Despite the widespread use of cell spheroids in tissue engineering for the regeneration of large tissues and organs and for high-throughput screening in pharmacology and toxicology, the clinical challenges include cellular heterogeneity, low structural stability, and uncontrolled cell differentiation. Using human bone marrow-derived mesenchymal stem cells (hBMSCs), we developed cell spheroids containing bioactive molecule (bone morphogenetic protein-2, BMP-2)-immobilized polycaprolactone (PCL) particles with a leaf-stacked structure (LSS). The LSS particles were fabricated via simple heating-cooling method, and the BMP-2 was continuously released from LSS particles for 19 days. Based on in vitro and in vivo observations of the cell spheroids, we found that (i) the porous LSS particles prevent cellular heterogeneity via sufficient diffusion of oxygen/nutrients, (ii) the cell adhesive surface on LSS particles improved the structural stability, (iii) the BMP-2 released from LSS particles induced effective osteogenic differentiation of stem cells, and (iv) the BMP-2-immobilized LSS particles induced new bone formation. Therefore, the cell spheroid containing bioactive molecule-immobilized LSS particles represent a potential strategy to overcome the inherent limitations of conventional cell spheroids. We further suggest that the cell spheroid containing bioactive molecule-immobilized LSS particles is an elegant platform for the regeneration of various tissues and organs as well as high-throughput screening in pharmacology and toxicology studies.

尽管细胞球体在组织工程中广泛用于大组织和器官的再生以及药理学和毒理学的高通量筛选，但临床挑战包括细胞异质性、低结构稳定性和不受控制的细胞分化。使用人骨髓间充质干细胞 ( h BMSC)，我们开发了含有生物活性分子（骨形态发生蛋白-2，BMP-2）-固定化聚己内酯（PCL）颗粒的细胞球体，具有叶堆叠结构（LSS）。LSS颗粒通过简单的加热-冷却方法制备，BMP-2从LSS颗粒中连续释放19天。基于体外和体内对细胞球体的观察，我们发现 (i) 多孔 LSS 颗粒通过氧气/营养物质的充分扩散防止细胞异质性，(ii) LSS 颗粒上的细胞粘附表面提高了结构稳定性，(iii) BMP-2 释放来自 LSS 颗粒诱导干细胞的有效成骨分化，和 (iv) BMP-2 固定化 LSS 颗粒诱导新骨形成。因此，含有生物活性分子固定化 LSS 颗粒的细胞球体代表了一种克服传统细胞球体固有局限性的潜在策略。我们进一步建议，含有生物活性分子固定化 LSS 颗粒的细胞球体是用于各种组织和器官再生以及药理学和毒理学研究中的高通量筛选的优雅平台。