Anatomy@Edinburgh, Edinburgh Medical School, Biomedical Sciences, University of Edinburgh, Edinburgh, United Kingdom.
Centre for Discovery Brain Sciences, College of Medicine and Veterinary Medicine, University of Edinburgh, Edinburgh, United Kingdom.
Front Endocrinol (Lausanne). 2023 Dec 19;14:1308604. doi: 10.3389/fendo.2023.1308604. eCollection 2023.
Bioassembly techniques for the application of scaffold-free tissue engineering approaches have evolved in recent years toward producing larger tissue equivalents that structurally and functionally mimic native tissues. This study aims to upscale a 3-dimensional bone model through bioassembly of differentiated rat osteoblast (dROb) spheroids with the potential to develop and mature into a bone macrotissue.
dROb spheroids in control and mineralization media at different seeding densities (1 × 10, 5 × 10, and 1 × 10 cells) were assessed for cell proliferation and viability by trypan blue staining, for necrotic core by hematoxylin and eosin staining, and for extracellular calcium by Alizarin red and Von Kossa staining. Then, a novel approach was developed to bioassemble dROb spheroids in pillar array supports using a customized bioassembly system. Pillar array supports were custom-designed and printed using Formlabs Clear Resin by Formlabs Form2 printer. These supports were used as temporary frameworks for spheroid bioassembly until fusion occurred. Supports were then removed to allow scaffold-free growth and maturation of fused spheroids. Morphological and molecular analyses were performed to understand their structural and functional aspects.
Spheroids of all seeding densities proliferated till day 14, and mineralization began with the cessation of proliferation. Necrotic core size increased over time with increased spheroid size. After the bioassembly of spheroids, the morphological assessment revealed the fusion of spheroids over time into a single macrotissue of more than 2.5 mm in size with mineral formation. Molecular assessment at different time points revealed osteogenic maturation based on the presence of osteocalcin, downregulation of Runx2 ( < 0.001), and upregulated alkaline phosphatase ( < 0.01).
With the novel bioassembly approach used here, 3D bone macrotissues were successfully fabricated which mimicked physiological osteogenesis both morphologically and molecularly. This biofabrication approach has potential applications in bone tissue engineering, contributing to research related to osteoporosis and other recurrent bone ailments.
近年来，用于无支架组织工程方法的生物组装技术已经发展到可以生产出更大的组织等效物，这些组织等效物在结构和功能上模仿天然组织。本研究旨在通过生物组装分化的大鼠成骨细胞（dROb）球体来放大 3D 骨模型，这些球体有可能发育和成熟为骨大组织。
在不同的接种密度（1×10、5×10 和 1×10 细胞）下，用台盼蓝染色法评估控制和矿化培养基中的 dROb 球体的细胞增殖和活力，用苏木精和伊红染色法评估坏死核心，用茜素红和 Von Kossa 染色法评估细胞外钙。然后，开发了一种新的方法，使用定制的生物组装系统在柱阵列支架中生物组装 dROb 球体。柱阵列支架由 Formlabs 通过 Formlabs Form2 打印机使用 Formlabs Clear Resin 定制设计和打印。这些支架被用作球体生物组装的临时框架，直到融合发生。然后去除支架，允许融合的球体无支架生长和成熟。进行形态和分子分析以了解它们的结构和功能方面。
所有接种密度的球体都增殖到第 14 天，并且在增殖停止时开始矿化。随着球体尺寸的增加，坏死核心的尺寸随时间增加。在球体生物组装后，形态学评估显示随着时间的推移，球体融合成一个超过 2.5 毫米大小的单个大组织，并有矿化形成。在不同时间点的分子评估显示，基于骨钙蛋白的存在、Runx2 的下调（<0.001）和碱性磷酸酶的上调（<0.01），存在成骨成熟。
使用这里使用的新型生物组装方法，成功制造了 3D 骨大组织，在形态和分子上都模拟了生理成骨。这种生物制造方法在骨组织工程中有潜在的应用，为骨质疏松症和其他复发性骨疾病的研究做出了贡献。
