Cryogel-based therapeutic platforms for disease modification in osteoarthritis

Abstract

Osteoarthritis (OA) is a leading cause of chronic pain and disability, and lacks disease-modifying therapies. Unlike conventional hydrogels, which suffer from poor mechanical robustness and limited retention, cryogels overcome existing limitations through their interconnected pore architecture, shape-memory behavior, and fatigue-resistant mechanics. Here, we review how cryogels are being engineered as platforms for stem cell delivery, bioactive molecule release, localized gene activation, and immunomodulation, and discuss key translation challenges.

Introduction

Osteoarthritis (OA) is a chronic, progressive joint disease and a cause of pain and disability worldwide. In 2021, an estimated 606.5 million individuals were living with OA, corresponding to an age-standardized prevalence of approximately 6967 per 100,000 individuals¹. The lifetime risk of developing OA has been reported at 14.21%, with knee OA, the most prevalent subtype, carrying a lifetime risk of 9.31%². The burden of OA is disproportionately borne by women, who exhibit higher age-standardized rates of incidence, prevalence, and disability-adjusted life years compared to men³. Between 1990 and 2021, the global burden increased substantially across all metrics (prevalence, incidence, and years lived with disability), and projections to 2050 suggest case numbers will grow by 60–100% depending on joint site, driven largely by aging populations and rising obesity rates. OA is associated with marked declines in quality of life and imposes substantial psychosocial burdens, including reduced mobility, loss of independence, and diminished participation in daily and social activities¹. Further, it ranks among the leading causes of musculoskeletal disability worldwide⁴. In high-income countries, OA also imposes a major economic burden: knee and hip OA together account for an estimated $89.1 billion in annual direct and indirect expenditures⁵. Together, these trends highlight an urgent need for therapeutic strategies that move beyond symptom management to directly target the coupled mechanical, inflammatory, and degenerative processes underlying OA progression.

Once considered a disease of isolated cartilage wear, OA is now recognized as a chronic whole-joint disorder driven by the interplay of structural, inflammatory, and biomechanical dysfunction. Disease progression involves deterioration of the articular cartilage extracellular matrix (ECM), remodeling of the subchondral bone, synovial inflammation, and alterations in joint biomechanics^6,7. Critically, the limited intrinsic repair capacity of cartilage renders these changes largely irreversible, underscoring the need for therapies that target the joint as an integrated biological system (Fig. 1A).

Fig. 1. Cryogel-based therapeutic platforms for osteoarthritis (OA): joint pathology and intervention strategies.

A Schematic overview of OA joint pathology, illustrating the coupled deterioration of articular cartilage, subchondral bone remodeling, and synovial inflammation that collectively drive progressive joint degeneration. B Therapeutic scope of cryogel-based intervention platforms for OA, encompassing stem cell delivery, bioactive molecule release, gene activation, and immunomodulation, each targeting distinct but interconnected aspects of OA pathophysiology. Figure created with BioRender.com.

Conventional clinical management remains largely palliative. The American Academy of Orthopaedic Surgeons recommends oral nonsteroidal anti-inflammatory drugs (NSAIDs) for patients without contraindications, with acetaminophen as an alternative when appropriate⁸. Intra-articular (IA) hyaluronic acid (HA) injections provide short-term analgesic benefit, with randomized trials showing transient reductions in WOMAC (Western Ontario and McMaster Universities OA Index) pain and stiffness within 2–4 weeks, although no improvements were observed in function or VAS (Visual Analog Scale) pain during activity in that same period. By 5–8 weeks, the only consistent change was a modest reduction in VAS pain at rest, with no measurable improvement in other symptom domains⁹. These findings indicate that IA HA injections produce limited short-term pain relief but do not meaningfully improve function or alter the course of disease. Non-pharmacologic interventions, including exercise therapy, weight management, physical therapy, and bracing, may reduce symptoms and slow functional decline, but do not halt disease progression^10,11. Surgical intervention is reserved for end-stage disease. Total joint arthroplasty reliably restores pain-free function in advanced OA, but is invasive, carries procedural risk, and often requires revision surgery over time^12,13. Collectively, these approaches alleviate symptoms but fail to prevent or reverse progressive joint degeneration. Furthermore, arthroplasty is particularly poorly suited for younger or more active patients given its invasiveness and revision burden, highlighting an unmet need for regenerative strategies capable of restoring joint structure and function rather than solely managing symptoms.

To overcome these limitations, a range of tissue engineering strategies have been explored as alternatives to conventional management, including biomaterial-based scaffolds¹⁴, stem cell-based therapies^15,16, and bioactive delivery systems designed to promote cartilage repair and modulate the joint microenvironment¹⁷. While nanoparticle systems enable localized delivery of anti-inflammatory or anabolic factors, these platforms lack the structural framework necessary to guide organized tissue regeneration¹⁸. Hydrogels have therefore received particular attention due to their hydrated three-dimensional (3D) matrix and ability to mimic key features of the native cartilage ECM. Advances in hydrogel design include natural and synthetic polymer systems, composite formulations, and diverse chemical crosslinking strategies aimed at tuning mechanical and biological performance^19–21. Injectable hydrogels permit minimally invasive defect filling with structural fidelity, support encapsulation of chondrocytes or mesenchymal stem cells (MSCs), and enable sustained delivery of bioactive molecules to degenerative sites^18,22. Efforts to address this have included hybrid gelatin/poly(vinyl alcohol) (PVA) hydrogels²³, double-network systems matching porcine cartilage in compressive strength and lubricity²⁴, and fiber-reinforced constructs incorporating melt electrowritten polycaprolactone (PCL) microfibers²⁵; yet each carries tradeoffs in cellular infiltration, fabrication complexity, or mechanical fidelity. Collectively, these findings highlight that effective OA biomaterial therapies must simultaneously address mechanical demands, inflammatory signaling, and the limited intrinsic repair capacity of cartilage: a combination no current hydrogel-based system has reliably achieved.

Cryogels have been extensively reviewed across tissue engineering applications, with prior work largely focusing on material properties or isolated therapeutic strategies. In the context of OA, however, the literature remains dominated by primary studies investigating cryogel-based platforms across individual therapeutic modalities, including cell delivery, bioactive molecule release, gene activation, and immunomodulation (Fig. 1B). While these studies demonstrate significant potential, a cohesive, disease-specific framework for cryogel design in OA has yet to emerge. Given that OA is a disease defined by coupled mechanical dysfunction, chronic inflammation, and progressive tissue degeneration, effective biomaterials strategies must be designed to address these interconnected processes simultaneously. Here, we present a disease-centered framework that integrates cryogel design principles with OA pathophysiology to synthesize existing findings and propose guiding principles for the development of multifunctional, disease-modifying cryogel platforms. Within this framework, we examine these therapeutic modalities in the context of their ability to address the coupled mechanical, inflammatory, and degenerative processes that define OA progression, while also highlighting key barriers to clinical translation, including long-term durability, biological control, and scalable manufacturing.

Pathophysiology of cartilage degeneration in arthritis

OA is a multifactorial and debilitating disease that affects more than 606.5 million individuals worldwide as of 2021¹. It is the leading cause of musculoskeletal disability and is characterized by chronic pain, stiffness, and progressive loss of mobility⁷. Once considered a consequence of “wear and tear,” OA is now recognized as a complex disease driven by mechanical overload, chronic low-grade inflammation, metabolic dysregulation, and progressive deterioration of the osteochondral unit. OA most commonly affects the knee, followed by the hand, hip, lumbar facet joint, and temporomandibular joint. The pathological mechanisms underlying OA vary by joint location: weight-bearing joints, such as the hip and knee, are primarily influenced by excessive mechanical loading, whereas OA of the hand, temporomandibular joint, and lumbar facet joint are associated with systemic inflammatory and metabolic factors²⁶.

Regardless of initiating factor and joint location, OA arises from an imbalance between anabolic and catabolic processes within cartilage. This imbalance may arise from abnormal loading on structurally intact joints or from physiological loading on maladaptive joints that have lost mechano-protective features due to compromised cartilage integrity, altered subchondral bone architecture, or impaired synovial lubrication²⁷. Collectively, these factors drive a self-reinforcing degenerative cascade involving progressive cartilage degradation, osteochondral unit damage, synovial inflammation, and abnormal remodeling of subchondral bone.⁵

Cartilage degradation

Articular cartilage consists of chondrocytes embedded within an ECM composed of type II collagen, proteoglycans, and other non-collagenous proteins, primarily aggrecans. In healthy tissue, chondrocytes maintain homeostasis between ECM synthesis and degradation. Within OA, this homeostasis is disrupted, shifting toward a predominantly catabolic phenotype²⁸.

Mechanical injury or biochemical perturbations stimulate chondrocytes to adopt a hypertrophic, pro-inflammatory phenotype, driving elevated secretion of matrix metalloproteinases (MMPs) and A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS) enzymes. MMPs break down ECM components (i.e., collagen), while ADAMTS proteases cleave aggrecan, the protein complex responsible for cartilage shock absorption through water retention. MMP-1 and MMP-13 mediate degradation of type II collagen, while ADAMTS-4 and ADAMTS-5 cleave aggrecan, leading to the progressive loss of cartilage structural integrity and shock-absorbing capacity. Reactivation of the canonical Wnt/β-catenin pathway, which regulates chondrocyte development and cartilage homeostasis, has also been implicated in OA, further amplifying MMPs and ADAMTS expression and accelerating cartilage breakdown²⁹.

ECM degradation products or fragments released into the synovial fluid act as damage-associated molecular patterns, activating toll-like receptors (TLRs) on synovial immune cells. Therefore, TLR-2 and TLR-4 are found to be upregulated in synovial tissue, cartilage lesions, and synovial membranes of OA patients, inducing an upregulation of MMPs, nitric oxide, and prostaglandin E2 (PGE2). This amplifies matrix breakdown, promoting angiogenesis, and contributes to pain sensitization via activation of the prostaglandin E₂ receptor 4 (EP4) on sensory neurons³⁰.

Osteochondral unit damage

Post-traumatic OA (PTOA) is a type of OA that develops following direct injury to the osteochondral unit and accounts for approximately 12% of OA cases. Intra-articular fractures alter joint biomechanics and trigger acute chondrocyte injury, characterized by calcium influx and mitochondrial cytochrome c release, leading to apoptotic cell death and subsequent inflammatory amplification³¹.

Subchondral bone undergoes profound structural and biochemical remodeling during OA progression. Mechanical stress initiates microdamage within the osteochondral unit, with subchondral changes often preceding visible cartilage damage. Osteocytes within the subchondral bone serve as mechanosensors, coordinating signaling among bone, cartilage, and inflammatory cells to regulate bone resorption and formation, maintaining homeostasis. In OA, however, osteocyte morphology and signaling become altered, contributing to dysregulated bone turnover³² and accelerated subchondral remodeling, which has been reported at up to fivefold above normal rates in early disease stages, leading to altered bone density and biochemical signaling³³.

Concurrent vascular invasion from subchondral bone into the calcified cartilage layer introduces angiogenic factors such as vascular endothelial growth factor (VEGF), which stimulates MMP synthesis/secretion of catabolic enzymes. They also degrade cartilage matrix and suppress tissue inhibitors of metalloproteases (TIMPs) that prevent inhibition of the MMPs, leaving cartilage degradation unchecked. As cartilage degrades and thins, mechanical stresses are increasingly transferred to the underlying subchondral bone, promoting osteophyte (i.e., bone spur) formation and sclerosis in an attempt to stabilize the joint³⁴.

OA-associated osteoblasts also secrete elevated levels of interleukin (IL)-6 and PGE2. Aberrant PGE2 production promotes subchondral sclerosis, bone remodeling, angiogenesis, and nociceptive signaling. It also binds to EP4 receptors on sensory nerves and contributes to increased OA pain³⁰. Through cAMP-mediated pathways, PGE2 inhibits chondrocyte proliferation and differentiation while promoting apoptosis, amplifying a degenerative microenvironment at the osteochondral interface³³.

Mechanotransduction pathways are also critical for preserving cartilage homeostasis. Integrin signaling, mechanosensitive ion channels including TRPV4 and Piezo1/2, primary cilia, and Hippo/YAP-associated pathways enable chondrocytes to convert physiologic loading into adaptive anabolic responses. In OA, however, abnormal mechanical stress and inflammatory mediators dysregulate these pathways, promoting catabolic signaling and progressive tissue degeneration^35–38. The effects of Hippo/YAP appear context dependent, with evidence supporting both injurious dedifferentiation under abnormal mechanical conditions and protective maintenance of chondrocyte phenotype in specific microenvironments³⁹.

Inflammation

Chronic low-grade inflammation is a key driver of OA progression. Mechanical stress and matrix damage activate chondrocytes, synoviocytes, and osteoblasts to release a broad spectrum of pro-inflammatory cytokines, lipid mediators, and reactive oxygen species (ROS). Pro-inflammatory cytokines, including IL-1β, IL-6, IL-15, IL-17, IL-18, tumor necrosis factor-α (TNF-α), and leukemia inhibitory factor (LIF), stimulate catabolic gene expression, suppress matrix synthesis, and upregulate MMP and ADAMTS activity, thereby accelerating cartilage matrix degradation.

Synovial macrophages are key modulators of OA inflammation and repair, polarizing into pro-inflammatory M1 macrophages and anti-inflammatory, reparative M2 phenotypes in response to local joint signaling. Polarization of these macrophages is regulated by local signaling within the joint. In OA, an elevated M1/M2 ratio drives sustained pro-inflammatory cytokine release and matrix-degrading enzyme production⁴⁰. Fibroblast-like synoviocytes further contribute to OA progression by producing pro-inflammatory cytokines, matrix-degrading enzymes, and mediators that reinforce synovial inflammation and cartilage degradation. ROS, typically produced at low levels by chondrocytes under homeostatic conditions, become elevated in OA, oxidizing lipids, proteins, and DNA while driving chondrocyte apoptosis, further amplifying inflammatory signaling⁴¹. In additional to oxidative stress, mitochondrial dysfunction compounds this pathology through reduced ATP production, loss of membrane integrity, impaired mitophagy, and the accumulation of damaged mitochondria⁴². Together, oxidative and mitochondrial stress can also drive chondrocyte senescence, which is characterized by cell-cycle arrest and a senescence-associated secretory phenotype. Senescent chondrocytes secrete pro-inflammatory cytokines and matrix-degrading enzymes, which reinforces chronic inflammation and ECM breakdown⁴³. These processes converge on NF-κB and MAPK pathway activation, creating a positive feedback loop that drives continued cytokine release, matrix breakdown, and joint deterioration⁴⁴.

Although anti-inflammatory cytokines such as IL-10, IL-4, IL-13 and TGF-β are present at the OA joint, their concentrations are insufficient to counteract chronic inflammatory signaling. As a result, the joint microenvironment remains persistently catabolic and hostile to tissue regeneration.

Understanding these interlinked degenerative mechanisms is critical for guiding the rational design of biomaterial-based therapies aimed at restoring cartilage structure, modulating inflammation, and re-establishing osteochondral homeostasis. The interconnected nature of these mechanical, inflammatory, and cellular processes motivates the development of multifunctional biomaterial platforms, such as cryogels, capable of addressing multiple aspects of OA pathology simultaneously.

Cryogels: defining features and biomaterials

Cryogels are a subclass of macroporous polymeric hydrogels formed via gelation or crosslinking under frozen conditions. Unlike conventional hydrogels, which are synthesized above freezing and characterized by nano- or microscale pore architectures, cryogels possess highly interconnected macropores (typically 10–300 µm) generated through ice crystal templating during the fabrication process^45,46. This unique architecture confers exceptional permeability^47,48, rapid mass transport^45,49,50, and notable mechanical resilience^51–53, rendering cryogels particularly attractive for biomedical applications that require load-bearing capacity, injectability, and cellular infiltration. These features are particularly relevant in OA, where biomaterials must function within a mechanically active and chronically inflamed joint environment.

Structurally, cryogels exhibit a sponge-like, elastic network that permits substantial compression and shear deformation with rapid recovery upon unloading^54,55. These distinguishing mechanical properties help distinguish cryogels from traditional hydrogels, and have driven growing interest in cryogels as scaffolds for tissue engineering, drug delivery, and injectable biomaterial platforms aimed at musculoskeletal disorders, such as OA⁵⁶.

Fabrication methods

Cryogels are fabricated through cryotropic gelation, or cryogelation, which involves the partial freezing of a polymer precursor solution to induce phase separation⁵⁷. As the temperature is decreased below the solvent freezing point, ice crystals form and drive polymers, crosslinkers, and initiators into a concentrated unfrozen microphase where polymerization or crosslinking occurs, as demonstrated in Fig. 2. Upon thawing, melting of the ice templates yields the characteristic interconnected macroporous network whose morphology reflects that of the ice crystals.

Fig. 2. Cryotropic gelation: from polymer solution to macroporous cryogel scaffold.

Schematic illustration of the cryogelation process, beginning with a liquid polymer precursor solution that undergoes controlled freezing. Ice crystals nucleate and grow within the freezing solution, concentrating polymer chains and crosslinkers into an unfrozen microphase where network formation occurs. Upon thawing, removal of the ice templates, typically via thawing or lyophilization, yields the characteristic interconnected macroporous architecture of the cryogel. This figure was reproduced with permission from Hixon et al.,¹⁰¹. Copyright © 2017 Elsevier.

Pore morphology and mechanical properties can be precisely tuned by modifying freezing temperature, cooling rate, polymer concentration, and crosslinking density, enabling control over pore morphology and mechanical behavior^45,57,58. Importantly, cryogels can be fabricated as preformed scaffolds or synthesized in situ, enabling minimally invasive injectable delivery followed by rapid shape recovery within confined anatomical spaces, such as the joint cavity^59,60.

Biomaterials

Cryogels can be fabricated from a wide range of natural and synthetic polymers selected to achieve desired biological compatibility, degradation kinetics, and mechanical performance. Naturally derived polymers such as gelatin, chitosan, alginate, and HA are widely used due to their inherent biocompatibility^61–64 and, in some cases, intrinsic bioactivity^65,66. HA-based cryogels are particularly relevant for OA applications, given the native presence of HA throughout synovial fluid and cartilage ECM, and its role in joint lubrication and mechanotransduction^67–69.

Synthetic polymers, including poly(ethylene glycol) (PEG), PVA, and methacrylate-based systems, provide greater control over network architecture and reproducibility, as well as tunable mechanical properties and degradation parameters^70–74. Hybrid cryogels combining natural and synthetic components are increasingly employed to balance biological functionality with mechanical robustness, a particularly important consideration for load-bearing joint environments^75,76.

Despite their widespread use, both natural and synthetic cryogel materials present important limitations in the context of OA. For example, HA-based hydrogels are susceptible to rapid enzymatic degradation in inflamed OA joints, potentially limiting durability and therapeutic efficacy. Similar considerations may apply to HA-based cryogels, where degradation kinetics must be carefully controlled to ensure sustained structural and functional performance. Conversely, synthetic polymer-based cryogels offer improved mechanical control and stability, but may elicit immune responses depending on composition and degradation byproducts. These considerations underscore the importance of carefully balancing biocompatibility, degradation kinetics, and host response in cryogel design for OA.

Biomechanical properties

Cryogels exhibit remarkable elasticity and compressibility arising from their interconnected macroporous architecture and thick pore walls. Under compressive loading, cryogels routinely tolerate large strains, often exceeding 80% without permanent deformation, with rapid and near-complete recovery of the original shape with unloading^45,54,77,78. This mechanical resilience stands in contrast to conventional hydrogels, which often exhibit brittle fracture or irreversible deformation under similar loading conditions. The ability of cryogels to undergo substantial deformation and recover elastically is particularly advantageous for injectable and minimally invasive applications, where materials must pass through narrow delivery devices (i.e., needles) and subsequently re-expand within confined anatomical spaces, such as in the joint cavity^60,79–81. Beyond compressive properties, parameters such as shear loading, frictional behavior, wear resistance, and interfacial mechanics are critical for joint function and long-term implant stability in OA, yet remain underexplored in the cryogel literature.

The mechanical stiffness of cryogels can be broadly tuned by adjusting polymer composition, concentration, crosslinking density, and freezing parameters that govern ice crystal size and pore morphology. Higher polymer concentrations and increased crosslinking generally produce stiffer cryogels with thicker pore walls, whereas lower concentrations yield more compliant structures with greater deformability^72,77,78. Freezing parameters influence ice crystal size and distribution which, in turn, affect pore morphology and bulk mechanical behavior. This high degree of tunability enables cryogels to be engineered to approximate the mechanical properties of cartilage and other musculoskeletal tissue^82,83, while preserving sufficient support for drug delivery and regenerative applications^76,84. Such adaptability is particularly relevant in OA, where materials must accommodate heterogeneous tissue mechanics and disease-stage-specific requirements.

Importantly, cryogels demonstrate substantial energy dissipation and fatigue resistance under cyclic mechanical loading, a behavior attributed to pore wall bending, viscoelastic network deformation, and interstitial fluid flow^45,85,86. Many cryogels retain structural integrity across repeated compression cycles with minimal loss of functional performance, a critical consideration for applications in mechanically active joint environments where repetitive loading occurs with daily activities. The ability of cryogels to endure cyclic stresses without catastrophic failure supports their potential utility in OA therapies requiring long-term mechanical reliability.

Their elastic, macroporous structure is also noteworthy, and enables them to function as space-filling scaffolds, load-distributing implants, or sustained drug delivery platforms capable of withstanding joint motion and wear. Additionally, the interconnected pore network facilitates cellular infiltration and nutrient transport for supporting regenerative and immunomodulatory approaches aimed at modifying disease progression. Collectively, these properties position cryogels as promising OA biomaterials capable of functioning simultaneously as space-filling scaffolds, load-distributing implants, and sustained drug delivery platforms, while supporting cellular infiltration, nutrient transport, and immunomodulation within the joint environment.

Advantages over conventional hydrogels

Conventional hydrogels are hydrophilic, 3D cross-linked polymeric networks composed of water and crosslinked natural or synthetic polymers^87–89. These scaffolds have been comprehensively explored in OA therapy as their nanoporous framework enables delivery of drugs or cells in a controlled manner while providing lubricity that can significantly reduce the joint inflammatory symptoms^90–93. By providing a sustained release of these additives, hydrogels are able to support tissue repair processes and help restore the microenvironment of damaged cartilage⁹⁴. However, the reduced polymer density inherent to porous hydrogel architectures compromises mechanical robustness, limiting their utility for sustained load-bearing cartilage repair^95–97. Additionally, the dense nanoscale pore architecture limits nutrient diffusion and cell infiltration^52,98.

In contrast, cryogels possess many characteristics that render them distinctly advantageous for cartilage repair⁹⁹. The cryogelation process produces polymer networks with interconnected macroporous architectures, superior elasticity, and cartilage-mimetic mechanical behavior that enable resistance to repetitive compressive loading^45,82,100. Their macroporosity facilitates deep cellular infiltration and better mass transport of nutrients, oxygen, and metabolites^{52,98,101,102}, while extensive internal surface area strengthens tissue-material interactions⁹⁹. A summary comparison between hydrogels and cryogels relevant to OA treatment is provided in Table 1.

Table 1.

Comparative biomechanical properties between conventional hydrogels and cryogels for use in OA and cartilage repair applications

	Conventional Hydrogels	Cryogels
Pore Architecture	Homogeneous gelation with nano-to microscale pores184	Heterogeneous, interconnected pores, typically 10–300 µm in diameter45,57,58
Fabrication Process	Chemical or physical crosslinking above freezing185	Cryotropic gelation at sub-freezing temperatures57,58
Mass Transport	Diffusion-limited exchange of nutrients and waste184	High permeability and convective transport45
Cell Infiltration	Pore size may limit cell infiltration, restricting cell-scaffold contact to the surface186	Macropores permit cell infiltration throughout the scaffold45
Injectability	Typically injected prior to crosslinking, may have inconsistencies related to in situ gelation187,188	Injectable after crosslinking via pore collapse and wall bending, generally without significant network fracture60,79–81
Load-Bearing Capabilities	Prone to fracture or creep under load-bearing conditions95–97,189,190	High elastic and compressive strength, similar to forces encountered in the native joint45,82,100
Elastic Recovery	Limited, irreversible deformation often occurs with high strain191,192	Rapid recovering even at >80% strain; excellent shape-memory behavior45,85,86
Fatigue Resistance	Generally lower resistance to cyclic loading testing189,193,194	High fatigue resistance under cyclic loading45,85,86
Degradation	Relatively rapid degradation195; better suited for short-term use or acute delivery of bioactive factors	Slower degradation kinetics196; better suited for longitudinal therapeutic application
Primary Limitations in OA	Lower mechanical stability; degradation kinetics limit long-term joint persistence; nanopore architecture restricts cellular integration and nutrient/waste exchange184	Manufacturing scalability and reproducibility remain challenging; translational evidence is very early-stage76,165,197

A recent published study by Qi et al. demonstrated the utility of a gelatin-methacryloyl/graphene oxide (GelMA/GO) composite cryogel as a cartilage-mimicking scaffold for OA repair⁹⁹. The cryogel supported chondrocyte adhesion and proliferation, and significantly improved cartilage regeneration in a 6–8-week-old Sprague Dawley rat knee defect model. Treated joints demonstrated increased expression of chondrogenic markers (i.e., SRY-box transcription factor 9 (SOX9), aggrecan, FAM20B) and reduced inflammatory and oxidative stress-related signaling, highlighting the therapeutic promise of cryogel-based cartilage mimetics.

Notably, recent advances in hydrogel design, including double-network and fiber-reinforced systems, have significantly improved their mechanical performance and functional capabilities, suggesting that distinctions between hydrogels and cryogels may be context-dependent rather than absolute, and that comparative evaluation in disease-relevant models will be important for establishing the added value of cryogel-based approaches.

Despite their many advantages, cryogels are not without limitations. Their macroporous architecture, while beneficial for cell infiltration and mass transport, may also permit vascular ingrowth and compromise the maintenance of a stable cartilage phenotype. Additionally, mechanical properties that are not carefully tuned may create environments that favor hypertrophic or osteogenic differentiation. Challenges related to manufacturing scalability, reproducibility, and regulatory approval further complicate clinical translation. These limitations underscore the need for more nuanced, disease-specific cryogel design strategies rather than assuming universal applicability across OA contexts.

Taken together, these features suggest that the utility of cryogels lies not only in their mechanical advantages, but in their ability to integrate this mechanical resilience with transport efficiency and therapeutic delivery within a single platform. In the context of OA, where mechanical dysfunction and inflammation are tightly coupled, this integration may be particularly important for enabling coordinated structural and biological repair. Collectively, these distinctions position cryogels not as incremental refinements of conventional hydrogels, but as a structurally and functionally distinct next-generation biomaterial class for joint disease applications.

Cryogels for cartilage regeneration

As discussed previously, OA encompasses multiple disease subsets with important implications for treatment development, as therapies effective for one patient subgroup may not be equally beneficial for others. Despite this, current management strategies for OA often remain generalized. In practice, treatment typically follows a stepwise increase in invasiveness, beginning with conservative interventions such as analgesics, bracing, injections, or exercise programs, and progressing to more invasive procedures as symptoms worsen. For many patients, conservative management is piecemeal, with interventions attempted sequentially until some degree of symptom relief is achieved. When these approaches fail, treatment may progress to surgical procedures such as osteotomies, microfracture, or debridement. However, these interventions are aimed at symptom management rather than restoring damaged cartilage. This raises the question of whether regenerative strategies should likewise move beyond a one-size-fits-all approach. In this context, cryogels represent tunable scaffolds with the potential to be adapted to distinct OA phenotypes. Current cryogel-based therapeutic modalities include stem cell-loaded scaffolds, bioactive molecule-loaded delivery systems, gene-activated matrices (GAMs), and immunomodulatory formulations.

Stem cell-loaded cryogels

Unlike conventional pharmacological therapies that deliver a single agent at a fixed dose, MSCs respond to their local environment and adjust their secretion of bioactive factors accordingly, resulting in variable and context-dependent signaling¹⁰³. MSCs have been widely investigated for cartilage repair^104–108 due to their ability to modulate cartilage metabolism, stimulate chondrogenic differentiation, and exert immunomodulatory effects^109–112. In a clinical proof-of-concept study, Jo et al. treated 18 patients with knee OA using intra-articular injections of autologous adipose-derived MSCs (AD-MSCs)¹¹³. The high-dose group (1.0 × 10⁸ cells) showed no treatment-related adverse events, along with improved WOMAC scores and evidence of reduced cartilage defects and hyaline-like cartilage regeneration. At a 2-year follow-up, WOMAC scores in the high-dose group decreased by 39.4% at 6 months, 70.0% at 1 year, and remained improved at 64.9% at 2 years¹¹⁴. Despite these encouraging findings, the direct intra-articular administration of MSCs remains constrained by important limitations, including rapid clearance from the joint space, poor cell survival within the inflammatory milieu, and limited control over post-delivery cell fate and differentiation¹¹⁵. Additionally, allogeneic MSC sources are generally preferred over autologous approaches due to concerns that OA patients, particularly in advanced stages, may not serve as optimal cell donors¹¹⁶. To address these limitations, one strategy has involved pre-conditioning MSCs prior to scaffold-based delivery. Gao et al. pre-conditioned MSCs with Mg²⁺ and the hypoxia-mimetic agent dimethyloxalylglycine (DMOG) before delivery within a self-healing HA-based hydrogel, of which cryogels are a subtype¹¹⁵. Mg²⁺ promoted anti-inflammatory macrophage polarization and bone preservation, while DMOG activated HIF-1α signaling to support chondrogenesis.

Despite promising early results, cell-based therapies have shown only modest success in restoring tissue function, in part because many transplanted cells frequently fail to survive or persist at the target site^117–119. A key limiting factor is the absence of structural support following delivery, leaving cells exposed to mechanical stress and an inflammatory microenvironment¹²⁰. Incorporating cells into microcryogels can help address these challenges by improving cell retention and providing a 3D cell culture environment more closely resembling physiological tissue than monolayer culture systems¹¹⁹. For example, Zhang et al. engineered gelatin microcryogels (GMs) for umbilical cord-derived MSC (UCMSC) delivery in a male 8-week-old C57BL/6 murine medial meniscus destabilization OA model¹¹⁹. Microcryogel-mediated delivery produced superior therapeutic effects compared with free MSC injection, attributed to improved intra-articular cell retention and prolonged modulation of cartilage homeostasis and synovial inflammation. This approach was similarly investigated by Xing et al., who loaded 1 × 10⁵ UCMSCs into microcryogels and administered them intra-articularly to 10-week-old Sprague Dawley rats with experimentally induced bilateral OA, via anterior cruciate ligament transection¹²¹. Treated animals showed significantly improved International Cartilage Repair Society macroscopic scores and pronounced proteoglycan deposition at 4 and 8 weeks, as well as reduced cartilage loss and radiographic changes (Fig. 3). The therapeutic benefits were attributed to the microcryogels microenvironment enhancing MSC paracrine activity while promoting cell-cell and cell-ECM interactions, increasing the release of immunomodulatory factors and exosomes while shielding cells from mechanical stress^122,123.

Fig. 3. Microcryogel-mediated MSC delivery improves macroscopic and radiographic outcomes in a rat osteoarthritis model.

A Macroscopic and B radiographic images of murine femoral condyles across treatment groups, demonstrating reduced cartilage loss and preserved joint architecture in microcryogel-treated animals relative to controls. Adapted from Xing et al., under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0)¹²¹.

Across studies, a consistent trend emerges: microcryogel-based systems improve MSC retention and prolong paracrine signaling, suggesting that structural support is a key determinant of therapeutic efficacy. However, these findings also highlight that the primary limitation of MSC-based therapies is not delivery efficiency alone, but also the inability to precisely control cell fate and function within an inflammatory OA microenvironment. Effective cryogel design must therefore extend beyond passive cell delivery to actively regulate biochemical and mechanical cues governing MSC behavior under pathological conditions.

Through these ideas, several design considerations emerge for stem cell-based cryogel systems in OA. First, scaffold architecture and macroporosity must be optimized to improve cell retention while preserving nutrient transport and enabling dynamic environmental cell-matrix interactions. Second, mechanical properties should be tuned to support chondrogenic differentiation while avoiding stiffness profiles that promote hypertrophy or osteogenic drift. Third, incorporation of bioactive or immunomodulatory cues will likely be necessary to stabilize cell phenotype and counteract the hostile inflammatory microenvironment. Finally, most current studies rely heavily on histological endpoints, with limited evaluation of long-term functional or biomechanical restoration, representing a critical barrier to clinical translation that will require more robust in vivo modeling to better recapitulate the human OA environment.

At this stage, long-term efficacy of cell-laden cryogels have not been reliably demonstrated for OA-specific outcomes. Given the hostile inflammatory environment of the OA joint, future work on cell-based cryogel therapies should prioritize integrated design strategies that couple structural support with precise control over cell fate and microenvironmental signaling, as simple delivery of cellular components is unlikely to achieve durable therapeutic benefit within the OA joint.

Bioactive molecule-loaded cryogels

Cryogels exhibit shape-memory behavior that permits compression-based minimally invasive delivery followed by rapid structural recovery in situ¹²⁴. Their durable polymer network provides mechanical resilience and swelling capacity to withstand repeated joint loading¹²⁵, enabling cryogels to function not only as passive scaffolds but also as local delivery platforms for therapeutic molecules within mechanically active joint environments¹²⁶.

To assess how these advantages translate in vivo, Chen et al. developed an injectable, acellular tri-layer cryogel scaffold with adaptable spatially segregated delivery of two small-molecule therapeutics, Y27632 and dexamethasone, incorporated into separate layers¹²⁴. Y27632, a Rho-kinase inhibitor, improves MSC chondrogenesis and suppresses hypertrophic differentiation, while dexamethasone serves as a potent in vitro osteoinductive and immunomodulatory factor. This platform was combined with scalp acupuncture to augment MSC mobilization and immune modulation, and tested in a 12-week-old male New Zealand white rabbit knee osteochondral defect model. Animals receiving the combined cryogel-based therapy exhibited significantly improved regeneration of both cartilage and bone, increased M2/M1 macrophage ratios, and elevated stromal cell-derived factor-1 (SDF-1) and TGF-β1 expression. Similarly, Yang et al. fabricated an injectable hydrogen-bonded hydrogel with dual release of tannic acid (TA; rapid) and kartogenin (KGN; sustained)¹²⁷. In a rabbit full-thickness articular cartilage defect model, they showed decreased ROS and pro-inflammatory markers (IL-1β, TNF-α), increased MSC recruitment and chondrogenic differentiation, formation of hyaline-like cartilage with elevated collagen II and glycosaminoglycan (GAG) deposition, and improved subchondral integration. A related approach by Zhang et al. employed an Arginine-Glycine-Aspartic acid (RGD)-modified HA methacryloyl (HAMA-RGD) hydrogel delivering KGN, a chondrogenic inducer, and Klotho, an anti-aging protein with antioxidant and anti-inflammatory activity, with sustained release¹²⁸. In a rat cartilage defect model, this reduced ROS, downregulated MMP13 and ADAMTS5, induced M2 macrophage polarization, improved MSC recruitment and KGN-driven chondrogenesis, and improved cartilage regeneration. Notably, sustained or supraphysiologic TGF-β1 signaling carries inherent risks of hypertrophic differentiation and ectopic mineralization, a concern revisited in the context of gene-based delivery strategies below. While these approaches demonstrate promising biological effects, their relevance to chronic OA remains uncertain, as many studies are conducted in acute or non-inflammatory models that do not reflect the native joint environment.

For broader applicability in cartilage repair, Huang et al. developed injectable gelatin-glucosamine cryogel microbeads for chondrocyte delivery¹²⁹. These squeezable cryogel beads were fabricated via emulsion cryogelation and supplemented with glucosamine to promote chondrocyte redifferentiation and cartilage-specific ECM synthesis. The resulting microbeads exhibited high porosity, supported chondrocyte viability and proliferation, and improved matrix deposition in a dose-dependent manner. When chondrocyte-seeded microbeads were injected subcutaneously into 5-week-old male BALB/c nude mice, ectopic neocartilage formation was observed within 21 days (Fig. 4).

Fig. 4. Progressive cartilage ECM deposition in chondrocyte-loaded cryogel microbeads over 21 days post-implantation.

A Hematoxylin and Eosin (H&E) staining of acellular cryogel microbeads. Comparison of acellular and cellular (chondrocyte-loaded) cryogel microbeads with B H&E, C Alcian Blue, D Safranin O, E Collagen Type II, and F Aggrecan staining at days 7, 14, and 21 post-implantation. Chondrocyte-loaded microbeads demonstrate sustained cell viability and progressive deposition of cartilage-specific ECM proteins, including collagen type II and aggrecan, consistent with maintenance of chondrocyte phenotype and function following implantation. This figure was reproduced with permission from Huang et al.,¹²⁹. Copyright © 2023 Elsevier.

Targeted growth factor delivery has also been achieved using cryogel systems designed to mimic native cartilage ECM composition and mechanics. Chen et al. fabricated triple-polymer macroporous cryogels composed of chondroitin sulfate, HA, and carboxymethylcellulose (CHC)¹³⁰. Through bioaffinity interactions with chondroitin sulfate, the cryogels were functionalized to anchor TGF-β1 and insulin-like growth factor-1 (IGF-1), achieving sustained release of both growth factors for over 21 days. ASCs seeded onto these growth-factor-loaded cryogels demonstrated improved chondrogenic differentiation, evidenced by elevated SOX9, COL2A1, aggrecan, and proteoglycan 4 (PRG4) expression, as well as elevated GAG and type II collagen deposition. When implanted into full-thickness cartilage defects in the knees of 6- to 8-week-old male New Zealand white rabbits, the optimized cryogels promoted cartilage regeneration and restored mechanical properties comparable to native articular cartilage. However, bioaffinity-mediated retention may restrict timely growth factor release and bioavailability, while the reliance on ASC seeding in an acute defect model limits the translational relevance of these findings.

Across the bioactive molecule-loaded cryogel literature, a consistent finding is that cryogel-based systems improve delivery precision and prolong presentation of bioactive factors, improving chondrogenic signaling and inflammatory modulation. However, most studies focus on single-delivery evaluated in models that do not adequately represent the complexity of OA, such as short duration, in vitro, or small animal in vivo designs. Given that OA pathology features persistent catabolic signaling, single-factor and short-duration delivery strategies are unlikely to achieve durable, disease-modifying treatment outcomes. Future designs will likely require multi-factor delivery with temporally controlled release profiles to more adequately address OA disease pathophysiology.

Gene therapy cryogels

Gene therapy has gained attention as a strategy for cartilage repair due to its ability to enable localized, sustained expression of therapeutic proteins directly at the injury site^131–133. Compared with recombinant protein delivery, gene-based approaches offer prolonged biological activity while reducing the need for repeated dosing. Non-viral vectors are particularly attractive given their avoidance of risks associated with viral carriers, including insertional mutagenesis immunogenicity and regulatory complexity^134,135. However, their therapeutic efficiency is limited by extracellular degradation, inefficient cellular uptake, endosomal entrapment, and poor nuclear trafficking^136,137. Importantly, GAMs address these limitations by providing a localized microenvironment that concentrates vectors at the defect site, protects genetic cargo, and promotes cell-matrix interactions that facilitate uptake and transfection. Cryogels represent an ideal scaffold class for GAM development due to their macroporosity, injectability, and capacity for sustained release of genetic material.

Carballo-Pedrares et al. developed gene-activated HA cryogels (G-HACGs) designed to locally transfect MSCs using niosome-based non-viral vectors (P80PX) loaded with SOX9-encoding plasmid DNA¹³⁸. Incorporation of the SOX9 plasmid enabled in situ chondrogenic differentiation of MSCs. The cryogels exhibited suitable porosity, shape-memory behavior, and mechanical properties compatible with joint loading resulting in supported sustained gene release, efficient MSC transfection with low cytotoxicity, and improved expression of hyaline cartilage markers in an ex vivo human osteochondral defect model (Fig. 5). More recently, Liu et al. developed an alginate hydrogel system incorporating recombinant adeno-associated virus (rAAV) vectors to deliver SOX9 and TGF-β genes to human OA articular chondrocytes and cartilage explants, achieving sustained transgene expression for up to 21 days¹³⁹. In vitro and ex vivo, SOX9 and TGF-β overexpression increased GAG/proteoglycan and type II collagen deposition, improved cell proliferation (TGF-β), reduced type I and type X collagen expression, and promoted a chondrogenic phenotype, though matrix mineralization was also observed.

Fig. 5. Gene-activated hyaluronic acid cryogels restore cartilage matrix markers in an ex vivo human osteochondral defect model.

HACG (+) denotes the control group of HA cryogels seeded with human MSCs. G-HACG denotes gene-activated HA cryogels seeded with MSCs, further divided into two treatment groups: P80PX (niosomal gene-activated cryogels) and LPF (positive control containing a commercial transfection reagent). A Immunohistochemical analysis comparing SOX9, Type II Collagen, Safranin-O, Type I Collagen, and Type X Collagen staining across treatment groups. B Macroscopic images of the ex vivo defect model showing an unfilled (empty) defect (left) and a filled defect (right). C Quantification of immunohistochemical staining from (A), demonstrating improved expression of hyaline cartilage markers in gene-activated cryogel groups relative to controls. This figure was reproduced from Carballo-Pedrares et al., under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC BY-NC-ND 4.0)¹³⁸.

Similarly, Odabas et al. combined non-viral bone morphogenic protein (BMP)-7 gene delivery with gelatin-oxidized dextran cryogel scaffolds for auricular cartilage repair¹⁴⁰. Primary chondrocytes transfected ex vivo with a BMP-7 plasmid and implanted into 12-week-old male rabbit critical-sized ear defects demonstrated significantly improved matrix synthesis and elastic cartilage regeneration compared with non-transfected and scaffold-only controls. These findings underscore the capacity of cryogel-based GAMs to provide spatially confined, sustained gene delivery while minimizing systemic exposure.

Gene-activated cryogel systems represent an emerging and promising area of OA tissue engineering. Despite these encouraging outcomes, several limitations constrain translational potential. Non-viral vectors such as niosomes exhibit low and spatially heterogeneous transfection efficiency in dense cartilage matrices, where limited cell infiltration and ECM barriers restrict vector uptake^141,142. Moreover, control over transgene expression also remains limited¹⁴³; sustained SOX9 or TGF-β overexpression can promote hypertrophy and ectopic mineralization¹⁴⁴, as reflected by increased type X collagen deposition. Similar concerns apply to TGF-β overexpression, underscoring that growth factor-driven chondrogenesis strategies, whether delivered as recombinant protein or via gene vector, require downstream regulatory mechanisms to prevent hypertrophic drift. While cryogels have improved localization, retention, and uptake of delivered gene vectors, the primary concern lies with the gene therapy mechanism itself; specifically, the lack of downstream regulatory strategies to finely tune expression magnitude and duration. In the context of OA, where homeostatic disruption already exists, uncontrolled or prolonged transgene signaling could exacerbate OA features rather than provide stable cartilage regeneration. Future cryogel designs for gene-based therapies must prioritize spatiotemporal control of gene expression, potentially through tunable delivery systems or feedback-responsive mechanisms, while accounting for the dense and avascular nature of cartilage.

Immunomodulatory cryogels

OA progression is driven not only by mechanical cartilage degeneration but also by a dysregulated inflammatory microenvironment within the joint¹⁴⁵. Synovial macrophages polarized toward a pro-inflammatory M1-like phenotype secrete high levels of IL-1β, TNF-α, and IL-6^40,146,147, upregulating matrix-degrading enzymes and accelerating articular cartilage breakdown¹⁴⁸. Concurrently, inflammatory signaling suppresses anabolic pathways and promotes chondrocyte senescence and apoptosis, further amplifying tissue degeneration^43,149,150. Synovitis exacerbates OA progression by increasing immune cell infiltration and chemokine secretion within the joint space^151,152.

Exosomes have emerged as important regulators of immune and regenerative signaling in this context. Exosomal miRNAs with osteogenic and anti-inflammatory functions have gained interest in OA therapy; for example, miR-5106 from M2 macrophage-derived exosomes promote BMSC osteogenic differentiation via SIK2/3 targeting, while miR-1260b-containing exosomes inhibit osteoclastogenesis and improve M2 macrophage polarization¹⁵³. Exosomes mediate intercellular communication by delivering bioactive cargo including proteins, lipids, and nucleic acids¹⁵⁴. MSC-derived exosomes additionally promote angiogenesis and macrophage polarization, creating a pro-regenerative microenvironment¹⁵⁵.

Recent work has explored cryogels as localized immunomodulatory platforms capable of reshaping this pathological inflammatory milieu. Yang et al. developed an ECM-mimetic cryogel composed of gelatin, chondroitin sulfate, and HA, loaded with human UCMSC-derived exosomes^107,156. In vitro, exosome-loaded cryogels improved chondrocyte proliferation and increased sulfated GAG deposition. In a 12-week-old healthy male rabbit articular cartilage defect model, these exosome-loaded cryogels yielded organized chondrocyte layering and smooth restoration of the articular surface.

Similarly, Nikhil et al. fabricated chitosan-gelatin-chondroitin sulfate (CGC) cryogels for sustained delivery of chondrocyte-derived exosomes, releasing both chondroprotective chondroitin sulfate and adsorbed exosomes over several days, synergistically enhancing chondrocyte proliferation and accelerating wound closure in scratch assays¹⁵⁷. Advancing this approach, Nikhil et al. engineered a four-layer osteochondral cryogel comprising three CGC cartilage layers with defined pore alignments and a nanohydroxyapatite-gelatin bone layer, loaded with goat chondrocyte-derived exosomes¹⁰⁸. These were implanted into 8 × 10 mm critical-size femoral condyle defects in 1.5–2-year-old male bakerwal breed goats, followed by an intra-articular exosome booster in artificial synovial fluid. This treatment achieved robust long-term (6–12 month) regeneration of stratified hyaline cartilage and well-integrated subchondral bone, highlighting the capacity of cryogel-based immunomodulatory platforms to promote durable osteochondral regeneration. Importantly, variability in exosome composition and insufficient standardization in isolation and dosing protocols introduces significant challenges in reproducibility and cross-study comparison.

Across these preclinical studies, cryogel-based platforms consistently demonstrated evidence of inflammatory microenvironment modulation, including reduction of pro-inflammatory cytokines and promotion of M2-like macrophage phenotypes. However, the magnitude and consistency of these effects vary across systems, reflecting differences in biologic cargo, dosing strategies, and cellular uptake, which are challenges that must be addressed to achieve predictable immunomodulatory outcomes.

Stratified and layered cryogels

Articular cartilage exists as part of a complex osteochondral unit characterized by both abrupt and gradual compositional, mechanical, and biological gradients transitioning across hyaline cartilage, calcified cartilage, and subchondral bone¹⁵⁸. In advanced OA, significant alterations to both cartilage and subchondral bone must be considered in therapeutic design. Further recapitulating this complex pathologic process in early preclinical modeling remains challenging; as a result, acute osteochondral defect models are commonly employed as controlled proxies that accelerate the evaluation of structural repair and regenerative potential, though they do not capture the chronic inflammatory and degenerative features of established OA^159–161. Furthermore, osteochondral damage progresses in a spatially heterogeneous manner, necessitating biomaterial strategies that address multiple tissue compartments simultaneously. Failure to recapitulate these critical interfaces remains a persistent limitation of homogeneous scaffolds, frequently yielding inadequate mechanical integration and risk of delamination¹⁶². To address this, cryogel-based strategies are increasingly moving toward layered, spatially stratified designs that closely recapitulate native osteochondral architecture. By harnessing the versatility of cryogelation, layer-specific control can be achieved over polymer composition, mineral content, pore architecture, and therapeutic loading, supporting coordinated regeneration of cartilage and bone while preserving native interface integrity.

For example, Nikhil and Kumar’s bi-layered osteochondral cryogel was fabricated in a single cryogelation process, consisting of a CGC cartilage layer integrated with a nano-hydroxyapatite-gelatin layer¹⁵⁷. The CGC layer was designed to mimic cartilage ECM composition and support chondrocyte viability, while the hydroxyapatite-gelatin layer recapitulated the mineralized subchondral environment and promoted osteogenic activity. Notably, the bi-layered cryogel system also served as a bioactive delivery platform, releasing chondrocyte-derived exosomes and chondroitin sulfate to improve cell migration and proliferation. This bi-layered design enabled simultaneous support of chondrocyte and osteogenic cell populations while maintaining mechanical integrity across the interface.

Kang et al. explored further complexity through a functionally graded tri-layered scaffold comprising a biomineralized cryogel base, an anisotropic cryogel middle layer, and a hydrogel cartilage surface¹⁶³. The mineralized base was designed to recruit endogenous osteocytes and drive in situ bone formation, while the macroporous cryogel middle layer supported cell infiltration and load transfer. Lastly, the hydrogel layer was loaded with MSC aggregates to facilitate chondrogenesis and formation of a lubricin-rich cartilage surface. Implantation in 3-month-old immunodeficient mice yielded spatially organized osteochondral tissue with distinct cartilage and bone regions, highlighting the ability of cryogel-based architectures to mediate coordinated regeneration through spatially confined biochemical and mechanical cues¹⁶³. The integration of cryogels with conventional hydrogels in this design highlights the potential for hybrid approaches that leverage the complementary strengths of both scaffold types.

Though not explicitly multilayered at the tissue-level or macroscopic scale, directional cryogel architectures have also been explored to recapitulate the depth-dependent organization of native cartilage. Yamamoto et al. designed a directionally frozen collagen-HA double-network cryogel mimicking the arcade-like collagen fiber arrangement of native cartilage⁸³. A pin-based freezing setup was used to guide ice crystal growth for generation of anisotropic pore structures which corresponded to superficial, middle, and deep cartilage zones, improving mechanical performance and providing depth-dependent structural cues to chondrocytes⁸³. Such approaches may complement multilayer osteochondral systems by addressing hierarchical organization across multiple length scales.

Throughout the studies examined, spatial control over scaffold architecture has consistently improved tissue organization, though whether this translates to improved long-term functional outcomes remains to be established^83,157,163. Notably, these layered cryogel systems have been evaluated primarily in acute defect models rather than chronic OA environments, where persistent inflammation, matrix degradation, and altered biomechanics may limit scaffold performance and integration in ways that acute models cannot capture. While stratified scaffold designs are promising, the extent to which cryogels provide distinct functional advantages over other multilayered biomaterial systems remains unclear and will require comparative validation in disease-relevant models.

Nonetheless, layered and stratified cryogel systems retain notable value in their ability to spatially regulate mechanical properties, cellular interactions, and biochemical signaling within a single platform¹⁶⁴. For OA applications, future designs must move beyond architectural complexity alone and demonstrate functional advantages in disease-relevant models, including improved load distribution, native tissue integration, and sustained performance under inflammatory conditions. The optimal level of structural complexity remains an open question, as increasingly intricate designs may introduce manufacturing challenges without proportional therapeutic benefit¹⁶⁵, which is a consideration of particular relevance for clinical translation, where scalability, reproducibility, and cost-effectiveness will strongly influence adoption.

Design principles for cryogels in OA

Synthesizing the therapeutic modalities reviewed above, four overarching design principles emerge for cryogel-based OA therapies (Fig. 6). The first principle is mechanical-biological coupling: cryogels must be designed to approximate the mechanical characteristics of the native joint (i.e., stiffness, elasticity, porosity, degradation)^54,77,82,83 without compromising biologic function such as cellular and nutrient infiltration^52,98,101. The ideal cryogel will likely require a balanced middle ground, where neither mechanical nor biological performance is sacrificed at the expense of the other. This balance distinguishes cryogels from many other tissue engineering strategies in OA, which more commonly prioritize biological or pharmaceutical delivery over the mechanical behavior of the delivery system itself^{19,21,22,52,98,166}. Cryogels are uniquely positioned to address both simultaneously through thoughtful design⁹⁸.

Fig. 6. Four guiding design principles for cryogel-based OA therapy.

Schematic overview of the optimal design framework for cryogel platforms in OA, organized around four interconnected principles: mechanical-biological coupling, microenvironmental control, spatiotemporal precision, and multifunctionality. Together, these principles guide the development of next-generation, disease-modifying cryogel systems capable of addressing the coupled mechanical, inflammatory, and degenerative processes underlying OA progression. Figure created with Biorender.com.

The second principle is microenvironmental control: it is insufficient to simply incorporate cells or bioactive factors; the design must anticipate how these additives will behave within the diseased joint. For example, MSCs delivered into an OA joint are likely to encounter persistent inflammatory signaling^40–43 that may impair survival, promote phenotypic instability, or limit efficacy^{115,117–119} without additional protective design features. Future cryogel systems must therefore actively modulate inflammation and provide a protective niche with regulated microenvironmental signaling, as passive delivery of cellular or biological components is likely to be insufficient to achieve or maintain a clinical therapeutic effect in an OA joint.

The third principle is spatiotemporal precision: thoughtful, anticipation-driven design must determine where and how long a cryogel-delivered therapeutic exerts its effect^124,125. This is especially important for stage-specific therapy, as early-stage OA may benefit from localized, shorter-acting delivery for focal repair, whereas late-stage OA may require broader, sustained modulation of joint inflammation and microenvironment^27,167.

The fourth principle is multifunctionality: cryogel platforms are uniquely positioned to integrate cellular, biological, and immunomodulatory strategies within a single scaffold, yet this potential remains underrepresented in current OA-focused studies. Most current cryogel studies in OA focus on a single modification such as cells or growth factors but not typically both^119,121,124, and future designs would benefit from multipronged design approaches, to achieve multiplicative effects, as has been demonstrated with hydrogel systems¹⁶⁸. For example, achieving successful integration and function of MSC-laden cryogels would be substantially improved through co-delivery of growth factors and immunomodulatory agents to more predictably control MSC behavior within the diseased joint space^169–171. Cryogel-based OA therapies are well-positioned to serve as multifunctional platforms for disease modification, rather than single monotherapy delivery systems.

While most cryogel-based studies in OA have demonstrated promising histological and molecular outcomes, comprehensive evaluation of long-term functional and biomechanical restoration remains lacking^{117–119,121}. Significant and promising preclinical research is nonetheless ongoing, and larger in vivo studies are anticipated as a critical next step as cryogels gain broader acceptance in OA tissue engineering¹⁷². Given the heterogeneity of OA disease^27,167, it remains unlikely that a single, unifying cryogel solution will be optimal across all patients and disease states. Rather, future advances will depend on tailoring platforms according to the four guiding design principles, supported by robust preclinical evidence.

Challenges, limitations, and future directions

Despite their considerable promise, cryogel-based platforms for OA therapy remain at an early stage of translational development, and several scientific, technical, and regulatory challenges must be addressed before widespread clinical adoption can be achieved. Although hydrogels have dominated OA biomaterials research for more than two decades, providing valuable insights into intra-articular delivery, cell encapsulation, and bioactive signaling, persistent limitations in mechanical robustness, long-term retention, and cellular infiltration suggest that conventional hydrogel platforms are approaching a translational plateau in mechanically demanding joint environments^{93,126,173,174}. Cryogels should therefore be recognized not as incremental variations of hydrogels, but as a structurally and functionally distinct second-generation biomaterial class capable of overcoming these longstanding barriers.

A central challenge for cryogel-based OA therapy lies in the biological and clinical heterogeneity of OA itself¹⁷⁵. OA encompasses a spectrum of disease phenotypes driven by mechanical overloading, inflammatory dysregulation, metabolic dysfunction, aging, and trauma; consequently, no single biomaterial strategy is likely to be universally effective across patient populations or disease stages¹⁷⁶. Precision cryogel designs that tailor scaffold mechanics, degradation kinetics, and therapeutic agents (e.g., cells, biological factors, pharmaceuticals) to specific joint location, disease phenotypes, or stages of progression will be essential. Given the tunability of cryogels via pore architecture, crosslinking density, and polymer composition, these systems are well-positioned to support personalized regenerative strategies.

From a biomechanical perspective, cryogels offer clear conceptual advantages over traditional hydrogels by coupling injectability with elasticity and fatigue resistance^45,56,76,177. However most studies evaluate cryogel performance over relatively short experimental windows, whereas OA is a chronic condition characterized by decades of cyclic joint loading. Larger animal models and longitudinal studies are required to establish long-term fatigue resistance, functional integration, and degradation behavior in vivo. Future cryogel designs may incorporate adaptive or dynamic mechanical properties, including mechanosensitive crosslinking, self-reinforcing polymer networks, and load-responsive degradation profiles, enabling materials to remodel in response to evolving joint biomechanics. Although promising early work has emerged in related biomaterial fields, adaptive cryogels remain largely unexplored in OA applications^85,178.

Cell-based cryogel therapies present translational challenges related to donor variability, manufacturing scalability, storage stability, and regulatory complexity. While stem cell-laden cryogels demonstrate better cell retention, viability, and immunomodulatory capacity compared with free-cell or hydrogel-based delivery, the field is likely to increasingly favor acellular or minimally cellular strategies that harness endogenous repair mechanisms^{59,60,95,100,179}. Such strategies may offer a more clinically tractable and regulatory-friendly pathway without sacrificing the mechanical advantages of cryogels, focusing instead on recruitment of host progenitor cells^122,180, modulation of macrophage polarization^181–183, and localized delivery of gene-activated¹³⁸ or exosome-based therapies^107,108,157.

A critical frontier lies in achieving spatiotemporal control over therapeutic signaling. Native articular cartilage exhibits depth-dependent gradients in ECM composition, stiffness, oxygen tension, and cellular phenotype, which are features that remain difficult to replicate using homogeneous hydrogel constructs. Advanced cryogel architectures incorporating zonal organization, anisotropic pore alignment, and bioactive gradients may better recapitulate osteochondral complexity. Emerging fabrication strategies, such as directional freezing⁸³ and multilayer cryogel assembly^108,124,163, offer promising routes toward structurally complex implants capable of guiding organized tissue regeneration rather than disordered matrix deposition.

Beyond structural repair, cryogels have the potential to transform OA treatment paradigms from symptom management to disease modification. Future cryogel systems may function as “smart” intra-articular platforms capable of sensing and responding to pathological cues. Incorporation of stimuli-responsive elements that release therapeutics in response to enzymatic activity, inflammatory mediators, or mechanical stress could enable on-demand delivery, minimizing off-target effects and improving long-term efficacy.

Finally, clinical translation of cryogel-based OA therapies will require substantial progress in manufacturing, standardization, and regulatory alignment. Cryogels are sensitive to processing variables such as freezing rate, temperature gradients, and polymer concentration, necessitating tightly controlled and reproducible production pipelines. Development of GMP-compliant manufacturing workflows, standardized mechanical and biological performance benchmarks, and early regulatory engagement, recognizing cryogels as multifunctional combination products rather than conventional scaffolds, will be essential for safe and scalable clinical deployment¹⁶⁵.

Conclusion

Cryogels represent a powerful and versatile class of biomaterial scaffolds uniquely suited to address the multifactorial pathophysiology challenges of OA. Their macroporous architecture, injectability, and mechanical resilience enable functions that extend beyond those of conventional hydrogels, supporting deep cellular infiltration, efficient mass transport, and sustained therapeutic delivery within mechanically active joint environments.

As demonstrated by the emerging preclinical studies in this review, cryogel-based platforms can be engineered to deliver cells, bioactive molecules, genes, and immunomodulatory signals, providing opportunities to both regenerate articular cartilage and actively modulate the inflammatory milieu that drives disease progression. These systems, therefore, offer not merely symptomatic relief but the potential for true disease-modifying intervention.

While significant challenges remain, particularly with respect to long-term durability, scalability, and clinical translation, the tunability and multifunctionality of cryogels position them as compelling next-generation platforms for disease-modifying OA therapies. Continued interdisciplinary research spanning biomaterials science, mechanobiology, and translational medicine will be essential to realize their full therapeutic potential. Through their ability to integrate mechanical support with biological instruction, cryogel-based systems have the capacity to move OA treatment beyond symptom control and toward durable joint preservation and functional restoration.

Author contributions

J.A.D., M.E.T.G., A.E.L.C., and A.R.P. contributed to investigation. J.A.D. and M.E.T.G. wrote the original draft. A.E.L.C. contributed to original draft preparation and manuscript revision. A.R.P. contributed to manuscript revision and visualization. K.R.H. conceived the study and led project administration, supervision, and resource acquisition, and contributed to investigation, visualization, and manuscript revision. All authors reviewed and approved the final manuscript.

Data availability

No datasets were generated or analyzed during this study.

Competing interests

The authors declare no competing interests.