Abstract
Bone tissue engineering has long been a focal point of research, aiming to address critical large segmental bone defects resulting from severe trauma, tumors, and other bone-related diseases. Despite significant advancements in conventional bone tissue engineering, the simulation of the intricate microenvironment characteristic of natural bone tissue remains inadequate. Natural bone is characterized by intricate macroscopic and microscopic architectures, along with a dynamic microenvironment that facilitates processes such as bone formation, remodeling, and repair. Bone organoids—three-dimensional structures that emulate natural bone tissue derived from stem cells—represent a substantial advancement in both bone tissue engineering and precision medicine. These organoids present a promising pathway for enhancing our understanding of bone biology and disease mechanisms. Their unique potential within precision medicine is underscored by their applications in personalized drug testing, disease modeling, and as platforms for regenerative therapies. As this field continues to progress, bone organoids are poised to play an essential role in developing tailored treatment strategies for disorders related to bones. In this review, we summarize the roles of cell types, biomaterials and culture techniques in the construction of bone organoids, and emphasize the key significance of microenvironment in guiding the maturation of bone organoids. In addition, we will discuss the standardization, current limitations, and future directions of bone organoids to provide insights for research and clinical applications.
Impact Statement
Bone organoids are a major breakthrough in bone tissue engineering, offering accurate models for studying bone biology, diseases, and therapies. This review summarizes their construction, culture techniques, and applications, emphasizing the roles of cell types, biomaterials, and microenvironments in guiding organoid maturation. It also discusses current challenges and future directions, highlighting their potential to advance personalized treatments and improve outcomes in bone repair and regeneration.
Introduction
The issue of large segmental bone defects arising from trauma, metabolic disorders, and tumors has garnered considerable attention within the academic research community.1,2 The process of natural bone regeneration is complex and highly organized, necessitating precise conditions regarding oxygen levels, vascular supply, immune response, and mineralization. 3 Traditional two-dimensional (2D) culture systems employed for bone regenerative materials exhibit a homogeneous distribution of cells and growth factors within the culture medium, which significantly deviates from the physiological conditions present in vivo. 4 Therefore, 2D cultivation is unable to synchronously simulate the dynamic balance and interaction of multiple factors such as cells, extracellular matrix, and growth factors under physiological conditions. Additionally, the vascularization and neural innervation in traditional bone tissue engineering are insufficient, making it difficult to precisely reconstruct the multilevel ordered structure of bone and achieve its functional activity. 5
Bone organoids are advanced three-dimensional (3D) biomimetic structures created from biomaterials and stem cells, which more accurately replicate the microenvironment essential for the differentiation of stem cells into osteogenic lineages under physiological conditions.6,7 Iordachescu et al. developed an innovative ex vivo bone model and introduced the concept of “organotypic bone” culture, which is acknowledged as the initial introduction of the “bone organoids” concept. This model employs a framework consisting of fibrin gel and calcium phosphate ceramic anchors to replicate the osteoblast network formation process observed in mature bone. It serves as a valuable tool for investigating the mechanisms of bone formation and for creating or inhibiting models of ossification phenomena in diverse pathological contexts. 8 Traditional bone tissue engineering scaffolds primarily serve as temporary physical templates for bone growth and regeneration, focusing on providing structural support and promoting cell proliferation and differentiation. They are often limited in their ability to fully replicate the complex microenvironment of natural bone, including the dynamic interactions between cells, the extracellular matrix, and biochemical signals. On the other hand, the design of bone organoids aims to be more in line with the actual conditions of the natural bone microenvironment. They can integrate multiple cell types and biological materials, thereby being able to simulate more complex biological processes, such as bone remodeling and the integration of blood vessels and neural networks. Additionally, in contrast to soft tissue organoids, bone organoids are expected to exhibit considerable mechanical strength, a substantial concentration of inorganic constituents, and diverse requirements for morphological changes. 9
Traditional theory posits that tissue engineering consists of three primary components: biomaterials, seed cells, and growth factors. 10 However, Gu et al. 11 have expanded this paradigm to include two additional factors: cell matrices and the regenerative microenvironment. The development of bone organoids is also influenced by these factors. Therefore, achieving an accurate replication of the cellular matrix characteristic of natural bone, along with the creation of a unique microenvironment that promotes bone regeneration, is critical factors that distinguish bone tissue organs from traditional bone scaffolds (Table 1). 12 Bone organoids have garnered significant interest owing to their considerable potential in elucidating osteogenic mechanisms, modeling various diseases, screening pharmaceutical compounds, and promoting advancements in regenerative medicine. Consequently, they hold substantial promise within the field of precision medicine. 7
The Differences between Bone Organoids and Bone Tissue Engineering Scaffold
BMSCs, bone marrow-derived mesenchymal stem cells; ESCs, embryonic stem cells; iPSC, induced pluripotent stem cells.
Summary of the Studies on Bone Organoids
3D, three-dimensional; AlgMA, alginate methacrylate; BMP, bone morphogenetic protein; ECM, extracellular matrix; GelMA, gelatin methacryloyl; hPDC, human periosteum-derived cells; PLLA, poly(
This review article aims to examine the contributions of biomaterials, stem cell biology, and culture methodologies in the development of bone organoids, emphasizing the critical importance of the microenvironment in influencing stem cell differentiation and maturation. Furthermore, we will address the standardization of bone organoids, identify existing limitations, and propose future directions to offer valuable insights for both research endeavors and clinical applications.
Macrostructure and Microenvironment of Natural Bone Tissue
Natural bone is a highly organized mineralized tissue characterized by intricate macro and microstructural features. 13 At the macroscopic level, bone is categorized into two primary types: cortical bone, also known as dense bone, and cancellous bone, or spongy bone. 14 Cortical bone constitutes the outer layer and is comprised of densely packed structural units known as the Haversian system, which primarily provide mechanical support. In contrast, cancellous bone is located in the inner layer, has a porous network structure, generally plays a role in stress and shock absorption, and is characterized by high metabolic activity and is involved in mineral storage, release, and hematopoietic function. 15 The outer surface of bone is enveloped by the periosteum, a tissue rich in blood vessels, nerves, and osteoblasts. Centrally located within long bones is the bone marrow cavity, which houses either red or yellow bone marrow. The microstructural composition of bone includes both organic and inorganic components, such as type I collagen and hydroxyapatite. These elements confer toughness and hardness to the tissue while also serving as a scaffold for cellular attachment and signaling. 16
At the microstructural level, bone tissue comprises bone cells, extracellular matrix (ECM), and mineral components. 17 Bone cells are classified into osteoblasts, which facilitate bone formation; osteoclasts, which are responsible for bone resorption; and osteocytes, which are embedded within the bone matrix and play a crucial role in maintaining bone homeostasis. The fundamental functional unit of bone is the Haversian system, which is organized in a cylindrical arrangement within cortical bone. This system consists of concentrically arranged lamellae surrounding a central Haversian canal, which contains blood vessels and nerves. These canals are interconnected with the periosteum and the bone marrow cavity through Volkmann’s canals, thereby supplying essential nutrients and oxygen to bone cells. In cancellous bone, trabecular structures are oriented along the lines of mechanical stress, contributing to the formation of an irregular network that enhances the bone’s structural integrity.
The microenvironment of bone tissue represents a dynamically regulated system comprising various cellular components, cytokines, growth factors, and physical factors. Noncollagenous proteins, such as osteopontin, which is secreted by osteoblasts, play a critical role in the regulation of the bone mineralization process.18,19 These cells and factors engage in interactions that contribute to the preservation of bone homeostasis. Bone homeostasis represents a dynamic equilibrium between the processes of bone formation and bone resorption within the phalangeal bone tissue, facilitated by the coordinated actions of osteoblasts and osteoclasts. This intricate process is modulated by various factors, including mechanical stress, hormonal influences (such as parathyroid hormone and calcitonin), cytokines (notably the nuclear factor κB receptor-activating factor ligand [RANKL]/OPG signaling pathway), and nutritional elements (including calcium, phosphorus, and vitamin D). Disruptions in this homeostatic balance may result in pathological conditions such as osteoporosis, osteosclerosis, or increased susceptibility to fractures. The activity of osteoclasts is modulated by the RANKL and macrophage colony-stimulating factor.20–22 Furthermore, mechanical stress is a significant factor influencing both the formation and remodeling of bone tissue. Adequate mechanical stimulation is known to enhance osteoblast activity, whereas insufficient mechanical stimulation can result in bone loss23,24 (Fig. 1).
Macrostructure and microenvironment of natural bone tissue; definition of bone organoids.
In contrast to conventional bone tissue engineering approaches, bone organoids more accurately replicate the 3D architecture and cellular heterogeneity of natural bone. They offer a microenvironment conducive to the dynamic regulation of cytokines and growth factors. Furthermore, bone organoids more effectively emulate the developmental and regenerative processes characteristic of natural bone. Consequently, it is imperative to compile a comprehensive overview of the composition and culture techniques associated with bone organoids to facilitate advancements in the field of bone tissue engineering.
Compositional Elements of Bone Organoids
Cellular origins of bone organoids
Bone marrow-derived mesenchymal stem cells
Bone marrow-derived mesenchymal stem cells (BMSCs) have been extensively utilized in bone regeneration research due to their multidirectional differentiation potential and immunomodulatory properties. These advantages underscore their suitability as functional cells in bone organoids. Recent studies have demonstrated that encapsulating BMSCs in self-mineralizing bioink for 3D printing can effectively facilitate the gradual formation of reticular structures by BMSCs, while also promoting their proliferation and osteogenic differentiation during long-term cultur.25–27 This streamlined self-mineralizing system enables the preparation of a large number of bone organoids in vitro.
The intrinsic ability of BMSCs to preferentially differentiate into osteogenic and chondrogenic lineages makes them the most representative functional cell type in the construction of bone organoids. Based on the multilineage differentiation characteristics of BMSCs, osteo-callus organoids cultivated in accordance with the physiological phenomenon of endochondral osteogenesis during bone development can significantly accelerate the healing of large segment bone defects.28,29 Osteo-callus organoids reproduce the longitudinal physiological process of BMSCs from chondrogenic differentiation, hypertrophic to osteogenic maturation. In addition, after modifying the gelatin matrix with hyaluronic acid and hydroxyapatite, BMSCs were loaded into microgel to induce chondrogenic differentiation and osteogenic differentiation, respectively, to achieve biomimetic structure and composition of natural bone and cartilage. 30
In addition, the crosstalk between different cell types is crucial for the development of organoids. A recent study examined the utilization of dental pulp stem cells (DPSCs) and BMSCs in generating vascularized bone organoids. DPSCs have been shown to enhance the survival rate of BMSCs through paracrine signaling and facilitate the integration of bone and blood vessels. 31
Osteoblasts and osteoclasts
Osteoblasts are cells responsible for bone formation and mineralization, originating from mesenchymal stem cells, while osteoclasts are specialized cells responsible for bone resorption, originating from precursor cells in the hematopoietic system. In bone homeostasis, the interaction between osteoblasts and osteoclasts is regulated through a fine feedback mechanism. This process is known as the bone remodeling unit, which involves the coupling of bone resorption and bone formation. Under normal circumstances, the activities of osteoblasts and osteoclasts are balanced to maintain the stability of bone mass and structure. When this balance is disrupted, it can lead to skeletal diseases such as osteoporosis and osteoarthritis.32–34 In a recent study, thin sections of demineralized cortical bone were employed to replicate the unmineralized extracellular matrix of bone tissue. This biomimetic scaffold was utilized to elucidate the regulatory mechanisms by which osteoblasts and osteoclasts modulate bone metabolism and the dynamics of bone remodeling. The researchers have designated this trabecular analog as demineralized bone paper (DBP). In this DBP trabecular organoid, osteoblasts not only participate in bone formation and mineralization but also regulate osteoclast function through phenotypic transformation of bone lining cells and paracrine of OPG and RANKL regulatory molecules, thus regulating bone metabolism and bone remodeling process. 35
Furthermore, Iordachescu et al. employed microscale trabecular bone derived from the femoral head as the structural basis and maintained the layered structure and surface morphology of the natural bone via thermal treatment. Subsequently, they implanted osteoblasts and osteoclasts into the structure to generate trabecular organoids. By situating the organoids in a microgravity bioreactor (NASA-Synthecon) to simulate the pathological state of bone loss induced by reduced mechanical stimulation, the experimental group exhibited distinct osteoclast-mediated bone resorption sites in contrast to the static control group. This model not only contributes to understanding the physiological and pathological mechanisms underlying bone loss and bone remodeling but also furnishes crucial experimental data and theoretical grounds for the development of drugs and other therapeutic approaches for osteoporosis and other bone-related disorders. 36
Human periosteum-derived cells
Human periosteum-derived cells (hPDCs) are specialized cell types originating from the periosteum. These hPDCs serve as integral components in bone formation and regeneration, demonstrate responsiveness to mechanical stress, and actively engage in the coupling of blood vessels with bone tissue. They play a pivotal role in the physiological processes underlying bone growth, development, and remodeling, thereby underscoring their significant potential in the field of bone tissue engineering and organoids. 37 Inspired by the “soft callus” intermediates during the natural fracture healing process, human periosteum-derived cells were employed to fabricate microspheres, which ultimately mature into cartilaginous bone marrow organoids with prehypertrophic properties under specific circumstances. By emulating the process of bone development, these organoids eventually form a structure analogous to the natural long bone in vivo, manifesting their potential in the repair of critical-size long bone defects. 38
Induced pluripotent stem cells
Induced pluripotent stem cells (iPSCs) are stem cells obtained through laboratory techniques by reprogramming mature somatic cells (such as skin or blood cells). These cells are reprogrammed to a state similar to that of embryonic stem cells and have the ability to differentiate into a variety of cell types.39,40 Studies have shown that iPSCs can be induced to differentiate into iPS-derived MSCs (iMSC) by chemical stimulation, biological stimulation and gene transduction in vitro. iMSCs exhibit superior performance compared to primary or natural MSCs in terms of cell proliferation, expansion capacity, immunomodulatory properties, paracrine signaling and exosome effects on cell-cell interactions.41–43 In addition, iMSCs genetically modified with overexpression of bone morphogenetic protein 6 (BMP6) were more effective in regenerating bone defects in mice than human BMSCs. 44
In addition to their application in regenerative bone organoids, iPSCs are also employed in disease model bone organoids. Frenz-Wiessner et al. introduced a novel approach for generating complex bone marrow organoids (BMOs) from iPSCs. The pluripotent differentiation potential of iPSCs facilitates the incorporation of key cell types present within the natural bone marrow microenvironment, thereby enhancing the fidelity of human hematopoietic microenvironment simulation in vitro. This represents an excellent model for investigating hematopoietic development and associated bone marrow pathologies. 45 Yao et al. generated self-organized neuromusculoskeletal organoids (hNMSOs) from human pluripotent stem cells (hPSCs). These organoids are capable of developing and self-organizing simultaneously, and establishing functional connections between neural, muscular, and skeletal tissues. This provides a new experimental model for studying the interactions of the human neuromusculoskeletal system and related diseases. 46
Embryonic stem cells (ESCs)
The utilization of embryonic stem cells (ESCs) in bone tissue engineering constitutes an advanced and promising domain, mainly attributed to the pluripotency, self-renewal, and directed differentiation of ESCs. 47 Jukes et al. employed ESCs to accomplish the regeneration of bone tissue via endochondral ossification. The team endeavored to induce the formation of a cartilage matrix from mouse ESCs and subsequently implanted this cartilage matrix beneath the skin of immunodeficient mice. The outcomes demonstrated that within 21 days, the cartilage layer underwent maturation, hypertrophy, calcification, and was eventually replaced by bone tissue. 48 Furthermore, Marolt et al. initially induced hESCs to differentiate into mesenchymal progenitor cells, and subsequently seeded these mesenchymal progenitor cells onto 3D osteoconductive scaffolds to construct large and dense bone tissues through ex vivo medium perfusion. After implanting the engineered bone tissue into immunodeficient mice for 8 weeks, the bone matrix was maintained and matured, without the formation of teratomas, which are typically observed when undifferentiated hESCs are implanted alone or in combination with bone scaffolds. 49 Despite the great potential of ESCs in bone tissue engineering, there are still many challenges, including how to improve differentiation efficiency and control the differentiation process, ethical controversies surrounding the use of embryonic stem cells, and the potential risk of teratoma formation. These controversial issues have led to a relatively low frequency of ESC use in the current field of bone tissue engineering.49,50
Biomaterials of bone organoids
The ECM serves as a supportive environment for cellular survival within the organism. In natural bone tissue, the ECM influences the behavior of osteocytes from both mechanical and biochemical standpoints, thereby playing an essential role in preserving the functionality and structural integrity of bone tissue. In contrast to the previous approach, ECM within bone organoids demonstrates a superior capacity to accurately regulate and replicate the natural microenvironment of bone.
The ECM of natural bone tissue consists of both organic and inorganic constituents. The organic portion typically includes collagen fibers, an amorphous matrix, and calcium-binding proteins, whereas the inorganic portion is primarily made up of hydroxyapatite crystals and various mineral salts. 7 Consequently, biomaterials employed in the fabrication of bone organoids are generally categorized into three classifications: inorganic synthetic biomaterials, organic synthetic biomaterials, and biomaterials sourced from natural bone tissue.
Inorganic synthetic biomaterial
Hydroxyapatite is an inorganic biomaterial that can be synthesized artificially or extracted from natural sources. Its composition and structural characteristics closely resemble those of the microenvironment of natural bone tissue, which contributes to its extensive application in the field of bone tissue engineering.50–52 Numerous studies have demonstrated that hydroxyapatite exhibits favorable biocompatibility, osteoconductivity, and plasticity,53,54 establishing it as a highly suitable biomaterial for the fabrication of bone organoids.25,30
Wang et al. integrated hydroxyapatite into a gelatin methacrylate (GelMA)/alginate methacrylate hydrogel to replicate the mineralization microenvironment characteristic of natural bone tissue. Subsequently, they introduced BMSCs into this composite system to formulate a bioink suitable for bioprinting applications. The findings demonstrated that this bioink successfully promoted the osteogenic differentiation of BMSCs, both at the site of bone defects in experimental animal models and in subcutaneous tissues distant from the bone.25,27 The most significant achievement of this bone organiods is its successful large-scale preparation in vitro. Nevertheless, it currently represents only a fundamental level of biomimicry concerning the composition and structure of the bone microenvironment. It does not adequately capture the intricate influences of the neural and vascular networks present in natural bone tissue on the bone microenvironment.
Li et al. incorporated hydroxyapatite nanowires (HAW), synthesized via the hydrothermal method, into osteoblast precursor (MC3T3-E1) cell spheroids. The findings of the study indicated that HAW significantly mitigated necrosis within the core of the cell spheroids and improved the osteogenic phenotype of the MC3T3-E1 cells. The findings indicate that the innovative utilization of existing biomaterials could also facilitate the development of large-scale, high-density biomimetic tissues and bone organoids. 55
Zheng et al. 56 formulated an injectable fully biomimetic bone organoid by combining calcium phosphate, calcium carbonate, magnesium phosphate, citric acid calcium, and sodium hydrogen phosphate in precise ratios within an extracellular matrix derived from the periosteum. This approach aimed to replicate the inorganic salt composition characteristic of natural bone.
Organic polymer biomaterial
Matrigel is the predominant matrix utilized in conventional organoid culture. It is abundant in extracellular matrix proteins and growth factors, thereby offering substantial support for the growth of organoids. 57 Frenz-Wiessner et al. employed collagen I in conjunction with Matrigel to encapsulate patterned embryonic bodies, thereby facilitating the self-assembly and subsequent maturation of bone marrow organoids. 45 Fuller et al. demonstrated that the 3D matrix provided by Matrigel not only enhances the osteogenic differentiation of MC3T3-E1 cells but also promotes the organized deposition of minerals within the Matrigel, resulting in the development of mineralized bone-like tissue. Furthermore, Matrigel contributes to the stability of these mineralized structures and supports the differentiation and functional activities of RAW 264.7 cells into osteoclasts, thereby mimicking the comprehensive processes of bone regeneration and resorption. Consequently, Matrigel is instrumental in the investigation of bone organoids and serves as an effective experimental model for the formation and reconstruction of bone tissue. 59 However,the high cost, xenogeneic properties, complex chemical characteristics, and inadequate mechanical performance of Matrigel limit its widespread application in the construction of bone organoids. 57
Considering the properties of bone organoids and the limitations associated with Matrigel, a range of natural synthetic polymers—such as gelatin, collagen, alginate, fibrin, hyaluronic acid, and chitosan—alongside artificial synthetic polymers including polyethylene glycol, polyvinyl alcohol, and poly(L-lactic acid) have been employed as alternatives to Matrigel.
Gelatin is extensively utilized in organoid culture owing to its superior biocompatibility, degradability, capacity for bioprinting, and potential for chemical modification. 59 Dai et al. conducted an experiment in which they implanted gelatin sponges loaded with BMP-2 into the muscle pouch adjacent to the femur of murine subjects. This intervention resulted in the formation of bone organoid structures that progressed through three distinct developmental phases: fibrous proliferation, chondro-osseous differentiation, and the formation of bone marrow. 60 Zhang et al. combined alginate, graphene, and human mesenchymal stem cells (hMSCs) with gelatin to formulate a bioink suitable for 3D printing bone organoids. Their findings indicate that this bioink effectively facilitates the osteogenic differentiation of hMSCs. 26 In addition to conventional gelatin, methylacrylylated gelatin is also extensively utilized in the fabrication of bone organoid ECM owing to its superior photocuring characteristics and mechanical properties.25,27 In summary, gelatin can effectively mimic the ECM of bone organoids in 3D environment, which provides the possibility for large-scale preparation of bone organoids.
Toni et al.
61
covered the surface of poly(
Natural bone biomaterial
De-mineralized Bone Matrix (DBM) is an allograft material derived from natural bone tissue through a demineralization process. The primary constituents of DBM include Type I collagen, noncollagenous proteins, and a limited quantity of calcium phosphate. 35 The production of DBM involves a series of chemical treatments that demineralize and defat allogeneic bone, resulting in a composite that contains Type I, IV, and X collagens, noncollagenous proteins, growth factors, a minor amount of calcium phosphate, and cellular debris. 63 DBM preserves the porous and reticular architecture of the original bone and exhibits osteoinductive properties, thereby demonstrating both osteoinductivity and osteoconductivity.64–67 Bone morphogenetic proteins and other growth factors play a significant role in the osteoinduction process associated with DBM. As a biological scaffold material, DBM not only autonomously releases bone morphogenetic proteins and growth factors but also has the capacity to transport various exogenous substances, thereby providing osteogenic biological stimulation to the recipient site through controlled release mechanisms. The 3D spatial configuration and mechanical conduction properties of DBM render it suitable as a scaffold material for bone tissue engineering, allowing for its combination with osteogenic-related cells or growth factors as an alternative to autologous bone grafts. These attributes have contributed to the extensive utilization of DBM in the repair of bone defects and in the field of bone tissue engineering. 68 Park et al. extracted and prepared a DBP from bovine femur, which can effectively simulate the coexistence of active and resting bone tissue surfaces and the process of local bone remodeling in bone trabecular cavity. Mouse osteoblasts obtained better ductility and tendency after surface osteogenesis induced differentiation of DBP. Therefore, this demonstrates the unique applicability of this bone trabecular organoid in the study of bone tissue dynamic processes and the creation of bone-related disease models. 36 (Fig. 2C).
In addition to DBM, various extracts derived from natural materials significantly contribute to the simulation and maintenance of the microenvironment of bone tissue.Hu et al. demonstrated that that oyster mantle-derived exosomes can regulate bone homeostasis and delay the progression of osteoporosis 69 (Fig. 2A). Wang et al. synthesized hydrogel composites exhibiting significant anisotropy, remarkable strength and stiffness, along with osteoconductive properties. This was accomplished by infusing biocompatible hydrogels into delignified wood, subsequently followed by the in situ mineralization of hydroxyapatite nanocrystals 70 (Fig. 2B). Li et al. developed a novel bone graft material that integrates antler acellular cancellous bone (antler-DCB) with extracellular vesicles (EVs) derived from antler bud stromal progenitor cells (ABPCs).The findings indicate that the material facilitated the phenotypic conversion of bone marrow stromal cells into cells resembling ABPCs, while also markedly enhancing new bone mass in vivo. Furthermore, it played a crucial role in the coordination of angiogenesis, neurogenesis, and immune regulatory processes 71 (Fig. 2D).In comparison to synthetic materials, natural extracts possess the potential to enhance the compositional and structural resemblance to the bone regeneration microenvironment found in living organisms, which makes them more competitive in regulating bone homeostasis and accelerating bone regeneration ability. This presents a novel strategy for the development of bone organoids.
Nevertheless, Natural bone extracts exhibit certain limitations, including constraints related to their source, potential risk of disease transmission, and insufficient mechanical strength. Furthermore, when compared to synthetic biological materials, natural bone extracts demonstrate inferior processability and pose challenges in terms of shaping and forming. 72 These deficiencies significantly restrict their applicability in the development of bone organoids.
Culture Techniques for Bone Organoids
The integration of bone organoids with other advanced technologies, such as organ-on-a-chip (OoC) systems, 3D bioprinting, and multicellular coculture, could further enhance their functionality. For example, combining bone organoids with OoC systems could allow for the simulation of dynamic physiological conditions, such as mechanical stress and fluid flow, which are critical for bone remodeling. Similarly, 3D bioprinting could enable the precise fabrication of complex bone structures, while multicellular coculture technology can better simulate the complexity of natural bone tissue (Fig. 3).
In vivo cultivation
In vivo cultivation technology plays a crucial role in bone organoid development by implanting 3D-bioprinted bone organoids into animal models (e.g., subcutaneously in nude mice) to leverage the biological environment for enhanced mineralization and maturation. This methodology significantly reinstates the intricate physiological conditions observed in vivo and promotes both vascularization and multicellular differentiation within bone organoids. 25 For instance, studies have shown that bone organoids printed with bioinks containing BMSCs and implanted in nude mice undergo spontaneous mineralization and develop bone-like structures after 40 days of in vivo cultivation. 28 However, this method is currently only used to verify the self-mineralization ability of bone organoids far from bone tissue. Subsequent research endeavors may focus on enhancing the mineralization of bone organoids through initial subcutaneous culture, followed by their implantation at the site of bone defects, with the aim of expediting the bone regeneration process.
Multicellular coculture
Multicellular coculture technology has emerged as a pivotal strategy in bone organoid engineering to recapitulate the complexity of native bone tissues. Recent studies highlight its applications in enhancing vascularization, osteogenic differentiation, and remodeling processes. For instance, tomographic volumetric bioprinting enables rapid fabrication of heterocellular bone-like constructs by coencapsulating hMSCs and human umbilical vein endothelial cells (HUVECs) in GelMA bioresins. This approach demonstrated that a 5% GelMA/0.05% LAP formulation supports high cell viability (>90%) and promotes osteogenic differentiation, with cocultures significantly upregulating early osteocytic markers after 21 days. 73 Bukhari et al. demonstrated the influence of estrogen on bone tissue vascularization and mineralization through the utilization of bone organoids constructed via the coculture of BMSCs and HUVECs. This research offers novel insights into the pathophysiological mechanisms underlying osteoporosis. 74
One notable approach involves the use of dental pulp stem cells (DPSCs) to enhance vascularization in bone organoids. Li et al. demonstrated that DPSCs, when cocultured with BMSCs, can differentiate into endothelial cells and form capillary-like structures within the organoid. This vascularization not only improves cell viability by enhancing nutrient and oxygen delivery but also supports the osteogenic differentiation of BMSCs, leading to increased mineralized matrix deposition. The study highlights the potential of DPSCs as a vascular source in bone organoid engineering, offering a scaffold-free method to create vascularized bone-like tissues for regenerative medicine and drug development. 32 In another study, Fuller et al. (2024) developed a murine-derived 3D bone organoid model using preosteoblast (MC3T3-E1) and osteoclast precursor (RAW 264.7) cell lines. This model successfully recapitulated bone homeostasis by allowing osteoblasts to mineralize the ECM and osteoclasts to resorb the mineralized matrix. The coculture system revealed that osteoclast differentiation and activity were enhanced in the presence of osteoblasts, suggesting a synergistic interaction between these cell types. The study also demonstrated that the organoids could be used to study the effects of mechanical unloading, such as in microgravity, on bone resorption and formation, providing insights into pathological bone loss conditions like osteoporosis. 59 Furthermore, Iordachescu et al. explored the use of primary human osteoblasts and osteoclasts cocultured on microtrabecular scaffolds to create a bone organoid prototype. This model allowed for the observation of osteoclastic resorption and osteoblastic matrix deposition in a physiologically relevant 3D environment. The organoids were subjected to simulated microgravity, which induced changes in osteoclast activity and resorption patterns, mimicking the bone loss observed in disuse osteoporosis. The study emphasized the importance of multicellular coculture in replicating the dynamic bone remodeling process and highlighted the potential of organoids for studying bone-related diseases and therapeutic interventions. 36
Bioprinting technologies
3D bioprinting is a technique for printing biomedical structures using living cells, biomolecules and biomaterials (bioinks, such as hydrogels). This methodology facilitates the sequential deposition of biological materials to fabricate 3D structures, including tissues and organs, and is widely employed in the construction of organoids.59,75–77 Zhang et al. utilized a pneumatic light-curing 3D printer to construct anisotropic bicellular living hydrogels (loaded with ACPCs and BMSCs) for osteochondral regeneration. This approach facilitates spatiotemporal regulation of cell-driven regeneration and serves as a valuable reference for the development of anisotropic living materials aimed at repairing intricate osteochondral tissue defect 80 (Fig. 4B).
3D bioprinting technology has the ability to precisely control complex structures and porous designs when constructing bone organiods, enabling personalized customization; it can simultaneously use multiple materials and precisely distribute cells, better simulating natural bone tissue; it possesses the advantages of rapid manufacturing and high reproducibility, which is conducive to standardization and large-scale production.6,7,25 The challenge of this technique is to ensure the stability of the overall structure after photocrosslinking while ensuring the survival and state of the cells. Living cells loaded with bioinks are very sensitive to external air pressure, light intensity and duration. The high air pressure and ultraviolet light itself have adverse effects on cell activity, so the parameters need to be repeatedly adjusted during the bioprinting process to obtain the best state of the scaffold function.
Bone organoid on-chip technologies
Bone organoid-on-chip technologies have emerged as a powerful tool for studying bone physiology, disease mechanisms, and therapeutic interventions by recapitulating the complex microenvironment of bone tissue in vitro. These platforms integrate microfluidic systems with biomimetic materials and cellular components, allowing for dynamic control of mechanical stimuli, biochemical gradients, and fluid flow, which are critical for bone health and disease.79–82
Traditional bone research has relied heavily on static cell cultures and animal models, both of which have significant limitations. Static cultures fail to replicate the 3D architecture and dynamic interactions within bone tissue, while animal models may not fully translate to human pathophysiology due to interspecies differences. 79 In contrast, bone organoid-on-chip technologies offer a more accurate and controllable environment for studying bone biology. These platforms enable the coculture of multiple bone cell types, such as osteoblasts, osteoclasts, and osteocytes, in a spatially organized manner, allowing researchers to investigate their interactions and signaling pathways in real-time. 82
Recent advancements in bone organoid-on-chip technologies have focused on modeling bone diseases such as osteoporosis and cancer metastasis. For instance, Lee et al. developed a bone-on-a-chip platform that simulates bone metastasis in osteoporotic conditions. 81 This platform recreates the bone microenvironment by coculturing osteoblasts, osteocytes, and osteoclasts in an ECM environment. The study demonstrated that osteoporotic conditions, characterized by increased osteoclast activity, promote bone metastasis by enhancing vascular permeability, which facilitates cancer cell invasion into the bone microenvironment. This finding highlights the importance of bone organoid-on-chip technologies in elucidating disease mechanisms and identifying potential therapeutic targets.
Another significant application of bone organoid-on-chip technologies is in drug development. These platforms allow for high-throughput screening of drug candidates and evaluation of their efficacy and toxicity in a physiologically relevant environment. For example, Zhang et al. reviewed the integration of biosensors into bone-on-a-chip systems, enabling real-time monitoring of cellular responses to drug treatments. 84 This capability is crucial for accelerating the drug discovery process and reducing the reliance on animal models.
Bone organoid-on-chip technologies also offer insights into the role of bone innervation and vascularization in health and disease. Neto et al. developed a microfluidic platform that models the neurovascular unit in inflammatory bone conditions. 85 This platform integrates sensory neurons, endothelial cells, and osteoclasts, allowing researchers to study the crosstalk between these components and their impact on bone homeostasis and pain perception. This study underscores the potential of bone organoid-on-chip technologies to advance our understanding of complex biological processes and develop targeted therapies for bone-related diseases.
The bone organiod chip technology simulates the physiological and pathological processes of bones on the chip, reducing the reliance on animal experiments and enabling a large number of experimental studies to be conducted in vitro. Furthermore, this technology supports personalized medicine by simulating the specific pathological conditions of patients and formulating personalized treatment plans for them. These advantages make the bone organ chip technology have great potential in bone research and clinical applications.
Prospect and Challenge
The field of bone organoids holds immense promise for advancing the understanding of bone biology, disease mechanisms, and regenerative medicine (Table 2). As a cutting-edge technology, bone organoids offer a unique platform for modeling bone-related diseases, screening drugs, and developing personalized therapeutic strategies. They provide a more physiologically relevant model compared to traditional 2D cell cultures and animal models, replicating the complex microenvironment of bone tissue, including interactions between osteoblasts, osteoclasts, and other cell types. This makes them invaluable for studying diseases like osteoporosis, osteoarthritis, and bone cancer, as well as for high-throughput drug screening, enabling more accurate and cost-effective testing of therapeutic compounds (Fig. 5).
Application prospect of bone organoids.
One of the most exciting prospects of bone organoids lies in personalized medicine. By using patient-derived stem cells, researchers can create organoids that mimic individual bone tissue, enabling tailored treatment strategies. This approach could revolutionize the treatment of bone diseases, particularly where standard therapies are ineffective or cause adverse effects. Additionally, bone organoids have significant potential in regenerative medicine, offering bioengineered bone grafts that closely mimic natural bone structure and function. These grafts could benefit patients with large segmental bone defects from trauma, tumors, or congenital disorders. However, for bone organoids to be effective, they must integrate with host tissue and promote functional bone regeneration, requiring a deep understanding of cell-matrix interactions, vascularization, and immune response.
Despite their potential, several challenges hinder the full realization of bone organoids capabilities. A major issue is the lack of standardization in their construction and culture, as variability in stem cell sources, biomaterials, and culture conditions leads to inconsistencies. Developing standardized protocols is essential for reproducibility and reliability. Wang et al. recently developed the first expert consensus and standard of bone organoids, which provides an important reference for the subsequent development of this field. However, compared with other tissue organoids, bone organoids are still a new field, and many problems still need to be further studied and solved. 12
Additionally, replicating the complexity of the bone microenvironment in vitro remains a significant challenge. Current bone organoids often lack key features such as vascular and neural networks, which are crucial for bone development and regeneration. Bone’s unique properties, combining rigid mineralized structures and softer bone marrow, further complicate the integration of tissues with varying stiffness requirements. Therefore, the mechanical properties of the 3D microenvironment provided by biomaterials for seed cells should be the focus of future research on bone organoid construction.26,87
Furthermore, DNA hydrogels exhibit considerable benefits in the development of bone organoids, attributable to their biomimetic properties, programmability, versatility, osteogenic potential, capacity for vascularization, and immunomodulatory effects. These characteristics present novel opportunities for advancements in bone tissue engineering and regenerative medicine.88–90
Scalability and cost are also limiting factors, as the production and long-term maintenance of bone organoids are resource-intensive. Developing cost-effective and scalable methods is crucial for widespread adoption. Ethical concerns, particularly regarding the use of ESCs, and the need for rigorous regulatory approval further complicate the translation of bone organoids from the lab to the clinic.
In conclusion, while bone organoids hold great promise for advancing bone research and regenerative medicine, addressing challenges such as standardization, microenvironment complexity, scalability, and ethical considerations is essential. Overcoming these hurdles will require interdisciplinary collaboration, innovative technologies, and a commitment to ethical and regulatory standards. With continued advancements, bone organoids are poised to play a transformative role in treating bone-related diseases and developing personalized therapies.
Construction and culture techniques of bone organoids.
Authors’ Contributions
Z.W., J.L., and J.C. conceived and designed the study, secured the funding, and supervised the entire project. K.M. and Y.W. conducted data visualization, drafted the initial article, and coordinated revisions. S.T., B.L., and Z.H. contributed to visual design and technical validation. C.W., C.Z., and X.W. critically reviewed the article, provided methodological feedback, and improved data interpretation. All the authors participated in article proofreading, final approval of the content, and ensured academic integrity. Z.W. and J.L. additionally acted as guarantors of the research.
Footnotes
Acknowledgment
The authors would like to thank the National Natural Science Foundation of China (82372401), the Beijing Natural Science Foundation (L202033), and the Key Project of the National Natural Science Foundation of China (21935011).
Funding Information
This work was financially supported by the National Natural Science Foundation of China (82372401), the Beijing Natural Science Foundation (L202033), and the Key Project of the National Natural Science Foundation of China (21935011).
Disclosure Statement
The authors declare no potential conflicts of interest concerning the research, authorship, or publication of this article.