Spagnuolo Francesca D, Kronemberger Gabriela S, 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.
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; Department of Anatomy and Regenerative Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland; Advanced Materials and Bioengineering Research Centre (AMBER), Royal College of Surgeons in Ireland and Trinity College Dublin, Dublin, Ireland.
Acta Biomater. 2025 Mar 1;194:109-121. doi: 10.1016/j.actbio.2025.01.024. Epub 2025 Jan 14.
Functional cartilaginous tissues can potentially be engineered by bringing together numerous microtissues (µTs) and allowing them to fuse and re-organize into larger, structurally organized grafts. The maturation level of individual microtissues is known to influence their capacity to fuse, however its impact on the long-term development of the resulting tissue remains unclear. The first objective of this study was to investigate the influence of the maturation state of human bone-marrow mesenchymal stem/stromal cells (hBM-MSCSs) derived microtissues on their fusion capacity and the phenotype of the final engineered tissue. Less mature (day 2) cartilage microtissues were found to fuse faster, supporting the development of a matrix that was richer in sulphated glycosaminoglycans (sGAG) and collagen, while low in calcium deposits. This enhanced fusion in less mature microtissues correlated with enhanced expression of N-cadherin, followed by a progressive increase in markers associated with cell-extracellular matrix (ECM) interactions. We then engineered larger constructs with varying initial numbers (50, 150 or 300 µTs per well) of less mature microtissues, observing enhanced sGAG synthesis with increased microtissue density. We finally sought to engineer a scaled-up cartilage graft by fusing 4,000 microtissues and maintaining the resulting constructs under either dynamic or static culture conditions. Robust and reliable fusion was observed between microtissues at this scale, with no clear benefit of dynamic culture on the levels of matrix accumulation or the tensile modulus of the resulting construct. These results support the use of BM-MSCs derived microtissues for the development of large-scale, engineered functional cartilaginous grafts. STATEMENT OF SIGNIFICANCE: Microtissues are gaining attention for their use as biological building blocks in the field of tissue engineering. The fusion of multiple microtissues is crucial for achieving a cohesive engineered tissue of scale, however the impact of their maturation level on the long-term properties of the engineered graft is poorly understood. This paper emphasizes the importance of using less mature cartilage microtissues for supporting appropriate cell-cell interactions and robust chondrogenesis in vitro. We demonstrate that tissue development is not negatively impacted by increasing the initial numbers of microtissues within the graft. This biofabrication strategy has significant translation potential, as it enables the engineering of scaled-up cartilage grafts of clinically relevant sizes using bone marrow derived MSCs.
通过将大量微组织(µTs)聚集在一起,并使其融合和重新组织成更大的、结构有序的移植物,有可能构建出功能性软骨组织。已知单个微组织的成熟水平会影响它们的融合能力,然而其对最终形成的组织的长期发育的影响仍不清楚。本研究的首要目标是调查源自人骨髓间充质干细胞(hBM-MSCSs)的微组织的成熟状态对其融合能力以及最终工程化组织表型的影响。发现不太成熟(第2天)的软骨微组织融合得更快,这有助于形成富含硫酸化糖胺聚糖(sGAG)和胶原蛋白、而钙沉积较少的基质。不太成熟的微组织中这种增强的融合与N-钙黏蛋白表达的增强相关,随后与细胞-细胞外基质(ECM)相互作用相关的标志物逐渐增加。然后,我们用不同初始数量(每孔50、150或300个µTs)的不太成熟的微组织构建了更大的结构,观察到随着微组织密度的增加,sGAG合成增强。我们最终试图通过融合4000个微组织并将得到的结构维持在动态或静态培养条件下,来构建放大的软骨移植物。在这个规模下,微组织之间观察到了强大且可靠的融合,动态培养对所得结构的基质积累水平或拉伸模量没有明显益处。这些结果支持使用源自骨髓间充质干细胞的微组织来开发大规模的工程化功能性软骨移植物。重要性声明:微组织作为组织工程领域的生物构建模块正受到关注。多个微组织的融合对于实现具有一定规模的凝聚性工程化组织至关重要,然而人们对它们的成熟水平对工程化移植物长期特性的影响了解甚少。本文强调了使用不太成熟的软骨微组织来支持体外适当的细胞-细胞相互作用和强大的软骨形成的重要性。我们证明,增加移植物内微组织的初始数量不会对组织发育产生负面影响。这种生物制造策略具有显著的转化潜力,因为它能够使用源自骨髓的间充质干细胞构建临床相关尺寸的放大软骨移植物。
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