Department of Immunology, Oslo University Hospital, Oslo, Norway.
Department of Physics, Norwegian University of Science and Technology, Trondheim, Norway.
Cartilage. 2021 Dec;13(1_suppl):1237S-1249S. doi: 10.1177/19476035211007923. Epub 2021 Apr 15.
Despite new strategies in tissue engineering, cartilage repair remains a major challenge. Our aim is to treat patients with focal lesions of articular cartilage with autologous hyaline cartilage implants using a scaffold-free approach. In this article, we describe experiments to optimize production of scaffold-free cartilage discs.
Articular chondrocytes were expanded , seeded in transwell inserts and redifferentiated using established chondrogenic components. Experimental variables included testing 2 different expansion media, adding bone morphogenetic protein 2 (BMP2), insulin-like growth factor 1 (IGF1), growth/differentiation factor 5 (GDF5), or fibroblast growth factor 18 (FGF18) to the differentiation medium and allowing the disc to float freely in large wells. Cartilage discs were analyzed by weight and thickness, real-time RT-qPCR (reverse transcriptase qualitative polymerase chain reaction), fluorescence immunostaining, transmission electron microscopy, second harmonic generation imaging, and measurement of Young's modulus.
Addition of BMP2 to the chondrogenic differentiation medium (CDM) was essential for stable disc formation, while IGF1, GDF5, and FGF18 were redundant. Allowing discs to float freely in CDM on a moving platform increased disc thickness compared with discs kept continuously in transwell inserts. Discs cultured for 6 weeks reached a thickness of almost 2 mm and Young's modulus of >200 kPa. There was abundant type II collagen. Collagen fibrils were 25 nm thick, with a tendency to be organized perpendicular to the disc surface.
Scaffold-free engineering using BMP2 and providing free movement in CDM produced firm, elastic cartilage discs with abundant type II collagen. This approach may potentially be used in clinical trials.
尽管在组织工程学方面有了新的策略，但软骨修复仍然是一个主要的挑战。我们的目标是使用无支架方法治疗患有关节软骨局灶性病变的患者。在本文中，我们描述了优化无支架软骨盘生产的实验。
关节软骨细胞经过扩增后，接种于 Transwell 插入物中，并使用已建立的软骨生成成分重新分化。实验变量包括测试 2 种不同的扩增培养基，向分化培养基中添加骨形态发生蛋白 2（BMP2）、胰岛素样生长因子 1（IGF1）、生长/分化因子 5（GDF5）或成纤维细胞生长因子 18（FGF18），以及让圆盘在大培养皿中自由漂浮。通过重量和厚度、实时 RT-qPCR（逆转录定量聚合酶链反应）、荧光免疫染色、透射电子显微镜、二次谐波成像以及杨氏模量测量来分析软骨盘。
向软骨分化培养基（CDM）中添加 BMP2 对于稳定的圆盘形成是必不可少的，而 IGF1、GDF5 和 FGF18 是多余的。让圆盘在 CDM 中自由漂浮在移动平台上，与将圆盘保持在 Transwell 插入物中连续培养相比，增加了圆盘的厚度。培养 6 周的圆盘厚度几乎达到 2 毫米，杨氏模量>200 kPa。有丰富的 II 型胶原蛋白。胶原纤维厚 25nm，有组织成垂直于盘表面的趋势。
使用 BMP2 进行无支架工程，并在 CDM 中提供自由运动，产生了坚固、有弹性的软骨盘，其中含有丰富的 II 型胶原蛋白。这种方法可能有潜力用于临床试验。
