Development of ECM-inspired supramolecular cryogels with innate mineralization and compression-resistance for 3D culture of mini-bone trabeculae tissue analogs

Bone microtissue grafts mimicking skeletal features and organogenesis are an emerging strategy, different from the traditional tissue engineered bone grafts using the 3D cell encapsulation and the top cell seeding, and remain challenging in regenerative medicine and drug discovery, because the existing scaffold-free and microcarrier-based microtissue systems are difficult to manipulate the microtissue morphologies towards native bone microstructures limited by their mechanical weakness and the absence of interconnected inner cavities. Herein, we synthesized a supramolecular cryogel by an ice-templated freezing-polymerization process, acquiring a promising microcarrier resembling native bone trabecular morphology for 3D culture of trabecular bone microtissue grafts. In our strategy, a macromolecular chitosan monomer and two supramolecular monomers including glycinamide and phytic acid constituted the supramolecular cryogel, the modification using glycinamide and phytic acid components enables the cryogel microcarrier with porous cavities and compression-resistant abilities like the native trabecular bone tissues. Moreover, the mineralization of the cryogel microcarriers was also improved by the modification of phytic acid monomers, consequently strengthening osteogenic differentiation of bone-marrow-derived mesenchymal stem cells (BMSCs) and in vitro microtissue biomineralization. The in vivo results also revealed that a trabeculae-like bone microtissue forms on the cryogel microcarriers, with vessel invasion into inner cavities of the trabecular bone microtissues. After in situ implantation of the prepared trabecular bone microtissues, the bone regeneration characterized by raising bone mineral density and remodeling bone trabecular microstructures was observed on a rat femur condyle defect model. Last but not least, we also discovered the intestinal bacterial communities and compositions are closely related to the bone regeneration after implantation of the engineered bone microtissue grafts, which is the first evidence focusing on the intestinal microbiota response to the bone injury and bone regeneration events representing a feasible approach to bone regeneration examination. In brief, the supramolecular cryogels we developed in this study have been proved to be a promising microcarrier for the construction and 3D culture of trabecular bone microtissues, and this work offers a novelty insight into microtissue engineering and bone regeneration.