3D bioprinting bone/cartilage organoids: construction, applications, and challenges

Abstract

Orthopaedic disorders, such as osteoporosis and osteoarthritis, impose substantial suffering upon an increasing population, driving demand for accurate disease models. Bone/cartilage organoids offer a promising solution by replicating complex 3D microstructures and multi-cellular niches, overcoming limitations of 2D models and animal experiments. 3D bioprinting, an additive manufacturing technology, enables the spatially precise deposition of cells and bioactive materials, facilitating efficient construction of organoids with enhanced structural fidelity. Therefore, this review specifically focuses on bone and cartilage organoids constructed using 3D bioprinting technologies. We summarize the prevailing 3D bioprinting techniques and biomaterials employed, critically analyze the unique advantages of bioprinting for creating these organoids, explore current technical challenges, such as standardization and scalability, and discuss future research directions. By addressing current progress and key issues in bioprinting bone/cartilage organoids, this review aims to accelerate their standardization and application as powerful platforms for multiscale disease modeling, drug screening, and regenerative medicine strategies. The translational potential of this article: Bone/cartilage organoids constructed via 3D bioprinting, through precise recapitulation of bone and cartilage tissue microenvironment and physiology, enable multiscale disease modeling from localized pathologies to systemic responses, despite persisting unresolved challenges in reproducibility and stability. This review highlights their clinical translational value and elucidates the driven role of 3D bioprinting in accelerating their clinical adoption, particularly in regenerative medicine.

Graphical abstract

1. Introduction

Over recent decades, propelled by continuous breakthroughs in modern medicine and the progressive extension of human lifespan, degenerative disorders including osteoporosis and osteoarthritis, as well as bone defects, have emerged as a global health challenge, exacerbated by aging populations [[1], [2], [3]]. Epidemiological data from the World Health Organization reveal that osteoarthritis alone affects over 500 million individuals globally, with prevalence rates surpassing 20 % in populations aged 65 years and older [4]. While the skeletal system exhibits unique self-regenerative capacities enabling spontaneous healing of most fractures without scar tissue formation, 5 %–10 % of fractures demonstrate impaired autonomous healing [5], mandating clinical intervention. Given the structural complexity and dynamic functional properties of osseous tissues, contemporary disease models developed through two-dimensional (2D) cellular culture systems or animal experimentation display significant limitations in replicating authentic human physiological conditions, particularly in reconstructing three-dimensional (3D) cell–cell interactions and cell-extracellular matrix crosstalk. Thereby, they inadequately reflect patients' intricate pathophysiological states and hinder comprehensive disease comprehension as well as drug development and screening [6]. Although artificial bone grafts and tissue engineered scaffolds have attained notable success in therapeutic applications, persistent challenges such as immune rejection, constrained cellular adhesion, and limited proliferative potential remain unresolved [7,8].

Organoids, 3D culture systems, utilize stem cells or progenitor cells with self-renewal and differentiation capacities to form miniature tissue analogs possessing spatial architectures and physiological functionalities, which partially recapitulate native organs in both structure and function while maintaining long-term stable passaging capabilities. Thereby, organoids provide an effective method to address the aforementioned challenges [9]. So far, successful cultivation has been achieved for the majority of physiological organs including intestine [10], brain [11], lung [12], liver [13], and kidney [14], and they consequently find extensive applications in disease modeling, drug screening, biocompatibility evaluation of implantable devices, and regenerative medicine [[15], [16], [17]]. For instance, Alizadeh et al. reported patient-derived lymphoma organoids (PDLOs) enabling disease-specific modeling and high-throughput pharmacological screening [18]. Furthermore, bone/cartilage organoids have garnered increasing attention due to their unique advantages in simulating complex osseous and chondral structures and functionalities, providing reliable models for investigating dynamic equilibrium of bone resorption-remodeling processes and pathogenesis of osteoporosis, while simultaneously serving as effective personalized drug screening platforms. Notably, bone organoids reconstructed through reprogramming of patient-derived adult stem cells circumvent immune rejection risks, thereby expanding their applications into regenerative medicine for bone defect remediation [[19], [20], [21]]. Nevertheless, given the structural complexity of osseous tissues, intricate vascular/neural networks, and stringent mechanical strength requirements, significant challenges persist in bone/cartilage organoid engineering [22]. Consequently, researchers are actively exploring novel construction technologies, including 3D bioprinting [23].

3D printing or additive manufacturing, distinguished from traditional subtractive and formative manufacturing methods, is a technology that fabricates complex objects through layer-by-layer deposition of bondable materials guided by computer aided design (CAD) digital model files, facilitating rapid prototyping and personalized customization [24]. In recent years, bioprinting, integrating 3D printing with tissue engineering, has demonstrated unique value in clinical treatments and medical research. It constructs intricate biomimetic living structures through precise deposition of cell-laden bioinks. Bioinks, comprising living cells, hydrogels, and cellular regulatory factors, can simulate the extracellular matrix microenvironment (ECM) to support cellular adhesion, proliferation, and differentiation [25]. The primary distinction from 3D printing lies in the incorporation of living cells as a constituent material, thereby conferring biological activity upon fabricated constructs. It should be noted that cells intrinsically lack printability; consequently, careful selection and modulation of hydrogel carriers within bioinks are critical for effective cellular printing and construction of complex 3D biomimetic structures. For bone/cartilage organoids, 3D bioprinting not only can optimally simulate the intricate multilayered microstructure of osseous/chondral tissues, but also achieve integration of vascular networks within bone/cartilage organoids. Furthermore, compared with conventional organoid construction methods, this technology demonstrates distinctive advantages including high precision, high-throughput capacity, automation compatibility, and enhanced reproducibility, thereby providing viable solutions for both scaled production and personalized customization of bone/cartilage organoids [26]. However, few previous reviews have focused on the advantages and application prospects of 3D bioprinting in bone/cartilage organoid construction. Therefore, within this review, a systematic elucidation is provided regarding the applications of 3D bioprinting in bone/cartilage organoid engineering. Additionally, critical challenges associated with organoid biofabrication via this technology are analyzed, along with prospective trajectories for its future development.

2. Construction strategies for bone/cartilage organoids

Compared to well-established hepatic, renal, pulmonary, and cerebral organoids with over a decade of developmental history, bone/cartilage organoid research remains in its nascent exploratory phase. The overarching objective of bone/cartilage organoid engineering lies in fully or highly recapitulating the intricate structural and functional characteristics of osseous/chondral tissues, including cell–cell and cell-ECM interactions, thereby establishing reliable platforms for disease modeling, pharmacological screening, and regenerative medicine, etc. Critically, patient-derived stem cells enable the personalized fabrication of bone/cartilage organoids, facilitating precise dissection of genetic background variations on disease phenotypes and advancing tailored therapeutic strategies [27]. Bone and cartilage are structurally complex, functionally dynamic tissues under homeostasis. To highly simulate their biological attributes, meticulous consideration must be given to three vital elements: stem/progenitor cells, ECM-mimetic biomaterials, and fabrication methods.

2.1. Seed cells

Osteogenic and chondrogenic developmental or remodeling processes involve osteoblasts, osteoclasts, and supportive stromal cells. However, their limited proliferative capacity renders them unsuitable as seed cells for organoid construction. Pluripotent or multipotent stem/progenitor cells, characterized by self-renewal competence, expansive proliferation potential, and multilineage differentiation capacity, serve as indispensable cellular reservoirs for tissue regeneration [28]. These attributes sustain their widespread application in bone/cartilage organoid engineering, with commonly utilized types including embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), and periosteum derived cells (PDCs).

Isolated from blastocyst-stage embryos, ESCs possess unlimited self-renewal and developmental totipotency. Their undifferentiated state permits directed differentiation into all somatic lineages, including osseous/chondral tissues. By employing activin-like kinase 5 (ALK-5) inhibitors to induce chondrogenic differentiation, researchers have achieved in vitro repair of articular defects in human cartilage explants [29,30]. ESC-derived small extracellular vesicles (ESC-sEVs) demonstrate therapeutic potential in advanced osteoarthritis by rejuvenating senescent chondrocytes via forkhead box O1 (FOXO1A)-autophagy axis modulation [31]. Nevertheless, ethical controversies persist due to the irreversible embryo destruction required for ESC isolation and maintenance, driving increasing adoption of iPSCs as alternatives.

iPSCs refer to cellular lineages reprogrammed from somatic cells to regain embryonic stem cell-like properties. These cells can be generated from any somatic cell type through reprogramming protocols, primarily employing Yamanaka factors. Patient-derived iPSCs retain donor-specific genomic profiles, conferring minimal immunogenicity to engineered bone/cartilage organoids and enabling personalized therapeutic applications. Limraksasin et al. [32] demonstrated murine iPSCs derived from gingival fibroblasts self-organized into rudimentary bone/cartilage organoids via osteogenic (Os) and osteochondrogenic (Os-Chon) induction. Similarly, Kondo et al. [33] established a mineralized 3D construct from human iPSCs for pathological analysis of hereditary dentinogenesis and osteogenesis imperfecta. Despite their pivotal role in organoid engineering, iPSCs face challenges including low reprogramming efficiency, batch variability, and tumorigenic risks [34]. Furthermore, incomplete reactivation of pluripotency-associated genes and deficient expression of ESC-specific markers hinder their full functional equivalence to ESCs [35]. Recent advancements in clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) genome editing enable targeted gene knockout, knockdown, or knock-in, offering strategies to enhance iPSC reprogramming fidelity and ESC functional mimicry [36,37].

MSCs, multipotent stromal cells isolatable from umbilical cord-derived (UC-MSCs), placenta-derived (PL-MSCs), bone marrow-derived (BMSCs), and adipose tissue-derived (AD-MSCs), exhibit excellent tissue-specific heterogeneity in gene expression and differentiation potential [38]. BMSCs, with well-characterized osteochondrogenic capacity, remain predominant in early-stage bone/cartilage organoid modeling [39]. UC-MSCs, isolated from neonatal cord blood, surpass BMSCs in proliferative capacity, multilineage differentiation potential, and reduced immunogenicity, while circumventing ethical concerns and donor morbidity [40]. For example, Pievani et al. [41] utilized cord blood-derived stem cells to construct bone/marrow organoids in vivo. Emerging evidence further indicates UC-MSCs effectively attenuate osteoarthritis-induced pain and inflammation while restoring knee function [42]. However, limited availability of autologous UC-MSCs—constrained by sparse cord blood banking infrastructure and neonatal donors—currently restricts clinical scalability. Nevertheless, UC-MSCs represent a promising candidate for future personalized bone/cartilage organoid biofabrication.

PDCs, multipotent stem cells isolated from the periosteum, have been extensively utilized in bone tissue engineering. The periosteum—a vascularized and innervated flexible fibrous connective tissue membrane covering osseous surfaces—exhibits unique mechanoresponsive properties and piezoelectric effects, playing critical roles in bone nutrition and injury repair [43]. Consequently, PDCs inherently possess mechanoresponsive characteristics and robust proliferative/differentiation capacities, enabling directed differentiation into osteoblasts or chondrocytes while demonstrating higher osteogenic activity than BMSCs under mechanical stimulation [44]. Studies reveal that mechanical forces activate the Piezo1 pathway in PDCs, triggering Ca²⁺ influx and subsequent upregulation of osteogenic factors and Ca²⁺/calmodulin-dependent protein kinase (CaMKII) expression, thereby enhancing osteogenic differentiation [45]. These attributes render PDCs uniquely advantageous for constructing osteochondral interface organoids. Dai et al. [46] engineered a bone morphogenetic protein 2 (BMP-2)/chondroitin sulfate (CS)-loaded scaffold capable of recruiting endogenous PDCs to form periosteum-like tissue in vivo, demonstrating superior osteogenic and osseointegration efficacy for complex craniofacial defect repair.

Given the intricate vascular/neural networks and multicellular-ECM interactions inherent to osseous/chondral tissues, co-culturing two or more cell types enables precise recapitulation of their structural and functional complexity [47]. Li et al. [48] incorporated dental pulp stem cells (DPSCs) into BMSC constructs at defined ratios, leveraging DPSCs vasculogenic differentiation potential to develop vascularized scaffold-free bone organoids. These findings underscore the promise of synergistic multicellular approaches for advancing bone/cartilage organoid engineering.

2.2. ECM-mimetic biomaterials

Within physiological contexts, osseous and chondral cells reside in specialized bone tissue microenvironments where ECM serves as a core constituent. The ECM comprises structural proteins, such as collagen and elastin, providing mechanical support, glycosaminoglycans (e.g., chondroitin sulfate, hyaluronic acid) establishing hydrogel microenvironments, adhesion proteins (e.g., fibronectin, laminin) mediating cell-ECM interactions, and signaling factors such as transforming growth factor-β (TGF-β) and vascular endothelial growth factor (VEGF) regulating cellular behavior. Additionally, osseous ECM is highly mineralized with abundant hydroxyapatite nanocrystals. Given the ECM's crucial role in cellular survival, proliferation, and differentiation, the selection of ECM-mimetic biomaterials constitutes an essential consideration in bone/cartilage organoids.

Matrigel, a basement membrane matrix derived from murine tumors, contains collagen, laminin, and multiple growth/signal factors, capable of recapitulating the native osseous/chondral microenvironment to support stem cell adhesion, proliferation, and lineage-specific differentiation. Its exceptional biocompatibility facilitates stem cell-derived secretion of tissue-specific ECM components such as chondroitin sulfate and hydroxyapatite, thereby enabling self-organization into organoids [21]. This substrate has been instrumental in developing diverse organoid models, including cerebral organoids for neurodevelopmental studies [49], hepatic organoids [50], and endometrial organoids [51]. As another animal-derived matrix material, decellularized extracellular matrix (dECM), obtained through perfusion/immersion-based cellular removal, preserves the native 3D ECM architecture with tissue-specific protein/glycan compositions and reduced immunogenicity, making it an ideal scaffold material [52,53]. Jiang et al. [54] engineered a PLA-templated, low-dose BMP-2 doped dECM periosteal analog, demonstrating efficacy in segmental bone defect reconstruction. However, both Matrigel and dECM are animal-derived biomaterials that inherently carry immunogenicity risks, while their compositional complexity and batch-to-batch variability further compromise experimental reproducibility and clinical translation.

Owing to the limitations of Matrigel and dECM in practical applications, researchers are actively pursuing alternative ECM-mimetic materials, with hydrogels emerging as a promising solution. Hydrogels for bone/cartilage organoid construction are categorized into natural hydrogels and synthetic hydrogels, which will be introduced in subsequent section. Particularly, synthetic hydrogel matrices enable precise control over composition and molecular architecture through standardized chemical synthesis and modification processes, effectively circumventing the batch variability and reproducibility issues inherent to Matrigel and dECM. Furthermore, the mechanical adaptability of hydrogels—achievable via composite crosslinking strategies—addresses the insufficient mechanical performance of Matrigel/dECM. For instance, the Matthias team engineered a dual-crosslinked polyethylene glycol (PEG) hybrid network hydrogel that provides stable mechanical support through reversible hydrogen bond-mediated dynamic rearrangement while facilitating stem cell differentiation and organoid formation [55]. Similarly, Matthew et al. [56] replaced Matrigel with tunable gelatin hydrogels, constructing mechanically adaptable inner ear organoids by modulating crosslinking density. Collectively, natural and synthetic hydrogels represent ideal ECM alternatives with substantial potential for bone/cartilage organoid engineering.

The highly mineralized nature of osseous/chondral ECM necessitates the incorporation of hydroxyapatite nanoparticles (HAp)—the major inorganic component in bone tissue. HAp activates the focal adhesion kinase/protein kinase B (FAK/AKT) signaling pathway to enhance MSCs osteogenic differentiation and bone matrix mineralization [57], providing osteoconductive and osteoinductive properties for hydrogel matrices. Wang et al. [58] developed a novel bioink comprising gelatin methacryloyl/alginate methacryloyl/HAp (GelMA/AlgMA/HAp), utilizing extrusion bioprinting to fabricate bone trabecula organoids. HAp doping conferred self-mineralization capacity and superior mechanical resilience. Additionally, HAp-incorporated hydrogels exhibit bioactive calcium ion release that promotes angiogenesis [59], highlighting their dual osteogenic/vasculogenic potential. In summary, the integration of calcium phosphate nanoparticles into hydrogel systems synergistically emulates bone-specific ECM characteristics, offering a transformative strategy for high-fidelity bone/cartilage organoid construction.

Similarly, tricalcium phosphate (TCP) and calcium silicate (CS) have been demonstrated to release multiple bioactive ions promoting the regeneration of multiple tissues, including bone and cartilage. Calcium ions and silicon ions can induce osteogenic differentiation, axonal growth, and neuropeptide secretion, while enhancing cellular migration capacity to recruit more cells for accelerating bone repair processes [60,61]. Therefore, bioinks doped with TCP or CS can simulate the inorganic components of natural bone tissue and enhance mechanical strength, while simultaneously inducing osteogenesis and neurogenesis. Zhang et al. [62] employed triaxial bioprinting technology to construct a vascularized bone tissue structure mimicking osseous and vascular microenvironments, using bioinks doped with nano β-TCP. The addition of β-TCP effectively modulated mechanical properties of this structure and promoted osteogenic differentiation and angiogenesis. Zhang et al. [63] developed a CS nanowire-doped GelMA-based hydrogel bioink, subsequently constructing it into bone organoids with enhanced restoration of innervation and innervated bone tissues. CS nanowires can activate ERK1/2-FOS/JUN pathway to promote osteogenic differentiation of BMSCs, while activating the neuroactive ligand–receptor interaction pathway to regulate neuronal differentiation.

2.3. Developmental regulation of bone/cartilage organoids

Organoid development is primarily governed by the directed differentiation of stem cells, achieved through precise modulation of signaling pathways and microenvironmental cues to induce lineage-specific cellular specialization and subsequent organotypic architecture. For instance, spatiotemporal activation/inhibition of wingless-related integration (Wnt), TGF-β, BMP, and Shh pathways enables differentiation of human embryonic stem cells into parathyroid cells for parathyroid organoid construction [64]. Similarly, coordinated regulation of fibroblast growth factor (FGF), Wnt/β-catenin, BMP, and TGF-β directs human pluripotent stem cells toward ureteric bud and collecting duct organoid formation [65]. Frenz-Wiessner et al. [66] further demonstrated bone marrow organoid generation via Wnt pathway modulation and activin/lymph node signal transduction in iPSCs.

During the development of bone tissue, the signal pathways involved in osteoblast differentiation, bone formation and bone homeostasis mainly include insulin-like growth factors (IGF), FGF, BMP, TGF-β, phosphoinositide 3-kinase/protein kinase B (PI3K/AKT), Notch and Piezo 1/2; the principal signaling pathways regulating chondrocyte development and differentiation include TGF-β, FGF, Hippo/yes-associated protein (Hippo/YAP), PI3K/AKT, BMP, and protein kinase A (PKA) [67,68]. Meanwhile, there are many vital transcription factors involved in the regulation of these differentiation processes, including activating transcription factor 4 (ATF4), Runx 1/2, Osterix, and transcriptional coactivator with a PDZ-binding domain (TAZ)/YAP [[67], [68], [69]]. These pathways are intricately interconnected through spatiotemporal coordination with transcriptional regulators to ensure proper skeletal tissue morphogenesis. Therefore, achieving spatiotemporal regulation of these signaling factors and pathways in vitro holds promise for the maturation of bone/cartilage organoids.

The spatiotemporal regulation of relevant signaling pathways to modulate cellular behaviors has been extensively applied in tissue engineering. Shukry et al. [70] demonstrated that covalent immobilization of Wnt3a onto functionalized surfaces provides localized Wnt signaling, establishing direct control over human skeletal stem cell (hSSC) fate through asymmetric cell division (ACD). Wu et al. [71] engineered hiPSCs carrying blue light-responsive TGF-β receptors to establish a photoactivatable TGF-β signaling platform. This system demonstrates that optical stimulation drives classic TGF-β signal transduction, enabling spatiotemporally precise control over stem cell differentiation, such as chondrogenic differentiation. Additionally, the development of novel scaffolds and carriers in tissue engineering facilitates more controllable release of relevant signaling factors, thereby enabling more precise spatiotemporal regulation of cellular behaviors. Feng et al. [72] prepared three gelatin/hyaluronic acid hybrid hydrogel (Gel-HA) microgels with distinct crosslinking densities and demonstrated that hydrogels of varying crosslinking densities can regulate the Hippo and TGF-β/Smad pathways, thus affecting the differentiation tendency of BMSCs. Low-density hydrogels promote chondrogenic differentiation, while high-density hydrogels promote osteogenic differentiation. Similarly, researchers developed a biomimetic scaffold encapsulating BMP-2 and nerve growth factor (NGF) mimetic peptides within poly(lactic-co-glycolic acid) PLGA microspheres [73]. This system facilitates spatiotemporally sequential release of NGF/BMP-2 mimetic peptides to promote neurotized bone repair. In vitro, rapid release of NGF-mimetic peptides significantly stimulates migration of RSC96 cells and axonal elongation of PC12 cells, while co-delivery of NGF/BMP-2 mimetic peptides synergistically enhances calcitonin gene-related peptide (CGRP)-dependent signaling pathways mediating osteogenic differentiation of BMSCs. The above studies demonstrate that in vitro regulation of diverse signaling pathways can mediate stem cell differentiation or proliferation behaviors, which has guiding implications for promoting the maturation of bone/cartilage organoids in vitro.

Sustained research continues to discover novel cytokines and refine signal pathways governing osteochondral development. These advancements not only deepen molecular understanding of skeletal morphogenesis but also establish critical theoretical frameworks for engineering structurally and functionally sophisticated bone/cartilage organoids in vitro.

2.4. Organoid construction techniques

The fabrication of high-fidelity bone/cartilage organoids is critically contingent upon the optimal selection of construction methods, with distinct approaches exhibiting significant divergence in emulating the structural and functional complexity of native tissues. These technical variations directly determine the biomimetic accuracy, experimental reproducibility, and clinical translatability of engineered organoids. Nowadays, diverse construction technologies have been developed for organoid systems, including: self-organizing suspension culture, microfluidic chip-based dynamic culture, electrospun scaffold composite culture, 3D bioprinting. Table 1 summarizes the commonly used techniques in organoid construction.

Table 1.

Comparative analysis of organoid construction strategies.

Preparation technology	Introduction	Advantage	Disadvantage	Ref.
Self-organizing	Stem cells can spontaneously aggregate to form certain structures and functions, relying on intrinsic pattern-forming ability of cells	ⅰ)Mature technology; ⅱ)Easy operation; ⅲ)Capable to construct and develop numerous organoids microspheres at once	ⅰ)Labor-intensive; ⅱ)Variable and complex morphology; ⅲ)Incomplete individual organs simulation	[74,75]
Microfluidics	Incorporating intricately engineered microchannels and microchambers, it enables precise manipulation of liquid volumes from nL to mL.	ⅰ)More efficient nutrient transport; ⅱ)Higher organoid generation efficiency and lower medium reduction; ⅲ)Uniform and high-throughput organoid arrays	ⅰ)The flow from medium to the cells impeded by bubbles; ⅱ)The channels are easily blocked; ⅲ)Difficult larger sized organoid formation	[[76], [77], [78]]
Electrospinning	Electrospinning refers to a category of highly porous fiber-forming methods using electrostatic forces, which can guide stem cell self-organization and generate high-throughput, scaffold-cultured organoids.	ⅰ)Achievable vascularization (co-culturing ECs) ⅱ)Uniform and high-throughput organoid arrays; ⅲ)Multi-factor tunability ⅳ)Promoting nutrients/gas diffusion	ⅰ)Lacking standardization; ⅱ)Uneven spatial cells distribution; ⅲ)Restricted Industrial production	[79,80]
3D bioprinting	3D bioprinting technology enables layer-by-layer spatial programming of cellular/biomaterial deposition to fabricate organoids, recapitulating native organs' structural-functional complexity	ⅰ)Printable directly vascular network; ⅱ)Highly controllable organoid structure; ⅲ)Personalized customization; ⅳ)High-throughput automated manufacture	ⅰ)Lacking standardization; ⅱ)Restricted Industrial production; ⅲ)Resolution insufficient for micron structure such as the capillary network.	[81,82]

At the microscale, bone tissue exhibits hierarchical structural characteristics, wherein cortical bone is composed of periodically arranged lamellae, while trabecular bone is characterized by a network of bone trabeculae. Similarly, the closely adjacent articular cartilage displays distinct spatial stratification. As the core functional unit of bone, the osteon, also known as the Havers system, which is constituted by central Haversian canal, Haversian lamellae, and embedded osteocytes, bone lacuna and canaliculi within lamellae, forms the structural basis of bone function [69]. Crucially, the dense neurovascular network within Haversian canals facilitates not merely nutrient delivery but also transmission of essential physiological signals. Simultaneously, the maintenance of dynamic microenvironmental homeostasis through continuous bone remodeling cycles is of crucial importance for bone integrity [83]. Therefore, bone/cartilage organoids should urgently reproduce such spatially graded hierarchical architectures and complex tubular networks while faithfully simulating dynamic physiological microenvironments. Nevertheless, conventional organoid fabrication methods encounter certain limitations in fulfilling these multidimensional biomimetic requirements.

Stem cell self-organization techniques, including hanging drop, microwell arrays, and rotational culture methods, are generally employed for organoid microsphere construction [84]. Although 3D structures are formed, hierarchical tissue organization cannot be achieved due to the lack of biological scaffolds, thereby impeding simulation of bone and cartilage physiological structures, whose capacity to imitate natural organs remains constrained. Microfluidic technology enables simulation of dynamic culture conditions, which may be utilized for emulation of the homeostatic microenvironment inherent to bone [85]. However, organoids generated by microfluidics predominantly exhibit spherical morphology and similarly represent scaffold-free constructs. Concurrently, high-viscosity and high-cell-density bioinks tend to cause microchannel blocking, thereby restricting material selection. Electrospinning technology permits construction of scaffold-based organoids capable of simulating organotypic physiological structural features. However, electrospun fiber layers typically demonstrate disordered or weakly aligned configurations with limited resolution, making precise spatial distribution control of distinct cell types within bone/cartilage organoids difficult. Additionally, electrospun scaffolds predominantly manifest as solid or lamellar structures, preventing direct fabrication of complex vascular network necessary for vascularized bone organoid development. 3D bioprinting technology provides effective solutions through its unique advantages, which will be discussed in detail in the next section.

3. Three advantages of using 3D bioprinting to construct bone/cartilage organoids

3.1. Precise construction and personalized customization for complex bone/cartilage organoids

Significant progress has been achieved in scaffold-free organoid spheroid construction leveraging the self-organizing capacity of stem/progenitor cells, which partially recapitulate organ-specific architectures and physiological functions. For instance, Parent et al. [86] demonstrated hPSC-derived self-assembled cerebral organoids with single-rosette 3D neural tube-like structures and dorsal forebrain characteristics, yet failed to achieve uniform laminar organization during later developmental stages. Similarly, Tan et al. [87] established murine testicular organoids with rudimentary seminiferous tubule-like compartmentalization, lacking complete spermatogenic epithelial stratification and functional spermatogenesis. Sun et al. [88] further developed cutaneous organoids mimicking hair follicle morphogenesis and sweat gland secretion. While self-organizing organoids partially replicate macroscopic morphology and basic functionality, they inadequately emulate full organ functionality. The functional realization of organs is inherently dependent on their structural integrity, as specialized architectures provide the foundation for physiological performance—a synergistic relationship. However, the stochastic nature of cellular self-organization impedes precise reconstruction of microarchitectural complexity. 3D bioprinting overcomes this limitation through computer-aided modeling and spatially precise deposition, ensuring accurate cellular positioning to facilitate intercellular communication while enabling directed fabrication of intricate microstructures, including trabecular bone porosity and vascular channel networks. These capabilities permit high-fidelity functional emulation of native tissues.

Nevertheless, interindividual heterogeneity in bone and cartilage tissue—driven by genetic and environmental variability—renders homogeneous self-organized organoids insufficient for personalized therapeutic applications. 3D bioprinting addresses this challenge by integrating patient-specific magnetic resonance imaging/computed tomography (MRI/CT) imaging data to achieve microstructure-specific customization, thereby establishing a transformative paradigm for precision medicine in orthopaedic disorders.

3.2. Direct vascularization potential for bone/cartilage organoids

It is inevitable to construct highly vascularized bone organoids due to the rich vascular network in Haversian canals. Conventional organoids relying solely on passive diffusion face metabolic limitations, as oxygen/nutrient diffusion distances cannot exceed ∼200 μm from capillaries [80]. Increasing organoid size exacerbates diffusion inefficiency, inevitably causing core necrosis. Vascularization is therefore critical for organoid viability and maturation. Current vascularization strategies include co-culture with vasculogenic cells, integration of vascular organoids, in vivo vascularization, organ-on-chip systems, and 3D bioprinting.

Co-culturing organoids with vasculogenic cells such as human umbilical vein endothelial cells (HUVECs), endothelial progenitor cells (EPCs) and iPSCs, remains the main vascularization approach. Smirani et al. [89] co-cultured human gingival fibroblasts (hGFs) with HUVECs to engineer vascularized connective tissue substitutes for oral rehabilitation. However, a single vasculogenic cell type inadequately recapitulates complex vascular architectures. Advanced protocols now employ multicellular co-culture systems (e.g. HUVECs/fibroblasts/pericytes) or incorporate micro-vessel fragments to enhance microvascular complexity [90]. Vascular organoid integration during early culture stages enables endogenous vascular network infiltration [91], though divergent culture requirements hinder co-culture optimization. Based on the strong regenerative potential and angiogenic propensity of the human body, it is possible to achieve vascular development within organoids in vivo [92]. Some studies have transplanted kidney organoids under the renal capsule of immunodeficient mice and achieved functional vascularization of kidney organoids [93]. Due to the use of autologous cells, this vascularization strategy can effectively weaken the immunogenic response and disease transmission, and has stronger clinical transformation potential.

The spontaneously generated blood vessels relied upon by traditional organoid construction techniques have drawbacks such as disordered and inefficient perfusion networks, and require extended maturation periods [94], whereas 3D bioprinting technology that permits direct fabrication of vascular structures has attracted substantial attention. Inspired by tissue engineering strategies employing materials such as salt particles, polysaccharides, and agar microbeads to generate porosity within biological scaffolds, Jennifer Lewis and colleagues pioneered a novel bioink system termed sacrificial ink [95]. The most commonly utilized sacrificial inks include easily removable materials such as gelatin, water-soluble polyethylene glycol, and Pluronic F127, which typically exhibit water solubility or temperature sensitivity [96]. Through the implementation of multi-nozzle bioprinting or coaxial bioprinting strategies, sacrificial ink is generally deposited separately from the bioinks, thereby creating complex hollow and perfusable internal architectures within constructs following its removal. This approach enables direct fabrication of vascular networks within organoids, which overcomes limitations associated with spontaneously formed vascular networks derived from HUVECs. Pan et al. [97] established interconnected circular microchannels within GelMA hydrogels using polyvinyl alcohol (PVA) as a sacrificial template, significantly enhancing oxygen/nutrient transport for long-term cellular viability. Federico et al. [98] employed GelMA as the bioink and Pluronic F127 as the sacrificial material to fabricate a biomimetic skin construct, which is applicable to in vitro investigations and wound repair applications. Using gelatin as a sacrificial bath, Zhang et al. [62] further engineered a vascularized bone model encapsulating mouse embryo osteoblast precursor cells (MC3T3-E1) and HUVECs within GelMA/alginate/β-TCP hydrogel. This methodology provides a robust technical framework for precision fabrication of large-scale vascularized bone/cartilage organoids.

3.3. High-throughput and automation for standardized bone/cartilage organoids

Conventional organoid construction methods remain labor-intensive, prone to human error, and limited in scalability and automation, severely hindering clinical translation. The absence of standardized protocols and widespread use of Matrigel and dECM have resulted in poor reproducibility of organoids from different batches, further exacerbating these challenges. In contrast, 3D bioprinting enables computer-automated spatial patterning of cells/ECM through microfluidic integration, significantly enhancing production efficiency and inter-batch/inter-laboratory consistency [99]. Kang et al. [100] developed a microarray 3D bioprinting platform with a 384/36-pillar plate system, achieving rapid (within 3 min) and reproducible deposition of foregut cells within alginate/Matrigel matrices for high-throughput fabrication of 384 human liver (HLO) and intestinal (HIO) organoids per batch with minimal manual intervention. Similarly, Yang et al. [101] engineered the OrgFab integrated bioprinter, which utilizes 5 μL bioink to non-contact print ∼30 organoids' precursors into 384-well plates with automated precision. These advancements position 3D bioprinting as the most viable strategy for industrial-scale organoid production, addressing critical bottlenecks in standardization and clinical translation.

4. Existing 3D bioprinting techniques for organoid construction

Compared to conventional organoid construction approaches, 3D bioprinting offers unique advantages in simulating the layered structure and dynamic microenvironment of osteochondral tissues, which can be used to construct higher quality bone/cartilage organoids to accelerate clinical translation. In this section, we will discuss prevalent 3D bioprinting technologies and hydrogel matrix materials for organoid construction.

4.1. 3D bioprinting strategies

In the construction of bone/cartilage organoids, the selection and optimization of 3D bioprinting technologies constitute a critical determinant, as distinct bioprinting modalities exert substantial impacts on the mechanical properties of matrix materials, cellular viability and spatial distribution, controlled release kinetics of growth factors, and architectural precision of organoids. Through the strategic selection of bioprinting technologies aligned with osseous/chondral tissue characteristics and the optimization of compatible processing parameters, precise regulation of microstructural configurations and spatial patterning of seed cells can be achieved, ultimately enabling the fabrication of high-fidelity bone/cartilage organoids. This section provides a systematic review of 3D bioprinting technologies routinely employed in bone/cartilage organoids (Fig. 1), followed by a critical comparative analysis of their technical specifications, advantages, and limitations.

Fig. 1.

3D bioprinting technology commonly used in bone/cartilage organoid construction [[102], [103], [104], [105], [106]].

4.1.1. Inkjet bioprinting

Inkjet bioprinting technology, characterized by high resolution, high-throughput capacity, and non-contact operation, has emerged as one of the pioneering 3D printing modalities for organoid construction [107]. This additive manufacturing technique employs micron-scale droplet ejection, wherein bioink microdroplets are generated through thermal or piezoelectric crystal deformation and subsequently deposited layer-by-layer onto a substrate to form 3D architectures [108]. Professor Xu's research team pioneered the conceptualization of progenitor cells derived from four major human tissue types as “biopixels”, which can be spatially assembled and differentially induced via inkjet bioprinting to generate diverse tissues or organs [107]. Liang et al. [109] employed electro-assisted inkjet bioprinting technology to construct disse space organoids (DOs), using alginate/laminin as the bioink, differentiating healthy donor and alcoholic liver disease (ALD) patient-sourced hiPSCs into hepatic endoderm/endothelial progenitor cells, which were co-assembled with hepatic stellate cells into microspheres. DOs generalized liver sinusoidal endothelial cell phenotypes and metabolic functions, providing a personalized assessment platform for environmental toxicology. Although the technology is widely adopted for fabricating intricate organoid structures owing to its micron-scale resolution (50–100 μm) and rapid printing velocity, cellular viability is inevitably compromised by shear stress and thermal effects attributable to nozzle and actuator mechanisms. Concurrently, the prerequisite for low-viscosity bioinks to prevent nozzle clogging imposes constraints on the construction of high cell density bone/cartilage organoids. Recent advancements in nozzle-free acoustic droplet generation techniques have garnered significant attention, with their inherent advantages of eliminating thermal exposure and no mechanical nozzles positioning them as promising candidates for high-fidelity organoid biofabrication [102,110].

4.1.2. Extrusion bioprinting

Extrusion bioprinting technology constructs spatial architectures through continuous deposition of bioink filaments extruded from micron-scale nozzles via pneumatic or mechanical piston-driven mechanisms [111]. It can accommodate bioinks with substantially higher viscosities and cellular densities, typically ranging from 10⁶ to 10⁷ cells/mL. Wu et al. developed a multiple myeloma organoid model with high cellular density (5 × 10⁶ cells/mL) utilizing coaxial extrusion bioprinting, demonstrating its utility in disease-specific drug development and personalized therapeutic strategies [112]. Zhang et al. [113] utilized human keratinocytes, fibroblasts, and endothelial cells to construct spherical skin organoids via self-assembly, which exhibited a stratified structure with a stromal core surrounded by surface keratinocytes. To ensure the overall mechanical properties of 3D bioprinting skin organoids, they innovatively combined extrusion bioprinting technology with dual-photo source cross-linking technology, achieving precise spatial organization and high cell viability, which can accelerate full-thickness wound healing in mice. Furthermore, the broad material compatibility of extrusion bioprinting enables the fabrication of bone/cartilage organoids with diverse matrix compositions. Nevertheless, shear stress induced at nozzle tips frequently causes cellular damage, resulting in diminished viability within engineered organoids. Implementation of low-shear nozzles (e.g., microfluidic coaxial nozzles) or combination with shear-thinning hydrogels as matrix materials has been demonstrated to mitigate shear forces and enhance cellular survival rates, which can exceed 90 % [114]. However, moderate shear stress during extrusion has been shown to activate the transcriptional coactivators TAZ/YAP and their associated signaling pathways, thereby promoting osteogenic differentiation of stem/progenitor cells [115].

4.1.3. Vat-based bioprinting

Light, serving as a non-invasive trigger, enables precise spatiotemporal control and property manipulation of photoactive materials, which has been widely utilized in the fields of tissue engineering and organ fabrication. Stereolithography (SLA) and digital light processing (DLP), as two common vat-based bioprinting technologies, operate through selective crosslinking of photosensitive materials under ultraviolet (UV) or visible light irradiation, fabricating intricate 3D biomimetic constructs within a dynamic range of size and resolution via layer-by-layer solidification of liquid resins [116]. SLA is the earliest-developed, most mature, and most widely applied 3D printing technology, typically employing a laser beam with a wavelength of 355 nm. The laser beam can be manipulated across a broad spatial range, rendering SLA the sole vat-based printing technology capable of fabricating large-scale models. However, resolution in SLA is generally constrained by laser beam dimensions [117]. DLP utilizes a projector to irradiate cross-sectional images of objects into photosensitive liquid resin, commonly adopting a 405 nm LED light source. Compared with the point-by-point laser scanning approach of SLA, the projection-based exposure strategy highly enhances printing velocity, which facilitates the construction of millimeter or centimeter scale biomimetic structures. The core component of DLP technology is the DLP chip, an optical switch recognized as the world's most advanced to date, which achieves switching frequencies of thousands of times per second. This chip reflects grayscale images with 1024-pixel resolution, converting input signals into rich grayscale images, thereby endowing DLP with exceptional resolution [118]. Nevertheless, to ensure high precision, both the exposure area and projection dimensions are restricted, confining DLP to the fabrication of small-sized objects. Therefore, DLP is widely used to construct high-precision, miniature organoids, owing to its superior printing resolution, printing velocity, and biocompatibility. Xie et al. [119] constructed engineered osteo-callus organoids for rapid bone regeneration using DLP bioprinting technology and stepwise-induction.

Although layer-by-layer printing technologies, such as SLA and DLP, have achieved significant accomplishments in tissue engineering, their prolonged fabrication processes required for constructing centimeter-scale organoids may compromise cellular viability and structural integrity, thereby hindering clinical translation. Volumetric bioprinting (VBP) enables single-step bulk solidification and fabrication of low-defect, freeform, centimeter-scale 3D biological constructs with high cell viability (>95 %) within seconds, offering a promising solution to the limitations of layer-by-layer printing [120]. In VBP, dynamically evolving light patterns constructed from back projections of the object's radon transforms are projected onto rotating photosensitive resin [121]. Solidification occurs nearly simultaneously throughout the volume when the accumulated 3D light-dose exceeds the photocrosslinking threshold, thereby fabricating the desired structure. During this process, the development of an ideal VBP photoresin is critical, requiring high transparency and appropriate viscosity. Bernal et al. [122] investigated a cell-laden, optically tuned hydrogel, utilizing VBP to construct centimeter-scale complex structures embedding human liver organoids without layers within 20 s. This preserved high cell viability (>93 %) and native-like polarization, while effectively modulating their metabolic function (e.g., enhanced ammonia detoxification) through designed geometrical configurations of porous scaffolds, thereby successfully building functional liver-like metabolic biofactories.

4.1.4. Other bioprinting methods

As an emerging technology, ultrasonic bioprinting leverages the high penetration capacity of acoustic waves and rapid acoustic thermal induced free radical polymerization to achieve non-contact bioink solidification into predesigned geometries. Xiao et al. developed a specialized bioink system that enabled deep-tissue printing of heterogeneous structures via ultrasonic bioprinting, successfully facilitating bone defect regeneration and cardiac valve repair [106]. Chen et al. [123] utilized acoustic bioprinting to spatially arrange patient-derived colorectal tumor organoids and healthy organoids, forming patient-derived microtissues (PDMs) that recapitulate primary tissue structure, which enabled in vitro drug screens and modeling of tumor invasion dynamics, providing a quantitative indicator to help doctors make better decisions on ultimate anus-preserving operation for colorectal cancer patients.

Microfluidic-assisted bioprinting technology integrates microfluidic chips with bioprinting heads to achieve precise co-deposition of multiple biomaterials and cell types [124]. By utilizing microfluidic technology to manipulate microdroplets, molecules, or cells for modulating bioinks, the combined use of 3D bioprinting techniques such as extrusion, inkjet, and vat-based, can achieve higher precision and resolution in organoid construction, for example, facilitating the construction of tiny capillary networks. Therefore, this method enables real-time modulation of cellular concentrations during printing—a critical parameter for controlling spatial cellular distribution in organoids—thereby standardizing organoid construction processes and enhancing experimental reproducibility. Ludovic Serex et al. [125] demonstrated microfluidic bioprinting of bioinks with ultrahigh cellular density (10⁷ cells/mL), establishing functional bladder organoids.

Aspiration-assisted bioprinting (AAB) is an innovative bioprinting technology through precise regulation of aspiration forces achieving grasping, transfer, and 3D spatial positioning of individual bioactive units (such as spheroids, tissue strands, or single cells) onto hydrogels. AAB utilizes negative pressure generated by custom-made glass pipettes, overcoming liquid–gas interface energy barriers and maintaining viscoelastic states of biological materials, and combined with microvalve systems depositing functional or sacrificial hydrogels, achieving scaffold-free or scaffold-based precise construction of complex biological structures [126]. Therefore, it can process organoids into specific 3D geometric shapes due to its precise positional accuracy (∼15 % with respect to the spherical size), flexible organoid microsphere size selection (80–800 μm), and high cell viability [126]. Heo et al. [127] used AAB to fabricate scaffold-free 3D bone tissue constructs, which realized the precise positioning of the spheroids onto hydrogels, and induced uniform bone formation in the whole spheroids. They utilized pre-differentiated co-cultured spheroids to achieve minimized post-printing shape deformation.

4.2. Biomaterials and bioinks

The judicious selection of bioinks constitutes a critical prerequisite in bone/cartilage organoid construction. Ideal bioinks must simultaneously fulfill biocompatibility criteria and 3D bioprinting process requirements. From a biological perspective, they should demonstrate superior biocompatibility and bioactivity to provide a physiologically relevant ECM microenvironment that supports cellular survival, growth, proliferation, and differentiation. From a manufacturing standpoint, adequate printability and mechanical robustness are essential to ensure rapid prototyping fidelity and long-term structural stability. Hydrogels represent the most prevalent bioink matrix in bioprinting applications. This section introduces several hydrogels applicable to bone/cartilage organoid engineering, encompassing natural and synthetic polymers (Fig. 2), followed by a critical analysis of their respective characteristics and limitations.

Fig. 2.

Commonly utilized biomaterials for 3D Bioprinting Bone/Cartilage Organoids [[128], [129], [130]].

4.2.1. Natural hydrogels

Silk fibroin (SF), a natural polymer extracted from Bombyx mori silkworms, comprises disulfide-linked 26 kDa light chains and 360 kDa heavy chains [131], characterized by a β-sheet crystalline structure that confers exceptional mechanical strength, flexibility, and processability. Its superior biocompatibility and enrichment in arginine-glycine-aspartic tripeptide (RGD) sequences facilitate osteogenic/chondrogenic cell adhesion, migration, and proliferation. These attributes render SF particularly suitable as a bioink, extensively employed in 3D-printed osseous/chondral tissue engineering scaffolds [132]. However, standalone SF application for bone/cartilage scaffold fabrication remains challenging due to low-concentration viscosity-induced structural collapse post-printing and high-concentration nozzle clogging tendencies. Furthermore, SF inherently lacks bone and cartilage inductivity. Consequently, SF is typically chemically modified, crosslinked with polymeric additives (e.g., collagen, gelatin), or compounded with inorganic particulates such as HAp to enhance tissue repair efficacy. For instance, Shen et al. [133] developed and utilized SF-DNA dual-network hydrogels to construct a cartilage organoid precursor (COP), which highly enhanced cartilage regeneration and provided a novel therapeutic strategy for osteoarthritis treatment.

Collagen, a predominant component of mammalian ECM, constitutes approximately 25 % of total body weight, with type I collagen being the most abundant variant [134]. Compared to silk fibroin, collagen possesses higher densities of cell-adhesive RGD motifs, rendering it excellent materials for constructing bone/cartilage organoids [7]. Owing to its exceptional biocompatibility, biodegradability, bioactivity, and processability, collagen is extensively utilized in organoid construction. Shi et al. [135] developed a low-concentration collagen I based bioink, which demonstrates favorable printability within an SF hydrogel-based supporting bath. Utilizing embedded bioprinting technology, breast tumor organoid models were constructed, facilitating enhanced comprehension of breast cancer and development of new drugs. However, it should be emphasized that inadequate mechanical strength and structural integrity of collagen restrict its application in load-bearing bone defect repair. Consequently, the development of composite reinforcement strategies with other materials is necessitated for bone/cartilage organoid construction.

Gelatin, a hydrolyzed derivative of collagen, retains abundant RGD sequences, conferring exceptional biocompatibility and cell adhesion. However, its inferior mechanical strength and thermal stability restrict standalone use in 3D bioprinting, often relegating it to sacrificial/support baths or requiring crosslinking with other polymers. Skylar-Scott et al. [136] employed gelatin as a sacrificial ink to fabricate personalized organs with embedded vasculature. GelMA, a photosensitive derivative containing methacrylamide and methacrylate groups, undergoes rapid UV-initiated free radical polymerization [137], making it the most prevalent photocrosslinkable hydrogel. Zhu et al. [138] utilized GelMA encapsulating DPSCs to construct stem cell spheroids via DLP bioprinting, demonstrating robust dentin and neovascular regeneration capabilities.

Alginate, a linear polysaccharide comprising β-D-mannuronic acid (M-blocks) and α-L-guluronic acid (G-blocks) linked via (1 → 4) glycosidic bonds, is typically extracted from cheap brown algae [139,140]. Its hydrogel formation relies on Ca²⁺-mediated ionic crosslinking with G-blocks. High M-block content yields elastic but mechanically weak hydrogels, whereas G-block enrichment enhances rigidity. Alginate's low cost, rapid gelation, and excellent printability have enabled widespread applications in tissue/organ engineering [141]. However, limitations persist, including inadequate mechanical strength, mismatched degradation rates, and poor bioactivity (insufficient cell adhesion or proliferation sites). Chemical modification or composite formulations are often required. For instance, Ding et al. [15] employed a composite strategy of alginate and gelatin with the doping of magnetic nanoparticles (MNPs) to construct advanced cartilage organoids, which partially replicated the intermediate layer structure of native cartilage and demonstrated markedly enhanced chondrogenic differentiation capacity and mechanical properties. In vivo experiments indicated that these advanced cartilage organoids significantly expedited tissue repair progression in cartilage defect regions, with restoration of normal structural characteristics in the chondral layer being observed. Jian et al. [142] employed a hybrid strategy utilizing the sodium alginate-based bioink and dipeptide-based bioink, through which liver organoids with biomimetic lobule structure were constructed using a multicellular 3D droplet-based bioprinting strategy.

Hyaluronic acid (HA), a linear natural non-sulfated glycosaminoglycan composed of repeating units of N-acetylglucosamine and D-glucuronic acid, is widely distributed throughout the human body as a core component of ECM [143]. HA exhibits exceptional biocompatibility, non-immunogenicity, and tunable degradability, thereby serving as an excellent bioink to support cellular growth, adhesion, and proliferation. However, its low viscosity and weak gelation capability limit printability. To address these shortcomings, HA is normally modified with UV-curable methacrylate (MA) to enhance mechanical properties and modulate degradation rate of 3D printing structures. Wang et al. [144] developed a 3D-printed islet organoid using hyaluronic acid methacrylate (HAMA)/pancreatic extracellular matrix (pECM) as a specific bioink. In diabetic mice, the islet organoids increased insulin levels, maintained normal blood glucose levels for 90 days, and promoted neovascularization, which offered a potential strategy to improve islet transplantation outcomes. As a vital constituent of cartilage ECM, HA has gained significant attention in bone and cartilage repair. HA has been demonstrated to effectively enhance tissue mechanical properties in cartilage repair, while inducing osteogenic differentiation of MSCs through signaling pathways such as TGF-β/Smad to support bone morphogenesis during bone repair [145]. Therefore, HA hydrogels have significant potential for bone/cartilage organoid construction, enabling the regulation of stem cell fate and facilitating maturation of tissue-specific functions.

Chitosan (CS), a linear natural polysaccharide composed of glucosamine and N-acetylglucosamine units linked by β-(1–4) glycosidic bonds, is derived from chitin deacetylation. Owing to its exceptional biocompatibility and biodegradable properties, CS plays a significant role in biomedical domains, including bone, chondral, and wound repair [146]. The relatively low mechanical strength, inadequate printing precision, and absence of shear-thinning behavior in original CS restrict its application as a bioink for 3D bioprinting of organoids. Chemical or physical modifications, such as methacrylation or incorporation of nano-hydroxyapatite, are commonly employed to enhance the printability and mechanical stability of CS. Nasiripour et al. [147] developed an N-carboxyethyl CS-based hydrogel bioink incorporated with 5 w/v% bioactive glass, which is applicable for constructing 3D-printing biomimetic bone structures.

dECM, obtained through perfusion or immersion for cellular removal, preserves the native 3D ECM architecture with tissue-specific protein/glycan compositions and reduced immunogenicity, which has been utilized as a hydrogel-based bioink in various 3D cell printing processes [148]. Dai et al. [149] developed a composite bioink using porcine adipose tissue-derived dECM and GelMA for the culture of colorectal cancer organoids, which demonstrated superior printability and shape fidelity post-printing. However, the use of dECM is subject to limitations. The quantity of ECM available for research is constrained by the availability of donor animals or humans, and the quality of the ECM can be influenced by the health status of the donor [150]. Furthermore, inter-batch variations persist even when the donor tissue is healthy. In contrast, Matrigel, another animal-derived material, is widely used for constructing and maturing microspheroid-based organoids. Nevertheless, its application in the 3D bioprinting of organoids remains relatively limited due to its formation of a temperature-dependent hydrogel [151]. This property renders Matrigel unsuitable for chemical modification aimed at modulating its mechanical properties, making it incompatible with 3D bioprinting [152].

4.2.2. Synthetic hydrogels

PEG, a synthetic polymer composed of repeating ethylene oxide units with molecular weights ranging from 200 to 8000 Da, has garnered significant attention in bone/cartilage organoid development owing to its hydrophilicity, biocompatibility, and tunable degradation kinetics [153]. PEG's structural diversity—including linear, 4-arm, 6-arm, and 8-arm topological configurations [154], which provides molecular versatility for functionalization. Incorporation of methacryloyl groups enables UV-mediated crosslinking for photocuring bioprinting. The mechanical properties and degradation rates of PEG-based materials can be precisely modulated by adjusting molecular weight and crosslinking density to meet osseous/chondral tissue requirements. However, inherent limitations such as inadequate mechanical strength and lack of bioactivity necessitate composite strategies with polymeric/inorganic components (e.g., HAp, β-TCP). Queralt et al. [155] developed a seamlessly integrated hybrid hydrogel of PEG and HA utilizing a transglutaminase crosslinked system, thereby enabling modulation and optimization of their physical and biological properties, subsequently leading to the construction of bone marrow organoids. Another innovative study developed a transglutaminase FXIII (TG-PEG) hydrogel encapsulating human amniotic fluid-derived stem cells (hAFSCs), achieving ectopic bone organoid formation in murine models [156]. These hAFSC-based organoids demonstrated bone regeneration efficacy comparable to BMSC-derived constructs, offering novel therapeutic paradigms.

Other categories of synthetic polymers, including PLGA, polylactic acid (PLA), and polycaprolactone (PCL), generally cannot directly incorporate or mix with cells, as the elevated temperatures and solvents required for their preparation into bioinks severely compromise cellular viability. Nevertheless, compared with natural polymeric materials, they possess superior mechanical strength and processability, which can be utilized as doping components to regulate various properties of bioinks, such as mechanical performance. For instance, to enhance the compressive resistance of cellular microspheres, Adrien et al. [157] incorporated porous PLGA micro-scaffolds into the bioinks, which provided increased surface area for cell adhesion and proliferation, thereby accelerating cellular growth and spreading, to construct an organoid. Similarly, Fisher et al. [158] developed an alginate-based bioink with suspended PLA nanofibers and human adipose-derived stem cells (hASCs) for constructing human medial knee meniscus structures through pneumatic extrusion-based 3D bioprinting. PLA nanofibers can induce local mechanical anisotropy and enhance metabolic and proliferative vitality of hASCs.

4.2.3. Critical challenges and limitations in bioinks development

During 3D bioprinting, biomimetic structures, such as organoids, are constructed by bioinks via specific crosslinking mechanisms. Consequently, the successful construction and subsequent application of bone/cartilage organoids critically hinge on the critical properties of bioinks, including printability, mechanical stability, cytocompatibility, and biomimicry, which currently present limitations requiring further optimization.

A critical challenge in bioinks is achieving optimal printability. As cell-laden materials, bioinks, typically existing in liquid or gel states, lead to flow/spreading during printing which compromises structural fidelity. Many current bioinks fail to meet the rheological requirements for precise extrusion, rapid crosslinking, and shape retention, resulting in limited resolution and stability of bioprinted constructs [159]. Furthermore, balancing printability with other properties, such as biocompatibility, complicates bioink design [160]. To enhance printability, increased precursor viscosity is necessary to reduce flow, however, excessive viscosity or high mechanical forces during extrusion would damage cell viability. Consequently, bioinks should exhibit rapid crosslinking and shear-thinning behavior (reduced viscosity under shear stress) to ensure printability. Optimizing bioinks thus requires meticulous balancing of viscosity, shear-thinning capacity, structural fidelity, and biocompatibility. Bioinks require adequate mechanical strength to maintain the biomimetic structure and match native organ properties, especially bone and cartilage. However, natural materials exhibit limited mechanical properties, which require using nanomaterials to enhance bioinks or developing dynamic hydrogels to improve mechanical adjustability [161,162], but challenges remain in achieving the necessary robustness without compromising other properties. Cytocompatibility remains a major limitation. The bioprinting process surpresses cells to continuous mechanical stress, compromising membrane integrity. Additionally, photoinitiators essential for photocrosslinking exhibit dose-dependent cytotoxicity, further restricting cytocompatibility. While many bioinks demonstrate favorable cell viability, they often lack bioactive molecules inherent to native tissues. dECM-based bioinks have been developed to address this challenge, yet introduce new limitations such as xenogeneic risks, batch-to-batch variability, and ethical concerns [143]. Furthermore, high-density incorporation of multiple cell types is essential for organoid bioinks, yet challenges remain in rapid large-scale expansion of cells and suppression of antagonistic interactions among distinct cellular microenvironments, requiring further breakthroughs. The achievement of biomimicry remains a significant hurdle, as most current bioinks cannot fully mimic the heterogeneous and dynamic nature of biological tissues. While advanced dynamic and multi-component bioinks enable more physiologic microenvironments, microscale biomimicry is still evolving.

5. Advances in 3D bioprinting bone/cartilage organoids

5.1. Bone organoids

The application of 3D bioprinting in organoid engineering enables precise replication of osseous tissue microarchitecture and physiological functionality. Given that bone is a highly mineralized, dense connective tissue, the construction of bone organoids via 3D bioprinting necessitates meticulous selection of cellular components and bioinks to emulate its unique structural properties. Tavares et al. [163] developed a nanocomposite hydrogel bioink (GelMA/MSNCaPDex) that sustains high viability of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and facilitates spontaneous osteogenic differentiation without exogenous biochemical or mechanical stimulation. Similarly, Wang et al. [58] engineered a large-scale self-mineralizing bone organoids using a GelMA/AlgMA/HAp composite bioink (Fig. 3A). These constructs demonstrated sustained cellular viability, autonomous mineralization capacity, and multicellular differentiation potential during in vitro/in vivo culture, ultimately forming mature trabecular structures in vivo. Thereby, it establishes a robust platform for modeling cancellous bone pathologies in vitro.

Fig. 3.

Current construction strategies for bone organoids. (A) Large-Scale self-mineralizing bone organoids constructed with HAp-incorporated bioink achieve effective osseous defect regeneration [58]. Copyright 2024 Wiley; (B) Self-Mineralizing bone organoids, cultivated in murine modelse, engineered through 3D bioprinting-based construction strategies [164]. Copyright 2025 Elsevier; (C) A neuro-bone construct is engineered via multicellular 3D bioprinting, enabling innervation-mediated bone regeneration [63]. Copyright 2022 Elsevier; (D) A highly vascularized bone organoid is engineered utilizing granular aggregate-prevascularized (GAP) bioink [166]. Copyright 2023 American Chemical Society; (E) Prevascularized bone organoids, doped with GO, enable rapid cranial bone regeneration [167]. Copyright 2025 Wiley.

Beyond advanced bioink formulations, optimized fabrication protocols are equally critical. A novel strategy employing projection-based photopolymerization 3D printing was recently proposed to generate bone organoid precursors, which matured into fully differentiated, mineralized, and vascularized osseous tissues following extended subcutaneous implantation in nude mice (Fig. 3B) [164]. Although not entirely in vitro, this approach validates the capacity of bone organoids to recapitulate dynamic skeletal development, positioning them as invaluable models for bone biology research. Furthermore, Zhang et al. [165] introduced a compression bioreactor system simulating skeletal mechanophysiological microenvironments. Integrated with 3D bioprinting and micro-CT monitoring, this platform provides mechanical loading to guide in vitro bone organoid formation. Their findings confirm that mechanical stimulation serves as a critical determinant in the self-organization of 3D bioprinting bone organoids, offering pivotal insights for in vitro bone tissue engineering.

Building upon the critical role of vascular and neural networks in bone remodeling, current endeavors focus on 3D bioprinting-mediated neurovascular integration. Zhang et al. [63] developed a gelatin-based bioink incorporating calcium silicate nanowires to fabricate biomimetic neuro-osseous constructs via dual-channel 3D bioprinting, which enables neurotized bone regeneration (Fig. 3C). This innovation not only provides a novel therapeutic strategy for bone defect repair but also holds significant potential for elucidating neuro-osseous regenerative mechanisms. Furthermore, a pre-vascularized gelatin-alginate-PCL bioink with exceptional self-organization and angiogenic capacities was engineered to construct highly vascularized osseous tissues, demonstrating superior vasculogenesis and osteogenic performance compared to conventional bioinks (Fig. 3D) [166]. Duan et al. [167] developed prevascularized bone organoids, which can be loaded into GelMA hydrogels for 3D bioprinting, by integrating MSCs, HUVECs, and osteogenic graphene oxide microparticles (GO MPs), enabling the fabrication of centimeter-scale constructs with high cell density for bone regeneration (Fig. 3E). GO primarily promoted osteogenesis through the focal adhesion and PI3K/AKT pathway.

Despite these advancements, contemporary bone organoids remain structurally and functionally rudimentary relative to native bone, with functional vascularization representing a persistent challenge. However, the convergence of 3D bioprinting advancements and the development of bioactive and biocompatible bioinks is progressively refining organoid construction paradigms, laying a robust foundation for clinically translatable, mature bone organoids.

5.2. Cartilage organoids

Mature cartilage tissue exhibits limited self-repair capacity due to its lack of vascular and neural properties and complex regeneration mechanisms, positioning cartilage organoids as critical models for chondropathology research and tissue replacement. Ding et al. [15] developed a magnetic nanoparticle (MNP)-loaded BMSC/alginate/gelatin hydrogel bioink, producing cartilage organoids with exceptional mechanical robustness and chondrogenic differentiation in vitro, while restoring lamellar cartilage architecture in defect models in vivo (Fig. 4A). A study successfully engineered 3D-printed cartilage constructs with histological and mechanical properties akin to native cartilage using adipose-derived mesenchymal stem cells (ADSCs), demonstrating fully mature chondrogenic characteristics even in deep zones (Fig. 4B) [168]. Shi et al. [169] engineered an antioxidant multifunctional hydrogel bioink for 3D bioprinting, creating reactive oxygen species (ROS)-resistant constructs that enhance cartilage regeneration under chronic inflammatory microenvironments. Emerging as an innovative biofabrication strategy, cartilage-derived microspheres serve as modular building blocks for constructing 3D bioprinting cartilage organoids. Burdis et al. [170] devised a modular biofabrication strategy to generate osteochondral tissues recapitulating native articular cartilage structure/function, achieving superior articular defect repair in vivo (Fig. 4C). Similarly, Gabriela et al. [171] fabricated scaffold-free cartilage tissues via extrusion-based 3D bioprinting of MSC-derived microtissues as modular units, which can be used for cartilage regeneration (Fig. 4D). To address limitations including post-printing maldistribution of cellular microspheres in conventional approaches, Gabriella et al. [172] developed a laser-assisted bioprinting methodology (LIPMO) utilizing multicellular spheroids as building blocks, which is applicable to efficient construction of high-cell-density cartilage organoids (Fig. 4E). These studies demonstrate significant translational potential of cartilage organoids for clinical applications in cartilage regeneration and repair.

Fig. 4.

Current construction strategies for Cartilage organoids. (A) A magnetically responsive cartilage organoids was constructed using MNP-BMSCs, demonstrating significantly enhanced chondrogenic regenerative capacity [15]. Copyright 2025 Published by Tsinghua University Press; (B) Using ADSCs to replace MSCs, thick cartilage constructs with fully mature deep layers are successfully fabricated [168]. Copyright 2022 Springer Nature; (C) A biomimetic microtissue with native osteochondral architectures has been engineered via modular biomanufacturing strategies, thereby achieving functional articular surface regeneration [170]. Copyright 2022 Elsevier; (D) Cartilage grafts are constructed from cartilage micro-tissues via 3D bioprinting [171]. Copyright 2024 American Chemical Society; (E) A novel laser-assisted bioprinting strategy enables the construction of high-density chondrospheres [172]. Copyright 2024 IOP Publishing Ltd.

Because of the unique composition and structural characteristics of cartilage, cartilage organoids can also be constructed in combination with other technologies. A microcryogel-based platform exploiting MSC self-organization generated bifunctional osteochondral organoids with robust regenerative capacity in vitro/in vivo, offering novel regenerative paradigms [173]. Shen et al. [133] combined microfluidics with RGD-silk fibroin-DNA hydrogel microspheres (RSD-MS) to cultivate long-term viable cartilage organoid precursors, demonstrating significant chondral defect repair in rat femoral models. These technologies demonstrate significant potential for integration with 3D bioprinting platforms toward the construction of 3D bioprinting cartilage organoids that more faithfully recapitulate native cartilage structure.

While 3D bioprinting plays vital roles in both bone and cartilage organoid engineering, cartilage organoid construction progresses more rapidly due to simple physiological structure of cartilage and reduced material dependency, whereas bone organoid construction remains technically challenging.

5.3. Other bone organoids

Callus, a transitional connective structure formed during fracture healing, bridges bone fragments and stimulates osteoblast activity to facilitate localized ossification. As a critical phase in bone defect repair, callus organoid research holds significant potential for elucidating healing mechanisms and accelerating osseous regeneration. Xie et al.[119] utilized DLP technology with stepwise induction to fabricate methacrylated silk fibroin (MS) hydrogel microspheres, which promoted rapid bone reconstruction in rabbit large-defect models via callus-mimetic organoids (Fig. 5A). Similarly, Bolander et al. [174] successfully engineered 3D-bioprinted cellularized regenerative implants mimicking native fracture callus, validating their osteogenic capacity in vitro and in vivo (Fig. 5B). Despite the relative simplicity of callus organoid fabrication, challenges persist in scaling to clinically relevant dimensions. Concurrently, bone marrow organoids are being actively explored as 3D models to investigate hematopoietic development and immunomodulatory mechanisms [[175], [176], [177], [178]]. These advancements not only underscore the versatility and precision of 3D bioprinting but also provide new possibilities for regenerative medicine, disease modeling, and personalized therapeutics.

Fig. 5.

Current construction strategies for osteo-callus organoids. (A) A osteo-callus organoids is engineered via DLP and stepwise-induction, achieving bone defect repair within 4 weeks [119]. Copyright 2022 Elsevier; (B) hPDCs undergo differentiation into osteogenic and chondrogenic lineages, subsequently self-organizing to generate osseous callus tissue [174]. Copyright 2021 Elsevier.

6. Applications of 3D bioprinting bone/cartilage organoids

Bone/cartilage organoids indicate innovative therapeutic orientation for bone defect repair. Current clinical management of critical-sized bone defects relies predominantly on autografts or allografts, which are constrained by secondary surgical trauma, donor scarcity, severe immune rejection, and potential disease transmission [179]. Patient-specific transplantable organoids derived from autologous stem/tissue cells via iPS technology avoid immunological barriers. Notably, 3D bioprinting's inherent advantages enable standardized organoid manufacturing platforms. Such “in vitro organ factories” offer high-throughput, automated, and quality-controlled production systems to address global shortages of bone implant materials.

Bone/cartilage organoids provide a powerful tool for the construction of bone and cartilage disease models. Precision medicine necessitates preclinical models capable of recapitulating histopathological alterations across disease progression. Conventional 2D cultures lack spatial heterogeneity, while animal models are limited by interspecies genetic disparities and ethical controversies, both failing to mirror human pathophysiology [180,181]. In contrast, stem cell self-organized organoids, as a 3D culture model, can simulate vital 3D structural features of bone and cartilage tissues and eliminate species differences. Now, osteoarthritis models, osteoporosis models, bone tumor models, bone defect models, genetic bone disease models and other bone/cartilage disease models have been developed. Nevertheless, traditional self-organized organoids face inherent limitations—stochastic cellular distribution, inadequate microarchitectural control, and necrotic cores. 3D bioprinting overcomes these constraints through micron-scale spatial patterning of cells/biomaterials, enabling precise replication of osteochondral laminar structures, mineralization gradients, and vascular channel networks. This precision not only resolves metabolic challenges in large-scale organoids but also models pathological vascular infiltration. Such engineered organoid systems permit dynamic observation of disease progression, thereby elucidating comprehensive mechanism and personalized therapeutic development.

Bone/cartilage organoids provide robust platforms for high-throughput drug screening and toxicity assessment. The conventional drug development paradigm—entailing decade-long timelines and multi-billion-dollar investments—faces temporal and financial constraints. 3D bioprinting organoids enable precise emulation of osseous/cartilaginous pathophysiological features, generating high-fidelity data for drug efficacy/toxicity evaluation [17,182]. Furthermore, standardized “in vitro organ factories” permit mass production of organoids, significantly enhancing screening throughput while reducing reliance on animal testing, thereby slashing preclinical costs and ultimately lowering drug prices. For instance, Wei et al. [183] identified α2-adrenergic receptor signaling as a therapeutic target for cartilage regeneration through cartilage organoids, validating their utility in drug screening. In precision medicine, patient-derived organoids retaining disease heterogeneity and individualized phenotypes enable predictive modeling of drug responses to guide tailored therapies, minimizing adverse effects such as osteosarcoma chemotherapy toxicity. Additionally, these systems facilitate biocompatibility evaluation of orthopaedic implants. Although biobanking for bone/cartilage organoids remains underdeveloped [184], limiting high-throughput applications, their transformative potential in high-throughput drug screening and personalized medicine is unequivocal.

Bone/cartilage organoids exhibit unique scientific value in aerospace medicine. Microgravity induces acute/chronic bone loss, bone microenvironmental damage, and articular degeneration in astronauts, elevating fracture risks [185,186]. During extended deep-space missions such as lunar bases and Mars exploration, prolonged microgravity exposure will exacerbate osteochondral deterioration [187]. 3D bioprinting organoids exposed to simulated microgravity offer ideal in vitro models to unravel these pathologies. Meanwhile astronaut-specific iPSC-derived organoids could predict individual adaptability to space environments and inform personalized countermeasures. Pioneering work has been achieved on-orbit 3D printing of tumor organoids aboard the International Space Station (ISS), where Professor Jamieson's team demonstrated microgravity-induced adenosine deaminase acting on RNA1 (ADAR1) activation driving cancer stem cell rapid multiplication, while validating Fedratinib and Rebecsinib efficacy [188,189]. Building on this foundation, on-orbit bioprinting of bone/cartilage organoids could accelerate anti-bone loss drug discovery and regenerative material development, greatly shortening the transformation cycle from laboratory to clinic, while facilitating civilian application of space science and technology.

7. Challenges in 3D bioprinting bone/cartilage organoids

While 3D bioprinting bone/cartilage organoids demonstrate enormous potential across diverse applications, critical challenges must be resolved prior to clinical translation.

7.1. Vascularization

Bone is highly vascularized, with medullary and periosteal vascular networks providing essential nutritional support for bone development and remodeling, making vascularization vital for accelerated bone repair [190]. Current coaxial bioprinting strategies using sacrificial materials have achieved preliminary organoids vascularization. However, existing printing resolutions (typically 50–200 μm) remain insufficient to replicate blood capillary networks with 6–8 μm luminal diameters [191]. Furthermore, incomplete sacrificial material removal and vascular collapse due to inadequate mechanical support influence perfusion efficiency, ultimately impairing organoid viability. To address this, Zhang et al. [62] employed triaxial bioprinting to deposit HUVECs within intermediate bioink layers, simulating vascular niches to induce endothelial wall formation. Another study engineered a tough dual-network hydrogel bioink combining ionically crosslinked alginate with enzymatically crosslinked gelatin, fabricating mechanically robust vascular analogs resistant to collapse [192].

7.2. Systemic integration

3D bioprinting bone/cartilage organoids excel in mimicking microstructural and microenvironmental features for disease modeling and drug screening compared to self-organized organoids. Nevertheless, native osteochondral tissues are dynamically regulated by neural innervation, immune modulation, and multi-organ crosstalk—features absent in current organoid systems. Additionally, orthopaedic drugs undergo hepatic metabolism, renal clearance, and immunomodulatory effects, which cannot be assessed using single-organoid platforms. Future directions involve multi-cellular bioprinting strategies (e.g., co-depositing BMSCs, neural stem cells, HUVECs, and immune cells) to construct multi-tissue organoid system, enabling systemic investigation of musculoskeletal pathogenesis and therapeutic interventions.

7.3. Standardization and scalability

The absence of standardized protocols for bone/cartilage organoid construction introduces significant difficulty in scalable production. Current research lacks consensus on critical parameters including organoid dimensions, cellular density, and performance criteria, compounded by batch-to-batch variability in raw materials and operator-dependent errors, collectively undermining data reproducibility and cross-study comparability. Although 3D bioprinting serves as an in vitro “cellular foundry” for mass production, inter-batch inconsistencies persist due to divergent bioprinting parameters and culture conditions. Establishing internationally recognized guidelines for standardized manufacturing and quality control is imperative to ensure reproducibility across laboratory-to-industrial transitions, thereby accelerating clinical translation.

7.4. Additional technical barriers

Inherent limitations of 3D bioprinting further challenge the construction of bone/cartilage organoids. Conventional extrusion/inkjet bioprinting places cells under deleterious shear stresses and thermal exposures, reducing stem cell viability and impeding high-density cellular deposition. Bioink development faces dual constraints: bone substitutes demand high strength/toughness materials, while the cartilage requires elastomeric properties. Current natural/synthetic hydrogels fail to concurrently satisfy mechanical and bioactive requirements of native tissues. Emerging AI technology may optimize bioprinting parameters and screen ideal bioinks through large model screening. Despite iPSCs' vital role in personalized organoid construction, prolonged reprogramming timelines and low efficiencies hinder high-throughput applications [193]. Chen et al. [194] engineered a fast chemical reprogramming (FCR) system, reducing human somatic cell-to-iPSC conversion from 40 days to 7–12 days. Wang et al. [195] further refined FCR to achieve iPSC induction within 10 days. However, the stability of iPSC-derived osteochondral differentiation necessitates rigorous validation to ensure clinical-grade reproducibility.

8. Conclusion

3D bioprinting has achieved significant milestones in bone/cartilage organoid engineering, with its high precision and automation enabling precise replication of bone/cartilage microarchitectures, biomechanical properties, and physiological microenvironments. This technology demonstrates transformative potential in disease modeling, high-throughput drug screening, and personalized medicine. Nevertheless, 3D-bioprinted bone/cartilage organoids remain in their beginning stage, with reproducibility and stability yet to meet clinical translation requirements. Future advancements should focus on refining bioprinting technologies and construction strategies to establish a standardized, systemically integrated organoid platform. The development of adjustable universal bioinks—coupled with innovations in efficient iPSCs induction protocols—will enhance organoid construction efficiency and batch consistency. Furthermore, multi-axis co-printing methods integrating vascular/neural networks and other relevant organs will enable systematic evaluation of musculoskeletal pathologies. We anticipate that the integration of 3D bioprinting will markedly accelerate the clinical translation of bone/cartilage organoids, particularly in advancing regenerative medicine applications.