The skeletal system, composed of bones, joints, muscles, tendons, and ligaments, plays a crucial role in human movement and support, working in concert to facilitate bodily motion, maintain posture, and preserve balance. However, with ageing, sports injuries, or the progression of diseases, these components of the skeletal system are prone to degenerative changes or damage, leading to a spectrum of conditions such as bone fractures, intervertebral disc degeneration, osteoarthritis, muscle atrophy, and ligament or tendon tears (Figure 1). These diseases not only impair patients’ mobility but also greatly impacting their quality of life and mental health, presenting significant challenges to healthcare systems. Traditional treatments often have limitations in efficacy and face high recurrence rates, underscoring the necessity for developing more advanced therapeutic approaches to safeguard human health.

Organoids are three-dimensional (3D), in vitro cultured micro–organ models capable of self–assembly, reproducing the structure and function of natural tissues. In recent years, organoid technology has attracted significant attention in the field of biomedical research. As studies have progressed, organoids have provided novel methodologies for investigating tissue development, disease mechanisms, and drug screening. Research on skeletal organoids has also emerged, with successful construction of models for bones, muscles, and joints, demonstrating tremendous potential in disease research and regenerative medicine. For instance, “mini–joints” synthesised through co–culturing bone and cartilage organoids have been utilised to test drugs for arthritis treatment,¹ while gene–editing techniques have been employed to create organoid models of muscular dystrophy, aiding in the exploration of disease progression and the identification of therapeutic strategies.² These findings indicate that skeletal system organoids not only facilitate a more comprehensive understanding of disease onset and progression but also hold significant promise in personalised medicine and regenerative therapy.

Given the clinical importance of skeletal organoids, there remains a lack of comprehensive reviews on the subject. This review aims to address this gap by first providing an overview of the definition and technological basis of organoids, followed by a systematic summary of the developmental history and current research progress of skeletal organoids. Subsequently, this article will review various organoid models, including those of bones, muscles, joints, ligaments, and tendons, discussing in detail their construction methods, application areas, and future potential. In addition, this review will provide a thorough discussion on the future prospects, challenges, and research directions of skeletal organoids, providing valuable references for researchers and promoting further advancement in this emerging field.

Organoids are 3D cell clusters cultured in vitro. Through the induced differentiation of pluripotent stem cells or organ–derived cells, these clusters grow and self–assemble on supporting substrates, such as hydrogels, ultimately forming organ–like structures with certain physiological functions that can be maintained in long–term cultures. As a 3D culture model, organoids exhibit significant advantages in many aspects. Compared to traditional two–dimensional (2D) cultures, organoids more closely mimic natural organs regarding gene and protein expression, metabolic function, and micro–architecture. Furthermore, organoids can be maintained for extended periods, display structures more similar to human tissues, and can be employed to model disease onset, offering immense potential in drug screening and organ development research (Figure 2).

The study of organoids can be traced back to 1907 when Henry and colleagues successfully cultured isolated sponge tissues in vitro, proving the feasibility of ex vivo culture. This experiment also demonstrated that organoids could self–organise into multicellular structures without external intervention, which is a hallmark feature of organoids.³ However, there was still a significant gap between ex vivo culture and the construction of self–organising organoids, as cells derived from organs rarely differentiate and proliferate into complete organoids. This led researchers to consider the induction and cultivation of pluripotent stem cells. However, obtaining human pluripotent stem cells raised ethical concerns, necessitating an alternative approach in organoid research.

A breakthrough came in 2006 when Takahashi and Yamanaka⁴ successfully induced the formation of induced pluripotent stem cells (iPSCs) by reprogramming somatic cells into pluripotent stem cells, opening up a new direction for organoid research based on pluripotent stem cell induction. In 2009, Sato et al.⁵ successfully constructed intestinal epithelial organoids, marking the beginning of formal organoid research. The following decade witnessed rapid advancements in the organoid field. In 2011, Eiraku et al.⁶ constructed retinal organoids using mouse embryonic stem cells; in 2013, brain organoids were successfully created from human pluripotent stem cells;^6,7 in 2020, venom gland organoids were successfully developed;⁸ in 2021, scaffold-free cartilage organoids were developed;⁹ and by 2024, bone organoids for large bone repair were successfully engineered.¹⁰ With the continuous improvement of scaffold materials and construction methods, the development of the organoid field is expected to accelerate further (Figure 3).

As self–assembled, 3D cell clusters cultivated in vitro, organoids share certain similarities with traditional 2D cell culture methods, such as the need for selecting suitable cells, a supportive matrix, and inductive agents or physical cues that enhance cell growth and differentiation. The establishment of stable, long–term organoids requires the precise spatial and temporal integration of these elements.

The construction of organoids depends on an appropriate cell source, with easily culturable, highly differentiated cells serving as the foundation. Suitable cells for organoid formation are typically stem cells, such as mesenchymal stem cells, iPSCs, adipose–derived mesenchymal stem cells, embryonic stem cells, and organ–specific stem cells. Under suitable growth conditions, these stem cells can create ideal organoid models. Adipose–derived mesenchymal stem cells, which possess multi–lineage differentiation potential, are commonly found in adipose tissue, bone marrow, and umbilical cords. They have the potential to differentiate into osteocytes, chondrocytes, and adipocytes and have advantages in ease of cultivation and reduced immune rejection, making them ideal for bone organoid construction.¹¹

iPSCs, produced by reprogramming adult somatic cells to regain pluripotency, resemble embryonic stem cells in their capacity to differentiate into multiple human cell types.¹² As artificially induced cells, iPSCs can be used to generate personalised cellular and tissue models for disease research and drug screening, facilitating personalised treatments and minimising immune rejection in tissue transplantation.¹³ Moreover, iPSCs avoid the use of foetal tissue, thus addressing the ethical concerns associated with embryonic stem cells.¹⁴

Scaffold materials are essential in bone organoid construction, providing physical support for 3D cellular growth and mimicking the in vivo tissue microenvironment, which influences cell growth, differentiation, and function. Ideal scaffold materials should offer biocompatibility, mechanical strength, biodegradability, and the capacity to promote cell adhesion and proliferation.¹⁵ Hydrogels, which create a 3D hydrated environment that supports cell adhesion and proliferation and promotes differentiation, are widely used in organoid construction due to their biomimetic properties.¹⁶ Hydrogels can be classified as natural, synthetic, or composite, depending on their source, and they effectively simulate the natural structure of bone tissue to support cell growth and differentiation.

Natural hydrogels consist of 3D network structures made from natural polymers with high water–retention and biocompatibility.¹⁷ Owing to their resemblance to the in vivo extracellular matrix (ECM), natural hydrogels provide cellular support that promotes adhesion, proliferation, and differentiation.^16,18 Examples include collagen, Matrigel, and fibrin:

Collagen, the predominant structural protein in human tissues, is biocompatible and biodegradable, facilitating cell attachment, proliferation, and differentiation, which makes it widely used in organoid construction.¹⁹

Matrigel is a complex ECM mimic derived from mouse tumour cells, composed of various proteins, glycoproteins, and growth factors. It provides structural support, promoting cell adhesion, proliferation, migration, and differentiation.^20,21

Fibrin, a natural protein important in tissue repair and coagulation, offers excellent biocompatibility. Fibrin scaffolds provide mechanical support to organoids, stimulate vascularisation, and enhance cell adhesion, making them ideal for tissue regeneration.

Synthetic hydrogels are engineered polymers with 3D network structures that absorb and retain substantial water content.²² The adjustability of synthetic hydrogels allows for precise adjustments in structure and composition to meet different cell growth and differentiation needs. Common synthetic hydrogels include poly(lactic–co–glycolic acid) (PLGA), polyethylene glycol (PEG), and polyacrylamide. Synthetic hydrogels offer enhanced stability and tunability over natural hydrogels and avoid immunogenic responses, thus providing excellent tissue compatibility.²³ For instance, PEG is extensively utilised in tissue engineering due to its biocompatibility, low immunogenicity, tunability, and ease of processing. PEG hydrogels simulate the ECM environment, promoting cellular interactions with the scaffold.²⁴ Another notable synthetic hydrogel, PLGA, enables precise control over degradation rates in vivo by adjusting the lactic-to-glycolic acid ratio, thus facilitating customised therapies.²⁵

The creation of a supportive microenvironment is vital to organoid success. Beyond scaffold materials, the microenvironment also involves chemical and biological signal transmission. Cell proliferation and differentiation are critical in bone organoid development, with growth factors and signalling pathways determining cell fate. While selecting appropriate cells and scaffold materials is the first step, guiding cells to form functional multicellular clusters is essential for successful organoid construction.

Various growth factors play crucial roles during cell growth and differentiation, including bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), insulin–like growth factors, platelet–derived growth factor, transforming growth factor–β (TGF–β), and vascular endothelial growth factor.²⁶ By selecting suitable growth factors in organoid culture, researchers can direct stem cell differentiation and promote tissue formation. Additionally, nanoparticles or microspheres delivering growth factors enable localised release of signalling molecules, which regulates tissue development and enhancing organoid construction.

Physical stimuli are also critical in organoid development. Stem cells are responsive to chemical signals and physical forces such as tension, electrical stimulation, and compression. These forces are transmitted through the ECM to cell surface receptors and are further relayed to the nucleus, affecting gene expression and cellular activities. In organoid construction, physical stimuli are introduced using various methods, such as rotating bioreactors and microfluidic systems, which provide fluid shear forces that simulate blood flow. These mechanical cues promote vascularisation within the organoid and facilitate tissue maturation, thereby improving functionality and viability.²⁷

Bone is a densely mineralised connective tissue that primarily functions to support and protect vital organs in the body, as well as to participate in calcium storage, release, and haematopoiesis.²⁸ The basic structural unit of bone tissue is the bone matrix, which mainly comprises collagen fibres and inorganic minerals (primarily hydroxyapatite). These components confer both toughness and hardness to the bone.²⁹ The primary cellular components of bone include osteocytes, osteoblasts, and osteoclasts. Osteoblasts synthesise, secrete, and mineralise bone matrix, serving as the main functional cells in bone formation, while osteoclasts are involved in the resorption and degradation of the bone matrix. The coordinated actions between osteoclasts and osteoblasts facilitate bone remodelling, thereby maintaining the dynamic equilibrium of bone tissue, which is essential during bone development.³⁰ Additionally, osteoprogenitor cells in the periosteum can differentiate into various bone cell types, playing an essential role in bone repair and regeneration.³¹ Bone tissue is highly vascularised, providing necessary nutrients and oxygen to bone cells while supporting haematopoietic activities within the bone marrow. These characteristics make bone not only the hardest organ in the body but also one with strong regenerative capacity.³²

In the skeletal system, bone tissue serves multiple functions. First, bones form the skeletal framework of the body, supporting other soft tissues within the skeletal system and maintaining body shape. Additionally, bones, in conjunction with muscles, ligaments, and joints, form complex lever systems that facilitate various physical activities such as walking, running, and jumping. Furthermore, bone tissue acts as a buffer, absorbing and dispersing external forces exerted on the body, thereby protecting internal organs from damage.³³ Bone also serves as a major reservoir of substances like calcium and phosphorus, playing an essential role in maintaining calcium ion balance in the blood. Furthermore, bones are responsible for the renewal of the body’s blood system, as the bone marrow within the medullary cavity is the primary site of haematopoiesis, producing red blood cells, platelets, and white blood cells, which are essential for oxygen supply and energy provision during physical activities.³⁴ Therefore, bone tissue is not only the structural core of the skeletal system but also significantly impacts the maintenance of human homeostasis through its metabolic and physiological functions. In cases of disease or injury, dysfunction in bone tissue can severely impair mobility and lead to systemic health issues, such as osteoporosis and regenerative anaemia. Thus, constructing bone organoid models that imitate the physiological functions and structures of bone is of great significance for advancing research and treatment of bone-related diseases.

Bone organoids can be constructed using various cell types. In addition to the commonly used iPSCs and adipose–derived mesenchymal stem cells, human bone marrow–derived osteoprogenitor cells are also ideal for bone organoid construction. Similar to mesenchymal stem cells, human bone marrow–derived osteoprogenitor cells are derived from human bone marrow and can differentiate into various types of bone cells. These cells exhibit a rapid differentiation capability towards osteoblasts and demonstrate strong tissue–forming potential in bone organoid construction.³⁵

Studies have shown that composite hydrogels formed by combining PEG and hyaluronic acid can be used for bone marrow organoid construction. PEG provides mechanical strength and stability, while hyaluronic acid significantly enhances cell proliferation and maintains stem cell properties. This composite hydrogel shows superior performance in bone generation and tissue regeneration, promoting human cell integration and creating a bone marrow–like microenvironment.³⁶

PLGA also demonstrates excellent tissue compatibility and promotes bone regeneration, making it a key scaffold material in bone organoid research and applications. PLGA can serve as a scaffold for bone repair or as a drug delivery carrier. When combined with inorganic calcium phosphate, PLGA’s osteoconductive and differentiation–inducing properties are significantly enhanced, with this composite material more effectively promoting osteoblast differentiation than single–material options.³⁷

Signalling molecules play a key role in inducing cell differentiation during bone organoid construction. For instance, BMPs can induce the migration and differentiation of osteoprogenitor cells, facilitating bone tissue regeneration and repair, making BMPs key proteins in bone formation.³⁸ Insulin–like growth factors stimulate osteoblast synthesis and secretion of bone matrix proteins, promoting bone matrix generation, inhibiting osteoblast apoptosis, and accelerating bone injury repair by acting synergistically with other growth factors. FGF and vascular endothelial growth factor are essential for angiogenesis, supporting the formation of blood vessels, ensuring adequate blood supply, and thereby aiding bone tissue regeneration and repair.³⁹

Bioink has recently emerged as a focal research area in bone organoid technology. Designed for 3D bioprinting, bioink can carry cells during the printing process and simulate biological tissue through precise 3D structures. Bioinks typically consist of biocompatible materials, cells, and bioactive factors that support cell growth, enabling the creation of complex tissue structures such as skin, bone, and cartilage.⁴⁰ Wang and collaborators¹⁰ developed a composite bioink composed of gelatin methacrylate, alginate methacrylate, and hydroxyapatite, which exhibits self–mineralising properties, enhances multicellular differentiation, and supports long–term culture and maturation of bone organoids. Bioink has opened new pathways for organoid culture, allowing for precise control over scaffold shape and internal structure to better imitate the complex morphology of natural bone tissue.

With continuous advancements in organoid culture techniques, strategies for constructing bone organoids are increasingly refined (Table 1).^{41⇓⇓⇓-45} The trend in bone organoid cultivation is moving toward creating larger, vascularised organoids through co-culture of multiple cell types. Technologies like 3D printing have opened new avenues for bone organoid applications, laying the foundation for more accurate modelling of complex bone structures and potentially enhancing clinical translation for bone tissue engineering.

The above construction method makes bone organoids have the morphology and chemical composition of bone tissue, but in order to test whether bone organoids have the mechanical properties and functional characteristics of bones, we still need to characterise their structure and function.

The construction of bone organoids not only involves culturing bone cells but also recreating their complex functional characteristics. The morphology of bone organoids should resemble the microstructure and macroscopic features of natural bone, such as a porous trabecular structure and a dense cortical bone layer. This morphological feature provides sufficient mechanical support, promotes the growth of blood vessels and nerves, and enhances the biological function of bone tissue.²⁹

In terms of functional characteristics, bone organoids should possess osteogenic capability, vascularisation potential, and mechanical responsiveness. Osteogenic capability is demonstrated by the cells’ ability to continuously secrete bone matrix and form mineralised bone tissue. Vascularisation is crucial for functional bone tissue, as an adequate blood supply is needed to maintain cell viability and facilitate metabolism. By introducing vascular endothelial cells or applying pro–angiogenic growth factors, a vascular network can be formed within the organoid, enhancing its physiological functions.²⁵ Additionally, bone organoids must exhibit mechanical responsiveness, with the ability to withstand external stress and perform self–repair. This feature is particularly important for applications in bone grafting and repair.

As a 3D in vitro model, bone organoids simulate the complex microenvironment and functional characteristics of bone tissue, demonstrating significant potential in the basic research of orthopaedic diseases. Traditional 2D culture systems and animal models have contributed to the study of orthopaedic diseases; however, they are limited in replicating the structure of human bone tissue, the ECM, cell–to–cell interactions, and the mechanical environment. Bone organoids address these limitations by providing a more physiologically relevant research platform (Figure 4). They have proven particularly valuable in studying osteoporosis, poor bone fracture healing, bone tumours, and genetic bone disorders.³² By simulating different pathological conditions, researchers can reproduce disease progression and pathogenesis in vitro. For example, bone organoids constructed with bone marrow mesenchymal stem cells and Matrigel can recreate the process of osteolytic lesions in multiple myeloma, facilitating the study of the disease’s biological mechanisms and identifying potential therapeutic strategies.⁴⁶ Additionally, micron–scale bone organoids can be used to study changes in bone structure under microgravity, offering valuable insights into bone loss and remodelling processes, and providing a feasible method for osteoporosis research.⁴⁷

Bone organoids offer significant advantages in drug screening and toxicological studies, particularly in developing and evaluating drugs related to bone tissue. Compared to traditional 2D cell culture models, bone organoids provide a more realistic cellular environment and biomechanical stress, which are crucial for assessing drug efficacy, cytotoxicity, and biometabolism.⁴⁸ In drug screening, bone organoids can be used for high–throughput screening, especially for drug development targeting diseases such as osteoporosis, bone tumours, and osteoarthritis. By using trabecular bone organoids constructed with demineralised bone paper made from bone biomaterials, in vitro mineralisation can be achieved, significantly enhancing clinical predictive power and reducing the time required for osteoporosis drug screening.⁴⁴ Furthermore, the 3D structure of bone organoids offers more targets for identifying potential drug molecules and their mechanisms of action.

Bone organoids exhibit tremendous potential in regenerative medicine, particularly in bone tissue repair and reconstruction. Traditional bone grafting and repair techniques face challenges, including limited donor availability, immune rejection, and long–term complications. Utilising bone organoids for personalised treatment may help overcome these limitations. Bone organoids constructed from a patient’s own cells can be stimulated to differentiate into bone tissue for transplantation or repair. This personalised bone grafting strategy not only reduces immune rejection but also better replicates the specific bone structure and function of the patient. Additionally, bone organoids can be combined with 3D printing technology to create bone tissue of specific shapes using scaffold materials, facilitating the repair of large or complex bone defects.⁴⁵

In bone regeneration and skeletal tissue engineering, bone organoids can also serve as an ideal platform for evaluating new scaffold materials, growth factors, and cell therapy strategies. Researchers can use bone organoids to test the biocompatibility and the mechanical properties of various materials in vitro and assess their effects on promoting bone tissue regeneration. Leveraging this advantage, future developments may lead to safer and more effective bone regeneration techniques for clinical bone defect repair and other orthopaedic applications in regenerative medicine.

Skeletal muscle is a highly specialised striated muscle attached to bones, responsible for body movement and posture control. By contracting, it converts chemical energy into mechanical energy, generating the force needed to drive skeletal motion. Comprising approximately 40% of body weight, skeletal muscle consists of multinucleated muscle fibres that exhibit a distinct striped appearance, with lengths reaching up to 1 cm and diameters of about 100 μm. Skeletal muscle is wrapped in three layers of connective tissue: the epimysium surrounding the entire muscle, the perimysium enclosing muscle bundles, and the endomysium covering individual muscle fibres.⁴⁹ Within each muscle fibre are myofilaments, which can be categorised into thick and thin filaments based on their protein composition. Thick filaments are mainly composed of myosin, while thin filaments are composed of actin, both forming the fundamental contractile unit known as the sarcomere.⁵⁰ Muscle contraction is achieved through the sliding filament theory, where neural signals trigger membrane depolarisation, resulting in the release of calcium ions that bind to myosin, causing conformational changes. Through the hydrolysis of ATP, actin and myosin filaments slide past each other, resulting in sarcomere shortening and inducing muscle contraction. Additionally, the ECM surrounding muscle fibres serves a vital role in providing structural support, integrating biochemical signals, and maintaining tissue integrity.⁵¹

Skeletal muscle plays a key crucial in the skeletal system, as it connects to bones through tendons and acts as the main executor of movement. By converting chemical energy into mechanical work, skeletal muscle produces force through contraction, enabling complex bodily movements ranging from fine motor skills to large-scale coordinated activities. The interaction between skeletal muscles and bones is crucial for maintaining body balance and supporting vital functions. Beyond its mechanical function, skeletal muscle also serves as an essential glucose reservoir, helping to maintain glucose homeostasis in the body through glycogen synthesis and breakdown.⁵² Mechanical stimulation of muscle fibres leads to hypertrophy, enabling muscles to adapt to increased mechanical loads, enhancing strength and endurance—explaining why physical training increases muscular strength.⁵³ This process also underscores the importance of mechanical stimulation in promoting muscle tissue growth, a factor integral to organoid cultivation.

There are multiple cell options available for muscle organoid construction, among which human pluripotent stem cells are most commonly used. These cells can be induced to differentiate into muscle–related cell lineages such as myogenic progenitor cells or satellite cells through specific differentiation strategies.⁵⁴ Scaffold materials are one of the core components in muscle organoid construction, with biocompatible materials such as collagen, gelatin, and fibrin commonly selected to provide a microenvironment conducive to cell adhesion, growth, and differentiation.⁵⁵ Alternatively, collagen can be combined with Matrigel, where the cells are mixed with the scaffold material and gelled in a mould to create a 3D muscle organoid structure.⁵⁶

A 3D culture system offers conditions closer to the in vivo environment for muscle organoid construction. By mimicking mechanical tension found in muscle tissue, researchers can promote the alignment, fusion, and formation of muscle fibre–like structures in vitro.⁵⁷ Additionally, some studies employ bioreactors to dynamically regulate the culture environment, providing mechanical stretching stimuli that enhance the functionality of muscle organoids, such as contractile force and muscle fibre alignment.⁵⁸

The cultivation of muscle organoids also requires the activation and inhibition of relevant signalling pathways to simulate the muscle development process. During differentiation, key molecules such as Wnt signalling pathway, BMP, and FGF are used to induce mesodermal progenitor cells to further differentiate into myogenic cells. The Wnt protein family plays a critical regulatory role in adult skeletal muscle regeneration, with Wnt signalling regulating satellite cell proliferation and differentiation through β–catenin. When Wnt signalling is successfully activated, β–catenin translocates from the cytoplasm to the nucleus, activating satellite cell proliferation.⁵⁵

BMP has a specific role in skeletal muscle regeneration. At certain concentrations, BMP induces mesodermal cells to differentiate towards bone tissue while inhibiting muscle differentiation.^59,60 On the other hand, BMP signalling promotes growth within muscle tissue through the Smad1/5/8 pathway, supporting muscle hypertrophy and preventing muscle atrophy.⁶⁰ The FGF family, composed of 22 conserved members, is extensively involved in embryonic development, tissue homeostasis, and injury repair. Among them, FGF2 and FGF6 play significant roles in skeletal muscle regeneration, where FGF signalling maintains satellite cells in a proliferative state by inhibiting myogenic differentiation, thus supporting muscle regeneration.⁶¹ In summary, selecting appropriate induction and differentiation strategies is crucial in muscle organoid culture (Table 2). ^{62⇓⇓⇓-66}

Functional characterisation of muscle organoids is crucial for evaluating their biological properties and functional integrity. Firstly, morphological analysis is the most fundamental characterisation method, involving the observation of muscle tissue structures, such as muscle fibre alignment, nuclear positioning, and ECM distribution. Using immunofluorescence staining and microscopy, researchers can detect specific muscle markers, such as α-actinin and myosin, to assess the maturation level of muscle organoids.⁴⁹

Secondly, functional assessment of muscle organoids, including measurements of their contractile ability, is a key indicator to determine if they possess the functions of normal muscle tissue. By applying electrical stimulation to muscle organoids in a 3D culture environment, researchers can induce contraction responses and subsequently measure the magnitude and persistence of contraction forces.⁵⁸

Finally, gene expression analysis is another important method for characterising the developmental and functional status of muscle organoids. Techniques such as real–time polymerase chain reaction and RNA sequencing enable researchers to examine the expression of muscle–specific genes, such as Pax genes 7 and myogenin, to evaluate the differentiation and maturation status of muscle organoids.⁵⁷

Overall, the construction of muscle organoids relies on various cell sources and differentiation strategies, combined with advanced tissue engineering technologies and 3D culture systems, to emulate the structure and function of in vivo muscle tissue. Functional characterisation methods, including morphological analysis, functional assessment, and gene expression analysis, provide a reliable model system for advancing studies on muscle development, regeneration, and disease mechanisms.

Muscle organoids play a crucial role in modelling muscle degenerative diseases, particularly those that cannot be adequately replicated using traditional animal models. By differentiating patient–derived iPSCs into muscle organoids, researchers can recreate the microenvironment of diseased muscle tissue in vitro, providing an ideal platform for studying the pathogenesis of muscle degenerative diseases (Figure 5). For instance, Duchenne muscular dystrophy, caused by mutations leading to the absence of dystrophin on the muscle cell membrane, cannot be accurately simulated using conventional cell culture models.⁶⁷ By utilising clustered regularly interspaced palindromic repeats (CRISPR) CRISPR–associated (Cas) nucleases 9 technology to perform gene editing on iPSCs, researchers can construct muscle organoid models with Duchenne muscular dystrophy gene mutations, offering new pathways for understanding the pathogenesis of the disease and identify potential therapeutic strategies.²

Moreover, muscle organoid models can also be employed to study other muscle degenerative diseases, such as amyotrophic lateral sclerosis and mitochondrial myopathies. Amyotrophic lateral sclerosis is an idiopathic, fatal neurodegenerative disease that affects the human motor system, often leading to respiratory failure in patients.⁶⁸ By inducing specific pathological states within organoids, researchers can observe disease progression at the cellular level and explore abnormalities in related signalling pathways. More importantly, these organoid models can be used for high-throughput screening of potential drugs, providing new hope for treating muscle degenerative diseases.⁶⁹

Muscle organoids serve as a reliable in vitro model for mechanical performance testing, which is of great significance in studying muscle function and drug development. By applying electrical stimulation to muscle organoids to induce contraction responses, researchers can measure their mechanical properties, such as contractile force, endurance, and dynamic responsiveness. These indicators reflect the maturity and functional state of the muscle organoids and can be used to assess the effects of exogenous compounds (e.g., growth factors or drugs) on muscle function.⁷⁰

In drug development, muscle organoids provide an efficient and cost–effective platform for assessing the efficacy and safety of new drugs on muscle tissue. For example, researchers can observe the effects of drugs on muscle fibre proliferation, differentiation, and contraction by culturing muscle organoids in a medium containing the drug, as well as analyse changes in related signalling pathways. Furthermore, muscle organoids can also be used to evaluate the effectiveness of gene therapy strategies. By introducing gene delivery vectors into muscle organoids, researchers can assess the effects of restoring or knocking down specific gene expressions on muscle function, providing reliable data support for the gene therapy of muscle diseases.⁵⁶

Muscle regeneration is a key research area in clinical and regenerative medicine, where muscle organoids hold unique advantages in studying muscle regeneration and rehabilitation. Satellite cells play a critical role in muscle regeneration during muscle injury and repair. Muscle organoid models can simulate the activation, proliferation, and differentiation of satellite cells in damaged muscle, enabling the study of muscle regeneration mechanisms.⁵⁵

Additionally, muscle organoids have promising applications in rehabilitation research. They can simulate the effects of different rehabilitation strategies (e.g., mechanical stretching, cyclic electrical stimulation) on muscle tissue to explore the mechanisms at the cellular and molecular levels.⁷¹ Furthermore, muscle organoids can be used to evaluate the effectiveness of tissue–engineered muscle transplantation. Researchers can combine various scaffold materials, cell types, and growth factors within muscle organoids to assess their impact on muscle tissue formation and functional recovery. By observing muscle organoids’ integration, vascularisation, and functional recovery post–transplantation, valuable scientific evidence can be provided for clinical muscle regeneration therapies.⁷²

In conclusion, muscle organoids have extensive applications in muscle degenerative disease research, mechanical performance testing, drug development, muscle regeneration, and rehabilitation research. They not only provide an advanced platform for basic research in muscle biology but also offer new perspectives and methods for optimising clinical treatments and rehabilitation strategies for muscle–related disease.

Joints are vital components of the human skeletal system, playing a critical role in biomechanics. The structure of a joint typically includes the joint capsule, articular cartilage, joint cavity, and synovial fluid. Articular cartilage, primarily composed of hyaline cartilage, has low-friction characteristics that support the free movement and weight–bearing capacity of joints. The primary cells in cartilage are chondrocytes, which are embedded within an abundant ECM.⁷³ The ECM contains a high concentration of type II collagen and proteoglycans that work together to ensure the elasticity and wear resistance of cartilage.⁷⁴ Moreover, the biomechanical properties of joints are also influenced by the joint capsule and synovial fluid. The joint capsule, made of fibrous tissue, provides protection and support during movement. Synovial fluid, acting as a lubricant, fills the joint cavity, reducing friction between joint surfaces and enhancing joint stability and smoothness of movement. This structural design allows joints to maintain a high degree of flexibility and stability while bearing external forces, helping prevent joint wear and damage.⁷⁵

The primary function of joints within the skeletal system is to facilitate relative movement between bones, thus enabling the execution of various motions. Through their biomechanical properties, joints can not only move freely in different directions but also effectively absorb external impacts, protecting other fragile bone structures. Articular cartilage, as an essential component of the skeletal system, has unique viscoelastic properties, allowing it to remain stable under cyclic loading. Its primary function is to provide smooth, low–friction joint movement and to resist compressive and tensile forces, thereby protecting bones from injury.⁷⁶

The joint capsule, ligaments, and tendons surrounding the joint play key roles in joint function. The joint capsule provides passive stability by limiting unwanted motion and promotes active stability through its proprioceptive nerve endings. Additionally, specialised structures within the joint capsule are involved in the formation of joint surfaces and can adapt to age-related and pathological changes.⁷⁷ From a regenerative medicine perspective, understanding the structure and function of joints is crucial for repairing or replacing damaged joints.

Joint organoids, comprising cells such as osteocytes, chondrocytes, and synoviocytes, represent a multicellular structured tissue. Consequently, constructing joint organoids requires cells with high pluripotency and differentiation potential. Besides iPSCs and mesenchymal stem cells, chondroprogenitor cells are also considered ideal due to their diversity in developmental differentiation.⁷⁸ Additionally, leucine–rich repeat–containing G protein–coupled receptor 5 positive embryonic joint progenitor cells have been identified as potential cell sources, capable of forming cartilage–functional organoids under suitable conditions.⁷⁹

The choice of scaffold material is crucial for cell proliferation, differentiation, and functional maintenance in joint organoid construction (Table 3). ^{80⇓⇓⇓-84} In recent years, decellularised ECM has gained popularity in joint organoid construction due to its excellent biocompatibility and biomechanical properties.^73,85 Decellularised ECM, derived by removing cells from natural tissues while retaining the ECM, preserves structural proteins and bioactive factors found in natural joint tissues. This material provides essential biological signals that support the regeneration of cartilage and bone tissue.⁸⁵ Other natural materials, such as alginate, gelatin, and collagen–based hydrogels, are also commonly used as matrix materials for joint organoids to create a supportive 3D environment for cell growth and differentiation. To better mimic the microenvironment of cartilage tissue, researchers have explored biomaterials modified with specific signalling molecules, such as peptide hydrogels incorporating Wnt signalling molecules to guide chondrocyte differentiation.⁸⁶

The cultivation of joint organoids involves regulation by cytokines and signalling pathways. Similar to bone organoid construction, joint organoids undergo differentiation and proliferation regulated by the TGF–β family, BMP, FGF family, and the Wnt/β–catenin signalling pathway, among others. By adding TGF–β3 and BMP–2, iPSCs can be successfully induced to differentiate into organoids with cartilage and bone characteristics, replicating signalling pathway changes observed in osteochondral development.⁷⁸

Simulating a 3D microenvironment is essential in joint organoid construction. The ECM of articular cartilage comprises collagen, proteoglycans, and other components that provide mechanical support and facilitate signal transmission. Hydrogels are commonly used as 3D scaffolds in research, along with cell embedding techniques and bioprinting technology, to construct organoids with structures similar to natural cartilage tissue. To more accurately simulate the joint environment, microfluidic chip technology (joint–on–a–chip) has been developed, introducing fluid shear stress and pressure during organoid cultivation to simulate mechanical stresses experienced in joint movement. This approach enables the study of the effects of mechanical stimuli on organoid development and function.⁸⁷

To verify whether joint organoids possess the desired structure and function, a series of biological and biomechanical tests are conducted. Histological and immunohistochemical staining are common methods used to evaluate the distribution and morphology of chondrocytes and the composition of the matrix within the organoids. For example, haematoxylin–eosin staining and immunofluorescence staining can be employed to observe the distribution of type II collagen and proteoglycans in the organoids, determining if the formation of the cartilage matrix resembles that of natural cartilage tissue.⁸⁶

To validate the biomechanical properties of joint organoids, mechanical tests such as compression and shear stress assessments are performed to evaluate the organoids’ response and resilience under mechanical stimuli. Additionally, some studies transplant organoids into animal models to observe their integration with host tissues and regenerative effects, thereby assessing the clinical application potential of the organoids.⁷⁴ Through these validation methods, the structure, function, and biomechanical characteristics of joint organoids can be evaluated, providing a solid foundation for their application in drug screening, disease modelling, and regenerative medicine.

Osteoarthritis is a common degenerative joint disease characterised by cartilage degeneration and joint dysfunction. It frequently occurs in the elderly population, causing pain and mobility issues, thereby posing an economic burden on society. The aetiology of osteoarthritis is complex, involving multiple factors such as obesity, ageing, and physical trauma.⁸⁸ Current treatment methods mainly involve medication, including oral non–steroidal anti–inflammatory drugs and intra–articular steroid injections to alleviate pain, as well as hyaluronic acid injections for treating joint inflammation.⁸⁹ However, these treatments primarily address symptoms without altering the disease’s progression and may have adverse side effects.

Organoid technology enables the dynamic simulation of cartilage degeneration and repair processes in vitro, enabling researchers to investigate the molecular mechanisms leading to cartilage matrix destruction and inflammation. For instance, joint organoids can be exposed to senescence and inflammation–related factors to study their impact on cartilage tissue. By inducing senescent joint organoids to express pro–inflammatory cytokines (e.g., interleukin–1β, interleukin–6) and tumour necrosis factor–α, the cartilage degradation observed in osteoarthritis patients can be replicated, thereby modelling the disease progression.⁷⁹

Furthermore, joint organoids can simulate the process of intervertebral disc degeneration, where changes in chondrocytes and the ECM are crucial features. During disc degeneration, chondrocytes undergo various changes, including senescence, altered secretion, and functional impairment. The ECM shows a marked decrease in collagen and proteoglycan content, leading to reduced elasticity and cushioning capacity of the disc.⁸⁷ By constructing degeneration models, researchers can study the molecular mechanisms of chondrocyte and matrix changes within the disc, providing new perspectives for treatment strategies of intervertebral disc degeneration (Figure 6).

Traditional drug screening methods are often based on 2D cell culture models, which lack the complex signalling and tissue specificity of the in vivo joint microenvironment. This discrepancy can result in significant differences between the drug’s actual efficacy in the body and the outcomes observed in vitro. Joint organoids provide a highly physiological 3D environment, better simulating the pathological state of in vivo joint tissues and the interactions between cells.

By constructing joint organoids, researchers can conduct high–throughput screening of candidate drugs for their efficacy and toxicity. For example, in osteoarthritis models, simulating the inflammation and matrix degradation process of joint tissue allows for evaluating the efficacy of anti–inflammatory drugs and matrix-regenerating agents, providing reliable data for the development of osteoarthritis treatments.⁸⁰ Additionally, utilising microRNA/organoid composite hydrogels enables the screening and assessment of anti-ageing drugs’ effects in improving chondrocyte homeostasis and inhibiting joint degeneration.⁸¹ The diversity and controllability of joint organoids make personalised drug screening possible, allowing drug testing based on specific pathological features of different patients, thereby enhancing the efficiency and precision of drug development.⁹⁰

Joint organoids have broad application prospects in tissue engineering and regenerative medicine, especially in cartilage and bone tissue repair and regeneration. Since cartilage tissue has limited self–repair capabilities, treating osteoarthritis and osteochondral defects has been a clinical challenge. Organoid technology offers a new strategy for cartilage regeneration by simulating the development of joint tissues and generating 3D structures similar to natural joint tissues.

For instance, researchers use mesenchymal stem cells and iPSCs to generate joint organoids that mimic the formation of cartilage and bone tissue, exploring the mechanisms of cartilage and bone regeneration.⁷⁸ These organoids can further be combined with biomaterials to construct grafts for joint repair. By optimising culture conditions and growth factor combinations during in vitro culture, researchers can induce chondrocyte differentiation and matrix deposition, producing functional and stable organoids. The combination of 3D bioprinting technology with organoids also provides new possibilities for constructing joint tissues with complex structures and functions, enabling precise design of the organoid’s shape, cell arrangement, and mechanical properties to better simulate natural joints.⁷⁴

Moreover, studies have found that organoids can regulate chondrocyte homeostasis, thereby promoting cartilage regeneration. For example, introducing microRNAs (e.g., miR–24) and specific signalling pathways can inhibit chondrocyte senescence, enhance their proliferation and differentiation capabilities, and ultimately improve cartilage repair outcomes.⁸¹ This offers a novel approach for developing organoid–based precision therapies to repair damaged joints.

Ligaments and tendons are connective tissues characterised by their complex structures and biomechanical properties. Both are primarily composed of collagen fibres (mainly type I collagen) and elastin embedded in a proteoglycan–water matrix. Tendons primarily function to transmit the force generated by muscles to bones, facilitating joint movement. Tendons are flexible but inelastic, capable of resisting tension and compression forces. For instance, the Achilles tendon, the largest tendon in the body, can withstand loads up to 17 times the body’s weight.^91,92 At the microscopic level, the collagen fibres in tendons and ligaments are not only aligned longitudinally but also arranged transversely and horizontally, forming a helical cross-linked structure. This unique 3D organisation grants tendons and ligaments high mechanical strength and elasticity, allowing them to resist forces from multiple directions during movement.⁹²

Ligaments connect bones to bones, providing stability and support to the joints. While their structure is similar to that of tendons, they differ slightly in cell types, cellular activity, collagen content, and fibre arrangement. Ligaments contain a higher proportion of type III collagen, proteoglycans, and water, and their collagen fibres exhibit a more irregular arrangement. Ligaments are primarily composed of ligamentocytes, which play a critical role in maintaining tissue homeostasis and facilitating healing after injury.^91,93 Moreover, ligaments and tendons exhibit different morphological and biochemical characteristics depending on their physiological states and developmental stages. For example, ligaments are more metabolically active, containing more DNA, a higher level of reducible cross-links, more type III collagen, and more proteoglycans.⁹³

Ligaments and tendons are essential components of the skeletal system. Tendons connect muscles to bones, transmitting the force generated by muscles to bones to facilitate joint movement. For example, the quadriceps and triceps have short and broad tendons to withstand greater forces, while finger flexors possess long and thin tendons to enable fine motor control.⁹² During force transmission, tendon fibres can absorb and release energy, effectively buffering external forces and preventing joint damage.

Ligaments, on the other hand, connect bones to bones, providing joint stability and guiding the range of motion during activity. When exposed to external forces, ligaments stabilise the joint, preventing abnormal bone displacement and protecting it from injury. In the knee joint, for instance, the anterior cruciate ligament and posterior cruciate ligament work together to maintain joint stability. Due to the critical roles of ligaments and tendons, their injuries often lead to severe mobility impairments, affecting the quality of daily life.^91,94

Research indicates that the tensile strength of tendons and ligaments largely depends on collagen fibre cross-linking.⁹⁵ Damage to ligaments and tendons can result in serious complications, such as joint instability, osteoarthritis, pain, and limited function. Traditional repair methods, such as surgical suturing and tissue grafting, often fail to restore the original structure and function completely. For certain tendons and ligaments, the re-rupture rates after surgical repair may be high.⁹⁶ Consequently, tissue engineering and regenerative medicine for ligaments and tendons are becoming promising therapeutic strategies, aiming to promote the repair and regeneration of damaged tissues by combining cells, scaffold materials, and bioactive molecules.⁹⁴

In tissue engineering, multiple cell sources are often utilised to simulate the functions of ligaments and tendons (Table 4).^97⇓-99 Commonly used cell sources include bone marrow-derived mesenchymal stem cells, adipose-derived stem cells, and stem cells derived from tendons or ligaments. These cells can be cultured on suitable biomaterial scaffolds and induced to differentiate into cells that exhibit ligament or tendon tissue characteristics, forming organoid structures.¹⁰⁰

The choice of matrix material is crucial in constructing ligament and tendon organoids, as its properties must align with the mechanical and biological characteristics of these tissues. Commonly used matrices include natural materials such as collagen, hyaluronic acid, gelatin, and alginate, as well as synthetic materials like polylactic acid and polycaprolactone. Collagen and elastin, the main components of the ECM in tendon and ligament tissues, are ideal choices for constructing organoids.¹⁰¹ Petrigliano attempted to use polycaprolactone nanofibres to reconstruct rodent ligaments, but the resulting organoids lacked sufficient strength and exhibited low biocompatibility.¹⁰² Matrix materials not only provide a scaffold for cell growth and differentiation but also influence cell behaviour and tissue formation by modulating material stiffness, porosity, and degradation rates.⁹⁴ For example, multilayered nanofibre scaffolds produced via electrospinning, coated with tendon or ligament-derived ECM, can promote cell differentiation towards tendon/ligament lineages, thereby enhancing the mechanical properties and biological function of the organoids.¹⁰³

Creating ideal matrix materials that mimic ECM is essential to establish a microenvironment similar to the natural biochemical composition of tendons and ligaments, which can initiate specific cellular responses to promote faster tissue regeneration.¹⁰⁴ Cell-scaffold composites combine cells with biomaterial scaffolds to form functional tissues. By introducing ligament- or tendon-specific cells into the scaffold, along with growth factors (such as TGF-β, BMP-12, and early growth response factor-1 (EGR-1)), cells can be induced to differentiate toward ligament or tendon lineages, forming organoids.¹⁰⁵ For instance, EGR-1 plays a role in early tendon differentiation; in tendon cells, EGR-1 expression correlates with increased collagen expression during embryonic tendon cell differentiation.¹⁰⁶ Additionally, to more exactly simulate the natural structure and function of ligaments and tendons, researchers apply mechanical stress (e.g., stretching or compression) using bioreactors during organoid construction to promote cellular characteristics that mirror in vivo tissue properties.^100,107

In cell self-assembly strategies, cells can autonomously form organoids with ligament or tendon characteristics under appropriate culture conditions. Alternatively, 3D bioprinting technology uses bioinks to layer cells and biomaterials in a designed structure, enabling the construction of complex 3D tissue structures.¹⁰⁸ These methods, which combine different cell sources, growth factors, and scaffold materials, allow precision control over the morphology and function of organoids, simulating the microenvironment of natural ligament and tendon tissue and providing ideal tissue replacements for clinical repair.

The mechanical properties and functional evaluation of organoids are crucial indicators for assessing their quality and potential applications. Ideal ligament and tendon organoids should possess mechanical strength, elastic modulus, and fracture elongation similar to natural tissues, ensuring their capability to support and transmit forces in the body. Studies have shown that regulating the fibre alignment and density of electrospun scaffolds can significantly enhance the tensile strength and elasticity of organoids, mimicking the biomechanical properties of natural tendons.¹⁰³

To evaluate the functionality of organoids, it is essential to assess their biological characteristics both in vitro and in vivo, including cell viability, collagen synthesis capacity, ECM deposition, and adaptability to mechanical stimuli. The performance of organoids after in vivo transplantation is also an important functional evaluation criterion, which includes their role in tissue repair, the degree of integration with surrounding tissues, and long-term biological stability. Among these evaluations, the mechanical performance of the organoids is the most critical parameter, as it directly impacts the organoids’ efficacy and safety in clinical applications.

Ligament and tendon organoids show great potential in the research of sports injury repair (Figure 7). As the intensity of physical activity increases among athletes and the general population, ligament and tendon injuries, especially anterior cruciate ligament and Achilles tendon injuries, have become more prevalent. Traditional treatment methods such as surgical repair and grafting often fail to fully reestablish the structure and functionality of damaged tissue and carry a high risk of complications and long-term functional impairment.⁹⁴ Organoid technology offers a highly biomimetic 3D structure that can promote the regeneration of damaged ligament and tendon tissues. In experimental studies, tissue-engineered ligament organoids are used as an alternative to traditional grafting techniques. By combining biomaterials and stem cells, and through the regulation of growth factors (e.g., EGR-1, FGF) and the application of mechanical loading, it is possible to better facilitate cellular repair and functional recovery.¹⁰⁹

Ligament and tendon organoids provide an innovative platform for drug testing. Compared to traditional 2D cell cultures, organoids can better simulate the in vivo 3D environment, providing a more realistic tissue response. By testing anti-inflammatory drugs, tissue repair promoters, or growth factors on organoids, researchers can assess the efficacy and safety of these drugs in a more realistic setting, thereby reducing the reliance on animal experiments.

Moreover, ligament and tendon organoids have extensive applications in regenerative medicine. They can be used to repair damaged skeletal tissues and assess their effectiveness in accelerating tissue regeneration and reducing scar tissue formation. Additionally, organoid models constructed from aged tendon stem cells allow researchers to study the effects of ageing on tendon structure. Comparative studies have shown that aged cells exhibit significant defects in forming 3D tendon tissue organoids, with marked degeneration in cell adhesion, migration, and self-renewal capabilities.¹¹⁰

Ligament and tendon organoids play a crucial role in preclinical models, particularly demonstrating significant potential in personalised medicine. By utilising a patient’s autologous stem cells or iPSCs to construct specific organoids, researchers can create tissue models that reflect the patient’s unique pathological characteristics or evaluate the tissue response following surgery.

In personalised medicine, organoid construction can be tailored to the patient’s injury severity, genetic background, and biomechanical needs. This approach is particularly suitable for complex ligament and tendon injury repair since the organoids can be designed based on the patient’s specific condition, using customised combinations of biomaterials and cells to ensure optimal tissue regeneration outcomes.

In summary, ligament and tendon organoids, as advanced tissue engineering tools, are transforming existing methods of sports injury repair, drug testing, and personalised medicine. With the ongoing advancement of technology, the potential for these organoids in clinical applications will be further expanded and validated.

The skeletal system is composed of various tissues, including bones, muscles, cartilage, ligaments, and tendons, whose coordinated functioning is essential for human activity. Focusing solely on a single tissue organoid model may not comprehensively reflect the overall function and interaction within the skeletal system. To replicate the complex functions of these tissues in vitro, researchers have begun constructing and integrating multiple organoids into a unified system. Through 3D cell culture and microfluidic technologies, researchers have developed multi-organoid models, including those for the intestine and heart.¹¹¹ Additionally, bone microfluidic chips have been successfully constructed, showing potential in clinical diagnostics.¹¹² By integrating multiple tissue organoids, researchers can more realistically simulate the skeletal system’s complex biological environment more realistically, providing more accurate disease models and platforms for therapeutic evaluation.

Understanding the interactions among different organoids is crucial in multi-tissue organoid research. In the skeletal system, various tissues interact in complex ways under both physiological and pathological conditions. For instance, muscle force transmission influences bone growth, and joint inflammation can affect surrounding ligaments and tendons. Organoid technology enables researchers to systematically study these interactions in a controlled environment. Advancing research on multi-tissue organoids will further reveal the complexities of skeletal system diseases and aid in developing more targeted treatment strategies.

Precision medicine and personalised therapies have become crucial in modern medicine, focusing on tailoring treatments according to individual differences in genetics, environment, and lifestyle. Organoid technology provides an essential tool for precision medicine, as organoids can be generated from a patient’s own cells, creating 3D tissue models that closely match the patient’s physiological state. These models can be used in the laboratory for drug screening, toxicity testing, and disease research, thereby facilitating the formulation of personalised treatment strategies. Particularly for skeletal diseases, personalised organoid models can accurately predict a patient’s response to specific treatments, avoiding ineffective or harmful interventions and improving treatment success rates.¹¹³

In personalised medicine applications of skeletal organoids, bone organoids have shown potential in predicting osteoporosis patients’ responses to medications. By generating bone organoids from patient-derived cells, researchers can evaluate the impact of different drugs on bone density and structure, thereby determining the most appropriate drug therapy for each patient.⁴⁷ Similarly, personalised muscle organoid models can be employed to study rare muscle diseases, such as Duchenne muscular dystrophy, and to test the effectiveness of gene therapies or drug treatments.² The construction of joint organoids offers new diagnostic and therapeutic approaches for arthritis patients, enabling the use of patient-derived chondrocytes to assess drug efficacy.⁸³ Additionally, patient-specific cancer organoids have shown promising results in personalised drug screening, facilitating tailored treatment approaches with potentially improved therapeutic outcomes.^114,115 These applications not only improve treatment outcomes but also reduce unnecessary clinical trials, offering new pathways for the advancement of personalised medicine (Figure 8).

Although organoid technology has shown immense potential in skeletal research, technical challenges remain. One primary issue is the standardisation of organoid construction, as the current processes are complex and yield variable results. Achieving higher reproducibility and standardisation is crucial. Additionally, simulating the organoid microenvironment needs further refinement to more accurately reflect the physical and biochemical conditions in vivo. Another challenge is the large-scale production and long-term functional maintenance of organoids. However, these challenges present opportunities for innovation. For instance, constructing anti-ageing organoid scaffolds through 3D bioprinting technology can effectively mitigate cellular senescence and alleviate symptoms of osteoarthritis.¹¹⁶ Alternatively, combining microfluidic technology with DNA hydrogels to construct cartilage organoids can enhance tissue regeneration.¹¹⁷ Furthermore, multi-omics analysis of organoids can reveal the gene regulatory mechanisms underlying diseases, enabling more precise, personalised therapeutic approaches.¹¹⁵

The future of skeletal organoid construction is expected to move towards greater complexity and precision. Co-culturing multiple cell types, utilising dynamic culture systems, and integrating biomechanical signals will further enhance the physiological relevance of organoids. The integration of tissue engineering and regenerative medicine will also drive the application of organoids in damaged tissue repair. Furthermore, the introduction of artificial intelligence and machine learning into the organoid generation process may optimise construction parameters and predict the developmental processes of organoids. With technological advancements, the functionality of organoids will increasingly resemble that of natural tissues, and their application range will continue to expand.

The rapid development of organoid technology raises ethical and regulatory challenges. Particularly in research involving patient-derived cells, ensuring patient privacy and data security is of utmost importance. Furthermore, the application of organoids may extend the ethical boundaries of human experimentation, necessitating a clear legal and ethical framework. Regulatory agencies need to keep pace with technological advancements, formulating appropriate standards and guidelines to ensure the safety and efficacy of organoid use in research and clinical applications. Additionally, public acceptance and understanding of organoid technology need to be enhanced. Through science education and transparent communication, concerns can be alleviated, promoting the widespread application of organoid technology.

In this review, we systematically summarised the progress, construction strategies, and applications of skeletal organoids in disease research and medical fields. While we discussed the construction of skeletal organoids, a standardised strategy for organoid construction has not yet been established due to the variability in construction approaches chosen by researchers based on specific application goals. Future research may allow for quantifying the influence of different factors within organoid construction strategies, thereby standardising organoid construction practices.

While we summarised the components of skeletal organoids, examples of combining these components in practical applications are lacking. Complex organoid construction involving multiple organoid types may present a promising research direction. Although we provided various examples of organoid construction, most of these remain small-scale, limited to laboratory environments, and large-scale industrial production of organoids is a challenge yet to be addressed. Establishing standardised organoid construction protocols may propel advancements in this area.

In summary, skeletal organoids offer significant practical value and developmental potential. Skeletal organoids provide a 3D model of tissues like bone, muscle, joints, ligaments, and tendons, creating new platforms for studying skeletal system disease mechanisms, drug screening, and tissue regeneration. However, challenges remain in the field, including standardisation, the complexity of multi-organoid integration, and barriers to clinical application. Therefore, further optimisation of organoid construction techniques is necessary to enhance organoid stability, facilitating their translation into personalised medicine, complex disease modelling, and clinical applications.

Author contributions

YJ and JW conceptualised the review; ZC drafted the manuscript; CZ, LB, YJ, JS, LY, and ZX revised the manuscript. All authors reviewed and approved the final version of the manuscript.

Financial support

This work was financially supported by National Natural Science Foundation of China (Nos. 32471396, 82230071, 82172098), Integrated Project of Major Research Plan of National Natural Science Foundation of China (No. 92249303), Shanghai Committee of Science and Technology (No. 23141900600, Laboratory Animal Research Project), Shanghai Clinical Research Plan (No. SHDC2023CRT01), Shanghai Municipal Demonstration Project for Innovative Medical Device Applications (No. 23SHS05700), and Young Elite Scientist Sponsorship Program by China Association for Science and Technology (No. YESS20230049).

Acknowledgement

None.

Conflicts of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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