Black Phosphorus Nanosheets Incorporated Mesenchymal Stem Cell Spheroids for Accelerated Bone Regeneration

Scaffold-free spheroids constitute a promising therapeutic strategy for bone defect repair because of their tissue homology. However, applying mesenchymal stem cell (MSC) spheroids for bone defect repair remains challenging due to the lack of organ-specific functionality. Black phosphorus nanosheets (BPNS) were incorporated into MSC spheroids to enhance their bone regeneration capacity. Here, we propose a synergic bone microtissue construction strategy based on reprogramming MSCs with YAP1 gene overexpression, amplifying the osteoinductive effect of BPNS. Results demonstrated that either BPNS incorporation or YAP1 overexpression in MSC spheroids promoted osteogenesis and matrix formation in engineered bone microtissues. Furthermore, bone microtissues reprogrammed with synergistic YAP1 overexpression and incorporating BPNS could significantly enhance their osteogenic differentiation, further accelerating bone regeneration in rat calvarial defect models. Meanwhile, the reprogrammed bone microtissues established a bone-regenerative microenvironment by facilitating vascularization and macrophage polarization. In summary, these findings demonstrate that reprogrammed bone microtissues with YAP1 overexpression and BPNS incorporation within MSC spheroids amplified BPNS-mediated osteogenic differentiation and mineralization of MSC spheroids, indicating that the direct reprogramming of MSC spheroids may offer a promising strategy for further functionalizing spheroids for bone regeneration application.