The Influence of Glycation- and Protein Denaturation- induced Biochemical Changes on Dynamic Mechanical Properties of Intervertebral Disc

Purpose: To investigate the biochemical influence of protein denaturation and glycation on the dynamic mechanical properties of intervertebral disc (IVD).

Introduction: With the increasing ageing population, the impact of protein denaturation and glycation, which may result in increased level of disc degeneration, are becoming an important field of intense interest. The protein denaturation is likely to lead to degradation of extracellular matrix, where as glycation would lead to consummation of protein as well as accumulation of advanced glycation end-product (AGE). Nevertheless, the relationship between these biochemical influences and the biomechanical change is still poorly understood. Among many mechanical testing methods, dynamical mechanical analysis (DMA) is a sensitive tool to investigate the minor changes in IVD with altered biochemical property. Understanding the relationship of biochemical and biomechanical in degenerated IVD is helpful for clarification of the mechanism of IVD degeneration, which can also be beneficial for the selection of appropriate material in the development of artificial disc in the future.

Material and method: 24 IVDs from porcine thoracic spine were randomly assigned to intact (n=8), trypsin (n=8), and glycation (n=8) groups. After a standard sterilization processing, the specimens were dissected and cultured in a whole organ culture system for 1 day. Dynamic mechanical analysis in compression (frequency 0.031~10Hz, stress control of 0.45±0.35MPa), rotation (frequency 0.01~1 Hz, strain control of 0°±2°), shear (frequency 0.031~3.1Hz, stress control of 0.25±0.15MPa) and biochemical tests of water, AGE, glycosaminoglycan (GAG), collagen content from nucleus pulposus (NP), inner anulus fibrosus (IAF), outer anulus fibrosus (OAF) as well as histological and scanning electron microscopic analysis were then subsequently conducted. One-way repeated ANOVA, student’s t-test and one-way ANOVA were used to calculate the differences in frequency of the dynamic mechanical tests, disc height, dynamical mechanical and biochemical properties, respectively. LSD test was used as the post-hoc analysis.

Results: In trypsin group, the stiffness and storage modulus decreased significantly in compression, with decreased water and GAG content in NP as well as decreased water content in OAF. As the frequency increased, the stiffness and storage modulus increased in within-group analysis of compression test. The boundary between lamella became less obvious and the structure became loose in histological analysis. In the glycation group, the IVD showed increased storage modulus and loss modulus in rotation, and increased storage modulus in shear. AGE content was increased in IAF, and water content was increased in both IAF and OAF. Gap boundary between lamella became more obvious with increased porosity in glycation group.

Discussion: The results of the current study demonstrated that Trypsin can potentially cleave the peptide bonding of the GAG in the NP and collagen structure in the AF. Subsequently, due to the decrease of GAG content and structure defect of the collagen, the swelling pressure in NP and hoop stress in AF decreased, which resulted in decreased stiffness and storage modulus in compression. Ribose can induce glycation by interacting with collagen, causing accumulation of AGEs in AF, which can lead to hardening and thickening as well as enlarged gap in lamella and increased porosity in AF. As the result, the storage modulus increased in rotation and shear.

Conclusion: There are two different patterns of degeneration mechanism in protein denaturation induced- and glycation induced- disc degeneration. The effect of protein denaturation from trypsin mainly reacted within NP, causing decreased stiffness and storage modulus in compression. In contrast, the effect of glycation mainly acted on AF, causing increased storage modulus in rotation and shear.