Effect of Nucleus Pulposus Denaturation andxogenous Crosslinking on the Dynamic Properties of Intervertebral Disc

目的：探討椎間核變性水解與外生性交聯對椎間盤動態性質的影響。景：椎間盤受傷後，會造成椎間盤的生化組成的變化，因而影響椎間盤的力學功能，若椎間盤的力學特性產生不可逆的變化，在日經月累的負重之下可能引起其他脊椎疾病的發生。然而椎間盤受傷於急性期基質降解與亞急性期交聯產生後的力學反應仍不清楚。料與方法：使用六個月大的豬隻腰椎運動單元作為試樣(L1-L2，L3-L4，共45副)，椎間核水解組有27組，椎間環損傷組有18組。在椎間核水解組，18組試樣為注射1毫升胰蛋白酶入椎間盤降解椎間核細胞基質，並施予第一次30分鐘循環負載，隨後給予24小時休息修復，然後在施於第二次30分鐘循環負載。第一次30分鐘循環負載後，將9組試樣直接浸泡生理食鹽水，另9組為注射1毫升梔子素溶液後在浸泡生理食鹽水，進行24小時休息修復。其餘9組試樣為沒有注射任何溶液的健康椎間盤，直接施予第一次30分鐘循環負載，隨後給予24小時浸泡生理食鹽水中休息修復，然後在施於第二次30分鐘循環負載。在循環負載其間每十分鐘間隔會施予一次衝擊測試。椎間環損傷組為施予第一次2小時循環負載產生椎間環傷害，隨後給予24小時休息修復，然後再施於第二次2小時循環負載。第一次2小時循環負載後，將9組試樣直接浸泡生理食鹽水，另9組為浸泡梔子素溶液中，進行24小時休息修復。在2小時循環負載期間在0、0.5、1和2小時時間點上會施予一次衝擊測試。椎間盤勁度係數K(N/mm)及阻尼係數C(Ns/mm)由衝擊過程的資料求的。果：椎間核降解椎間盤阻尼係數則顯著性的小於健康椎間盤。降解椎間盤經由外生性交聯後，降解椎間盤的動態性質都有顯著性上升，勁度初始值與健康椎間盤的無顯著性差異，但在循環負載其間都顯著小於健康椎間盤；而阻尼係數雖顯著性的小於健康椎間盤，但都已顯著的大於降解椎間盤。椎間環斷裂組中，本實驗重現了文獻相同的實驗結果，在0.5小時循環負載後，椎間環斷裂椎間盤的動態性質就到達穩態。而經由外生性交聯後，勁度初始值與健康椎間盤無顯著性差異，則阻尼係數初始值時明顯沒有恢復；但在循環負載其間勁度都顯著小於健康椎間盤，而阻尼係數則與健康椎間盤相同。論：本實驗的模擬結果顯示，當椎間盤受傷處於急性期時，因椎間核基質降解，使的椎間盤的緩衝能力下降，且椎間盤孔洞性也變大。而當椎間盤受傷來到亞急性期時，因膠原蛋白交聯的增生，使的受傷椎間盤孔洞收縮，且緩衝能力提升，但增生交聯組織卻會降低膠原纖維對循環負載的耐受力，最後使椎間盤整體強度降低。Objective: The purpose of this study is to evaluate the effect of nucleus denaturation and exogenous crosslinking on disc dynamic properties. ummary of Background Data: Fatigue loading can damage disc structure integrity. In early stage of tissue healing post disc injury, disc matrix crosslinkings, especially nucleus pulposus, are denatured by disc enzymes. In the following subacute stage, new crosslinking forms with the growth of fibrosis tissue from the injured sites. Howerer, the interaction of nucleus denaturation and crosslinking generation with the disc dynamic properties remains unclear.ethods: In total of 45 porcine lumbar body-disc-body constructs (L1-L2, L3-L4) were assigned to “nucleus pulposus (NP) denaturation protocol” (n=27) and “anular fibrosus (AF) damage protocol” (n=18). For the “NP denaturation protocol”, 9 specimens were selected as “healthy discs”, receiving no injection, and applied with a 30 min fatigue loadings twice. These discs rehydrated in saline solution for 24 hr before and after the first fatigue loading. The other 18 specimens were injected with 1ml trypsin solution, immersed in saline solution for 24 hr and then loaded with a 30 min fatigue loading. After the fatigue loading, 9 discs out of these 18 specimens were immersed in saline bath for 24 hr, while the other 9 discs were injected with 1ml 0.33% genipin solution before saline bath immersion. Each disc was then loaded with another 30 min fatigue loading. An impulse test was applied to every disc in NP-denatured group at 0, 10, 20, and 30 min of second fatigue loading. For the “AF damage protocol”, each specimen was loaded with 2 hr fatigue loading first, followed by a 24 hr rest, and then applied with another 2 hr fatigue loading. After the first 2 hr fatigue loading, 9 specimens were immersed in saline bath, while the other 9 specimens were immersed in 0.33% genipin solution. During the second 2 hr fatigue loading, an impulse test was applied at time point of 0, 0.5, 1, and 2 hr. The stiffness (K, N/mm) and damping coefficient (C, Ns/mm) of disc was calculated using the one-dimension spring-damping model and impulse test loading information.esults: (1) NP denaturation protocol: compared to the healthy disc, NP denaturation did not change the disc stiffness but significantly decreased the damping coefficient (P= 0.000) at the end of fatigue loading. NP degeneration also increased the change rate of disc stiffness and damping coefficient with the fatigue loading time. In comparison with the NP-denatured disc, after crosslinking generation the disc stiffness significantly decreased (P= 0.024) but the damping coefficient significantly increased (P= 0.024) at the end of fatigue loading. The change rates of disc stiffness and damping coefficient was the same as those of healthy discs. (2) AF damage protocol: the disc stiffness and damping coefficient reached plateau at 1 hr of the first fatigue loading but at 0.5 hr of the second fatigue loading. The plateau value of disc stiffness and damping coefficient were the same comparing the first and the second fatigue loading. After crosslinking, the disc stiffness and damping coefficient reached plateau at 1 hr of the second fatigue loading. The plateau value of disc stiffness significantly decreased (P= 0.042), while that of damping coefficient was not changed. onclusion: Faster fluid outflows during fatigue loading is caused by both of NP denaturation and AF damage based on the increased change rate of disc dynamic properties. Disc damping coefficient decreases after NP denaturation and recovers after crosslinking generation in NP, indicating positive relation between shock attenuation capacity and NP crosslinking level. The crosslinking generation in disc decreases disc stiffness, reducing disc strength to external loading.口試委員會審定書 i文摘要 ii文摘要 iv一章 前言 1-1 脊椎的基本構造 1-2 椎間盤的構造及功能 1-3 椎間盤傷害 5-4 組織癒合過程的急性期 5-5 組織癒合過程的亞急性期 7-6椎間盤力學性質與循環負載 9-7研究動機與實驗目的 9二章 實驗設備 11-1 連續式衝擊測試平台(Continuous Impact Testing Apparatus) 11-2 衝擊錘(Impactor) 12-3 撞擊承受器(Impounder) 12-4 往復式衝擊模組(Cyclic Loading) 12-5 線性電位計(Potentiometer) 14-6 線性位移計(Liner Variable Differential Transformer) 14-7 一維測力元(1-D Load Cell) 14-8 訊號擷取處理及控制系統 15三章 材料與方法 16-1試樣準備 16-2實驗流程 17-2-1 椎間核變性水解模型流程 17-2-2椎間環損傷模型流程 17-3實驗方法 18-4數學模型 20-5統計分析方法 21四章 結果 22-1椎間核變性降解/外生性交聯產生後的椎間盤之動態性質分析 22-2椎間環破裂/外生性交聯產生後的椎間盤之動態性質分析 26-3椎間盤切面觀察 28五章 討論 30-1椎間盤動態性質隨循環負載時間增加的變化 30-2椎間盤傷害對椎間盤動態性質的影響 30-3外生性交聯對椎間盤傷害的影響 31-4實驗限制 33六章 結論與未來展望 35-1結論 35-2未來展望 35考文獻 3