Liu Hao, Scherpe Lynn, Hummer Linnea B, Snedeker Jess Gerrit, Zenobi-Wong Marcy
Tissue Engineering + Biofabrication Laboratory, Department of Health Sciences & Technology, ETH Zürich, Otto-Stern-Weg 7, 8093 Zürich, Switzerland.
Laboratory for Orthopedic Biomechanics, Department of Health Sciences & Technology, ETH Zürich, Lengghalde 5, 8008 Zürich, Switzerland.
Biofabrication. 2025 Apr 28;17(3). doi: 10.1088/1758-5090/adce35.
One hallmark of healthy tendon tissue is the high confinement of tenocytes between tightly packed, highly aligned collagen fibers. During tendinopathy, this organization becomes dysregulated, leading to cells with round-shaped morphology and collagen fibers which exhibit crimping and misalignment. The elongated nuclei in healthy tendons are linked to matrix homeostasis through distinct mechanotransduction pathways, and it is believed that the loss of nuclear confinement could upregulate genes associated with abnormal matrix remodeling. Replicating the cell and nuclear morphology of healthy and diseased states of tendon, however, remains a significant challenge for engineeredtendon models. Here we report on a high throughput biofabrication of mini-tendons that mimick the tendon core compartment based on the filamented light (FLight) approach. Each mini-tendon, with a length of 4 mm, was composed of parallel hydrogel microfilaments (2-5m diameter) and microchannels (2-10m diameter) that confined the cells. We generated four distinct matrices with varying stiffness (7-40 kPa) and microchannel dimensions. After 14 d of culture, 29% of tenocytes in the softest matrix with the largest microchannel diameter were aligned, exhibiting an average nuclear aspect ratio (nAR) of 2.1. In contrast, 84% of tenocytes in the stiffest matrix with the smallest microchannel diameter were highly aligned, with a mean nAR of 3.4. When tenocytes were culturedthe FLight hydrogels (2D) as opposed to within the hydrogels three-dimensional (3D), the mean nAR was less than 1.9, indicating that nuclear morphology is significantly more confined in 3D environments. By tuning the stiffness and microarchitecture of the FLight matrix, we demonstrated that mechanical confinement can be modulated to exert control over the extent of nuclear confinement. This high-throughput, tunable platform offers a promising approach for studying the mechanobiology of healthy and diseased tendons and for eventual testing of drug compounds against tendinopathy.
健康肌腱组织的一个标志是，肌腱细胞高度局限于紧密排列、高度对齐的胶原纤维之间。在肌腱病期间，这种组织结构会失调，导致细胞呈圆形形态，胶原纤维出现卷曲和排列紊乱。健康肌腱中细长的细胞核通过独特的机械转导途径与基质稳态相关联，据信核局限性的丧失可能会上调与异常基质重塑相关的基因。然而，复制肌腱健康和疾病状态下的细胞及细胞核形态，对于工程化肌腱模型来说仍然是一项重大挑战。在此，我们报告一种基于丝状光（FLight）方法的、模拟肌腱核心区室的微型肌腱高通量生物制造技术。每个长度为4毫米的微型肌腱由平行的水凝胶微丝（直径2 - 5微米）和微通道（直径2 - 10微米）组成，这些微丝和微通道限制着细胞。我们生成了四种具有不同刚度（7 - 40千帕）和微通道尺寸的独特基质。培养14天后，在微通道直径最大的最软基质中，29%的肌腱细胞排列整齐，平均核纵横比（nAR）为2.1。相比之下，在微通道直径最小的最硬基质中，84%的肌腱细胞高度对齐，平均nAR为3.4。当肌腱细胞在FLight水凝胶（二维）中培养，而不是在三维水凝胶中培养时，平均nAR小于1.9，这表明在三维环境中细胞核形态的局限性明显更大。通过调整FLight基质的刚度和微结构，我们证明可以调节机械局限性，以控制核局限性的程度。这个高通量、可调节的平台为研究健康和患病肌腱的力学生物学以及最终测试针对肌腱病的药物化合物提供了一种很有前景的方法。
