2011-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/642972摘要：椎間盤退化是常見的脊椎病變，也是造成下背痛的主因之一，此問題隨著社會老化人口增多而增加。在退化早期給予恰當的治療希望能有助於緩解椎間盤的退化。椎間盤退化開始後，椎間盤內發炎物質及降解酵素活性增加，造成椎間盤基質化學組成改變、含水量減少，椎間環結構分解。目前具前瞻性的治療策略有給予天然交聯劑、含血小板之血漿（以下簡稱血漿）、間質幹細胞等三種，天然交聯劑已被證明可強化椎間環組織的力學強度，但是否有減緩椎間盤細胞凋亡的效果則不得而知。血漿及間質幹細胞已被證實能促進細胞生長與椎間盤基質合成，但其效果是否能達到恢復椎間盤力學功能的程度目前仍無法肯定。此外，退化性椎間盤的治療方法常藉由注射方式做為傳遞手段，針刺傷大小是否會影響治療的成效，也有待探討。驗證治療或預防椎間盤退化的方法的有效性主要分為兩大類，一為生物、生化測試，二為力學測試。生物與生化測試看的是細胞階段的反應，通常不需要完整的椎間盤結構即可進行檢驗，但其實驗成效僅限於細胞階段，無法延展至組織或是整個椎間盤；相反的，力學測試則是屬於椎間盤的功能性測試，但受測試樣必須是完整的椎間盤組織，待測的治療方法需經過較長的作用時間才有辦法對整體椎間盤形成顯著性的影響。若欲同時獲得可進行此兩種測試的試樣，最好的方式是進行動物活體實驗。但進行動物實驗常會遇到動物個體間差異，生理條件難控制，飼養費昂貴，與人道倫理等問題。體外全椎盤培養系統可利用生物反應器讓椎間盤於接近體內環境的狀態下維持活性，是在進行動物實驗之前較簡便的測試方法。本計畫將藉著比較天然交聯劑、血漿、間質幹細胞對退化初期椎間盤的生物、生化反應與生物力學影響，推論治療退化性椎間盤的可能策略。採用的生物反應測試有：細胞存活率與組織切片；生化反應測試有：蛋白多醣含量與膠原蛋白含量；力學測試有：椎間盤流變性質與動態性質。計畫將分成四個階段來進行。第一階段，建立一循環式體外全椎間盤培養系統；第二階段，使用豬隻椎間盤模型，評估針刺傷大小對培養天數的影響，並計算椎間盤生物、生化表現與力學功能的相關性；第三階段，使用第二階段實驗中所得到的建議針號與培養天數，將胰蛋白酶注射入豬隻椎間盤產生退化性變化後，分別將天然交聯劑、血漿、間質幹細胞注射入椎間盤，於培養時間結束後，測試椎間盤的生物、生化反應與力學功能的回復程度，並依回復程度加以評分，以得分最高者為「治療方式一」，將得分最高與得分次高者等比例混合後，為「治療方式二」。第四階段，先以經皮穿刺豬隻脊椎終板的方式，建立一個活體豬隻的初期退化性椎間盤模型。兩個禮拜後，分別將「治療方式一」與「治療方式一」注射入退化初期的椎間盤內，於三個月後將豬隻犧牲，測試退化性椎間盤之生物、生化反應與力學功能的回復程度，比較兩種治療策略的實際效果。<br> Abstract: Disc degeneration is a frequently observed spinal disorder, and one of the major reasons of low back pain. This problem isgetting popular in an aging society. It is hoped that the disc degeneration can be released during its early stage. During the stage ofearly disc degeneration, the inflammation factors and digestive enzymes are activated, and hence change the disc biochemicalcompositions, decrease the disc water content and decompose the disc structural integrity. The potential treatments at this momentfor the disc degeneration include the natural cross-linkers (NCL), platelet-rich plasma (PRP), and mesenchymal stem cell (MSC)therapy. The NCL was proved to enhance the strength of anular fibrosus. However, its effect on cell death inhibition remainsunknown. The PRP and MSC were proved to increase cell viability and disc matrix synthesis. Nevertheless, the effectiveness ofPRP and MSC were only observed on tissue level, but not to the level of whole disc or living animal. In addition, the treatments fordegenerated disc were often delivered through needle injection. The injury due the needle injection is also not clear.The treatments for disc degeneration are often assessed with biological, biochemical and mechanical tests. The biological andbiochemical tests focus on the cell reaction and thus only needs small amount of disc tissue. The conclusion of biochemical testmay not be easily applied to the whole disc since the treatment efficacy is limited to tissue level. The mechanical test reveals thedisc function and usually needs the whole disc as subjects. Since the structure of whole disc is much larger and more complicatedthan the disc tissue, it takes longer time to determine the efficacy of treatment. The in vivo experiments can be accessed by thebiological, biochemical and mechanical examinations at the same time. However, the in vivo experiments are difficult andexpensive because of the issues of physiological variations, cost of experiments and animal sacrifice ethics. The whole discculturing method maintains the disc integrity and cultures the disc cell at physiological condition, and thus can be an effectivemethod to screen the potential treatment before the large in vivo animal experiment.The purpose of this study is to investigate the effect of NCL, PRP and MSC on the biological, biochemical and biomechanicalbehaviors of early degenerated disc. The tests of biological response include the cell viability and histology change. The test ofbiochemical response includes the proteoglycan content and collagen content. The biomechanical tests include the examinations ofrheological and dynamic properties. The experimental result is expected to give reference for treatment selection of degenerateddisc. Four phases of experiment will be conducted. In the first phase, a whole disc culturing system will be established. In thesecond phase, porcine discs will be used to determine the size of needle gauge and duration of culturing for the whole discculturing system. The correlation of disc biological and biochemical reactions with respect to the biomechanical properties willalso be evaluated. In the third phase, the trypsin will be injected into porcine discs to create early disc degeneration. Then thebiological, biochemical and biomechanical properties of NCL, PRP, and MSC treated discs will be tested and graded. Thetreatment with highest score (Treatment 1, T1) and the combination of treatments with the highest and the second highest score(Treatment 2, T2) will be decided for the fourth phase of experiment, in which an in vivo large animal study will be conducted. Anearly disc degeneration model will be created by stabbing the disc endplate through percutaneous puncture. After two weeks ofsurgery, the T1 and T2 treatments will be injected into the degenerated discs. After three months, the pigs will be sacrificed, andthe treated disc will be evaluated using biological, biochemical and biomechanical examinations to determine the efficacy of T1and T2 treatments.椎間盤椎間盤培養系統天然交聯劑富含血小板之血漿間質幹細胞流變學性質動態性質Intervertebral discwhole organ culturing systemnatural crosslinkerplatelet-rich plasmamsenchyumal stem cellrheological propertydynamic property