A Comparison Between The Influence Of Needle Puncture And Abnormal Mechanical Compression To Annulus Fibrosus: A Preliminary Study At Nano-scale

Objective: The purpose of this study was to establish modified rat disc degeneration model to explore the alteration of collagen fibrils of annulus fibrosus(AF)induced by needle puncture and abnormal mechanical loading on AF at nano-scale.Methods:14 fully grown SD male rat were chosen in this study and the coccygeal vertebral discs from Co5-6 to Co9-Co10 were located(digital palpation)respectively under anesthesia.The Co6-Co7 intervertebral disc was penetrated with a 20 G needle to the center of Nucleus Pulposus(NP)(5mm in depth).Than the coccygeal vertebras(Co7-Co10)were drilled percutanously by four parallel K-wires and the wires were fixed with self-made braces to make Co8-Co9 under a constant compression environment.All rats were examined with Magnetic resonance imaging(MRI)4 weeks post-surgery and evaluated with MRI index.Then the 14 rats were randomly divided into two parts.Histological examination was performed in one part and the remaining samples were harvested integrally with scapel for frozen section and atomic force microscopy(AFM)test to image the tissue surface and calculate the elastic modulus(EM)of individual collagen fibrils.Results: Significant NP dehydration shown in MRI,remarkable fibril disorder and a decrease in cell number within the intervertebral disc(IVD)in histological analysis indicated the initiation of IVDD in abnormal mechanical loading group and needle puncture group.Individual collagen fibrils in out layers of AF were stiffer than those in the inner layers in all groups.Compared to the normal group,the EM of AF collagen fibrils underwent an increase from 142.89 ±17.15 MPa to 167.10± 35.05 MPa(P=0.0652)in the compression group,from 142.89±17.15 MPa to 225.73±25.39 MPa(P=0.0004)in the needle puncture group respectively when measured in the outer layer 4 weeks post-surgery.Conclusion: First,the self-made rat model have induced IVDD successfully.Second,the EM results indicated that the needle puncture can cause remarkable stiffening of individual fibril,which is different from that of abnormal mechanical loading pattern.This interesting change provides a new insight of the influence of needle puncture on the process of disc degeneration and may influence the choice and development of the treatment strategies in the future.