Osteogenic Activity of ADSCs Enhanced by CEFFE and a Novel Strategy for ADSCs-Osteogenic Microtissue

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.

骨组织工程被认为是修复大体积骨缺损的最优方案。本团队既往研究证实，采用骨髓间充质干细胞（Bone Marrow Mesenchymal Stem Cells, BMSCs）的“自下而上”微组织工程策略可提升种子细胞的利用效率。然而该策略需提取骨髓，伴随显著创伤且细胞获取难度较大。脂肪来源间充质干细胞（Adipose-derived Mesenchymal Stem Cells, ADSCs）可从抽吸获取的脂肪组织中分离，是一种创伤更小、更易获取的替代方案，但其成骨能力显著较弱，严重限制了其作为种子细胞在骨组织工程修复中的应用。无细胞脂肪提取物（Cell-free Fat Extract, CEFFE）是从脂肪组织中提取的无细胞液态组分，具备促进增殖、抗氧化、抗凋亡及促血管生成等有益特性。本研究在制备基于ADSCs的成骨性微组织过程中，首次发现CEFFE可显著增强ADSCs的成骨活性。此外，我们在成骨诱导第7天即可观察到致密成骨膜的形成。将经CEFFE预处理的ADSCs所构建的微组织植入裸鼠皮下后，我们发现其成骨活性显著提升。最后，为深入探究CEFFE增强ADSCs成骨分化的潜在机制，我们对经CEFFE处理的ADSCs开展了转录组学分析，并通过蛋白质印迹（Western blotting）验证了关键PI3K-Akt通路的激活。综上，本研究提出了一种利用CEFFE制备基于ADSCs的成骨性微组织的新方法，并阐明了其增强成骨活性的潜在机制。该研究成果对骨组织工程领域具有重要意义，同时为基于ADSCs的治疗方案的临床转化提供了潜在的技术突破。