醣化作用與酶解對椎間盤在生物力學與生物化學性質之影響

Abstract

目的：本研究目的在建立一套體外培養系統之椎間盤退化模型。本研究使用三種椎間盤退化方法，分別為醣化作用(Glycation)、膠原蛋白酶解(Collagenase)與多醣酶解(Chondroitinase)，研究之目的在觀察椎間盤在此三種退化方法處理後之生物力學與生物化學性質之影響。 背景介紹：椎間盤退化是人體老化的自然現象之一，也是引起脊椎病痛的來源之一。退化椎間盤的治療一直是學界與醫界共同努力的目標，一般而言，研究新的藥物須先經過生物實驗，在驗證藥物在生物體內的有效性與安全性之後，才能進入臨床實驗。一般動物實驗分成小動物實驗與大動物實驗，小動物生命週期短，較易取得退化性椎間盤，但與人體椎間盤的型態差異較大，所以在生物力學上的模擬差異較大；若使用大動物椎間盤進行研究，雖然型態與人體椎間盤較類似，但生命週期較長，若要取得退化性椎間盤，則須經長時間畜養，耗費較高。因此，若能建立一套離體(Ex Vivo)的大動物椎間盤退化模型，將有助於實驗資源的降低，並應用於大量篩選或測試新的治療方法。椎間盤退化受到許多複雜因子影響，當年齡老化，人體內源性(Endogenous)醣化作用增加，會導致椎間盤內醣類堆積與水分流失。惟，目前一般的椎間盤退化方法是利用注入酶解藥物，造成基質降解方式建立模型，但此一方法，無法模擬上述醣化現象。因此建立一適當且合理的椎間盤退化模型有其必要性。 材料與方法：本研究使用六個月大豬隻之胸椎第二至第七節(T2~T7)椎間盤，為保持試樣內細胞之活性，實驗於豬隻屠宰後四小時內進行。本研究使用三種方式建立椎間盤退化模型，分別是使用核醣溶液(Ribose)模擬基質醣化、第二型膠原蛋白酶(Type II collagenase)模擬第二型膠原蛋白降解、與硫酸軟骨酶(Chondroitinase ABC)模擬多醣降解。椎間盤退化模型會於試樣處理完分別打入0.5ml, 0.6M的核醣溶液與0.5ml, 50U/ml的第二型膠原蛋白酶以及0.5ml, 0.25U/ml的硫酸軟骨酶。各組別皆會放入生物培養箱中培養七天，在第二天與第七天進行潛變測試(Creep test)；衝擊測試(Impulse test)會在第七天潛變測試完12小時後進行，最後對生化性質包含椎間盤水分含量、組織切片、掃描式電子顯微鏡( Scanning electron microscope , SEM )、醣胺聚醣( Glycosaminoglycan , GAG )與膠原蛋白( Collagen )含量進行量測。 結果：椎間盤醣化組，在負載後椎間盤聚合模數下降，勁度無明顯改變，液體滲透性與阻尼係數皆下降，椎間環層與層排列無明顯改變，組織間隙與纖維孔隙增加，椎間盤內組織水分、醣胺聚醣與膠原蛋白含量下降；第二型膠原蛋白酶組，在負載後椎間盤聚合模數與勁度上升，而液體滲透度與阻尼係數下降，椎間環層與層排列不規則，纖維孔隙增加，內層椎間環明顯外突，椎間盤內組織水分、醣胺聚醣與膠原蛋白含量下降；硫酸軟骨酶組，在負載後椎間盤聚合模數下降，勁度無明顯改變，液體滲透度與阻尼係數下降，椎間環層與層排列不整齊，組織間隙與纖維孔隙增加，內層椎間環向外突出，椎間盤內組織水分與醣胺聚醣含量明顯下降。小結：椎間盤醣化作用增加，堆積於基質間的最終醣化產物隨之增多，使椎間盤水分以及吸震能力下降；第二型膠原蛋白酶切斷椎間盤內第二型膠原蛋白，使椎間盤的強度、保水能力、吸震能力下降；硫酸軟骨素酶破壞蛋白多醣的結構，使椎間盤保水能力、吸震能力下降。 結論：本研究將椎間盤退化模型實驗結果與文獻中人體退化椎間盤進行比較，在第二型膠原蛋白酶椎間盤退化模型上，與人體退化椎間盤的生物力學與生物化學趨勢相同，但結構上較人體退化椎間盤輕微。因此，本研究之結果較適合應用於輕微椎間盤退化之臨床應用。

Objective: To investigate the biomechanical and biochemical properties of intervertebral discs treated with ribose, collagenase and chondroitinase. The knowledge of this study is helpful for the development of an ex-vivo big-sized animal disc degeneration model. Summary of background data: Disc degeneration is a reason to induce low back pain in aging population. Treatments of degenerative disc diseases have been studied in both academic and medical communities. In general, the safety and efficacy of a new treatment has to be validated with in-vivo animal studies before clinical trials. Due to short life cycles, creating degeneration discs is easier in small-sized animals than in big-sized ones. However, discs of small-sized animals are less similar to human being’s, and thus the results of small-sized animal studies are less clinically applicable. On the other hand, creating degenerative discs in big-sized animals is time and money consuming. Therefore, screening treatment candidates with an ex-vivo big-sized animal degeneration disc model before in-vivo animal studies will help to cut down experimental expanses. As for now, most ex-vivo animal degeneration disc models are induced by digestive enzymes, such as collagenase and chondroitinase. Both of them are unable to simulate the accumulation of advanced glycation end-products (AGEs) in degenerative discs. Methods: Six-month old porcine thoracic discs (T2~T7) were used as specimens. In order to preserve viable cells in discs, the specimens were dissected within 4 hrs after pigs were slaughtered. To simulate matrix glycation, collagen degradation, and proteoglycan depletion in degenerative discs, ribose solution (0.5ml, 0.6M), Type II collagenase (0.5ml, 50U/ml) and chondrotinase ABC (ChABC, 0.5ml, 0.25U/ml) were individually injected into one of the three disc groups. A creep test was applied on all specimens on Day 2 and Day 7 to obtain the aggregate modulus and permeability of specimens. An impulse test was performed 12 hrs after the creep test on Day 7 to evaluate the stiffness and damping coefficient of specimens. In addition, biochemical properties (i.e. water, glycosaminoglycan (GAG) and collagen content), anulus histology and micro structure were inspected on Day 7. Results: After injection of ribose/ChABC, the aggregate modulus, permeability and damping coefficient of disc decreased. After injection of Type II collagenase, the aggregate modulus and stiffness of disc increased, but the permeability and damping coefficient of disc decreased. All chemical solutions increased pores in anulus, caused inner anulus delamination and decreased disc water and GAG content. Only ribose and Type II collagenase reduced collagen content of disc. Discussion and Conclusion: Both of AGEs accumulation and proteoglycan depletion of disc matrix decreases disc stiffness, energy dissipation capacity, water/GAG content and anulus fibrosus integrity. AGEs further reduced collagen content. Type II collagen degradation of disc matrix increases disc stiffness, but decreases energy dissipation capacity, water/GAG/collagen content, and anulus fibrosus integrity. Among the three degeneration-induced treatments in this study, the biomechanical and biochemical properties of the discs treated by Type II collagenase are more similar to those of human discs with mild-level degeneration.