Hydrogel Engineering Interventions for Precision Cartilage Regeneration: Bridging Pathological Mechanisms and Therapeutic Innovation

2.1 Cartilage part

Articular cartilage is a specialized connective tissue that covers the ends of bones in diarthrodial joints. Its intricate structure and composition are engineered to provide mechanical support and exceptional lubrication. The tissue is primarily composed of water, collagen, proteoglycans, and a sparse population of chondrocytes. Water constitutes 65% to 80% of the wet weight, conferring high hydration and elasticity, which enables stress dissipation through fluid movement during compression [1]. Type II collagen forms a 3D network, accounting for 25% to 35% of the dry weight. In the superficial zone, collagen fibers align parallel to the surface to resist shear forces, whereas in deeper zones, they adopt an interwoven, wavy configuration that enhances compressive strength [18]. Proteoglycans, such as aggrecan, which contains chondroitin sulfate and binds to hyaluronic acid, represent 10% to 15% of the dry weight. These molecules trap water to create a molecular sieve, regulate ion balance, and act as biological lubricants. The synergistic interaction between glycosaminoglycan chains and the collagen network confers the distinctive viscoelastic properties of cartilage.

Chondrocytes represent the functional core of cartilage but occupy only 1% to 5% of the tissue volume, residing in cavities known as lacunae. Superficial zone chondrocytes are flattened and metabolically active, synthesizing and renewing the ECM. In the deep zone, chondrocytes form isogenous groups and maintain matrix homeostasis by secreting type II collagen and proteoglycans. Structurally, articular cartilage is organized into four distinct layers [19, 20]. The superficial zone, comprising 10% to 20% of the thickness, contains densely aligned collagen fibers and a low cell density, providing a smooth, low-friction surface. The transitional zone features obliquely oriented collagen fibers and more rounded chondrocytes. The radial zone is characterized by collagen bundles arranged perpendicular to the joint surface, with chondrocytes aligned in columns. This structure culminates at the tidemark, a basophilic line that demarcates the transition to the calcified zone. The calcified zone anchors the cartilage to the underlying subchondral bone through a mineralized interface, thereby facilitating the transfer of mechanical loads (Figure 1A) [21]. This layered architecture and gradient composition provide articular cartilage with its remarkable biomechanical properties. A smooth collagen network and synovial fluid containing hyaluronic acid and lubricin maintain an extremely low coefficient of friction, which can be as low as 0.002 during movement. The deep calcified layer transmits mechanical loads to the subchondral bone [22]. Furthermore, the water-retaining capacity of proteoglycans allows the matrix to harden under compression by expelling fluid, thereby dispersing stress, and to recover its elasticity upon unloading as water is reabsorbed. It is critical to note that articular cartilage is both avascular and aneural. Nutrient supply occurs primarily via diffusion from the synovial fluid, supplemented by permeation from the subchondral bone, while metabolic waste is removed through cyclic loading and absorption [23, 24]. This passive diffusion-limited nutrition mechanism severely constrains the tissue’s innate repair capacity. While superficial injuries may see limited chondrocyte-mediated repair, deeper defects typically progress to fibrocartilage formation or calcification.

2.2 Bone part

The subchondral bone serves as a critical structural and metabolic foundation for the overlying articular cartilage. This complex tissue exhibits a distinct hierarchical organization, comprising a dense cortical bone plate at the surface and a porous trabecular bone network beneath it. The cortical bone, with its highly organized lamellar structure of collagen fibers, provides a solid mechanical anchor for the cartilage and effectively resists excessive deformation under load [25]. Underlying this, the trabecular bone forms an optimized mechanical system through its unique lattice of trabeculae. This network demonstrates a high porosity ranging from 50% to 90%, and the spatial orientation of the trabeculae aligns closely with the joint’s primary loading direction [26]. This refined architectural design allows the trabecular bone to absorb mechanical energy through elastic deformation while achieving high load-bearing efficiency with minimal weight [27].

A delicate cellular balance maintains the subchondral bone at the cellular level. Osteoblasts function as the primary bone-forming cells, continuously synthesizing key matrix components, including type I collagen and osteocalcin. In contrast, multinucleated osteoclasts are responsible for bone resorption, which they accomplish by secreting acids and proteolytic enzymes to degrade the bone matrix. Osteocytes, embedded within the mineralized matrix in the lacunar-canalicular system, act as the principal mechanosensors. Accounting for over 90% of all bone cells, osteocytes precisely regulate the dynamic balance between bone formation and resorption through an extensive communication network, thereby maintaining skeletal homeostasis [28].

From a materials science perspective, the mechanical properties of the bone matrix originate from its unique composite nature. The organic phase, predominantly type I collagen, constituting 20% to 30% of the dry weight, forms a 3D network that provides toughness and resistance to fracture. The inorganic phase, composed mainly of hydroxyapatite crystals, which account for 60% to 70% of the dry weight, impregnates this collagen network to impart stiffness and compressive strength [29]. This natural composite design enables the subchondral bone to meet the dual mechanical demands of high compressive strength and fracture toughness, making it ideally suited for the long-term weight-bearing function of joints.

Furthermore, the subchondral bone and the articular cartilage constitute a functionally integrated osteochondral unit. Mechanically, the subchondral bone dissipates stress through its controlled deformability, thereby preventing localized stress concentrations in the cartilage. Metabolically, its abundant vascular network provides essential nutrient support for the avascular cartilage via diffusion, helping to sustain chondrocyte viability. This profound structural and functional integration means that pathological changes in the subchondral bone, such as sclerosis or cyst formation, can directly disrupt the cartilaginous microenvironment and accelerate its degeneration. This tight functional coupling provides a critical histological basis for understanding the pathogenesis of osteoarthritis [29, 30].

2.3 Osteochondral defects

Injuries to the articular cartilage and subchondral bone are common orthopedic pathologies characterized by diverse types and complex mechanisms arising from the interplay of mechanical stress, biochemical imbalance, and a pathological microenvironment. Traumatic injury represents the most direct cause, where intense impact or falls during sports can induce superficial cartilage microcracks or subchondral bone bruising. These acute injuries often present with joint swelling and limited mobility and may progress to chronic degeneration without prompt intervention. Degenerative injury is strongly associated with aging and is characterized by reduced chondrocyte metabolic activity, decreased synthesis of type II collagen and proteoglycans, and elevated activity of matrix-degrading enzymes such as MMP-13. These changes lead to cartilage fibrillation and subchondral bone sclerosis, resulting in osteophyte formation and exacerbated joint pain [31]. Inflammatory injury, prevalent in conditions like rheumatoid arthritis, involves cytokines such as IL-1β and TNF-α activating chondrocyte apoptosis pathways. This process accelerates matrix degradation while inhibiting intrinsic repair mechanisms, ultimately causing full-thickness cartilage loss and subchondral bone erosion.

The pathological manifestations of cartilage injury can be categorized based on depth and severity. Superficial injuries involve surface softening or fibrillation, where chondrocytes retain partial metabolic function but possess limited regenerative capacity. Deep injuries extend into the radial and calcified zones, featuring collagen network disruption and abnormal calcification that can provoke stress fractures in the underlying bone. In full-thickness injuries, the complete loss of cartilage exposes the subchondral bone to synovial fluid, often leading to the formation of loose bodies that accelerate osteoarthritis progression. A distinct pathological entity, osteochondritis dissecans, frequently occurs in adolescent knees and stems from localized subchondral ischemia or repetitive microtrauma. This condition causes subchondral bone necrosis, with the affected bone fragment potentially detaching and causing mechanical symptoms like joint locking and chronic pain. For evaluation, magnetic resonance imaging clearly visualizes full-thickness cartilage defects and associated bone marrow edema, while computed tomography provides precise assessment of bony structural integrity, thereby supporting graded treatment planning [32]. The International Cartilage Repair Society grading system offers a quantitative clinical standard for assessing injury severity. This system ranges from grade I, indicating surface softening, to grade IV, representing full-thickness defects with exposed bone, where higher grades correlate with more advanced osteoarthritis and greater challenges for effective repair (Figure 1B) [33].

The clinical outcomes of cartilage injury are intrinsically linked to the selected repair strategy. Mild injuries may be managed conservatively through load reduction, physical therapy, or intra-articular injections, such as platelet-rich plasma (PRP), to modulate the local biological environment. Moderate to severe injuries typically necessitate surgical interventions, including microfracture, autologous chondrocyte implantation, or the transplantation of engineered biomaterial scaffolds. Recent advances in stem cell-based tissue engineering have focused on replicating the native cartilage’s mechanical gradient structure, which has significantly improved the functional integration of regenerated tissue and offers promising directions for treating complex defects. Nevertheless, significant challenges persist, including the prevention of abnormal subchondral vascularization and achieving a biomechanical match between the repaired and native tissue. The development of multimodal therapeutic strategies that address these multifaceted issues remains essential for optimizing long-term clinical outcomes.

3.1 Pathological mechanisms of cartilage injury

The pathological mechanisms of cartilage injury involve complex cellular and molecular interactions among cartilage, the synovium, and the subchondral bone. In the initial stage, mechanical stress or inflammatory stimuli trigger chondrocytes to release matrix-degrading enzymes such as matrix metalloproteinases and aggrecanases. This enzymatic activity progressively breaks down collagen fibers and proteoglycans, leading to structural disruption and functional impairment of the ECM. Concurrently, oxidative stress induces mitochondrial dysfunction in chondrocytes, resulting in the accumulation of reactive oxygen species. This oxidative damage activates caspase-mediated apoptosis and stimulates key signaling pathways, including NF-κB and MAPK, which promote the further release of pro-inflammatory cytokines like IL-1β and TNF-α [34]. These inflammatory factors then amplify the immune response by activating macrophages and synovial fibroblasts within the synovial cavity. This activation leads to the overexpression of destructive enzymes such as MMP-13 and ADAMTS-5, which accelerate the breakdown of the core cartilage components. Furthermore, matrix degradation products, including hyaluronic acid fragments and collagen peptides, function as damage-associated molecular patterns. These fragments persistently activate synovial immune cells through pattern recognition receptors such as TLR4, thereby establishing a self-perpetuating positive feedback loop that sustains chronic inflammation [35].

Simultaneously, the subchondral bone undergoes compensatory remodeling characterized by increased osteoclast activity and bone resorption, alongside attempted repair by osteoblasts. During this remodeling process, factors such as transforming growth factor-β and insulin-like growth factor are released. These factors not only drive bone changes but also permeate into the overlying cartilage layer, where they exert a direct influence on chondrocyte metabolism [36]. The pathology is further propagated as macrophages and synoviocytes in the synovial lining recognize cartilage debris, activating the NF-κB pathway and secreting more pro-inflammatory cytokines, which diffuse back into the cartilage.

As the disease advances, blood vessels from the subchondral bone penetrate the calcified cartilage layer, forming an invasive vascular pannus [37, 38]. This invasion disrupts the critical integrity of the osteochondral junction and introduces additional inflammatory cells and pro-angiogenic factors into the cartilage environment. This process establishes a vicious cycle intertwining inflammation, matrix degradation, and abnormal vascularization. Ultimately, this self-reinforcing tripartite pathological network among cartilage, synovium, and subchondral bone results in irreversible cartilage loss, chronic synovitis, and aberrant bone remodeling, collectively driving the progression of osteoarthritis (Figure 2).

3.2 Limitations of current pharmacological and surgical treatments

Current treatment strategies for osteoarthritis are designed to alleviate symptoms and slow disease progression, but they generally fail to reverse existing cartilage damage. These approaches exhibit significant limitations in their therapeutic precision, long-term safety, and broad clinical applicability. Pharmacological interventions primarily involve non-steroidal anti-inflammatory drugs and intra-articular injections [39, 40]. While these agents can suppress inflammation in the short term, for example, by reducing prostaglandin E2 production through COX-2 inhibition, their inability to penetrate the dense cartilage matrix prevents them from reaching the core of the injury. Furthermore, prolonged use of these drugs may paradoxically accelerate cartilage calcification. Biological agents, such as IL-1 receptor antagonists or anti-TNF-α monoclonal antibodies, offer more targeted modulation of inflammatory pathways. However, they carry a risk of immunogenicity and demonstrate limited efficacy in restoring an ECM that has already been degraded. Gene therapy approaches that utilize viral vectors to deliver anabolic genes like SOX9 or BMP aim to enhance matrix synthesis. Their clinical translation remains challenging due to low delivery efficiency, uncontrollable transgene expression, and persistent safety concerns [41]. A summary of typical drugs and small molecules used in cartilage injury treatment is provided in Table 1.

In the realm of surgical interventions, microfracture techniques promote the formation of fibrocartilage by recruiting bone marrow stem cells. The resulting repair tissue, however, lacks the biomechanically robust architecture of native hyaline cartilage and exhibits a high failure rate of up to 60% within two to three years [42]. Autologous osteochondral transplantation can provide genuine hyaline cartilage, but it is associated with donor site morbidity in 20% to 30% of cases. Furthermore, the repair of large defects often requires multiple complex procedures. Allograft transplantation carries inherent risks of immune rejection due to challenges in HLA matching and potential disease transmission, with five-year graft survival rates reported below 70%. Autologous chondrocyte implantation necessitates two separate surgical procedures and entails high costs, while the survival rate of the implanted chondrocytes is only 30% to 50%. These cells also frequently lose their stable phenotype over the long term. Emerging technologies, such as 3D bioprinted scaffolds, can mimic the mechanical gradients of the native ECM. However, the inadequate vascularization of these constructs often leads to central cell necrosis, and the engineered tissues frequently fail to integrate and adapt under dynamic mechanical loading in the joint [43, 44]. These collective shortcomings underscore a fundamental inadequacy of existing therapies to achieve targeted, durable, and functionally integrated cartilage repair. This persistent challenge highlights the urgent need for the development of multi-modal therapeutic strategies that effectively combine biological activity with biomechanical compatibility.

6.1 Drug-loaded hydrogel for cartilage repair

Drug-loaded hydrogels represent an advanced strategy for cartilage repair in osteoarthritis, capitalizing on their 3D network structure, excellent biocompatibility, and controllable drug release profiles. These biomaterial systems serve a dual function by acting as biomimetic scaffolds that replicate the native cartilage ECM to provide mechanical support for new tissue growth, while also enabling the targeted delivery of therapeutic agents to modulate the pathological microenvironment and enhance chondrocyte activity. This combined approach helps disrupt the self-perpetuating cycle of cartilage degradation and chronic inflammation.

Recent research has demonstrated substantial progress in utilizing drug-loaded hydrogels for cartilage regeneration. Scientists have engineered systems that incorporate small molecule drugs, targeted pathway inhibitors, and clinically relevant agents to achieve a coordinated therapeutic outcome combining microenvironment regulation with structural tissue repair. For instance, Chen et al. [74] developed an inflammation-responsive hydrogel loaded with bevacizumab for anti-inflammatory and anti-angiogenic effects in cartilage repair. This system employs a dynamic hydrogen-bonded network stabilized by phenolic hydroxyl groups to enable sustained release of the anti-angiogenic drug at the defect site, effectively inhibiting VEGF signaling and preventing pathological vascular invasion. Concurrently, released magnesium ions mitigated inflammation through suppression of the NF-κB pathway while promoting chondrogenic differentiation of resident bone marrow mesenchymal stem cells. In a rabbit model of full-thickness cartilage defects, this treatment generated regenerated tissue with collagen organization closely resembling that of native hyaline cartilage.

Addressing chondrocyte ferroptosis, Yang et al. created a folic acid-modified polydopamine nanoparticle hydrogel delivering leonurine [75]. This targeted system significantly reduced IL-1β-induced ferritinophagy by specifically modulating M1 macrophage polarization. The gelatin-polyethylene glycol diacrylate composite formed a durable drug reservoir within the joint space, extending the therapeutic duration to more than three times that of conventional delivery methods while restoring the elastic modulus of repaired cartilage to near-normal levels.

Huang et al. designed a gel responsive to reactive oxygen species that encapsulates cartilage growth factor (KGN) for cartilage repair [76]. This system underwent ultrasound-triggered in situ gelation with subsequent sustained KGN release, which activated the Smad5/mTOR pathway to drive chondrogenic differentiation of mesenchymal stem cells. In rabbit models, this treatment achieved 78% depth repair in full-thickness defects, with glycosaminoglycan content in the regenerated tissue reaching 92% of normal cartilage values. Furthermore, a calcium/strontium co-crosslinked hydrogel incorporating the Wnt pathway inhibitor LGK-974 effectively suppressed β-catenin signaling and reduced the proportion of pro-inflammatory M1 macrophages to 15% [77]. The incorporated strontium ions simultaneously stimulated chondrocyte anabolic activity, resulting in 68.5% type II collagen-positive areas in a porcine cartilage defect model, presenting a promising approach for osteoarthritis intervention.

These advancements highlight the multidimensional benefits of drug-loaded hydrogel platforms, where small molecule drugs provide prolonged therapeutic action through sustained release kinetics, targeted inhibitors precisely block pathological signaling cascades, and clinically approved compounds facilitate translational potential. From a technical perspective, dynamic crosslinked networks enable stimulus-responsive drug release, biomimetic mechanical gradients replicate zonal cartilage characteristics, and surface modifications enhance biocompatibility and integration. Future development will likely focus on sophisticated multi-drug co-delivery systems capable of spatiotemporally controlled sequential release, such as initially releasing anti-inflammatory agents followed by pro-regenerative factors, or designing intelligent responsive hydrogels that synchronize drug release profiles with the distinct phases of the tissue regeneration process.

6.2 Stem cell-loaded hydrogels for cartilage repair

Stem cell-loaded hydrogels represent a rapidly advancing frontier in cartilage tissue engineering, combining the regenerative potential of various stem cell sources with the supportive and biomimetic properties of hydrogel scaffolds. These integrated systems have demonstrated significant progress in achieving functional cartilage regeneration. Researchers have successfully incorporated multiple cell types, including bone marrow mesenchymal stem cells (BMSCs), adipose-derived mesenchymal stem cells (ADSCs), induced pluripotent stem cells (iPSCs), and human embryonic stem cells (hESCs), into hydrogel matrices to enhance repair outcomes. For instance, Zhong et al. [78] developed an injectable hydrogel derived from decellularized porcine meniscus ECM and loaded it with BMSCs for implantation in a rabbit model of full-thickness white-zone meniscus injury. The thermosensitive hydrogel, combined with the immunomodulatory properties of the ECM, synergistically enhanced chondrogenic differentiation of the stem cells, resulting in regenerated tissue with glycosaminoglycan content comparable to native cartilage. In another approach, Wang et al. created a methacrylated silk fibroin hydrogel featuring surface microgrooves to guide the oriented growth of BMSCs [79]. When combined with a decellularized cartilage matrix scaffold in a rabbit tracheal defect model, this construct supported the regeneration of neocartilage with a native-like elastic modulus after 12 weeks, without calcification or fibrosis.

Adipose-derived mesenchymal stem cells have gained prominence due to their accessibility and abundance. Yang et al. [80] designed a chitosan hydrogel composite loaded with ADSCs, in which an optimized crosslinked network enabled the sustained release of the cells. This system significantly enhanced type II collagen expression while reducing type I collagen deposition, indicating successful inhibition of fibrotic tissue formation. In a more sophisticated strategy, Chen et al. incorporated human urine-derived stem cells overexpressing basic fibroblast growth factor into a decellularized small intestinal submucosa hydrogel for tendon-bone interface repair [81]. This construct modulated local inflammation by promoting M2 macrophage polarization, resulting in extensive type II collagen deposition covering 68.5% of the defect area and mechanical properties restored to near-normal levels.

hESCs offer exceptional differentiation potential but face clinical limitations due to ethical concerns and tumorigenic risks. As a promising alternative, induced pluripotent stem cells have emerged as a viable cell source. Toh et al. [82] demonstrated that hESC-derived chondrocytes encapsulated in hyaluronic acid (HA) hydrogels formed ECM-rich cartilage structures. When implanted into critical-sized osteochondral defects in rats, these constructs closely mimicked native osteochondral tissue after 12 weeks, while control defects treated with hydrogel alone showed poor repair. Importantly, no tumor formation was observed, supporting the safety profile of this approach. Similarly, porcine model studies confirmed that transplanted iPSCs could regenerate cartilage at osteochondral defect sites without tumorigenesis [83]. A 3D microchamber hydrogel transition platform has further been developed to guide iPSC differentiation into chondrocytes, producing grafts rich in type II collagen. Notably, the repair quality achieved with iPSC-derived chondrocytes surpassed that mediated by BMSCs, potentially due to epigenetic modifications such as increased methylation at CpG sites in the COL10A1 promoter that suppress hypertrophic differentiation and promote stable cartilage phenotypes. Despite these advances, low survival rates of transplanted iPSCs remain a significant challenge.

In summary, stem cell-loaded hydrogels have demonstrated promising cartilage repair outcomes in animal models. However, clinical translation requires overcoming several obstacles, including mismatched degradation and regeneration kinetics, unstable chondrogenic differentiation of stem cells, and the establishment of standardized manufacturing protocols. Future research should focus on advanced material modifications, precise control of release kinetics, and optimized preclinical evaluation systems to accelerate the clinical application of these regenerative strategies.

6.3 Gene-loaded hydrogels for cartilage repair

Gene-loaded hydrogels represent an advanced therapeutic strategy for multi-dimensional intervention in cartilage degeneration through the incorporation of novel regulatory molecules, including miRNA, siRNA, and circRNA. These systems enable precise genetic modulation within the challenging joint environment. Li et al. [84] developed a streamlined zinc oxide hydrogel delivering miR-17-5p through a dynamically crosslinked network. This design reduced injection resistance by 40% due to its optimized hydrodynamic properties, while the responsively released miRNA effectively inhibited MMP-13 expression and activated the chondrogenic SOX9 pathway. In a rat model, this treatment restored the elastic modulus of regenerated cartilage to 85% of normal tissue values. To address cellular senescence, a self-assembling peptide hydrogel delivering miR-29b-5p was engineered to recruit synovial mesenchymal stem cells through incorporation of the homing peptide SKPPGTSS [85]. The sustained miRNA release suppressed P16INK4a expression via the TET1/PPARγ pathway, achieving 78% depth repair of full-thickness cartilage defects in an osteoarthritic rat model. Similarly, Long et al. created a nanocomposite hydrogel loaded with miR-455-3p that utilized a biomimetic gradient pore structure to enable sustained release [86]. This system improved miRNA delivery efficiency threefold, significantly enhanced type II collagen synthesis, and inhibited ADAMTS5 activity, resulting in glycosaminoglycan content recovery reaching 92% of normal levels in a porcine cartilage defect model.

In the domain of siRNA delivery, an inflammation-regulated self-assembling hydrogel microsphere system was developed to deliver siRNA targeting IL-1β [87]. By incorporating pH-responsive calcium phosphate co-precipitation, this system achieved 80% gene silencing efficiency in the weakly acidic microenvironment of cartilage while effectively anchoring to the cartilage matrix to prevent therapeutic diffusion. In a rabbit model, it reduced MMP-13 expression by 62%. Zhang and collaborators constructed a metal-organic framework co-loaded with HIF-2α siRNA and curcumin [87]. The released siRNA effectively cleaved HIF-2α mRNA, achieving potent gene silencing, while curcumin concurrently suppressed inflammation and cartilage degradation, demonstrating a valuable synergistic therapeutic effect.

The repair mechanisms of gene-loaded hydrogels primarily operate through three key pathways. The first pathway involves inflammation suppression, exemplified by microRNA-140-loaded hydrogels that significantly reduce intra-articular levels of pro-inflammatory factors, including IL-6, IL-1β, and TNF-α. The second pathway promotes chondrogenic differentiation, as demonstrated by systems containing small molecule inducers like TD-198946, which effectively drive mesenchymal stem cells toward chondrogenic commitment while inhibiting fibrotic changes. The third mechanism focuses on maintaining ECM homeostasis by regulating the expression of collagen-degrading enzymes, such as MMP-13 and ADAMTS-5, thereby reducing matrix breakdown.

Despite these significant advances, several challenges remain unresolved. Non-viral delivery systems frequently suffer from limited transfection efficiency, while viral vectors present potential safety concerns, including immunogenicity and insertional mutagenesis. Additionally, mismatched kinetics between hydrogel degradation and tissue regeneration rates often compromise clinical outcomes. Successful translation to clinical applications will require overcoming several hurdles, including validation in large animal models, comprehensive long-term safety assessments, and the development of standardized manufacturing protocols. Future research should prioritize optimizing vector design, developing smart, responsive hydrogels with precise release kinetics, and conducting multi-center clinical trials to advance this promising therapeutic approach toward clinical reality.