A Novel Strategy for Preparation of ADSCs ‐Osteogenic Microtissue Based on CEFFE and Mechanisms for Enhancing Osteogenic Activity

ABSTRACT Adipose‐derived mesenchymal stem cells (ADSCs) are more accessible than bone marrow mesenchymal stromal cells but have limited osteogenic differentiation capabilities in bone tissue engineering. Cell‐free fat extract (CEFFE) from adipose tissue shows promise in enhancing the osteogenesis of ADSCs. This study aimed to investigate the impact of CEFFE on ADSCs and its role in promoting ADSC osteogenic activity both in vitro and in vivo. CEFFE was obtained and characterized. In vitro, ADSCs were isolated, characterized, and analyzed using various assays. Osteogenic differentiation was quantified through staining and gene expression analysis. ADSC‐based osteogenic microtissues with CEFFE were created and tested in nude mice models. Molecular mechanisms were explored through transcriptomic analysis and western blotting. CEFFE enhanced various osteogenic aspects of ADSCs, including proliferation, calcium nodule formation, and expression of osteogenic markers. CEFFE‐treated ADSCs showed early dense osteogenic cell sheet formation and increased ectopic bone volume in mice. Transcriptomic analysis revealed upregulation/downregulation of genes and activation of the PI3K‐Akt signaling pathway by CEFFE. Western blotting confirmed that the PI3K‐Akt pathway played a crucial role in CEFFE‐induced acceleration of osteogenesis in ADSCs. The study confirms CEFFE's significant enhancement of ADSCs osteogenic potential and sheds light on the underlying mechanisms. These findings provide a preclinical foundation for potential applications in bone tissue engineering and regenerative medicine.