Construction of growth plate organoids via a layered induction based in vitro 3D cultivation system.

Serious injury to the growth plate often leads to bony bridge formation, resulting in halted long bone growth, angular deformities, and limb length discrepancies. These problems persist unaddressed in the clinic. In this study, we engineered a four-layered growth plate organoid by integrating three-dimensional culture with layer-specific induction techniques. A gelatin/alginate hydrogel scaffold was utilized to recapitulate the architecture of the native growth plate. In the three cartilage zones, bone marrow derived mesenchymal stem cells (BMSCs) and chondrocytes were co-cultured at a 3:1 ratio and directed toward chondrogenesis with gradient concentrations of TGF-β3, resulting in cartilage tissue similar to the native growth plate. In the calcified zone, BMP-2 directed BMSCs toward mineralization. Scanning electron microscopy (SEM) and energy-dispersive spectroscopy (EDS) were used to examine the microstructure of the gelatin/alginate hydrogel. Cell-based assays further confirmed the biocompatibility of the 3D culture system. A series of chondrogenic and osteogenic assays validated the successful formation of the organoid. In conclusion, by emulating the growth plate's distinct four-layered organization within a stratified hydrogel and applying targeted differentiation cues, we have established a highly biomimetic in vitro growth plate organoid. This model offers a novel platform for studying growth plate mechanisms and developing potential therapeutic strategies.