Endochondral ossification in a capsule

Abstract

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  Liquefied microcapsules enable multicellular interactions and self-organization
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  Key early events of endochondral ossification are recapitulated in vitro
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  Chondrocyte hypertrophy and bone-like matrix mineralization develop in microcapsules
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  Endothelial cells are retained within developing constructs

Traditional bone tissue engineering approaches have primarily focused on intramembranous ossification, which fails to fully capture the complexity of native bone formation. Endochondral ossification-based strategies offer a more biomimetic route, but replicating early cartilage-to-bone transitions in vitro remains a challenge. Here, we present a compartmentalized in vitro model of early endochondral ossification that integrates chondrogenically primed microtemplates with mesenchymal stem/stromal and endothelial cells within liquefied microcapsules. These liquefied microcapsules create a confined yet permissive microenvironment that enables dynamic cell rearrangement and multicellular interactions while maintaining construct integrity during culture. This design supports multicellular self-organization and the sequential progression of hallmark endochondral events, including matrix mineralization, while maintaining endothelial cell retention within the developing constructs. This modularity allows incorporation of bioactive signals, modulation of mechanical and chemical cues, and investigation of spatiotemporal regulation of endochondral ossification. Together, these features establish a versatile platform for studying skeletal development, mechanobiology, and multicellular interactions during endochondral ossification. In the longer term, the system may facilitate disease modeling, drug screening, and the development of donor-matched in vitro bone microtissues, thereby bridging developmental biology and translational bone tissue engineering.

Bone tissue engineering has traditionally focused on intramembranous ossification, whereas endochondral strategies remain less explored despite recapitulating the developmental pathway of most long bones. Here, we developed an in vitro model of early endochondral ossification by co-encapsulating cartilaginous microtemplates, umbilical cord-derived mesenchymal stem/stromal cells, and human umbilical vein endothelial cells within liquefied microcapsules. The semipermeable membrane maintained integrity during dynamic culture while permitting nutrient and oxygen exchange, whereas the liquefied core enabled cell migration, rearrangement, and matrix remodeling. This permissive three-dimensional environment promoted multicellular interactions and self-organization reminiscent of native tissues. The system supported sequential progression of hallmark events, including chondrogenic differentiation, chondrocyte hypertrophy, and early matrix mineralization while supporting endothelial cell retention and organization within the developing constructs. By enabling controlled recapitulation of these spatial and temporal events in vitro, this platform may advance developmental studies, disease modeling, and rational design of endochondral bone tissue engineering strategies.

Bone tissue engineering has traditionally focused on intramembranous ossification, whereas endochondral strategies remain less explored despite recapitulating the developmental pathway of most long bones. Here, we developed an in vitro model of early endochondral ossification by co-encapsulating cartilaginous microtemplates, umbilical cord-derived mesenchymal stem/stromal cells, and human umbilical vein endothelial cells within liquefied microcapsules. The semipermeable membrane maintained integrity during dynamic culture while permitting nutrient and oxygen exchange, whereas the liquefied core enabled cell migration, rearrangement, and matrix remodeling. This permissive three-dimensional environment promoted multicellular interactions and self-organization reminiscent of native tissues. The system supported sequential progression of hallmark events, including chondrogenic differentiation, chondrocyte hypertrophy, and early matrix mineralization while supporting endothelial cell retention and organization within the developing constructs. By enabling controlled recapitulation of these spatial and temporal events in vitro, this platform may advance developmental studies, disease modeling, and rational design of endochondral bone tissue engineering strategies.

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