3D printed HAMA/GelMA/lignosulfonate hydrogel integrating oxygen-releasing and antioxidative modules for liver regeneration

3D printed HAMA/GelMA/lignosulfonate hydrogel integrating oxygen-releasing and antioxidative modules for liver regeneration | 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 3D printed HAMA/GelMA/lignosulfonate hydrogel integrating oxygen-releasing and antioxidative modules for liver regeneration Sang Luo, Longbao Feng, Bingren Tian, Fang Wu, Wenjun Wu, Shuai Xiao, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8380365/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Feb, 2026 Read the published version in Journal of Nanobiotechnology → Version 1 posted 11 You are reading this latest preprint version Abstract A multifunctional polysaccharide-based hydrogel was developed for liver regeneration by integrating sodium alginate (SA), sodium lignosulfonate (SL), and 3D bioprinting technology. The hydrogel incorporated FH1@SA microspheres and CaO 2 @ZIF-8@SL nanoparticles to reconstruct the hepatic microenvironment through coordinated oxygen generation, antioxidant activity, and anti-inflammatory modulation. The FH1@SA microspheres enabled sustained release of FH1, facilitating hepatocyte adaptation to hypoxia and promoting functional recovery. Meanwhile, the CaO 2 @ZIF-8@SL nanoparticles achieved controlled oxygen release and pH-responsive degradation, while the lignosulfonate component scavenged reactive oxygen species and mitigated inflammatory stress. The 3D-printed hydrogel exhibited favorable mechanical strength, injectability, and cytocompatibility, effectively supporting hepatocyte proliferation and tissue regeneration. By simultaneously relieving hypoxia and oxidative stress, this polysaccharide hydrogel provides a synergistic strategy for enhancing hepatic repair, offering a promising platform for bioartificial liver construction and treatment of liver injury. sodium alginate sodium lignosulfonate hydrogel oxygen release liver regeneration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1 Introduction Liver injury caused by trauma, infection, drug toxicity, or metabolic disorders represents a growing global health challenge 1 . As the central organ responsible for detoxification, metabolism, and immune regulation, the liver plays indispensable roles in maintaining systemic homeostasis 2 . Severe hepatic injury causes hepatocyte necrosis and structural collapse, leading to persistent inflammation and, eventually, liver failure 3 , 4 . For patients with end-stage liver disease, the regenerative capacity of hepatocytes is largely exhausted, and liver transplantation remains the only effective treatment. However, donor shortage, high surgical cost, and immune rejection still constrain its clinical utility 5 . To meet this demand, hydrogels have been widely explored as promising biomaterial candidates for bioartificial liver construction. Hydrogel-based biomaterials have attracted increasing attention in hepatic tissue engineering owing to their three-dimensional, highly hydrated networks that mimic the extracellular matrix and provide structural and biochemical cues for hepatocyte growth, migration, and differentiation 6 , 7 . Nevertheless, conventional hydrogels provide limited control over the hepatic microenvironment, which is typically characterized by hypoxia, oxidative stress, and chronic inflammation 8 . Moreover, hydrogel-based drug delivery systems often suffer from burst release of bioactive molecules, resulting in transient over-concentration and potential cytotoxicity 9 . To overcome these limitations, polysaccharide-based hydrogels have emerged as versatile scaffolds due to their tunable chemical functionalities, intrinsic biocompatibility, and resemblance to native glycosaminoglycans 10 , 11 . Among them, sodium alginate (SA) is widely used to construct ionically cross-linked hydrogel microspheres for controlled drug release 12 , 13 , while sodium lignosulfonate (SL), a natural anionic polysaccharide rich in phenolic hydroxyl groups, exhibits strong antioxidant and anti-inflammatory activities that can mitigate oxidative stress within damaged tissues 14 – 16 . These inherent properties make polysaccharide networks an ideal foundation for developing multifunctional hydrogels capable of modulating the oxidative and inflammatory microenvironment during liver regeneration. Building upon these insights, a polysaccharide-based 3D-printed hydrogel scaffold was designed to reconstruct the hepatic microenvironment by integrating bioactive microspheres and oxygen-generating nanoparticles. Specifically, sodium alginate microspheres were utilized as controlled-release carriers for the liver-regenerative molecule functional small molecule 1 (FH1), forming FH1@SA microspheres capable of providing sustained hepatocyte stimulation and promoting differentiation 17 , 18 . Meanwhile, calcium peroxide (CaO 2 ) was employed as an oxygen source to alleviate local hypoxia 19 , 20 , and its release behavior was precisely regulated by encapsulation within zeolitic imidazolate framework-8 (ZIF-8), which enables pH-responsive degradation 21 , 22 and controlled oxygen generation. Furthermore, sodium lignosulfonate (SL) was introduced to modify the nanoparticle surface, imparting intrinsic antioxidant and anti-inflammatory functions by scavenging excessive reactive oxygen species and buffering oxidative stress 23 . The resulting hydrogel network, composed primarily of biocompatible polysaccharides, provided interconnected porosity, suitable mechanical strength, and tunable degradation, supporting 3D bioprinting and long-term hepatocyte viability. Overall, this study presents a polysaccharide-based biohybrid hydrogel integrating biochemical and structural functions for liver regeneration. By combining FH1@SA microspheres and CaO 2 @ZIF-8@SL nanoparticles within a 3D-printed polysaccharide matrix, the system establishes a synergistic microenvironment that simultaneously alleviates hypoxia, suppresses oxidative stress, and reduces inflammation, thereby enhancing hepatocyte survival and functional recovery. The hydrogel exhibits favorable printability, mechanical stability, and biocompatibility, offering both structural support and dynamic regulation of the hepatic niche. This polysaccharide-centered design not only provides a new strategy for reconstructing bioartificial liver systems but also highlights the potential of multifunctional polysaccharide hydrogels as versatile platforms for tissue regeneration and microenvironmental modulation. 2 Materials and methods Polysaccharides structural. Sodium alginate (SA, CAS 9005-38-3, Macklin, Shanghai, China) was an ultra-low-viscosity type (1% aqueous solution viscosity: 3–5 mPa·s, pH 6–8, ash 18–27%), which has a viscosity-average molecular weight of 100,000 g/mol and appears as an off-white to pale yellow solid.SA is a linear copolymer composed of β-D-mannuronic acid (M) and α-L-guluronic acid (G) residues linked by 1→4 glycosidic bonds, with an M/G ratio typically around 1:1 as provided by the manufacturer. Hyaluronic acid sodium (YuanYe Bio-Technology Co., Ltd., Shanghai, China) is a white powder composed of repeating disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine linked by β(1→3) and β(1→4) glycosidic bonds, soluble in water and DMSO. The product used in this study has a molecular weight of approximately 200–400 kDa (supplier data). Polysaccharide inks. Methacrylated gelatin (GelMA) and methacrylated hyaluronic acid (HAMA) were prepared as described (SI § 1.6–1.7). Sodium alginate (SA) was used to form drug-loaded microspheres, and sodium lignosulfonate (SL) served as an antioxidant/anti-inflammatory polysaccharide coating (SI § 1.1). Oxygen donor nanoparticles. Calcium peroxide (CaO 2 ) nanoparticles were synthesized by precipitation (SI § 1.2) and assembled with zeolitic imidazolate framework-8 (ZIF-8) followed by SL surface modification to obtain CaO 2 @ZIF-8@SL (CZS) (SI § 1.3–1.4). Physicochemical features were assessed by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), transmission electron microscopy (TEM), and dynamic light scattering (DLS) (SI § 1.9). Dissolved oxygen release and 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging were evaluated as in SI § 1.9.5–1.9.6. FH1-loaded microspheres. FH1-loaded sodium alginate microspheres (FH1@SA) were generated via a microfluidic SA/Ca–EDTA system (SI § 1.5). Morphology and size distribution were analyzed by optical microscopy and scanning electron microscopy (SEM) (SI § 1.10). FH1 release from GelMA/HAMA matrices was quantified in vitro (SI § 1.11.9). 3D-printed hydrogel scaffolds. Composite bioinks were prepared by dissolving GelMA/HAMA with lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) and tartrazine, followed by addition of FH1@SA and/or CZS (SI § 1.8). Four groups were printed: GHH (GelMA/HAMA), GHHF (GHH + FH1@SA), GHHC (GHH + CZS), and GHHFC (GHH + FH1@SA + CZS). Printed scaffolds were rinsed in phosphate-buffered saline (PBS) and post-cured (SI § 1.8). Bulk and microstructural properties were assessed by FTIR, SEM, unconfined compression, rheology, swelling, and enzymatic/degradation tests (SI § 1.11). In vitro assays. Human umbilical cord mesenchymal stem cells (HUCMSCs) and HepaRG hepatocytes (HepaRG) were used for cytocompatibility (CCK-8), live/dead staining, and cytoskeleton staining (SI § 1.12). Human umbilical vein endothelial cells (HUVECs) on Matrigel were used for tube formation analysis (SI § 1.13). Hepatic differentiation of printed constructs and expression of ALB, AFP, CK18, Hnf1α, Foxa2, Prkaca, Prkacb, and Prkx were evaluated by real-time reverse transcription quantitative PCR (RT-qPCR) (SI § 1.14–1.17). Bioinformatics analyses (differentially expressed genes, Gene Ontology/GO, Kyoto Encyclopedia of Genes and Genomes/KEGG, and Gene Set Enrichment Analysis/GSEA) followed SI § 1.18. In vivo evaluation. All animal procedures were conducted in accordance with institutional guidelines and were approved by the Laboratory Animal Ethical and Welfare Committee of laboratory Animal Center, Ningxia Medical University. Approval No. IACUC- 2025280. All procedures followed the principles of the Declaration of Helsinki and complied with the ARRIVE guidelines. Scaffolds were implanted in the mouse mesentery; vascularization (FITC-dextran), histology (hematoxylin and eosin/H&E, periodic acid–Schiff/PAS), and immunofluorescence (fumarylacetoacetate hydrolase/FAH, CD31) were performed per SI § 1.19–1.23. A subsequent extended hepatectomy model was used to assess survival and liver function (SI § 1.20). Statistics. Data are reported as mean ± standard deviation (SD). One- or two-way analysis of variance (ANOVA) with Tukey’s post-hoc test determined significance (p < 0.05) (SI § 1.24). 3 Results and discussion 3.1 Characterization of the Structure and Composition of Self-Produced Oxygen Nanoparticles (CaO 2 @ZIF-8@SL) In the CaO 2 sample, distinct characteristic diffraction peaks at 30.0° (002), 35.5° (110), 47.5° (112), and 52.5° (103) were observed, corresponding to the characteristic peaks of CaO 2 (Fig. 1 A), which align with the CaO 2 crystal ICDD card. The synthesized ZIF-8 exhibits a highly crystalline structure, and its diffraction peaks match well with the standard simulated XRD pattern of ZIF-8 24 . However, in the XRD spectrum of CaO 2 @ZIF-8@SL, some characteristic peaks of ZIF-8 are clearly visible, indicating that the ZIF-8 crystallinity attached in situ on CaO 2 is high. Moreover, due to the in-situ growth of ZIF-8 on its surface, no distinct CaO 2 characteristic peaks are observed in the XRD spectrum of CaO 2 @ZIF-8@SL. As shown in Fig. 1 B, the FTIR spectrum of CaO 2 shows an absorption peak at 873 cm − 1 , which corresponds to the O-O stretching vibration and is the most important characteristic peak of CaO 2 , indicating the successful synthesis of CaO 2 . Additionally, the asymmetric and symmetric stretching vibration absorption bands of O-Ca-O in CaO 2 appear at 1419 and 1483 cm − 1 , respectively. The absorption peak at 1594 cm − 1 corresponds to the CO 3 2− stretching vibration of calcium carbonate (CaCO 3 ), which is a byproduct formed by the reaction between CaO 2 and CO 2 during preparation and storage 25 . For the ZIF-8@SL sample, absorption bands at 3134 and 2925 cm − 1 represent the aromatic and aliphatic C-H stretches of the imidazole ring, while absorption bands at 687–758 and 948–998 cm − 1 are related to the in-plane and out-of-plane bending of the imidazole ring. In addition to the characteristic bands of unmodified ZIF-8, a broad absorption band around 3425 cm − 1 is associated with the -OH vibration in the SL structure, and the absorption band at 1424 cm − 1 corresponds to the C-C bond present in SL and ZIF-8. This indicates that SL and ZIF-8 are hydrogen-bonded, and the Zn 2+ (positively charged) tends to attach to the sulfonic groups in SL. In the FTIR spectrum of CaO 2 @ZIF-8@SL, both the O-O and imidazole ring stretching vibration peaks appear, indicating that ZIF-8@SL has successfully attached in situ to the surface of CaO 2 . Figure 1 G shows the SEM images of two nanoparticles (ZIF-8 and CaO 2 @ZIF-8@SL). As seen from the images, the unmodified ZIF-8 nanoparticles exhibit a uniform rhombic dodecahedral shape, with a smooth surface and an average particle size of approximately 100 nm. However, after loading with CaO 2 and the SL coating, the surface of CaO 2 @ZIF-8@SL becomes rough, with the average particle size reduced to approximately 70 nm, showing a more restricted particle size distribution. This suggests that CaO 2 and SL, as growth inhibitors, regulate the nucleation rate of ZIF-8 and limit its growth, thereby effectively controlling the size of ZIF-8 crystals during synthesis. EDS characterization (Figs. 1 H and 1 I) was used to analyze the elemental composition of ZIF-8@SL nanoparticles, confirming the presence of the required elements (Zn, Ca, S, O) in the structure. As shown in Fig. 1 C, in pure water, there are no peroxides or oxygen sources, so the dissolved oxygen remains near the saturation value of the natural environment and does not significantly increase or decrease. Hydrogen peroxide slowly decomposes in water to generate oxygen (2H 2 O 2 → 2H 2 O + O 2 ), so the dissolved oxygen content in this group increases. Due to the dual effect of CaO 2 @ZIF-8@SL and hydrogen peroxide, the CaO 2 @ZIF-8@SL group exhibits the highest dissolved oxygen level. Furthermore, because of the encapsulation effect of ZIF-8@SL, the CaO 2 @ZIF-8@SL group shows stability in oxygen release over an extended period. Particle size analysis of ZIF-8 and CaO 2 @ZIF-8@SL nanoparticles was performed using a laser nanoparticle size analyzer, as shown in Figs. 1 D and 1 E. The average particle size of ZIF-8 was 412.8 ± 14.08 nm, and that of CaO 2 @ZIF-8@SL was 484.3 ± 38.52 nm, which may be due to the loading of CaO 2 inside ZIF-8, increasing the particle size. Additionally, the SL layer surrounding the particles also directly increases the particle size. Furthermore, aggregation may occur during the synthesis process, leading to particle agglomeration and an increase in particle size. TEM observations revealed that ZIF-8 nanoparticles exhibited a polyhedral structure and were uniformly dispersed, while CaO 2 @ZIF-8@SL nanoparticles showed aggregation phenomena, with rough, irregular surfaces, leading to increased particle size. This result is consistent with the hydrated particle size results (Fig. 1 G). As shown in Fig. 1 F, the H 2 O group and H 2 O + H 2 O 2 group exhibited no significant radical scavenging activity, while the H 2 O + H 2 O 2 + CaO 2 @ZIF-8@SL group displayed a noticeable lightening of the purple color, likely due to the presence of multiple phenolic hydroxyl groups (-OH) in sodium lignosulfonate (SL). These hydroxyl groups can interact with DPPH radicals (2,2-diphenyl-1-picrylhydrazyl) through a hydrogen atom transfer (HAT) reaction, reducing them to the yellow diphenyl-picrylhydrazyl form 26 . Additionally, the aromatic rings in the lignosulfonate structure contain π electron systems that can participate in the radical scavenging reaction, providing electrons through an electron transfer (ETr) reaction to the radicals, thereby converting them into stable non-radical forms. 3.2. Structural and Compositional Characterization of FH1@SA Microspheres The morphology of the prepared microspheres can be observed under a microscope, as shown in Figure S1 A. Using microfluidic technology, sodium alginate microspheres loaded with FH1 were successfully prepared with uniform size and controllable dimensions. Meanwhile, the size of the microspheres was quantitatively analyzed (Figure S1 B). The results show that when the dispersed phase to continuous phase flow rate ratios were 1:2.5, 1:5, and 1:10, the sizes of the microspheres prepared were 260.2 µm, 225.2 µm, and 210.1 µm, respectively. As the flow rate ratio decreased, the size of the microspheres gradually decreased. This indicates that the faster the flow rate of the continuous phase, the greater the fluid shear force applied to the dispersed phase as it enters the continuous phase. This force promotes the emulsification process of the droplets and accelerates the rupture of the droplets into spheres, thereby resulting in smaller microspheres with a greater number formed per unit time. Figure S2 shows the SEM images of the microspheres. As can be seen from the image, the surface of the microspheres has a certain porous structure. 3.3 Structural and physicochemical characterization of polysaccharide-based hydrogels To elucidate the chemical architecture of the polysaccharide-based network, FTIR and 1 H NMR analyses were first performed (Fig. 2 A–D). Distinct amide I, II, and III peaks in GelMA 27 , 28 and new ester carbonyl signals in HAMA confirmed successful methacrylation 29 , 30 , while both materials preserved the abundant hydroxyl and carboxyl groups characteristic of natural polysaccharides. These functional moieties play a dual role: they ensure high hydrophilicity and enable multiple hydrogen-bonding or ionic interactions during gelation. Such chemical versatility provides the foundation for constructing an ECM-mimicking matrix capable of coordinating metal ions and anchoring bioactive components. The rheological and mechanical profiles (Fig. 2 E–I) revealed that this polysaccharide-dominated network exhibited multiscale crosslinking. The flexible glycosidic chains of HAMA and SA contributed to dynamic hydrogen bonding and water retention, while the Ca 2+ ions released from CaO 2 @ZIF-8@SL interacted electrostatically with sulfonic groups on SL, forming a reversible ionic framework 31 . This multi-interactive network endowed the hydrogel with remarkable elasticity (G′ > G″ throughout) and recoverability under compression, reflecting the intrinsic adaptability of polysaccharide scaffolds to mechanical stress 32 . Such viscoelastic resilience is essential for hepatocyte mechanotransduction and liver tissue remodeling. The swelling and degradation behaviors (Fig. 2 J–L) further highlighted the regulatory role of the polysaccharide domains. The SA microspheres introduced additional hydrophilic sites, markedly enhancing water absorption(Fig. 2 J). Meanwhile, as the proportion of GelMA increased, the overall swelling rate and degradation rate of the hydrogel decreased. This indicates that GelMA, as a protein backbone, has a significant impact on the overall structural properties (Fig. 2 K–L). These effects stem from the inherent responsiveness of polysaccharide networks, where hydrogen-bond dynamics and partial ionic dissociation govern water diffusion and polymer relaxation. This balance between hydration and degradation not only maintains the scaffold’s dimensional stability but also synchronizes its resorption rate with native hepatic regeneration. From a structural standpoint, the polysaccharide backbone serves not merely as a passive matrix but as an active biochemical modulator. The hydroxyl and sulfonic groups of lignosulfonate can scavenge reactive oxygen species and mediate protein adsorption 16 , while the carboxyl-rich SA domains facilitate sustained drug release and microenvironment buffering 33 . Together, these cooperative features construct a biofunctional hydrogel with hierarchical architecture—where molecular interactions translate into macroscopic mechanical robustness and controlled bioactivity. Collectively, the GelMA/HAMA/SA-based hydrogel represents a polysaccharide-governed supramolecular network integrating covalent, ionic, and hydrogen-bonded crosslinks. This architecture provides tunable viscoelasticity, hydration, and degradation, while the intrinsic redox and adsorption properties of lignosulfonate endow the system with microenvironment-modulating capability. Such synergy of chemistry and function forms the essential physicochemical basis for subsequent hepatocyte proliferation and tissue regeneration. 3.4 Evaluation of cell compatibility and angiogenic potential of 3D printed HUCMCS hydrogel The polysaccharide backbone, abundant in hydroxyl, carboxyl, and sulfonic groups, enables dynamic interactions with proteins, ions, and cellular membranes, thereby creating a hydrated and redox-buffered microenvironment. Such structural features are expected to modulate cell adhesion, viability, and angiogenic behavior. The CCK-8 method was used to systematically evaluate the effects of different concentrations of CaO 2 @ZIF-8@SL solution and FH1@SA hydrogel microspheres on the viability of HUCMSC and HepaRG cells (Figure S3). The results showed that CaO 2 @ZIF-8@SL exhibited a concentration-dependent effect on both cell types. In HUCMSC, cell viability reached its highest value (112.92%) at a drug concentration of 0.05 mg/mL, but significantly decreased to 94.37% at higher concentrations (5 mg/mL), while the lower concentration range (0.025-0.1 mg/mL) promoted cell proliferation (Figure S3A). A similar phenomenon was observed in HepaRG cells, where cell viability was highest (111.54%) at 0.05 mg/mL and decreased to 91.78% at 2.5 mg/mL, indicating that high doses had adverse effects on liver cells as well, while the lower concentration range (0.025–0.25 mg/mL) had a promoting effect (Figure S3B). However, overall cell viability was above 80%, indicating that CaO 2 @ZIF-8@SL nanoparticles had no cytotoxicity. In contrast, FH1@SA hydrogel microspheres had a milder effect on cell viability. In both HUCMSC and HepaRG cells, when the concentration reached 0.5 mg/mL, the viability increased to 107.19% and 107.60%, respectively, while cell viability remained stable at other concentrations (Figure S3C, D). This suggests that FHI@SA hydrogel microspheres have good biocompatibility across a wide concentration range and do not cause significant toxic side effects. The CCK-8 assay was used to perform a time-gradient evaluation of the 3D printed cell-loaded hydrogels. Overall, as the culture time extended from Day 1 to Day 5, the viability of HUCMSCs in all groups showed an upward trend, indicating that the GelMA/HAMA base framework (GHH) has good cell compatibility. Further comparison of different functionalized groups revealed that on Day 3, the GHHFC group, containing the dual modules of oxygen generation/ROS scavenging and pro-hepatocyte differentiation, was significantly higher than the other groups (p < 0.05), and the difference continued to expand on Day 5. GHHC (oxygen generation + antioxidation) and GHHF (FH-1 microspheres) showed moderate enhancement, while GHH exhibited the smallest improvement (Fig. 3 B). The results suggest that under the same printing and culture conditions, local oxygen supply and oxidative stress buffering, combined with FH-1 pro-differentiation signals, can more effectively support cell survival and proliferation, creating a more favorable microenvironment for early tissue formation. Calcein-AM/PI staining further confirmed the cell compatibility of different scaffolds for HUCMSCs. No significant PI-positive accumulation (dead cells) was observed in any group, indicating that the materials were overall safe (Fig. 3 C). Notably, the GHHFC group showed stronger and more uniformly distributed Calcein-AM signals, which became denser over time. Cell morphology transitioned from dispersed to clustered, with more abundant pseudopodia and protrusions (Fig. 3 D), indicating improved cell adhesion, spreading, and more active proliferation. The GHHC group also showed better performance than GHH, suggesting that moderate alleviation of hypoxia and ROS load can directly enhance cell status. The enhanced effect in GHHF was consistent with the pro-differentiation/maturation role of FH-1, but the effect was smaller than the synergistic effect seen in the dual-module GHHFC. In the HUVEC tube formation assay, the tubular network induced by GHHFC was the most dense and continuous (Fig. 3 E). The number of branches (Nb branches), junctions (Nb junctions), and total branching length (Total branching length) were all significantly higher than in the other groups (all p < 0.001, Fig. 3 F–H). This indicates that the dual-module material not only supports matrix cell growth but also creates a pro-vascularization microenvironment. Comparatively, GHHC showed a significant improvement over GHH, suggesting that continuous oxygen supply and ROS buffering facilitate endothelial cell migration and lumen stabilization. GHHF also showed some promoting effects, which may be related to the upregulation of paracrine factors (such as the VEGF family, HGF, etc.) by FH-1, ultimately leading to a synergistic effect in GHHFC, resulting in the most prominent angiogenic phenotype. 3.5 Transcriptomic Analysis of HUCMCS Cultured in GHHFC Hydrogel We performed transcriptome analysis on HUCMSCs cultured in GHHFC hydrogel, and the sequencing results revealed pronounced gene expression differences between the GHHFC and control groups, identifying 1,710 upregulated and 985 downregulated genes (Fig. 4 A). The heatmap of representative DEGs (Fig. 4 B) showed clear transcriptional remodeling across pathways associated with lipid metabolism, mitochondrial activity, extracellular matrix (ECM) organization, and signal transduction, indicating that the hydrogel microenvironment exerts a broad regulatory influence on cellular homeostasis. This result is consistent with the qRT-PCR results, indicating that that the GHHFC hydrogel most effectively promoted the hepatic differentiation of HUCMSCs (Figure S4A–H). Compared with the GHH group, the GHHFC scaffold markedly upregulated the expression of mature hepatocyte markers ALB (2.13-fold), AFP (1.78-fold), and CK18 (1.79-fold), indicating enhanced functional maturation. The transcription factors HNF-1α (2.26-fold) and FOXA2 (2.54-fold), which govern hepatocyte lineage commitment, were also significantly elevated, suggesting activation of hepatic developmental programs. In addition, genes associated with cAMP signaling and metabolic regulation, including PRKACA, PRKACB, and PRKX, increased by 2.10-, 2.50-, and 2.18-fold, respectively, reflecting improved cellular energy metabolism.Such coordinated gene activation can be attributed to the polysaccharide-dominated microenvironment constructed by the HAMA/SA backbone and lignosulfonate components. The abundant hydroxyl, carboxyl, and sulfonic groups maintain a hydrated and redox-buffered niche, while oxygen release from CaO2@ZIF-8 sustains mitochondrial activity under physiological conditions. This combination stabilizes intracellular ROS levels and supports AMPK-dependent signaling cascades, which in turn facilitates the expression of HNF-1α and FOXA2 and promotes the metabolic transition toward a mature hepatocyte phenotype. KEGG enrichment analysis (Fig. 4 C) demonstrated that pathways related to hepatic differentiation and metabolic reprogramming—such as AMPK, VEGF, Hippo, and Insulin signaling, together with glycolysis/gluconeogenesis, fatty acid degradation, and pyruvate metabolism—were significantly activated. These results suggest a shift toward enhanced energy metabolism and biosynthetic activity, both essential for hepatocyte maturation. GO analysis (Fig. 4 D) further highlighted biological processes including lipid metabolic regulation, glycogen synthesis, liver development, MAPK cascade activation, endothelial cell migration, and ECM remodeling, consistent with the cellular proliferation and angiogenic behavior observed in vitro. GSEA plots (Fig. 4 E) confirmed enrichment of the AMPK and VEGF pathways, while network mapping (Fig. 4 F) showed coordinated activation of multiple metabolic node genes, implying a systemic reinforcement of mitochondrial bioenergetics and fatty acid oxidation. This transcriptomic profile reflects the redox-balanced and oxygenated environment established by the dual-module hydrogel: oxygen generation from CaO₂@ZIF-8 alleviates hypoxia, whereas the phenolic hydroxyl and sulfonic groups of sodium lignosulfonate (SL) buffer excess reactive oxygen species and stabilize mitochondrial function. Within this microenvironment, the controlled release of FH1 provides persistent pro-differentiation signals that cooperate with metabolic regulation to activate HNF-1α, FOXA2, and downstream hepatic programs 34 . Meanwhile, the upregulation of ECM and adhesion-related gene sets corresponds to the 3D polysaccharide framework’s physical cues, which promote cytoskeletal polarization and intercellular communication. The concurrent enrichment of VEGF signaling and endothelial migration genes provides molecular evidence for the angiogenic phenotype observed in the tube-formation assays. Altogether, these transcriptomic findings indicate that the polysaccharide-based oxygen–antioxidant–differentiation cascade remodels cellular metabolism and transcriptional activity toward a hepatic phenotype. The synergistic interplay among oxygen supply, ROS buffering, and sustained biochemical signaling establishes a stable biochemical–biophysical niche that enhances energy metabolism, vascularization, and liver-specific gene expression within the 3D scaffold. 3.6 In vivo vascularization and liver-specific function after mesenteric transplantation of 3D printed hydrogel. Figure 5 A shows the schematic of vascularization 14 days post-transplantation. In Fig. 5 B, the GHHFC group exhibits longer and thicker blood vessel structures with a larger vascular area ratio. The CaO 2 @ZIF-8@SL component may continuously supply oxygen, activating pro-angiogenic signaling pathways. Additionally, the study found that SL can regulate the inflammatory response and stabilize the endothelial microenvironment, further supporting the formation of the capillary network. 14 days after transplantation into the mesentery, histological and immunological analyses were performed on the 3D printed hydrogel sections. H&E staining results (Fig. 5 C) showed significant cell infiltration and tissue remodeling in all groups, indicating that the materials possess good biocompatibility. In the immunofluorescence staining (Fig. 5 D,E,F), the control group (GHH) showed almost no noticeable vascular-related signals; the GHHF and GHHC groups displayed a certain number of CD31-positive newly formed blood vessels, while the GHHFC group showed the most prominent result, with dense distribution of CD31-positive vessels and well-formed lumen structures. Meanwhile, FAH signals were most intense in the GHHFC group, indicating more significant hepatocyte differentiation of HUCMSCs in this material microenvironment. Further PAS staining (Fig. 5 G and S5) revealed the most prominent glycogen deposition in the GHHFC group, with strong positive reaction in the area indicated by the black arrow, while other groups showed relatively weaker positive signals. Overall, the results indicate that GHHFC hydrogel not only promotes angiogenesis in vivo but also significantly enhances the liver-specific function of cells in the grafts. 3.7 GHHFC protects 90% liver resected mice from liver failure (B) Kaplan-Meier curve of cumulative survival rates after 90% liver resection in different groups (control group, sham surgery group, GHH group, GHHF group, GHHC group, GHHFC group). (C) H&E staining of the transplanted constructs/tissues at the endpoint, showing cell infiltration and tissue remodeling (scale bar: 50 µm). (D) Serum biochemistry of surviving rats 90 days after liver resection: albumin (ALB), alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), direct bilirubin (DBIL), total bile acids (TBA), total bilirubin (TBL), γ-glutamyl transferase (γ-GT), and total protein (TP). (E) Immunofluorescence staining of CD31 (green) and FAH (red) with DAPI nuclear staining (blue) in the transplanted area after liver resection; the GHHFC group shows the most abundant CD31⁺ vascular structures and strong FAH expression (scale bar: 50 µm). (F) Quantitative analysis of the density/area fraction of CD31⁺ vessels. (G) PAS staining of the transplanted area showing glycogen deposition (arrow); the strongest PAS positive signal is observed in the GHHFC group (scale bar: 50 µm). (H) Quantitative analysis of PAS positive area or integrated density. Preoperative blood routine tests were performed on animals from each group (Figure S6). The results showed no significant differences in indicators such as WBC, Lymph, Gran, RBC, HGB, HCT, and PLT among the groups, with all values falling within the normal range. This indicates that the baseline hematopoietic function and immunological status were consistent across the different hydrogel transplant groups before undergoing the 90% liver resection surgery, providing a reliable control for the subsequent postoperative differences among the groups. Preoperative blood biochemical tests were performed on animals from each group (Figure S7), including ALT, AST, ALP, γ-GT, TBIL, DBIL, TBA, ALB, and TP. The results showed that the levels of these indicators were within similar ranges across all groups, with no significant differences observed. This suggests that prior to hydrogel transplantation, the baseline liver function status was relatively consistent among the different groups, providing a comparable starting point for assessing the improvements in survival and functional recovery after surgery. Figure 6 A shows the transplantation of the hydrogel onto the mesentery of mice. After 28 days of mesenteric transplantation, 90% liver resection was performed, and follow-up results showed that animals pretreated with GHHFC had the highest survival probability (Fig. 6 B), while the control and single-module groups (GHHF or GHHC) had relatively lower survival rates. This suggests that the dual-module preconditioning of "oxygen generation-antioxidant + pro-differentiation" helps improve survival under extreme liver resection conditions. Histologically, H&E staining showed cell infiltration and remodeling in all groups (Fig. 6 C).Postoperative blood biochemistry results from 24–48 hours further confirmed this trend (Fig. 6 D): compared to the control/sham and GHH groups, the GHHFC group showed significantly lower injury and cholestasis-related indicators such as ALT, AST, ALP, γ-GT, TBL, DBIL, and TBA, while ALB and TP were significantly higher (p-values as shown), indicating reduced hepatocyte damage, improved bile metabolism, and better maintenance of synthetic function. However, in immunofluorescence, the GHHFC group had the most abundant and well-structured CD31-positive blood vessels, along with significantly enhanced FAH signals (Fig. 6 E, F), consistent with its better survival and biochemical improvement. PAS staining also showed the most prominent glycogen deposition in the GHHFC group (Fig. 6 H and S8), suggesting stronger glucose storage and metabolic capacity in the graft area (and its functional coupling with the host). Mechanistically, it is hypothesized that CaO 2 @ZIF-8 continuously provides oxygen and that lignosulfonate removes excess ROS, reducing acute ischemia-reperfusion injury and oxidative stress after surgery. Meanwhile, the sustained release of FH-1 promotes the differentiation of HUCMSCs into a hepatocyte-like phenotype and enhances functional secretion/synthetic capacity. The synergistic action of both in the 3D scaffold enhances graft vascularization and perfusion potential (increased CD31), as well as liver-specific functions (increased FAH and PAS positivity), thereby providing compensatory support during the "functional gap" period following 90% liver resection. Blood routine tests were performed on animals after the 90% liver resection to assess changes in hematopoietic and immune function (Figure S9). The results showed significant abnormalities in WBC, Gran, HGB, HCT, PLT, and other parameters in the control group and some of the single-module groups, indicating severe postoperative inflammation, hematopoietic dysfunction, and coagulopathy. In contrast, the blood parameters in the GHHFC group were closer to the normal range, with a moderate increase in white blood cell count, stable lymphocyte proportion, and maintained hemoglobin and hematocrit levels. Additionally, platelet count was significantly higher in the GHHFC group compared to the control and single-treatment groups. These results suggest that the dual-module hydrogel has advantages in improving the postoperative hematopoietic microenvironment, maintaining immune homeostasis, and alleviating postoperative inflammatory damage. 4 Conclusion In this work, a 3D-printed polysaccharide-based hydrogel scaffold integrating FH1@SA microspheres and CaO 2 @ZIF-8@SL nanoparticles was successfully constructed. The dual-network matrix composed of HAMA and SA provided a hydrated, ionically cross-linked framework resembling native extracellular matrix, while the incorporation of SL endowed the system with intrinsic antioxidant and redox-buffering capabilities. Through these polysaccharide-driven interactions, the scaffold achieved a balanced combination of mechanical resilience, moisture retention, and dynamic oxygen regulation. The integrated platform enabled controlled release of FH1 and sustained oxygen generation, jointly alleviating hypoxia and oxidative stress while promoting metabolic reprogramming and hepatocyte differentiation. Transcriptomic and in vivo results confirmed that this multi-polysaccharide microenvironment activated hepatic developmental and angiogenic pathways, resulting in enhanced tissue remodeling and functional recovery. By leveraging the chemical versatility and bioactivity of polysaccharides to orchestrate redox homeostasis, cell signaling, and matrix-cell crosstalk, this study provides a mechanistically informed and adaptable strategy for liver tissue regeneration. The modular design concept also lays a foundation for extending polysaccharide–inorganic hybrid systems to other metabolically demanding tissues and bioartificial organ construction. Declarations Ethics Statements This study was reviewed and approved by the Ethics Committee of the General Hospital of Ningxia Medical University. The approved project, titled "Application of Human Mesenchymal Stem Cell 3D Bioprinted Organoids with Slow-Release FH1 in Liver Injury Repair," was granted ethical clearance. The approval number for this project is KYLL-2023-0157, and the date of approval is November 7, 2023. Conflict of Interest The authors declare no conflict of interest. Fundings This work was supported by grants from Science and Technology Support Project of Yinchuan City (No.2024SF045). Author Contribution Sang Luo: Writing-review & editing, Writing-original draft, Validation, Investigation, Formal analysis, Conceptualization. Longbao Feng: Validation, Investigation. Bingren Tian: Methodology, Investigation. Fang Wu: Validation, Investigation. Shuai Xiao: Investigation, Formal analysis. Wenjun Wu: Software, Investigation. Xiaojun Yang: Supervision. 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Mater Today Bio. 2025;35:102461. 10.1016/j.mtbio.2025.102461 . Samanta HS, Ray SK. Synthesis, characterization, swelling and drug release behavior of semi-interpenetrating network hydrogels of sodium alginate and polyacrylamide. Carbohydr Polym. 2014;99:666–78. 10.1016/j.carbpol.2013.09.004 . Luo S, Wu F, Jin Y, Liu D. The Potential Hepatocyte Differentiation Targets and MSC Proliferation by FH1. J Cell Mol Med. 2025;29:e70601. 10.1111/jcmm.70601 . Schemes Scheme 1 is available in the Supplementary Files section Additional Declarations No competing interests reported. Supplementary Files Supportinginformation20251216.docx image1.png Scheme 1. Schematic illustration of the preparation of 3D-printed hydrogel scaffold and its mechanism for liver tissue repair. Cite Share Download PDF Status: Published Journal Publication published 28 Feb, 2026 Read the published version in Journal of Nanobiotechnology → Version 1 posted Editorial decision: Revision requested 09 Jan, 2026 Reviews received at journal 08 Jan, 2026 Reviews received at journal 05 Jan, 2026 Reviewers agreed at journal 05 Jan, 2026 Reviews received at journal 31 Dec, 2025 Reviewers agreed at journal 25 Dec, 2025 Reviewers agreed at journal 24 Dec, 2025 Reviewers invited by journal 24 Dec, 2025 Editor assigned by journal 17 Dec, 2025 Submission checks completed at journal 17 Dec, 2025 First submitted to journal 16 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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1","display":"","copyAsset":false,"role":"figure","size":950495,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructural and compositional characterization of CaO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e@ZIF-8@SL. \u003c/strong\u003e(A) XRD of CaO\u003csub\u003e2\u003c/sub\u003e, ZIF-8, and CaO\u003csub\u003e2\u003c/sub\u003e@ZIF-8@SL; (B) Fourier-transform infrared spectroscopy of CaO\u003csub\u003e2\u003c/sub\u003e, SL, ZIF-8@SL, and CaO\u003csub\u003e2\u003c/sub\u003e@ZIF-8@SL; (C) dissolved oxygen content; (D) particle size distribution of CaO\u003csub\u003e2\u003c/sub\u003e, ZIF-8, and CaO\u003csub\u003e2\u003c/sub\u003e@ZIF-8@SL; (E) Zeta potential of CaO\u003csub\u003e2\u003c/sub\u003e, ZIF-8, and CaO\u003csub\u003e2\u003c/sub\u003e@ZIF-8@SL; (F) DPPH radical scavenging activity; (G) SEM images of ZIF-8 and CaO\u003csub\u003e2\u003c/sub\u003e@ZIF-8@SL; (H) Mapping images of CaO\u003csub\u003e2\u003c/sub\u003e@ZIF-8@SL; (I) EDS analysis of CaO\u003csub\u003e2\u003c/sub\u003e@ZIF-8@SL.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8380365/v1/2c5f2988c8b9845c980ecc3e.png"},{"id":99024526,"identity":"e683ca02-e414-4a82-9dde-44853d20bf90","added_by":"auto","created_at":"2025-12-26 06:37:07","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":567066,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhysicochemical property characterization of hydrogel scaffold materials. \u003c/strong\u003e(A) FTIR spectra of Gel and GelMA; (B) FTIR spectra of HA and HAMA; (C) 1H NMR spectra of Gel and GelMA; (D) \u003csup\u003e1\u003c/sup\u003eH NMR spectra of HA and HAMA; (E) Storage modulus, loss modulus, and their relationship with frequency for HAMA/GelMA hydrogel; (F) Storage modulus, loss modulus, and their relationship with time for HAMA/GelMA hydrogel; (G) Stress-strain curve of HAMA/GelMA hydrogel; (H) Compressive strength of HAMA/GelMA hydrogel (strain at 30%); (I) Compressive images of HAMA/GelMA hydrogel; (J) Swelling curve of HAMA/GelMA/FH1@SA hydrogel in PBS; (K) Degradation curve of HAMA/GelMA/FH1@SA hydrogel in PBS; (L) Degradation curve of HAMA/GelMA/FH1@SA hydrogel in collagenase; (M) Printed liver lobule sample image; (N) Release curve of FH1; (O) SEM images of the hydrogel.\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8380365/v1/c28fed490d9d248123c869de.jpeg"},{"id":99024529,"identity":"df8dc43e-0e97-4060-ba47-7b2cd022ca6d","added_by":"auto","created_at":"2025-12-26 06:37:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":915970,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCytocompatibility and angiogenic potential of 3D-printed HUCMCS hydrogel constructs. \u003c/strong\u003e(A) Schematic illustration of the experimental procedure, including 3D bioprinting of cell-laden hydrogels, cell culture, staining, and imaging. (B) Quantitative analysis of cell viability in different hydrogel groups (GHH, GHHF, GHHC, GHHFC) on days 1, 3, and 5, measured by CCK-8 assay. (C, D) Live/Dead staining of encapsulated HUCMCS at days 1, 3, and 5, showing viable cells (green) and dead cells (red). (E) Representative tube-like network formation images of HUCMCS cultured in different hydrogel groups. (F–H) Quantification of angiogenic parameters, including number of branches (F), number of junctions (G), and total branching length (H). Data are presented as mean ± SD; *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8380365/v1/dec90840bd755352ec9f06b8.png"},{"id":99313000,"identity":"6393491b-e83a-4f5c-8339-62f8a1ad64d9","added_by":"auto","created_at":"2025-12-31 16:19:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":354440,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptomic analysis of HUCMCSs cultured in GHHFC hydrogels. \u003c/strong\u003e(A) Volcano plot showing differentially expressed genes (DEGs) between the control and GHHFC groups (1710 upregulated and 985 downregulated genes). (B) Heatmap of representative DEGs, highlighting gene expression changes induced by GHHFC treatment. (C) KEGG pathway enrichment analysis of DEGs, with significantly enriched pathways including focal adhesion, insulin, TNF, AMPK, Hippo, and VEGF signaling, as well as glycolysis/gluconeogenesis and fatty acid degradation. (D) GO enrichment analysis showing significant biological processes related to lipid metabolism, gap junction assembly, extracellular matrix organization, MAPK cascade activation, liver development, endothelial cell migration, and glycogen metabolism. (E) GSEA plots confirming enrichment of AMPK and VEGF signaling pathways in GHHFC-treated cells. (F) Gene network map of the AMPK signaling pathway, illustrating the interconnected DEGs involved in metabolic regulation.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8380365/v1/c8a418db8934ea63da239b04.png"},{"id":99313413,"identity":"b363c602-749c-472f-a9bf-05968e949677","added_by":"auto","created_at":"2025-12-31 16:20:08","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1121056,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003evascularization and liver-specific function after mesenteric transplantation of 3D printed hydrogel. \u003c/strong\u003e(A) Schematic of vascularization 14 days post-transplantation. (B) Formation of vascular networks within the artificial liver tissue. (C) H\u0026amp;E staining of the transplanted hydrogel 14 days post-transplantation, showing cell infiltration and tissue remodeling in all groups (scale bar: 50 μm). (D) Immunofluorescence staining of CD31 (green) and FAH (fumarate hydratase, red) in the grafts, with DAPI (blue) counterstaining of cell nuclei. The GHHFC group shows the most abundant CD31⁺ vascular structures and the strongest FAH expression, indicating enhanced vascularization and hepatocyte differentiation (scale bar: 50 μm). (E–F) Quantification of CD31 and FAH positive cells. (G) PAS staining of the grafts showing glycogen deposition (black arrow). The GHHFC group displays the most prominent PAS positive signal compared to other groups (scale bar: 50 μm).\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8380365/v1/1bc17497be0528f7313e0779.png"},{"id":99312770,"identity":"9a05f147-4016-4d61-acbc-b3d6d42f8989","added_by":"auto","created_at":"2025-12-31 16:19:28","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":861923,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctional recovery after pre-transplantation of 3D printed hydrogel following 90% liver resection in mice. \u003c/strong\u003e(A) Schematic of the experimental timeline: Four 3D printed hydrogels were transplanted onto the mesentery of mice; 28 days later, the rats underwent 90% liver resection, followed by survival and functional evaluation.\u003c/p\u003e\n\u003cp\u003e(B) Kaplan-Meier curve of cumulative survival rates after 90% liver resection in different groups (control group, sham surgery group, GHH group, GHHF group, GHHC group, GHHFC group). (C) H\u0026amp;E staining of the transplanted constructs/tissues at the endpoint, showing cell infiltration and tissue remodeling (scale bar: 50 μm). (D) Serum biochemistry of surviving rats 90 days after liver resection: albumin (ALB), alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), direct bilirubin (DBIL), total bile acids (TBA), total bilirubin (TBL), γ-glutamyl transferase (γ-GT), and total protein (TP). (E) Immunofluorescence staining of CD31 (green) and FAH (red) with DAPI nuclear staining (blue) in the transplanted area after liver resection; the GHHFC group shows the most abundant CD31⁺ vascular structures and strong FAH expression (scale bar: 50 μm). (F) Quantitative analysis of the density/area fraction of CD31⁺ vessels. (G) PAS staining of the transplanted area showing glycogen deposition (arrow); the strongest PAS positive signal is observed in the GHHFC group (scale bar: 50 μm). (H) Quantitative analysis of PAS positive area or integrated density.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-8380365/v1/c5b842018689d294ced28e21.png"},{"id":103765445,"identity":"4b71a2c6-3d6a-4884-86bd-0a4197dd9e9c","added_by":"auto","created_at":"2026-03-02 16:02:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5439077,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8380365/v1/1b5d6702-6963-4ae4-aba5-d4da3ca0d09b.pdf"},{"id":99312875,"identity":"7aa75ba9-5cad-4cf1-9e67-1344eaeaec71","added_by":"auto","created_at":"2025-12-31 16:19:33","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3089832,"visible":true,"origin":"","legend":"","description":"","filename":"Supportinginformation20251216.docx","url":"https://assets-eu.researchsquare.com/files/rs-8380365/v1/acab773e0e698a8fce269ae9.docx"},{"id":99313205,"identity":"49b2b263-9eff-41c6-a264-117445aaf2fc","added_by":"auto","created_at":"2025-12-31 16:19:54","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":634178,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1. \u003c/strong\u003eSchematic illustration of the preparation of 3D-printed hydrogel scaffold and its mechanism for liver tissue repair.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8380365/v1/4b4ba9e826cf2875710a50cc.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"3D printed HAMA/GelMA/lignosulfonate hydrogel integrating oxygen-releasing and antioxidative modules for liver regeneration","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eLiver injury caused by trauma, infection, drug toxicity, or metabolic disorders represents a growing global health challenge\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. As the central organ responsible for detoxification, metabolism, and immune regulation, the liver plays indispensable roles in maintaining systemic homeostasis\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Severe hepatic injury causes hepatocyte necrosis and structural collapse, leading to persistent inflammation and, eventually, liver failure\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. For patients with end-stage liver disease, the regenerative capacity of hepatocytes is largely exhausted, and liver transplantation remains the only effective treatment. However, donor shortage, high surgical cost, and immune rejection still constrain its clinical utility\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. To meet this demand, hydrogels have been widely explored as promising biomaterial candidates for bioartificial liver construction.\u003c/p\u003e \u003cp\u003eHydrogel-based biomaterials have attracted increasing attention in hepatic tissue engineering owing to their three-dimensional, highly hydrated networks that mimic the extracellular matrix and provide structural and biochemical cues for hepatocyte growth, migration, and differentiation\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Nevertheless, conventional hydrogels provide limited control over the hepatic microenvironment, which is typically characterized by hypoxia, oxidative stress, and chronic inflammation\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Moreover, hydrogel-based drug delivery systems often suffer from burst release of bioactive molecules, resulting in transient over-concentration and potential cytotoxicity\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. To overcome these limitations, polysaccharide-based hydrogels have emerged as versatile scaffolds due to their tunable chemical functionalities, intrinsic biocompatibility, and resemblance to native glycosaminoglycans\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Among them, sodium alginate (SA) is widely used to construct ionically cross-linked hydrogel microspheres for controlled drug release\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, while sodium lignosulfonate (SL), a natural anionic polysaccharide rich in phenolic hydroxyl groups, exhibits strong antioxidant and anti-inflammatory activities that can mitigate oxidative stress within damaged tissues\u003csup\u003e\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. These inherent properties make polysaccharide networks an ideal foundation for developing multifunctional hydrogels capable of modulating the oxidative and inflammatory microenvironment during liver regeneration.\u003c/p\u003e \u003cp\u003eBuilding upon these insights, a polysaccharide-based 3D-printed hydrogel scaffold was designed to reconstruct the hepatic microenvironment by integrating bioactive microspheres and oxygen-generating nanoparticles. Specifically, sodium alginate microspheres were utilized as controlled-release carriers for the liver-regenerative molecule functional small molecule 1 (FH1), forming FH1@SA microspheres capable of providing sustained hepatocyte stimulation and promoting differentiation\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Meanwhile, calcium peroxide (CaO\u003csub\u003e2\u003c/sub\u003e) was employed as an oxygen source to alleviate local hypoxia\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, and its release behavior was precisely regulated by encapsulation within zeolitic imidazolate framework-8 (ZIF-8), which enables pH-responsive degradation\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003eand controlled oxygen generation. Furthermore, sodium lignosulfonate (SL) was introduced to modify the nanoparticle surface, imparting intrinsic antioxidant and anti-inflammatory functions by scavenging excessive reactive oxygen species and buffering oxidative stress\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The resulting hydrogel network, composed primarily of biocompatible polysaccharides, provided interconnected porosity, suitable mechanical strength, and tunable degradation, supporting 3D bioprinting and long-term hepatocyte viability.\u003c/p\u003e \u003cp\u003eOverall, this study presents a polysaccharide-based biohybrid hydrogel integrating biochemical and structural functions for liver regeneration. By combining FH1@SA microspheres and CaO\u003csub\u003e2\u003c/sub\u003e@ZIF-8@SL nanoparticles within a 3D-printed polysaccharide matrix, the system establishes a synergistic microenvironment that simultaneously alleviates hypoxia, suppresses oxidative stress, and reduces inflammation, thereby enhancing hepatocyte survival and functional recovery. The hydrogel exhibits favorable printability, mechanical stability, and biocompatibility, offering both structural support and dynamic regulation of the hepatic niche. This polysaccharide-centered design not only provides a new strategy for reconstructing bioartificial liver systems but also highlights the potential of multifunctional polysaccharide hydrogels as versatile platforms for tissue regeneration and microenvironmental modulation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cp\u003e \u003cb\u003ePolysaccharides structural.\u003c/b\u003e Sodium alginate (SA, CAS 9005-38-3, Macklin, Shanghai, China) was an ultra-low-viscosity type (1% aqueous solution viscosity: 3\u0026ndash;5 mPa\u0026middot;s, pH 6\u0026ndash;8, ash 18\u0026ndash;27%), which has a viscosity-average molecular weight of 100,000 g/mol and appears as an off-white to pale yellow solid.SA is a linear copolymer composed of β-D-mannuronic acid (M) and α-L-guluronic acid (G) residues linked by 1\u0026rarr;4 glycosidic bonds, with an M/G ratio typically around 1:1 as provided by the manufacturer. Hyaluronic acid sodium (YuanYe Bio-Technology Co., Ltd., Shanghai, China) is a white powder composed of repeating disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine linked by β(1\u0026rarr;3) and β(1\u0026rarr;4) glycosidic bonds, soluble in water and DMSO. The product used in this study has a molecular weight of approximately 200\u0026ndash;400 kDa (supplier data).\u003c/p\u003e \u003cp\u003e \u003cb\u003ePolysaccharide inks.\u003c/b\u003e Methacrylated gelatin (GelMA) and methacrylated hyaluronic acid (HAMA) were prepared as described (SI \u0026sect;\u0026nbsp;1.6\u0026ndash;1.7). Sodium alginate (SA) was used to form drug-loaded microspheres, and sodium lignosulfonate (SL) served as an antioxidant/anti-inflammatory polysaccharide coating (SI \u0026sect;\u0026nbsp;1.1).\u003c/p\u003e \u003cp\u003e \u003cb\u003eOxygen donor nanoparticles.\u003c/b\u003e Calcium peroxide (CaO\u003csub\u003e2\u003c/sub\u003e) nanoparticles were synthesized by precipitation (SI \u0026sect;\u0026nbsp;1.2) and assembled with zeolitic imidazolate framework-8 (ZIF-8) followed by SL surface modification to obtain CaO\u003csub\u003e2\u003c/sub\u003e@ZIF-8@SL (CZS) (SI \u0026sect;\u0026nbsp;1.3\u0026ndash;1.4). Physicochemical features were assessed by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), transmission electron microscopy (TEM), and dynamic light scattering (DLS) (SI \u0026sect;\u0026nbsp;1.9). Dissolved oxygen release and 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging were evaluated as in SI \u0026sect;\u0026nbsp;1.9.5\u0026ndash;1.9.6.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFH1-loaded microspheres.\u003c/b\u003e FH1-loaded sodium alginate microspheres (FH1@SA) were generated via a microfluidic SA/Ca\u0026ndash;EDTA system (SI \u0026sect;\u0026nbsp;1.5). Morphology and size distribution were analyzed by optical microscopy and scanning electron microscopy (SEM) (SI \u0026sect;\u0026nbsp;1.10). FH1 release from GelMA/HAMA matrices was quantified in vitro (SI \u0026sect;\u0026nbsp;1.11.9).\u003c/p\u003e \u003cp\u003e \u003cb\u003e3D-printed hydrogel scaffolds.\u003c/b\u003e Composite bioinks were prepared by dissolving GelMA/HAMA with lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) and tartrazine, followed by addition of FH1@SA and/or CZS (SI \u0026sect;\u0026nbsp;1.8). Four groups were printed: GHH (GelMA/HAMA), GHHF (GHH\u0026thinsp;+\u0026thinsp;FH1@SA), GHHC (GHH\u0026thinsp;+\u0026thinsp;CZS), and GHHFC (GHH\u0026thinsp;+\u0026thinsp;FH1@SA\u0026thinsp;+\u0026thinsp;CZS). Printed scaffolds were rinsed in phosphate-buffered saline (PBS) and post-cured (SI \u0026sect;\u0026nbsp;1.8). Bulk and microstructural properties were assessed by FTIR, SEM, unconfined compression, rheology, swelling, and enzymatic/degradation tests (SI \u0026sect;\u0026nbsp;1.11).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro assays.\u003c/b\u003e Human umbilical cord mesenchymal stem cells (HUCMSCs) and HepaRG hepatocytes (HepaRG) were used for cytocompatibility (CCK-8), live/dead staining, and cytoskeleton staining (SI \u0026sect;\u0026nbsp;1.12). Human umbilical vein endothelial cells (HUVECs) on Matrigel were used for tube formation analysis (SI \u0026sect;\u0026nbsp;1.13). Hepatic differentiation of printed constructs and expression of ALB, AFP, CK18, Hnf1α, Foxa2, Prkaca, Prkacb, and Prkx were evaluated by real-time reverse transcription quantitative PCR (RT-qPCR) (SI \u0026sect;\u0026nbsp;1.14\u0026ndash;1.17). Bioinformatics analyses (differentially expressed genes, Gene Ontology/GO, Kyoto Encyclopedia of Genes and Genomes/KEGG, and Gene Set Enrichment Analysis/GSEA) followed SI \u0026sect;\u0026nbsp;1.18.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo evaluation.\u003c/b\u003e All animal procedures were conducted in accordance with institutional guidelines and were approved by the Laboratory Animal Ethical and Welfare Committee of laboratory Animal Center, Ningxia Medical University. Approval No. IACUC- 2025280. All procedures followed the principles of the Declaration of Helsinki and complied with the ARRIVE guidelines. Scaffolds were implanted in the mouse mesentery; vascularization (FITC-dextran), histology (hematoxylin and eosin/H\u0026amp;E, periodic acid\u0026ndash;Schiff/PAS), and immunofluorescence (fumarylacetoacetate hydrolase/FAH, CD31) were performed per SI \u0026sect;\u0026nbsp;1.19\u0026ndash;1.23. A subsequent extended hepatectomy model was used to assess survival and liver function (SI \u0026sect;\u0026nbsp;1.20).\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistics.\u003c/b\u003e Data are reported as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). One- or two-way analysis of variance (ANOVA) with Tukey\u0026rsquo;s post-hoc test determined significance (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (SI \u0026sect;\u0026nbsp;1.24).\u003c/p\u003e"},{"header":"3 Results and discussion","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Characterization of the Structure and Composition of Self-Produced Oxygen Nanoparticles (CaO\u003csub\u003e2\u003c/sub\u003e@ZIF-8@SL)\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the CaO\u003csub\u003e2\u003c/sub\u003e sample, distinct characteristic diffraction peaks at 30.0\u0026deg; (002), 35.5\u0026deg; (110), 47.5\u0026deg; (112), and 52.5\u0026deg; (103) were observed, corresponding to the characteristic peaks of CaO\u003csub\u003e2\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), which align with the CaO\u003csub\u003e2\u003c/sub\u003e crystal ICDD card. The synthesized ZIF-8 exhibits a highly crystalline structure, and its diffraction peaks match well with the standard simulated XRD pattern of ZIF-8\u003csup\u003e24\u003c/sup\u003e. However, in the XRD spectrum of CaO\u003csub\u003e2\u003c/sub\u003e@ZIF-8@SL, some characteristic peaks of ZIF-8 are clearly visible, indicating that the ZIF-8 crystallinity attached in situ on CaO\u003csub\u003e2\u003c/sub\u003e is high. Moreover, due to the in-situ growth of ZIF-8 on its surface, no distinct CaO\u003csub\u003e2\u003c/sub\u003e characteristic peaks are observed in the XRD spectrum of CaO\u003csub\u003e2\u003c/sub\u003e@ZIF-8@SL.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, the FTIR spectrum of CaO\u003csub\u003e2\u003c/sub\u003e shows an absorption peak at 873 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which corresponds to the O-O stretching vibration and is the most important characteristic peak of CaO\u003csub\u003e2\u003c/sub\u003e, indicating the successful synthesis of CaO\u003csub\u003e2\u003c/sub\u003e. Additionally, the asymmetric and symmetric stretching vibration absorption bands of O-Ca-O in CaO\u003csub\u003e2\u003c/sub\u003e appear at 1419 and 1483 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. The absorption peak at 1594 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e corresponds to the CO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e stretching vibration of calcium carbonate (CaCO\u003csub\u003e3\u003c/sub\u003e), which is a byproduct formed by the reaction between CaO\u003csub\u003e2\u003c/sub\u003e and CO\u003csub\u003e2\u003c/sub\u003e during preparation and storage\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. For the ZIF-8@SL sample, absorption bands at 3134 and 2925 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e represent the aromatic and aliphatic C-H stretches of the imidazole ring, while absorption bands at 687\u0026ndash;758 and 948\u0026ndash;998 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are related to the in-plane and out-of-plane bending of the imidazole ring. In addition to the characteristic bands of unmodified ZIF-8, a broad absorption band around 3425 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is associated with the -OH vibration in the SL structure, and the absorption band at 1424 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e corresponds to the C-C bond present in SL and ZIF-8. This indicates that SL and ZIF-8 are hydrogen-bonded, and the Zn\u003csup\u003e2+\u003c/sup\u003e (positively charged) tends to attach to the sulfonic groups in SL. In the FTIR spectrum of CaO\u003csub\u003e2\u003c/sub\u003e@ZIF-8@SL, both the O-O and imidazole ring stretching vibration peaks appear, indicating that ZIF-8@SL has successfully attached in situ to the surface of CaO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG shows the SEM images of two nanoparticles (ZIF-8 and CaO\u003csub\u003e2\u003c/sub\u003e@ZIF-8@SL). As seen from the images, the unmodified ZIF-8 nanoparticles exhibit a uniform rhombic dodecahedral shape, with a smooth surface and an average particle size of approximately 100 nm. However, after loading with CaO\u003csub\u003e2\u003c/sub\u003e and the SL coating, the surface of CaO\u003csub\u003e2\u003c/sub\u003e@ZIF-8@SL becomes rough, with the average particle size reduced to approximately 70 nm, showing a more restricted particle size distribution. This suggests that CaO\u003csub\u003e2\u003c/sub\u003e and SL, as growth inhibitors, regulate the nucleation rate of ZIF-8 and limit its growth, thereby effectively controlling the size of ZIF-8 crystals during synthesis. EDS characterization (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI) was used to analyze the elemental composition of ZIF-8@SL nanoparticles, confirming the presence of the required elements (Zn, Ca, S, O) in the structure.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, in pure water, there are no peroxides or oxygen sources, so the dissolved oxygen remains near the saturation value of the natural environment and does not significantly increase or decrease. Hydrogen peroxide slowly decomposes in water to generate oxygen (2H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e \u0026rarr; 2H\u003csub\u003e2\u003c/sub\u003eO\u0026thinsp;+\u0026thinsp;O\u003csub\u003e2\u003c/sub\u003e), so the dissolved oxygen content in this group increases. Due to the dual effect of CaO\u003csub\u003e2\u003c/sub\u003e@ZIF-8@SL and hydrogen peroxide, the CaO\u003csub\u003e2\u003c/sub\u003e@ZIF-8@SL group exhibits the highest dissolved oxygen level. Furthermore, because of the encapsulation effect of ZIF-8@SL, the CaO\u003csub\u003e2\u003c/sub\u003e@ZIF-8@SL group shows stability in oxygen release over an extended period.\u003c/p\u003e \u003cp\u003eParticle size analysis of ZIF-8 and CaO\u003csub\u003e2\u003c/sub\u003e@ZIF-8@SL nanoparticles was performed using a laser nanoparticle size analyzer, as shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE. The average particle size of ZIF-8 was 412.8\u0026thinsp;\u0026plusmn;\u0026thinsp;14.08 nm, and that of CaO\u003csub\u003e2\u003c/sub\u003e@ZIF-8@SL was 484.3\u0026thinsp;\u0026plusmn;\u0026thinsp;38.52 nm, which may be due to the loading of CaO\u003csub\u003e2\u003c/sub\u003e inside ZIF-8, increasing the particle size. Additionally, the SL layer surrounding the particles also directly increases the particle size. Furthermore, aggregation may occur during the synthesis process, leading to particle agglomeration and an increase in particle size. TEM observations revealed that ZIF-8 nanoparticles exhibited a polyhedral structure and were uniformly dispersed, while CaO\u003csub\u003e2\u003c/sub\u003e@ZIF-8@SL nanoparticles showed aggregation phenomena, with rough, irregular surfaces, leading to increased particle size. This result is consistent with the hydrated particle size results (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, the H\u003csub\u003e2\u003c/sub\u003eO group and H\u003csub\u003e2\u003c/sub\u003eO\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group exhibited no significant radical scavenging activity, while the H\u003csub\u003e2\u003c/sub\u003eO\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;CaO\u003csub\u003e2\u003c/sub\u003e@ZIF-8@SL group displayed a noticeable lightening of the purple color, likely due to the presence of multiple phenolic hydroxyl groups (-OH) in sodium lignosulfonate (SL). These hydroxyl groups can interact with DPPH radicals (2,2-diphenyl-1-picrylhydrazyl) through a hydrogen atom transfer (HAT) reaction, reducing them to the yellow diphenyl-picrylhydrazyl form\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Additionally, the aromatic rings in the lignosulfonate structure contain π electron systems that can participate in the radical scavenging reaction, providing electrons through an electron transfer (ETr) reaction to the radicals, thereby converting them into stable non-radical forms.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Structural and Compositional Characterization of FH1@SA Microspheres\u003c/h2\u003e \u003cp\u003eThe morphology of the prepared microspheres can be observed under a microscope, as shown in Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA. Using microfluidic technology, sodium alginate microspheres loaded with FH1 were successfully prepared with uniform size and controllable dimensions. Meanwhile, the size of the microspheres was quantitatively analyzed (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). The results show that when the dispersed phase to continuous phase flow rate ratios were 1:2.5, 1:5, and 1:10, the sizes of the microspheres prepared were 260.2 \u0026micro;m, 225.2 \u0026micro;m, and 210.1 \u0026micro;m, respectively. As the flow rate ratio decreased, the size of the microspheres gradually decreased. This indicates that the faster the flow rate of the continuous phase, the greater the fluid shear force applied to the dispersed phase as it enters the continuous phase. This force promotes the emulsification process of the droplets and accelerates the rupture of the droplets into spheres, thereby resulting in smaller microspheres with a greater number formed per unit time. Figure S2 shows the SEM images of the microspheres. As can be seen from the image, the surface of the microspheres has a certain porous structure.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Structural and physicochemical characterization of polysaccharide-based hydrogels\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo elucidate the chemical architecture of the polysaccharide-based network, FTIR and \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR analyses were first performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026ndash;D). Distinct amide I, II, and III peaks in GelMA\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003eand new ester carbonyl signals in HAMA confirmed successful methacrylation\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, while both materials preserved the abundant hydroxyl and carboxyl groups characteristic of natural polysaccharides. These functional moieties play a dual role: they ensure high hydrophilicity and enable multiple hydrogen-bonding or ionic interactions during gelation. Such chemical versatility provides the foundation for constructing an ECM-mimicking matrix capable of coordinating metal ions and anchoring bioactive components.\u003c/p\u003e \u003cp\u003eThe rheological and mechanical profiles (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE\u0026ndash;I) revealed that this polysaccharide-dominated network exhibited multiscale crosslinking. The flexible glycosidic chains of HAMA and SA contributed to dynamic hydrogen bonding and water retention, while the Ca\u003csup\u003e2+\u003c/sup\u003e ions released from CaO\u003csub\u003e2\u003c/sub\u003e@ZIF-8@SL interacted electrostatically with sulfonic groups on SL, forming a reversible ionic framework\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. This multi-interactive network endowed the hydrogel with remarkable elasticity (G\u0026prime; \u0026gt; G\u0026Prime; throughout) and recoverability under compression, reflecting the intrinsic adaptability of polysaccharide scaffolds to mechanical stress\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Such viscoelastic resilience is essential for hepatocyte mechanotransduction and liver tissue remodeling.\u003c/p\u003e \u003cp\u003eThe swelling and degradation behaviors (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ\u0026ndash;L) further highlighted the regulatory role of the polysaccharide domains. The SA microspheres introduced additional hydrophilic sites, markedly enhancing water absorption(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ). Meanwhile, as the proportion of GelMA increased, the overall swelling rate and degradation rate of the hydrogel decreased. This indicates that GelMA, as a protein backbone, has a significant impact on the overall structural properties (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK\u0026ndash;L). These effects stem from the inherent responsiveness of polysaccharide networks, where hydrogen-bond dynamics and partial ionic dissociation govern water diffusion and polymer relaxation. This balance between hydration and degradation not only maintains the scaffold\u0026rsquo;s dimensional stability but also synchronizes its resorption rate with native hepatic regeneration.\u003c/p\u003e \u003cp\u003eFrom a structural standpoint, the polysaccharide backbone serves not merely as a passive matrix but as an active biochemical modulator. The hydroxyl and sulfonic groups of lignosulfonate can scavenge reactive oxygen species and mediate protein adsorption\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, while the carboxyl-rich SA domains facilitate sustained drug release and microenvironment buffering\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Together, these cooperative features construct a biofunctional hydrogel with hierarchical architecture\u0026mdash;where molecular interactions translate into macroscopic mechanical robustness and controlled bioactivity.\u003c/p\u003e \u003cp\u003eCollectively, the GelMA/HAMA/SA-based hydrogel represents a polysaccharide-governed supramolecular network integrating covalent, ionic, and hydrogen-bonded crosslinks. This architecture provides tunable viscoelasticity, hydration, and degradation, while the intrinsic redox and adsorption properties of lignosulfonate endow the system with microenvironment-modulating capability. Such synergy of chemistry and function forms the essential physicochemical basis for subsequent hepatocyte proliferation and tissue regeneration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Evaluation of cell compatibility and angiogenic potential of 3D printed HUCMCS hydrogel\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe polysaccharide backbone, abundant in hydroxyl, carboxyl, and sulfonic groups, enables dynamic interactions with proteins, ions, and cellular membranes, thereby creating a hydrated and redox-buffered microenvironment. Such structural features are expected to modulate cell adhesion, viability, and angiogenic behavior. The CCK-8 method was used to systematically evaluate the effects of different concentrations of CaO\u003csub\u003e2\u003c/sub\u003e@ZIF-8@SL solution and FH1@SA hydrogel microspheres on the viability of HUCMSC and HepaRG cells (Figure S3). The results showed that CaO\u003csub\u003e2\u003c/sub\u003e@ZIF-8@SL exhibited a concentration-dependent effect on both cell types. In HUCMSC, cell viability reached its highest value (112.92%) at a drug concentration of 0.05 mg/mL, but significantly decreased to 94.37% at higher concentrations (5 mg/mL), while the lower concentration range (0.025-0.1 mg/mL) promoted cell proliferation (Figure S3A). A similar phenomenon was observed in HepaRG cells, where cell viability was highest (111.54%) at 0.05 mg/mL and decreased to 91.78% at 2.5 mg/mL, indicating that high doses had adverse effects on liver cells as well, while the lower concentration range (0.025\u0026ndash;0.25 mg/mL) had a promoting effect (Figure S3B). However, overall cell viability was above 80%, indicating that CaO\u003csub\u003e2\u003c/sub\u003e@ZIF-8@SL nanoparticles had no cytotoxicity. In contrast, FH1@SA hydrogel microspheres had a milder effect on cell viability. In both HUCMSC and HepaRG cells, when the concentration reached 0.5 mg/mL, the viability increased to 107.19% and 107.60%, respectively, while cell viability remained stable at other concentrations (Figure S3C, D). This suggests that FHI@SA hydrogel microspheres have good biocompatibility across a wide concentration range and do not cause significant toxic side effects.\u003c/p\u003e \u003cp\u003eThe CCK-8 assay was used to perform a time-gradient evaluation of the 3D printed cell-loaded hydrogels. Overall, as the culture time extended from Day 1 to Day 5, the viability of HUCMSCs in all groups showed an upward trend, indicating that the GelMA/HAMA base framework (GHH) has good cell compatibility. Further comparison of different functionalized groups revealed that on Day 3, the GHHFC group, containing the dual modules of oxygen generation/ROS scavenging and pro-hepatocyte differentiation, was significantly higher than the other groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and the difference continued to expand on Day 5. GHHC (oxygen generation\u0026thinsp;+\u0026thinsp;antioxidation) and GHHF (FH-1 microspheres) showed moderate enhancement, while GHH exhibited the smallest improvement (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The results suggest that under the same printing and culture conditions, local oxygen supply and oxidative stress buffering, combined with FH-1 pro-differentiation signals, can more effectively support cell survival and proliferation, creating a more favorable microenvironment for early tissue formation.\u003c/p\u003e \u003cp\u003eCalcein-AM/PI staining further confirmed the cell compatibility of different scaffolds for HUCMSCs. No significant PI-positive accumulation (dead cells) was observed in any group, indicating that the materials were overall safe (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Notably, the GHHFC group showed stronger and more uniformly distributed Calcein-AM signals, which became denser over time. Cell morphology transitioned from dispersed to clustered, with more abundant pseudopodia and protrusions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), indicating improved cell adhesion, spreading, and more active proliferation. The GHHC group also showed better performance than GHH, suggesting that moderate alleviation of hypoxia and ROS load can directly enhance cell status. The enhanced effect in GHHF was consistent with the pro-differentiation/maturation role of FH-1, but the effect was smaller than the synergistic effect seen in the dual-module GHHFC.\u003c/p\u003e \u003cp\u003eIn the HUVEC tube formation assay, the tubular network induced by GHHFC was the most dense and continuous (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). The number of branches (Nb branches), junctions (Nb junctions), and total branching length (Total branching length) were all significantly higher than in the other groups (all p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF\u0026ndash;H). This indicates that the dual-module material not only supports matrix cell growth but also creates a pro-vascularization microenvironment. Comparatively, GHHC showed a significant improvement over GHH, suggesting that continuous oxygen supply and ROS buffering facilitate endothelial cell migration and lumen stabilization. GHHF also showed some promoting effects, which may be related to the upregulation of paracrine factors (such as the VEGF family, HGF, etc.) by FH-1, ultimately leading to a synergistic effect in GHHFC, resulting in the most prominent angiogenic phenotype.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Transcriptomic Analysis of HUCMCS Cultured in GHHFC Hydrogel\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe performed transcriptome analysis on HUCMSCs cultured in GHHFC hydrogel, and the sequencing results revealed pronounced gene expression differences between the GHHFC and control groups, identifying 1,710 upregulated and 985 downregulated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The heatmap of representative DEGs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) showed clear transcriptional remodeling across pathways associated with lipid metabolism, mitochondrial activity, extracellular matrix (ECM) organization, and signal transduction, indicating that the hydrogel microenvironment exerts a broad regulatory influence on cellular homeostasis. This result is consistent with the qRT-PCR results, indicating that that the GHHFC hydrogel most effectively promoted the hepatic differentiation of HUCMSCs (Figure S4A\u0026ndash;H). Compared with the GHH group, the GHHFC scaffold markedly upregulated the expression of mature hepatocyte markers ALB (2.13-fold), AFP (1.78-fold), and CK18 (1.79-fold), indicating enhanced functional maturation. The transcription factors HNF-1α (2.26-fold) and FOXA2 (2.54-fold), which govern hepatocyte lineage commitment, were also significantly elevated, suggesting activation of hepatic developmental programs. In addition, genes associated with cAMP signaling and metabolic regulation, including PRKACA, PRKACB, and PRKX, increased by 2.10-, 2.50-, and 2.18-fold, respectively, reflecting improved cellular energy metabolism.Such coordinated gene activation can be attributed to the polysaccharide-dominated microenvironment constructed by the HAMA/SA backbone and lignosulfonate components. The abundant hydroxyl, carboxyl, and sulfonic groups maintain a hydrated and redox-buffered niche, while oxygen release from CaO2@ZIF-8 sustains mitochondrial activity under physiological conditions. This combination stabilizes intracellular ROS levels and supports AMPK-dependent signaling cascades, which in turn facilitates the expression of HNF-1α and FOXA2 and promotes the metabolic transition toward a mature hepatocyte phenotype.\u003c/p\u003e \u003cp\u003eKEGG enrichment analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) demonstrated that pathways related to hepatic differentiation and metabolic reprogramming\u0026mdash;such as AMPK, VEGF, Hippo, and Insulin signaling, together with glycolysis/gluconeogenesis, fatty acid degradation, and pyruvate metabolism\u0026mdash;were significantly activated. These results suggest a shift toward enhanced energy metabolism and biosynthetic activity, both essential for hepatocyte maturation. GO analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) further highlighted biological processes including lipid metabolic regulation, glycogen synthesis, liver development, MAPK cascade activation, endothelial cell migration, and ECM remodeling, consistent with the cellular proliferation and angiogenic behavior observed in vitro.\u003c/p\u003e \u003cp\u003eGSEA plots (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE) confirmed enrichment of the AMPK and VEGF pathways, while network mapping (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF) showed coordinated activation of multiple metabolic node genes, implying a systemic reinforcement of mitochondrial bioenergetics and fatty acid oxidation. This transcriptomic profile reflects the redox-balanced and oxygenated environment established by the dual-module hydrogel: oxygen generation from CaO₂@ZIF-8 alleviates hypoxia, whereas the phenolic hydroxyl and sulfonic groups of sodium lignosulfonate (SL) buffer excess reactive oxygen species and stabilize mitochondrial function.\u003c/p\u003e \u003cp\u003eWithin this microenvironment, the controlled release of FH1 provides persistent pro-differentiation signals that cooperate with metabolic regulation to activate HNF-1α, FOXA2, and downstream hepatic programs\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Meanwhile, the upregulation of ECM and adhesion-related gene sets corresponds to the 3D polysaccharide framework\u0026rsquo;s physical cues, which promote cytoskeletal polarization and intercellular communication. The concurrent enrichment of VEGF signaling and endothelial migration genes provides molecular evidence for the angiogenic phenotype observed in the tube-formation assays.\u003c/p\u003e \u003cp\u003eAltogether, these transcriptomic findings indicate that the polysaccharide-based oxygen\u0026ndash;antioxidant\u0026ndash;differentiation cascade remodels cellular metabolism and transcriptional activity toward a hepatic phenotype. The synergistic interplay among oxygen supply, ROS buffering, and sustained biochemical signaling establishes a stable biochemical\u0026ndash;biophysical niche that enhances energy metabolism, vascularization, and liver-specific gene expression within the 3D scaffold.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.6 \u003cem\u003eIn vivo\u003c/em\u003e vascularization and liver-specific function after mesenteric transplantation of 3D printed hydrogel.\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA shows the schematic of vascularization 14 days post-transplantation. In Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, the GHHFC group exhibits longer and thicker blood vessel structures with a larger vascular area ratio. The CaO\u003csub\u003e2\u003c/sub\u003e@ZIF-8@SL component may continuously supply oxygen, activating pro-angiogenic signaling pathways. Additionally, the study found that SL can regulate the inflammatory response and stabilize the endothelial microenvironment, further supporting the formation of the capillary network. 14 days after transplantation into the mesentery, histological and immunological analyses were performed on the 3D printed hydrogel sections. H\u0026amp;E staining results (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) showed significant cell infiltration and tissue remodeling in all groups, indicating that the materials possess good biocompatibility. In the immunofluorescence staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD,E,F), the control group (GHH) showed almost no noticeable vascular-related signals; the GHHF and GHHC groups displayed a certain number of CD31-positive newly formed blood vessels, while the GHHFC group showed the most prominent result, with dense distribution of CD31-positive vessels and well-formed lumen structures. Meanwhile, FAH signals were most intense in the GHHFC group, indicating more significant hepatocyte differentiation of HUCMSCs in this material microenvironment. Further PAS staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG and S5) revealed the most prominent glycogen deposition in the GHHFC group, with strong positive reaction in the area indicated by the black arrow, while other groups showed relatively weaker positive signals. Overall, the results indicate that GHHFC hydrogel not only promotes angiogenesis \u003cem\u003ein vivo\u003c/em\u003e but also significantly enhances the liver-specific function of cells in the grafts.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.7 GHHFC protects 90% liver resected mice from liver failure\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e(B) Kaplan-Meier curve of cumulative survival rates after 90% liver resection in different groups (control group, sham surgery group, GHH group, GHHF group, GHHC group, GHHFC group). (C) H\u0026amp;E staining of the transplanted constructs/tissues at the endpoint, showing cell infiltration and tissue remodeling (scale bar: 50 \u0026micro;m). (D) Serum biochemistry of surviving rats 90 days after liver resection: albumin (ALB), alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), direct bilirubin (DBIL), total bile acids (TBA), total bilirubin (TBL), γ-glutamyl transferase (γ-GT), and total protein (TP). (E) Immunofluorescence staining of CD31 (green) and FAH (red) with DAPI nuclear staining (blue) in the transplanted area after liver resection; the GHHFC group shows the most abundant CD31⁺ vascular structures and strong FAH expression (scale bar: 50 \u0026micro;m). (F) Quantitative analysis of the density/area fraction of CD31⁺ vessels. (G) PAS staining of the transplanted area showing glycogen deposition (arrow); the strongest PAS positive signal is observed in the GHHFC group (scale bar: 50 \u0026micro;m). (H) Quantitative analysis of PAS positive area or integrated density.\u003c/p\u003e \u003cp\u003ePreoperative blood routine tests were performed on animals from each group (Figure S6). The results showed no significant differences in indicators such as WBC, Lymph, Gran, RBC, HGB, HCT, and PLT among the groups, with all values falling within the normal range. This indicates that the baseline hematopoietic function and immunological status were consistent across the different hydrogel transplant groups before undergoing the 90% liver resection surgery, providing a reliable control for the subsequent postoperative differences among the groups. Preoperative blood biochemical tests were performed on animals from each group (Figure S7), including ALT, AST, ALP, γ-GT, TBIL, DBIL, TBA, ALB, and TP. The results showed that the levels of these indicators were within similar ranges across all groups, with no significant differences observed. This suggests that prior to hydrogel transplantation, the baseline liver function status was relatively consistent among the different groups, providing a comparable starting point for assessing the improvements in survival and functional recovery after surgery.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA shows the transplantation of the hydrogel onto the mesentery of mice. After 28 days of mesenteric transplantation, 90% liver resection was performed, and follow-up results showed that animals pretreated with GHHFC had the highest survival probability (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), while the control and single-module groups (GHHF or GHHC) had relatively lower survival rates. This suggests that the dual-module preconditioning of \"oxygen generation-antioxidant\u0026thinsp;+\u0026thinsp;pro-differentiation\" helps improve survival under extreme liver resection conditions. Histologically, H\u0026amp;E staining showed cell infiltration and remodeling in all groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).Postoperative blood biochemistry results from 24\u0026ndash;48 hours further confirmed this trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD): compared to the control/sham and GHH groups, the GHHFC group showed significantly lower injury and cholestasis-related indicators such as ALT, AST, ALP, γ-GT, TBL, DBIL, and TBA, while ALB and TP were significantly higher (p-values as shown), indicating reduced hepatocyte damage, improved bile metabolism, and better maintenance of synthetic function.\u003c/p\u003e \u003cp\u003eHowever, in immunofluorescence, the GHHFC group had the most abundant and well-structured CD31-positive blood vessels, along with significantly enhanced FAH signals (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, F), consistent with its better survival and biochemical improvement. PAS staining also showed the most prominent glycogen deposition in the GHHFC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH and S8), suggesting stronger glucose storage and metabolic capacity in the graft area (and its functional coupling with the host).\u003c/p\u003e \u003cp\u003eMechanistically, it is hypothesized that CaO\u003csub\u003e2\u003c/sub\u003e@ZIF-8 continuously provides oxygen and that lignosulfonate removes excess ROS, reducing acute ischemia-reperfusion injury and oxidative stress after surgery. Meanwhile, the sustained release of FH-1 promotes the differentiation of HUCMSCs into a hepatocyte-like phenotype and enhances functional secretion/synthetic capacity. The synergistic action of both in the 3D scaffold enhances graft vascularization and perfusion potential (increased CD31), as well as liver-specific functions (increased FAH and PAS positivity), thereby providing compensatory support during the \"functional gap\" period following 90% liver resection.\u003c/p\u003e \u003cp\u003eBlood routine tests were performed on animals after the 90% liver resection to assess changes in hematopoietic and immune function (Figure S9). The results showed significant abnormalities in WBC, Gran, HGB, HCT, PLT, and other parameters in the control group and some of the single-module groups, indicating severe postoperative inflammation, hematopoietic dysfunction, and coagulopathy. In contrast, the blood parameters in the GHHFC group were closer to the normal range, with a moderate increase in white blood cell count, stable lymphocyte proportion, and maintained hemoglobin and hematocrit levels. Additionally, platelet count was significantly higher in the GHHFC group compared to the control and single-treatment groups. These results suggest that the dual-module hydrogel has advantages in improving the postoperative hematopoietic microenvironment, maintaining immune homeostasis, and alleviating postoperative inflammatory damage.\u003c/p\u003e \u003c/div\u003e"},{"header":"4 Conclusion","content":"\u003cp\u003eIn this work, a 3D-printed polysaccharide-based hydrogel scaffold integrating FH1@SA microspheres and CaO\u003csub\u003e2\u003c/sub\u003e@ZIF-8@SL nanoparticles was successfully constructed. The dual-network matrix composed of HAMA and SA provided a hydrated, ionically cross-linked framework resembling native extracellular matrix, while the incorporation of SL endowed the system with intrinsic antioxidant and redox-buffering capabilities. Through these polysaccharide-driven interactions, the scaffold achieved a balanced combination of mechanical resilience, moisture retention, and dynamic oxygen regulation.\u003c/p\u003e \u003cp\u003eThe integrated platform enabled controlled release of FH1 and sustained oxygen generation, jointly alleviating hypoxia and oxidative stress while promoting metabolic reprogramming and hepatocyte differentiation. Transcriptomic and in vivo results confirmed that this multi-polysaccharide microenvironment activated hepatic developmental and angiogenic pathways, resulting in enhanced tissue remodeling and functional recovery.\u003c/p\u003e \u003cp\u003eBy leveraging the chemical versatility and bioactivity of polysaccharides to orchestrate redox homeostasis, cell signaling, and matrix-cell crosstalk, this study provides a mechanistically informed and adaptable strategy for liver tissue regeneration. The modular design concept also lays a foundation for extending polysaccharide\u0026ndash;inorganic hybrid systems to other metabolically demanding tissues and bioartificial organ construction.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics Statements \u003c/h2\u003e\n\u003cp\u003eThis study was reviewed and approved by the Ethics Committee of the General Hospital of Ningxia Medical University. The approved project, titled \u0026quot;Application of Human Mesenchymal Stem Cell 3D Bioprinted Organoids with Slow-Release FH1 in Liver Injury Repair,\u0026quot; was granted ethical clearance. The approval number for this project is KYLL-2023-0157, and the date of approval is November 7, 2023.\u003c/p\u003e\u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\u003ch2\u003eFundings\u003c/h2\u003e \u003cp\u003eThis work was supported by grants from Science and Technology Support Project of Yinchuan City (No.2024SF045).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eSang Luo: Writing-review \u0026amp; editing, Writing-original draft, Validation, Investigation, Formal analysis, Conceptualization. Longbao Feng: Validation, Investigation. Bingren Tian: Methodology, Investigation. Fang Wu: Validation, Investigation. Shuai Xiao: Investigation, Formal analysis. Wenjun Wu: Software, Investigation. Xiaojun Yang: Supervision. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data that support the findings of this study are available upon reasonable request from the authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhou R et al. Prevention of hepatic ischemia-reperfusion injury by reactive oxygen species-responsive nanozymes. J Control Release 385, 114057. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jconrel\u003c/span\u003e\u003cspan address=\"10.1016/j.jconrel\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. 2025.114057 (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi M, et al. Nanotuner targeting mitochondrial redox and iron homeostasis imbalance for the treatment of acute liver injury. 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J Cell Mol Med. 2025;29:e70601. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/jcmm.70601\u003c/span\u003e\u003cspan address=\"10.1111/jcmm.70601\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Schemes","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"sodium alginate, sodium lignosulfonate, hydrogel, oxygen release, liver regeneration","lastPublishedDoi":"10.21203/rs.3.rs-8380365/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8380365/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA multifunctional polysaccharide-based hydrogel was developed for liver regeneration by integrating sodium alginate (SA), sodium lignosulfonate (SL), and 3D bioprinting technology. The hydrogel incorporated FH1@SA microspheres and CaO\u003csub\u003e2\u003c/sub\u003e@ZIF-8@SL nanoparticles to reconstruct the hepatic microenvironment through coordinated oxygen generation, antioxidant activity, and anti-inflammatory modulation. The FH1@SA microspheres enabled sustained release of FH1, facilitating hepatocyte adaptation to hypoxia and promoting functional recovery. Meanwhile, the CaO\u003csub\u003e2\u003c/sub\u003e@ZIF-8@SL nanoparticles achieved controlled oxygen release and pH-responsive degradation, while the lignosulfonate component scavenged reactive oxygen species and mitigated inflammatory stress. The 3D-printed hydrogel exhibited favorable mechanical strength, injectability, and cytocompatibility, effectively supporting hepatocyte proliferation and tissue regeneration. By simultaneously relieving hypoxia and oxidative stress, this polysaccharide hydrogel provides a synergistic strategy for enhancing hepatic repair, offering a promising platform for bioartificial liver construction and treatment of liver injury.\u003c/p\u003e","manuscriptTitle":"3D printed HAMA/GelMA/lignosulfonate hydrogel integrating oxygen-releasing and antioxidative modules for liver regeneration","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-26 06:37:02","doi":"10.21203/rs.3.rs-8380365/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-10T02:24:23+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-08T09:37:25+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-05T09:18:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"112731221522558887373993930987562564895","date":"2026-01-05T07:15:17+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-31T06:19:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"41705461372853056227999307771754283250","date":"2025-12-25T07:24:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"150675837034466920975744919108464652423","date":"2025-12-24T08:16:35+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-24T08:02:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-17T07:48:44+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-17T07:46:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Nanobiotechnology","date":"2025-12-17T00:53:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"566b6a65-5147-4af8-beee-103e15a54503","owner":[],"postedDate":"December 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-02T16:00:41+00:00","versionOfRecord":{"articleIdentity":"rs-8380365","link":"https://doi.org/10.1186/s12951-026-04197-5","journal":{"identity":"journal-of-nanobiotechnology","isVorOnly":false,"title":"Journal of Nanobiotechnology"},"publishedOn":"2026-02-28 15:57:24","publishedOnDateReadable":"February 28th, 2026"},"versionCreatedAt":"2025-12-26 06:37:02","video":"","vorDoi":"10.1186/s12951-026-04197-5","vorDoiUrl":"https://doi.org/10.1186/s12951-026-04197-5","workflowStages":[]},"version":"v1","identity":"rs-8380365","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8380365","identity":"rs-8380365","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}