Shape‐Memory Injectable Cryogels: From Minimally Invasive Delivery to Multifunctional Tissue Regeneration

ABSTRACT

Injectable cryogels have emerged as a transformative class of biomaterials that combine macroporosity, mechanical resilience, and shape‐memory properties to enable minimally invasive therapeutic delivery. Unlike conventional hydrogels, cryogels are fabricated through cryogelation—a sub‐zero polymerization process that generates interconnected macropores through ice crystal templating—endowing them with sponge‐like elasticity and the ability to withstand extreme compression and recover their original architecture upon injection. Over the past decade, injectable cryogels have evolved from simple porous scaffolds to sophisticated multifunctional platforms incorporating nanocomposites, bioactive molecules, and stimuli‐responsive elements for applications spanning bone and cartilage regeneration, stem cell delivery, cancer immunotherapy, and hemostatic wound healing. This review provides a comprehensive and critical analysis of recent advances in shape‐memory injectable cryogels, organized around five interconnected themes: (i) the physicochemical mechanisms governing shape‐memory behavior and material design strategies, (ii) emerging fabrication approaches including three‐dimensional printing–cryogelation hybrids and nanocomposite reinforcement, (iii) cryogel‐mediated stem cell delivery and tissue regeneration, (iv) immunomodulatory and vaccine platforms, and (v) hemostatic and wound‐healing applications. We critically evaluate how macro‐architectural features, mechanical tunability, and biochemical functionalization collectively dictate biological outcomes, and identify key challenges—including scalability, sterilization, regulatory pathways, and clinical translation—that must be addressed to realize the full therapeutic potential of injectable cryogels.

Conventional hydrogels cannot combine injectability with an open, load‐bearing architecture. Shape‐memory injectable cryogels overcome this limit: ice‐templated macropores confer sponge‐like elasticity, allowing compression through a syringe and shape recovery after injection. This review connects cryogel design to four therapeutic frontiers—bone and cartilage regeneration, stem‐cell delivery, cancer immunotherapy, and hemostasis.

1. Introduction

The clinical management of tissue defects arising from trauma, degenerative disease, and surgical resection remains a formidable challenge in modern medicine. Conventional surgical approaches—including autologous grafting, allografts, and pre‐formed synthetic implants—are effective in many scenarios but are inherently limited by donor‐site morbidity, immune rejection, poor conformity to irregular defect geometries, and the invasiveness of open surgical procedures [1]. These limitations have motivated intense research into injectable biomaterials that can be delivered through minimally invasive routes, conforming to complex defect architectures while providing immediate structural support and sustained biological cues for tissue regeneration [2, 3].

Hydrogels have long occupied a central position in this landscape, owing to their high water content, tunable biochemistry, and structural resemblance to the native extracellular matrix (ECM) [4]. However, conventional hydrogels suffer from well‐documented limitations: their nanoscale porosity restricts cell infiltration and nutrient transport, their mechanical weakness under physiological loading conditions compromises structural integrity, and their inability to maintain shape after injection limits their utility as space‐filling scaffolds [5]. These shortcomings have catalyzed the emergence of cryogels—a distinctive class of macroporous polymeric materials formed through cryogelation—as compelling alternatives that address many of the fundamental limitations of conventional hydrogels (Figure 1A).

FIGURE 1.

Overview schematic of injectable cryogels — (A) Architecture comparison of a conventional nanoporous hydrogel (mesh size ∼nm) versus a macroporous cryogel (pore size 10–200 µm); (B) the cryogelation process, in which ice crystals formed during freezing act as porogens and, upon thawing, leave an interconnected macroporous network; and (C) the five major application domains covered in this review (bone, cartilage, stem‐cell delivery, vaccine/immunotherapy, and hemostasis).

It is important to position cryogels within the broader landscape of injectable hydrogel strategies that have emerged to overcome the limitations of conventional bulk hydrogels. Shear‐thinning and self‐healing hydrogels exploit reversible physical crosslinks to flow under injection and recover thereafter [6], while in situ‐gelling systems remain liquid before administration and solidify in response to physiological triggers [7]; granular and microgel‐based inks achieve injectability through the jamming of pre‐formed microparticles [8]. These approaches, however, generally rely on the gel transitioning between liquid‐like and solid‐like states, and their as‐injected porosity remains predominantly nanoscale [9]. Shape‐memory cryogels are mechanistically distinct: they are pre‐fabricated, covalently or physically stabilized macroporous sponges that are elastically compressed for needle passage and recover their predefined geometry and interconnected macroporosity upon rehydration, thereby uniquely combining injectability with immediate macroscale structural support and open pore networks for cell infiltration [10, 11]. A direct comparison of these injectable classes with respect to porosity, mechanical resilience, gelation requirements, and cell‐carrying capacity is therefore warranted to contextualize the specific advantages of cryogels.

Cryogels are synthesized by conducting polymerization or crosslinking reactions at sub‐zero temperatures, during which the solvent (typically water) forms ice crystals that serve as porogens [12, 13]. Upon thawing, these ice crystals melt to yield a highly porous scaffold characterized by large, interconnected macropores (typically 10–200 µm) surrounded by dense polymer walls (Figure 1B). This unique architecture endows cryogels with several extraordinary properties: (i) exceptional mechanical resilience, enabling them to withstand up to 90% compressive strain without permanent deformation; (ii) shape‐memory behavior, allowing complete morphological recovery after passage through hypodermic needles; and (iii) rapid fluid uptake kinetics through capillary action within the interconnected pore network [11, 14]. These properties collectively render injectable cryogels uniquely suited for minimally invasive tissue engineering applications.

The concept of injectable cryogels was pioneered in the early 2010s [10] and was subsequently extended to therapeutic applications, including cancer vaccine platforms [15]. Since then, the field has expanded rapidly, with injectable cryogels finding applications in bone regeneration [16, 17], cartilage repair [18], neovascularization [19], hemostatic wound healing [20, 21], and neural tissue engineering [22]. The breadth and pace of this expansion are reflected in the exponential growth of primary research articles. Yet, although cryogel technology has been reviewed broadly [23, 24] and injectable cryogels have been surveyed as a class [25, 26, 27], no prior review has focused specifically on shape‐memory injectable cryogels or integrated their material design principles, fabrication innovations, and biological applications within a single comprehensive framework. The field has since undergone transformative advances, including the integration of three‐dimensional (3D) printing with cryogelation [28, 29], nanocomposite‐reinforced systems [16, 30], and the emergence of multifunctional cryogels that simultaneously address hemostasis, infection control, and tissue regeneration [20, 31].

This review aims to bridge this gap by providing a comprehensive and critical analysis of shape‐memory injectable cryogels, spanning material design, fabrication innovation, and therapeutic applications. We organize the discussion around five thematic pillars: shape‐memory mechanisms and material design (Section 2), fabrication strategies (Section 3), stem cell delivery and tissue regeneration (Section 4), immunomodulatory and vaccine platforms (Section 5), and hemostasis and wound healing (Section 6). We conclude with an assessment of current challenges and future perspectives for clinical translation (Section 7). Throughout, we emphasize the structure–property–function relationships that connect cryogel architecture to biological performance, and we highlight critical gaps and emerging opportunities that define the frontier of this rapidly evolving field (Figure 1C).

2. Shape‐Memory Mechanism and Material Design

2.1. Fundamentals of Cryogelation

Cryogelation is a cryotropic gelation process in which polymer solutions or monomer mixtures undergo crosslinking at sub‐zero temperatures, typically between −10°C and −20°C [12, 32]. At these temperatures, the majority of the solvent freezes to form ice crystals, while the polymer and crosslinking agents concentrate in the unfrozen liquid microphase surrounding the ice crystal boundaries. This cryo‐concentration effect dramatically accelerates the local crosslinking reaction, producing dense polymer walls that faithfully template the ice crystal morphology (Figure 2A). Upon thawing, the ice crystals melt, leaving behind a scaffold with large, interconnected macropores whose dimensions, connectivity, and wall thickness are governed by freezing rate, initial polymer concentration, and crosslinker density [13, 33].

FIGURE 2.

Shape‐memory mechanism and material design of injectable cryogels. (A) Cryogelation process, showing ice‐crystal templating, cryo‐concentration of polymer and crosslinker within the unfrozen phase, and the interconnected macroporous network formed after thawing. (B) Molecular basis of the shape‐memory cycle: an elastic hydrated cryogel is compressed with water expulsion, delivered through a needle, and recovers its original shape upon rehydration. (C) Qualitative comparison of representative natural (GelMA, chitosan, hyaluronic acid, alginate) and synthetic/hybrid (PVA, PEG, PEG–alginate) polymer systems, summarizing elastic modulus, pore‐size range, representative biomedical applications, shape recovery, and degradation behavior. (D) Crosslinking strategies for cryogel networks: physical, chemical, and dual‐network approaches.

The freezing kinetics play a decisive role in determining pore architecture. Slow freezing rates promote the growth of large ice crystals and consequently larger pores (100–200 µm), while rapid freezing yields smaller, more numerous ice crystals and finer pore structures (10–50 µm) [32, 34]. This tunability is particularly valuable for tissue engineering applications, where optimal pore dimensions vary by target tissue: bone regeneration benefits from pores exceeding 100 µm to facilitate osteoblast ingrowth and vascularization, while neural applications may require finer architectures that guide axonal extension [22, 35].

2.2. Molecular Basis of Shape‐Memory Behavior

The shape‐memory behavior of cryogels—their ability to be compressed to a fraction of their original volume and subsequently recover their full dimensions—arises from the combination of their macroporous architecture and the elastic properties of their polymer walls [11, 14]. Under compression, the macropores collapse as water is expelled from the scaffold, while the dense, crosslinked polymer walls undergo elastic deformation without fracture. Upon release of the compressive force (or upon rehydration after injection), capillary forces drive rapid water re‐uptake through the interconnected pore network, and the elastic restoring force of the polymer walls drives the scaffold back to its equilibrium dimensions (Figure 2B).

This mechanism is fundamentally distinct from shape‐memory polymers based on thermal transitions or crystalline switching segments [36]. In cryogels, the shape‐memory effect is entirely mechanical and operates at physiological temperatures without external stimuli. The recoverability is governed by the crosslink density and the elastic modulus of the polymer walls: higher crosslink densities increase elastic recovery but may reduce the maximum compressible strain, while excessively low crosslink densities permit plastic deformation and irreversible pore collapse [11]. The optimization of this balance—achieving high compressibility (>80%) with near‐complete recovery (>95%)—represents a central material design challenge.

2.3. Natural Polymer‐Based Systems

Natural polymers have been extensively explored for injectable cryogel fabrication owing to their inherent biocompatibility, biodegradability, and capacity for cell‐interactive signaling (Figure 2C). Gelatin methacryloyl (GelMA) has emerged as one of the most widely used natural polymer platforms, as it combines the cell‐adhesive RGD motifs and matrix metalloproteinase‐degradable sequences of gelatin with the photocrosslinkable methacrylate groups that enable precise control over crosslink density [37]. GelMA cryogels have demonstrated excellent shape‐memory properties, supporting injection through 16‐gauge needles with >90% shape recovery, and have been functionalized with bioactive glasses and growth factors for bone regeneration applications [16, 38].

Chitosan‐based cryogels leverage the polycationic nature of chitosan to provide intrinsic antimicrobial activity alongside shape‐memory behavior. Physical crosslinking through glutaraldehyde or genipin, combined with the hydrogen‐bonding network inherent to chitosan chains, produces mechanically robust cryogels suitable for both tissue regeneration and hemostatic applications [20, 39]. Hyaluronic acid (HyA) cryogels have been specifically engineered for cartilage repair, exploiting the natural role of HyA in cartilage ECM to promote chondrocyte phenotype maintenance [18, 40]. Alginate cryogels, often reinforced through dual‐network strategies combining ionic crosslinking with covalent modification, have demonstrated particular promise as cancer vaccine platforms due to their biocompatibility and tunable degradation kinetics [15].

2.4. Synthetic and Hybrid Polymer Systems

Synthetic polymers offer superior batch‐to‐batch reproducibility and tunability compared to natural counterparts (Figure 2C). Polyvinyl alcohol (PVA) cryogels, fabricated through physical crosslinking via freeze–thaw cycling, represent the most established synthetic platform, with mechanical properties tunable through PVA molecular weight, concentration, and number of freeze–thaw cycles [41, 42]. However, the bioinertness of PVA necessitates surface functionalization or blending with bioactive components for tissue engineering applications.

Poly(ethylene glycol) (PEG)‐based cryogels have gained prominence due to the versatility of PEG chemistry, enabling incorporation of degradable linkages, cell‐adhesive peptides, and stimuli‐responsive moieties [43, 44]. The development of PEG‐alginate hybrid double‐network cryogels has demonstrated how the combination of synthetic and natural components can simultaneously optimize mechanical performance and biological activity [45]. Lee et al. reported a biomimetic macroporous hybrid scaffold system that combined synthetic and natural polymer networks with sustained drug delivery capability, achieving enhanced bone regeneration through the synergistic interplay of structural support and controlled bioactive molecule release [46].

2.5. Crosslinking Strategies and Mechanical Tuning

The crosslinking strategy fundamentally determines the mechanical properties, degradation behavior, and shape‐memory performance of injectable cryogels (Figure 2D). Physical crosslinking approaches—including freeze–thaw cycling for PVA, ionic crosslinking for alginate, and thermally induced gelation for gelatin—offer the advantage of avoiding potentially cytotoxic chemical crosslinkers but generally produce weaker networks with limited long‐term stability [32, 41]. Chemical crosslinking via glutaraldehyde, carbodiimide chemistry, or photoinitiated radical polymerization (as in GelMA) provides stronger, more tunable networks but requires careful optimization to minimize residual crosslinker cytotoxicity [13, 37].

Dual‐network and interpenetrating network (IPN) strategies have emerged as particularly effective approaches for achieving the mechanical robustness required for load‐bearing tissue engineering applications. These systems combine two complementary networks—typically one rigid and one flexible—to achieve synergistic improvements in toughness, fatigue resistance, and shape recovery that exceed the performance of either network alone [45, 47]. The concept of self‐healing crosslinks, which can dynamically reform after rupture, has been incorporated into cryogel designs to further enhance durability under cyclic loading conditions encountered in vivo [48].

3. Fabrication Strategies

3.1. Conventional Cryogelation

The standard cryogelation protocol involves preparing a polymer solution with crosslinking agents at low temperature (typically 0°C–4°C to prevent premature gelation), transferring the solution to a mold, and incubating at sub‐zero temperatures (−10°C to −20°C) for 12–24 h to allow complete crosslinking within the cryo‐concentrated phase [12, 13]. The resulting monolithic cryogel is then thawed and washed to remove unreacted components (Figure 3A). For injectable applications, cryogels are typically fabricated in cylindrical molds matching the internal diameter of the target syringe or needle, enabling direct loading and delivery [11, 14].

FIGURE 3.

Fabrication strategies for injectable cryogels — (A) Conventional cryogelation protocol with directional freezing variant; (B) 3D printing–cryogelation hybrid approaches showing modular design; (C) Nanocomposite reinforcement strategies (bioglass, Si₃N₄, MXene, nano‐whitlockite) with representative mechanical and biological data; (D) Micro‐cryogel fabrication and injectable microparticle systems.

While this approach is simple and scalable, it offers limited control over pore architecture beyond the modulation of freezing rate and polymer concentration. Directional freezing techniques, in which a controlled temperature gradient is applied during freezing, have been developed to create anisotropic pore structures that mimic the oriented architectures of native tissues such as nerve and muscle [49, 50].

3.2. Three‐Dimensional Printing–Cryogelation Hybrids

The convergence of 3D printing with cryogelation represents one of the most significant recent advances in cryogel fabrication. Lee et al. pioneered a transformative approach by developing 3D‐printed assemblable bespoke scaffolds that serve as versatile carriers for microcryogels, enabling site‐specific regenerative medicine [28]. This modular strategy decouples the macroscopic scaffold architecture (controlled by 3D printing) from the microscopic cryogel environment (controlled by cryogelation), allowing independent optimization of structural support and biological functionality (Figure 3B).

Subsequent work has demonstrated in‐bath 3D printing of anisotropic shape‐memory cryogels functionalized with bone‐bioactive nanoparticles, where the printing process enables patient‐specific geometries while directional cryogelation imparts characteristic macroporosity and anisotropic architecture [29]. These 3D‐printed nanocomposite cryogels exhibit excellent shape recovery (>95%) and enhanced pore connectivity, demonstrating suitability for minimally invasive bone tissue engineering applications [29].

Complementary to direct cryogel printing, light‐based printing of leachable salt molds has been demonstrated as a versatile indirect approach for shaping complex macroporous structures with high geometric fidelity, expanding the accessible design space for cryogel‐based scaffolds [51]. The integration of LEGO‐inspired modular design principles with cryogel technology has further expanded the design space, enabling the assembly of patient‐specific scaffolds from standardized building blocks that can be individually loaded with cryogels carrying distinct biological cargoes [52].

3.3. Nanocomposite Reinforcement

The incorporation of nanoscale reinforcing agents into cryogel matrices has proven highly effective for enhancing mechanical properties, introducing bioactivity, and enabling multifunctional performance. Bioglass nanoparticles have been particularly widely explored, with Kwon, Song et al. demonstrating that bioglass‐incorporated GelMA cryogels significantly enhance osteogenic differentiation and bone defect regeneration compared to pristine GelMA cryogels [16]. The bioactive glass releases calcium, phosphate, and silicate ions that stimulate osteoblast activity while simultaneously improving the compressive modulus of the cryogel network (Figure 3C).

Silicon nitride (Si₃N₄) represents an emerging bioceramic reinforcing agent with unique antibacterial properties. Lee et al. reported that Si₃N₄‐reinforced cryogel systems exhibit simultaneous antibiofilm and osteogenic effects, addressing the critical clinical challenge of infection‐associated bone graft failure [53]. This dual‐function approach exemplifies the trend toward designing cryogel nanocomposites that address multiple pathological processes simultaneously.

MXene nanosheets—two‐dimensional transition metal carbides with exceptional electrical conductivity and biocompatibility—have been recently incorporated into PVA cryogel scaffolds for neural tissue repair, where their electrical conductivity supports the transmission of bioelectrical signals essential for neural cell function [22]. Similarly, nano‐whitlockite (Ca₁₈Mg₂(HPO₄)₂(PO₄)₁₂) has been explored as a bone‐specific reinforcing agent, releasing magnesium and phosphate ions that promote osteogenesis [54, 55].

3.4. Micro‐Cryogels and Injectable Microparticles

The development of micro‐cryogels—cryogel particles with dimensions ranging from tens to hundreds of micrometers—has expanded the delivery paradigm beyond monolithic scaffolds (Figure 3D). Micro‐cryogels can be injected as suspensions through fine‐gauge needles, eliminating the need for the syringe‐compression approach required for monolithic cryogels, and can fill irregular defects by conforming to complex geometries through collective packing [28, 56].

Lee et al. demonstrated a particularly elegant approach in which 3D‐printed bespoke scaffolds serve as organized carriers for micro‐cryogels, combining the structural precision of additive manufacturing with the biological advantages of cryogel microenvironments [28]. This system enables spatially controlled delivery of distinct cell populations or growth factors by loading different micro‐cryogels into designated compartments of the printed scaffold, a capability with significant implications for engineering complex, multi‐tissue constructs.

4. Stem Cell Delivery and Tissue Regeneration

4.1. Cryogels as Stem Cell Carriers: Advantages Over Hydrogels

The macroporous architecture of injectable cryogels provides fundamental advantages over conventional hydrogels for stem cell delivery. The interconnected pores (typically 50–200 µm) enable efficient cell seeding with high efficiency (>80%), uniform spatial distribution, and unimpeded nutrient and waste exchange throughout the scaffold volume [10, 11]. In contrast, cells encapsulated in nanoporous hydrogels are confined within the polymer mesh, with limited motility and dependence on diffusion‐limited mass transport that restricts viability in constructs exceeding 200 µm in thickness [5].

The shape‐memory properties of cryogels enable a unique cell delivery paradigm: cells can be seeded onto the cryogel under optimal culture conditions, and the cell‐laden construct can then be compressed and injected through a needle without significant loss of cell viability or spatial organization (Figure 4A) [11, 19]. Studies have consistently demonstrated that mesenchymal stem cells (MSCs), adipose‐derived stem cells (ADSCs), and neural stem cells maintain >85% viability after injection through 16‐gauge needles when delivered within shape‐memory cryogels [10, 19, 29].

FIGURE 4.

Stem cell delivery and tissue regeneration applications of injectable cryogels — (A) Schematic of cell seeding → compression → injection → shape recovery → tissue integration cycle; (B) Bone regeneration: sequential growth factor release, nanocomposite strategies, and in vivo outcomes; (C) Cartilage repair: HyA cryogels for focal defect filling; (D) Specialized applications: neural repair (conductive cryogels), vocal fold (self‐healing), and muscle engineering; (E) Comparative table of key studies with material, cell type, animal model, and outcome metrics.

Furthermore, the mechanical stiffness and topographical features of cryogel walls provide mechanotransductive cues that influence stem cell fate. The dense polymer walls present an effectively stiffer microenvironment compared to bulk hydrogels, and the pore geometry creates three‐dimensional confinement effects that have been shown to promote osteogenic, chondrogenic, or neurogenic differentiation depending on the specific material composition and architectural parameters [35, 57].

4.2. Bone Regeneration

Bone regeneration represents the most extensively studied application domain for injectable cryogels, motivated by the clinical prevalence of bone defects and the limitations of current grafting approaches [1, 58]. Lee et al. developed a sequential growth factor‐releasing double cryogel system that mimics the temporal sequence of growth factor presentation during natural bone healing [59]. This innovative dual‐chamber design enables the early release of bone morphogenetic protein‐2 (BMP‐2) to initiate osteogenic signaling, followed by sustained release of vascular endothelial growth factor (VEGF) to promote neovascularization—a critical bottleneck in large bone defect healing. In vivo studies demonstrated significantly enhanced bone regeneration compared to single‐factor delivery systems, underscoring the importance of biomimetic temporal signaling in cryogel‐based approaches (Figure 4B).

Kim and Lee et al. further advanced cryogel‐mediated bone regeneration by developing heparin‐functionalized injectable cryogels with rapid shape‐recovery properties for neovascularization [19]. Heparin immobilization served the dual purpose of enhancing growth factor retention through electrostatic binding and promoting endothelial cell adhesion, resulting in robust angiogenic responses both in vitro and in vivo. The same group subsequently demonstrated that VEGF‐overexpressed human adipose‐derived stem cells (hADSCs) seeded on whitlockite‐reinforced cryogels synergistically promote osteogenesis and angiogenesis, achieving significantly enhanced bone formation in a calvarial defect model [54].

Cell reprogramming approaches have also been explored to augment cryogel‐mediated bone regeneration; ectopic transient overexpression of OCT‐4 has been shown to facilitate BMP4‐induced osteogenic transdifferentiation of human umbilical vein endothelial cells, offering a strategy to generate osteoprogenitor cells from readily accessible vascular cell sources for seeding into injectable scaffolds [60]. Nanocomposite strategies for cryogel‐mediated bone regeneration have leveraged bioactive glass [16], nano‐whitlockite [54, 55], hydroxyapatite [58], and silicon nitride [53] as osteoinductive reinforcing agents. Amirthalingam, Lee et al. demonstrated the combinatorial effect of nano‐whitlockite and nano‐bioglass in an injectable hydrogel system for craniofacial bone regeneration, where the synergistic release of bioactive ions (Mg^{2 +}, Ca^{2 +}, Si^{4 +}, PO₄ ^{3 −}) significantly outperformed single‐component formulations [55]. The biomimetic rationale underlying these approaches—mimicking the mineral composition and hierarchical structure of native bone—has been comprehensively reviewed in the context of scaffold design for bone tissue engineering [61, 62].

4.3. Cartilage Repair

Articular cartilage defects present unique challenges for injectable biomaterials due to the avascular, low‐cellularity nature of cartilage tissue and the demanding mechanical loading environment of synovial joints. HyA‐based shape‐memory cryogel scaffolds have been specifically engineered for focal cartilage defect repair, exploiting the natural role of HyA as a major cartilage ECM component to promote chondrocyte phenotype maintenance and reduce fibrocartilage formation [18, 40]. These cryogels can be injected arthroscopically to noninvasively fill cartilage defects, expanding to conform to irregular defect geometries through their shape‐memory behavior (Figure 4C).

PEG‐alginate hybrid double‐network cryogels have demonstrated tunable degradation rates that can be matched to the rate of neocartilage formation, addressing the critical challenge of scaffold persistence in cartilage repair applications [45]. Bioceramic‐mediated approaches have also shown promise, with recent work demonstrating that cryogel systems can induce controlled chondrocyte hypertrophy and calcified cartilage formation for osteochondral defect repair [63].

4.4. Soft Tissue and Specialized Applications

Beyond mineralized tissues, injectable cryogels have found applications in neural repair, vocal fold regeneration, and skeletal muscle engineering (Figure 4D). For neural tissue repair, electrically conductive cryogels incorporating polypyrrole, MXene, or graphene derivatives provide topographical guidance and electrical stimulation that enhance neural stem cell differentiation and axonal outgrowth [22, 64]. Chitosan‐gelatin‐polypyrrole cryogels have demonstrated promising results in peripheral nerve regeneration models, supporting Schwann cell proliferation and axonal regrowth across nerve gaps [64].

Lee et al. developed a self‐healing and adhesive artificial tissue implant for voice recovery that exemplifies the versatility of cryogel‐based approaches for soft tissue applications [48]. This system combines the macroporous, elastic properties of cryogels with self‐healing crosslinks and tissue‐adhesive functionality, enabling minimally invasive delivery to the vocal folds with sustained mechanical performance under the cyclic vibratory loading characteristic of phonation.

Recent advances in stem cell biology have expanded the cellular toolkit available for cryogel‐mediated tissue regeneration. Kim, Lee et al. demonstrated a self‐renewing biomimetic skeletal muscle construct engineered using induced myogenic progenitor cells, establishing that cryogel‐like macroporous scaffolds can support the long‐term maintenance and differentiation of lineage‐specific progenitors for functional tissue reconstruction [65]. The integration of cryogel technology with organoid engineering—including vascularized brain organoids—represents an emerging frontier with significant implications for both fundamental neuroscience and regenerative medicine [66]. These advances, combined with recent progress in bridging stem cells with 3D printing technologies for orthopedic applications [67], suggest that the next generation of cryogel‐based therapies will increasingly leverage sophisticated cell engineering approaches (Figure 4E).

5. Immunomodulatory and Vaccine Platforms

5.1. Cryogel‐Based Cancer Vaccines

The application of injectable cryogels as cancer vaccine platforms represents one of the most innovative and clinically impactful developments in the field. Pioneered by Bencherif, Mooney, and colleagues, cryogel‐based cancer vaccines exploit the unique combination of injectability, macroporosity, and sustained release capability to create subcutaneous immune niches that recruit and activate dendritic cells (DCs) in situ [10, 15]. The foundational work demonstrated that alginate cryogels co‐loaded with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), cytosine‐phosphoguanine oligodeoxynucleotides (CpG‐ODN), and irradiated tumor cells can elicit potent, durable anti‐tumor T‐cell responses in melanoma models, significantly reducing tumor burden and providing prophylactic protection against tumor rechallenge [10].

The macroporous architecture of cryogels is central to their efficacy as vaccine platforms (Figure 5A). The large, interconnected pores (>50 µm) permit the rapid infiltration of host immune cells—a process that is severely limited in nanoporous hydrogels—while the dense polymer walls provide sustained release of encapsulated immunomodulators over days to weeks [10, 15]. This spatio‐temporal control over immune cell recruitment and activation mimics key aspects of the germinal center reaction, enabling in situ priming of adaptive immune responses without the need for ex vivo cell manipulation.

FIGURE 5.

Immunomodulatory and vaccine platforms — (A) Mechanism of cryogel cancer vaccine: subcutaneous injection → DC recruitment via GM‐CSF → antigen presentation → T‐cell activation → anti‐tumor immunity; (B) Evolution of material designs: alginate → tough alginate → zwitterionic → stimuli‐responsive; (C) Ultrasound‐triggered release concept; (D) Immune niche engineering: pro‐inflammatory vs. anti‐inflammatory configurations; (E) Key preclinical outcomes summary.

5.2. Evolving Material Designs for Immune Engineering

Since the seminal reports, the material design of cryogel‐based immunotherapeutic platforms has undergone substantial evolution (Figure 5B). Injectable, tough alginate cryogels with enhanced mechanical resilience have been developed to improve the durability of the immune niche following injection [15]. Zwitterionic cryogels—prepared using zwitterionic monomers that resist protein adsorption and non‐specific cell adhesion—have been developed as degradable cancer vaccine platforms specifically designed to prevent cancer recurrence after surgery [68]. The anti‐fouling properties of the zwitterionic surface ensure that the immune response is directed exclusively toward the co‐delivered tumor antigens rather than the scaffold material itself.

Stimuli‐responsive release mechanisms have been integrated into cryogel vaccine platforms to achieve on‐demand delivery of immunomodulators (Figure 5C). Shih and colleagues demonstrated ultrasound‐triggered release of CpG‐ODN from cryogel vaccines, revealing that the timing of adjuvant release critically determines the magnitude and quality of the anti‐tumor immune response [69]. This finding has important implications for vaccine design, suggesting that temporal control over adjuvant presentation—enabled by the stimuli‐responsive capabilities of advanced cryogels—may be as important as the choice of adjuvant itself.

5.3. Dendritic Cell Recruitment and Immune Niche Engineering

The concept of engineering an immune niche through biomaterial‐mediated control of the local immune microenvironment extends beyond cancer vaccination (Figure 5D). Cryogels have been designed to recruit and reprogram specific immune cell populations, creating localized immunomodulatory environments that can be tuned from pro‐inflammatory (for cancer immunotherapy) to anti‐inflammatory (for regenerative medicine) through the choice of incorporated cytokines and adjuvants [70, 71]. Studies have demonstrated that cryogel vaccines effectively induce immune responses independent of proximity to draining lymph nodes, suggesting that the engineered immune niche can function autonomously as a decentralized immune activation site [72].

The integration of cryogel‐based immune niches with cell therapy approaches—such as the use of cryogels as stem cell factories for the production of bispecific antibodies—points toward a convergence of regenerative medicine and immunotherapy that represents a particularly promising future direction [73]. Key preclinical outcomes of representative cryogel‐based immunotherapy platforms are summarized in Figure 5E.

6. Hemostasis and Wound Healing

6.1. Shape‐Memory Cryogels for Non‐Compressible Hemorrhage

Uncontrolled hemorrhage is a leading cause of preventable death in both military and civilian trauma. Shape‐memory cryogels have emerged as a uniquely effective hemostatic platform because their rapid expansion upon contact with blood mechanically compresses the bleeding vessel from within the wound cavity—a capability that is critically important for non‐compressible hemorrhages in junctional or intracavitary wounds where external pressure cannot be applied [20, 21, 74].

The hemostatic mechanism of shape‐memory cryogels involves multiple synergistic pathways (Figure 6A): (i) rapid blood absorption through capillary action within the macroporous network, concentrating coagulation factors and platelets at the wound site; (ii) mechanical compression of bleeding vessels as the cryogel expands to fill the wound cavity; and (iii) activation of the intrinsic coagulation cascade through contact with the cryogel surface, which can be engineered to present pro‐coagulant functional groups [20, 31, 74]. In mouse liver puncture and rat liver cruciate incision models, shape‐memory cryogels have demonstrated significantly faster hemostasis and reduced blood loss compared to commercial hemostatic agents such as Surgicel and gelatin sponges [20, 21].

FIGURE 6.

Hemostasis and wound healing applications — (A) Mechanism of shape‐memory hemostatic cryogel: injection into wound cavity → expansion → vessel compression → blood absorption → clot formation; (B) Multifunctional integration: antibacterial (photothermal, electroactive, intrinsic) + hemostatic + regenerative properties; (C) Diabetic wound healing: ROS scavenging, O₂ release, angiogenesis promotion; (D) Comparative performance data vs. commercial hemostatic agents; (E) Future directions: smart wound monitoring integration.

6.2. Antibacterial and Multifunctional Wound Dressings

Wound infection is a major complication that delays healing and increases morbidity, particularly in the context of traumatic injuries and chronic wounds. The integration of antibacterial functionality into hemostatic cryogels has been achieved through multiple strategies (Figure 6B), including the incorporation of silver nanoparticles, quaternary ammonium compounds, photothermal agents, and inherently antibacterial polymers such as chitosan [20, 39, 74].

Photothermal antibacterial cryogels represent a particularly elegant approach, in which near‐infrared (NIR)‐responsive agents (such as polydopamine, MXene, or reduced graphene oxide) are incorporated into the cryogel matrix to enable on‐demand bactericidal activity through localized heating [31, 75]. Recent multifunctional cryogel systems have achieved approximately 98% killing ratios against both Escherichia coli and methicillin‐resistant Staphylococcus aureus (MRSA) after just 3 min of NIR irradiation, while simultaneously providing hemostatic and tissue‐regenerative functions [31].

A particularly notable recent development is the network‐remodeling, electroactive, and antibacterial cryogel with tissue‐sealing and pro‐coagulant activity for hemostasis and wound healing [76]. This system integrates electroactive polypyrrole for electrical stimulation of cell proliferation, dynamic crosslinks for tissue‐adaptive mechanical behavior, and antimicrobial functionality into a single injectable platform, exemplifying the trend toward “all‐in‐one” multifunctional cryogel wound dressings.

6.3. Diabetic and Chronic Wound Healing

Chronic wounds, particularly diabetic foot ulcers, represent a major clinical burden characterized by impaired angiogenesis, persistent inflammation, excessive reactive oxygen species (ROS), and susceptibility to biofilm formation [77]. Multifunctional cryogel dressings have been specifically designed to address these multiple pathological features simultaneously (Figure 6C).

An “all‐in‐one” composite biomimetic cryogel was recently reported that integrates hemostasis, photothermal antibacterial activity, ROS scavenging, oxygen release, and angiogenesis promotion for coagulation disorder hemostasis and diabetic wound healing [31]. The cryogel achieves this multifunctionality through the incorporation of calcium peroxide (for oxygen generation), manganese dioxide (for ROS decomposition), and polydopamine (for photothermal antibacterial activity) within a chitosan‐gelatin cryogel matrix. In a diabetic rat model, this system significantly accelerated wound closure compared to single‐function controls, demonstrating the clinical relevance of the multifunctional approach.

Anti‐swelling polysaccharide‐based cryogel sponges with rich positive charge have also been developed for non‐compressive hemostasis and wound healing, leveraging the electrostatic interaction between cationic cryogel surfaces and negatively charged blood cell membranes to enhance blood cell adhesion, platelet activation, and fibrin network formation [78]. Comparative performance data for shape‐memory cryogel hemostats and commercial hemostatic agents are compiled in Figure 6D, and the prospective integration of smart wound‐monitoring functionality into such dressings is outlined in Figure 6E.

7. Challenges and Future Perspectives

7.1. Manufacturing Scalability and Quality Control

Despite the remarkable properties and therapeutic potential demonstrated in preclinical studies, the clinical translation of injectable cryogels faces significant challenges in manufacturing scalability and quality control. Conventional cryogelation is a batch process that requires precise control of freezing temperature, freezing rate, and incubation duration, all of which can vary significantly between laboratory‐scale and production‐scale operations [12, 32]. The sensitivity of pore architecture to freezing conditions means that small variations in thermal history can produce significant batch‐to‐batch variability in pore size distribution, mechanical properties, and consequently biological performance.

The integration of 3D printing with cryogelation introduces additional manufacturing complexity, as it requires the sequential execution of two distinct fabrication processes—each with its own set of critical process parameters—and the compatibility constraints between them [28, 29]. However, this same integration also offers potential advantages for scalability, as 3D printing inherently enables digital manufacturing with high reproducibility, and the modular design philosophy enabled by printed‐cryogel hybrids may facilitate quality‐controlled mass production of standardized building blocks.

7.2. Sterilization Challenges

Terminal sterilization of cryogel scaffolds presents particular challenges due to their polymeric nature and macroporous architecture. Autoclaving (steam sterilization at 121°C) is incompatible with most polymer‐based cryogels due to thermal degradation, while ethylene oxide (EtO) sterilization raises concerns about residual cytotoxic agents trapped within the macroporous network [79, 80]. Gamma irradiation and electron beam sterilization can induce crosslink scission or additional crosslinking, altering the mechanical and degradation properties of the cryogel in unpredictable ways [79].

Supercritical CO₂ sterilization has emerged as a promising alternative for macroporous biomaterials, as it can effectively penetrate the interconnected pore network without causing thermal damage or leaving toxic residues [80]. However, standardized protocols for cryogel sterilization that maintain both sterility assurance and material integrity remain to be established, representing a critical gap in the translational pathway.

7.3. Regulatory Pathway Considerations

Injectable cryogels intended for tissue regeneration or cell delivery fall under complex regulatory classifications that depend on the specific composition, mechanism of action, and intended use. Scaffolds without incorporated biologics may be classified as medical devices, while cryogels loaded with cells or growth factors are likely to be classified as combination products or advanced therapy medicinal products (ATMPs), subjecting them to more stringent regulatory requirements [81, 82].

The demonstration of adequate safety, efficacy, and quality in accordance with regulatory standards requires well‐defined specifications for critical quality attributes—including pore size distribution, mechanical properties, degradation kinetics, sterility, and bioburden—that can be consistently achieved at manufacturing scale. The development of standardized characterization methods and acceptance criteria for these attributes represents an important prerequisite for regulatory submissions.

7.4. Toward Smart and Stimuli‐Responsive Cryogels

The next generation of injectable cryogels will likely incorporate stimuli‐responsive functionalities that enable dynamic, context‐dependent therapeutic responses. Temperature‐responsive cryogels incorporating poly(N‐isopropylacrylamide) (PNIPAAm) or related polymers can modulate drug release rates in response to local temperature changes associated with inflammation [83, 84]. pH‐responsive systems can exploit the acidic microenvironment of tumors or infection sites to trigger targeted drug release [84]. The integration of conductive and piezoelectric components enables electrically responsive cryogels for applications in neural and cardiac tissue engineering where bioelectrical signaling is essential [22, 64].

The convergence of cryogel technology with emerging paradigms in tissue engineering—including organoid culture [66], induced pluripotent stem cell (iPSC)‐based therapies, and immunoengineering—promises to further expand the application landscape. The development of biodegradable natural polymer‐based drug delivery systems integrated with cryogel platforms represents a particularly promising direction for sustained therapeutic delivery in bone tissue engineering applications [85]. As the field matures, the combination of computational design tools, machine learning‐guided optimization, and high‐throughput screening approaches may accelerate the identification of optimal cryogel formulations for specific clinical applications.

7.5. Clinical Translation Prospects

Several favorable attributes of injectable cryogels position them well for clinical translation: they can be fabricated from FDA‐approved polymers (e.g., alginate, PVA, PEG), delivered through minimally invasive procedures using standard clinical tools (syringes and needles), and stored in a dehydrated state with extended shelf life [11, 14]. The hemostatic cryogel platform is arguably closest to clinical translation, given the clear unmet medical need, the relatively straightforward regulatory pathway for hemostatic devices, and the strong preclinical evidence of efficacy [20, 21, 74].

For cell‐laden cryogel therapies, the path to clinical translation is longer and more complex, requiring demonstration of consistent cell viability after manufacturing, storage, shipping, and injection—the so‐called “clinic‐ready” cryogel concept. The development of cryopreservation protocols that enable long‐term storage of cell‐laden cryogels without loss of cell viability or scaffold integrity represents a critical enabling technology for this vision [86].

Notwithstanding these prospects, it should be acknowledged that injectable hydrogels are considerably more advanced than cryogels along the clinical‐translation pathway: multiple injectable hydrogel products have already reached clinical use or late‐stage trials, whereas injectable cryogels remain almost entirely at the preclinical stage [87]. Several cryogel‐specific factors underlie this gap. The sub‐zero fabrication step complicates aseptic, large‐scale manufacturing and is less familiar to regulators than the mild, often in situ gelation of injectable hydrogels [25, 88]. Terminal sterilization is also more challenging, as common methods (autoclaving, gamma irradiation) can damage the macroporous network or alter mechanical and shape‐recovery properties [89]. In addition, batch‐to‐batch reproducibility of pore architecture is sensitive to cooling rate and freezing geometry, which govern pore formation [32, 34], raising quality‐control burdens that hydrogels—whose gelation is more readily standardized—do not face to the same degree [90]. Addressing these manufacturing, sterilization, and reproducibility challenges, rather than the intrinsic biological performance of the scaffolds, is likely to be the rate‐limiting step for clinical translation of injectable cryogels.

8. Conclusions

Injectable cryogels have evolved from a niche biomaterial concept into a versatile and powerful technological platform that addresses critical unmet needs across multiple domains of tissue engineering and regenerative medicine. The fundamental attributes that distinguish cryogels from conventional hydrogels—interconnected macroporosity, mechanical resilience under extreme compression, and shape‐memory‐driven recovery after needle injection—arise from the elegant simplicity of the cryogelation process and its ice‐crystal templating mechanism. These properties collectively enable a paradigm of minimally invasive scaffold delivery that preserves both the structural integrity of the scaffold and the viability of encapsulated cells.

The material design space for injectable cryogels has expanded dramatically, encompassing natural polymers (gelatin, chitosan, hyaluronic acid, alginate), synthetic polymers (PVA, PEG), and increasingly sophisticated hybrid and nanocomposite formulations that synergize the advantages of multiple components. The integration of 3D printing with cryogelation has opened new possibilities for patient‐specific scaffold geometries and modular, multi‐compartment designs that enable spatially controlled delivery of distinct biological cargoes. Nanocomposite reinforcement with bioactive glasses, silicon nitride, MXene, and other functional nanomaterials has extended the capabilities of cryogels beyond passive structural support to active modulation of the biological microenvironment.

In the realm of tissue regeneration, injectable cryogels have demonstrated compelling efficacy for bone healing through sequential growth factor delivery, nanocomposite‐mediated osteoinduction, and stem cell transplantation. For cartilage repair, shape‐memory HyA cryogels enable arthroscopic delivery of chondro‐supportive scaffolds. Emerging applications in neural, vocal fold, and skeletal muscle regeneration further underscore the versatility of the platform. The cryogel cancer vaccine paradigm—in which injectable macroporous scaffolds recruit and activate dendritic cells to elicit potent anti‐tumor immunity—represents a conceptual breakthrough that bridges biomaterial science with immunology. Hemostatic cryogels, with their unique ability to mechanically compress bleeding vessels through shape‐memory expansion, address a critical unmet need in trauma care, and multifunctional variants that combine hemostasis with antibacterial activity, ROS scavenging, and tissue‐regenerative properties exemplify the cutting edge of the field.

Looking forward, the realization of the full clinical potential of injectable cryogels will require coordinated advances in manufacturing scalability, sterilization methodology, regulatory science, and clinical trial design. The integration of stimuli‐responsive elements, smart sensing capabilities, and computational design tools will further enhance the precision and efficacy of cryogel‐based therapies. As the field continues to mature, injectable cryogels are well‐positioned to become a foundational technology in minimally invasive regenerative medicine.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.