Osteoarthritis (OA) is a chronic whole joint disorder characterized by the degeneration and malfunction of various parts of joint tissues, including the wear and tear of articular cartilage, the formation of osteophytes, and inflammatory responses in the synovial membrane and other tissues surrounding the joint (Glyn-Jones et al., 2015; Hügle and Geurts, 2017). OA can be found in all joint structures, including knee joints, hip joints, shoulder joints, and temporomandibular joint (TMJ), etc. (Coaccioli et al., 2022). OA causes joint pain, swelling and stiffness. In severe cases, OA can limit mobility and even disability (Abramoff and Caldera, 2020). OA is a common regenerative disease among elderly and the number of patients with OA worldwide had reached 595 million in 2020 (Collaborators, 2023). It is estimated that by 2050, the number of global patients with knee OA will continue to increase to 642 million (Collaborators, 2023; Safiri et al., 2020). The rising prevalence of OA, driven by global demographic aging, poses a substantial medical and economic burden on individuals and represents a growing public health challenge worldwide.

Currently, great efforts and progresses have been made to decipher the underlying mechanism of OA. However, the specific challenge of OA include two aspects: the precise molecular mechanism of the pathogenesis of OA is incompletely defined and the existing treatment methods for OA mainly alleviate symptoms which hardly achieves satisfactory results. Therefore, innovative therapies are needed for OA treatment. In response to this challenge, cartilage organoids, as a powerful tool, have been developed to simulate the microenvironment of articular cartilage, reproduce the occurrence and development process of OA, and utilize in potential drug screening. This emerging in vitro approach provides a unique platform for in-depth exploration of the disease mechanism, screening of and evaluation of the effectiveness of new therapies. Utilizing cartilage organoids for basic research and therapy exploration is expected to bring new hope for ultimately overcoming OA (Maharjan et al., 2024). In this review, we summarized our current understanding of OA pathogenesis and treatment, highlighted the construction of cartilage organoids, and discussed their current applications and future directions in OA treatment.

OA is a complex pathological process driven by multiple factors, involving the cellular apoptosis of chondrocytes, remodeling of subchondral bone, muscle weakness, inflammation, and interaction between neuro-immunity (Umoh and de Oliveira, 2024; Guilak et al., 2018; Yao et al., 2023) (Figure 1).

At the early stage, the X-ray film shows a mild narrowing of the joint space with obvious small osteophytes. Mild wear and roughness cause cell damage of chondrocytes, releasing inflammatory mediators, such as interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α) and matrix metalloproteinase (MMP), into the joint environment (Woodell-May and Sommerfeld, 2020; Chen et al., 2020a). After the synovial membrane is stimulated by these inflammatory factors, the activated macrophages in the synovial membrane regulate the chondral microenvironment by secreting pro-inflammatory factors such as IL-1β and TNF-α, triggering systemic inflammation and activating NF-κB and MAPK pathways in chondrocytes, resulting in excessive production of degradation enzymes like MMP-3, MMP-13 and ADAMTS-5 by chondrocytes, thereby causing the degradation of collagenous extracellular matrix (ECM) (Mantovani et al., 2004; Lopes et al., 2017; Zhang et al., 2019; Shibo et al., 2024). Meanwhile, the levels of chemokines such as IL-6 and IL-8 in synovial fluid increase, forming a chronic inflammatory microenvironment (Samavedi et al., 2017). During the progress stage, X-ray films show definite narrowing of the joint space, moderate osteophytes, mild hardening of the subchondral bone, and possible bony deformity of the knee joint. The progressed hard bone changes are ly resulted from the increases in reactive bone formation. During OA progress, the chondrocyte apoptosis is an important pathological process. Reported studies indicated that some cytokines such as IL-1, TNF-α and TGF-β2, and the p38 MAPKs pathway and the Fas/FasL pathway link to chondrocyte apoptosis in OA pathogenesis (Hwang and Kim, 2015; Fazio et al., 2024; Sun et al., 2020). In the advanced stage, x-ray films show severe joint space stenosis, a large number of osteophytes, obvious subchondral bone sclerosis, and obvious bony deformity of the knee joint. During this stage, new bone grows at the edges of the cartilage, joint capsule and ligament junctions, forming osteophytes. This development of subchondral bone sclerosis and osteophytes is driven by the imbalance of Wnt/β-catenin and RANK/RANKL signaling pathways, which may gradually lead to overall joint dysfunction (Kovács and Nagy, 2019; Hongbo Ai et al., 2025). Moreover, abnormal loading promotes bone formation through the TGF-β/Smad pathway, resulting in excessive mineralization and a decrease in the mechanical stability of subchondral bone (Ting-Hsuan Wang et al., 2024).

During the OA pathogenesis, the pain is highly associated with nerve innervation and synovium inflammation in the affected joints. The joint is a highly innervated organ. In humans, the joint branches of the tibial nerve that innervate the posterior capsule of the knee joint contain 70%–80% unmyelinated C-fibres and sympathetic nerves. Pain receptors are widely distributed in multiple tissues such as the joint capsule, ligaments, periosteum, meniscus, subchondral bone, and synovium (Malfait and Schnitzer, 2013). Recent reported showed that netrin-1, an axon-guiding factor, may play essential roles in OA pain and OA progress. During OA progress, the metabolism of the subchondral bone altered and osteoclasts enhance the secretion of molecular netrin-1 (Zhu et al., 2019; Suri et al., 2007). Netrin-1 contribute to OA pain by two distinct approaches. On one hand, netrin-1 induces angiogenesis by promoting the function of endothelial cells at the bone-cartilage junction. On the other hand, it directly stimulates the growth of sensory nerve axons through the netrin-1 receptor, which in turn leads to abnormal changes in the surrounding nerve distribution. These processes mediated by netrin-1 jointly drive the progression of OA and triggers pain in the degenerated joint. In addition to nerve innervation during the progression of OA, macrophages accumulate in the joints and synovium, leading to inflammation, pain and cartilage degradation. Studies showed that pro-inflammatory macrophages (M1 macrophages) not only directly release pro-inflammatory factors to stimulate the injured nerve endings, but also enhance the sensitivity of sensory neurons to prostaglandin E2 (PGE2) by secreting cytokines such as TNF-α, thereby promoting hyperalgesia (Zou et al., 2024). Furthermore, nerve growth factor (NGF) can increase joint pain by stimulating neuronal growth and enhancing the sensitivity of peripheral nociceptive neurons. Aso K quantified the histopathology of OA tissues, NGF expression, and neuropeptide-calcitonin gene-related peptide (CGRP) nerves in OA model rats induced by medial meniscus resection before and after the operation at different time points. Their results showed that NGF expression had tissue specificity and time dependence: it increased early in the synovium and then decreased, but continued to increase in the bone-cartilage channels and bone marrow. Correspondingly, the CGRP nerve innervation, which is a marker of sensory nerves, also increased along with NGF expression, confirming that NGF is the core molecule driving the growth of joint nerves and pain associated with OA (Aso et al., 2022).

The therapeutic aim of OA treatment is to alleviate the pain symptoms, protect joint structure, and maintain or improve joint function. Depending on the different disease stages and patients’ conditions, clinical therapeutic treatment varies (Figure 1). At the early onset of OA, patients are suggested with basic treatment, such as weight control, exercise therapy, and physical therapy (Van Doormaal et al., 2020). During the moderate stage of OA, patients experience cartilage wear, narrowing of joint space and obvious osteophytes. If there is mild pain, one can consider using painkillers such as non-steroidal anti-inflammatory drugs, anti-anxiety and anti-depressants, anti-inflammatory drugs, and Chinese patent medicines, etc. (Hermann and Muller-Ladner, 2018; Wang et al., 2018; Khan et al., 2025). Moreover, intra-articular injections of hyaluronic acid and platelet-rich plasma (PRP) can be used as an additional treatment for OA patient to lubricate the joints and reduce the pain (Du et al., 2025; Sewell and Ö, 2022; Karaborklu et al., 2024). If these painkilling strategies cannot reach patients’ requirement, minimally invasive interventional procedures will be applied to alleviate symptoms. The common minimally invasive surgeries for treating OA include arthroscopy, unicompartmental knee arthroplasty (UKA), and high tibial osteotomy (HTO). Arthroscopy helps delay the progression of the disease by repairing the meniscus and ligaments (Yang et al., 2023a). UKA preserves the lateral compartment, anterior and posterior cruciate ligaments, and the patellofemoral joint, allowing it to bear the natural biomechanics of the knee joint (Lim et al., 2014). HTO reduces cartilage wear by correcting the lower limb alignment. These surgeries all have the advantages of minimal trauma and quick recovery (Liu et al., 2019). When OA has reached a severe stage and the patient’s joint movement becomes extremely painful with limited joint functions, the doctor mainly adopts joint replacement surgery (Anne, 2015).

In short summary, the current treatments for OA only relieve symptoms and the specific challenges remains, including the detailed molecular regulation and precise tissue-specific pathology during OA progress. Moreover, the current OA studies widely utilized traditional animal models, which have certain limitations such as high cost, long modeling time, ethical controversies and limitation to fully recapture human OA pathology. Therefore, there is an urgent need for innovative therapies to achieve cartilage regeneration for OA therapy.

The development of tissue culture technology has been demonstrated with significant scientific potential as in vitro models to explore OA pathogenesis. Tissue culture helps to replicate tissues and organs in vitro, thereby facilitating drug screening and research on disease mechanisms. In general, two-dimensional (2D) and three-dimensional (3D) culture techniques are applied to chondrocyte studies. Traditional 2D culture technology, which allows stem cells or adult cells to grow in a two-dimensional manner on the surface of a culture dish, is simple to operate and has been widely used as a mature technique. However, this cultivation method cannot maintain the original characteristics of cells over the long term, nor can it simulate the complex microenvironment and processes within the body, such as signal transduction and changes in spatial structure (Jensen and Teng, 2020). Compared to 2D culture, 3D culture technology simulates the three-dimensional environment inside the body and enables cell proliferation and differentiation. Its advantages include the ability to preserve the in vivo microenvironmen and the ability of being intuitive and controllable.

In 1907, H.V. Wilson’s observations reveal the self-organization and regeneration of isolated sponge cells, which lays the foundation for the development of organoid (Wilson, 1907). The concept of organoids can be traced back to 1946. Smith and Cochrane first described it as “cystic organoid teratoma” when conducting research on cystic teratoma (Smith and Cochrane, 1946). In 2009, Hans Clevers transplanted Lgr5 (+) intestinal adult stem cells from mice into a matrix gel and cultivated intestinal organoids. This was the first description of the process of generating organoids from adult stem cells, and this technique has now become the basis for various organ and cancer research as well as personalized medicine (Sato et al., 2009). Kuo CJ upgraded the organoids from a “tumor morphology simulator” to a “tumor immunotherapy testing platform,” and designed a Patient-Derived Organoids (PDO) system with an air-liquid interface (ALI): the lower layer is a collagen gel providing support, where the organoids absorb nutrients; the upper layer is exposed to the air and directly acquires sufficient oxygen. Thus, cells grow at the interface between gas and culture medium. This system provides a living in vitro model for the study of tumor immune microenvironment and personalized immunotherapy testing (Neal et al., 2018). These have laid a solid foundation for the research and application of organoids. In 2014, Lancaster and Knoblich systematically proposed the concept of organoids and defined organoids as “collections of organ-specific cells that develop from stem cells or organ-progenitor cells and are capable of self-organizing through cell division and spatially restricted line differentiation in a manner similar to in vivo” (Lancaster and Knoblich, 2014). In the early 21st century, with the rapid development of regenerative medicine and stem cell technology, the discovery of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) has opened up new avenues for the research of organoids (Takahashi and Yamanaka, 2006; Oldershaw et al., 2010).

Organoids are self-assembling 3D tissues generated from pluripotent stem cells or adult cells, which have been recognized as a emerging powerful tool for developmental biology and disease modeling (Dutta and Clevers, 2017; Hu et al., 2018). Moreover, organoids are formed to replicate the cellular, structural and functional characteristics of a real organ, which have been developing rapidly to narrow the gap between two-dimensional cultivation and animal models (Chen et al., 2022; Corrò and Li, 2020; Takasato et al., 2016; Zhang et al., 2024a). In addition, organoids possess the characteristic of being constructed from multiple types of cells, which is suitable for in vitro studies of various tissues and organs, thus promoting drug discovery and disease modeling efforts (Sato et al., 2009; Rossi and Lutolf, 2018).

The pathogenesis of OA involves complex multi-factor interactions. Cartilage organoids, with their highly biomimetic characteristics, provide an innovative platform for multifunctional applications in OA pathogenesis and treatment, which includes in vitro disease models, drug screening, regenerative medicine and biomechanical studies (Aikang et al., 2025) (Figure 4).

Even though there are promising application prospects of cartilage organoids in the treatment of OA, remaining challenges limit the scope and effectiveness of cartilage organoids in clinical applications. The construction of cartilage organoids requires precise control of culture conditions, including the selection of biomaterials, the addition of cytokines, and the simulation of physical environments. The fine regulation of these conditions is crucial for the successful construction and functional maintenance of organoids (Hu et al., 2025; Bajic et al., 2020).

First, since OA is a whole joint disease, study with cartilage organoids only answer part of the question. Mature organoids need to replicate “Mini-joint” in vitro which contain multiple types of cells. However, different cells require different conditions for in vitro cultivation, which poses a challenge for the construction of multi-cellular organoids. Currently, single-cell organoid cultivation has become relatively mature, and dual-cell co-culture has also made progress. However, the construction of mature organoids containing multiple types of cells still requires overcoming the technical difficulties of multi-cellular co-culture and exploring compatible cultivation conditions. Furthermore, during the cultivation process, the internal cells may encounter problems of insufficient nutrient and oxygen supply. Due to the 3D nature of organoids, if they are too large (up to millimeter or centimeter levels), the internal cells are prone to being isolated from the external cells, resulting in a lack of sufficient nutrients and ultimately death. Therefore, how to effectively prevent cell aging and death during the construction process is an important challenge (He et al., 2022).

Second, the preparation and optimization of culture conditions or physical microenvironment is still a challenge in the construction of cartilage organoids for OA treatment. Among all the factors, the biocompatibility and mechanical properties of scaffolds still need to be optimized (Yi et al., 2021). On the one hand, ideal scaffold materials are difficult to obtain: natural materials have good biocompatibility but high processing costs, while synthetic materials are easy to process but lack cell-inducing properties. On the other hand, the structural design of the scaffold (such as porosity, mechanical properties) needs to precisely match the cartilage tissue, making it very difficult to achieve a complex structure with uniform and controllable degradation. Moreover, integrating bioactive molecules to promote cell behavior also faces challenges in effective delivery and maintaining activity (Faeed et al., 2024). Although the biochemical regulatory factors are powerful tools for construction cartilage organoids, their application faces multiple challenges. These factors have pleiotropic effects and complex interaction networks, and different factors have synergistic, antagonistic or overlapping effects. Therefore, it is difficult to precisely control their dosage and concentration. At the same time, different individuals or different animal models may have different responses to the factors, and the microenvironment in which the cells are located (such as ECM, co-cultured cell types, etc.) can significantly affect the effect of factors. These issues all require more basic research and clinical practice to explore.

Last but not least, there is a lack of unified standards in the production and validation of cartilage organoids. Independent studies reported diverse cell sources, culture conditions and treatment in the laboratory, which are validated mainly with in vitro methods including chondrocyte-specific mRNA and protein expression test, cartilage staining. However these in vitro validation neither provide good histological evidence of real cartilage, nor cartilage repair or regeneration trails in vivo. Moreover, to achieve clinical application, it is difficult to produce functional and fully structured cartilage organoids at a low cost, safely, and on a large scale. Therefore, it is necessary to develop efficient, scalable, and standardized production technologies that comply with Good Manufacturing Practice (GMP) to meet clinical and commercial needs (Desai and Gishto, 2015).

The complex pathogenesis of OA continues to present significant challenges, leaving critical gaps in the existing body of research. While current pharmacological and surgical interventions offer some benefits to patients, a deeper understanding of the underlying mechanisms, as well as the development of novel therapeutic strategies, remains imperative. Cartilage organoids, with their biomimetic properties, adaptability, and efficiency, have the potential to revolutionize both fundamental research and therapeutic applications in OA.

With the fast development of new techniques, several groundbreaking technologies have been applied for further enhancement and expansion of cartilage organoids. For example, organ-on-a-chip is an engineered or micro-manufactured culture system that incorporates microfluidic channels, enabling it to naturally simulate the dynamic shear stress of in vitro culture, thereby enabling the simulation of organ functions (Zhang et al., 2024b). The dynamic integration of cartilage organoids and organ-on-a-chip will make great advantages of cell variability of organoids and device complexity of organ-on-a-chip, which shall bring new insights to clinical studies (Yimu et al., 2024). Moreover, micro-manufacturing and micro-fluidic technologies enable the controllable size of organoids, facilitating the construction of precisely sized organoids structures. These technologies can enhance the reproducibility of organoids, offer various environmental control and monitoring capabilities, and support large-scale production (Velasc et al., 2020; Shyam and Palaniappan, 2023). In addition, the tissue clearing technique enables high-resolution confocal imaging of 3D architectures of transparency tissues. Ni Y introduced an optimized solution for 3D analysis of bone tissue, achieving efficient visualization of hematopoietic and stromal cells in transparent bone samples, providing a reliable tool for studying rare cell populations and their spatial dynamics within organs (Ni et al., 2025). Hence, these technologies provide more accurate and efficient solutions for the basic research and clinical application of organoids.

By facilitating interdisciplinary integration of cutting-edge technologies, cartilage organoids not only provide new insights into the pathophysiology of OA but also hold promise for accelerating the translation of therapeutic innovations from preclinical studies to clinical practice.