Immobilization can lead to intervertebral disc degeneration. The biomechanical characteristics of such discs have not so far been investigated at the micro- or nanoscale, the level at which cells sense and respond to the surrounding environment. This study aimed to characterize changes in the elastic modulus of the collagen fibrils in the nucleus pulposus at the nanoscale and correlate this with micro-biomechanical properties of the nucleus pulposus after immobilization, in addition to observation of tissue histology and its gene expressions. An immobilization system was used on the rat tail with an external fixation device. The elastic modulus was measured using both nano and micro probes for atomic force microscopy after 4 and 8 weeks of immobilization. Histology of the tissue was observed following hematoxylin and eosin staining. Gene expression in the annulus fibrosus tissue was quantified using real-time reverse transcription-polymerase chain reaction. The elastic modulus of the collagen fibrils in the nucleus pulposus at the nanoscale increased from 74.07 ± 17.06 MPa in the control to 90.06 ± 25.51 MPa after 8 weeks (P = 0.007), and from 33.51 ± 9.33 kPa to 43.18 ± 12.08 kPa at the microscale (P = 0.002). After immobilization for 8 weeks, a greater number of cells were observed by histology to be aggregated within the nucleus pulposus. Collagen II (P = 0.007) and aggrecan (P = 0.003) gene expression were downregulated whereas collagen I (P = 0.002), MMP-3 (P < 0.001), MMP-13 (P < 0.001) and ADAMTs-4 (P < 0.001) were upregulated. Immobilization not only influenced individual collagen fibrils at the nanoscale, but also altered the micro-biomechanics and cell response in the nucleus pulposus. These results suggest that significant changes occur in intervertebral discs at both scales after immobilization, a situation about which clinicians should be aware when immobilization has to be used clinically.

固定可能导致椎间盘退变。迄今为止，尚未在微米或纳米级研究这种盘的生物力学特征，即细胞对周围环境的感知和响应水平。这项研究旨在表征髓核中胶原纤维的弹性模量在纳米尺度上的变化，并将其与固定后髓核的微生物力学特性相关联，此外还观察组织组织学及其基因表达。固定系统用于带有外部固定装置的大鼠尾巴。固定4周和8周后，使用纳米探针和微型探针进行原子力显微镜测量弹性模量。苏木精和曙红染色后观察组织的组织学。使用实时逆转录-聚合酶链反应对纤维环组织中的基因表达进行定量。8周后，在纳米级的髓核中胶原纤维的弹性模量从对照组的74.07±17.06 MPa增加到90.06±25.51 MPa（P = 0.007），并且在微尺度下从33.51±9.33 kPa到43.18±12.08 kPa（P = 0.002）。固定8周后，通过组织学观察到大量细胞聚集在髓核内。胶原II（P = 0.007）和蛋白聚糖（P = 0.003）基因表达下调，而胶原I（P = 0.002），MMP-3（P <0.001），MMP-13（P <0.001）和ADAMTs-4（P<0.001）上调。固定化不仅影响纳米级的单个胶原纤维，而且改变了髓核中的微生物力学和细胞反应。这些结果表明，固定后两种尺寸椎间盘均发生显着变化，这是临床上必须在临床上使用固定时应注意的一种情况。