S3). The thickness of the top layer film of the channel was measured to characterize the cured depth of the hydrogel solution, and the relationship between the cured depth and the energy density was fitted (Fig. 3a, D p = 44.4 μm, ln( E c ) = 4.97 mJ/cm 2 ). According to this model, the ideal exposure time for 10-40 μm thick film is 4-6 s. Utilizing these optimized process parameters, micro lines with a width ranging from 10-50 μm (Fig. 3b) were printed, achieving the upper limit resolution of the DLP printer. The application of a higher precision UV source could further improve the resolution. Furthermore, hydrogel samples with complex 2D/3D patterns were printed, including the Xiamen University emblem (Fig. 3c) and a 3D octopus (Fig. 3d), which reproduced the details of the circular design and characters with fidelity. To further validate the printability of the material, a flexible sample containing double-helix internal channels was printed with a diameter of 1 mm (Fig. 3e). The two channels were infused with different liquids, which demonstrated smooth flow without any obstruction (Video S1). These features endow this hydrogel formula with the capability to manufacture integrated flexible and complex 3D microfluidic chips. Mechanical Properties of the Hydrogels with Adjustable Modulus Alginate in the hydrogel formula formed an adjustable ion crosslinking network in the environment of multivalent cations. Different multivalent ions exhibited varying effects on crosslinking alginate, such as Na + , Ca 2+ , Sr 2+ , Ba 2+ , Al 3+ , or Fe 3+ 37,46 . Besides, the concentration of the cations was also related to the crosslinking density of the polymer chains 47 . By controlling the Fe 3+ concentration in the ion bath, the crosslinking density of the PAAm-Alg double network hydrogel was manipulated, which allowed Young's modulus adjustment to hydrogel samples. After Fe 3+ ion bath treatment, the color of the samples intensifies with the rising Fe 3+ concentrations. The introduction of Alg into the hydrogel formula led to a subtle increase of modulus in untreated samples pre- and post-swelling (Fig. 4b), due to the physical double network hydrogels of PAAm-Alg 48 . Following the Fe 3+ ion bath treatment, the hydrogel modulus exhibited a proportional increase with the ascending ion concentration, ranging from 15.8-345 kPa (Fig. 4a). Such a large modulus adjustable range allows the printed hydrogel scaffold to simulate the modulus of almost all human soft tissues. As shown in Fig. 4c and Fig. S4, the elastic deformation portion of the stress-strain curves in Fig. 4a was used to calculate Young’s modulus of hydrogel samples, revealing its relationship with the Fe 3+ concentration in the ion bath. The modulus increased rapidly and proportionally with the ion concentration, which indicated a rise in crosslinking density. However, with further increasing the Fe 3+ ion concentration (>0.1 M), the modulus value gradually flatten. This deceleration was attributed to a reduction in ion crosslinking sites within the alginate segments. Cyclic stretching tests on hydrogel samples (0.1 M Fe 3+ ) showed that the stress-strain curves of the samples had good repeatability under 15%-30% strain (Fig. 4d). Patterned Tissue Induced by Hydrogel Scaffolds. Various soft tissues within the human body exhibit inherent orientations, such as the organization of cardiac tissue and the intricate patterns in vascular networks. Utilizing patterned hydrogel scaffolds offers a promising method for inducing well-organized or patterned tissue growth. The cell viabilities of hydrogels crosslinked with 0 M and 1 M Fe 3+ were 1.01 and 0.98, as shown in Fig. S5, compared to the control group, indicating no obvious toxicity. Cardiac tissue scaffolds with periodic H-shaped grooves (width of 100 μm, depth of 200 μm, gap of 110 μm) were manufactured. Upon immersion in DI water, the printed H-shaped grooves experienced controlled expansion. To facilitate communication between induced cardiac tissues within each groove, additional vertical grooves were incorporated into the H-shaped grooves (Figs. 5a,c). This design allowed the seeding of cells onto the hydrogel scaffold, inducing the formation of a cohesive and organized tissue. Cardiac tissue is characterized by its softness, with a modulus of ~10 kPa 5-10 . Therefore, the cardiac tissue scaffold was prepared without Fe 3+ bath treatment to maintain a minimum modulus of ~16 kPa. A mixed suspension of self-fluorescent cardiomyocyte and fibroblast cell (2:1) was seeded onto the flexible scaffolds. Due to the gravity, a considerable number of cells were settled down and distributed within the grooves. Cardiomyocytes gradually migrated and aggregated, forming organized and continuous tissue (Fig. 5a). As shown in Fig. 5d, cardiomyocytes transitioned from an initial spherical shape to an organized tissue structure induced by H-shaped grooves. The tissue in different lines was connected through the horizontal connections. Therefore, an organized mesh-like tissue was formed through topographical constraint. Comparatively, cardiac tissue cultured on a flat substrate (Fig. 5e) failed to form an organized structure and tended to grow in clusters (Fig. 5b,f). Those cells underwent arbitrary clustering, displaying uncontrollable boundaries between tissue clusters, which is more apparent in the stained images (Fig. S6). The organized cardiac tissue could be driven to contract under an electric field generated by the electrical stimulation device. Under a pulse field with frequencies ranging from 0.5 to 1.5 Hz, the cardiac tissue exhibited synchronous contractions that followed the applied frequency (Video S2). However, at 2 Hz, although the tissue demonstrated contractions, it failed to keep pace with the frequency of the electric field. Compared with cardiac tissue, blood vessels possess a higher modulus and exhibit a more intricate macroscopic structure. Vasculature-like hydrogel scaffolds were printed and treated with 1 M Fe 3+ to obtain Young's modulus of ~345 MPa, mimicking that of native blood vessels as shown in Fig. 6a. The vascular tissue scaffold was designed with semi-open channels of varying diameters. After swelling, the diameter raised from the designed 1.2, 0.3, and 0.1 mm to 1.52, 0.47, and 0.16 mm, respectively. HCAECs were seeded onto the scaffold, forming a semi-open vascular network. Tissues grown on scaffolds exhibited apparent vascular morphological features. Upon staining HCAECs across the entire scaffold, it was evident that HCAECs could be well-distributed on the scaffold and microchannels (Fig. 6b-d). Within those 0.16 mm grooves (Fig. 6e), HCAECs exhibited distinctive vascular pattern. Conversely, endothelial cells cultured on a substrate devoid of grooves did not exhibit any noticeable pattern (Fig. 6f). Conclusion In conclusion, a simple and versatile two-step process, combining DLP and post-treatment. Ion bath treatment is performed after the DLP printing. Using a single AAm-Alg hydrogel formula, hydrogel structures with biocompatible, high-resolution, and adjustable modulus can be fabricated. This two-step process makes bioprinting more cost-effective and straightforward. The UV-curable hydrogel solution exhibited excellent printability, achieving a printing feature of 10 μm (limited by the resolution of the light source) that resembles the characteristic morphology of biological tissues. The ion bath treatment enables a wide range of modulus adjustments (15.83-344.67 kPa) in a simple way, with the ability to customize the modulus of hydrogel samples. Patterned hydrogel scaffolds with low or high moduli were respectively designed and manufactured for cultivating cardiac and vascular tissue. The cultivated tissues were induced by the scaffold morphology, even resembling a biomimetic multilevel vascular network. The cultured organized cardiac tissue exhibits synchronous beating under electrical stimulation. We believe that this two-step process provides an easily feasible method for manufacturing tailored scaffolds for a broad range of tissues in the human body. Declarations Data availability All data analyzed during this study are included in this published article. Acknowledgements The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publicaiton of this article:This work was supported by the National Natural Science Foundation of China (No. U2005214) and the National Key Research and Development Program of China (2022YFB4600600). Author contributions Feng Xu: conceptualization, methodology, data analysis and writing (original draft). Hang Jin, Huiquan Wu and Acan Jiang: investigation, validation and data analysis. Bin Qiu, Lingling Liu and Qiang Gao: validation and data analysis. Bin Lin, Weiwei Kong: investigation and data analysis. Songyue Chen, and Daoheng Sun: conceptualization, writing (review and editing) and funding acquisition. Conflict of interest The authors declare no conflicts of interest. References Zhang, F. & King, M. W. 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Supplementary Files Video1AflowchanneldoublehelicalinshageS1.mp4 Video2ElectricalStimulationS2.mp4 supplementaryinformation.pdf Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 07 May, 2024 Reviews received at journal 27 Apr, 2024 Reviewers agreed at journal 17 Apr, 2024 Reviewers agreed at journal 16 Apr, 2024 Reviews received at journal 15 Apr, 2024 Reviews received at journal 04 Apr, 2024 Reviewers agreed at journal 29 Mar, 2024 Reviewers agreed at journal 29 Mar, 2024 Reviewers invited by journal 29 Mar, 2024 Editor assigned by journal 29 Mar, 2024 Editor invited by journal 26 Mar, 2024 Submission checks completed at journal 25 Mar, 2024 First submitted to journal 12 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4083780","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":284227421,"identity":"2d75025a-fc31-4f8f-bf91-1558e8ae48f3","order_by":0,"name":"Feng Xu","email":"","orcid":"","institution":"Xiamen University","correspondingAuthor":false,"prefix":"","firstName":"Feng","middleName":"","lastName":"Xu","suffix":""},{"id":284227422,"identity":"bcdb08eb-bd46-4f66-9dd2-5df0bfa66d6b","order_by":1,"name":"Hang Jin","email":"","orcid":"","institution":"Xiamen University","correspondingAuthor":false,"prefix":"","firstName":"Hang","middleName":"","lastName":"Jin","suffix":""},{"id":284227423,"identity":"8f981672-e9ac-41fd-9058-5ba90d136038","order_by":2,"name":"Huiquan Wu","email":"","orcid":"","institution":"Xiamen University","correspondingAuthor":false,"prefix":"","firstName":"Huiquan","middleName":"","lastName":"Wu","suffix":""},{"id":284227424,"identity":"48a2f1a1-63af-471f-a4a1-62e6a274e8f2","order_by":3,"name":"Acan Jiang","email":"","orcid":"","institution":"Xiamen University","correspondingAuthor":false,"prefix":"","firstName":"Acan","middleName":"","lastName":"Jiang","suffix":""},{"id":284227425,"identity":"11a301d9-44c7-4ffd-b1d5-17796f538c07","order_by":4,"name":"Bin Qiu","email":"","orcid":"","institution":"Xiamen University","correspondingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Qiu","suffix":""},{"id":284227426,"identity":"674c9335-9171-48c2-824f-c257c7fed69e","order_by":5,"name":"Lingling Liu","email":"","orcid":"","institution":"Xiamen University","correspondingAuthor":false,"prefix":"","firstName":"Lingling","middleName":"","lastName":"Liu","suffix":""},{"id":284227427,"identity":"99deff89-b2b1-4c78-81bc-cf605981f55a","order_by":6,"name":"Qiang Gao","email":"","orcid":"","institution":"Guangdong Provincial People’s Hospital","correspondingAuthor":false,"prefix":"","firstName":"Qiang","middleName":"","lastName":"Gao","suffix":""},{"id":284227428,"identity":"714983ad-74c5-48c9-9fac-ed38cb22435a","order_by":7,"name":"Bin Lin","email":"","orcid":"","institution":"Guangdong Provincial People’s Hospital","correspondingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Lin","suffix":""},{"id":284227429,"identity":"175a628c-d73c-41af-8f17-3f6d84244402","order_by":8,"name":"Weiwei Kong","email":"","orcid":"","institution":"Guangdong Provincial People’s Hospital","correspondingAuthor":false,"prefix":"","firstName":"Weiwei","middleName":"","lastName":"Kong","suffix":""},{"id":284227430,"identity":"a28689b0-b34f-4179-a91b-f8ca05988682","order_by":9,"name":"Songyue Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAo0lEQVRIiWNgGAWjYHACNiC2gTB5SNCSRrqWwyRokW9gfvbg447ziWtnJDA+eNtGhBaDA2zmhjPP3DY2u5HAbDiXKC0MPGzSvG235YBaQAyiHAbU8rftHA9QC/tvorQwHABqYWw7ALaFmSgtBofZzCR725KNzc48bJacc44Yh7U3P5P42WaXuO148sEPb8qIcRgznMXYQIz6UTAKRsEoGAXEAAAYUzCVRDykYwAAAABJRU5ErkJggg==","orcid":"","institution":"Xiamen University","correspondingAuthor":true,"prefix":"","firstName":"Songyue","middleName":"","lastName":"Chen","suffix":""},{"id":284227431,"identity":"9b9cd222-e982-42e1-b438-f2dfc7fb6124","order_by":10,"name":"Daoheng Sun","email":"","orcid":"","institution":"Xiamen University","correspondingAuthor":false,"prefix":"","firstName":"Daoheng","middleName":"","lastName":"Sun","suffix":""}],"badges":[],"createdAt":"2024-03-12 12:07:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4083780/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4083780/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53549947,"identity":"59604371-20a7-4ba0-8a63-cc9bcdedb0f7","added_by":"auto","created_at":"2024-03-27 11:06:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1076844,"visible":true,"origin":"","legend":"\u003cp\u003eTwo-step process: DLP printing and modulus adjustment. a) Schematic diagram of top-down DLP printing. b) Treatment of the sample in an ion bath. c) Crosslinking process of the hydrogel solution: First step, the solution was cured under 405 nm light to form a long-chain polymer network during the DLP process, and the sample was 3D molded at this stage. Second step, the secondary crosslinking of alginate in the samples was performed using Fe\u003csup\u003e3+\u003c/sup\u003e ion bath treatment. The sample modulus can be regulated by controlling the Fe\u003csup\u003e3+\u003c/sup\u003e concentration.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4083780/v1/697509b700fe0a67a8ef60e7.png"},{"id":53549948,"identity":"940735b6-e447-414f-879f-77e94f5a4ada","added_by":"auto","created_at":"2024-03-27 11:06:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":855569,"visible":true,"origin":"","legend":"\u003cp\u003eViscosity and swelling properties of hydrogel. a) Viscosity curves of hydrogel solutions with Alg/AAm ratios of 0-6%. b) Swelling properties of cured hydrogel samples with different Alg/AAm ratios subjected to 1 M Fe\u003csup\u003e3+\u003c/sup\u003e crosslinking (dashed) or without crosslinking (solid), compared by the lengthwise deformation of the testing samples. c) The swelling of a 4% Alg/AAm ‘Hydrogel Ruler’ in different conditions.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4083780/v1/f8f0adc410d94803d8496f56.png"},{"id":53549955,"identity":"93cb0dc1-5a32-4c53-8b11-48d5dd3f7afd","added_by":"auto","created_at":"2024-03-27 11:06:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1202494,"visible":true,"origin":"","legend":"\u003cp\u003ePrinting parameter optimization. a) Jacobs equation fitting of the curing depth of the UV-curable hydrogel solution. b) Printing of a single line. c) Xiamen University micro emblem and its schematic. d) 3D octopus sample and its schematic. e) Printed sample with double-helical flow channels.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4083780/v1/758824cf423ff13c06f9d810.png"},{"id":53549953,"identity":"510ccfc8-32a4-43e1-8e0c-58467ac71ba6","added_by":"auto","created_at":"2024-03-27 11:06:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":806636,"visible":true,"origin":"","legend":"\u003cp\u003eMechanical properties of Fe\u003csup\u003e3+\u003c/sup\u003e-treated PAAm-Alg hydrogel samples. a) Stress-strain curves of hydrogel samples treated with 0-1 M Fe\u003csup\u003e3+\u003c/sup\u003e ion bath treatment. b) Stress-strain curves of untreated (0, 4 wt% Alg) hydrogel samples, where the solid line represents the sample without swelling and exhibits significantly higher fracture strain than the swollen samples. c) Relationship between Fe\u003csup\u003e3+\u003c/sup\u003e concentration in the ion bath and the Young's modulus of the treated samples. d) Cyclic tensile testing (with 15%, 20%, 25%, and 30% strain) of hydrogel samples treated with 0.1 M ion bath, with the top left inset showing the sample after 100 cycles of 30% strain.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4083780/v1/36fa3c1dbc3f1718a307046b.png"},{"id":53549952,"identity":"92f0d765-db40-4593-aa8c-5f4172e13d59","added_by":"auto","created_at":"2024-03-27 11:06:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2254049,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescent cardiomyocytes were cultured on cardiac tissue scaffolds. a) Schematic illustration of the cultivation process on the scaffold with H-shaped grooves: i. Cell seeding in H-shaped grooves, ii. Induction of cardiac tissue by grooves. b) Schematic illustration of cultivation process on the flat substrate: i. Cell seeding on a flat substrate, ii. Formation of cardiac tissue clusters. c) Cardiac tissue hydrogel scaffold with H-shaped grooves, images of the printed sample and the swollen scaffold. and e) flat substrates. Observing cardiac tissue cultivates on d) scaffolds with H-shaped grooves and f) flat substrates at 0, 4, 8, 24, 48, and 72 hours reveals distinct patterns. In the H-shaped grooves, tissues were induced to form an organized sheet. On the flat substrate, cardiomyocytes gradually aggregate, forming noticeable boundaries between clusters.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4083780/v1/2eca5dc688a822fb3fd64f93.png"},{"id":53550598,"identity":"cbcc1eaf-f2e3-43b1-8c64-83069342fafb","added_by":"auto","created_at":"2024-03-27 11:14:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2517752,"visible":true,"origin":"","legend":"\u003cp\u003eHCAECs on the vasculature-likehydrogel scaffold were stained. a) The Vasculature-like hydrogel scaffold, the crosslinked sample (above) and the printed sample (below). b-e) HCAECs seeded onto the scaffold, growing along the grooves to form vasculature-like structures. b) grooves with width of 1.52 mm, c) grooves with width of 0.47 mm, d) grooves with width of 0.16 mm. e) HCAECs were seeded at the bottom of the grooves, inducing tissue formation. f) HCAECs cultured on a flat substrate exhibited disorganized distribution.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4083780/v1/75b2576344f207c442ddd06c.png"},{"id":53550883,"identity":"fd44693d-5682-4781-a15f-139e116b3160","added_by":"auto","created_at":"2024-03-27 11:22:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5891894,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4083780/v1/35f0b415-e810-4b9d-9025-ae759ca8f80a.pdf"},{"id":53549950,"identity":"754616b4-87bc-4004-86f0-e2e1b56afdfa","added_by":"auto","created_at":"2024-03-27 11:06:48","extension":"mp4","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4851074,"visible":true,"origin":"","legend":"","description":"","filename":"Video1AflowchanneldoublehelicalinshageS1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-4083780/v1/aecbd92fc9eb678a818f6c74.mp4"},{"id":53549956,"identity":"55813717-369f-4eab-90fc-fa12946cddd6","added_by":"auto","created_at":"2024-03-27 11:06:51","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":68122230,"visible":true,"origin":"","legend":"","description":"","filename":"Video2ElectricalStimulationS2.mp4","url":"https://assets-eu.researchsquare.com/files/rs-4083780/v1/a0f984c4587e5f4e131ebab1.mp4"},{"id":53550597,"identity":"e192ff9e-7706-4721-9fe3-461a8c292159","added_by":"auto","created_at":"2024-03-27 11:14:48","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1074257,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryinformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4083780/v1/fd5419408508ee4e34dc5cf2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Digital Light Processing Printed Hydrogel Scaffolds with Adjustable Modulus","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTissue scaffold plays an important role in tissue engineering, providing structural support for the biomimetic characteristics of tissues \u003csup\u003e1\u003c/sup\u003e. The main motivation is to replicate the specific structures and mechanical environments of human tissues, thereby promoting effective tissue remodeling \u003csup\u003e2\u003c/sup\u003e. However, the morphology and modulus of soft tissues vary widely within the human body, e.g. liver (modulus\u0026thinsp;\u0026lt;\u0026thinsp;10 kPa \u003csup\u003e3,4\u003c/sup\u003e) possesses functional hepatic lobules arranged in a radial distribution, cardiac tissue (modulus\u0026thinsp;~\u0026thinsp;10 kPa \u003csup\u003e5\u0026ndash;10\u003c/sup\u003e) exhibits an organized orientation, blood vessels (modulus\u0026thinsp;~\u0026thinsp;10\u003csup\u003e3\u003c/sup\u003e kPa \u003csup\u003e11\u003c/sup\u003e) feature network-like structures. Moreover, the modulus of cardiac tissue increases with development, starting at approximately 12 kPa in the embryonic stage and reaching around 30 kPa in adulthood \u003csup\u003e12\u003c/sup\u003e. This makes the manufacturing of diverse tissue scaffolds challenging while aiming to match the complexed structural and mechanical properties.\u003c/p\u003e \u003cp\u003eHydrogels find extensive applications in tissue engineering, due to their excellent properties including flexibility, stretchability, and biocompatibility \u003csup\u003e13\u0026ndash;16\u003c/sup\u003e. UV-curable hydrogels, in particular, offer a versatile solution for creating high-resolution 3D structures through techniques like Stereolithography or Digital Light Processing (DLP) \u003csup\u003e17\u0026ndash;21\u003c/sup\u003e. It is feasible to efficiently and rapidly manufacture bionic tissue scaffolds with macro-microscopic structures. For example, tissue scaffolds with structures like networked blood vessel scaffolds \u003csup\u003e22,23\u003c/sup\u003e, tendon-like scaffolds \u003csup\u003e24\u003c/sup\u003e, and irregular-shaped bone and cartilage engineering scaffolds \u003csup\u003e25,26\u003c/sup\u003e can be manufactured using UV-curable hydrogel in DLP fabrication \u003csup\u003e27\u003c/sup\u003e. And DLP-printed hydrogel scaffolds can easily replicate the specific structures of diverse tissue.\u003c/p\u003e \u003cp\u003eCurrently, the mechanical properties of hydrogel scaffolds can be generally modulated through UV exposure control \u003csup\u003e28,29\u003c/sup\u003e, structural design \u003csup\u003e30\u003c/sup\u003e, and formula adjustment \u003csup\u003e23,31\u003c/sup\u003e. UV energy intensity and exposure time have a pronounced effect on the mechanical behavior of hydrogels \u003csup\u003e28,29\u003c/sup\u003e. However, unavoidable scattering, transmission, and refraction of light occur during printing, resulting in varying crosslinking densities across different parts of the scaffold (where monomers are not fully crosslinked). This becomes more pronounced in scaffolds with complex structures \u003csup\u003e27,29\u003c/sup\u003e. Structural design is another important way to modify the modulus of a scaffold. Incorporating hollow structures into the scaffold reduces the overall stiffness of a sample \u003csup\u003e28,30\u003c/sup\u003e. The cells sense the local modulus of scaffolds, which indicates a structural resolution of micrometer scale is required. Such methods would greatly reduce the efficiency and raise the cost of printing \u003csup\u003e32,33\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBy adjusting the components or ratios in the formula, the polymer network and the modulus of the DLP-printed sample can be regulated remarkably \u003csup\u003e23,31\u003c/sup\u003e. An increase in the monomer content or the number of unsaturated bonds in the formula can raise the crosslinking density of the polymer network rapidly \u003csup\u003e29,34\u003c/sup\u003e. On the contrary, if components prone to hydrolysis are added to the polymer network, the crosslinking density of the polymer network decrease, which results in a reduction of the modulus \u003csup\u003e35\u003c/sup\u003e. The presence of additives in solid form into the polymer network will also enhance the sample modulus. The addictive and hydrogels form composite materials, and the cured sample exhibited higher modulus with increasing the additive contents \u003csup\u003e36\u003c/sup\u003e. However, scaffolds produced with the same formula have a limited modulus adjusting range, making it challenging to match the mechanical environment required by a variety of tissues. On the other hand, altering the formula reduces versability, raises printing costs, and lowers its maneuverability.\u003c/p\u003e \u003cp\u003eHerein, we report a two-step process to realize simple adjustment of mechanical modulus over a broad range, by combining DLP and post-processing steps. Through this two-step process, hydrogel scaffolds with exceptional spatial resolution, biomimetic microstructural, and adjustable modulus were fabricated. These attributes represent a distinctive advantage not attainable through other bioprinting processes. The fabricated scaffold, mirroring the morphology and modulus of native tissue, effectively induced the cultivation of well-organized, synchronized beating cardiac tissue or branching coronary artery. Importantly, this manufacturing process is user-friendly and broadly applicable to ion-crosslinked double network hydrogel systems, and is expected to provide an important method for biomimetic tissue engineering.\u003c/p\u003e"},{"header":"Experimental","content":"\u003cp\u003eMaterials\u003c/p\u003e\n\u003cp\u003eAcrylamide (AAm) (Macklin, Shanghai, China) as the monomer; poly(ethylene glycol) diacrylate (PEGDA, Mw = 1000) (Macklin, Shanghai, China) as crosslinker. Sodium alginate (Alg) (Rhawn, Shanghai, China) was introduced as a second crosslinking network, Lithiumpheny-2,4,6-trimethyl-benzoylphosphinate (LAP) (Energy Chemical, Shanghai, China) photoinitiator, Tartrazine (Aladdin, Shanghai, China) as a UV absorber. FeCl\u003csub\u003e3\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO (Acmec, Shanghai, China) was used as ionic crosslinks to adjust the modulus of the sample. Deionized water (DI water) was produced for laboratory pure water systems (Master-Q30UT, HHitech, Shanghai, China).\u003c/p\u003e\n\u003cp\u003ePreparation of UV-curable Hydrogel Solutions\u003c/p\u003e\n\u003cp\u003eThe proposed UV-curable hydrogel formula was a mixture of AAm, PEGDA, and Alg precursors. The basic printing solution was developed with a composition of AAm: PEGDA: LAP: Tartrazine: DI water = 1: 0.03: 0.03: 0.015: 4. The hydrogel solution was prepared at five concentrations of Alg (0, 1, 2, 4, and 6% Alg/AAm ratios). Alg solution was prepared by dissolving quantitative powder \u0026nbsp;in 100 g of DI water under magnetic stirring for 12 hours at 35\u0026deg;C. 0.75 g of LAP and 0.375 g of Tartrazine were dissolved in the Alg solution by stirring for 2 hours at 25\u0026deg;C. 25 g AAm and 0.75 g PEGDA were added to the above mixture solution and stirred for 5 hours to obtain the desired UV-curable hydrogel solution. It is worth noting that stirring should be conducted in the dark, and the resulting hydrogel solution should be stored in a refrigerator.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHydrogel Samples Manufacturing\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe samples were fabricated using a 3D printer (S240, BMF Precision Tech Inc., Chong Qin, China) based on digital light processing technology, which provides a resolution of 10 \u0026mu;m (Fig. 1a). A 405 nm light source was used, and the light energy density was adjusted to 43.1 mW/cm\u003csup\u003e2\u003c/sup\u003e for our experiments. The 3D digital model was sliced using CHITUBOX V1.9.4, with 10-40 \u0026mu;m for each layer, corresponding to an exposure time of 4-6 s.\u003c/p\u003e\n\u003cp\u003eThe printed samples were immersed in a 40 wt% ethanol solution for 15 min to dissolve any hydrogel solution on the surface. All sample surface was dried using a high-pressure air gun. Then the samples were post-cured under UV light (1000 mW, 15 min). Subsequently, the samples were soaked in a corresponding concentration of Fe\u003csup\u003e3+\u003c/sup\u003e solution for 24 h to guarantee a complete ion exchange and crosslinking with alginate in the ion bath (Fig. 1b)\u0026nbsp;\u003csup\u003e37\u003c/sup\u003e. To ensure uniform crosslinking, a sufficient amount of Fe\u003csup\u003e3+\u003c/sup\u003e ion bath solution is provided so as to maintain the stability of the ion concentration during the soaking (Fig. 1c).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCharacterization of Hydrogels\u003c/p\u003e\n\u003cp\u003eThe rheological properties of hydrogel solutions were measured using a rotational viscometer (MCR302, Anton Paar, Graz, Austria). All measurements were performed at 25\u0026deg;C (except for temperature scans) and low viscosity solutions were tested using a cone-and-plate geometry of CP50-1 (cone angle: 1\u0026deg;, diameter: 50 mm).\u003c/p\u003e\n\u003cp\u003eThe swelling behavior of hydrogel samples was evaluated using rectangular specimens measuring 16 mm\u0026nbsp;\u0026acute;\u0026nbsp;10 mm\u0026nbsp;\u0026acute;\u0026nbsp;2 mm. The lengthwise deformation of the samples was recorded for 7 days after crosslinking with Fe\u003csup\u003e3+\u003c/sup\u003e at concentrations of 0, 0.005 M, 0.01 M, 0.02 M, and 1 M, for alginate content of 0 wt%, 1 wt%, 2 wt%, 4 wt%, and 6 wt% (Alg/AAm).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAt room temperature, the mechanical properties of hydrogel samples were measured using an electromechanical universal testing machine (E43, Meters Industrial Systems, USA) @2 mm/min with a 50 N load cell. Each sample was tested and recorded with a dial caliper with a precision of 0.01 mm. Unless otherwise specified, the hydrogel samples were subjected to ion bath treatment for 1 day and soaked in DI water for 7 days. According to the diffusion formula of ions in an aqueous solution, prolonged soaking allows for the uniform diffusion of Fe\u003csup\u003e3+\u003c/sup\u003e into the hydrogel samples\u0026nbsp;\u003csup\u003e37\u003c/sup\u003e. The structures of testing models and hydrogel scaffolds were characterized and measured using a digital microscope (DSX1000, Olympus Corporation, Japan).\u003c/p\u003e\n\u003cp\u003eAfter ion bath treatment, PAAm-Alg hydrogels were cut into equal-sized pieces (~6 mm\u0026nbsp;\u0026acute;\u0026nbsp;6 mm\u0026nbsp;\u0026acute;\u0026nbsp;3 mm) for biocompatibility testing. After rinsing thoroughly with phosphate-buffered saline (PBS), the hydrogel samples were incubated in quantitative DMEM medium (the ratio of surface area to medium volume is 3 cm\u003csup\u003e2\u003c/sup\u003e/mL) for 72 hours @37\u0026deg;C. The biocompatibility was tested on fibroblast cells cultured in the extract of hydrogels using Enzyme Markers (K3 Touch, Thermo Fisher, USA), and a CCK-8 kit (Shanghai Beyonc\u0026eacute; Bio, C0043) after 72 hours.\u003c/p\u003e\n\u003cp\u003eGeneration of HiPSC Derived Cardiomyocytes and Endothelial Cells\u003c/p\u003e\n\u003cp\u003eHiPSC (Beijing Cel-lapy Biological Technology Co. Ltd., Beijing, China) were cultured on matrigel-coated (0.15%, Corning, 356234) six-well plates in StemFlexTM medium (Gibco, A3349401) at 37℃ in 5% CO\u003csub\u003e2\u003c/sub\u003e. The hiPSCs were labeled with green fluorescent by introducing an EGFP expressional element in an AAVS1 locus. HiPSC-CMs were cultured according to previous publications\u0026nbsp;\u003csup\u003e38,39\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHuman Coronary Artery\u0026nbsp;Endothelial Cells\u0026nbsp;(HCAEC) (FengHuiShengWu, CL0117) were cultivated as adherent cells in Endothelial Cell Medium (ECM, Sciencell, 1001). When the cell confluence\u0026nbsp;reached 80%, a 0.05% trypsin solution was employed for a digestion period of 8-10 minutes. To halt the enzymatic digestion, a serum-containing culture medium was used. Subsequently, the cells were gently detached using a pipette, transferred to a centrifuge tube, and subjected to centrifugation at 200 g for 5 minutes. After discarding the supernatant, the cell pellet was resuspended in ECM to achieve a concentration of 5\u0026times;10\u003csup\u003e6\u003c/sup\u003e/mL and then seeded onto the hydrogel substrates.\u003c/p\u003e\n\u003cp\u003eSeeding and Culture of the Cardiomyocytes and Endothelial Cells\u003c/p\u003e\n\u003cp\u003eAll samples underwent sterilization through \u0026gamma;-ray irradiation. The hydrogel scaffolds were immersed in the culture medium three consecutive times, with each immersion lasting no less than 3 hours. Subsequently, the samples were immersed in the culture medium for more than 72 hours and stored in a refrigerator. Before cell seeding, all hydrogel scaffolds were coated with porcine gelatin (Sigma, V900863) at 2% w/v in phosphate-buffered saline (PBS) for 2 days @37\u0026deg;C.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCardiac tissue scaffolds: a cell suspension was prepared by mixing cardiomyocytes and fibroblasts with a ratio of 2:1. 500 \u0026mu;l cell suspension (3\u0026nbsp;\u0026acute;10\u003csup\u003e5\u003c/sup\u003e/well) was seeded on a hydrogel scaffold, and all samples were incubated for 12 h @37\u0026deg;C to allow cell attachment. Subsequently, the devices were cultured in the RPMI 1640 Medium with 3% KnockOut Serum Replacement @37℃, and 5% CO\u003csub\u003e2\u003c/sub\u003e every 2-3 days changes of media for the duration of the experiment.\u003c/p\u003e\n\u003cp\u003eVasculature-like hydrogel scaffolds: 15 \u0026mu;L of endothelial cell suspension (7.5\u0026nbsp;\u0026acute;10\u003csup\u003e4\u003c/sup\u003e/well) were pipetted onto the central groove of the samples. Culture medium was added 4 hours later.\u0026nbsp;For immunofluorescence staining, cells were fixed with 4% paraformaldehyde for 20 min, followed by permeabilization with 0.25% Triton-X 100 (Sigma-Aldrich) in PBS for 20 min at room temperature.\u003c/p\u003e\n\u003cp\u003eThe morphology of tissues cultured on the scaffold was recorded using a fluorescence microscope (MF52-N, Mshot, China). Furthermore, rhythmic contractions of tissue were induced using a custom-made electrical stimulation device, employing a pair of electrodes to generate a uniform electric field. Unless otherwise specified, the electrical field strength applied to the cardiac tissue by the stimulation device was set at 8 V/cm. The cardiac tissue contractions were recorded through video documentation.\u003c/p\u003e\n\u003cp\u003eStatistical analysis\u003c/p\u003e\n\u003cp\u003eThe data were indicated by the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). We conducted swelling, surface morphology, and mechanical testing calculations for each experimental group at least three times. We used OriginPro2017 software for statistical analysis was applied to compare the mean values within each group. The data of Stress-strain curves in the paper has undergone smoothing processing.\u003c/p\u003e\n\u003cp\u003eEthical approval\u003c/p\u003e\n\u003cp\u003eThe cardiomyocytes and endothelial cells used in the study were commercially cells. All cells experiment in the manuscript were approved by the Declaration of Helsinki guidelines.\u003c/p\u003e"},{"header":"Results and discussions","content":"\u003cp\u003ePrintability and Swelling Behaviors of the Hydrogel\u0026nbsp;Formula\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo meet the desired printability and swelling behaviors, the AAm-Alg hydrogel formula was carefully designed, as shown in Fig. 1. AAm has been extensively studied as a UV-curable hydrogel solution. To minimize the toxicity of printed samples, the hydrogel formula was improved by using LAP and Tartrazine, which have superior biocompatibility. And PEGDA (Mw = 1000) was added as a crosslinker to form rigid chains connecting the flexible chains of polyacrylamide (PAAm), enhancing the mechanical properties of the polymer network\u0026nbsp;\u003csup\u003e40,41\u003c/sup\u003e. Among the variety of choices, a formable formula (AAm: PEGDA: LAP: Tartrazine: DI water =1: 0.03: 0.03: 0.015: 4) was selected.\u003c/p\u003e\n\u003cp\u003eA sufficient fluidity of the UV-curable formula is crucial to reduce the difficulty of the recoating process in DLP printing, thereby improving both the printing efficiency and quality.\u0026nbsp;A hydrogel solution with low viscosity enhances printability, including continuity and thickness control\u0026nbsp;\u003csup\u003e42,43\u003c/sup\u003e. However, to create a dual-network hydrogel with an adjustable modulus, Alg was added into the formula. The addition of alginate increased the viscosity and brought distinct non-Newtonian fluid behavior to the hydrogel solution, as shown in Fig. 2a. At a low shear rate of 10 s\u003csup\u003e-1\u003c/sup\u003e, the viscosity of the hydrogel solution (0-6 wt% Alg/AAm) increased from 3.58 mPa\u0026middot;s to 168 mPa\u0026middot;s (Fig. 2a),. Besides, the rheological properties of this formula are temperature-sensitive. In a temperature scan ranging from 25 to 60℃, the 4 wt% solution viscosity decreased from 61.4 mPa\u0026middot;s to 26.4 mPa\u0026middot;s @50 s\u003csup\u003e-1\u003c/sup\u003e (Fig. S1).\u003c/p\u003e\n\u003cp\u003eIn addition, the swelling behaviors of hydrogels are closely related to Alg. Cured PAAm-Alg hydrogels exhibit hydrophilicity due to the abundant hydrophilic functional groups in the polymer chains. Therefore, those cured samples swelled at immersion in solutions, resulting in undesirable shape changes \u003csup\u003e44\u003c/sup\u003e. Excessive deformation is not conducive to printing scaffolds with high resolution.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAdvanced dual-network hydrogel formula design helps the swelling control.\u0026nbsp;The deformation extent of 0-6 wt% (Alg/AAm) UV-curable hydrogel samples in DI water, 0.005 M, 0.01 M, 0.02 M, and 1 M Fe\u003csup\u003e3+\u003c/sup\u003e environments was recorded for 7 days. At the same Alg content, the samples treated in different ion baths exhibit similar deformations (Fig. S2). However, the swelling deformations ratio of the samples decreased with a higher content of alginate in the same ion bath (Fig. 2b). Notably, the addition of alginate created a double network hydrogel that limited swelling even in DI water\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;without ionic crosslinking. After 1 M Fe\u003csup\u003e3+\u003c/sup\u003e bath treatment, the swelling deformation of 4wt% and 6wt% samples (~130%, and ~120%, respectively) were comparable and acceptable. Ultimately, balancing the viscosity and swelling requirements of the formula, a UV-curable hydrogel solution containing 4wt% alginate was selected in this study. To visualize the swelling deformation of hydrogel samples, a 10 mm hydrogel ruler (4wt% Alg/AAm) was printed. In Figure 2c, the state of the hydrogel ruler was shown directly after printing, either with 1 M Fe\u003csup\u003e3+\u003c/sup\u003e bath treatment, or after immersion in DI water. The ratio of their length to width was nearly equal (2.67, 2.53, 2.67 respectively). It could be confirmed that, after swelling, the manufactured hydrogel samples undergo consistent deformation on a macroscopic scale.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt is worth mentioning that the cost-effective UV-curable hydrogel (priced at approximately $10 for 100 g solution) exclusively incorporates readily accessible commercial-grade raw materials, thereby diminishing the entry threshold for 3D bioprinting.\u003c/p\u003e\n\u003cp\u003eOptimization of DLP Parameters\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;To achieve high-resolution printing, the optimal printing parameters for the UV-curable hydrogel solution were investigated. Based on the Beer-Lambert law, the curing behavior of photocurable inks can be described by Jacobs equation \u003csup\u003e43,45\u003c/sup\u003e:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"312\" height=\"47\"\u003e\u003c/p\u003e\n\u003cp\u003ewhere\u0026nbsp;\u003cem\u003eC\u003csub\u003ed\u003c/sub\u003e\u003c/em\u003e is the depth of cure at a given exposure\u0026nbsp;\u003cem\u003eE\u003c/em\u003e,\u0026nbsp;\u003cem\u003eD\u003csub\u003ep\u003c/sub\u003e\u003c/em\u003e is the transmission depth of UV-curable solutions, and\u0026nbsp;\u003cem\u003eE\u003csub\u003ec\u003c/sub\u003e\u003c/em\u003e is the critical exposure intensity. Printing hollow structures with a transverse channel using different energy densities inputted on the top layer was performed (Fig. S3). The thickness of the top layer film of the channel was measured to characterize the cured depth of the hydrogel solution, and the relationship between the cured depth and the energy density was fitted\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(Fig. 3a,\u0026nbsp;\u003cem\u003eD\u003csub\u003ep\u003c/sub\u003e\u003c/em\u003e = 44.4 \u0026mu;m, ln(\u003cem\u003eE\u003csub\u003ec\u003c/sub\u003e\u003c/em\u003e) = 4.97 mJ/cm\u003csup\u003e2\u003c/sup\u003e). According to this model, the ideal exposure time for 10-40 \u0026mu;m thick film is 4-6 s.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eUtilizing these optimized process parameters, micro lines with a width ranging from 10-50 \u0026mu;m (Fig. 3b) were printed, achieving the upper limit resolution of the DLP printer. The application of a\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;higher precision UV source could further improve the resolution. Furthermore, hydrogel samples with complex 2D/3D patterns were printed, including the Xiamen University emblem (Fig. 3c) and a 3D octopus (Fig. 3d), which reproduced the details of the circular design and characters with fidelity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo further validate the printability of the material, a flexible sample containing double-helix internal channels was printed with a diameter of 1 mm (Fig. 3e). The two channels were infused with different liquids, which demonstrated smooth flow without any obstruction (Video S1). These features endow this hydrogel formula with the capability to manufacture integrated flexible and complex 3D microfluidic chips.\u003c/p\u003e\n\u003cp\u003eMechanical Properties of the Hydrogels with Adjustable Modulus\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAlginate in the hydrogel formula formed an adjustable ion crosslinking network in the environment of multivalent cations.\u0026nbsp;Different multivalent ions exhibited varying effects on crosslinking alginate, such as Na\u003csup\u003e+\u003c/sup\u003e, Ca\u003csup\u003e2+\u003c/sup\u003e, Sr\u003csup\u003e2+\u003c/sup\u003e, Ba\u003csup\u003e2+\u003c/sup\u003e, Al\u003csup\u003e3+\u003c/sup\u003e, or Fe\u003csup\u003e3+\u003c/sup\u003e \u003csup\u003e37,46\u003c/sup\u003e. Besides, the concentration of the cations was also related to the crosslinking density of the polymer chains\u0026nbsp;\u003csup\u003e47\u003c/sup\u003e.\u0026nbsp;By controlling the Fe\u003csup\u003e3+\u003c/sup\u003e concentration in the ion bath, the crosslinking density of the PAAm-Alg double network hydrogel was manipulated, which allowed Young\u0026apos;s modulus adjustment to hydrogel samples. After Fe\u003csup\u003e3+\u003c/sup\u003e ion bath treatment, the color of the samples intensifies with the rising Fe\u003csup\u003e3+\u003c/sup\u003e concentrations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe introduction of Alg into the hydrogel formula led to a subtle increase of modulus in untreated samples pre- and post-swelling (Fig. 4b), due to the physical double network hydrogels of PAAm-Alg\u0026nbsp;\u003csup\u003e48\u003c/sup\u003e. Following the Fe\u003csup\u003e3+\u003c/sup\u003e ion bath treatment, the hydrogel modulus exhibited a proportional increase with the ascending ion concentration, ranging from 15.8-345 kPa (Fig. 4a). Such a large modulus adjustable range allows the printed hydrogel scaffold to simulate the modulus of almost all human soft tissues. As shown in Fig. 4c and Fig. S4, the elastic deformation portion of the stress-strain curves in Fig. 4a was used to calculate Young\u0026rsquo;s modulus of hydrogel samples, revealing its relationship with the Fe\u003csup\u003e3+\u003c/sup\u003e concentration in the ion bath. The modulus increased rapidly and proportionally with the ion concentration, which indicated a rise in crosslinking density. However, with further increasing the Fe\u003csup\u003e3+\u003c/sup\u003e ion concentration (\u0026gt;0.1 M), the modulus value gradually flatten. This deceleration was attributed to a reduction in ion crosslinking sites within the alginate segments. Cyclic stretching tests on hydrogel samples (0.1 M Fe\u003csup\u003e3+\u003c/sup\u003e) showed that the stress-strain curves of the samples had good repeatability under \u0026nbsp; 15%-30% strain (Fig. 4d).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePatterned Tissue Induced by Hydrogel Scaffolds.\u003c/p\u003e\n\u003cp\u003eVarious soft tissues within the human body exhibit inherent orientations, such as the organization of cardiac tissue and the intricate patterns in vascular networks. Utilizing patterned hydrogel scaffolds offers a promising method for inducing well-organized or patterned tissue growth. The cell viabilities of hydrogels crosslinked with 0 M and 1 M Fe\u003csup\u003e3+\u003c/sup\u003e were 1.01 and 0.98, as shown in Fig. S5, compared to the control group, indicating no obvious toxicity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCardiac tissue scaffolds with periodic H-shaped grooves (width of 100 \u0026mu;m, depth of 200 \u0026mu;m, gap of 110 \u0026mu;m) were manufactured.\u003cem\u003e\u0026nbsp;\u003c/em\u003eUpon immersion in DI water, the printed H-shaped grooves experienced controlled expansion. To facilitate communication between induced cardiac tissues within each groove, additional vertical grooves were incorporated into the H-shaped grooves (Figs. 5a,c). This design allowed the seeding of cells onto the hydrogel scaffold, inducing the formation\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;of a cohesive and organized tissue. Cardiac tissue is characterized by its softness, with a modulus of ~10 kPa \u003csup\u003e5-10\u003c/sup\u003e. Therefore, the cardiac tissue scaffold was prepared without Fe\u003csup\u003e3+\u003c/sup\u003e bath treatment to maintain a minimum modulus of ~16 kPa. A mixed suspension of self-fluorescent cardiomyocyte and fibroblast cell (2:1) was seeded onto the flexible scaffolds. Due to the gravity, a considerable number of cells were settled down and distributed within the grooves. Cardiomyocytes gradually migrated and aggregated, forming organized and continuous tissue (Fig. 5a). As shown in Fig. 5d, cardiomyocytes transitioned from an initial spherical shape to an organized tissue structure induced \u0026nbsp; by H-shaped grooves. The tissue in different lines was connected through the horizontal connections. Therefore, an organized mesh-like tissue was formed through topographical constraint. Comparatively, cardiac tissue cultured on a flat substrate (Fig. 5e) failed to form an organized structure and tended to grow in clusters (Fig. 5b,f). Those cells underwent arbitrary clustering, displaying uncontrollable boundaries between tissue clusters, which is more apparent in the stained images (Fig. S6). The organized cardiac tissue could be driven to contract under an electric field generated by the electrical stimulation device. Under a pulse field with frequencies ranging from 0.5 to 1.5 Hz, the cardiac tissue exhibited synchronous contractions that followed the applied frequency (Video S2). However, at 2 Hz, although the tissue demonstrated contractions, it failed to keep pace with the frequency of the electric field.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCompared with cardiac tissue, blood vessels possess a higher modulus and exhibit a more intricate macroscopic structure. Vasculature-like hydrogel scaffolds were printed and treated with 1 M Fe\u003csup\u003e3+\u003c/sup\u003e to obtain Young\u0026apos;s modulus of ~345 MPa, mimicking that of native blood vessels as shown in Fig. 6a. The vascular tissue scaffold was designed with semi-open channels of varying diameters. After swelling, the diameter raised from the designed 1.2, 0.3, and 0.1 mm to 1.52, 0.47, and 0.16 mm, respectively. HCAECs were seeded onto the scaffold, forming a semi-open vascular network. Tissues grown on scaffolds exhibited apparent vascular morphological features. Upon staining HCAECs across the entire scaffold, it was evident that HCAECs could be well-distributed on the scaffold and microchannels (Fig. 6b-d). Within those 0.16 mm grooves (Fig. 6e), HCAECs exhibited distinctive vascular pattern. Conversely, endothelial cells cultured on a substrate devoid of grooves did not exhibit any noticeable pattern (Fig. 6f).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, a simple and versatile two-step process, combining DLP and post-treatment. Ion bath treatment is performed after the DLP printing. Using a single AAm-Alg hydrogel formula,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ehydrogel structures with biocompatible, high-resolution, and adjustable modulus can be fabricated. This two-step process makes bioprinting more cost-effective and straightforward. The UV-curable hydrogel solution exhibited excellent printability, achieving a printing feature of 10 \u0026mu;m (limited by the resolution of the light source) that resembles the characteristic morphology of biological tissues. The ion bath treatment enables a wide range of modulus adjustments (15.83-344.67 kPa) in a simple way, with the ability to customize the modulus of hydrogel samples. Patterned hydrogel scaffolds with low or high moduli were respectively designed and manufactured for cultivating cardiac and vascular tissue. The cultivated tissues were induced by the scaffold morphology, even resembling a biomimetic multilevel vascular network. The cultured organized cardiac tissue exhibits synchronous beating under electrical stimulation. We believe that this two-step process provides an easily feasible method for manufacturing tailored scaffolds for a broad range of tissues in the human body.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data analyzed during this study are included in this published article.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author(s) disclosed receipt of the following financial support for the research, authorship, and/or publicaiton of this article:This work was supported by the National Natural Science Foundation of China (No. U2005214) and the National Key Research and Development Program of China (2022YFB4600600).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFeng Xu: conceptualization, methodology, data analysis and writing (original draft). Hang Jin, Huiquan Wu and Acan Jiang: investigation, validation and data analysis. Bin Qiu, Lingling Liu and Qiang Gao: validation and data analysis. Bin Lin, Weiwei Kong: investigation and data analysis. Songyue Chen, and Daoheng Sun: conceptualization, writing (review and editing) and funding acquisition.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhang, F. \u0026amp; King, M. W. Biodegradable Polymers as the Pivotal Player in the Design of Tissue Engineering Scaffolds. Adv. Healthc. 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Horizons 8, 1173\u0026ndash;1188, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1039/d0mh01514h\u003c/span\u003e\u003cspan address=\"10.1039/d0mh01514h\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Digital Light Processing, Double Network Hydrogels, Hydrogel Scaffolds, Adjustable Modulus","lastPublishedDoi":"10.21203/rs.3.rs-4083780/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4083780/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHydrogels are extensively explored as biomaterials for tissue scaffolds, and their controlled fabrication has been the subject of wide investigation. However, the tedious mechanical property adjusting process through formula control hindered their application for diverse tissue scaffolds. To overcome this limitation, we proposed a two-step process to realize simple adjustment of mechanical modulus over a broad range, by combining digital light processing (DLP) and post-processing steps. UV-curable hydrogels are 3D printed via DLP, with the ability to create complex 3D patterns. Subsequent post-processing with Fe\u003csup\u003e3+\u003c/sup\u003e ions bath induces secondary crosslinking of hydrogel scaffolds, tuning the modulus as required through soaking in solutions with different Fe\u003csup\u003e3+\u003c/sup\u003e concentrations. This innovative two-step process offers high-precision (10 \u0026micro;m) and broad modulus adjusting capability (15.8\u0026ndash;345 kPa), covering a broad range of tissues in the human body. As a practical demonstration, hydrogel scaffolds with tissue-mimicking patterns were printed for cultivating cardiac tissue and vascular scaffolds, which can effectively support tissue growth and induce tissue morphologies.\u003c/p\u003e","manuscriptTitle":"Digital Light Processing Printed Hydrogel Scaffolds with Adjustable Modulus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-27 11:06:43","doi":"10.21203/rs.3.rs-4083780/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-05-07T04:24:32+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-27T19:17:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"731b877f-de58-4336-8997-0efaf18fff15","date":"2024-04-17T13:43:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"f496fec4-9fb2-4a05-9988-47c1fb460177_SNPRID","date":"2024-04-16T08:14:34+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-15T05:12:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-04T21:06:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"965a2907-7a04-43a4-b8cd-d1243d135c7f","date":"2024-03-29T17:28:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"664e6269-6737-4c99-84f0-f12a272fd514","date":"2024-03-29T17:24:06+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-29T17:21:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-29T12:08:13+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-03-26T16:17:44+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-25T04:33:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-03-12T11:00:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b2f040fd-9888-4b5b-a2be-8b21302aea17","owner":[],"postedDate":"March 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":29922354,"name":"Physical sciences/Engineering/Biomedical engineering"},{"id":29922355,"name":"Physical sciences/Materials science/Soft materials"},{"id":29922356,"name":"Biological sciences/Biotechnology/Tissue engineering"}],"tags":[],"updatedAt":"2024-07-02T05:46:06+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-27 11:06:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4083780","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4083780","identity":"rs-4083780","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}