Legerlotz Kirsten, Dorn Jonas, Richter Jens, Rausch Martin, Leupin Olivier
Musculoskeletal Disease Area, Novartis Institutes for Biomedical Research, Basel, Switzerland.
Development, Novartis Pharma AG, Basel, Switzerland.
Acta Biomater. 2014 Oct;10(10):4447-55. doi: 10.1016/j.actbio.2014.05.029. Epub 2014 Jun 4.
The black-and-white patterning of tendon fascicles when visualized by light microscopy, also known as crimp, is a well-known feature of fiber-forming collagens. However, not much is known about its development, function and response to strain. The objective of this study is to investigate the interaction of tenocyte and crimp morphology as well as their changes with increasing age and acute strain. In contrast to previous studies, which used indirect measures, such as polarized light, to investigate the crimp structure, this study visualizes internal crimp structure in three dimensions without freezing, sectioning, staining or fixing the tissue, via two-photon imaging of green fluorescent protein expressing cells within mouse tail tendon fascicles. This technique further allows straining of the live tissue while visualizing changes in crimp morphology and cell shape with increasing specimen length. Combining this novel microscopy technique with computational image and data analysis revealed a complex relationship between tenocytes and the extracellular matrix that evolves with increasing age. While the reduction of crimping with strain was observed as expected, most of the crimps were gone at 0-1% strain already. Even relatively low strains of 3% led to pronounced changes in the crimp structure after relaxation, particularly in the young animals, which could not be seen with bright-field imaging. Cell length and gap width increased with strain. However, while the cells were able to return to their original length even after high strains of 6%, the gaps between the cells widened, which may imply modified cell-cell communication after overstretching.
在光学显微镜下观察到的肌腱束黑白相间的图案，也称为卷曲，是成纤维胶原的一个众所周知的特征。然而，关于其发育、功能以及对应变的反应，人们了解得并不多。本研究的目的是调查腱细胞与卷曲形态之间的相互作用，以及它们随年龄增长和急性应变的变化。与以往使用偏振光等间接测量方法来研究卷曲结构的研究不同，本研究通过对小鼠尾腱束中表达绿色荧光蛋白的细胞进行双光子成像，在不冷冻、切片、染色或固定组织的情况下，三维可视化内部卷曲结构。该技术还允许在对活组织施加应变的同时，观察卷曲形态和细胞形状随标本长度增加的变化。将这种新颖的显微镜技术与计算图像和数据分析相结合，揭示了腱细胞与细胞外基质之间随着年龄增长而演变的复杂关系。正如预期的那样，观察到卷曲随着应变而减少，但在0-1%的应变下，大多数卷曲已经消失。即使是3%的相对低应变，在松弛后也会导致卷曲结构发生明显变化，尤其是在幼小动物中，这在明场成像中是看不到的。细胞长度和间隙宽度随应变增加。然而，即使在6%的高应变后，细胞仍能恢复到原来的长度，但细胞间的间隙变宽了，这可能意味着过度拉伸后细胞间通讯发生了改变。
