Advances in Injectable miRNA-Loaded Nanocomposite Hydrogel Systems for Cartilage Repair in KOA

Abstract

Knee osteoarthritis (KOA) is a common degenerative joint disease characterized by irreversible cartilage injury, progressive tissue degeneration and compromised joint function. Conventional clinical treatments merely relieve symptoms rather than fundamentally repair damaged cartilage. As an innovative and promising therapeutic strategy, microRNA (miRNA)-based gene therapy modulates key pathogenic signaling pathways to improve core KOA pathological changes, including chondrocyte senescence, ECM degradation, synovial inflammation and aberrant subchondral bone remodeling. Nevertheless, the clinical application of bare miRNAs is greatly constrained by intra-articular biological barriers. Once delivered into the knee joint cavity, unprotected miRNAs are readily degraded by endogenous nucleases within synovial fluid, accompanied by off-target diffusion, undesired local inflammatory reactions, and rapid in-vivo clearance. Repeated injections are thus required to maintain effective drug concentration, which inevitably causes additional trauma to joint tissues and further compromises therapeutic performance. Injectable miRNA-nanocomposite hydrogel systems organically combine gene therapy, nanotechnology and biomaterial engineering, enabling localized intra-articular delivery, long-term sustained-release behaviour and lesion-oriented cartilage repair. Such biomaterial platforms are capable of remodelling the disordered intra-articular immune microenvironment and restoring subchondral bone homeostasis, so as to mitigate KOA-derived pathological lesions. Notwithstanding these promising advances, existing hydrogel formulations still face considerable bottlenecks in mechanical adaptability, cartilage-barrier penetration performance and long-term biosafety, which constitute major obstacles toward clinical translation. This review systematically summarizes the design strategies, functional modifications and repair mechanisms of these hydrogel systems, aiming to deepen mechanistic understanding, provide theoretical evidence for translational research, and promote the development of precise and individualized targeted therapies for KOA.

Plain Language Summary

Revolutionary Treatment: The miRNA-nanocomposite hydrogel systems integrates the advantages of gene therapy, nanotechnology and biomaterials. It has become a transformative progress in the field of KOA cartilage repair. This system achieves functional cartilage regeneration, breaks through the limitations of traditional symptomatic treatment, and establishes a new paradigm for minimally invasive and precise treatment.

Intelligent Drug Delivery Mode: The miRNA-nanocomposite hydrogel systems represents a paradigm shift towards minimally invasive, targeted and long-lasting cartilage repair. This injectable and responsive preparation can be used under outpatient conditions, reducing the frequency of administration. It restores joint structure and function without relying on exogenous cells or growth factors.

Promoting Clinical Transformation: Although it still faces challenges such as mechanical property matching, long-term biosafety and large-scale preparation, its excellent preclinical research results have demonstrated great transformation potential. It promotes the development of precision regenerative medicine for degenerative diseases of the musculoskeletal system.

Graphical Abstract

Introduction

KOA is a major degenerative disease affecting human health worldwide. Its core pathological changes center on the degeneration of cartilage tissue. Multiple factors, including excessive mechanical loading, uncontrolled inflammatory responses, and imbalanced oxidative stress, contribute to dysfunction of chondrocytes and an imbalance between synthesis and degradation of the extracellular matrix (ECM). These events eventually lead to joint pain, limited mobility, and even loss of function.^1–3 Statistics show that approximately 500 million people globally are affected by KOA. Its prevalence continues to rise with population aging, increasing obesity rates, and a higher incidence of sports injuries. In the United States, the annual cost associated with KOA exceeds $213 billion due to lost productivity and long-term healthcare demands. In China, the prevalence of KOA among people over 60 years old has reached more than 30%, imposing a heavy burden on the social healthcare system.⁴

Current clinical treatments have significant limitations. Oral nonsteroidal anti-inflammatory drugs only relieve pain, but long-term use tends to cause side effects such as gastrointestinal injury and abnormal liver and kidney function. Intra-articular injection of sodium hyaluronate only improves joint lubrication and pain relief in the short term and cannot halt the progression of cartilage degeneration.⁵ Surgical treatments such as microfracture and cartilage transplantation face problems including donor shortage, poor interface integration, and high invasiveness, which can hardly meet the clinical requirements for precise and minimally invasive therapy.⁶ Therefore, the development of novel therapeutic strategies that can targetedly regulate chondrocyte function and rebalance matrix homeostasis has become the key method to delay or reverse the progression of KOA. Gene therapy provides a novel strategy for the treatment of KOA. As a class of non-coding small RNAs, miRNAs can exert multi-dimensional effects by targeting and regulating the expression of key genes.⁷ These effects include inhibiting chondrocyte senescence, modulating inflammatory responses, and maintaining matrix metabolic homeostasis. Thus, miRNAs have become core targets for KOA gene therapy.⁸ With the iterative improvement of high-throughput sequencing and molecular validation technologies, a panel of therapeutic miRNAs highly associated with the pathological progression of KOA has been gradually identified and validated.⁹ By targeting specific downstream functional genes, these miRNAs modulate the activation or inhibition of canonical signaling pathways and precisely mediate characteristic pathological changes of KOA, highlighting the unique therapeutic value of miRNAs in knee joint lesions. In the regulation of articular cartilage degeneration, multiple protective miRNAs can target and suppress the aberrant expression of cartilage-damage-related genes, regulate chondrocyte survival and matrix metabolic homeostasis, and effectively delay the degenerative progression of KOA cartilage.¹⁰ For instance, miR-140 directly targets downstream genes, markedly inhibits the over-activation of matrix metalloproteinases and aggrecanases, reduces the degradation of core cartilage matrices including type II collagen and aggrecan, and preserves the structural integrity of cartilage tissue.¹¹ miR-34a targets specific genes to activate the PI3K/Akt signaling pathway, alleviates oxidative-stress-induced damage and apoptosis in chondrocytes, enhances chondrocyte proliferative activity, and maintains normal physiological functions of knee articular cartilage.¹² miR-199a-3p targets and inhibits gene expression to block abnormal chondrocyte calcification and ossification, restrain aberrant osteophyte formation, and specifically ameliorate advanced osteoarticular degenerative injuries in KOA. In terms of synovial inflammation and joint microenvironment regulation, specific miRNAs target core genes of inflammatory pathways, modulate synoviocyte activation and macrophage polarization, mediate local chronic inflammatory responses in the knee joint, and directly influence KOA progression.¹³ As a key molecule governing KOA-related inflammation, miR-155 targets and inhibits gene expression to activate the NF-κB inflammatory signaling pathway. It triggers massive release of pro-inflammatory factors such as TNF-α, IL-6 and IL-1β in synovial tissue, induces synovial hyperplasia, inflammatory infiltration and joint swelling, and aggravates local inflammatory injuries of the knee joint. For subchondral bone remodeling regulation, miRNAs target bone-metabolism-related functional genes and modulate canonical bone-remodeling signaling pathways to balance osteoblast and osteoclast activities, thereby ameliorating typical pathological alterations in KOA such as subchondral bone sclerosis and trabecular disorganization. miR-34a targets and inhibits gene expression to activate the MAPK signaling pathway. It promotes excessive osteoclast activation and bone resorption, induces disorganized subchondral bone architecture and impaired mechanical stability, indirectly increases mechanical load-related injuries to the overlying cartilage, and further accelerates KOA pathological progression. Different from the broad physiological regulatory roles of miRNAs, their functions in KOA exhibit distinct disease- and tissue-specific features, enabling precise targeting of characteristic pathological injuries in the knee joint.^14–16

However, direct intra-articular injection of miRNAs faces multiple physiological barriers. Degradation by ribonucleases, clearance by synovial macrophages, and rapid excretion through synovial capillaries and lymphatic vessels lead to an extremely short half-life.¹⁷ The dense and avascular nature of the chondrocyte ECM further hinder the efficient delivery of miRNAs to chondrocytes. In addition, anionic components in synovial fluid (such as hyaluronic acid and albumin) competitively bind to cationic nanocarriers, reducing their transport efficiency into cartilage tissue.¹⁸ Furthermore, miRNAs have inherent drawbacks, including poor targeting and low intracellular delivery efficiency, which severely limit their direct clinical application.

The emergence of the miRNA-nanocomposite hydrogel systems provides an ideal solution to the above challenges. This system uses injectable hydrogel as the carrier framework. It possesses both the ability to mimic the biomimetic ECM microenvironment and the advantage of minimally invasive delivery, enabling precise filling of irregular cartilage defects. Nanocarriers (such as lipid nanoparticles, peptide nanofibers, inorganic nanoparticles, etc) can effectively protect miRNA from degradation, while improving cellular uptake efficiency and targeting ability. This system integrates three core functions: minimally invasive delivery, biomimetic support, and targeted regulation. It provides a brand-new therapeutic modality for KOA cartilage repair. Based on previous studies, this review further expands the in-depth analysis of pathological mechanisms, the research progress of novel materials and carriers, the application of interdisciplinary technology integration, and the key breakthroughs in clinical translation. It comprehensively presents the current research status and future directions in this field.

Design Strategies of Nanocomposite Hydrogel Systems

Design Strategies of Hydrogel Matrices

As the major scaffold of the entire system, the hydrogel matrix undertakes key functions including loading of miRNA-nanocomposites, local retention in the joint, sustained drug release, mechanical support, and biocompatibility adaptation. Its design is systematically developed around material selection, structural modification, and performance optimization. The core principles are to mimic the articular cartilage ECM, adapt to the pathological microenvironment of KOA, and enable efficient loading compatible with nanocarriers.¹⁹ For material selection, polymeric materials with excellent biocompatibility, degradability, easy modification, and tunable mechanical properties are preferred. They are mainly divided into three categories:²⁰ natural polymers, synthetic polymers, and natural-synthetic composite polymers.²¹ Natural polymers are mainly intrinsic components of cartilage ECM, such as hyaluronic acid, collagen, gelatin, and chitosan. They have outstanding biocompatibility, degradability, and adaptability to the joint microenvironment, and are rich in active groups for chemical modification. Among them, hyaluronic acid is the preferred matrix material because it can bind to cartilage CD44 receptors;²² collagen and gelatin provide a biomimetic growth microenvironment; chitosan has antibacterial, anti-inflammatory, and pH-responsive properties, matching the local acidic microenvironment of KOA. Synthetic polymers, such as polyethylene glycol (PEG), polyacrylamide (PAM), and polylactic-co-glycolic acid (PLGA), have controllable mechanical properties, stable structures, and are easy for large-scale preparation.²³ They can precisely regulate swelling, degradation, and mechanical behaviors, making up for the insufficient mechanical strength of natural materials. Natural-synthetic composite hydrogels combine the advantages of both components, integrating the high biocompatibility and targeting ability of natural polymers with the mechanical stability and structural controllability of synthetic polymers,²⁴ effectively solving the contradiction that single materials can hardly balance biological adaptability and mechanical properties.

On this basis, key performance optimizations are carried out for the hydrogel matrix. For example, mechanically, it matches the elastic modulus of articular cartilage (0.1–10 MPa). By regulating crosslinking density, constructing double-network/triple-network structures, and adding inorganic nanofillers such as hydroxyapatite, the compressive performance, elasticity, and fatigue resistance are improved to prevent damage and loss under dynamic joint loads.²⁵ For degradation, Feng G, Zha Z, Huang Y, et al have discovered through exploration that the degradation rate is synchronized with the cartilage repair cycle. Through degradable materials, enzymatic/hydrolytic/responsive crosslinking methods, and crosslinking density regulation (introducing MMPs-sensitive crosslinking bonds), rapid degradation at lesion sites and slow degradation in normal areas are achieved,²⁶ avoiding burst release of miRNA or material accumulation. The swelling ratio is controlled at a moderate range of 10–50 times, ensuring effective loading and sustained release while preventing excessive swelling that increases intra-articular pressure and causes discomfort.²⁷

Beyond structural optimization, the hydrogel can also be functionally modified through chemical approaches. Adhesive modification introduces dopamine, RGD peptides, Schiff base bonds, to enhance binding to the cartilage surface and prolong retention time in the joint. Stimuli-responsive modification introduces pH-, ROS-, or MMPs-responsive groups or peptide sequences according to the pathological characteristics of KOA (high ROS, high MMPs, acidity, etc), realizing lesion-specific release of miRNA-nanocomposites. Targeting modification grafts cartilage-targeting peptides or cell membrane receptor ligands to accurately recognize chondrocytes,²⁸ improving delivery efficiency and reducing off-target effects. Ultimately, a biomimetic scaffold and sustained-release carrier that adapts to the joint microenvironment and enables efficient miRNA delivery is constructed.

Design Strategies of Nanocarriers

As the essential delivery unit for miRNA, nanocarriers mainly undertake the key functions of stable miRNA loading, promoting target cell uptake, and facilitating intracellular escape. The core design principles are “high-efficiency miRNA loading + protection of miRNA activity + promotion of target cell uptake + low toxicity”.^29–31 Meanwhile, nanocarriers must be highly compatible with the hydrogel matrix without affecting the crosslinking, structure, or properties of the hydrogel, and enable controlled release within the hydrogel.

For material selection, nanomaterials with good biocompatibility, degradability, high miRNA loading efficiency, and easily modifiable surfaces are preferred. They are mainly classified into three categories: inorganic, organic, and novel biomimetic nanocarriers. Inorganic nanocarriers, such as mesoporous silica nanoparticles, hydroxyapatite nanoparticles, magnetoelectric-responsive Fe₃O₄nanoparticles,³² and zinc oxide (ZnO) nanoparticles, exhibit large specific surface area, high loading efficiency, stable structure, and easy modification.³³ Among them, ZnO nanoparticles show strong targeting ability and can form rigid supporting structures.³⁴ However, inorganic nanocarriers have a degradation cycle of several months. Incomplete degradation tends to cause their accumulation in chondrocyte lacunae and synovial tissues, triggering oxidative stress and chronic cytotoxicity. Degraded metal ions (silicon, manganese, iron) continuously disrupt the homeostasis of the cartilage microenvironment. Existing studies only examine short-term organ toxicity in the heart, liver and kidneys, without evaluating cumulative damage to local joint tissues.²⁹ Organic nanocarriers, including liposomes, polymeric nanoparticles, micelles, and exosomes, represent the mainstream materials for miRNA delivery. They possess excellent biocompatibility and degradability, and can effectively encapsulate and protect miRNA.³⁵ Exosomes, as natural extracellular vesicles, are ideal biomimetic carriers due to their non-immunogenicity and high ability to cross biological membranes.³⁶ Novel nanocarriers, such as stem cell homing peptide nanofibers and nanohydrogel particles, demonstrate both high loading capacity and controlled release properties. Among them, stem cell homing peptide nanofibers feature a self-assembled network structure and enable both stem cell homing and miRNA loading³⁷ (Table 1).

Table 1.

Functional Properties of Nanocarriers

Types of Nanocarriers	Core Advantage	Key Modification Strategy	Performance Features	References
Liposomes	High biocompatibility and non-toxicity, capable of carrying hydrophobic/hydrophilic drugs	Chemical modification of disulfide pentacyclic compounds for structural construction	Direct transmembrane transport significantly enhances intracellular delivery efficiency	[35]
Peptide nanofiber	Self-assembling network structure with dual capabilities for stem cell homing and loading	Self-assembled peptides, integrated homing peptide sequences	No exogenous cell transplantation is required	[37]
Fe3O4nanoparticles	Magnetic-electric response characteristics enable synergistic repair through physical stimulation	A piezoelectric phase with an integrated dual-network semi-cross-linked entanglement structure	Respond to joint movement to generate electromagnetic stimulation, regulating Ca2⁺ influx	[32]
Zinc oxide nanoparticles	High targeting specificity and capable of forming rigid support structures	Surface grafting with hyaluronic acid to optimize the streamlined structure	Penetrating cartilage microcracks and hardening upon water contact to form an adaptive scaffold	[34]

For miRNA loading strategies, the essential objectives are to improve loading efficiency, ensure loading stability, and maintain miRNA bioactivity. According to the physicochemical properties of nanocarriers, the strategies are divided into three categories. Physical adsorption achieves mild loading through electrostatic interactions, hydrogen bonding, and hydrophobic interactions without impairing miRNA activity, although stability is relatively low and can be improved by surface modification. Covalent conjugation attaches miRNA to the carrier surface via responsive covalent bonds, providing extremely high stability but with relatively complex operations. Encapsulation embeds miRNA inside the carrier, which effectively shields miRNA from nuclease degradation and offers the best protection. This is the most widely used strategy at present.³⁸

Beyond material selection and loading, functional modification of nanocarriers is critical for improving targeting, cellular uptake efficiency, and biocompatibility.^39,40 The core approaches are “surface modification and functional group grafting”, which specifically include: Biocompatibility modification using PEG, HA, and similar polymers to achieve “stealth” properties, reduce toxicity and immunogenicity, and prevent aggregation. Targeting modification by grafting cartilage-targeting peptides, CD44 receptor ligands, etc, to enhance uptake by target cells and reduce off-target effects. Intracellular escape modification by introducing cell-penetrating peptides or lysosomal escape peptides to help miRNA avoid degradation in endosomes/lysosomes. Stimuli-responsive modification by introducing pH-, ROS-, or MMPs-responsive groups, which match the responsiveness of the hydrogel to achieve dual-precision release of miRNA.^41,42 Ultimately, an ideal “protective shell and transporter” for efficient miRNA delivery is constructed, which significantly improves the overall delivery efficiency.

However, single-functional modification only endows nanocarriers with one single property such as targeted accumulation, environmental responsiveness or drug loading. It cannot simultaneously meet multiple requirements including efficient delivery, stable circulation and safe therapy. In contrast, dual-functional combined modification integrates multiple complementary functional modules into the nanocarrier structure in a synergistic manner.^43,44 It effectively compensates for performance limitations of single-modified systems and greatly improves the comprehensive therapeutic potential of nano-delivery systems.⁴⁵ Rational dual-functional combined modification can produce prominent synergistic therapeutic benefits. Two classic modification combinations include targeted accumulation-microenvironment-responsive release and drug loading-immune evasion.⁴⁶ Among them, targeted modification provides nanocarriers with lesion-targeting recognition capacity.⁴⁷ It effectively improves the specific accumulation efficiency of carriers at diseased sites and reduces non-specific drug distribution in normal tissues. Auxiliary modifications such as responsive release and immune evasion optimize the in-vivo circulation behavior and drug-release properties of nanocarriers. The two types of functional modules cooperate and produce synergistic effects. They not only raise local drug concentration in lesion regions and reduce off-target drug loss, but also avoid carrier clearance triggered by the in-vivo microenvironment and continuously improve drug delivery efficiency.⁴⁸ Consequently, synergistic therapeutic outcomes superior to those of single-functional modification are achieved. Nevertheless, blind multi-component composite modification and excessive introduction of functional groups can induce obvious antagonistic effects and exert negative impacts on the structural and physicochemical properties of nanocarriers.⁴⁹ The skeleton structure of nanocarriers relies on stable cross-linked networks to maintain overall morphology and mechanical features. Excessive modification with functional modules breaks the original structural balance and destroys the ordered micro-structural network. Such structural defects further degrade mechanical properties. Modified nanocarriers thus exhibit sharply reduced stability under complex in-vivo physiological conditions. Problems such as structural rupture, morphological deformation and particle aggregation tend to occur. These greatly impair the in-vivo circulation stability and structural integrity of nanocarriers and hinder long-term targeted delivery. Apart from impaired physicochemical performance, excessive modification also markedly damages drug delivery performance and biocompatibility of nanocarriers and weakens their in-vivo therapeutic efficacy.^50,51 Massive redundant functional groups occupy valid loading sites on the surface and inside nanocarriers. This greatly lowers the loading capacity and loading efficiency of miRNA and directly weakens the effective therapeutic dose advantage of the system. Meanwhile, excessive modification alters surface physicochemical properties of nanocarriers and significantly increases their immunogenicity in synovial tissues. When modified carriers act on joint synovium, they are easily recognized as foreign substances by the host immune system and rapidly cleared.^52,53 This drastically shortens the in-vivo retention time of carriers and reduces targeted delivery efficiency.^54,55 Therefore, nanocarrier modification design shall follow the principle of precise regulation. Researchers should reasonably select functional module types and control modification quantity and degree. On the premise of guaranteeing favorable structural stability, mechanical properties and low immunogenicity of nanocarriers, the synergistic therapeutic advantages of dual-functional or multi-functional modification can be maximized. Finally, an optimal balance among structural performance, delivery efficiency and therapeutic effect of nano-delivery systems can be realized.⁵⁶

Design Strategies of Mirna

During the progression of KOA, degradation of cartilage matrix, local inflammatory infiltration, chondrocyte senescence, and aberrant hypertrophic differentiation constitute the core pathological mechanisms driving joint degeneration and joint functional impairment. Based on the biological functional characteristics of therapeutic miRNAs, they are classified into four categories: matrix-protective, anti-inflammatory, Anti-aging, and Anti-hypertrophic degenerative types. This classification system precisely corresponds to the major pathological features of KOA and covers all key damage mechanisms throughout the osteoarthritis course. (Table 2) To achieve precise effects of miRNA in regulating inflammation and repairing cartilage for osteoarthritis treatment, and to adapt to the loading and sustained-release characteristics of nanocomposite hydrogels, a multi-dimensional optimized design strategy has been developed. First, focusing on pathological targeting, miR-140-5p and miR-212-5p, which are significantly downregulated in OA, are selected. Dual-luciferase reporter assays confirm that they specifically bind to target genes MMP-3/13 and ELF3, respectively, enabling precise regulation of cartilage degeneration and inflammatory pathways. To improve stability and loading efficiency, MON-PEI mesoporous organosilica nanoparticles are used for complex formation. Due to their high positive charge (Zeta potential 34.1 mV), stable complexes with miRNA are formed. Agarose gel electrophoresis verifies that complete miRNA encapsulation is achieved at an N/P ratio > 20, effectively resisting nuclease degradation. Meanwhile, miRNA-nanoparticle complexes are incorporated into hyaluronic acid-based self-healing hydrogels.⁵⁷ An injectable, self-healing, and biodegradable 3D delivery scaffold is constructed via dynamic Schiff base bonds. SEM and rheological tests confirm that the hydrogel has a uniform porous structure and favorable mechanical stability, allowing sustained release of miRNA in the articular cavity environment. In vitro cell experiments show that this system significantly promotes chondrogenic differentiation of hBMSCs, enhances proliferation of C28/I2 chondrocytes, reverses IL-1β induced apoptosis and cell cycle arrest, and effectively reduces levels of IL-6, TNF-α, ROS, and MMPs. In vivo rabbit femoral condyle defect models further confirm that miR-140-5p loaded hydrogels significantly promote cartilage defect repair, while miR-212-5p exosomes inhibit cartilage degradation and inflammation by targeting ELF3.⁵⁸ These experiments collectively demonstrate that the miRNA design strategy integrating sequence targeting, nanoparticle stabilization, hydrogel adaptation, and functional synergy enables efficient and precise gene therapy for KOA.

Table 2.

Functional Classification of miRNAs

Functional Classification	Representativeness Miana	Core Mechanism of Action	Targeted Pathological Process	Treatment Effect	References
Matrix-protective Type	miR-27b, miR-197-3p	Balancing the synthesis and catabolism of the extracellular matrix	Cartilage degeneration	Relieves progressive cartilage injury and maintains the structural integrity of articular cartilage.	[59,60]
Anti-inflammatory type	miR-214-3p	Targeted modulation of classical inflammatory signaling pathways such as NF-κB and MAPK to inhibit the secretion of pro-inflammatory cytokines including IL-1β, TNF-α, and IL-6.	Inflammatory infiltration	Amelioration of inflammation-mediated cartilage injury	[61]
Anti-aging type	miR-29b-5p	Inhibits chondrocytic cell cycle arrest and downregulates the expression of aging-related molecules.	Chondrocyte senescence	Restoration of the proliferative capacity and normal biological functions of aged chondrocytes	[36]
Anti-hypertrophic degenerative type	miR-140-5p, miR-199a-5p	Inhibits abnormal hypertrophy, calcification, and ectopic ossification of chondrocytes	Chondrocytic abnormal hypertrophy	Prevents late-stage pathological changes such as cartilage sclerosis and joint deformities	[62,63]

In addition to these protective miRNAs that alleviate chondrocyte injury and delay the progression of KOA, multiple pathogenic miRNAs are abnormally overexpressed in the articular cartilage, synovium and synovial fluid of KOA patients. These up-regulated miRNAs serve as key negative regulators of chondrocyte homeostasis. Accordingly, targeted inhibition of such pathogenic miRNAs represents an important strategy for precise intervention of KOA. Several pathogenic miRNAs markedly up-regulated in KOA have been validated, including miR-146a and miR-34a. miR-146a acts as a core molecule driving chronic joint inflammation and cartilage microenvironment destruction in KOA.^64,65 Under physiological conditions, endogenous anti-inflammatory pathways maintain immune homeostasis in joints.⁶⁶ In KOA, highly expressed miR-146a targets and inhibits critical anti-inflammatory regulatory molecules. It blocks the body’s anti-inflammatory negative-feedback mechanism and persistently activates classic pro-inflammatory pathways such as NF-κB and MAPK. Consequently, synoviocytes and chondrocytes secrete large amounts of pro-inflammatory and pro-degenerative factors including TNF-α, IL-6, IL-1β and matrix metalloproteinases (MMPs).^67,68 Sustained stimulation by abundant inflammatory mediators triggers synovial hyperplasia and edema, induces persistent chronic synovitis, and forms a vicious cycle of amplified inflammatory cascades. Meanwhile, miR-146a directly impairs chondrocytes, suppresses their proliferation and repair capacity, initiates cartilage matrix degradation, and disrupts the joint microenvironment. It is a major driver for early-stage inflammatory initiation and continuous disease progression of KOA. MiR-34a is a key pro-degenerative mediator of chondrocyte apoptosis, pyroptosis and repair failure in KOA.⁶⁹ Normal chondrocytes maintain survival and proliferation via the PI3K/Akt and Bcl-2 pathways to support intrinsic cartilage repair. Aberrantly up-regulated miR-34a in KOA silences core target genes such as SIRT1, Bcl-2 and CyclinD1.^12,70 It blocks survival-signal transduction and chondrocyte proliferation, and abrogates endogenous cartilage repair.^71,72 Moreover, miR-34a activates the Caspase-1/3 inflammasome to trigger chondrocyte pyroptosis, accompanied by massive release of inflammatory mediators that further aggravate local tissue damage. Ultimately, the rates of chondrocyte apoptosis and pyroptosis exceed those of cell repair and proliferation. The number of functional chondrocytes declines sharply, and cartilage repair capacity is severely compromised. These changes result in thinned cartilage layers with increased brittleness, and mediate irreversible chondrocyte injury in moderate-to-late-stage KOA.

Two parallel and complementary strategies dominate miRNA-targeted therapy for KOA: supplementation of protective miRNAs and silencing of pathogenic miRNAs.⁷³ The protective-miRNA supplementation strategy restores the reduced levels of endogenous protective miRNAs in KOA tissues. It activates cartilage-repair and anti-inflammatory signals to rebuild cartilage homeostasis. By contrast, the pathogenic-miRNA silencing strategy targets abnormally activated pathogenic-miRNA signaling axes. It exerts specific competitive inhibition to continuously block the pro-inflammatory and pro-degenerative effects of pathogenic miRNAs and eliminate pathological drivers of KOA progression. The two therapeutic strategies differ in mechanisms and applicable scenarios. Their combined application enables multi-dimensional regulation of KOA pathological processes. Protective-miRNA supplementation is more suitable for early-stage KOA characterized by mild cartilage damage and insufficient endogenous repair capacity. Inhibition of pathogenic miRNAs achieves better intervention effects for moderate-to-late-stage KOA with severe inflammatory responses and sustained activation of pathogenic miRNAs. Systematic comparison of the two therapeutic approaches expands the research scope of miRNA-targeted KOA therapy. It provides solid theoretical evidence for designing miRNA-combination therapeutic regimens, and lays a foundation for developing efficient and precise clinical intervention techniques for KOA.⁷⁴

Composite Design Strategies of Nanomaterial–Hydrogel Systems

The nanocomposite-hydrogel integration strategy is the core of constructing an efficient miRNA delivery system. Its key design principle is to achieve uniform dispersion of nanocarriers, stable loading of miRNA, and structural integrity of the composite system, while ensuring functional synergy between the two components and effectively avoiding potential risks such as nanoparticle aggregation, uneven miRNA loading, impaired hydrogel crosslinking, and reduced mechanical properties⁷⁵ (Figure 1).

Figure 1.

Various Composite Design Strategies for Nanomaterial-Hydrogel Systems.

At present, three mainstream strategies are widely used for nanocomposite-hydrogel integration: The first is the in-situ integration strategy. In this approach, miRNA-loaded nanocomposites are uniformly dispersed in a hydrogel precursor solution, which is then crosslinked in-situ at the target site (eg, joint cavity) via photo-, chemical-, or temperature-induction to form a hydrogel. The nanocomposites are evenly embedded in the three-dimensional network of the hydrogel.⁷⁶ This method has broad applicability and simple operation, and enables uniform distribution of nanocomposites in the hydrogel. It is especially suitable for injectable hydrogel systems to achieve local long-term sustained release of miRNA. The second is the blend crosslinking strategy. Nanocarriers are blended with hydrogel monomers or polymers, and interactions (hydrogen bonds, electrostatic interactions, or covalent bonds) between molecules synchronously complete hydrogel crosslinking and nanocarrier loading.⁷⁷ miRNA can be loaded into nanocarriers before blending or during the blending process. Due to the tight binding between nanocarriers and the hydrogel, this strategy effectively inhibits burst release of miRNA, and is suitable for nanocarriers with well-defined surface active groups. The third is the layer-by-layer self-assembly strategy. Using hydrogel as the core substrate, miRNA-nanocomposites with opposite charges are assembled layer by layer on the hydrogel surface through electrostatic interactions, hydrogen bonds, or antigen-antibody binding to form a core-shell structured nanocomposite hydrogel.⁷⁸ This strategy allows precise regulation of miRNA loading capacity and release rate. In addition, the surface nanocomposite layer can rapidly respond to the pathological microenvironment of KOA and significantly improve the uptake efficiency of miRNA by target cells.

Regardless of the integration method, compatibility optimization of the composite system is critical for ensuring delivery efficiency. In terms of size matching, the particle size of nanocarriers should be compatible with the pore size of the hydrogel. This ensures that nanocarriers can be uniformly embedded in the three-dimensional network of the hydrogel and smoothly released during hydrogel degradation, enabling controllable delivery of miRNA. In terms of surface properties, the surface characteristics of nanocarriers must be compatible with the hydrogel precursor solution to avoid aggregation or delayed gelation.⁷⁹ Ultimately, the primary functions of the hydrogel, including adhesiveness, mechanical properties, and stimuli responsiveness, should remain intact after integration. This achieves the synergistic goal of “enhancing efficacy without introducing adverse effects” and provides a stable and reliable carrier support for efficient miRNA delivery.

Despite their respective advantages, all three composite strategies also have limitations. The in-situ composite strategy has inherent drawbacks. Uniform dispersion of nanocarriers leads to uncontrollable miRNA release, obvious initial burst release, and insufficient long-term sustained-release performance, which cannot match the chronic repair cycle of cartilage defects in KOA.^29,80 Meanwhile, its homogeneous non-gradient structure fails to mimic the layered mechanical properties of native articular cartilage, resulting in poor mechanical adaptability to layered cartilage defects.⁸¹ In addition, high-loading nanocarriers may reduce the compactness and structural stability of scaffolds, and further impair the overall mechanical properties of composites.⁸² The blending-crosslinking strategy also has non-negligible shortcomings. Interlayer cross-linking interfaces act as structurally weak regions. Under cyclic frictional and compressive loads in the knee joint, interlayer delamination and fracture readily occur, shortening the in-vivo service life of scaffolds.^83,84 Furthermore, the layer-by-layer cross-linking assembly process is time-consuming and demands high preparation precision.⁸⁵ Its low fabrication efficiency cannot meet the requirements for large-scale clinical production. Moreover, the independent release pattern of each functional layer cannot achieve sequential and synergistic miRNA release, which limits the synergistic repair efficacy for layered cartilage defects.⁸⁶ For the layer-by-layer self-assembly strategy, although it achieves favorable miRNA activity retention and sustained-release performance, the multi-layer assembled structure increases the mass-diffusion barrier. This results in a slow initial miRNA release rate, which fails to rapidly suppress acute inflammation in progressive KOA.⁸⁷ Long-term mechanical compression and joint friction may cause slippage and damage of multi-layer assemblies, triggering abrupt miRNA leakage and failure of the sustained-release system.⁵⁷ Meanwhile, the high-precision layer-by-layer self-assembly technique involves complicated procedures and low product yield, restricting its clinical translation and large-scale application.⁸⁸ These three composite fabrication strategies exhibit respective advantages and disadvantages in structural design, mechanical properties, drug-release behavior, material compatibility and clinical-adaptable scenarios, and can complement one another.⁸⁹ Therefore, in practical clinical practice, the in-situ composite strategy is preferred for early-stage mild KOA with low mechanical-load requirements and demands for short-term anti-inflammatory intervention. Its moderate mechanical strength satisfies the basic load-bearing requirements of minor cartilage defects. Rapidly released miRNA can effectively block early inflammatory activation.⁹⁰ Featuring simple fabrication procedures and low cost, this strategy is suitable for popularized interventional therapy for early-stage KOA. The blending-crosslinking strategy serves as the optimal option for moderate KOA with layered defects and gradient mechanical-adaptation demands.⁹¹ Its biomimetic gradient cross-linked structure matches the mechanical environment of native knee joints and avoids structural damage induced by mechanical mismatch.⁹² The layered targeted drug-release pattern meets the differentiated repair requirements for cartilage defects at different depths, realizing precise and efficient treatment for moderate KOA.⁹³ The layer-by-layer self-assembly strategy shows the highest applicability for severe advanced KOA that requires high mechanical stability and long-term repair intervention.⁸⁸ The orderly stacked multi-layer assembly guarantees scaffold structural integrity under long-term joint friction and compression. Its precise hierarchical assembly enables stable miRNA release over an ultra-long duration, which perfectly fits the chronic repair cycle of full-thickness cartilage and subchondral bone defects in severe KOA.⁹⁴ It can continuously maintain miRNA biological activity under complex joint inflammatory microenvironments.⁹⁵

Functional Characteristics of Nanocomposite Hydrogel Systems

Biocompatibility: The Cornerstone of System Performance

For biomedical applications, biocompatibility and biodegradability are the key essential functional characteristics of nanocomposite hydrogels.^96,97 Nanocomposite hydrogel systems usually employ natural or synthetic polymers with high biosafety, such as chitosan, gelatin, sodium alginate, and polyethylene glycol, as gel matrices. Through mild cross-linking methods, biomimetic ECM structures are constructed, which can provide a favorable microenvironment for cell adhesion, spreading, and proliferation, and exhibit extremely low cytotoxicity in in vitro cell experiments. Meanwhile, nanocomponents including hydroxyapatite, silica, nanoclay, and graphene oxide can effectively reduce agglomeration and biointerface stress after surface functional modification, avoiding excessive oxidative stress and inflammatory responses. They improve the mechanical properties of hydrogels without compromising biosafety. In the in vivo environment, the degradation rate of nanocomposite hydrogels can be regulated by chemical structure and cross-linking density.⁹⁸ Most degradation products are non-toxic small molecules that can be metabolized without accumulation in vivo. Implantation only induces mild and transient local inflammatory responses, with no obvious tissue damage, calcification, or long-term foreign body reactions. Thus, nanocomposite hydrogels possess good histocompatibility and hemocompatibility.^99,100

High Loading Capacity: Adaptable to Diverse Delivery Requirements

This delivery system combines the high encapsulation properties of hydrogels with the high loading efficiency of nanocarriers, enabling efficient loading and uniform dispersion of miRNAs to meet the dosage requirements for the treatment of various diseases.³³ The hydrogel matrix possesses a loose and porous three-dimensional network structure, whose pore size can be flexibly tuned by adjusting cross-linking conditions. It can accommodate numerous types of nanocarriers, providing sufficient space for miRNA loading.^101,102 Nanocarriers can be endowed with positive charges through surface chemical modification (eg, cationization), forming stable nanocomplexes with negatively charged miRNA molecules via electrostatic interactions. This greatly improves the loading capacity of miRNAs and effectively prevents aggregation, leakage, or loss of activity during the delivery process.¹⁰³

In addition, precise regulation of the cross-linking density of hydrogels, as well as the particle size and surface chemical properties of nanocarriers, allows flexible adjustment of miRNA loading amount and efficiency.¹⁰⁴ This system can achieve low-dose precise delivery required for local tissue therapy and high-dose stable loading suitable for systemic therapy.^20,105 It is adaptable for the treatment of various diseases including tumors, inflammation, and tissue injury repair.^20,106

Controllable Sustained Release: Achieving Long-Acting and Precise Drug Delivery

Nanocomposite hydrogels can achieve controllable and sustained release of miRNA through two synergistic mechanisms: passive release and active responsive release, which effectively avoids the “burst effect” commonly observed in traditional delivery systems.¹⁰⁷ This not only improves therapeutic efficacy but also reduces the toxic and side effects of drugs on normal tissues.^104,108

Passive release is mainly achieved through the slow swelling and gradual degradation of hydrogels in the physiological environment in vivo, as well as the slow diffusion of nanocarriers from the gel network. By adjusting key parameters such as molecular weight, cross-linking density, and hydrophilicity/hydrophobicity of the hydrogel, the release duration of miRNA can be precisely regulated from several days to dozens of days.^21,107 For example, the delivery system constructed from PCEC (polycaprolactone-polyethylene glycol-polycaprolactone) triblock copolymer hydrogel composite nanocarriers enables continuous and stable release of miRNA for more than 35 days.¹⁰⁹

Active responsive release is realized by designing stimuli-sensitive hydrogels (eg, pH-responsive, enzyme-responsive, light-responsive, temperature-responsive, etc).¹¹⁰ The microenvironmental differences between target tissues and normal tissues (such as the acidic microenvironment of tumor tissues, high enzyme activity at inflammatory sites, and temperature differences in specific tissues) are used as trigger signals to achieve on-demand release of miRNA,¹¹¹ further improving the accuracy of drug delivery.¹¹² For instance, pH-responsive hydrogels can rapidly undergo structural dissociation and release loaded miRNA in the acidic microenvironment of myocardial infarction sites (approximately pH 6.8), significantly increasing local drug concentration in diseased tissues and enhancing therapeutic effects.

Targeting: Improve the Delivery Efficiency to Target Cells

In the construction of miRNA-targeted delivery systems, precise and controllable targeted modification of nanocarriers and hydrogel matrices is a key step to achieve efficient drug enrichment and improve therapeutic efficacy.^21,113 Through rational molecular design and material modification, the specific distribution of miRNA at lesion sites can be significantly enhanced, non-specific exposure to normal tissues reduced, off-target effects and toxic side effects decreased,¹¹⁴ and the uptake and intracellular release efficiency of nucleic acid drugs by target cells greatly improved, ultimately enhancing the specificity and effectiveness of treatment.

At present, mainstream targeted modification strategies can be mainly divided into two categories: active targeting and local passive targeting. Active targeting focuses on covalently or non-covalently conjugating targeting molecules on the surface of nanocarriers to achieve precise homing through specific receptor-ligand recognition.¹⁰⁶ Common targeting molecules include targeting peptides (such as RGD peptide, CD44-targeting peptide, TAT cell-penetrating peptide), monoclonal antibodies, and small-molecule specific ligands.¹¹⁵ These molecules can specifically recognize receptors highly expressed on the surface of target cells and enter cells via receptor-mediated endocytosis, realizing hierarchical targeted delivery from tissues to cells.¹¹⁶

The other strategy relies on the local retention property of hydrogel matrices to achieve spatially targeted enrichment.²⁰ Hydrogels exhibit good biocompatibility, degradability, and three-dimensional network structure. They can be implanted into lesion areas via in-situ injection, local smearing, and other methods,¹¹⁷ prolonging the residence time of miRNA delivery systems locally, reducing systemic circulation diffusion and rapid metabolic clearance, thereby achieving drug enrichment at injured or inflammatory sites.

In practical applications, the two strategies are often combined synergistically to form a multi-level targeted delivery system. For example, RGD peptide-modified nanocarriers can specifically recognize integrin αvβ3 receptors highly expressed on the surface of macrophages and vascular endothelial cells. Combined with the local retention effect of hydrogels, a triple-targeted delivery system of “hydrogel retention–nanocarrier targeting–receptor-mediated endocytosis” can be constructed,¹¹⁸ which significantly improves the uptake efficiency of miRNA by cells involved in wound healing. In addition, targeted nanocarriers loaded in hyaluronic acid (HA)-based hydrogels can specifically bind to CD44 receptors on the surface of macrophages via HA, accurately deliver miRNA,¹¹⁹ regulate the polarization of macrophages towards an anti-inflammatory phenotype, effectively modulate the local immune microenvironment, and accelerate tissue injury repair and regeneration. Such multi-level targeting strategies provide a stable, efficient and safe delivery platform for the translational application of miRNA in the fields of inflammation, trauma, degenerative diseases and others.^29,45

Multi-Dimensional Functional Modification Strategy

Multi-Response Coordinated Release Design: Adapting to Complex Pathological Microenvironments

Single-response release mechanisms are limited by the complexity of the pathological microenvironment¹²⁰ in KOA and fail to achieve precise and efficient miRNA delivery.^104,117,121 Therefore, multi-responsive synergistic release systems have become the research focus of miRNA-loaded hydrogels for KOA therapy.¹²² Such designs integrate two or more response mechanisms, enabling hydrogels to accurately recognize specific microenvironmental signals at KOA inflammatory sites, realize on-demand release of miRNAs, and greatly improve delivery efficiency and therapeutic specificity.¹²³

Among dual-responsive systems, MMP/pH dual-responsive hydrogels are typical representatives. The hydrogel network is co-modified with MMP-sensitive cross-linking bonds and pH-sensitive groups, which perfectly matches the microenvironmental characteristics of KOA inflammatory sites: highly expressed matrix metalloproteinases (MMPs) at inflammatory sites can specifically degrade the cross-linking bonds, while the acidic environment caused by inflammation triggers the cleavage of pH-sensitive groups.¹²⁴ The dual effects synergistically achieve precise and efficient release of miRNAs.¹²⁵ Experimental validation demonstrated that its release efficiency was significantly improved compared to a single-response system, effectively solving the problems of insufficient release and weak targeting in single-response systems.¹²⁶ Lan et al designed a pH/ROS dual-responsive smart microgel miRNA delivery system. It combines phenylboronic acid-functionalized microgels with strontium sulfite nanoparticles to load miR-155, efficiently deliver miR-155, reduce cellular apoptosis and improve delivery efficiency (Figure 2).

Figure 2.

A pH/ROS dual-responsive smart microgel miRNA delivery system. (A) CCK-8 to evaluate the biocompatibility of the microgel drug system. (B) Dead staining at 48 h verified the viability of the NPCs. (C) The DPPH test showed that the microgel drug system exerted a strong antioxidative effect. (D)The in vitro degradation process of the microgel under different conditions suggested that the pathological acidic environment in IDD accelerates microgelcollapse. (E) The release result of miR-155 indicated that the inflammatory environment (low-pH and high-ROS) promotes miR-155 release from microgels. (F) Representative immunofluorescent staining for IL-10, TNF-a, BCL-2, BAX, COL-II, and MMP-13 (scale bars: 20 mm).¹²⁷ * 0.01 ≤ p < 0.05, ns: not significant Copyright 2025, The Royal Society of Chemistry.

In triple-responsive systems, redox/pH/mechanical triple-responsive hydrogels further break through the limitations of single-response systems by integrating three responsive units: disulfide bonds (redox response), hydrazone bonds (pH response), and dynamic boronate bonds (mechanical response).^123,127 This system can simultaneously respond to multiple physiological and pathological signals in KOA joints, including mechanical stimulation generated by joint movement, the acidic environment at inflammatory sites, and high redox potential. Under the synergistic effect of the three stimuli, on-demand release of miRNAs is achieved, which not only ensures efficient drug release at inflammatory sites but also avoids off-target release in normal tissues, significantly improving therapeutic specificity and reducing side effects caused by off-target effects.^128,129

Precise Construction of Bionic Microenvironment: Promoting Cartilage Regeneration and Functional Repair

The central pathological features of KOA include cartilage injury and reduced regenerative capacity, and the growth, proliferation, and differentiation of chondrocytes are highly dependent on the natural ECM microenvironment.¹³⁰ Therefore, the precise construction of a biomimetic ECM microenvironment to provide suitable growth conditions for chondrocytes has become an important direction for the optimization of miRNA-loaded hydrogels. The core objective is to achieve efficient regeneration and functional recovery of cartilage tissue by simulating the natural cartilage microenvironment in combination with miRNA.¹³¹

ECM-derived hydrogels are important carriers for biomimetic design. Prepared through decellularization techniques, these hydrogels can maximally preserve the bioactive components of natural cartilage ECM, including collagen, proteoglycans, growth factors, etc. These components can accurately simulate the natural growth microenvironment of chondrocytes and provide sufficient signal support for the adhesion, proliferation, and differentiation of chondrocytes.^132–134 After compounding with synthetic polymers, the mechanical stability of the hydrogels is further enhanced. When loaded with miRNA and applied in a rabbit cartilage defect model, the amount of regenerated cartilage reached 95% of that in normal tissue after 8 weeks of treatment, which was significantly superior to traditional biomimetic hydrogels, confirming its excellent efficacy in cartilage regeneration.^36,135

To address the problems of insufficient nutrient supply and slow maturation of regenerated cartilage caused by the avascular nature of cartilage tissue, vascularization-inducing modification has become an important supplementary strategy for biomimetic microenvironment construction.¹³⁶ This strategy co-loads vascular endothelial growth factor (VEGF) and miR-126 into the hydrogel. By using the precise regulation of VEGF expression by miR-126, it induces angiogenesis at the cartilage defect site and establishes a complete nutrient transport network.¹³⁷ This effectively solves the problem of insufficient nutrient supply in cartilage tissue, guarantees the maturation and functional stability of newborn cartilage tissue, and further improves the overall effect of cartilage repair.¹³⁸

Integration of Antibacterial Functions: Enhancing Treatment Safety

Intra-articular injection is the main administration route for miRNA-loaded hydrogels in KOA treatment. However, this approach may introduce exogenous bacteria and cause intra-articular infection, which severely compromises therapeutic efficacy and even exacerbates the disease.²⁰ Therefore, integrating antibacterial function into the miRNA-loaded hydrogel system to achieve dual effects of “therapy + antibacterial activity” has become a key design for improving the safety of KOA treatment.^117,139 Such a strategy can accomplish cartilage repair and anti-inflammatory therapy, while effectively avoiding infection risks.¹³⁰

Silver nanoparticle (AgNP)-composited hydrogels represent a classic strategy for integrating antibacterial function. In this system, AgNPs are loaded into the hydrogel. Benefiting from the broad-spectrum antibacterial activity of AgNPs, the system can efficiently inhibit the growth of potential bacteria in the joint cavity.^140–142 Meanwhile, AgNPs can also exert synergistic anti-inflammatory effects with miRNAs, achieving combined antibacterial and anti-inflammatory enhancement.¹⁴³ In vitro antibacterial experiments confirm that the hydrogel exhibits an antibacterial rate of over 99% against Staphylococcus aureus and Escherichia coli, with no obvious cytotoxicity. It ensures antibacterial efficacy without damaging chondrocytes, balancing safety and therapeutic performance.^144,145

Antimicrobial peptide-modified hydrogels achieve antibacterial activity via surface grafting of antimicrobial peptides (such as LL-37), with the advantages of high antibacterial specificity and favorable biocompatibility.¹⁴⁶ Antimicrobial peptides can directly target bacterial cell membranes, effectively inhibiting bacterial adhesion and proliferation, and preventing infection caused by bacterial colonization on the hydrogel surface.¹⁴⁷ Meanwhile, antimicrobial peptides also possess certain anti-inflammatory activity, which can synergistically regulate the inflammatory response of KOA joints with miRNAs. This approach improves therapeutic safety and further enhances the overall therapeutic effect,^148,149 providing a novel technical route for the safe and effective treatment of KOA.

The Pivotal Mechanism of System-Mediated OA Cartilage Repair

Regulate the Balance of the Immune Microenvironment

The pathological progression of KOA is closely associated with the disruption of the local immune microenvironment in joints. Imbalance between pro-inflammatory and anti-inflammatory responses is a key trigger leading to continuous cartilage damage.¹⁵⁰ Injectable miRNA-nanocomposite hydrogel systems can precisely deliver specific miRNAs, targetedly regulate immune cell functions, restore homeostasis of the joint immune microenvironment, and inhibit the progression of cartilage damage from the source.^20,151

On the one hand, this system can promote macrophage phenotypic polarization and amplify anti-inflammatory effects. As core cells in the joint immune microenvironment, macrophages are classified into pro-inflammatory M1 phenotype and anti-inflammatory M2 phenotype.¹⁵² In patients with KOA, the proportion of M1 macrophages in joints is significantly increased. These cells secrete large amounts of pro-inflammatory factors such as IL-1β and TNF-α, activate the NLRP3 inflammasome, induce chondrocyte pyroptosis, and accelerate cartilage matrix degradation. MiRNAs delivered by the hydrogel system, including miR-223, can specifically inhibit the NF-κB signaling pathway, block the release of pro-inflammatory factors, and promote the transition of macrophages from M1 to M2 phenotype. M2 macrophages secrete anti-inflammatory factors such as IL-10 and TGF-β, which not only directly suppress local inflammatory responses but also provide a favorable microenvironment for chondrocyte proliferation and ECM synthesis, thus alleviating the vicious cycle of inflammation-pyroptosis-injury.¹⁵³

On the other hand, this system can inhibit abnormal T-cell activation and reduce immune-mediated cartilage damage. Excessive activation of T cells in the joints of KOA patients results in massive secretion of pro-inflammatory factors, further aggravating chondrocyte apoptosis and matrix degradation.^154–156 A breakthrough was achieved by Li et alMiR-155 delivered by the hydrogel system can effectively inhibit the activation and proliferation of T cells by targeting the STAT3 signaling pathway, reduce the release of pro-inflammatory factors, decrease immune cell-mediated attack on cartilage tissue, thereby protecting chondrocyte function and delaying the pathological progression of osteoarthritis (OA)^157,158 (Figure 3).

Figure 3.

Hydrogel-delivered miR-155 protects cartilage and delaying OA. The relative expression of (A) M1 and (B) M2 markers in PBMC‑derived macrophages cultured with either KOA SF or CM for 12 h. CD86, iNOS and CD206 expression levels in PBMC-derived macro‑phages cultured with KOA SF or CM for 24 h were (C) determined by flow cytometry and quantified. The relative expression of (D) IL-1β, IL-6, iNOS and (E) IL-10 mRNA in PBMC-derived macrophages. (F) Levels of STAT1 and STAT6 phosphorylation in PBMC‑derived macrophages treated and/or transfected with CM, KOA SF, CM + NC or CM + miR‑155-5p mimic. (G) The potential interaction between miR‑155‑5p and SOCS1 3’-UTR-WT or SOCS1 3’-UTR-MUT. (H) The interaction between miR‑155 and SOCS1 was confirmed using a dual-luciferase reporter assay.¹⁵⁷ * P<0.05, **p < 0.01,***P<0.001. Copyright 2021, Spandidos Publications.

Abbreviations: KOA, knee osteoarthritis; SF, synovial fluid; PBMC, peripheral blood mononuclear cells; CM, control medium; iNOS, inducible nitric oxide synthase; IL, interleukin; ARG1, arginase 1; Ym1, chitinase-like 3; NS, not significant; NC, negative control; p, phosphorylated; 3’-UTR, 3’-untranslated region; WT, wild-type; MUT, mutant; SF, synovial fluid; p, phosphorylated; MUT, mutant.

Promote Subchondral Bone Repair

Cartilage repair is not limited to the regeneration of cartilage tissue alone. The repair of subchondral bone injury and the tight integration of the osteochondral interface are critical for maintaining the stability of neo-cartilage and preventing the shedding of repaired tissue. The injectable miRNA-nanocomposite hydrogel systems achieves subchondral bone repair and osteochondral integration through gradient structural design and synergistic delivery of multiple miRNAs,¹⁵⁹ providing solid structural support for cartilage regeneration.^139,160,161

First, this system can precisely regulate the balance between osteoblasts and osteoclasts to repair subchondral bone injury.¹⁶² In the pathological state of KOA, subchondral bone exhibits an imbalance between bone resorption and bone formation. Enhanced osteoclast activity and weakened osteoblast function lead to decreased bone mass and destroyed bone microstructure in subchondral bone, which fails to provide effective mechanical support for cartilage. Using a layered loading strategy, the hydrogel system loads bone repair-related miRNAs (such as miR-21 and miR-146a) in the bottom layer, which can specifically promote osteoblast proliferation, differentiation and bone matrix synthesis, while inhibiting osteoclast activity and reducing bone resorption,¹⁶³ gradually repairing subchondral bone injury and restoring its normal mechanical properties. Meanwhile, Zhu et al made a significant discovery. The top layer is loaded with cartilage repair-related miRNAs, such as miR-29b-5p, focusing on promoting chondrocyte proliferation and ECM synthesis to achieve simultaneous repair of cartilage and subchondral bone³⁶ (Figure 4).

Figure 4.

miR-29b-5p-loaded top layer repairs cartilage/subchondral bone via chondrocyte proliferation and ECM synthesis. (A) Schematic illustration of ACLT-induced OA model and in vivo imaging of Cy5.5-labeled agomir-29b-5p in mice joints at 1, 3, 5, 7, 9, and 14 days. (B) Schematic illustration of the recruitment process pattern of SMSCs during cartilage repair. (C) Three-dimensional and planar view reconstruction images of rat knee joints showingthe abnormal growth of osteophytes (indicated by arrow) in sham, PBS, miR, SKP, and SKP@miR groups at 7 and 10 weeks. (D) Morphological analysis of the synovium at7 and 10 weeks indicated by H&E staining. (E) Synovia thickness and total synovia scores of enlargement of the synovial lining cell layer, inflammatory infiltrates, and density of the resident cells. (F) qRT-PCR analysis of genes associated with cell migration, cell adhesion, cartilage development, and cellular senes-cence. n=3. (G) SA-Gal staining images of rat chondrocytes cultured on RAD, RAD@miR, SKP, and SKP@miR for 7 days. (H) Quantification of SA-Gal positivity in normal chondrocytes, doxorubicin-treated chondrocytes, and chondrocytesat passage 3(P3). n=3. (I) Toluidine blue and Alcian blue staining of chondrocyte micromasses and monolayer chondrocytes cultured with hydrogels for 3 days. (J) KEGG enrichment analysis (focal adhesion, TGF-signaling pathway, signaling pathways regulating pluripotency of stem cells, cellular senescence, adherens junction, and ECM-receptor interaction). AGE-RAGE, age range; HIF-1, hypoxia inducible factor-1.³⁶ Data are presented as means ± SD. Statistical analysis was performed using one-way ANOVA. *P < 0.05, **P < 0.01, and ***P < 0.001 and ^#P < 0.05. Copyright 2022, AAAS.

Second, this system can activate the Wnt/β-catenin signaling pathway to achieve synergistic osteochondral repair. As a key regulator of this pathway, miR-21 can activate the Wnt/β-catenin signaling pathway by targeting and inhibiting the expression of the PTEN gene.¹⁶⁴ This pathway not only promotes osteoblast proliferation and bone matrix mineralization, but also regulates chondrocyte differentiation and maturation to facilitate cartilage tissue regeneration. Animal experiments have confirmed that after OA cartilage repair using this system, the integration strength of the bone-cartilage interface was significantly improved in the short-term compared to the single cartilage repair group.⁸³ This effectively solves the problems of osteochondral interface separation and poor repair efficacy in traditional repair methods,¹⁶⁵ achieving the goal of integrated osteochondral repair. However, clinical translation faces challenges such as significant differences in gene expression profiles between animal and human chondrocytes, as well as patient heterogeneity. Future designs must integrate patient-specific mechanisms and feedback-regulated drug administration to bridge the gap between preclinical promises and clinical realities.

Regulate Extracellular Matrix Remodeling

ECM is the main component of cartilage tissue. Imbalanced synthesis and degradation, as well as abnormal remodeling of ECM, are the core characteristics of OA cartilage degeneration. Injectable miRNA-nanocomposite hydrogel systems achieve precise ECM remodeling by regulating the synthesis, degradation and cross-linking processes of ECM, thereby improving the mechanical strength and functional stability of neo-cartilage and ensuring long-term repair efficacy.^166–168

On one hand, this system can promote ECM cross-linking maturation and enhance cartilage mechanical strength. The mechanical properties of ECM mainly depend on the cross-linking degree of collagen. Lysyl oxidase (LOX) is a key enzyme regulating collagen cross-linking, and its expression level directly affects ECM stability.^169,170 Li et al demonstrated through experiments that MiRNAs delivered by the hydrogel system can upregulate related signaling pathways, promote cross-linking between collagen molecules, and improve the mechanical strength and anti-degradation ability of ECM¹⁷¹ (Figure 5). We can make bold speculations, miR-140 can remove the inhibitory effect on LOX by targeting and inhibiting Smad gene expression,¹⁷² thus significantly upregulating LOX levels and promoting cross-linking maturation of cartilage matrix.⁶² Experiments have confirmed that after repair with this system, the compressive modulus of neo-cartilage is increased by more than 40% compared with the control group, which is close to the mechanical properties of normal cartilage.¹¹

Figure 5.

MiR-140 Modulates Cartilage Mechanical Environment via Surface Stress Homeostasis. (A) The immunohistochemistry image revealed the expression levels of RhoA in articular cartilage. (Black arrows indicate chondrocytes with positive RhoA immunohistochemical staining). (B) Fluorescent in situ hybridization of miRNA140-5p (green) in tibial plateau articular cartilage (×200). Nuclei were labeled with DAPI (blue). (C and D) Quantitative analysis of normalized fluorescence intensity of miRNA-140-5p expression within human and mouse tibial plateau articular cartilage. (E) Immunofluorescence staining image of F-actin with or without transfection under mechanical pressure with 1.925 g/cm2 (×200). Stress+ means chondrocytes cultured under 1.925 g/cm2; stress- represents cells cultured without mechanical load; MiRNA-140+ represents cells cultured with miRNA-140 agomir transfection. (F and G) Quantitative analysis of AOD of RhoA expression to total interested area within mouse tibial plateau articular cartilage. (H) Quantitative analysis the influence of miRNA-140-5p overexpression on chondrocyte cytoskeleton circularity and roundness under pressure.¹⁷¹ Value was presented as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001 Copyright 2025, SAGE Publications.

On the other hand, this system can inhibit excessive ECM calcification and maintain normal cartilage function. Cartilage calcification often occurs in the late stage of KOA, leading to increased stiffness, decreased elasticity of cartilage and seriously impaired joint mobility.¹⁷³ This phenomenon is closely related to the differentiation of chondrocytes into osteoblast-like cells and abnormal expression of Runx2 gene.^174,175 MiR-378 delivered by the hydrogel system can specifically target and inhibit Runx2 gene expression, block the differentiation of chondrocytes into osteoblast-like cells, reduce calcium salt deposition in ECM, and inhibit cartilage calcification,¹⁷⁶ so as to maintain the normal structure and function of cartilage tissue and avoid dysfunction of neo-cartilage.^177,178

Correct the Abnormal Physiological State of Chondrocytes

Chondrocytes are the functional core of cartilage tissue. Increased pyroptosis and apoptosis, as well as dysfunctional autophagy in chondrocytes, are important cellular mechanisms underlying cartilage degeneration in KOA.^179,180 Injectable miRNA nanocomposite hydrogel systems can precisely deliver miRNAs to target and correct the abnormal physiological state of chondrocytes, restore their proliferation, differentiation, and matrix synthesis functions, and provide sufficient functional cells for cartilage repair.^181,182

First, this system can inhibit chondrocyte pyroptosis and reduce cell loss.⁷ In cartilage tissue from KOA patients, the expression of pyroptosis-related markers such as Gasdermin D, IL-1β, and IL-18 is significantly elevated. Pro-inflammatory factors including IL-1β and TNF-α can induce chondrocyte pyroptosis by activating the NLRP3 inflammasome, leading to massive chondrocyte loss and aggravated cartilage damage.¹⁸³ Anti-inflammatory miRNAs (eg, miR-124, miR-223) delivered by the hydrogel system can reduce the release of pro-inflammatory factors and block the activation of the NLRP3 inflammasome by inhibiting the NF-κB signaling pathway,¹⁸⁴ thereby suppressing chondrocyte pyroptosis, reducing chondrocyte loss, and preserving sufficient cell sources for cartilage repair (Figure 6).

Figure 6.

Hydrogel-delivered anti-inflammatory miR-223 suppresses chondrocyte pyroptosis and preserves cartilage repair. (A) Surface markers on BMSCs were detected by flow cytometry. (B) miR-223 expression in BMSCs, BMSCs-secreted exosomes, and co-cultures of BMSCs-secreted exosomes and RAW 264.7 cells were detected by qRT-PCR after BMSCs transfected with miRNA-223 inhibitor/miRNA-NC in-hibitor. (C) NLRP3 expression in RAW 264.7 cells were detected by qRT-PCR after transfection of si-NLRP3/si-NC. (D–F). The effect of BMSCs-secreted exosomes on the levels of IL-1β, TNF-α and IL-18 in RAW 264.7 cells. (G) Different miRNAs expression levels in BMSCs-secreted exosomes were measured by RT-qPCR. (H) The binding site between NLRP3 and miR-223 was predicted by Targetscan. (I) The miR-223 expression was measured by RT-qPCR.¹⁵² Data are described as mean ± SD; ** P < 0.01, *** P < 0.001, compared with control group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, compared with LPS + ATPgroup. Copyright 2022, Elsevier Ltd.

Second, this system can restore chondrocyte autophagy and maintain cellular homeostasis.¹⁸⁵ Autophagy is an important mechanism for chondrocytes to remove abnormal proteins, damaged organelles, and respond to oxidative stress, and plays a key role in maintaining the functional stability of chondrocytes.¹⁸⁶ Under KOA pathological conditions, chondrocyte autophagy is significantly decreased. The expression of autophagy-related genes such as Beclin-1 and LC3 is reduced, while the expression of autophagy inhibitors such as mTOR is increased, resulting in the accumulation of damaged substances in cells and aggravated cell senescence and apoptosis.¹⁸⁷ The hydrogel system can deliver autophagy-regulating miRNAs to restore chondrocyte autophagy by inhibiting the mTOR signaling pathway or upregulating the expression of Beclin-1 and LC3, promote the removal of intracellular damaged substances, inhibit cell senescence and apoptosis, and maintain the normal physiological functions of chondrocytes.¹⁸⁸

In addition, this system can further optimize chondrocyte function by regulating epigenetic processes. Non-coding RNAs such as lncRNAs and circRNAs participate in the regulation of chondrocyte proliferation, apoptosis, and ECM synthesis by adsorbing miRNAs or directly binding to target genes. MiRNAs delivered by the hydrogel system can form regulatory networks with these non-coding RNAs.¹⁸⁹ For example, by competitively binding lncRNA HOTAIR,¹⁹⁰ the adsorption of miR-124 is relieved, the expression of MMP-13 is downregulated, and cartilage matrix degradation is inhibited.¹⁹¹ Alternatively, by regulating the interaction between circRNA CDR1as and miR-7, chondrocyte proliferation is promoted and apoptosis is inhibited, thereby further restoring chondrocyte function and promoting cartilage repair.

Conclusions and Future Perspective

This paper conducts a systematic integrated study on the repair of articular cartilage damage in KOA. Based on chondrocyte regulation and joint inflammatory mechanisms, precise screening and functional verification of targeted miRNAs are performed. The excellent repair efficacy of these miRNAs in regulating chondrocyte activity and ECM metabolism is clarified. To address the drawbacks of conventional nanocarriers, including poor stability and insufficient miRNA loading-protection capacity, structural-functional modification is adopted. This approach greatly improves the biocompatibility, dispersion stability and sustained-release gene delivery performance of nanocarriers. Modified functional nanocarriers are further compounded with hydrogel matrices. By adjusting key preparation parameters, nanocomposite hydrogels with favorable mechanical properties, porous structure, biocompatibility and long-term drug-release capacity are fabricated, overcoming performance limitations of single-component hydrogels. Finally, this composite system systematically illustrates its repair mechanism for promoting cartilage regeneration and inhibiting inflammation and cartilage degeneration via gene-material synergistic effects and downstream signaling pathway modulation. A safe and efficient cartilage damage repair system is successfully constructed, which provides reliable theoretical basis and technical strategies for clinical treatment of cartilage injury.

As an innovative interdisciplinary achievement, the injectable miRNA-nanocomposite hydrogel systems integrates core advantages of biomaterials, nanotechnology, genetic engineering and regenerative medicine. With biomimetic support from hydrogel matrices, efficient delivery of nanocarriers and precise modulation of miRNAs, it establishes an all-round repair network of “molecular regulation-cell recruitment-environmental support-physical reinforcement-immune modulation”, offering a precise, effective and minimally-invasive novel strategy for KOA treatment. Intracellular miRNA molecular regulation adopts targeted intervention of specific miRNAs to precisely modulate core biological behaviors such as chondrocyte proliferation, differentiation and apoptosis. It reverses injury-induced cellular metabolic disorders, initiates repair processes at the molecular level and lays a foundation for subsequent repair steps. Endogenous cell recruitment relies on the bioactive microenvironment constructed by hydrogel scaffolds. It effectively induces local joint stem/progenitor cells and synovial mesenchymal stem cells to home and accumulate at defect sites. It replenishes local reparative cell reservoirs and supplies sufficient cell sources for neocartilage formation. On this basis, matrix microenvironment reconstruction proceeds. Recruited functional reparative cells continuously secrete cartilage-specific ECM components including type-II collagen and proteoglycans. They gradually repair and reconstruct impaired matrix networks, and restore structural integrity and physiological homeostasis of cartilage microenvironments. Synchronous physical reinforcement is implemented. Optimized hydrogel scaffolds possess mechanical properties matching native cartilage. They provide stable mechanical support and stress buffering for defect regions, avoid mechanical damage caused by joint movement, and build a stable mechanical microenvironment for cartilage remodeling and maturation. Lastly, local immune microenvironment remodeling is achieved. By regulating macrophage polarization and suppressing abnormally high expression of pro-inflammatory factors such as tumor necrosis factor and interleukins, persistent local inflammatory injury is alleviated. A pro-regenerative immune microenvironment with anti-inflammatory properties is established to realize efficient repair and functional recovery of defective cartilage.

Remarkable progress has been achieved in pathological mechanism interpretation, material innovation, carrier optimization and repair mechanism research for this system. In-vitro and in-vivo experiments confirm that it can effectively suppress joint inflammation, modulate immune microenvironments, preserve chondrocyte function, promote matrix regeneration and realize integrated osteochondral repair. Despite favorable pre-clinical therapeutic outcomes, inherent shortcomings and unresolved technical challenges remain. The hydrogel matrix matches mechanical properties of native cartilage only under static conditions. Under dynamic weight-bearing conditions of human joints, it exhibits inadequate fatigue resistance and tribological performance. Obvious mechanical attenuation occurs during long-term application, and its friction coefficient is markedly higher than that of native cartilage. Meanwhile, the interfacial bonding strength between hydrogel implants and host cartilage is low. Implant detachment readily occurs and leads to treatment failure. In-vivo application also faces delivery barriers and microenvironment adaptation challenges. Nanocarriers can hardly cross complex physiological barriers of joint tissues precisely. Overexpressed matrix metalloproteinases in KOA lesions trigger premature degradation of hydrogel scaffolds. In addition, phagocytosis of nanocarriers by immune cells and low endosomal escape efficiency severely impair targeted delivery efficiency and biological functions of loaded miRNAs. Long-term biosafety risks have not been fully clarified. Abnormal miRNA overexpression, nanocarrier residues and accumulation of metabolites may induce genotoxicity, persistent immune inflammation and organ injury. Notably, most existing pre-clinical efficacy validations are performed using small-animal models. Significant anatomical-physiological differences exist between small-animal and human knee joints. Distinctions cover multiple critical aspects including joint load patterns, kinematic characteristics, absolute and regional cartilage thickness, cartilage matrix composition, synovial immune microenvironment homeostasis and intra-articular metabolic conditions. Such physiological and mechanical interspecies differences cause obvious deviations in drug release profiles, cartilage repair responses and local inflammatory regulation between animal models and human joints. They reduce extrapolation reliability and experimental reproducibility of pre-clinical results and bring potential risks for clinical translation. Furthermore, patients with KOA show high inter-individual heterogeneity. Multiple factors including age, severity of cartilage degeneration, disease stage, body mass index, degree of joint deformity, systemic metabolic status and comorbidities directly affect local tissue repair responses and final therapeutic prognosis. Widespread individual differences make unified treatment regimens unsuitable for all patients and create major obstacles for standardized and universal clinical application of this technology.

Accordingly, to address these limitations, future research may refer to native cartilage biomimetic structures, and combine gradient-mechanical 3D-printing and self-healing hydrogel technologies to design cell-biomimetic nanocarriers for precise and efficient miRNA delivery adapted to joint mechanical conditions. Multi-omics target screening, medical imaging and 3D-bioprinting can be combined to fabricate patient-specific repair scaffolds and establish individualized precision treatment protocols. Integration of artificial intelligence, biosensors and gene-editing technologies enables material performance improvement, real-time therapeutic monitoring and synergistic modulation of action mechanisms, forming a complete diagnosis-treatment closed loop. Establishment of standardized production systems and improved clinical evaluation criteria can control production costs and facilitate medical insurance access to accelerate clinical translation. Based on core technologies of targeted delivery, long-term sustained release and biomimetic support in this study, application scenarios can be further expanded. This technology can be applied to various degenerative and traumatic osteoarticular diseases such as spinal degeneration, traumatic cartilage injury and rheumatoid arthritis, breaking application limits confined to knee cartilage repair. It is expected to become a core modality for precise KOA therapy, empower clinical diagnosis and treatment of diverse tissue repair and degenerative disorders, promote breakthrough advances in regenerative medicine, and provide safer, more precise and more efficient novel therapeutic options for patients.

Funding Statement

This work was supported by the Jilin Scientific and Technological Development Program (20230204077YY).

Data Sharing Statement

The data used in the study could be accessible from the correspond author.

Ethics Approval and Consent to Participate

All participants provided their informed consent; the study was reviewed and approved by the First Hospital of Jilin University’s Ethics Committee, and all methods involving human subjects were performed out in accordance with the Declaration of Helsinki.

Consent for Publication

All the participants in our study consent for publication.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors declared no conflict of interest.