由于难以诱导间充质干细胞（MSCs）空间、定向和可控地分化成软骨和骨分区，因此分级骨软骨单位的再生具有挑战性。新兴的器官样技术为骨软骨再生提供了新的机遇。在本研究中，我们开发了基于明胶的微小冰冻凝胶，并分别使用透明质酸（HA）和羟基磷灰石（HYP）进行定制，以通过在体内自组装成骨软骨器官（分别表示为CH-Microcryogels和OS-Microcryogels）诱导软骨和骨再生。 定制的微型冰冻凝胶显示出良好的细胞相容性，诱导 MSCs 的软骨和骨形成分化，并展示了自组装成双相软骨-骨结构中不脱层的骨软骨器官的能力。 mRNA-seq分析表明，CH-Microcryogels促进了软骨形成分化并抑制了炎症，而OS-Microcryogels通过调节特定的信号通路促进了骨形成分化并抑制了免疫反应。最后，在犬骨软骨缺损的体内移植预分化的定制微冰冻凝胶导致软骨和下软骨骨的同时再生。总之，利用量身定制的微冰冻凝胶生成自组装骨软骨器官的新方法，为组织工程领域的进展提供了高度有前途的道路。©2023年作者。

The regeneration of hierarchical osteochondral units is challenging due to difficulties in inducing spatial, directional and controllable differentiation of mesenchymal stem cells (MSCs) into cartilage and bone compartments. Emerging organoid technology offers new opportunities for osteochondral regeneration. In this study, we developed gelatin-based microcryogels customized using hyaluronic acid (HA) and hydroxyapatite (HYP), respectively for inducing cartilage and bone regeneration (denoted as CH-Microcryogels and OS-Microcryogels) through in vivo self-assembly into osteochondral organoids. The customized microcryogels showed good cytocompatibility and induced chondrogenic and osteogenic differentiation of MSCs, while also demonstrating the ability to self-assemble into osteochondral organoids with no delamination in the biphasic cartilage-bone structure. Analysis by mRNA-seq showed that CH-Microcryogels promoted chondrogenic differentiation and inhibited inflammation, while OS-Microcryogels facilitated osteogenic differentiation and suppressed the immune response, by regulating specific signaling pathways. Finally, the in vivo engraftment of pre-differentiated customized microcryogels into canine osteochondral defects resulted in the spontaneous assembly of an osteochondral unit, inducing simultaneous regeneration of both articular cartilage and subchondral bone. In conclusion, this novel approach for generating self-assembling osteochondral organoids utilizing tailor-made microcryogels presents a highly promising avenue for advancing the field of tissue engineering.© 2023 The Authors.