Nano and micro biomechanical alterations of annulus fibrosus after in situ immobilization revealed by atomic force microscopy.

Annulus fibrosus is critical to bear loads and resist fluid flow in the intervertebral disc. However, the detailed biomechanical mechanism of annulus fibrosus under abnormal loading is still ambiguous, especially at the micro and nano scales. This study aims to characterize the alterations of modulus at the nano scale of individual collagen fibrils in annulus fibrosus after in-situ immobilization, and the corresponding micro-biomechanics of annulus fibrosus. An immobilization model was used on the rat tail with an external fixation device. The elastic modulus of annulus fibrosus at both the nano- and micro-scale was examined using atomic force microscopy after fixation for 4 and 8 weeks, respectively. The fibrils in inner layer showed an alteration in elastic modulus from 91.38 ± 20.19 MPa in the intact annulus fibrosus to 110.64 ± 15.58 MPa (p < 0.001) at the nano scale after immobilization for 8 weeks, while the corresponding modulus at the micro scale also underwent a change from 0.33 ± 0.04 MPa to 0.47 ± 0.04 MPa (p < 0.001). The fibril disorder after immobilization was observed by hematoxylin/eosin staining. The gene expression of annulus fibrosus was also measured by real-time reverse transcription-polymerase chain reaction, which showed the upregulation of collagen II (p = 0.003) after immobilization. The results indicated that the immobilization not only influenced the individual fibril at the nanoscale, but also the micro-biomechanical property of annulus fibrosus which is critical to define the cell response to surrounding biomechanical environment. These alterations may also lead to the change in the mechanical property of the whole disc and the load-bearing function.