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methodologies predominantly employ stem cell-derived the microporous structures, fabricating micron-scale
osteoblasts, with BMSCs representing the most widely trabecular bone organoids. This model successfully
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utilized progenitor cell source due to their well-characterized recapitulated microgravity-induced osteopenia, providing
osteogenic differentiation capacity. In addition, attempts a valuable platform for studying bone loss under simulated
have also been made to construct bone organoids from space conditions. Alternative processing methods for DBM
pluripotent stem cells (PSCs), including induced PSCs have further expanded its applications. For instance, Gai
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(iPSCs), periosteum-derived cells (PDCs), and embryonic et al. developed an innovative biomimetic matrix hydrogel
stem cells (ESCs). 126,127 These alternative cell sources offer by incorporating calcium phosphate oligomers into bone
distinct advantages in terms of scalability and patient- decellularized ECM, enabling continuous construction of
specific applications. The critical role of osteoclasts in bone bone organoids with vascularization and mineralization
remodeling processes has prompted innovative co-culture functions. At the same time, the Matrigel remains a widely
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approaches. Iordachescu et al. developed an advanced utilized commercial ECM derived from Engelbreth-Holm-
model system utilizing decellularized bovine femoral Swarm (EHS) mouse sarcoma. However, the high cost,
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cancellous bone scaffolds to support the 3D co-culture of undefined chemical nature, and significant batch-to-
osteoblasts and osteoclasts, thereby better mimicking the batch variability of EHS lead to poor reproducibility and
dynamic equilibrium of bone formation and resorption. limited application in the construction of bone organoids.
Osteocytes, representing approximately 90–95% of all bone Therefore, lots of organic bioactive materials, including
cells, serve as the primary mechanosensory cells responsible natural polymers such as silk fibroin, gelatin, chitosan, and
for maintaining bone homeostasis and orchestrating hyaluronic acid, as well as synthetic polymers including
remodeling processes in response to mechanical stimuli. polycaprolactone (PCL), polylactic acid, and polyethylene
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The growth and maintenance of osteocytes require long- glycol (PEG), have been developed for the construction of
term mechanical loading, which presents significant bone organoids. 123
technical challenges for utilizing organoids. To address 3D bioprinting has emerged as a transformative
this challenge, Zhang et al. fabricated a compression technology for bone organoid engineering, which offers
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bioreactor to deliver long-term mechanical loading to precise spatial control of cell-matrix deposition, enhanced
osteocyte bone organoid (Figure 3A-G). This technological structural complexity, and improved reproducibility. For
advancement represents a crucial step toward recreating the example, methacrylated gelatin (GelMA) microspheres
native osteocyte microenvironment in 3D culture systems. containing BMSCs were prepared using digital light
Despite these developments, the application of osteocytes processing 3D printing technology, and efficient bone
in bone organoid construction remains at an early stage of regeneration-like callus tissue was cultivated through
investigation. Several critical aspects, including mechanical chondrogenic induction and osteogenic differentiation.
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loading parameters, long-term maintenance of osteocyte Another study created self-mineralizing large-sized bone
networks, and standardized characterization protocols, still organoids by combining GelMA, alginate methacryloyl,
need further exploration. and hydroxyapatite (HA) in a bioprinting process,
Beyond cellular composition, the extracellular mimicking the complexity of the ECM and constructing
matrix (ECM) represents a fundamental component bone-like organs with similar functions and mechanical
in bone organoid engineering, serving both structural properties to natural bone tissue. Innovative in vivo
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and regulatory functions. The ECM not only provides approaches have also been developed, such as BMP-2-
essential support for cell adhesion, growth, proliferation, loaded scaffolds implanted in murine muscle pouches that
and differentiation but also plays a crucial role in the generate functional bone organoids capable of treating liver
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spatiotemporal control of organs. The ECM-derived injury. Genome editing technology, such as clustered
materials have emerged as particularly effective substrates regularly interspaced short palindromic repeats (CRISPR)
for accelerating bone formation. The earliest biomaterial and CRISPR-associated protein 9 (Cas9), also plays a
used in the design of bone organoids is demineralized critical role in the construction of bone tumor organoids.
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bone matrix (DBM), which is advantageous for its Gerardo-Ramirez et al. successfully established CD44-
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wide availability and low immunogenicity. Park et al. knockout OS organoids with validated genetic stability
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constructed a trabecular bone organoid to simulate local using CRISPR/Cas9 technology. Zhang et al. developed
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bone remodeling using a demineralized bone paper (DBP) OS organoid models using CRISPR/Cas9 technology
made from biomaterials, which highlights the potential and evaluated the combination therapy of protein kinase,
of ECM-based materials to recapitulate complex bone DNA-activated, catalytic subunit inhibitor, 7-methyl-
microenvironments in vitro (Figure 3H-J). In another 2-([7-methyltriazolo{1,5-a}pyridin-6-yl]amino)-9-
study, Iordachescu et al. utilized trabecular bone as (tetrahydro-2H-pyran-4-yl)-7,9-dihydro-8H-purin-8-one,
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scaffolds to co-culture osteoblasts and osteoclasts within with doxorubicin, pioneering novel experimental models

Volume 1 Issue 3 (2025) 7 doi: 10.36922/OR025280024