Kronemberger Gabriela S, Spagnuolo Francesca D, Karam Aliaa S, Chattahy Kaoutar, Storey Kyle J, Kelly Daniel J
Trinity Centre for Biomedical Engineering, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland.
Department of Mechanical, Manufacturing and Biomedical Engineering, School of Engineering, Trinity College Dublin, Dublin, Ireland.
Tissue Eng Part C Methods. 2025 Jan;31(1):36-48. doi: 10.1089/ten.tec.2024.0309. Epub 2024 Dec 30.
Scaffold-free tissue engineering strategies using cellular aggregates, microtissues, or organoids as "biological building blocks" could potentially be used for the engineering of scaled-up articular cartilage or endochondral bone-forming grafts. Such approaches require large numbers of cells; however, little is known about how different chondrogenic growth factor stimulation regimes during cellular expansion and differentiation influence the capacity of cellular aggregates or microtissues to fuse and generate hyaline cartilage. In this study, human bone marrow mesenchymal stem/stromal cells (MSCs) were additionally stimulated with bone morphogenetic protein 2 (BMP-2) and/or transforming growth factor (TGF)-β1 during both monolayer expansion and subsequent chondrogenic differentiation in a microtissue format. MSCs displayed a higher proliferative potential when expanded in the presence of TGF-β1 or TGF-β1 and BMP-2. Next, the chondrogenic potential of these human MSCs was explored in a medium-high throughput microtissue system. After 3 weeks of culture, MSCs stimulated with BMP-2 during expansion and differentiation deposited higher levels of glycosaminoglycans (GAGs) and collagen, while staining negative for calcium deposits. The fusion capacity of the microtissues was not impacted by these different growth factor stimulation regimes. After 3 weeks of fusion, it was observed that MSCs stimulated with TGF-β1 during expansion and additionally with BMP-2 during chondrogenic differentiation deposited the highest levels of sulfated GAGs. No increase in type X collagen deposition was observed with additional growth factor stimulation. This study demonstrates the importance of carefully optimizing MSC expansion and differentiation conditions when developing modular tissue engineering strategies (e.g., cellular aggregates and microtissues) for cartilage tissue engineering applications.
使用细胞聚集体、微组织或类器官作为“生物构建块”的无支架组织工程策略可能潜在地用于扩大规模的关节软骨或软骨内骨形成移植物的工程化。这种方法需要大量细胞；然而，关于细胞扩增和分化过程中不同的软骨生成生长因子刺激方案如何影响细胞聚集体或微组织融合并生成透明软骨的能力，人们知之甚少。在本研究中，人骨髓间充质干/基质细胞（MSCs）在单层扩增和随后以微组织形式进行软骨生成分化过程中，额外用骨形态发生蛋白2（BMP - 2）和/或转化生长因子（TGF）-β1进行刺激。当在TGF -β1或TGF -β1与BMP - 2存在的情况下扩增时，MSCs表现出更高的增殖潜力。接下来，在中高通量微组织系统中探索这些人MSCs的软骨生成潜力。培养3周后，在扩增和分化过程中用BMP - 2刺激的MSCs沉积了更高水平的糖胺聚糖（GAGs）和胶原蛋白，同时钙沉积染色为阴性。这些不同的生长因子刺激方案并未影响微组织的融合能力。融合3周后，观察到在扩增过程中用TGF -β1刺激且在软骨生成分化过程中额外用BMP - 2刺激的MSCs沉积了最高水平的硫酸化GAGs。额外的生长因子刺激未观察到X型胶原蛋白沉积增加。本研究表明，在开发用于软骨组织工程应用的模块化组织工程策略（如细胞聚集体和微组织）时，仔细优化MSC扩增和分化条件的重要性。
