A Novel Strategy for Preparation of ADSCs-Osteogenic Microtissues Based on CEFFE and Mechanisms for Enhancing Osteogenic Activity
Bone tissue engineering is considered the optimal solution for the repair of large bone defects. Previous studies by our team have demonstrated the effectiveness of a "bottom-up" microtissue engineering strategy using bone marrow mesenchymal stem cells (BMSCs) to improve the utilization efficiency of seed cells. However, this strategy requires the extraction of bone marrow, which is associated with significant trauma and difficulties in obtaining the cells. Adipose-derived mesenchymal stem cells (ADSCs), obtained from aspirated adipose tissue, offer a less invasive and more accessible alternative, but their osteogenic capacity is noticeably weaker, severely limiting their application as seed cells in bone tissue engineering repair.Cell-free fat extract (CEFFE) is a cell-free liquid component extracted from adipose tissue, which exhibits beneficial properties such as promoting proliferation, antioxidation, anti-apoptosis, and angiogenesis. In this study, during the preparation of ADSC-based osteogenic microtissues, we first discovered that CEFFE significantly enhanced the osteogenic activity of ADSCs. Furthermore, we observed the formation of dense osteogenic membranes as early as day 7 of osteogenic induction. When the microtissues based on CEFFE-treated ADSCs were implanted subcutaneously in nude mice, we found a significant enhancement in their osteogenic activity. Finally, to further investigate the mechanism underlying the enhancement of ADSC osteogenic differentiation by CEFFE, we performed transcriptomic analysis of CEFFE-treated ADSCs and validated the activation of the key PI3K-Akt pathway using Western blotting.In conclusion, this study presents a novel approach for the preparation of ADSC-based osteogenic microtissues using CEFFE and provides insights into the mechanisms underlying the enhancement of osteogenic activity. These findings hold significant implications for the field of bone tissue engineering and offer potential advancements in the clinical translation of ADSC-based therapies. Overall design: In this study, we aimed to investigate the effects of CEFFE on ADSCs and its role in enhancing osteogenic activity. Firstly, we obtained ADSCs and CEFFE and validated their characteristics. Subsequently, we examined the changes in proliferation and differentiation abilities of ADSCs when exposed to CEFFE during the osteogenic induction process. Furthermore, we utilized CEFFE to enhance the osteogenic activity of ADSCs during the preparation of osteogenic microtissues, resulting in the formation of denser osteogenic membranes as early as day 7. These osteogenic microtissues were then implanted subcutaneously in nude mice, and the specimens were evaluated through macroscopic observation, micro-CT imaging, and histological staining. Finally, transcriptomic analysis was conducted on ADSCs treated with CEFFE to investigate its effects, and the main pathways were validated using Western blotting (WB) analysis.