Phase-separated hydrogels for advanced biomedical engineering: From material design to applications.

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Learn more: PMC Disclaimer | PMC Copyright Notice Mater Today Bio . 2026 Apr 3;38:103096. doi: 10.1016/j.mtbio.2026.103096 Search in PMC Search in PubMed View in NLM Catalog Add to search Phase-separated hydrogels for advanced biomedical engineering: From material design to applications Junpeng Mu Junpeng Mu a Research Institute of General Surgery, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, 210093, China Find articles by Junpeng Mu a, 1 , Sha Li Sha Li b Nanjing University of Chinese Medicine, Nanjing, 210023, China Find articles by Sha Li b, 1 , Zherui Zhang Zherui Zhang a Research Institute of General Surgery, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, 210093, China Find articles by Zherui Zhang a, 1 , Ze Li Ze Li a Research Institute of General Surgery, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, 210093, China Find articles by Ze Li a , Sicheng Li Sicheng Li a Research Institute of General Surgery, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, 210093, China Find articles by Sicheng Li a , Zongan Li Zongan Li c Jiangsu Key Laboratory of 3D Printing Equipment and Manufacturing, NARI School of Electrical and Automation Engineering, Nanjing Normal University, Nanjing, 210042, China Find articles by Zongan Li c , Haohui Li Haohui Li b Nanjing University of Chinese Medicine, Nanjing, 210023, China Find articles by Haohui Li b , Lili Yu Lili Yu b Nanjing University of Chinese Medicine, Nanjing, 210023, China Find articles by Lili Yu b , Huajian Ren Huajian Ren a Research Institute of General Surgery, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, 210093, China Find articles by Huajian Ren a, ⁎ , Jianan Ren Jianan Ren a Research Institute of General Surgery, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, 210093, China b Nanjing University of Chinese Medicine, Nanjing, 210023, China Find articles by Jianan Ren a, b, ⁎⁎ , Xiuwen Wu Xiuwen Wu a Research Institute of General Surgery, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, 210093, China b Nanjing University of Chinese Medicine, Nanjing, 210023, China Find articles by Xiuwen Wu a, b, ⁎⁎⁎ , Jinjian Huang Jinjian Huang a Research Institute of General Surgery, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, 210093, China b Nanjing University of Chinese Medicine, Nanjing, 210023, China Find articles by Jinjian Huang a, b, ⁎⁎⁎⁎ Author information Article notes Copyright and License information a Research Institute of General Surgery, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, 210093, China b Nanjing University of Chinese Medicine, Nanjing, 210023, China c Jiangsu Key Laboratory of 3D Printing Equipment and Manufacturing, NARI School of Electrical and Automation Engineering, Nanjing Normal University, Nanjing, 210042, China ⁎ Corresponding author. Research Institute of General Surgery, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, 210093, China. [email protected] ⁎⁎ Corresponding author. Research Institute of General Surgery, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, 210093, China. [email protected] ⁎⁎⁎ Corresponding author. Research Institute of General Surgery, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, 210093, China. [email protected] ⁎⁎⁎⁎ Corresponding author. Research Institute of General Surgery, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, 210093, China. [email protected] 1 These authors contribute to this paper equally. Received 2025 Dec 2; Revised 2026 Mar 28; Accepted 2026 Apr 2; Collection date 2026 Jun. © 2026 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). PMC Copyright notice PMCID: PMC13090737  PMID: 42006717 Abstract Hydrogels are extensively employed in wound healing, drug delivery, and the manufacturing of medical devices due to their distinctive physical and chemical properties and biocompatibility. The development of manufacturing platforms such as bioprinting and microfluidics has further advanced the clinical application of hydrogels, improving precision, efficiency, and cost-effectiveness. However, the constrained mechanical and biological properties of hydrogels have hindered their broader use, prompting research into new modification methods. Phase separation is one promising way to address this problem. Here, the mechanisms of phase separation and the recent progress in the fabrication of phase-separated hydrogels is reviewed to elucidate the relationship between hydrogel properties and phase separation. This review subsequently highlights four main biomedical applications of phase-separated hydrogels, including tissue engineering, drug delivery, biosensing, and bioprinting. Finally, the challenges and future prospects of phase-separated hydrogels in the biomedical field are discussed, offering new insights for further improvements and exploration of novel directions. Keywords: Hydrogel, Phase separation, Biomedical engineering, Bioprinting, Regenerative medicine Graphical abstract Open in a new tab 1. Introduction The combination of materials science and medicine is a significant force driving progress in biomedical engineering, where the development and application of novel materials continue to propel innovations in areas such as diagnosis, treatment, and tissue repair. Among these materials, hydrogels are polymeric substances with a three-dimensional (3D) network structure capable of absorbing and retaining large amounts of water. This characteristic allows them to mimic the microenvironment of the natural extracellular matrix (ECM), creating ideal conditions for cell growth and differentiation [ 1 ]. Moreover, their soft and moist properties closely resemble those of biological tissues, while their tunable degradability and functional diversity make them ideal candidates for implants and in vitro models [ 2 ]. Consequently, since their introduction in the 1960s [ 3 ], hydrogels have been widely used in drug delivery, wound dressings, tissue engineering, and smart sensors, among other fields. The application of hydrogels in the biomedical field relies on various advanced platforms, including bioprinting, microfluidic technology, electrospinning, and others. For instance, bioprinting, a crucial branch of 3D printing, is responsible for fabricating tissue scaffolds, artificial organs, and medical devices [ 4 ]. The combination of hydrogels and bioprinting technology is a key research direction in the field of biomaterials. By optimizing parameters such as printing speed, nozzle diameter, and layer thickness, researchers can regulate the porosity, mechanical strength, and morphological structure of hydrogel scaffolds, thereby enhancing their biological performance [ 5 , 6 ]. Compared to traditional methods like mold casting, which suffer from inefficiency and inconsistency, bioprinting technology can efficiently construct complex structures such as porous networks and vascularized channels, effectively overcoming these restrictions [ 5 ]. However, the manufacturing of hydrogels faces numerous challenges in practical applications. Firstly, the mechanical properties of hydrogels are often insufficient (e.g., low strength, susceptibility to deformation), making it difficult to simultaneously meet the structural stability requirements during production and the mechanical demands after implantation [ 7 ]. Secondly, manufacturing precision and resolution are constrained by the material's mechanical properties, such as viscosity and elasticity, constraining the fabrication of complex structures [ 8 ]. In addition, to adapt to specific application scenarios, it is often necessary to optimize the biological functions of hydrogels (e.g., cell adhesion, controlled drug release). Among various modification strategies, phase separation technology has garnered significant attention due to its unique advantages. This method can optimize both the bulk properties of the material and its compatibility with manufacturing platforms. For example, phase separation technology can achieve high-resolution bioprinting by improving printability [ 9 ], and enable the one-step formation of high-throughput, monodisperse hydrogel microspheres in microfluidics [ 10 ]. Therefore, the potential of phase separation technology in hydrogel production within the field of biomedical engineering deserves further exploration. In this review, we systematically explore the role of phase-separated hydrogels in biomedical engineering. First, based on recent studies, we analyze the interaction mechanisms between phase separation and hydrogels, focusing on the commonly used raw materials, process parameters, and formed structures for manufacturing phase-separated hydrogels. Next, we comprehensively elaborate on the latest advancements in phase-separated hydrogels in the field of biomedical application, and the discussion covers four aspects, including tissue engineering, drug delivery, biosensing, and bioprinting. Prior to elaborating on these two sections, detailed introductions to the thermodynamic mechanisms and kinetic mechanisms of phase separation are also provided respectively to facilitate understanding. In summary, through a systematic review of this research area, we aim to inspire new ideas for the deep integration of bioprinting and phase-separated hydrogels and uncover further potential value. 2. Overview of phase separation 2.1. History of phase separation Phase separation refers to the process by which an initially homogeneous mixture spontaneously divides into two or more compositionally distinct and immiscible phases. The study of phase separation dates back to observations in liquid mixtures in the early 20th century [ 11 ]. By 1950-1960, scholars conducted extensive explorations on the phase behavior of polymer solutions and gels [ 12 ], and further confirmed the existence of this phenomenon during the protein crystallization process in the 1970s [ 13 , 14 ]. At the theoretical level, the physical model of polymers represented by the Flory-Huggins theory provides a thermodynamic and kinetic basis for understanding the micro/nano structures formed when phase separation occurs in multi-component systems [ 15 ]. These theories not only laid the basic rules for the formation of material structures, but also became the theoretical source for the design of functional materials such as porous scaffolds and smart hydrogel. In 2009, Hyman's team found that the dynamic behavior of P particles in Caenorhabditis elegans was related to liquid-liquid phase separation (LLPS). This breakthrough introduced the concept of LLPS into cell biology and revealed that it may be a universal physicochemical mechanism for the formation of a variety of membraneless organelles, thus stimulating the research boom in this field [ 16 ]. It also showed materials scientists that living systems can achieve precise programming of intracellular microregions by regulating the interactions and "valence" of biological macromolecules. In 2015, one study further revealed that pathological protein fibrillization in neurodegenerative diseases can be driven by aberrant liquid-liquid phase separation, which leads to the formation of solid, pathological inclusions [ 17 ]. This finding indicates that our understanding of disease mechanisms has evolved from "chemical abnormalities" to "physical state disorders," namely, the incorrect transformation of intracellular biomaterials from a functional liquid to a pathogenic solid gel, a process highly consistent with the polymer sol-gel transition. 2.2. Mechanism of phase separation Phase separation is commonly observed in polymer systems, colloidal suspensions, and biomacromolecular assemblies. In materials science, LLPS and solid-liquid phase separation are key mechanisms for regulating the microstructures of materials [ 18 , 19 ]. Phase separation in polymer gels is a spontaneous evolution of a system from a homogeneous state to a heterogeneous structure, driven by thermodynamic principles and governed by complex kinetic paths. 2.2.1. Thermodynamic driving forces At the thermodynamic level, the fundamental driving force for phase separation stems from the thermodynamic pursuit of minimizing the Gibbs free energy (Δ G ) of the system [ 20 ]. The enthalpy term (Δ H ) in the free energy change (Δ G = Δ H - T Δ S ) competes with the entropy term (- T Δ S ) [ 21 ]. The entropy term (- T Δ S ) tends to promote molecular mixing to increase the disorder degree of the system, while the enthalpy term (Δ H ) reflects the polymer-solvent interaction energy. On this basis, the Flory-Huggins solution theory provides a more rigorous quantitative framework for the thermodynamic principles of phase separation [ 15 ]. The free energy of mixing per lattice site is expressed as: Δ G m i x k B T = Φ N ln Φ + ( 1 − Φ ) ln ( 1 − Φ ) + χ Φ ( 1 − Φ ) , where k B is the Boltzmann constant, T is the absolute temperature, Φ is the polymer volume fraction, N is the degree of polymerization, and χ is the Flory-Huggins interaction parameter. The first two terms represent the combinatorial entropy favoring mixing, while the third term represents the enthalpic contribution governed by polymer-solvent interactions [ 22 , 23 ]. Experimentally, the occurrence of phase separation can be controlled by tuning χ . For example, in poly (N-isopropylacrylamide) (PNIPAM) hydrogels, χ increases with temperature due to the hydrophobic effect. Heating the system above its Lower Critical Solution Temperature (LCST, approximately 32 °C) shifts the system into the two-phase region [ 24 , 25 ], which is important for forming pores in hydrogels. 2.2.2. Kinetic mechanisms At the kinetic level, the final porous morphology of hydrogels is primarily governed by the path and rate of phase separation. This process primarily occurs through nucleation and growth (NG) and spinodal decomposition (SD) [ 26 ], which exhibit distinct characteristics ( Table 1 ), such as their formation mechanisms and morphological evolution [ [27] , [28] , [29] ]. In the NG mechanism, nuclei of the new phase are formed through random concentration fluctuations. To initiate phase separation, the initial stage must overcome an energy barrier, which manifests as a large-amplitude local concentration fluctuation. Subsequently, the new phase gradually grows and coalesces by consuming the surrounding parent phase. This process typically leads to the formation of discrete, granular or droplet-like phase domain morphologies. It is noteworthy that NG can be categorized into homogeneous (bulk initiation) and heterogeneous (surface-assisted). As illustrated in Fig. 1 a, Heterogeneous nucleation dominates practical systems by leveraging preexisting interfaces to significantly reduce the thermodynamic energy barrier. By contrast, the system of SD does not have to overcome a nucleation barrier, and concentration fluctuations (initially of small amplitude) with a characteristic fixed wave length are spontaneously amplified, which leads to the rapid decomposition of the system into two interpenetrating continuous phases. Table 1. Comparison between nucleation and growth and spinodal decomposition. Feature Nucleation and growth Spinodal decomposition Refs Thermodynamic Region Metastable (between binodal and spinodal) Unstable (inside spinodal curve) [ 18 , 27 ] Concentration Evolution Large amplitude initially Small amplitude initially, fixed wavelength [ [27] , [28] , [29] ] Energy Barrier Requires overcoming nucleation barrier Barrier free (spontaneous) [ [27] , [28] , [29] ] Typical Morphology Discrete, spherical droplets ("Sea-Island") Bicontinuous, interpenetrating networks ("Worm-like") [ 30 ] Scattering Signature Monotonic decay and no distinct peak initially Distinct correlation peak ("spinodal ring") [ 31 , 32 ] Key Tunable Parameter Shallow quench Deep quench [ 29 , 33 ] Open in a new tab Fig. 1. Open in a new tab The kinetic mechanisms of phase separation mainly include nucleation-growth, spinodal decomposition, and destabilization processes. (a) Nucleation-growth diagram. (b) Schematic phase diagram and typical morphologies of the two kinetic mechanisms. Reproduced with permission [ 18 ]. 2019, CELL. (c) Four destabilization processes during phase separation of hydrogels, with Ostwald ripening and coalescence being the most prominent. Created with Biorender.com . Morphologically, NG is marked by the formation of discrete, spherical droplets (“sea-island” morphology), which increase in size over time while maintaining a constant composition. By comparison, SD produces a bicontinuous, "worm-like" interconnected structure [ 30 ]. These mechanisms can also be rigorously distinguished using scattering techniques (eg., small-angle X-ray/neutron scattering). SD tends to exhibit a distinct and often sharp correlation peak known as the "spinodal ring", which shifts to lower scattering vectors as the domains coarsen. In contrast, NG usually shows a monotonic decay without a characteristic peak in the early stages of phase separation [ 31 , 32 ]. Phase diagrams, serving as a classical representation of the interplay between thermodynamics and kinetics, provide a critical framework for understanding phase separation pathways. In the thermodynamic phase diagram characterizing binary mixtures, the ordinate typically represents temperature, while the abscissa represents the composition or volume fraction of the components. This plot delineates the stability limits of the system using two critical boundaries: the binodal and spinodal curves [ 18 , 27 ]. The region located between these two curves defines the metastable state, where phase separation proceeds via NG. In contrast, the region enclosed within the spinodal curve corresponds to the unstable state, characterized by spontaneous SD ( Fig. 1 b). Rational control of parameters can guide the system into different regions of the phase diagram. For example, shallow quenching into the metastable region ( T spinodal < T < T binodal ) favors NG into isolated precipitates, while deep, rapid quenching into the unstable region ( T < T spinodal ) enables direct SD, forming bicontinuous structures [ 29 , 33 ]. While thermodynamics defines the boundary conditions for miscibility, the final morphological outcome is increasingly recognized to be governed by kinetic control. Recent efforts have demonstrated that kinetic control serves as a powerful approach to tune hydrogel architecture without altering chemical composition [ [34] , [35] , [36] ]. By modulating the photopolymerization rate, specifically through adjustments in light intensity, the phase separation process can be arrested at precise time points. This kinetic trapping allows for the programmable tuning of pore sizes ranging from micrometers to hundreds of micrometers and the fabrication of hierarchical, multi-scale porosities essential for engineered vasculature and cell guidance. In addition, the two kinetic mechanisms of phase separation can also be influenced by the competition between deformation and relaxation rates. To be specific, polymerization or crosslinking reduces diffusion and prolongs relaxation. When relaxation is slower than deformation, elastic forces dominate over interfacial tension, arresting phase separation and leading to stable network or sponge-like structures [ 37 ]. Subsequently, the phase domain undergoes coarsening primarily through the Ostwald ripening or droplet coalescence. Ostwald ripening is a thermodynamic, diffusion-limited process where larger droplets grow at the expense of smaller ones, whereas coalescence involves the fusion of droplets following collisions driven by Brownian motion. While Ostwald ripening often characterizes dilute regimes, coalescence typically dominates in systems with higher volume fractions, leading to accelerated phase growth [ 33 , 38 , 39 ]. These coarsening phenomena represent significant drivers of system instability. Additionally, flocculation and sedimentation serve as alternative destabilization pathways. The former involves the aggregation of droplets into clusters without fusion, while the latter is characterized by the gravitational settling of the dispersed phase driven by density contrasts. The distinct mechanisms of these four destabilization processes are summarized in Fig. 1 c. In brief, phase separation in polymer gels is the result of competition and coupling between thermodynamic driving forces and kinetic paths. Thermodynamics provides the internal driving force and end point for phase separation, while kinetics describes the specific path to the end point, and ultimately determines the scale and morphology of phase separation structure, so as to realize the precise regulation of material properties. 3. Phase-separated hydrogels At the end of the last century, the introduction of phase separation principles brought renewed significance to hydrogels [ 40 ]. These hydrogels are porous polymeric networks formed through phase separation mechanisms. Crucially, their structural features, including pore size, porosity, and mechanical properties, can be precisely tailored by modulating phase separation parameters such as polymer concentration, crosslinking density, and solvent choice [ 41 ]. Unlike traditional homogeneous hydrogels, their heterogeneous porous architecture can effectively mimic the topological characteristics of the natural ECM, thereby promoting cell adhesion, proliferation, and mass transport [ 42 ]. Among the various mechanisms driving these architectures, LLPS has emerged as a particularly versatile strategy. Driven by thermodynamic instabilities, which often arise from complex coacervation or segregative incompatibility between polymers, LLPS generates a polymer-dense phase enriched with solutes and a polymer-dilute phase [ 27 , 43 , 44 ]. This dynamic demixing process allows for the spontaneous formation of membrane-less compartments that can sequester specific biomolecules, creating localized micro-reactors within the hydrogel matrix. The transient nature of the interactions within the dense phase imparts unique viscoelastic properties, enabling the hydrogels to dissipate energy efficiently while maintaining structural integrity under physiological conditions [ 27 , 45 ]. Beyond constructing heterogeneous architectures, the phase separation process endows hydrogels with crucial physicochemical properties, which include temperature responsiveness, injectability, and self-healing capabilities, to these hydrogels, demonstrating their significant potential for biomedical applications [ [46] , [47] , [48] ]. Collectively, this section focuses on the common effects of phase separation in terms of material selection, process parameters, and structural construction ( Fig. 2 ), which facilitates the understanding of the role of phase-separated hydrogels in biomedical applications in subsequent sections. Fig. 2. Open in a new tab Common raw materials, process parameters, and formation results for fabricating phase-separated hydrogels (PVA, polyvinyl alcohol; PEG, polyethylene glycol; SA, sodium alginate; CS, chitosan; PNIPAM, poly (N-isopropylacrylamide); TIPS, thermally induced phase separation; LCST, lower critical solution temperature; UCST, Upper Critical Solution Temperature; UV, ultraviolet). Created with Biorender.com . 3.1. Material selection To design excellent phase-separated hydrogels, the first step is to identify suitable polymer materials. Therefore, this section reviews six commonly used polymers related to phase-separated hydrogels, specifically their characteristics and application directions when combined with phase separation ( Table 2 ). Additionally, the role of 3D printing in these polymers is also discussed separately, as it enables precise control over structure and functionality. Table 2. Polymers commonly employed in hydrogel phase separation. Material Chemical structure Advantages Applications 3D printing Refs PVA Non-toxicity Biodegradability Hydrophilicity Thickening Wound dressing Mechanical enhancement Hydrogel matrix Sacrificial material rheological modifier High printability High cell viability [ [49] , [50] , [51] , [52] , [53] ] PEG High water solubility Pore-forming agent Hydrogel matrix Hydrogel shaping Pore formation 4D printing High printability [ [54] , [55] , [56] , [57] , [58] , [59] ] Gelatin Degradability High modifiability Cell adhesion Hydrogel modification Cell encapsulation Interpenetrating network High printability Matrix material [ 53 , [60] , [61] , [62] , [63] ] CS Antibacterial Positively charged High modifiability Hydrophobicity Wound dressing Hydrogel modification 4D printing High printability [ [64] , [65] , [66] ] SA Metal-ion affinity Rapid crosslinking Lipase detection Cell encapsulation Drug delivery Cell encapsulation Molecular encapsulation [ [67] , [68] , [69] , [70] ] PNIPAM Thermosensitivity Reversible phase separation Drug delivery Mechanical enhancement 4D printing [ [71] , [72] , [73] , [74] , [75] ] Open in a new tab 3.1.1. Polyvinyl alcohol (PVA) Polyvinyl alcohol (PVA) is widely used as a hydrogel matrix material owing to its biodegradability, non-toxicity, biocompatibility, and hydrophilicity [ 49 ]. However, the broader application of pure PVA hydrogels faces challenges due to their inadequate antibacterial activity and mechanical strength [ 76 ]. To address this, researchers have constructed composite hydrogels with oriented structures for synergistic enhancement. Lan et al. [ 50 ], for example, combined PVA with chitosan (CS) and polyethylene glycol (PEG). Using a bidirectional freezing method with phase separation, they fabricated a macroporous PVA/CS hydrogel with PEG as a pore-forming agent, and the resulting pore size was approximately 5 μm. Subsequent infusion of this scaffold with nanofillers (derived from reduced graphene oxide, polydopamine, and zeolite imidazolate framework-8) produced an antibacterial dressing that effectively promotes wound healing ( Fig. 3 a). Fig. 3. Open in a new tab Six common polymers used to create phase-separation hydrogels, including PVA, PEG, gelatin, CS, SA, and PNIPAM. (a) Schematic illustration of the synthetic route for rGO-PDA@ZIF-8/PVA/CS hydrogel. Reproduced with permission [ 50 ]. 2024, ELSEVIER. (b) PEG/gelatin polymer 3D microparticles via induced phase separation and applications. Reproduced with permission [ 56 ]. 2021, American Chemical Society. (c) Schematic diagram of the h-GelMA hydrogel crosslinking mechanism based on the PTPC reaction and the traditional free radical polymerization-based mechanism for GelMA hydrogel. Reproduced with permission [ 62 ]. 2025, John Wiley & Sons Inc. (d) Comparison of drug loading and release kinetics between CS hydrogel and aerogel/cryogel forms of dexamethasone sodium phosphate Reproduced with permission [ 77 ]. 2023, American Chemical Society. (e) Schematic diagram of an alginate-based paper sensor detecting lipase through viscosity changes induced by phase separation. Reproduced with permission [ 69 ]. 2022, American Chemical Society. (f) Composite porous structure of PNIPAM ((I) PNIPAM monomer molecular structure. Nanochannel top (II) and side (III) views. (IV) Water-gel composite system at 275 K) Reproduced with permission [ 71 ]. 2024, American Chemical Society. (rGO, reduced graphene oxide; PDA, polydopamine; ZIF-8, zeolite imidazolate framework-8; PVA, polyvinyl alcohol; CS, chitosan; PEG, polyethylene glycol; 3D, three-dimensional; UV, ultraviolet; PTPC, persistent-radical coupling; GelMA, gelatin methacrylamide; h-GelMA, high-performance GelMA; GelNB, o-nitrobenzyl alcohol modified gelatin; PNIPAM, poly (N-isopropylacrylamide)). Meanwhile, in a study focused on mechanics, Ma et al. [ 51 ] employed a synergistic "freeze-concentration–annealing crystallization" phase separation to uniformly immobilize flexible SiO 2 nanofibers within a PVA matrix, resulting in the formation of dense crystalline domains and a highly entangled network. This approach simultaneously improved strength, toughness, stiffness, and elasticity, thus establishing a versatile design paradigm for creating high-strength, tough, and recoverable hydrogels, such as artificial ligaments. In another investigation, Liu et al. [ 52 ] adopted an alternative strategy by immersing PVA xerogels in a kosmotropic anions solution to induce hydrophobic aggregation and facilitate the phase separation of PVA polymer chains through a salting-out effect. This process rapidly generated nanoscale crystalline domains, which served as physical cross-linking points to enhance mechanical performance. Consequently, this method enables the fabrication of hydrogels with tunable mechanical properties that can effectively address diverse medical requirements. In phase-separated hydrogel bioprinting, it primarily serves as a sacrificial material or rheological modifier, where its excellent thickening property helps stabilize the oil-water interface and enable the formation of intricate structures. For instance, Maciel et al. [ 53 ] observed that a mixture of PVA and gelatin undergoes microphase separation upon cooling, causing the formation of PVA-rich liquid inclusions within an elastic gelatin network. These inclusions act as a lubricating phase, promoting wall slip during extrusion, thereby reducing shear stress on encapsulated cells and improving both print resolution and cell viability. Comparable phase separation can also be induced by mixing nanoclay laponite with the gel, which creates dense microdomains that similarly reduce cellular shear damage and enhance printing quality. 3.1.2. Polyethylene glycol (PEG) Valued for​ its good water solubility and biocompatibility, Poly (ethylene glycol) (PEG) is also a common hydrogel matrix material [ 54 ]. This polymer's water solubility also enables flexible structural control via aqueous two-phase system (ATPS), which is a vital type of LLPS and usually formed by mixing incompatible polymers or a polymer and a salt [ 20 , 55 , 78 ]. Lee et al. [ 56 ] achieved a temperature-responsive PEG-gelatin ATPS using a parallel stepped emulsification device under low-temperature induction. As a photocrosslinkable backbone, PEG provided structural stability to the particles and allowed precise control over internal cavity morphology and surface functionalization by regulating interfacial tension and interactions with gelatin ( Fig. 3 b). Using this approach, the researchers achieved the efficient production of over 40 million particles per hour while maintaining minimal cross-interference in single-cell secretion assays. Similarly, Cheng et al. [ 55 ] utilized a PEG-dextran (PEG-DEX) ATPS on superhydrophobic surfaces to produce morphologically programmable Janus droplets in one step, without organic solvents. Furthermore, PEG serves as an excellent porogen. Roudsari et al. [ 57 ] found that low-molecular-weight PEG200 migrates out of the hydrogel matrix during phase separation, forming numerous mesopores (2–20 nm) alongside limited micropores. This significantly increases porosity and specific surface area, suggesting a potential strategy for improving mass transfer efficiency in bioseparation. In neuroscience, Broguiere et al. [ 58 ] rapidly formed transparent macroporous hydrogels with tunable pore sizes (0.5–50 μm) via dynamic phase separation between PEG and high-molecular-weight polysaccharides under physiological conditions. In a rat sciatic nerve defect model, this system maintained a stable 3D electroactive network without adhesive peptides and achieved approximately 70% of the autograft level of axon regeneration. In 3D printed phase-separated hydrogels, PEG typically forms the continuous phase. By tuning its compatibility with other components, PEG can induce microphase separation, a process that significantly enhances the hydrogel's mechanical strength and toughness. This is exemplified by the work of Wang et al. [ 59 ], who used PEG as a good solvent for the polymerizing poly (2-hydroxyethyl methacrylate) network. When combined with poly (propylene glycol), this formulation triggers in situ phase separation, creating a micro-scale soft-rigid phase-separated structure. This structure results in exceptional strength (8.0 MPa), toughness (17.0 MJ m −3 ), and shape memory properties, enabling the four-dimensional (4D) printing of hydrogels with high printability and mechanical performance. 3.1.3. Gelatin As a collagen-based biomaterial, gelatin is derived from animal skin, bones, tendons, and connective tissues, and frequently serves as a key modifier in the fabrication of phase-separated hydrogels [ 60 ]. Its excellent biocompatibility and cell adhesiveness (e.g., via RGD sequences) make it ideal for cell encapsulation, while its multiple modifiable groups and thermoreversible gelation provide broad scope for hydrogel customization [ 61 ]. In the bioprinting study by Maciel et al. [ 53 ] mentioned earlier, gelatin acted as the primary matrix. During gelation, its interactions with PVA or nanoclay (Laponite) induced microphase separation, yielding controlled microheterogeneous structures. These structures significantly improved printability by mitigating issues like wall slip and brittle fracture. Interestingly, Ren et al. [ 62 ] successfully printed a pure gelatin hydrogel with phase-separated characteristics. They achieved this by chemically modifying gelatin with methacryloyl (gelatin methacrylamide, GelMA) and o-nitrobenzyl groups, then using a photo-triggered transient-to-persistent radical coupling reaction. This process induced microphase separation and strengthened the interfacial bonding, significantly enhancing the mechanical properties ( Fig. 3 c) while fully retaining the inherent biocompatibility, cell adhesion, and degradability. However, gelatin's applicability is constrained by its inherent properties. As noted by Van Nieuwenhove et al. [ 79 ], pure gelatin hydrogels possess a narrow range of mechanical strength due to their lack of polysaccharide components. The combination of starch (representing polysaccharides) and gelatin (representing proteins) can form an interpenetrating network that better mimics the dual chemical composition of natural ECM. During this process, phase separation leads starch to form isolated microdomains. Although this may slightly reduce the gel fraction and mechanical performance, it also allows fine-tuning of physicochemical properties such as swelling ratio and stiffness. Another inherent characteristic of gelatin hydrogels is their dense network structure and small pore size, which can hinder cell migration and vascularization. To address this, Nishiguchi et al. [ 63 ] chemically modified tendon-derived gelatin by introducing hydrogen-bonding units (UPy). Using LLPS, they constructed a microporous hydrogel with a microfiber network, providing a superior 3D environment for cell migration and vascular ingrowth. 3.1.4. Chitosan (CS) Chitosan (CS) is widely used in wound repair hydrogels. Its inherent cationic nature provides broad-spectrum antibacterial activity by disrupting microbial cell membranes [ 64 ]. Furthermore, its abundant reactive functional groups—such as amino and hydroxyl groups—make it highly amenable to chemical modification [ 80 ]. Combining this chemical versatility with phase separation strategies significantly broadens the scope for tailoring high-performance CS-based hydrogels. For instance, Bankoti et al. [ 65 ] blended CS with an aqueous polyurethane diol dispersion (PUD). Inter- and intramolecular hydrogen bonding between the amino/hydroxyl groups of CS and the hydroxyls of PUD induced phase separation. This led to the self-assembly of a macroporous hydrogel scaffold upon drying at room temperature. Serving as a structural matrix, CS not only conferred significant antibacterial properties but also, through this dynamic hydrogen-bond network, effectively modulated the material's swelling behavior, degradation rate, and protein adsorption capacity. These features collectively contributed to accelerated healing of full-thickness skin wounds in vivo. CS's superior modifiability also extends to 3D-printed phase-separated hydrogels. As demonstrated by Zhang et al. [ 66 ], CS chains can form hydrogen bonds with poly (acrylamide-co-acrylic acid) chains and simultaneously coordinate with Zr 4+ ions to create dynamic metal-coordination bonds. This hybrid dual-network structure enhances the hydrogel's mechanical strength, toughness, and self-recovery, while also promoting rapid solvent-responsive behavior during phase separation. This enables programmable actuation in 3D-printed shape-memory hydrogels. However, the inherent insolubility of CS in water necessitates solvent considerations. To address this, Chartier et al. [ 77 ] employed a non-solvent induced phase separation (NIPS) method, using a 4 M NaOH aqueous solution to induce phase separation in a CS solution, forming a stable 3D network hydrogel ( Fig. 3 d). This NIPS process, which avoids chemical crosslinkers, serves as a versatile precursor for aerogels and cryogels, ensuring safety for biomedical applications. The NIPS strategy is also evident in the work of Kang et al. [ 81 ], who used tetrahydrofuran as a non-solvent with chloroform to prepare a poly (3-hydroxybutyrate) organogel. This approach circumvented the need for rare and expensive solvents traditionally required for poly (3-hydroxybutyrate)/CS composites. Combined with a solvent exchange process, they successfully loaded CS into the PHB hydrogel, achieving a low-cost and efficient method for composite preparation. 3.1.5. Sodium alginate (SA) Alginate chains contain abundant carboxyl groups, which form an "egg-box" cross-linked network via ionic interactions with Ca 2+ and other divalent metal ions (e.g., Mg 2+ ) [ 67 ]. This rapid gelation avoids high temperatures or harmful reactions and stabilizes 3D-printed structures. However, SA hydrogel precursor solutions are typically low in viscosity and lack stackability, hindering printability. To address this, Zhou et al. [ 68 ] reported that ethanol disrupts the hydrogen-bonding network in SA aqueous solutions, inducing local dehydration. At ethanol concentrations above 25% and temperatures below a critical transition point, the mixture forms a thermosensitive printable ink. This ink exhibits low viscosity at room temperature for easy extrusion, rapid gelation on a cooled print bed, shear-thinning behavior, and high yield stress to maintain structural stability. After Ca 2+ cross-linking and ethanol removal, the printed structure undergoes approximately 34% isotropic shrinkage, significantly improving resolution (>10%). However, at ethanol concentrations ≥40%, excessive dehydration causes SA chain aggregation and macroscopic phase separation, leading to instability and turbidity. The rapid cross-linking between alginate and metal cations also inspires innovations in relevant platforms. For example, Xia et al. [ 69 ] developed a lipase sensor based on lipase-catalyzed triglyceride hydrolysis, producing oleic acid. The oleic acid binds with calcium ions to form calcium oleate particles, while excess Ca 2+ induces "egg-box" gelation of alginate, triggering phase separation. Lipase concentration is quantified by measuring the flow distance of the upper aqueous phase on pH paper, achieving a detection limit of 0.052 U/mL. This low-cost, instrument-free sensor shows potential for point-of-care diagnosis of acute pancreatitis ( Fig. 3 e). Additionally, Tokarev et al. [ 70 ] developed a stimuli-responsive nanoporous film by blending alginate with amine-terminated PEG. During spin-coating, the polymer blend phase-separates into a 3D fibrous network. Cross-linking alginate with Ca 2+ and removing PEG yielded a pH-responsive hydrogel membrane. This membrane exhibits reversible pore opening/closing around pH 4.5, with applications in smart filtration and controlled drug release. Additionally, the average pore diameters are tunable from 74 nm to 170 nm depending on the polymer ratio. 3.1.6. Poly (N-isopropylacrylamide) (PNIPAM) The most prominent advantage of PNIPAM is its sensitive thermoresponsiveness. Below the LCST, PNIPAM chains remain hydrophilic and swollen through hydrogen bonding between amide groups and water molecules. Above the LCST, hydrogen bonds are disrupted, hydrophobic interactions dominate, and the chains dehydrate and collapse, enabling rapid sol–gel transition [ 72 ]. By using molecular dynamics simulations, Chen et al. [ 71 ] demonstrated that above the LCST, PNIPAM chains inside nanochannels undergo phase separation, dehydration, and contraction, leading to reduced hydrogen bonding and accelerated desorption of water molecules. External heat flow intensifies this effect, promoting the release of hydrated water into the bulk phase and forming a distinct water layer ( Fig. 3 f). It is noteworthy that PNIPAM exhibits a LCST around 32 °C, close to human body temperature [ 72 ]. This property enables drug loading into the swollen polymer at lower temperatures. When the temperature rises near body temperature, PNIPAM undergoes a phase transition and contracts, forming a barrier that slows drug release while simultaneously exerting a squeezing effect that may facilitate rapid release. This dual mechanism highlights its significant potential for controlled release applications. According to Lee et al. [ 73 ], PNIPAM was incorporated into polymer vesicles formed by poly (ethylene glycol)-b-poly (D,L-lactide) block copolymers. At 37 °C, PNIPAM underwent phase separation, partially transforming into a hydrogel phase enriched near the vesicle membrane, creating an additional barrier. This membrane-associated hydrogel structure effectively slowed the diffusion of the model drug FITC-DEX, extending its release duration to 30 days while reducing the initial burst release. In contrast, He et al. [ 74 ] utilized microfluidics to prepare thermoresponsive PNIPAM microcapsules. When heated above the volume phase transition temperature, the capsule shells rapidly contracted, expelling encapsulated lipophilic drugs within 4–6 min. Upon cooling, the shells re-expanded within 2–3 min, enabling drug re-uptake and achieving reversible rapid release. Release performance can be optimized by adjusting the monomer-to-crosslinker ratio to control crosslinking density. As a temperature-sensitive polymer, PNIPAM also enables programmable shape morphing in 4D printed phase-separation hydrogels. Liu et al. [ 75 ] combined polymerization-induced phase separation (PIPS) with 3D printing to fabricate hierarchically porous PNIPAM hydrogels with enhanced thermal response speed. When ethylene glycol was used as the solvent, its poor compatibility with PNIPAM monomers induced strong phase separation, yielding a microstructure with high porosity and thin polymer walls. This facilitated rapid water diffusion, resulting in significantly faster swelling/deswelling rates in 25 °C water. Moreover, the synergy between 3D-printed macropores and PIPS-generated submicron pores formed interconnected water channels. Without sacrificing mechanical strength, the hydrogel achieved 45.5% volumetric change within 10 s and exhibited programmable anisotropic actuation. Therefore, the use of this hydrogel enabled​ 4D printing via the 3D printing process. 3.2. Adjustment of process parameters The previous section has reviewed polymer materials closely associated with hydrogel phase separation. However, intermediate factors such as temperature, concentration, and crosslinking density also significantly influence phase separation behavior during hydrogel formation. Therefore, this section will focus on the significance of process parameter regulation in preparing phase-separated hydrogels. 3.2.1. Thermally induced phase separation (TIPS) Thermally induced phase separation (TIPS) refers to the phase separation of a polymer solution due to temperature changes. In TIPS, the polymer solution reaches a critical temperature through cooling or heating, causing the polymer and solvent to separate into distinct phases. Typically, TIPS is an entropy-driven process. Elevated temperatures enhance hydrophobic interactions (entropy gain) and disrupt polymer-water hydrogen bonds, initiating phase separation [ 82 , 83 ]. In rare cases, TIPS is enthalpy driven. In these systems, the stronger interactions between the components, the enthalpy gain, stabilize the phase separation state at low temperatures [ 84 ]. Heating disrupts this favorable interaction, allowing the component-solvent interaction to dominate and leading to dissolution or homogeneous mixing. Using thermoresponsive polymers to fabricate phase-separated hydrogels is a common approach, where temperature changes reversibly trigger liquid–liquid or sol–gel transitions in the hydrophilic polymer network. For typical LCST-type hydrogels like PNIPAM, heating enhances hydrophobic interactions and weakens hydrogen bonds, leading to polymer dehydration, chain collapse, and aggregation, ultimately causing phase separation and a macroscopic transition from sol to gel [ 72 , 85 ]. In contrast, UCST (Upper Critical Solution Temperature)-type hydrogels phase-separate upon cooling, but this is less common [ 86 ]. TIPS strategies involving thermoresponsive polymers are important for drug delivery. For instance, near body temperature, PNIPAM undergoes a phase transition and contracts. This behavior can both act as a barrier to slow drug release and produce a "squeezing" effect to facilitate rapid drug release, as demonstrated by Lee et al. [ 73 ] and He et al. [ 74 ]. It should be noted that some thermoresponsive substances lack LCST or UCST behavior. Gelatin, for instance, undergoes phase transition governed by its unique helix-coil transition mechanism [ 87 ]. Recent studies highlight the relevance of such polymers in the fabrication of biological scaffolds. Their structures formed via TIPS can be fixed by cross-linking or rapid cooling, followed by solvent extraction or drying, converting the solvent-occupied space into a porous 3D scaffold that mimics the extracellular matrix [ 88 ]. For example, Xu et al. [ 42 ] dissolved PU-PEG-PVCL polymer in dimethyl sulfoxide (DMSO) at 80 °C, injected it into a mold, and froze it at −80 °C for 3 h to induce crystallization and solvent solidification. After 7 days of aqueous solvent exchange to remove DMSO and subsequent freeze-drying, a porous scaffold was obtained. Its cross-section showed a honeycomb-like pore structure, while the longitudinal section exhibited channels aligned with the solidification direction ( Fig. 4 a). The scaffolds had pore sizes ranging from approximately 50 to 120 μm. When mixed with a heart tissue-derived gel, the new material showed strength and flexibility similar to natural heart muscle, making it a promising patch for repairing heart attacks without using cells. Similarly, in the context of neurological disorders, Firouzian et al. [ 89 ] reported that cryogenically cooled poly (lactic-co-glycolic acid) (PLGA) solutions form uniaxially porous scaffolds whose mechanical properties closely match those of spinal cord tissue ( Fig. 4 b). The PLGA solution was first cooled at 4 °C for 30 min, then frozen at −80 °C for 5 h to induce the formation of an oriented microporous structure, and finally solidified by lyophilization for 72 h. This approach effectively combines the rigidity inherent in TIPS-based scaffolds with the flexibility provided by hydrogels, making it applicable for complex cell assembly and studies on neural regeneration. Fig. 4. Open in a new tab The role of temperature, concentration, cross-linking, and light in the preparation of phase-separated hydrogels. (a) Fabrication of anisotropic polyurethane porous scaffolds through TIPS. Reproduced with permission [ 42 ]. 2020, American Chemical Society. (b) Spinal cord scaffolds were produced by TIPS casting with a template of spaced steel rods. Reproduced with permission [ 89 ]. 2020, John Wiley & Sons Inc. (c) The process of preparing PEG microgels through mixing aqueous PEG with dextran solution, spinodal decomposition, and polymerization reaction, along with material characterization. Reproduced with permission [ 90 ]. 2023, American Chemical Society. (d) Crosslinking of CS-related hydrogels after 3D printing. Reproduced with permission [ 91 ]. 2024, ELSEVIER. (e) Laser-induced phase separation for the fabrication and micropatterning of PEDOT:PSS hydrogels. Reproduced with permission [ 92 ]. 2022, American Association for the Advancement of Science (TIPS, thermally induced phase separation; 3D, three-dimensional; DMSO, dimethyl sulfoxide; PEG, polyethylene glycol; ATPS, aqueous two-phase system; PEDOT, poly (3,4-ethylenedioxythiophene); PSS, poly (styrene sulfonate); AuNPs, gold nanoparticles). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 3.2.2. Concentration Polymer concentration is equally critical in controlling the formation and structure of phase-separated hydrogels. At low concentrations, large intermolecular distances and weak interactions result in a homogeneous solution, inhibiting phase separation. As concentration increases, enhanced interchain interactions beyond the critical overlap concentration trigger liquid–liquid phase separation, forming polymer-rich dense phases and polymer-poor dilute phases that constitute a porous network [ 93 ]. Thus, precise concentration control allows tailored design of phase separation behavior, pore structure, and mechanical properties. This principle is exemplified by Zauchner et al. [ 94 ], who reported a biodegradable synthetic microporous hydrogel formed via PIPS in the presence of cells. The material developed interconnected micropores (5–20 μm), enabling human osteocytes to rapidly spread and form functional networks in 3D. Increasing DEX concentration enhanced phase separation, enlarged pore size and porosity, and improved cell spreading and network formation. This degradable and structurally tunable hydrogel provides a controllable in vitro model for studying early bone development and bone-on-a-chip systems. The regulation of hydrogel phase separation by dual-polymer concentrations is another key focus, with DEX/PEG concentrations being critical factors in ATPS. Below critical concentrations (binodal), the system remains homogeneous. Above this threshold, polymer incompatibility dominates, leading to separation into DEX-rich and PEG-rich phases. Adjusting concentration ratios alters phase volumes and interfacial tension. Generally, higher polymer concentrations enhance compositional differences between phases and promote more complete separation. For instance, Cheng et al. [ 55 ] produced Janus droplets and hydrogels with controlled morphology by merging PEG and DEX aqueous droplets on superamphiphobic surfaces. When concentrations exceeded the binodal threshold, liquid–liquid phase separation yielded a PEG-rich upper phase and a DEX-rich lower phase. Varying initial polymer ratios precisely tuned phase equilibrium and interfacial curvature—all without organic solvents. However, Tigner et al. [ 90 ] demonstrated that homogenization during PEG/DEX mixing governs microgel properties. Microgel polymer concentration correlates linearly with the average PEG concentration in ATPS, not the predicted phase-separated composition. Mechanical properties are co-influenced by average PEG concentration and phase volume ratio. Complete phase separation occurs above 9.3 wt% PEG or 14.5 wt% dextran, but agitation disrupts phase equilibrium, thus inducing spinodal decomposition that dominates microgel formation ( Fig. 4 c). While ATPS offers a benign, solvent-free environment for bio-fabrication, its application is governed by strict physicochemical constraints that must be critically evaluated. First, the polymer concentrations required for ATPS are typically safe. However, in certain cases, high polymer concentrations may lead to significant osmotic stress, which can compromise cell viability or alter protein conformation [ 20 , 95 ]. Second, the ultra-low interfacial tension characteristic of ATPS creates a narrow stability window. Without precise control, the dispersed phase is prone to rapid coalescence, destroying the intended microarchitecture [ 20 , 96 , 97 ]. Furthermore, there is a kinetic competition between thermodynamic shape relaxation and network fixation. If the crosslinking rate is insufficient, the structural fidelity of the constructs cannot be maintained [ 98 , 99 ]. Finally, the practical "costs" of component residues and the difficulty of downstream purification remain major challenges in minimizing interfacial contamination [ 100 ]. Salt concentration also significantly influences phase-separated hydrogel structure and properties by modulating polymer hydration and interactions. Salting-out ions (e.g., SO 4 2− ) induce polymer dehydration and collapse, triggering phase separation and interconnected porous structures. Accordingly, Li et al. [ 101 ] found that sulfate ion concentration modulates the phase separation degree in poly (2-hydroxyethyl methacrylate)-gelatin hydrogels via the Hofmeister effect. High salt concentrations (>1 M) promoted salting-out, forming dense microphase-separated structures with enhanced mechanical properties, while low concentrations (<0.5 M) caused swelling. This mechanism imparted shape-memory functionality, suitable for bio-actuators. 3.2.3. Crosslinking In designing and fabricating phase-separating hydrogels, the choice of crosslinking strategy is as critical as polymer composition and reaction conditions. Common methods include Michael addition reaction, Schiff base formation, and photo-crosslinking. Proper crosslinking can establish permanent or dynamic networks, stabilizing phase-separated structures and controlling their dimensions. For instance, Yang et al. [ 102 ] utilized host–guest interactions between γ-cyclodextrin and an anthracene dimer to construct a weakly reversible supramolecular network, endowing the assembly with liquid-like rheology. This dynamic crosslinking promoted swelling-induced phase separation in aqueous media, leading to the formation of large-pore condensates that evolved into hydrogels with hierarchical porosity. Michael addition, a conjugate addition reaction between an electrophilic conjugated system and a nucleophilic carbanion, can also result in dynamic crosslinking. Similarly, Michael-addition crosslinking between 4-arm PEG vinylsulfone and dithiols not only formed a 3D network but also triggered PIPS, yielding controlled micropores of 5-20 μm under cytocompatible conditions at 37 °C [ 94 ]. Both studies underscore the role of dynamic crosslinking in governing phase separation. Conversely, Zhu et al. [ 91 ] revealed a negative correlation between Schiff base crosslinking and phase separation. The Schiff base reaction is a condensation process in which aldehydes or ketones react with amines to form imines (C=N). At a high active carbonyl-to-carboxyl ratio (ACR, e.g., 2:1), extensive Schiff base crosslinking between oxidized starch (OS) and CS formed a dense homogeneous network, inhibiting phase separation. Lower ACR (e.g., 2:3) reduced crosslinking, worsened compatibility, and intensified phase separation, impairing printability and mechanical properties. By tuning ACR, they produced OS–CS hydrogels suitable for thermal extrusion 3D printing ( Fig. 4 d). Meanwhile, Kotsiras et al. [ 103 ] found that photopolymerization crosslinking with N,N′-methylenebisacrylamide and acrylamide could stabilize lyotropic liquid crystal-templated nanostructures at optimal crosslinker concentrations (0.5–1.0 wt%), suppressing phase separation to preserve ordered pores. Higher crosslink density (e.g., 2.0 wt%) disrupted template stability, intensified phase separation, and degraded structure. 3.2.4. Other key approaches In recent years, researchers have uncovered more unique pathways to induce hydrogel phase separation. For example, Chee et al. [ 104 ] utilized UV-initiated photopolymerization 3D printing to copolymerize GelMA and hydroxyethyl methacrylate monomers within confined spaces. As the reaction proceeded, hydrophobic polymer chains separated from the aqueous phase, forming enriched regions, while water molecules were expelled to create pores. The pores were controlled by the printing layer thickness, ranging from about 25 μm to 280 μm​ This resulted in a hierarchical porous structure resembling cuttlebone. In this process, the phase separation enhanced hydrogen bonding and entanglement among polymer chains, thereby improving the mechanical strength and anti-swelling properties of the hydrogel. Furthermore, Won et al. [ 92 ] employed a laser-induced phase separation strategy, where laser interaction with gold nanoparticles (AuNPs) in poly (3,4-ethylenedioxythiophene):poly (styrene sulfonate) (PEDOT:PSS) generated strong electric fields and photothermal effects. This prompted the separation of conductive hydrophobic PEDOT regions from hydrophilic soft PSS domains ( Fig. 4 e). The AuNPs enhanced laser absorption, amplifying thermal and electric field effects to accelerate phase separation. Rapid cooling after scanning fixed the microstructure, yielding a hydrogel with high conductivity and stability in water. Additionally, Ma et al. [ 105 ] reported moisture-induced phase separation of PVA in a DMSO solution. In a humid environment, DMSO absorbed moisture and diffused into the PVA solution, triggering PVA gelation to form an organogel. Subsequent solvent exchange dissolved some low-molecular-weight PVA into the H 2 O/DMSO solution, ultimately forming a porous PVA hydrogel. This structure provided a mechanical interlocking foundation for subsequent incorporation of a poly (acrylic acid)-based wet-adhesive layer, synergistically enhancing the hydrogel's anti-swelling and burst-pressure resistance. It is pertinent to note that pH regulation and magnetic field intervention are equally critical factors in modulating phase separation. Given their intimate connection with biomedical engineering applications, this review will elaborate on these mechanisms in Sections 4.2.2.1 and Section 4.2.2.3 . Ultimately, future research must continue to uncover additional determinants of phase separation, as expanding the repertoire of induction methods is essential for achieving precise control over hydrogel microstructures and tailoring their properties for increasingly complex application scenarios. 3.3. Effects of phase separation on hydrogels In the field of biomaterials, hydrogels closely resemble the native cellular microenvironment, particularly in their ability to mimic the physicochemical properties and biological functions of the ECM. However, homogeneous hydrogels often oversimplify these environments and fail to capture the inherent complexity required for cellular interactions. To address this challenge, researchers have actively investigated various strategies, among which phase separation has become as an effective approach. Phase-separated hydrogels can create a variety of specific structures, including porous configurations, hierarchical architectures, and fibrous networks. These characteristics are essential for enhancing biocompatibility and enabling controlled drug release. Crucially, the functional versatility of these systems is not merely a result of their chemical composition, but is intrinsically governed by the characteristic length scales of the phase-separated domains. This hierarchical architecture allows for the decoupling of material properties. At the nanoscale, the phase morphology, which is often characterized by hard or crystalline domains dispersed within a softer matrix, serves as the primary driver for mechanical reinforcement through physical crosslinking and energy dissipation. Furthermore, these nanoscopic channels dictate molecular transport phenomena, controlling diffusion kinetics and permselectivity. In contrast, as we scale up to micro-to-macroscopic structures, the focus shifts to biological interaction. These larger-scale features regulate surface topography and porosity, which play a decisive role in modulating cellular behaviors such as adhesion, proliferation, and differentiation via mechanotransduction pathways [ 85 , 106 ]. Consequently, understanding how these multiscale architectures translate into specific material performance establishes a coherent framework for optimizing hydrogel design. In this review, the specific manifestations of these length-scale effects are further elucidated within the context of distinct fabrication strategies and applications. 3.3.1. Porous structure Porous hydrogels are highly favored in tissue engineering because their 3D porous structure closely simulates the natural ECM [ 107 ]. This architecture not only promotes cell adhesion and proliferation but also enables controlled and targeted drug release due to their high porosity and tunable pore size [ 108 , 109 ]. A convenient and highly controllable method for creating such porous structures is phase separation. To better define the operational niche of phase separation, it is instructive to compare it with sacrificial templating and cryogelation. As primary alternatives, these methods generate porosity through fundamentally different mechanisms. Sacrificial templating relies on removable templates [ 110 , 111 ], while cryogelation depends on ice crystal formation [ 112 ]. These distinct approaches lead to significant performance trade-offs. While templating methods offer precise control over defined pore architectures, they often involve complex removal steps or harsh solvents that may compromise biocompatibility [ 110 ]. Similarly, although cryogelation effectively generates sponge-like, interconnected macropores, it can lack the tunable control over micro-porosity that phase separation provides [ 112 ]. Consequently, phase separation stands out as a superior strategy when tunable micro-porosity and seamless interconnectivity are critical for nutrient transport [ 113 ]. It also usually serves as a robust alternative when the harsh processing conditions of other methods must be avoided to preserve bioactivity [ 114 ]. However, it may be less effective for creating large macro-channels needed for immediate vascularization or strictly defined constructs, where 3D printing or macro-templating would be more appropriate [ 115 , 116 ]. Fundamentally, when a polymer solution undergoes phase separation, it splits into polymer-rich and polymer-lean phases [ 117 ]. The polymer-rich phase forms the continuous hydrogel skeleton upon crosslinking, while the polymer-lean phase acts as a porogen, leaving behind an interconnected porous network [ 48 ]. By adjusting experimental conditions, pore size and porosity can be controlled, as demonstrated by Shao et al. [ 118 ]. Shao's group mixed GelMA with polyethylene oxide (PEO) to form a viscous bioink. During rest, the mixture phase-separated due to viscosity differences, and PEO was expelled. After photo-crosslinking the GelMA network, PEO was dissolved, yielding a hydrogel with tunable pore size and porosity. Specifically, this structure allows for the adjustment of pore size and porosity with increasing PEO concentration, thereby improving material transport and optimizing the cell growth environment, without affecting the subsequent formation of shear-induced highly aligned structures. In hydrogel porous structures, macropores (tens to hundreds of micrometers) and micropores (nanometers to a few micrometers) serve distinct roles. Macropores provide space for cell migration and mass transport of nutrients, and influence mechanical properties such as elasticity [ 119 ]. Guo et al. [ 120 ], for instance, found that PVA and SA undergo enhanced phase separation during Ca 2+ crosslinking. PVA promotes SA aggregation for toughness and introduces water-filled gaps, forming macropores of 50–700 μm with over 85% porosity ( Fig. 5 a). This structure improves permeability and stress dissipation, overcoming the brittleness of conventional porous materials. Similarly, Xue et al. [ 123 ] used LLPS between PEG and DEX to form DEX droplets as soft templates. Photopolymerization of a PEG-containing precursor created hydrogels with 50 μm macropores, enabling high-density 3D cell culture without contact inhibition. In contrast, micropores form dense mini-networks that provide high surface area. This supports water retention, bioactive factor loading, and cell adhesion, and enables fine control of the local microenvironment [ 124 ]. For example, through LLPS, Wang et al. [ 125 ] found that soluble sodium polyphosphate (polyP) and aragonite (nacre powder) react in wound exudate containing calcium. Ca 2+ released from aragonite complexes with polyP, forming coacervates that expel water and create pores. With aragonite, pores shrink to 5 μm, yielding a denser structure that enhances mechanical stability promotes cell proliferation and microvascular formation, and traps bacteria. Additionally, humidity-induced phase separation combined with solvent exchange can produce PVA hydrogels with surface micropores averaging 2.21 μm, improving layer adhesion and making the hydrogel tougher and more compression-resistant [ 105 ]. Fig. 5. Open in a new tab Phase separation promotes the formation of porous, hierarchical, and fibrous structures in hydrogels. (a) Single-step fabrication of SMP hydrogels from a PVA-dispersed and alginate-framed structure. Reproduced with permission [ 120 ]. 2024, John Wiley & Sons Inc. (b) The process for constructing bPVA/SIS + -NP layered patches. Reproduced with permission [ 121 ]. 2025, John Wiley & Sons Inc. (c) Methodology for producing nanofibrous hybrid hydrogels through phase separation techniques (stages I and II) and for creating core–shell structured nanofibrous polypeptide-polysaccharide composite hydrogels using electrostatic crosslinking (stages III to IV). Reproduced with permission [ 122 ]. 2013, ELSEVIER. (PVA, polyvinyl alcohol; SMP, super microporous; PDA, polydopamine; PEI, polyethyleneimine; bPVA, polyvinyl alcohol molecular brush; DMSO, dimethyl sulfoxide; NP, nicotinamide phosphoribosyltransferase; SIS, small intestinal submucosal decellularized matrix). 3.3.2. Hierarchical structure Hierarchical hydrogels possess multi-layered or gradient structures, wherein physicochemical properties such as chemical composition, crosslinking density, or pore size vary spatially across different layers in an ordered manner. These hydrogels not only mimic the complex structures of natural biological tissues (e.g., skin, cartilage) but also simultaneously fulfill multiple functional requirements, such as enabling time-controlled drug release or providing differential support for directional growth and differentiation of various cells. Consequently, they exhibit broad application prospects in the biomedical field [ 126 ]. Phase separation technology is an effective approach for constructing hierarchical hydrogels, as it can both promote stable integration between layers and directly participate in forming hydrogel systems with gradient structures. In terms of layer integration, Huang et al. [ 121 ] employed a self-induced phase separation strategy to successfully stabilize the composite of a zwitterionic polymer brush-modified polyvinyl alcohol (bPVA) hydrogel layer with a cationized small intestinal submucosa decellularized matrix (SIS + ). During the phase separation process, water from the SIS + layer diffused downward into the DMSO solution of bPVA, inducing the crosslinking of bPVA molecular chains via dense hydrogen bonds at the interface, thereby forming a robust mechanical interlocking structure ( Fig. 5 b). This method endowed the bilayer patch with high interfacial bonding strength and anti-swelling properties, laying the foundation for its application in repairing infectious abdominal wall defects by providing sustained mechanical support and anti-contamination functionality. Additionally, the moisture-induced phase separation can also be utilized to promote the gelation of a PVA solution in a humid environment, resulting in a porous topological structure with micron-scale pores (average diameter 2.21 μm) on the surface as a dissipative layer, and the pore was further refined to 0.43 μm after the hydrogel was fully structured. This layer created a mechanically interlocked interface for subsequent wet-adhesive layers, significantly enhancing both the interlayer bonding strength and the overall burst pressure resistance of the hydrogel [ 105 ]. In the fabrication of gradient-structured hydrogels, TIPS and induced ATPS are two important methods. For instance, Tialiou et al. [ 127 ] ingeniously utilized the TIPS behavior of hydroxypropyl cellulose in aqueous solution. By controlling the temperature gradient during crosslinking (with some regions below the LCST and others near or above the LCST), they successfully induced varying degrees of phase separation at different depths of the material. This process was "fixed" through subsequent crosslinking and freeze-drying steps, thereby constructing, in a single step, a gradient porous structure transitioning from large pores (220 μm) at the top to micropores (3 μm) at the bottom. This gradient structure can flexibly adapt to mechanical pressure changes in complex environments. On the other hand, Chen et al. [ 128 ] reported a one-step method for constructing biphasic gels with multi-scale heterogeneity. Their research demonstrated that the spontaneous separation of incompatible polymers (e.g., PEG and dextran) in aqueous solutions can form immiscible aqueous phases with distinct physicochemical properties. This process enables the pre-construction of macroscopic multilayered structures, mesoscopic cellular patterns, and microscopic molecular compartmentalization prior to gelation, thereby simplifying the complex procedures of traditional layer-by-layer methods and significantly enhancing the interfacial mechanical properties between different gel phases. 3.3.3. Fibrous structure Fiber hydrogels are elastic materials with a three-dimensional network structure formed through physical entanglement or chemical cross-linking of fibers or polymers, combining excellent biocompatibility with multifunctionality. Their internal fibrous structure not only exhibits outstanding flexibility, high elasticity, and anti-swelling properties but also promotes cell adhesion, proliferation, differentiation, and migration, making them an ideal new-generation biomimetic material [ 129 ]. Phase separation also plays a crucial structural-directing role in the fabrication of fibrous hydrogels. By regulating the spontaneous separation and aggregation behavior of molecules or polymer chains in a solution, it lays the foundation for the formation of fibrous networks with finely hierarchical structures. In the study by Zhou et al. [ 130 ], a rapid phase separation process induced by solvent exchange served as the core driving force for constructing a uniformly cross-linked network. This process facilitated the formation of evenly distributed microcrystalline regions within a highly entangled polymer solution, acting as cross-linking points in the network. Subsequently, under constrained entanglement conditions, phase separation promoted the homogeneous nucleation and spatial distribution of microcrystals, effectively preventing structural defects and thereby significantly enhancing the strength and toughness of the hydrogel fibers. Similarly, Cheng et al. [ 131 ] leveraged the core constructive role of phase separation. Using an ATPS of SF/alginate, they achieved uniform dispersion of SF as micron-sized particles within the alginate continuous phase. This process mimics the mechanism by which sericin envelops SF in natural silk, enabling the mild and continuous spinning of meter-long SF hydrogel fibers in a microfluidic chip. The alginate shell effectively protected the SF, allowing it to withstand subsequent mechanical stretching and alignment, thereby regulating the internal structure and mechanical properties of the fibers. Furthermore, Chen et al. [ 122 ] developed a nanofibrous gelatin/bioactive glass composite hydrogel via phase separation and electrostatic crosslinking. This hydrogel forms a 3D nanofibrous network in an ethanol/water solvent and is crosslinked with chitosan-hyaluronic acid polyelectrolytes, significantly enhancing its structural and thermal stability in physiological environments. The core-shell structure not only preserves the fibrous morphology but also imparts bioactivity and antibacterial properties through ion release from the bioactive glass ( Fig. 5 c). Moreover, phase separation is deeply involved in the property enhancement of fibrous hydrogels. According to Shao et al. [ 118 ], phase separation provides a key pore-regulation mechanism for constructing hydrogel microfibers. Due to viscosity differences, high-molecular-weight compounds such as GelMA/PEO spontaneously phase-separate during standing. After photo-cross-linking and removal of PEO, an interconnected porous structure is formed. This structure enables pore size and porosity adjustment via PEO concentration. Zhang et al. [ 132 ] also observed a coordination between PEO and phase separation. Spontaneous phase separation between PEO and CS induced the formation of a "sea-island" structure, where the CS-rich phase acts as the "sea" and the PEO-rich phase as the "island." Subsequently, the researchers removed PEO to create micro-scale fibrillar structures within the CS fibers, successfully constructing a spider-silk-like fibrous hierarchical structure. Thus, phase separation serves as a critical bridge connecting macroscopic fibers with microscopic fibrils, providing the core microstructural foundation for the formation of a spider-silk-like multi-level structure and endowing the hydrogel with exceptional mechanical properties, such as a fracture strength as high as 47.0 ± 2.4 MPa. 4. Applications of phase-separated hydrogels in biomedical engineering Compared to conventional hydrogels, phase-separated hydrogels exhibit superior structural controllability, biocompatibility, and functional integration, offering a new platform for constructing complex biological interfaces and advancing regenerative medicine. In tissue engineering, they can better mimic the extracellular matrix microenvironment, supporting directed cell migration, proliferation, and differentiation. For drug delivery, their controllable channels and stimulus-responsive properties enable time-programmed drug release. In biosensing and disease modeling, their multi-scale heterogeneous structures facilitate biomolecular recognition and the simulation of pathological processes. In bioprinting, their tunable rheological behavior and rapid phase transition mechanisms ensure high printing fidelity and structural stability, enabling the precise fabrication of complex vascularized tissue constructs with high cell survival rates. Systematically summarizing their application progress holds significant importance for promoting clinical translation ( Fig. 6 ). Fig. 6. Open in a new tab The biomedical applications of phase-separated hydrogels. Created with Biorender.com . 4.1. Tissue engineering 4.1.1. Bone tissue engineering The application of traditional bulk scaffolds in repairing large bone defects is severely restricted by their uncontrollable pore diameter and connectivity, which impedes nutrient transport and cell infiltration. To address this challenge, various engineering strategies, such as templating, microgel assembly, and 3D printing, have been developed to introduce physiological void spaces into hydrogels [ 133 ]. Among these strategies, phase separation technology has emerged as a bioinspired approach that, by precisely regulating thermodynamic and kinetic processes, enables the construction of hydrogel scaffolds with multi-level pore structures [ 134 ]. Building on this potential, researchers have focused on optimizing polymer compositions to enhance biological functionality within these porous networks. For instance, Zauchner et al. [ 94 ] developed an interconnected microporous network through PIPS based on PEG. By incorporating matrix metalloproteinase (MMP)-sensitive degradable peptides, they enabled cells to actively remodel the extracellular matrix. Moreover, the addition of hyaluronic acid and dextran allowed for precise control over the phase separation process, resulting in a cell-compatible, interconnected porous architecture. This structure not only enhances fluid permeability but also effectively mimics the native bone developmental microenvironment. Beyond simple connectivity, achieving precise control over pore size and hierarchical distribution represents a further advancement in mimicking the complexity of native bone. Through adjusting phase separation parameters, pore sizes can be tuned to support cell encapsulation and substance exchange. Muller et al. [ 36 ] achieved this by manipulating the photopolymerization-induced phase separation process through light intensity and composition, creating hydrogels with cell-guiding micropores (2–40 μm) alongside perfusion-supporting macrochannels, which serves as an optimal design for vascularized bone regeneration. Another example is from Xue et al. [ 123 ], who combined LLPS with interfacial supramolecular self-assembly. They used dextran droplet templates as scalable modulators to precisely control pore size and create hierarchical porous structures, and these structures enable both cell guidance and perfusion pathways ( Fig. 7 a). Fig. 7. Open in a new tab Application of phase separation hydrogel in bone tissue engineering. (a) The preparation process of the hydrogel scaffold (P-G-C-MgO 2 ) doped with MgO nanoparticles developed by freeze-induced phase separation method was used for bone defect repair. Reproduced with permission [ 123 ]. 2025, Nature. (b) 3D printing schematic dasiagram of the PNASC-PCBAA-6-40 hydrogel meniscus scaffold and its application as a meniscus substitute. Reproduced with permission [ 135 ]. 2021, Royal Society of Chemistry. (c) Macroporous hydroxyl gels through liquid-liquid phase separation and interfacial supramolecular self-assembly of protein fibers for stem cell-driven bone regeneration. Reproduced with permission [ 136 ]. 2025, ELSEVIER. (Alp, alkaline phosphatase; Bmp2, bone morphogenetic protein 2; Osx, osterix; Opn, osteopontin; PVA, polyvinyl alcohol; BMSCs, bone marrow-derived mesenchymal stem cells; NASC, N-acryloyl-semicarbazide; CBAA, carboxybetaine acrylamide; RGD, arginine-glycine-aspartic acid; PEG, polyethylene glycol). While optimizing the internal pore structure is crucial, phase separation is equally important for improving bone or cartilage repair platforms. Taking 3D printing platforms in cartilage repair as an example, Fan et al. [ 135 ] introduced a hydrophilic comonomer (carboxybetaine acrylamide) into a supramolecular hydrogel ( Fig. 7 b). This modification enhanced hydrophilicity to mitigate phase separation while maintaining reversible gel-sol transitions, yielding a hydrogel with high stiffness, lubricity, and excellent 3D printability. Furthermore, bone graft platforms constitute a significant avenue as well. Liu et al. [ 136 ] developed a cryogel scaffold (P-G-C-MgO 2 ) with a biomimetic trabecular architecture for the repair of critical-sized bone defects ( Fig. 7 c). They used MgO nanoparticles to precisely control phase separation during freezing. The MgO nanoparticles, electrostatically adsorbed by gelatin, acted as heterogeneous nucleation sites, which guided the second phase to preferentially precipitate during freezing, producing a bicontinuous structure with an average pore size of 565.7 ± 53.62 μm. This effectively overcame the pore size limitations of conventional cryogels and closely mimicked natural cancellous bone. Beyond direct defect repair, recapitulating this native microenvironment is equally pivotal for the scalable production of therapeutic cells. Yin et al. [ 137 ] developed bone marrow-inspired macroporous hydrogels via a soft-templating strategy based on LLPS. By precisely tuning the pore size (200–600 μm) and stiffness to resemble the native marrow niche, this platform preserved critical-ECM interactions often lost in conventional expansion. The resulting MSC-ECM spheroids showed significantly higher expression of skeletal development-related genes and superior osteogenic potential, confirming that replicating bone tissue microstructure is essential for scaffold performance and functional cell sourcing. 4.1.2. Corneal tissue engineering While penetrating keratoplasty is a primary treatment for severe corneal perforation—a leading cause of blindness—it faces significant challenges including donor shortages, immune rejection, and ethical concerns [ 138 ]. Consequently, phase-separated hydrogels have emerged as a promising class of materials for corneal bioengineering, as their tunable microstructure allows them to function either as optical prostheses or regenerative scaffolds. However, developing such multifunctional materials is non-trivial, as an ideal construct must simultaneously exhibit optical clarity and mechanical strength while also possessing high permeability, machinability, and recyclability [ 139 ]. Rational control over arrested phase separation enables the creation of a multi-scale architecture that addresses a central challenge in conventional pore-forming methods. Demonstrating this principle, Pan et al. [ 140 ] developed a novel hydrophobically-associated hydrogel (HAH) from PVA grafted with octyl side chains. By subjecting the HAH to hydrothermal treatment at 90 °C for varying durations ( Fig. 8 ), they induced a structural evolution governed by thermodynamic instability. Specifically, the heating triggers an LCST-type liquid-liquid phase separation, where enhanced hydrophobic interactions drive the polymer chains to aggregate into a dense phase while expelling water into emerging voids. Crucially, this coarsening process is kinetically arrested by the vitrification of the polymer-rich domains, effectively "freezing" the porous network at a non-equilibrium state before macroscopic demixing can occur. This mechanism allowed for the meticulous tuning of pore size to the sub-micrometer scale (about 100 nm). The resulting optimized structure achieves high optical transparency while simultaneously enhancing gas permeability and nutrient diffusivity, thereby fulfilling the critical requirements for an advanced corneal prosthesis. Fig. 8. Open in a new tab Application of phase separation hydrogel in Corneal Tissue Engineering. (a) By controlling the degree of phase separation through hydrothermal treatment and generating an optimized multi-scale structure, the contradiction between the permeability and transparency of corneal transplants can be coordinated and resolved. Multi-scale structural evolution during hydrothermal treatment. (b) The potential mechanism of irreversible phase transition proposed under isovolumic conditions. Reproduced with permission [ 140 ]. John Wiley & Sons Inc. (HAH, hydrophobic association hydrogel; ht-HAH, hydrothermal treatment-hydrophobic association hydrogel). Furthermore, phase separation strategies extend beyond enhancing mechanical strength to creating bioactive environments for treating corneal defects, particularly limbal stem cell deficiency—a severe disorder leading to opacity and blindness [ 141 ]. Conventional therapies like cultivated limbal epithelial transplantation are complex, invasive, and suffer from poor cell retention. To address this, Guo et al. [ 142 ] developed a non-invasive delivery system using a porous GelMA hydrogel. By inducing phase separation between GelMA and an immiscible PEO phase within an aqueous emulsion, they engineered a scaffold with a highly interconnected porous architecture. This phase-separated structure is functionally critical. It not only facilitates nutrient exchange but, more importantly, provides physical channels that enable the effective release and migration of encapsulated stem cells onto the corneal surface. Furthermore, this specific microenvironment activates the non-canonical Wnt signaling pathway, thereby enhancing stem cell self-renewal. Integrated with a corneal bandage lens, this system achieved avascular and scar-free repair in a rabbit LSCD model. 4.1.3. Vascular tissue engineering Adequate vascularization is essential for tissue homeostasis and regeneration by ensuring oxygen and nutrient supply. While the delivery of bioactive factors such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) remains a standard strategy to stimulate angiogenesis [ 143 ], recent efforts have shifted towards engineering perfusable architectures that physically support vessel development [ 144 , 145 ]. In this context, phase separation techniques have been leveraged to fabricate construct-spanning vascular networks with tunable transport properties. For example, Gu et al. [ 111 ] integrated 3D printing of sacrificial caramel templates with TIPS to generate nanofibrous poly (L-lactide)/poly (ε-caprolactone) scaffolds featuring interconnected perfusable microchannel networks. The phase-separation process generated hierarchical microporous channel walls with an average pore size of 34.3 ± 12.8 μm and porosity over 90 percent, which significantly reduced perfusion resistance and facilitated the intraluminal fixation of a VEGF-loaded Schiff-base crosslinked hydrogel. In another approach, Dinh et al. [ 146 ] exploited the immiscibility of poly (ethylene glycol) diacrylate and dextran within microfluidic droplets to engineer reservoir microcapsules characterized by a distinct liquid-core and hydrogel-shell architecture. Rather than forming continuous channels, phase separation here sequestered VEGF and PDGF into a hydrated dextran core, achieving approximately 80 % encapsulation efficiency, while organizing the poly (ethylene glycol) diacrylate phase into a surrounding permeable shell. By precisely tuning the shell thickness from about 12 to 29 μm to modulate the diffusive resistance of the capsule wall, the system successfully decoupled drug loading from matrix stiffness. This ensured sustained factor bioavailability, which significantly reduced cardiac fibrosis and promoted vascularization in vivo. Given the increased propensity for arterial diseases in vessels smaller than 6 mm, there is an urgent need for small-diameter vascular grafts that mimic native vessels. In this context, phase separation techniques offer a robust solution to the challenge of mechanical mismatch and leakage. For instance, Tang and colleagues utilized [ 147 ] TIPS to infiltrate bacterial nanocellulose tubes with PVA solution. This process established an interpenetrating network where the polymer phase crystallized within the fibrous skeleton to significantly enhance the burst pressure to approximately 0.065 MPa. The resulting composite structure effectively eliminated transmural leakage thereby optimizing perfusion resistance while simultaneously providing a compliant substrate that supported endothelial cell proliferation. 4.1.4. Skin tissue engineering The application of hydrogels in skin tissue engineering has evolved from static substitutes to dynamic repair systems and, more recently, to intelligent responsive platforms [ 148 ]. Effective wound repair requires scaffolds that not only offer biomimetic multi-hierarchical pore structures to guide cell differentiation, but also deliver bioactive factors to promote angiogenesis [ 149 ]. However, integrating these distinct functionalities into a single construct remains a challenge. Addressing this, phase separation strategies, like ATPS ( Fig. 9 a), have been developed to generate heterogeneous structures in a one-step process [ 128 ]. This approach provides a versatile platform for incorporating diverse functional units, such as nanofibers, inorganic particles, or cytokines. Fig. 9. Open in a new tab Application of phase separation hydrogel in Skin Tissue Engineering. (a) TAP gel imitating stratified heterogeneous biological tissues. (I) Schematic diagram of the heterogeneous structure of biological tissues at different length scales: the distinct layers of human skin at the macroscopic scale; Cell patterns within each tissue layer at mesoscale; Cell partitioning at the microscopic scale. (II) Schematic phase diagram of the ATPS composed of polymer A and Polymer B. The deep orange bimodal curve is outlined as the critical boundary between the single-phase region and the two-phase region. At an initial point in the two-phase region, two thermally balanced aqueous phases are separated, one rich in polymer A and the other rich in polymer B, following a line. (III) Schematic diagram of multi-scale two-water phase gel, as an imitation of biological tissue, including multilayer structure at the macroscopic scale, programmable printable structure at the mesoscale, and molecular partitioning at the microscopic scale. Reproduced with permission [ 128 ]. 2023, Nature. (b) Multihydrophobic hydrogel mimicking skin for diabetic wound treatment (QL@MAB). Reproduced with permission [ 150 ]. 2025, John Wiley & Sons Inc. (ATPS, Aqueous Two-Phase System; TAP gels, two-aqueous phase hydrogel; MA,methyl acrylate; AAPBA, (3-acrylamidophenyl)boronic acid). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) Through using phase separation, researchers can synergistically enhance the mechanical and biological properties of hydrogel composites, enabling precise adaptation to the complex microenvironment of wound healing [ 151 , 152 ]. For instance, Wang et al. [ 125 ] exploited LLPS to transform thermodynamically unstable aragonite (pearl powder) into a bioactive coacervate. The phase transition, triggered by calcium ions in wound exudate, converts solid minerals into a porous, energy-generating fluid matrix that mimics the dynamic nature of biological systems. By facilitating the transfer of calcium ions, this coacervate system not only suppresses bacterial growth but also significantly accelerates angiogenesis and tissue granulation in diabetic wounds. By sensing acidic or alkaline changes in the wound microenvironment, pH-responsive hydrogels can adjust their state and drug release behavior for precise treatment. For example, Nakipoglu et al. [ 153 ] applied NIPS to fabricate an asymmetric polycaprolactone membrane as a protective top layer, coupled with a chitosan-carrageenan hydrogel that exhibits pH-responsive swelling and degradation to dynamically adapt to the physiological changes of the wound environment. Similarly, Bankoti et al. [ 65 ] utilized a polyurethane diol dispersion blended with CS to induce phase separation through hydrogen bonding, leading to the self-assembly of a macroporous hydrogel scaffold. This scaffold exhibits pH-responsive degradation characteristics and demonstrated excellent efficacy in promoting healing in a full-thickness skin defect model. Furthermore, the integration of stimuli-responsiveness with biomimetic design represents a significant advancement in phase-separation-based hydrogels. Inspired by the structure and function of the skin, Liu et al. [ 150 ] developed a hydrophobic hydrogel dressing (QL@MAB) for diabetic wounds ( Fig. 9 b). During the solvent exchange process, the hydrophobic methyl acrylate (MA) segments undergo rapid phase separation at the interface to self-assemble into a condensed, dense "epidermis-like" surface layer. This structure not only locks in substantial moisture by generating high internal osmotic pressure, but also functions as a semi-permeable barrier that significantly retards water loss and ensures prolonged drug retention. Simultaneously, boronic acid monomers form dynamic borate ester bonds with quercetin, creating "sweat pore"-like structures. This biomimetic "epidermis–sweat pore" system enables glucose-responsive self-regulated drug release and provides long-lasting moisturization. 4.2. Drug delivery 4.2.1. Controlled-release carrier design A controlled drug release system is a drug-loaded platform comprising various polymeric carriers and active pharmaceutical agents. Upon injection at the lesion site, this system facilitates targeted and controlled drug release, thereby enhancing therapeutic efficacy [ 154 ]. Phase-separated hydrogels exhibit inherent advantages as carriers for such systems. Specifically, phase separation can establish a "reservoir-matrix" architecture, wherein coacervate microdroplets function as reservoirs while the high-water-content gel phase acts as the matrix. This inherent architecture is ideal for achieving sustained and regulated release [ 155 , 156 ]. Moreover, the exceptional structural and functional tunability afforded by phase separation allows for precise modulation of thermodynamic driving forces and kinetic parameters through molecular design, enabling customized release profiles. Researchers have leveraged phase separation to engineer diverse carrier architectures, ranging from fundamental homogeneous systems to complex structured designs [ 123 ]. To optimize release kinetics, core-shell structures fabricated via coaxial printing or self-assembly are employed to ensure stability, while porous scaffolds combine macropores for tissue ingrowth with phase-separated micropores for effective drug loading [ 157 ]. On the micro- and nanoscale, discrete carriers like vesicles and polymer particles play a pivotal role [ 158 , 159 ]. Vesicles utilize amphiphilic bilayers to co-deliver hydrophilic and lipophilic agents ( Fig. 10 a), whereas polymer particles can encapsulate a wide range of therapeutics for precise control ( Fig. 10 b). Both systems offer significant advantages in encapsulation capacity, biocompatibility, and targeting potential. Fig. 10. Open in a new tab Application of phase separation hydrogel in Drug Delivery. (a) Thermosensitive hydrogel polymer vesicles for controlled release of drugs. Reproduced with permission [ 73 ]. 2010, ELSEVIER. (b) The process of preparing loaded particles by phase separation method. Reproduced with permission [ 159 ]. 2012, Taylor and Francis. (c) Schematic diagram of the preparation of NIPS-induced RSF hydrogel and porous foam. Reproduced with permission [ 160 ]. 2016, Royal Society of Chemistry. (d) Mechanism of action of non-solvent induced phase separation on RSF hydrogels. Reproduced with permission [ 160 ]. 2016, Royal Society of Chemistry. (N/Ps, PNIPAM-containing polymersomes; LCST, lower critical solution temperature; mPEG-PDLLA, poly (ethylene glycol)-b-poly (D,L-lactide); PNIPAM, poly (N-isopropylacrylamide); RSF, regenerated silk fibroin; NIPS, nonsolvent-induced phase separation). In terms of specific fabrication strategies, NIPS acts as a critical method for manipulating the thermodynamic equilibrium of polymer-solvent-non-solvent ternary systems to construct macromolecular hydrogel carriers. This strategy triggers the desolvation of polymer chains by introducing a non-solvent, thereby inducing phase separation and forming a stable gel network. For example, Kasoju et al. [ 160 ] utilized the NIPS principle to fabricate silk fibroin hydrogels with highly interconnected porous structures by regulating the ratio of methanol (non-solvent) to aqueous regenerated silk fibroin solution ( Fig. 10 c). Their study demonstrated that the demixing of solvent and non-solvent phases promoted the transition of silk fibroin from a disordered structure to an ordered β-sheet conformation. The resulting hydrogels not only possessed tunable porosity and mechanical strength but also exhibited the potential to co-load hydrophilic and hydrophobic drugs ( Fig. 10 d). Furthermore, to overcome the mechanical and functional limitations of single polymer networks, researchers have combined phase separation with solvent exchange strategies. Kang et al. [ 81 ], for instance, proposed a composite strategy involving the preparation of PHB hydrogel via NIPS, followed by the introduction of CS during the solvent exchange process. In this process, the non-solvent induced liquid-liquid phase separation of PHB to form an organogel skeleton. The subsequent exchange with a chitosan-containing solution not only achieved homogeneous composite formation, but also leveraged the hydrophilicity of chitosan to endow the hydrophobic PHB network with excellent thixotropy and injectability. Notably, chitosan itself also contributed notable antibacterial properties to the system. This approach significantly enhanced the compressive modulus in the aerogel state, and enabled pH-responsive synergistic delivery of both hydrophilic and hydrophobic drugs. 4.2.2. Stimuli-responsive release Stimuli-responsive hydrogels possess the ability to undergo reversible structural and physical transformations in response to fluctuations in external environmental factors, such as pH, temperature, and magnetic field. Notably, these environmental factors can trigger swelling, collapse, phase separations, or changes in crosslinking density. Among the transformations, phase separation is of particular interest as it can modulate the thermodynamic stability of the polymer solution, leading to the formation of hierarchical microstructures that define the material's macroscopic behavior. These processes enable on-demand drug release and tunable performance. 4.2.2.1. pH-responsive release In responsive drug delivery systems, pH is a widely utilized external stimulus due to the significant acid-base variations found in pathological environments such as the gastrointestinal tract, tumor tissues, and infection sites. pH-responsive hydrogels typically contain ionizable functional groups (e.g., carboxyl, sulfonic, or amino groups) or dynamic covalent bonds. Upon exposure to specific pH environments, the ionization of these groups disrupts existing hydrogen bonds and triggers electrostatic repulsion, driving the hydrogel to undergo structural changes such as swelling, collapsing, and inducing phase separation. For instance, Han et al. [ 161 ] constructed a conductive hydrogel electrode with a bicontinuous structure via an acid-induced in situ phase separation strategy. In this system, acid treatment partially protonates PSS, weakening the electrostatic interaction between PEDOT and PSS while enhancing hydrophobic interactions. This transition promotes the transformation of PEDOT:PSS from a colloidal state into a fibrous conductive network, which subsequently integrates with the PVA-polyvinylpyrrolidone network via hydrogen bonding to form a bicontinuous structure. Ultimately, this enables the material to achieve rapid drug release at low pH while maintaining a high drug loading capacity. Moreover, Wang et al. [ 162 ] developed a novel pH-dependent antisolvent phase separation strategy. Using methanol to program the dehydration aggregation and conformational changes of sodium polyacrylate, they fabricated single-component hydrogel fibers with outstanding mechanical properties. These fibers form a rigid network through carboxyl protonation at low pH, while transitioning to a highly elastic network via deprotonation at high pH. This approach not only highlights applications in soft electronics but also offers new insights for designing pH-responsive drug carriers. Beyond phase separation directly driven by protonation, pH can also modulate properties by altering crosslinking density. For instance, a supramolecular composite hydrogel constructed through pH-induced UPy tautomerization uses this tautomerism to mediate physical crosslinking while forming microphase-separated structures, significantly enhancing the hydrogel's strength, toughness, and fatigue resistance [ 163 ]. Despite their remarkable functionality, the fabrication of pH-responsive hydrogels is often constrained by the lack of universal inks that can simultaneously accommodate multiple printing techniques. For instance, digital light processing requires low-viscosity, highly transparent liquid inks, whereas Direct Ink Writing necessitates high-viscosity, semi-solid gel-like inks. To resolve this contradiction, Wu et al. [ 164 ] has proposed a two-phase 3D printable hydrogel based on a controllable microphase separation strategy. Through pH modulation, this material achieves reversible transitions in microphase domains, enabling its viscosity to be widely tuned from fluid-like to solid-like states, thereby meeting the extreme rheological requirements of different printing technologies. Additionally, this hydrogel combines excellent mechanical properties with high-resolution 3D shaping capability, making it suitable for high-precision manufacturing ranging from 2D filaments to complex 3D structures. 4.2.2.2. Thermally responsive release Thermosensitive hydrogels are characterized by tunable sol-gel phase transitions. They are primarily classified into LCST types which undergo gelation upon heating for sustained release, and UCST types which exhibit swelling upon heating for triggered release [ 165 ]. By adjusting polymer chemical structures—such as hydrophilic/hydrophobic balance and functional group polarity—transition temperatures can be precisely tuned to specific physiological environments [ 166 ]. These hydrogels offer significant advantages for drug delivery, including superior biocompatibility and the elimination of toxic initiators often required by pH-sensitive systems. Temperature variations regulate drug release by shifting the material's hydrophilic-hydrophobic balance. For example, in a study by Lee et al. [ 73 ], a thermosensitive polymer, PNIPAM (LCST = 32 °C), was encapsulated inside degradable mPEG-PDLLA polymer vesicles. At physiological temperature (37 °C), PNIPAM underwent phase separation and formed a hydrogel core whose hydrophilic-to-hydrophobic transition delayed drug release, highlighting the critical role of thermosensitivity. Furthermore, thermosensitive transitions can enhance mechanical and adhesive properties. For instance, one research conducted by Zhang et al. incorporated cellulose nanofibers to leverage temperature-mediated hydrogen bonding, triggering reversible phase separation. This resulted in a 24-fold increase in interfacial toughness (to 117.2 J/m 2 ) and stiffness tunable from 0.02 to 0.14 MPa, highlighting the material's potential for smart medical devices [ 46 ]. Notably, pH/temperature dual-responsive hydrogels can also enable highly specific drug release through logic-gated responses to two critical environmental stimuli. In the field of separation science, Kobayashi et al. [ 167 ] exploited the temperature/pH-responsive characteristics of a smart polymer-based stationary phase. Under alkaline conditions, this negatively charged stationary phase retains protonated catecholamines through electrostatic attraction. At elevated temperatures (50 °C), increased polymer hydrophobicity prolongs the retention time of hydrophobic analytes like tyramine. This synergistic modulation of electrostatic and hydrophobic interactions enables efficient separation of catecholamines even under purely aqueous conditions. 4.2.2.3. Magnetism-responsive hydrogels Magnetic fields offer a distinct advantage as a contactless stimulus that provides homogeneous processing without compromising the material's chemical or mechanical stability. By inducing alignment in anisotropic constituents, magnetic fields can effectively modulate macroscopic properties like optical clarity and mechanical strength. The stimuli-responsiveness of phase-separated hydrogels can thus be extended to these physical fields through strategic material design, enabling spatiotemporally controlled functions such as drug release [ 168 , 169 ]. This point is exemplified by research into thermoresponsive polymers like PNIPAM. While their phase behavior can be modulated by magnetic fields, the underlying mechanism has been unclear. Investigating this, Neal et al. [ 170 ] examined the thermodynamic, optical, and rheological properties of aqueous PNIPAM solutions containing hydrophilic silica nanoparticles (NPs) under magnetic fields. They determined that while both magnetic fields and NPs lower the phase separation energy barrier and optical transition temperatures, they do so via distinct mechanisms. Magnetic fields alter solvent polarization, whereas NPs serve as hydrogen bonding sites. Furthermore, combining NPs with magnetic fields yields unique field-dependent rheological behavior, such as strengthened hydrogel moduli at elevated temperatures, which is not observed in polymer-only solutions. 4.3. Biosensing Phase separated hydrogels have attracted considerable attention for sensing applications due to their tunable polymer networks and customizable responsive properties. Since many sensing mechanisms rely on changes in electrical signals, conductivity becomes one of the keys to achieving high-performance sensing functions. Among various conductive fillers, conjugated polymers stand out for their flexibility, biocompatibility, and high conductivity. Particularly notable is PEDOT:PSS, which can achieve an electrical conductivity exceeding 4000 S cm −1 after processing. For instance, Tropp et al. [ 171 ] employed acid-induced phase separation to fabricate highly conductive and dispersible PEDOT:PSS nanoparticles. This process selectively removes excess insulating PSS and promotes π-π stacking crystallization within the PEDOT domains, thereby significantly enhancing intrinsic conductivity while maintaining aqueous dispersibility. These particles were successfully integrated into hydrogels for the application of soft organic bioelectronic materials. Meanwhile, Won et al. [ 92 ] utilized laser-induced phase separation to achieve localized conductive domain crosslinking in a gold nanoparticle-doped PEDOT:PSS system. By leveraging photothermal energy, this technique effectively redistributes the polymer network to form expanded and interconnected PEDOT-rich domains, ensuring robust electrical pathways and structural stability in aqueous environments. This resulted in a hydrogel structure with high precision, superior electrical conductivity, and excellent charge injection capability, thus providing new avenues for flexible bioelectronics. Inspired by biological phase separation, stimuli-responsive hydrogels can serve as "engineered phase-separation systems" for biosensing. They function by converting subtle biochemical signals into macroscopic physical changes through volume phase transitions. As illustrated in Fig. 11 a, one representative design involves coating a nanodiamond sensor with thermosensitive PNIPAM hydrogel and anchoring magnetic particles [ 172 ]. When temperature variations trigger the "swelling–collapse" phase transition of the hydrogel, the distance between the magnetic particles and the sensor is altered, enabling highly sensitive and reversible temperature detection via quantum magnetometry. Fig. 11. Open in a new tab Application of phase separated hydrogels in Biosensing. (a) ND@PNIPAM–Ni hybrid sensor synthesis scheme and TEM characteristics. Reproduced with permission [ 172 ]. 2018, Nature. (b) Schematic diagram of the preparation process and network design of P (AA-PEA X )−Zr 4+ supramolecular hydrogel, a high-strength, rapid self-recovery bionic anti-swelling hydrogel sensor for underwater information transmission. Reproduced with permission [ 173 ]. 2025, American Chemical Society. (TEOS, tetraethyl orthosilicate; MPS, 3-(methacryloyloxy)propyl trimethoxysilane; NIPAM, N-isopropylacrylamide; MBA, N-isopropylacrylamide; KPS, potassium persulfate; PEI, polyethyleneimine; ND, nanodiamond; pNIPAM, poly (N-isopropylacrylamide); DMSO, dimethyl sulfoxide; P (AA-PEA) chains, poly (acrylic acid-co-phenethyl acrylate) chains; AA, acrylic acid; PEA, phenethyl acrylate). Additionally, engineering multi-network hydrogels for microphase separation is also a sophisticated strategy to augment sensor functionality [ 174 ]. These self-assembled, heterogeneous structures efficiently dissipate mechanical stress while enhancing strength and toughness, ensuring device durability. More critically, they facilitate the creation of functionally diversified microenvironments, providing an elegant platform for integrating essential sensing attributes such as electrical conductivity and frost resistance [ 175 ]. While photocurable hydrogels derived from highly polymerized low-molecular-weight monomers exhibit excellent processability, their structural homogeneity often causes stress concentration and brittle fracture, impeding their mechanical performance [ 176 ]. To address this, Pan et al. [ 173 ] developed a photocurable supramolecular hydrogel via water-induced phase separation, creating hydrophobic PEA-rich regions with π–π and cation–π interactions as “soft” energy-dissipating domains, alongside a “rigid” quadridentate carboxyl–Zr 4+ coordination network. This heterogeneous architecture enabled a tensile strength of 1.42 MPa, complete self-recovery within 3 min, and an ultra-low swelling ratio ( Fig. 11 b). The hydrogel was successfully applied as an underwater strain sensor for real-time Morse code transmission, demonstrating the potential of phase-separated networks for stable sensing in aqueous environments. 4.4. Bioprinting Bioprinting is a technology based on additive manufacturing principles that constructs 3D tissue engineering structures using cell-laden bioinks [ 177 ]. With development, this technology has gradually evolved into multiple types [ 178 ], including stereolithography bioprinting, inkjet bioprinting, laser-assisted bioprinting, extrusion bioprinting and electrospinning-based bioprinting ( Fig. 12 a). Hydrogels have become the most commonly used bioink matrix due to their excellent biocompatibility and ability to mimic the ECM [ 180 ]. However, traditional bioprinting hydrogels still face multiple challenges in practical applications. First, hydrogels suitable for a single printing strategy struggle to possess universality across different printing processes. Second, traditional designs often fail to replicate the microscale topological cues of natural bone ECM, making it difficult to provide the physical microenvironment required for cell differentiation. Furthermore, although progress has been made in microchannel fabrication, achieving microvascularization remains a difficulty. Fig. 12. Open in a new tab Classification of bioprinting techniques and applications of phase-separated hydrogels in bioprinting. (a) Schematic illustration of five bioprinting technologies. (b) Material composition of TP-3DPgel and its application schematic in DLP and DIW-3D printing. Reproduced with permission [ 164 ]. 2024, American Chemical Society. (c) PIPS regulates the microporous structure within the GelMA hydrogel, while the overall shape of the scaffold is precisely controlled through DLP based 3D printing. Reproduced with permission [ 179 ]. 2025, John Wiley & Sons Inc. (UV, ultraviolet; P-gel, AAm, acrylamide; MBAA, N,N′ -methylenebis (acrylamide); GelMA, gelatin methacrylamide; PEG, polyethylene glycol; nHAP, nano-hydroxyapatite; PIPS, polymerization-induced phase separation; DLP, digital light processing; 3D, three-dimensional). Created with Biorender.com . To overcome these limitations, phase separation technology, a strategy capable of regulating material properties across scales, is becoming a focal point of innovation in the bioprinting field. The primary intervention of this technology in bioprinting lies in breaking the restrictions imposed by single materials on printing processes, thereby endowing inks with broader rheological adaptability. For instance, Wu et al. [ 164 ] proposed a two-phase 3D printing hydrogel based on a controllable micro-phase separation strategy. Under pH regulation, this material undergoes reversible micro-phase transitions, allowing its viscosity to be tuned over a wide range from liquid to solid states, thus making it applicable to both liquid-phase DLP and solid-phase Direct Ink Writing technologies ( Fig. 12 b). Moreover, phase separation technology not only improves process adaptability but also offers an effective pathway for constructing biomimetic microstructures with physiological functions. According to Dudaryeva et al. [ 35 ], combining phase-separating cell-interactive gelatin-norbornene hydrogels with volumetric bioprinting allows for the fabrication of centimeter-scale structures. These structures contain printed channels of 0.1–1 mm and interpenetrating micron-scale pores (1-200 μm). This method achieves the freeform construction of light-controlled micron scales and hierarchical porosity, promoting the infiltration of endothelial cells into deep tissues and the formation of microvasculature. Another microscopic advantage of phase separation technology is the ability to precisely replicate the topological structure of natural tissues, which is crucial for structure-sensitive bone regeneration. For example, Yu et al. [ 179 ] combined DLP 3D printing with PIPS to fabricate methacrylated gelatin hydrogel scaffolds, precisely reproducing ECM-like topological morphologies in patient-specific mandibular repair structures ( Fig. 12 c). The PIPS technology achieved precise control over pore morphology while maintaining the high resolution of DLP. Furthermore, to enhance fluid perfusion efficiency within tissues, phase separation can be combined with other micro/nano-fabrication methods to construct more ordered microchannel networks. Gu et al. [ 111 ] employed a method combining phase separation with sacrificial templates to prepare poly (L-lactide)/poly (ε-caprolactone) nanofiber scaffolds containing interconnected perfusable microchannel networks via 3D printing. The microchannel structure of the scaffold could be regulated through a caramel-based template strategy, which, when combined with angiogenic factors, significantly promoted angiogenesis and bone regeneration. Simultaneously, phase separation technology has been extended to microgel platforms to meet the demands of high-throughput screening and in vitro models. In response to this, Ort et al. [ 181 ] developed a stiffness-tunable 3D tissue culture platform based on a type I collagen-alginate interpenetrating network. When blended with polymers that induce phase separation, this system can utilize standard liquid handling equipment to achieve microliter-level bioprinting, yielding microgels with good reproducibility and tunable mechanical properties that maintain the viability of various printed cells. 5. Conclusion The past few decades have seen continuous progress in regenerative medicine driven by innovations in biomaterials. Among these, hydrogels have been widely used in tissue engineering, drug delivery, biosensing, and bioprinting due to their tunable physicochemical properties, injectability, and excellent biocompatibility. Phase separation overcomes the limitations of homogeneous networks by engineering unique microarchitectures and functionalities, thereby expanding the scope of hydrogel applications. However, current phase-separated hydrogels still face limitations that warrant further investigation. There remains significant room for improvement in the design of polymer systems. Although some hydrogels show considerable promise in medical applications, the robustness of their fabrication strategies remains suboptimal, limiting their reproducibility. Specifically, the structural stability at the interface between separated phases is often compromised, leading to mismatched degradation rates and potential immunogenic responses. In addition, hydrogels with high strength and toughness face challenges in maintaining adaptability and stability within dynamic physiological environments, such as the mechanical deformations involved in intestinal peristalsis. Moreover, multifunctional hydrogels need further development to effectively treat complex pathologies involving multiple tissue layers or heterogeneous tissue interfaces. To tackle these issues, elucidating the interaction mechanisms between polymer chemistry and cellular signaling is essential for achieving major breakthroughs in future biomaterial research. Advancing phase separation technology requires simultaneous innovation in both process parameters and hydrogel design. Regarding process parameters, precise control of variables such as ambient temperature and material concentration can modulate phase separation to occur at specific sites, enabling functions like autonomous targeting of pathological regions and site-specific drug delivery. In the future, regulating phase separation will increasingly rely on dynamic factors within the human body. Such control is particularly important for healing processes in organs like the intestines, which experience complex environmental fluctuations. Additionally, reconciling the slow kinetics of phase separation with the rapid solidification required for bioprinting fidelity is another major challenge. In terms of hydrogel design, porous hierarchical structures have already demonstrated various practical benefits while improving mechanical properties. Future research should focus on exploring novel hydrogel structures, particularly leveraging 4D bioprinting concepts to resolve the insufficient adaptability of current materials in dynamic biological microenvironments. Improvements in hydrogel manufacturing platforms are equally important. Notably, bioprinting enables not only the construction of precise architectures using specific hydrogels but also the integration of thermodynamically incompatible materials, thereby advancing the development of multifunctional hydrogels. Looking ahead, the transition from macro-scale printing to micro-scale printing will amplify these advantages, which is crucial for developing intelligent hydrogels capable of precisely targeting tumor sites and overcoming the biological barriers of heterogeneous tissues. In conclusion, the future development of bioprinted phase-separated hydrogels should focus on the synergistic enhancement of three key dimensions—hydrogel materials, phase separation technology, and manufacturing platforms. The design of high-quality biomaterials should transcend existing theoretical frameworks. For clinical applications, factors such as long-term storage stability, sterilization compatibility, and regulatory standardization are paramount for successful translation. CRediT authorship contribution statement Junpeng Mu: Writing – original draft. Sha Li: Writing – original draft. Zherui Zhang: Writing – original draft. Ze Li: Writing – review & editing. Sicheng Li: Funding acquisition, Writing – review & editing. Zongan Li: Writing – review & editing. Haohui Li: Writing – review & editing. Lili Yu: Writing – review & editing. Huajian Ren: Conceptualization. Jianan Ren: Conceptualization. Xiuwen Wu: Conceptualization. Jinjian Huang: Conceptualization, Funding acquisition, Writing – original draft. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments The authors are grateful for financial support from the National Natural Science Foundation of China (Nos. 82300648, 82500613, and 82570668), Natural Science Foundation of Jiangsu Province (Nos. BK20231091, and BK20251673), and Funding of Jinling Hospital (Nos. 2024JSYXZD042, 2024JCYJQN111, and 22LCZLXJS8). Contributor Information Huajian Ren, Email: [email protected]. Jianan Ren, Email: [email protected]. 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