Bone and cartilage tissues are integral to the human musculoskeletal system, providing essential structural integrity and function. However, bone diseases, particularly those related to cartilage such as articular cartilage injuries, are prevalent across all age groups, with a clinical incidence rate of 20–33%.¹ These injuries, often caused by degenerative diseases or sports injuries, are detected in 61–63% of arthroscopy examinations.² Millions suffer from varying degrees of cartilage damage annually, which can lead to severe joint diseases like osteoarthritis (OA), affecting 595 million people globally.³ In China, the number is also significant, with 10,681,311 cases of OA in 2019, a 132.66% increase from 1990,⁴ and the prevalence continues to rise with the growing rates of obesity and an ageing population.⁵

Given the importance of bone and cartilage disease models in disease research and drug development, current methods such as two–dimensional cell culture and animal experiments have limitations as they cannot fully simulate real human tissues and systems.⁶ The development of more advanced in vitro biological models is therefore urgently needed.⁷ Advances in tissue engineering and regenerative medicine, especially organoid technology, have provided new hope for cartilage repair.⁸

Cartilage injury diseases like OA are characterised by chondrocyte inflammation and extracellular matrix (ECM) destruction, and similar pathological processes occur in bone injuries. The lack of vascularity in cartilage and certain bone areas limits their self–repair capabilities. The pathogenesis of bone and cartilage diseases, such as OA, is driven by a complex interplay of genetic, mechanical, inflammatory, and enzymatic factors that lead to tissue degeneration.³ While two–dimensional cell cultures have traditionally been used to study these diseases, they often fall short in capturing the intricate cellular interactions and ECM influences present in vivo. Three–dimensional (3D) organoid models, however, offer a superior system that better mimics the tissue’s 3D architecture and multicellular dynamics, allowing for a more accurate investigation of disease mechanisms and therapeutic responses. This advanced model facilitates a holistic study of cellular communication and signaling, providing a deeper understanding of pathogenesis and the discovery of new therapeutic targets.⁸ Although traditional treatments such as artificial cartilage or synthetic materials have been applied in repairs, they still pose challenges in terms of durability, post–implant rejection, and tissue compatibility.⁹ Fortunately, advances in tissue engineering and regenerative medicine, particularly organoid technology, offer new hope for the repair of bones and cartilage.⁸

The development of organoid technology, based on the self–renewal and self–organising capabilities of stem cells, provides a new platform for the study and treatment of bone and cartilage diseases. Bone or cartilage organoids can not only mimic the physiological microenvironment of cartilage tissue but also rapidly expand in vitro, offering new strategies for bone or cartilage bio–tissue engineering.^10,11 In the construction process of organoids, stem cells are used as the basic material, which can be induced to differentiate into specific types of cells and assembled into 3D structures in appropriate scaffold materials.^12,13 This mutually beneficial relationship makes organoid technology not only advance our understanding of human biology but also provide new strategies for disease treatment, especially in the field of regenerative medicine for tissues and organs such as bones and cartilage, showing great application potential.^14⇓-16

With the continuous development of bioengineering, we believe that organoid technology will become a more effective method for treating bone or cartilage injury–related diseases in the future. In this review, we discuss the role and development of bone/cartilage organoids prepared by various types of cells, biomaterials, and external factors through various assembly methods.

Stem cell technology plays an essential role in organoid construction due to its distinctive diversity and regenerative capacity.^17,18 The evolution of organoid technology, especially through the utilisation of pluripotent stem cells (PSCs) and adult stem cells, has created new opportunities for simulating and regenerating bone and cartilage tissues. Comprising both embryonic stem cells (ESCs) and induced PSCs (iPSCs), along with adult stem cells such as bone marrow mesenchymal stem cells (BMSCs), adipose–derived mesenchymal stem cells (ADSCs), and synovium–derived mesenchymal stem cells, these cells form a crucial cellular resource in organoid research.^19⇓-21 Their abilities for self–renewal and self–organise have forged new avenues in the study and treatment of bone and cartilage diseases,²² showing significant potential in facilitating cartilage regeneration.²³ Stem cell–derived organoids can mimic the key functions of tissues or organs, presenting extensive biomedical application prospects in the field of bone and cartilage regeneration.^24,25

Mesenchymal stem cells (MSCs) are highly valued in regenerative medicine for their self–renewal and multi–lineage differentiation potential.²⁶ Harvested from various tissues including bone marrow, adipose, and synovium, MSCs exhibit unique biological traits and robust regenerative capabilities.^18,27

Yang et al.²⁵ developed novel microcryogels to induce and induce chondrogenesis and osteogenic differentiation of MSCs, enabling the regeneration of cartilage and bone tissues to form osteochondral organoids through self–assembly in vivo (Figure 1A). The osteogenic or chondrogenic differentiation of MSCs encompasses multiple stages, including proliferation, maturation, and terminal differentiation.²⁸ By employing in vitro culture methods alongside specific growth factors and ECM molecules, it is possible to generate organoids that mimic bone or cartilage.²⁹ The advantage of using MSCs to develop bone or cartilage organoids lies in their accessibility, rapid expansion, and ability to differentiate into diverse mesenchymal–derived tissues.^30,31 Notably, cells derived from bone marrow, synovium, and periosteum demonstrate the highest potential for cartilage generation.^32,33 Xie et al.³⁴ have utilised digital light processing and distribution induction techniques to load BMSCs hydrogel microspheres, which, after induction, form cartilage and then transform into osteoid organoids, thus recapitulating the endochondral ossification process in vitro. Scotti et al.³⁵ have seeded BMSCs onto a collagen scaffold to serve as a developmental template and added interleukin (IL)–1β to the culture medium to promote cartilage reshaping. The resulting organoids contain haematopoietic stem cells and various types of progenitor cells, resembling natural bone in structure and function.

In the field of tissue engineering and regenerative medicine, MSCs have a wide range of applications. They can differentiate into bone or cartilage cells and also modulate the local microenvironment by secreting soluble factors and exosomes, which facilitate tissue repair and regeneration.¹⁸ ADSCs and BMSCs are widely studied due to their similar biological characteristics.^33,36 However, research indicates that BMSCs in osteogenic differentiation compared to ADSCs.³⁷ In organoid construction, the combination of MSCs with biomaterials can create 3D bone or cartilage organoids, providing a powerful tool for studying disease pathogenesis, drug screening, and therapeutic strategy assessment. Dai et al.²⁶ have shown that bone morphogenetic protein (BMP)–2 loaded scaffolds can promote the formation of bone organoids, offering an acellular strategy that fosters an ecological environment for the exchange between stem cells and immune cells, thus harvesting a rich source of autologous cells such as osteoblasts, fibroblasts, and haematopoietic cells (Figure 1B). A novel polymer–modified deoxyribonucleic acid hydrogel combined with tetrahedral framework nucleic acids has been shown to accelerate the osteogenic differentiation of BMSCs and the angiogenesis of human umbilical vein endothelial cells with in vivo rat experiments demonstrating their efficacy in promoting new bone formation³⁸ (Figure 1C). Lin et al.³⁹ have also proposed that embryonic joint progenitor cells expressing leucine–rich repeat–containing G–protein–coupled receptor 5–green fluorescent protein, generated through gel embedding, have the potential to form cartilage organoids (Figure 1D). Despite the potential of MSCs in cell–based tissue engineering and regenerative medicine, challenges remain in addressing the variability of MSC characteristics from different sources and in creating organoids that more closely resemble native bone or cartilage.^39,40

PSCs, comprising both ESCs and iPSCs, have demonstrated immense potential in regenerative medicine and disease modelling due to their remarkable pluripotency and self–renewal capabilities.^19,41 In recent years, iPSCs have garnered particular interest for their application in generating bone and cartilage organoids.^14,42

While iPSCs share similarities with ESCs, such as unlimited self–renewal, pluripotency, surface marker expression, and telomerase activity,^43⇓-45 they offer a promising alternative cell source as they circumvent the ethical and political issues associated with ESCs by not requiring the destruction of human embryos.⁴⁴ Moreover, when compared to MSCs, iPSCs have shown greater evidence of long–term potential in bone and cartilage formation. They tend to express higher levels of types I and X collagen.^46,47 Despite the advantages of iPSCs, challenges remain in generating functional chondrogenic organoids from iPSCs, including large–scale production and maintenance of their functionality. Additionally, the potential for dedifferentiation or tumour formation from iPSCs could impact clinical applications.^44,48 However, iPSCs have an edge over MSCs in addressing issues such as cell quantity, fibrocartilage formation, or loss of phenotype during passaging.⁴⁷

iPSCs have become a versatile tool in the study of chondrogenic organoid formation.^46,49,50 The development of chondrogenic regenerative therapies based on iPSCs is a rapidly evolving field. iPSCs have shown effectiveness in modelling monogenic cartilage diseases and have achieved positive results in simulating skeletal developmental abnormalities.⁵¹ A significant advantage of iPSCs is their ability to reprogram mature somatic cells back to a pluripotent state akin to ESCs, thus bypassing the ethical concerns associated with the use of ESCs.^49,52 Nilsson Hall et al.⁵³ proposed that periosteum–derived cells have similar characteristics to MSCs and can be used to produce chondrogenic microspheres that differentiate into cartilage organoids, which can be used to prepare engineered bone organoids for the regenerative treatment of long bone defects. The height of new bone formation and the part connected with the old bone are comparable to natural long bones (Figure 2A). A recent study showed that allogeneic iPSC–derived chondrogenic organoids transplanted into the knee joints of primate models with cartilage defects integrated with the host articular cartilage and prevented further degradation of the surrounding cartilage⁵² (Figure 2B), highlighting the clinical potential of iPSC technology in the treatment of articular cartilage defects. Tsumaki et al.⁵⁴ also raised the possibility of selecting iPSC–induced chondrocytes from the iPSC repertoire for transplantation, which demonstrated their potential for preparing cartilage organoids. O’Connor and colleagues⁴⁶ used mouse iPSCs to establish bone–cartilage organoids by exposing them to a time–dependent series of transforming growth factor–β3 and BMP–2, simulating the natural skeletal development process through endochondral ossification. The resulting organoids displayed dual tissues composed of cartilage and calcified bone regions. Furthermore, Hall et al.⁵⁰ found that the implantation of iPSC–derived chondrogenic microtissues combined with hypertrophic chondrogenic organoids in nude mice led to the formation of cartilage and bone regions. Tam et al.⁴⁹ investigated the potential of human–iPSCs (hiPSCs)–derived cartilage–like organoids in bone defect repair, showing that IL–1β could promote bone healing in human PSC osteoid organoids (Figure 2C). In addition, Rodríguez Ruiz et al.⁵⁵ constructed cartilage and bone organoids using human iPSCs expressing osteoprotegerin extra–long (OPG–XL) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, which could reduce osteoclast activity and overcome the effects of chondrogenic fibrosis and high mineralisation of bone matrix on OA. It has been reported that iPSC–derived chondrogenic organoids can also recruit osteogenic precursors for bone repair and have demonstrated the ability to repair cartilage defects in mouse models.⁴⁹ Xiahou et al.²⁴ developed a hydrogel with a cell response switch to induce ADSCs to spontaneously and sequentially proliferate and aggregate, producing engineered columnar cartilage microtissues with histological characteristics and biomechanical properties very similar to natural transparent cartilage. Han et al.⁵⁶ successfully made organoids by culturing synovial fluid–derived cells in a 3D culture environment, optimising the use of synovial fluid–derived cells and enhancing their application potential in cartilage repair. These findings have opened new horizons for the development of complex tissue–engineered implants, using pre–differentiated organoids as building blocks to promote region–specific functions for the establishment of articular cartilage grafts.

In addition to MSCs and iPSCs, other cell types such as immortalised cells and adult cells have also shown potential in constructing organoids for bone and cartilage repair.

Osteoblasts, derived from MSCs, are the cells responsible for matrix deposition and migrate to areas of bone remodelling via the local soft tissue or peripheral vascular system. Osteoblasts are pivotal for bone remodelling and offer targeted advantages for bone organoid construction, such as their direct role in bone matrix deposition and their ability to form mature bone cells. This makes them ideal for creating organoids that accurately represent bone tissue. However, their limited plasticity and reliance on specific maturation factors can be more restrictive compared to the broader differentiation potential of MSCs and the extensive self–renewal capabilities of iPSCs.⁵⁷ Additionally, osteoblasts’ lack of self–renewal and susceptibility to senescence may impact the long–term vitality of organoids. These osteoprogenitor cells subsequently differentiate into mature osteoblasts under a multitude of influences, including BMPs and phosphorus–rich compounds that act as substrates for alkaline phosphatase––a key enzyme expressed by both developing and mature osteoblasts.^57⇓-59 Fuller et al.⁶⁰ established a 3D bone organoid model using mouse osteoblasts in hydrogels, providing an economical and efficient platform for bone research (Figure 3A). Iordachescu’s team⁶¹ constructed an organoid model composed of osteoblasts and osteoclasts seeded on the trabecular surface of the femoral head. This model effectively mimics the process of bone loss and overcomes the limitations of traditional laboratory simulations of the bone microenvironment, offering an ex vivo platform (Figure 3B). Similarly, Park et al.⁶² found that using bone biomaterial to guide the structural mineralisation of osteoblasts and constructing bone organoids through repeated bone remodelling cycles can locally regulate the trabecular bone to maintain physiological bone homeostasis. Knowles et al.⁶³ successfully created a mature 3D human osteocyte organoid by culturing primary human osteoblasts in fibrinogen/thrombin gels, providing a valuable tool for drug screening and disease mechanism research (Figure 3C).

Bone/cartilage organoids are constructed from immortalised cells such as chondrocytes obtained from osteoblast culture, which makes organoid construction easier and more economical. Kleuskens et al.⁶⁴ successfully made cartilage organoids from human chondrocytes derived from OA and non–degenerative sources by using a 3D culture environment with matrices from porcine embryonic axial cells, showing good potential for repair. More notably, Crispim and Ito⁶⁵ successfully produced cartilage organoids from bovine chondrocytes through a new suspension expansion protocol. These organoids not only showed significant cell proliferation and vitality but also formed structures similar to transparent cartilage through self–assembly, with matrix content and structure very similar to natural cartilage, which is expected to completely change the research methods in the field of cartilage research and related fields.

In summary, stem cells play a fundamental role in the development of bone or cartilage organoids. They not only provide the necessary cellular sources for the formation of bone or cartilage organoids but also promote the self–organisation and functional simulation of tissues.

Biomaterials play a pivotal role in advancing stem cell research and organoid technology. They are crucial for replicating the mechanical properties and 3D architecture of bones in organoid construction. Beyond selecting the appropriate cells, scaffolds and signalling factors—encompassing biochemical, chemophysical, and physical cues—are vital in the fabrication of bone or cartilage organoids. The resulting organoids closely mimic natural tissues in terms of chemistry, physics, and functionality, which is essential for successful research outcomes.⁶⁶ Biomaterials, with or without growth factors, offer a promising platform for achieving spatial complexity within the microenvironment to fully harness cellular capabilities.⁶⁷

Generally, ideal natural or synthetic scaffolds demand biocompatibility, bioactivity, protective mechanical strength, adhesive morphological ability, the capacity to support cell proliferation and/or differentiation, the ability to mimic the natural ECM, biointegration, and biodegradability.^68,69 Additionally, scaffolds designed for bone or cartilage organoids require osteoinductivity, osteoconductivity, and specific mechanical properties.^70,71

The construction of bone or cartilage organoids employs a variety of biocompatible materials that provide an optimal environment for stem cell differentiation.^65,72 These materials include natural materials such as alginate,^73,74 chitosan,⁷⁵ collagen,^64,76 and gelatin,⁷⁷ as well as synthetic materials like poly(lactic–co–glycolic acid),⁷⁸ polycaprolactone,⁷⁹ and polyethylene glycol (PEG).⁸⁰ These biomaterials are significant because they not only possess excellent biocompatibility and adjustable physicochemical properties that support cell growth and tissue regeneration but also effectively simulate the ECM, guide cell differentiation, and construct complex tissue engineering scaffolds. They enable scientists to simulate the microenvironment of human tissues and organs more accurately, providing powerful tools for disease modelling, drug screening, and the development of regenerative medical strategies.

Through the application of these natural biomaterials, the potential of organoid technology in regenerative medicine has been further demonstrated. They not only provide researchers with a platform for simulating complex bone/cartilage biological microenvironments and pathological states but also provide new strategies and directions for future clinical treatment. Especially in cartilage and bone regeneration, by precisely controlling the structure and function of organoids, a better understanding of tissue repair mechanisms can be achieved, opening new avenues for bone/cartilage repair.

Alginate–based hydrogels have become invaluable in the field of regenerative medicine due to their biocompatibility and ability to encapsulate cells, providing a supportive environment for tissue regeneration. They have shown significant efficacy in promoting cartilage and bone regeneration and have been widely applied in the engineering of complex tissue constructs.^81,82

In the realm of cartilage regeneration, alginate constructs high–interconnectivity scaffolds that foster an environment conducive to chondrocyte proliferation and matrix production, ensuring good shape retention and cellular activity. Kleuskens et al.⁶⁴ combined alginate gel with a matrix from porcine notochord cells to create cartilage organoids composed of human chondrocytes. The use of alginate gel not only promotes the proliferation and matrix deposition of organoids but also fuses organoids into a homogeneous tissue similar to natural cartilage. Shehzad et al.⁷⁷ proposed the combination of alginate with other biocompatible materials such as gelatin to prepare hydrogel scaffolds that not only promote cell spreading and survival but also provide structural support for the fabrication of bone organoids (Figure 4A). The design of these scaffolds balances shape fidelity with cellular viability, especially their large pore size, which is crucial for cell migration and tissue regeneration.

As a natural biomaterial, chitosan plays a key role in regenerative medicine, particularly in the construction of organoid models and the regeneration of cartilage and bone tissues.⁸³ It provides a 3D scaffold environment for cells and promotes the proliferation and differentiation of specific cell types by mimicking the characteristics of the ECM, showing great potential in organoid development. Functionalised chitosan further enhances its anti–inflammatory and metabolic regulatory roles in organoid models by combining with specific bioactive molecules, such as the A–type endothelin receptor antagonist (BQ–123–CHI) and the B–type bradykinin receptor antagonist (R–954–HA).⁷⁵ In a horse joint cartilage inflammation model induced by IL–1β, this functionalised chitosan showed significant therapeutic effects, effectively reducing markers of inflammation and metabolic degradation, thereby demonstrating its potential in organoid research and clinical applications. Moreover, the degradation products of sulfated chitosan can specifically increase the number of therapeutic haematopoietic stem/progenitor cells in bone organoids⁸⁴ (Figure 4B), providing a potential new cell source for bone marrow transplantation. These findings not only enrich our understanding of the application of chitosan in regenerative medicine but also provide new directions and opportunities for future therapeutic strategies.

Hyaluronic acid (HA), with its excellent biocompatibility and ability to regulate cell behaviour, plays a key role in organoid construction.⁸⁵ New biocompatible materials, especially hydrogels, provide a favourable microenvironment for stem cell differentiation due to their high water content and adjustable physicochemical properties that mimic the ECM.¹² Biomimetic hydrogels, such as composites of HA and hydroxyapatite (HYP), have been proven to be highly effective in replicating tissue microenvironments.^13,86 Yang et al.²⁵ successfully developed a customised gelatin microgel based on HA and HYP. This innovative material not only enhances the ability of MSCs to spontaneously assemble into bone–cartilage organoids through bone–cartilage units in vivo but also demonstrates the broad application prospects of organoid technology in the field of bone–cartilage repair by promoting the regeneration of cartilage and bone tissues (Figure 4C).

Collagen, as a major component of the ECM, plays an essential scaffold role in tissue engineering to support cell growth and simulate a natural environment for tissue regeneration.⁶⁸ Cardier et al.⁷⁶ used allogeneic BMSCs from healthy donors, collagen microbeads (CM), and platelet–rich plasma clots to successfully develop an innovative composite material. As an osteogenic organoid, this material was applied to non–healing tibial segments and promoted osteogenesis in patients with bone formation failure (such as congenital pseudoarthrosis of the tibia). The application of collagen in cartilage and bone tissue engineering is also remarkable. In hydrogel studies containing non–degenerate organoids, Kleuskens et al.⁶⁴ observed the distribution of type I collagen among organoids, which formed a continuous tissue composed of cells, proteoglycans, and type II collagen around the organoids, highlighting the key role of collagen in simulating the natural cartilage environment.

Gelatin, a partially hydrolysed form of collagen, has been widely used in the field of tissue engineering due to its excellent biocompatibility and scaffold–forming ability. Studies have confirmed that gelatin–based hydrogels perform exceptionally well in the field of tissue engineering, especially in bone tissue engineering and the manufacture of bone organoids. The large pore size and highly interconnected scaffold structure of these hydrogels significantly promote cell diffusion and activity.⁷⁷ Deng et al.⁸⁷ also used a gelatin sponge scaffold loaded with recombinant human BMP–2 to create osteoid organoids, successfully constructing mature osteoid organoids with a bone marrow–like structure. These organoids, through the separation of MSCs from bone organs in the body to replace autologous MSCs, show superior proliferation ability and tri–lineage differentiation potential, becoming a new source of obtaining stem cells with excellent performance. Wang et al.⁸⁸ innovatively invented a new type of bio–ink, composed of gelatin methacrylate (GelMA), alginate methacrylate, and HYP, to print highly complex bone ECM analogues. This scaffold helps to culture and mature bone organoids and promotes multicellular differentiation, and its self–mineralisation characteristics are similar to natural bone, effectively enhancing bone repair ability and providing new possibilities for clinical applications (Figure 4D). Not only for bone repair ability, but organoids also have great potential in the treatment of OA. Han et al.⁵⁶ used transglutaminase–crosslinked gelatin to make osteoid organoids and successfully constructed tissue models with good structural stability. Transglutaminase–crosslinked gelatin has excellent mechanical properties and biocompatibility, which enables it to support cell growth and tissue function maintenance. These osteoid organoids show good application potential in simulating joint cartilage degeneration and related arthritis, especially in the research and treatment of OA. Coincidentally, Sun et al.⁸⁹ designed a type of organoid hydrogel (miR–24μS/SMSC organoid hydrogel [MSOH]) for ageing synovial MSCs. This hydrogel, by regulating the agieng–related miR–24/tau kinase 1 signalling pathway, slows down the ageing of chondrocytes and promotes cartilage regeneration. In a rat animal experiment, MSOH showed excellent cartilage repair ability, indicating its potential to become a new therapy for treating arthritis cartilage defects (Figure 4E, and F). Shehzad successfully made bone tissue organoids using gelatin–based hydrogels. These hydrogels combine gelatin, GelMA, and alginate, a combination that optimises the printing accuracy, stability, and biocompatibility of hydrogels, making them suitable for 3D bioprinting. Experimental results show that the developed hydrogels have good cell survival rates and biodegradability and can effectively support the mineralisation and differentiation of bone tissue. This method provides an efficient and economical solution for bone tissue engineering and in vitro models.⁷⁷ Zhang et al.⁹⁰ developed a new type of bio–ink, using granular hydrogels to simplify the production and 3D printing process of stem cell spheroids. It can spontaneously form stem cell spheroids at temperature changes to adsorb chondrocytes and form cartilage–like tissue.

Synthetic materials play a vital role in the field of biomaterials, offering superior biocompatibility and broad applications in bone and cartilage repair.

Polylactic acid (PLA), as a biodegradable material, serves not only as a scaffold for stem cell growth and differentiation but also demonstrates immense potential in bone remodelling. A hydrogel developed by Xiahou et al.²⁴ combines PLA with poly(L–glutamic acid)/fatty acid diazide, utilising dual crosslinking mechanisms—photo–induced and ionic bonding—to effectively support the spontaneous proliferation and aggregation of ADSCs. This hydrogel can automatically regulate cell adhesion, detachment, and aggregation through a cell response switch mechanism, leading to the formation of native–like cartilage microtissues. It exhibits excellent histological and biomechanical properties in cartilage tissue engineering (Figure 5A). Toni’s team⁹¹ constructed a 3D bone organoid model using poly–L–lactic acid and rat MSCs, replicating the micro–anatomical structure and vascular network of flat and short bones. By combining the decellularised adult male rat scapula matrix with poly–L–lactic acid–reconstructed vasculature and functionalising it with rat tail collagen, they successfully simulated the cortical and trabecular structures. This organoid can guide the osteogenic process of MSCs and support the attachment and survival of MSC–like cells on the reconstructed vascular walls, offering the potential for studying bone remodelling mechanisms in specific skeletal regions. Moreover, Ma et al.⁹² showed that poly(lactic–co–glycolic acid) scaffolds support osteogenic differentiation and mineralisation, making them promising candidates for bone tissue engineering applications.

PCL is widely used to construct bone tissue engineering scaffolds due to its excellent biocompatibility and biodegradability. PCL scaffolds can mimic the natural bone matrix, promoting vascularisation and nerve innervation, thereby reducing inflammation and promoting osteointegration⁷⁹ (Figure 5B). This makes PCL an ideal material choice for promoting bone healing and regeneration.

The development of PEG–based hydrogels provides an alternative to traditional Matrigel models for organoid culture. PEG hydrogels are biocompatible and have adjustable mechanical properties, making them suitable for organoid tissue engineering models.^80,93 These studies reveal the potential of PEG–based hydrogels in providing physiologically relevant models and promoting cell differentiation. Shen et al.⁹⁴ developed arginine–glycine–aspartic acid (RGD)–silk fibroin–DNA hydrogel microspheres (RSD–MS) for cartilage organoid fabrication. These microspheres combine the excellent mechanical properties of silk fibroin–DNA double network hydrogels with the cell adhesion capability of RGD peptides. RSD–MS can load BMSCs, promoting their chondrogenic differentiation and forming cartilage organoid precursors. Both in vitro and in vivo experiments show that RSD–MS significantly promotes cartilage regeneration and repair. This research provides new materials and strategies for cartilage regeneration and tissue engineering.

HYP) has good osteoconductive properties, promoting direct cell–matrix interactions, and is a key material for bone regeneration, widely used in bone tissue engineering.⁹⁵ Li et al.⁹⁶ used MSCs embedded in autocrine ECM and, with the help of osteogenic HYP nanoparticles, directly differentiated MSCs into osteogenic precursor cells and induced mineralisation within the cell–ECM structure. Concurrently, they also used iPSCs to generate multipotent mesenchymal precursor cells and further constructed organoids mimicking joint tissue. Wang et al.⁸⁸ used a mixed bioink of GelMA/alginate methacrylate/HYP to successfully fabricate bone organoids. This bioink simulates the complex ECM of bone tissue, with the ability for self–mineralisation and cell differentiation. Through 3D bioprinting technology, the constructed scaffolds can be long–term cultured and promote the maturation of bone organoids, with mechanical properties close to natural bone. This research is of great significance in the field of bone tissue engineering, especially in bone regeneration and repair, with broad application potential (Figure 5C). Li et al.⁹⁷ embedded HYP nanoparticles into MSC spheroids to create bone microtissues, which further self–organised into bone organoids. This method can enhance the osteogenic differentiation ability of MSCs, promote the formation and mineralisation of bone matrix, and generate bone organoids with a trabecular structure. HYP nanoparticles play a key role in promoting the development of bone microtissues, including improving cell survival rates and osteogenic capabilities. The method also shows good application prospects in the treatment of bone defects. He et al.⁹⁸ constructed an organoid model using β–tricalcium phosphate/HYP), encapsulating it in rat muscle tissue or implanting it in a muscle pouch for culture, resulting in a system that supports tissue survival, angiogenesis, and osteogenic gene expression, potentially generating bone tissue suitable for clinical transplantation. This provides new ideas for future bone tissue transplantation engineering applications.

Graphene oxide (GO), derived from graphite oxidation, is a promising nanomaterial for tissue engineering and regenerative medicine due to its atomically thin sheet, large surface area, and hydrophilic functional groups that facilitate chemical modifications and absorption of various molecules and cations.⁹⁹ Studies have shown that GO enhances human MSC (hMSC) adhesion, proliferation,¹⁰⁰ cell growth,^101,102 and osteogenic differentiation.¹⁰³ In 3D studies, GO has been integrated into hydrogel scaffolds such as alginate,^104,105 collagen,¹⁰⁶ and GelMA,¹⁰⁷ creating 3D GO hybrid scaffolds. Shin et al.¹⁰⁷ demonstrated that GO–GelMA hybrid hydrogels support fibroblast spreading and alignment with improved viability and proliferation. Choe et al.¹⁰⁵ found that adding GO to alginate enhanced the bioink’s structural stability and reduced swelling. However, higher alginate concentrations in 3D bio–printed scaffolds led to increased mechanical properties, inhibiting cell spreading and osteogenic differentiation of hMSCs.¹⁰⁸ Incorporating GO at an optimal concentration into low–density alginate/gelatin–based bioink could improve scaffold fidelity, enhance osteogenic differentiation of hMSCs, and increase ECM mineralisation for bone tissue engineering. Furthermore, GO/alginate/gelatin composite bioink materials not only enhance scaffold mechanical strength and stability but also promote cell adhesion and proliferation, thereby improving osteogenic differentiation and mineralisation efficiency.¹⁰⁹ Zhang et al.¹¹⁰ successfully created functional bone organoids by applying mechanical loading to 3D bioprinted hMSCs with GO composite scaffolds, which increased mineral density, stiffness, and osteoblast differentiation, forming bone cell–like structures (Figure 5D). These organoids offer an in vitro human model for studying skeletal pathophysiology and drug screening, potentially replacing animal experiments.

The advancement of organoid technology provides a unique model for simulating the complex biology of cartilage and bone. The formation of these organoids relies on the precise regulation of signalling factors that activate at specific time points, guiding cells to self–organise along particular developmental pathways.¹¹¹ Although various protocols promoting bone or cartilage formation have been developed, each involves different biofactors, intermediate steps, and culture durations, and no consensus on the most effective method has been reached.¹¹² Therefore, systematically comparing the factors and preparation methods that play a role is crucial for optimising the formation of bone or cartilage organoids.

The differentiation of bone or cartilage formation is a complex process regulated by multiple signalling pathways that control the aggregation, differentiation, and ultimate formation of mesenchymal progenitor cells into bone or cartilage.¹¹² Key signalling molecules such as BMP, growth factor, transcription termination factor B, Wnt, and cell adhesion molecules (neural cell adhesion molecule, N–cadherin, β–catenin) play an essential role in this process.^113⇓-115 Sox9, a cartilage–specific transcription factor, is crucial for maintaining the chondrocyte phenotype and promoting bone or cartilage formation differentiation.¹¹² Moreover, BMP–2, as an osteogenic growth factor, has been widely used to promote the differentiation of stem cells into osteoblasts.¹¹⁶ Osteomodulin can actively coordinate the osteogenic process through the BMP–2/suppressor of mother against decapentaplegic signalling pathway.¹¹⁷ In a scaffold–free micro mass environment, finely tuning the concentration of growth factors such as transforming growth factor–β can effectively guide iPSCs to differentiate into chondrocytes.¹¹⁸ These studies highlight the importance of biomolecules in mimicking the in vivo microenvironment and promoting organoid formation. Using specific chemical small molecule inhibitors can regulate the Wnt and melanocyte inducing transcription factor signalling pathways, thereby improving the efficiency and uniformity of hiPSC chondrogenic formation.¹¹⁹

Physical stimulation such as mechanical forces, electrical stimulation, and hypoxia are crucial for the formation and function of cartilage organoids. For instance, cartilage is often subject to compression and shear forces, which can promote the synthesis of ECM and tissue formation.¹²⁰In vitro studies that mimic these physical stimuli can better simulate the in vivo environment, thereby enhancing the maturity and functionality of organoids. Mechanical forces can promote organoid maturation by affecting cell morphology and the synthesis of ECM.¹²¹ Electrical stimulation is another physical stimulus that affects the regeneration of bone and cartilage tissues. A study has shown that Electrical stimulation can regulate cell behaviour, including proliferation, differentiation, and the synthesis of ECM.⁹¹ The use of electroactive biomaterials can mimic endogenous bioelectric phenomena, thereby promoting the regeneration process of bone and cartilage.⁹⁸ Hypoxia is an essential factor in cartilage tissue engineering. Chondrocytes can better maintain their phenotype and function in a hypoxic environment.¹²²In vitro culture that simulates a hypoxic environment can improve the formation efficiency and quality of cartilage organoids. The stable expression of hypoxia–inducible factor–1α under hypoxic conditions is significant for maintaining the homeostasis and function of chondrocytes.^123⇓-125

Self–assembly techniques offer a scaffold–free method for cellular self–organisation, utilising specific biomaterials and culture conditions to induce stem cell differentiation and form organoids. For instance, Yang et al.²⁵ employed gelatin–based microcryogels customised with HA and HYP to induce cartilage and bone regeneration, respectively, assembling into osteochondral organoids in vivo. Nilsson Hall’s team⁵³ allowed human periosteum–derived cells to self–assemble into microspheroids within specially designed microwell plates made from 3% agarose. These microspheroids then differentiated into mini–tissues with chondrogenic properties in vitro and further fused to form larger tissue structures, eventually generating bone organoids in vivo and healing critical–sized long bone defects in rats ex vivo. Tam’s group⁴⁹ successfully cultivated chondrogenic organoids using induced hiPSCs and self–assembly techniques, demonstrating stable chondrogenic characteristics upon in vitro culture and in vivo implantation. Stimulation with factors such as BMP–4, BIO, and 3,3,5–triiodo–L–thyronine induced the maturation and mineralisation of these organoids, particularly after the addition of IL–1β, which effectively bridged critical–sized long bone defects in vivo models, confirming the feasibility of using hiPSCs and self–assembly strategies to promote bone repair. This approach not only allows for precise control over the size and characteristics of bone or cartilage organoids but also enables accurate simulation of the cartilage intermediates in bone tissue engineering by mimicking the natural fracture healing process.

The application of 3D printing technology in organoid construction enables researchers to precisely control the shape and structure of organoids. Wang et al.⁸⁸ used bioinks such as GelMA/ alginate methacrylate /HYP to print scaffolds that mimic the microstructure of bone tissue, on which bone organoids were cultivated. Xie et al.³⁴ utilised digital light processing 3D printing technology to load BMSCs into hydrogel microspheres, printing and inducing the formation of osteo–callus organoids, effectively simulating the endochondral ossification process and significantly accelerating bone defect repair. Bolander’s team¹²⁶ combined human bone membrane–derived cells pre–treated ex vivo with thiol–ene alginate hydrogel to successfully fabricate callus organoids that mimic fracture–healing tissue. These not only verified cell viability and the ability to maintain chondrogenic and osteogenic differentiation in vitro but also showed that these biomaterials provided the necessary 3D cues for cells to organize tissue formation without additional stimulatory molecules in vivo. Li et al.¹²⁷ also used 3D printing technology with digital light processing printing methods to create a mini joint system simulating the knee joint using human BMSCs. This system integrates an engineered osteochondral complex, synovial fibro–like tissue, and adipose tissue, capable of simulating the interactions and inflammatory responses between joint tissues, providing an effective in vitro model for joint disease research and drug testing. This method offers new possibilities for the precise repair of bone defects.

Adding specific growth factors to 3D culture systems such as hydrogels can effectively induce stem cells to differentiate into osteoblasts or chondrocytes. For example, Fuller et al.⁶⁰ used a hydrogel ECM to culture a pre–osteoblastic cell line, controlling its differentiation into functional osteoblasts, thus forming bone organoids. Iordachescu et al.⁶¹ developed a trabecular bone organ model to study the regulatory mechanisms of local bone remodelling. This model assembles trabecular bone organoids by inducing cell differentiation simulating the bone formation process in bone tissue engineering. These constructs serve as multicellular, organotypic units, forming large bone cell protrusions and tubular structures that develop on a millimeter scale and initiate new matrix formation away from the original structure. This strategy not only aids in studying the differentiation mechanisms of bone cells but also provides a new cellular source for bone tissue engineering.

Demineralised bone matrix (DBM) technology provides a natural scaffold for cells by removing cellular components while retaining the original structure and bioactive molecules of the ECM.⁶¹ With excellent biocompatibility in constructing bone and cartilage organoids, DBM aids in cell adhesion, proliferation, and differentiation.⁴⁹ Park et al.⁶² found that inducing cell differentiation within DBM using demineralised bone paper biomaterial simulates unmineralised bone matrix, guiding osteoblasts to deposit mineralised bone tissue and activating quiescent osteocytes and inducing osteoclastogenesis under chemical stimulation such as vitamin D3 and prostaglandin E2. The use of the DBM matrix offers an effective strategy for tissue engineering, especially in the repair of bone and cartilage defects.

Microfluidic technology provides a highly controllable environment for organoid culture by precisely controlling fluid flow. Shen et al.⁹⁴ developed a novel RSD–MS using a microfluidic system combining photopolymerisation and self–assembly techniques, promoting the proliferation, adhesion, and chondrogenic differentiation of BMSCs, offering innovative strategies and material choices for cartilage regeneration and tissue engineering. Paggi’s team¹²⁸ created a “cartilage–on–chip” system using microfluidic technology, which can simulate the microenvironment of chondrocytes in vitro. This system allows researchers to apply precisely defined compression and multi–directional mechanical stimuli to a 3D agarose hydrogel filled with chondrocytes. This microenvironment simulation method enables researchers to explore how different types of mechanical stimuli affect the phenotype of chondrocytes and the production of ECM. Mechanical stimulation increases the expression of specific chondrocyte markers and promotes the production of glycosaminoglycans, a major component of the native cartilage ECM (Figure 6). Additionally, Alamán–Díez et al.¹²⁹ embedded pre–differentiated chondrogenic stem cells in a microfluidic chip and provided suitable culture conditions to simulate the cartilage tissue microenvironment, promoting cell maturation and matrix formation, thus providing an efficient in vitro model for cartilage tissue engineering. This technology is particularly useful in cartilage tissue engineering as it can simulate the microenvironment of cartilage, facilitating the formation of chondrogenic organoids. Bahmaee et al.¹³⁰ designed a microfluidic chip with a 3D microenvironment, combining fluid shear stress to simulate the growth conditions of cartilage tissue, promoting the differentiation and matrix formation of chondrogenic cells, thus providing a controllable and efficient in vitro model for cartilage tissue engineering.

Organoid technology has revolutionised the field of biomedical research, offering unprecedented opportunities for studying complex biological systems and advancing clinical applications. Particularly, bone and cartilage organoids have emerged as invaluable tools due to their ability to recapitulate the intricate microenvironments of human tissues.¹⁵ The rapid advancement of organoid technology has established it as a vital platform for studying the self–renewal and self–organising capabilities of stem cells, including ESCs, iPSCs, and adult stem cells.²² The in vitro models of organoids, with their ability to simulate complex natural tissue structures, have become crucial tools in both fundamental biological research and clinical applications. Particularly in the realm of bone and cartilage organoids, these models have successfully emulated the physiological characteristics of human cartilage at a microscopic level and have filled the gap for high–fidelity models of cartilage diseases.^14⇓-16

MSCs have made significant advances in the field of tissue engineering, particularly in bone regeneration.²³ By integrating MSCs with a gelatin sponge scaffold loaded with human BMP–2, Deng et al.⁸⁷ successfully constructed bone organoids in vivo to promote bone regeneration. This innovative approach not only addresses the limitation of autologous cell supply but also minimises the associated risks of using allogeneic cells in stem cell transplantation. Furthermore, Zhang et al.¹³¹ discovered that cells derived from bone organoids can rapidly and effectively rebuild the compromised peripheral and solid immune organs of irradiated mice. This finding presents a potential alternative source of haematopoietic stem/progenitor cells for transplantation, potentially benefiting a broader range of patients with bone regeneration disorders. Qiao et al.’s⁷⁹ neuro–vascularised bone organoid, constructed from a double small interfering RNA loaded cell membrane coated scaffold, utilised the synergistic effects of angiogenesis and neurogenesis to not only improve the microenvironment of bone defects, but also promote bone tissue repair and integration.

The regeneration of transparent cartilage has posed a significant challenge in the field of tissue engineering. However, innovative organoid cultivation strategies have led to groundbreaking progress. These strategies harness the self–organising capabilities of dissociated cells to successfully simulate the natural regeneration process of cartilage, effectively recapturing classic models of cartilage regeneration.^132,133 Notably, by utilising fibroblasts derived from terminal phalangeal elements as a source of chondrogenic progenitor cells, researchers have successfully facilitated the regeneration of transparent cartilage.^132,134 This breakthrough not only presents a fresh viewpoint on cartilage regeneration but also highlights the pivotal role of cellular self–organisation. The crux of this innovative method is its reliance on the cells’ intrinsic potential to catalyze tissue formation and healing, thus minimising the need for external growth factors or invasive procedures. This paradigm shift in cartilage regeneration heralds a new era of regenerative medicine, offering hope for the treatment of cartilage–related diseases and injuries.

The rapid development of organoid technology has revolutionised the field of drug screening, demonstrating immense potential, particularly in the study of bone disease treatments.^16,135 Li et al.¹³⁶ indicates that organoid–on–a–chip technology provides an innovative method for constructing in vitro models that simulate the physiological environment of specific organs, such as cartilage. This approach successfully overcomes the limitations of traditional two–dimensional cell culture models and animal models in the drug screening process. By constructing bone organoids, researchers can assess the efficacy of drugs in a more physiologically relevant environment. This not only reduces the time and cost associated with drug screening but also decreases the toxicity of drug testing. Moreover, organoid technology enhances intercellular interactions, allowing for a more accurate simulation of human physiological and pathological conditions. This highly mimetic method significantly improves the efficiency and accuracy of drug development and provides a new model system for a deeper understanding of drug mechanisms of action.

Furthermore, organoid technology has shown unique advantages in simulating disease histological characteristics and heterogeneity. It offers important tools for precision medicine and personalised therapy, assisting researchers in tailoring treatment plans based on the specific pathological features of patients.

Precision medicine, as an emerging trend in disease treatment, has shown particular importance in the field of bone disease therapy.¹⁶ By utilising patient–derived organoids, researchers can delve into the genetic heterogeneity among individuals, offering a novel strategy for personalised healthcare. This approach not only aids in predicting patients’ responses to specific treatments but also simulates the differences between tumours and individual patients, providing a robust supportive tool for clinical decision–making. As research progresses and develops, patient–specific bone organoids are expected to play an increasingly critical role in the realm of precision medicine. Human OA cartilage organoids have become vital tools for uncovering new molecular mechanisms of cartilage degeneration and provide a new platform for assessing targeted treatment pathways.¹³⁷ In the exploration of novel treatment strategies, iPSC–derived organoids have also been employed to identify pathogenic mutations in families with early–onset OA and to conduct functional studies on exon–edited hiPSCs, offering key insights into the gene expression of chondrocytes.¹³⁸ Furthermore, organoids and organoid extracellular vesicles have demonstrated tremendous potential as nanocarriers due to their acellular nature, stable drug–loading capacity, nanoscale size, and excellent biocompatibility. Particularly in the repair of osteoporotic fractures, the application of organoid extracellular vesicles has already shown its significance.¹³⁹

These in vitro models, capable of intricately simulating natural tissue structures, have become indispensable tools in basic biological research and clinical applications. Especially in the study of bone and cartilage organoids, these models not only mimic the micro–physiology of human cartilage to a certain extent but also fill the gap for high–fidelity cartilage disease models, which is crucial for understanding the pathogenesis of cartilage diseases and the development of new drugs.^14⇓-16

The potential of organoid technology in advancing the treatment of bone/cartilage injuries and diseases, particularly OA, is immense. This regenerative medicine approach has opened new avenues for disease modelling and therapeutic development. However, as we look to the future, it is essential to recognise the challenges and limitations that organoid research may encounter. One of the primary challenges is the discrepancy between organoids and human tissues, which may impact the accuracy of drug screening and disease studies. Maintaining the long–term stability and functionality of organoids for extended use in disease research and therapy is another significant hurdle. Achieving consistency and replicability in organoid production for large–scale applications is a technical challenge that needs to be addressed. Economic considerations are also crucial, as the high costs associated with organoid generation and maintenance could limit their broad application. Potential clinical risks, such as immune rejection and safety concerns, must be thoroughly investigated before organoid technology can be translated into clinical practice. Furthermore, despite the availability of various natural and synthetic materials, identifying an ideal biomaterial for organoid construction remains a challenge. In vitro organoid models have limitations in mimicking the complexity of in vivo physiological and pathological processes, which affects their predictive power for clinical outcomes.

Despite these challenges, the future of organoid technology holds great promise. The integration of organoid technology with advanced bioengineering techniques, such as 3D printing, microfluidics, and gene editing, presents a promising frontier. These interdisciplinary advancements could lead to more sophisticated disease models and tailored treatment strategies, enhancing both the efficiency and accuracy of medical interventions. In the realm of bone/cartilage regeneration, organoids offer a unique advantage by providing a controlled microenvironment for stem cell differentiation and tissue development. This controlled system allows researchers to study the intricate processes of tissue regeneration and to test the efficacy of potential therapeutics in a physiologically relevant context.

The implications of organoid technology for precision medicine are particularly noteworthy. By utilising patient–derived organoids, researchers can explore the genetic heterogeneity among individuals, leading to a more personalised approach to treatment. This strategy has the potential to predict patient responses to specific treatments and to model the variability between tumours and individual patients, thus informing clinical decision–making. As we look to the future, the role of organoid technology in addressing the challenges faced in translational medicine is clear. The development of more complex tissue–engineered implants, using pre–differentiated organoids as building blocks, could revolutionise the way we approach cartilage repair and bone regeneration.

Exploring the clinical potential of bone/cartilage organoids in disease modelling reveals several key limitations. These include discrepancies between organoids and human tissues that may impact drug screening and disease studies, as well as challenges in maintaining the long–term stability and functionality of organoids for extended use in disease research and therapy. Additionally, the technical challenge of achieving consistency and replicability in organoid production for large–scale applications, the high costs associated with organoid generation and maintenance, and potential clinical risks such as immune rejection and safety concerns must be addressed. Furthermore, despite the availability of various natural and synthetic materials, identifying an ideal biomaterial for organoid construction remains a challenge. Lastly, in vitro organoid models have limitations in mimicking the complexity of in vivo physiological and pathological processes, affecting their predictive power for clinical outcomes. Addressing these challenges is essential for enhancing the physiological relevance of organoid models and unlocking their full potential in regenerative medicine and precision healthcare.

In conclusion, the advancement of organoid technology holds great promise for the future of medicine, particularly in the fields of bone and cartilage regeneration. As research continues to push the boundaries of this technology, we can anticipate a future where organoids play a critical role in medical research, drug development, and clinical treatment, offering patients a wider range of therapeutic options and improved outcomes. The ongoing evolution of organoid technology is a testament to the power of interdisciplinary collaboration in driving biomedical innovation.

Author contributions

Concept and design: JH, CL, XZ, JL; acquisition, analysis, or interpretation of data: JH, AL, RL, JL; critical revision of the manuscript for important intellectual content: JH, XW, CL, SJ, JC; administrative, technical, or material support: JL, XZ, CL; supervision: JL, XZ, JH. All authors drafted of the manuscript and read and approved the final version of the manuscript.

Financial support

This work was supported by National Natural Science Foundation of China (No. 82272568), Sanming Project of Medicine in Shenzhen (No. SZSM202211019), Guangdong Basic and Applied Basic Research Foundation (Nos. 2023A1515220019 & 2022A1515220056), “Merro” Young Physician Innovation and Development Project (No. GSKQNJJ–2023–004), and Peking University Shenzhen Hospital Scientific Research Fund (No. KYQD2023297).

Acknowledgement

None.

Conflicts of interest statement

All authors declared no conflicts of interest.

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