醣化作用與蛋白質變性作用的生化性質改變於椎間盤動態力學性質之影響

Abstract

目的：探討蛋白質變性作用及醣化作用產生之生物化學性質對於椎間盤動態生物力學性質之影響。 背景介紹：隨著人體年紀的增加，蛋白質變性作用及醣化作用對於椎間盤內部的傷害會逐漸累積，導致椎間盤退化程度增加。蛋白質變性作用主要造成細胞外間質的降解，醣化作用則會消耗蛋白質、增加有害物質醣化最終產物(Advanced glycation end-product, AGE)的累積，但二者對於椎間盤生物力學性質的直接影響仍少有證據直接支持。在所有研究材料黏彈性質的方法中，動態力學分析是ㄧ種相當具有敏感度的方法，可測得生化性質改變後椎間盤於黏彈性質上之細微變化。了解椎間盤退化要素中生物化學性質與生物力學性質之間的關聯，將有助於釐清椎間盤退化的機制，亦有助於未來人工椎間盤材料之選擇與設計開發。 材料與方法：本研究使用24個豬隻胸椎椎間盤，隨機分配至健康組(n=8)、蛋白質變性組(n=8)、與醣化組(n=8)，經無菌處理取下試樣後，以全椎間盤培養系統培養一天，並進行壓縮(頻率0.031~10Hz，應力範圍0.45±0.35MPa)、旋轉(頻率0.01~1Hz，應變範圍0°±2°)、剪切(頻率0.031~3.1Hz，應力範圍0.25±0.15MPa)三個方向之動態力學測試，再依髓核、內側纖維環、外側纖維環三部分進行包括水分含量、醣化最終產物含量、醣胺聚醣含量、膠原蛋白含量之生化測試，最後利用染色切片與掃描式電子顯微鏡進行微觀結構觀察。統計部分，動態力學性質中，同組別不同頻率之測試採用單因子重覆變異數分析法分析；椎間盤於培養前後之高度變化採用學生t檢驗分析，事後檢定採用LSD法；不同組間的動態力學性質及生化性質採用單因子變異數分析，事後檢定採用LSD法。 結果：胰蛋白酶組相對於健康組在壓縮方向之勁度及儲存模數下降，髓核之水分與醣胺聚醣含量下降、外側纖維環之水分含量下降；以組內差異分析，可知勁度及儲存模數隨著頻率的增加而上升。結構觀察方面，胰蛋白酶組層與層間分界不明、結構較鬆散。醣化組相對於健康組在旋轉方向之儲存模數及損失模數上升、在剪切方向之儲存模數上升，內側纖維環的醣化最終產物含量上升、水分在內外側纖維環之含量均下降。結構觀察方面，髓核細胞外間質變緻密，層與層間分界清楚但距離增大、纖維環孔洞變大。 討論：胰蛋白酶能破壞主要位於髓核中的醣胺聚醣，及主要位於纖維環中的膠原蛋白。由於醣胺聚醣量下降，造成椎間盤髓核之澎潤壓下降，再加上纖維環變得較為鬆散，產生的環向應力下降，因此在壓縮方向的動態力學測試中勁度與儲存模數均下降；核糖能與纖維環內的膠原蛋白發生醣化作用，產生醣化最終產物，因此能讓纖維環變硬、變黏，在結構上使得層與層間之距離變大、孔洞變大，因此在旋轉及剪切方向的動態力學測試中，造成低頻的勁度及儲存模數上升。 結論：造成椎間盤退化的兩大成因─蛋白質變性作用與醣化作用，對於椎間盤的生化與生物力學性質作用機制並不相同。胰蛋白酶造成的蛋白質變性作用主要作用於髓核，造成椎間盤壓縮方向的勁度、儲存模數下降；而核糖溶液造成的醣化作用主要作用在纖維環，造成椎間盤於旋轉與剪切方向的低頻勁度、儲存模數上升。

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.