椎間核變性與椎間環損傷對椎間盤之黏彈性質影響

Abstract

目的: 探討椎間核變性與椎間環損傷對於椎間盤黏彈性質的影響 背景: 椎間盤具有黏彈性質，是由膠質纖維、蛋白多醣及水分所組成的器官。椎間盤的主要力學作用是抵抗外力與吸收震盪所帶來的能量。抵抗外力的力量主要藉由膠原纖維網與水分所提供。震盪能量的吸收由流體與固體間產生的摩擦力去消耗，而水分和蛋白多醣的結合力則提供了這種摩擦力。椎間盤功能下降易使椎間盤受外部壓力而受傷，加速椎間盤的退化。年紀與過度的疲勞負載被視為引起椎間盤退化的刺激物。變性酵素與機械性疲勞負載是兩種主要被使用來創造椎間盤退化模型的方法。當年紀增長，如同基質金屬蛋白酶的水解酵素被活化，分裂了膠原纖維和蛋白多醣，而高重複性的動作與待在震動的環境下會使椎間盤累積機械性的微小傷害。藉由量化椎間盤黏彈性質可表現出椎間盤的功能，當理解損傷椎間盤黏彈性質的改變可提升對於椎間盤退化原因的知識。 材料與方法: 32組新鮮豬的胸椎椎間盤從連接的椎骨中切割下來，將軟組織與後方關節小心地移除。將椎間盤試樣平均分成四組，分別為健康組、變性組、低程度疲勞負載組與高程度疲勞負載組，測量最初的椎間盤高度與面積。健康組椎間盤不經過任何型式的破壞。變性組的椎間盤注射濃度為0.5 %的胰蛋白酶溶液0.5 毫升。低程度疲勞負載組與高程度疲勞負載組的椎間盤分別施加0.1-0.8 MPa與0.8-1.6 MPa的疲勞負載兩小時，疲勞負載的頻率為5 Hz。所有的試樣測試前，以紗布包覆浸泡於生理食鹽水中並放置於攝氏-20度中。使用Bose ElectroForce 3510的材料測試機台，藉由動態機械分析技術測量椎間盤的黏彈性質。將裝有生理食鹽水之容器固定在機台上，把椎間盤放置於容器之正中央。將自製的鐵氟龍圓盤固定在材料測試機台的驅動器上，將週期性外力施加在試樣上。所有的椎間盤承受0.1-0.8 MPa的週期外力，其頻率範圍由0.01 Hz到10 Hz。在頻率0.01、0.063、0.1、0.63、1.0、6.3與10 Hz下根據力量與位移的結果分析椎間盤的勁度、阻尼係數、儲存模數、損失模數以及相位角。變性酵素與疲勞負載對於健康椎間盤之勁度、阻尼係數、儲存模數、損失模數以及相位角的影響以一方變異數(One-way ANOVA)來檢驗是否有顯著差異。當p<0.05視為統計上有顯著差異。 結果: 變性組椎間盤的勁度、儲存模數與損失模數顯著低於健康組，而阻尼係數與相位角在頻率大於0.63 Hz後即無顯著改變。當頻率升高，變性組的勁度顯著上升，但阻尼與相位角卻都顯著下降。頻率升高至0.1 Hz之後，變性組椎間盤的儲存模數不再顯著升高，而損失模數不隨頻率有顯著改變。在頻率小於0.01 Hz時，低程度疲勞負載組之阻尼係數、損失模數與相位角都有小於健康組之趨勢，但當頻率逐漸變大後，卻有大於健康組之趨勢。高程度疲勞負載組之勁度有大於健康組之趨勢，但阻尼係數、相位角、儲存模數與損失模數有小於健康組的趨勢。隨頻率升高，低程度疲勞負載組的勁度顯著增加，阻尼係數顯著減少，當頻率超過0.1 Hz後，儲存模數與相位角改變不顯著，而損失模數不受頻率增加而改變。隨頻率升高，高程度疲勞負載組的阻尼係數與相位角顯著減小，儲存模數顯著上升，而損失模數不受頻率增加而改變；在頻率超過0.63 Hz後，勁度不再顯著增加。高程度疲勞負載組其勁度、阻尼係數、儲存模數(E’)、損失模數(E’’)和相位角(δ)皆小於低程度疲勞負載組。 結論： (1)與健康組椎間盤相比，變性組椎間盤結構較鬆散、吸震能力較差、彈力和黏滯力較低。但當頻率超過0.63 Hz後，變性組之吸震能力與健康組椎間盤相比已無顯著差異。 (2)疲勞負載會造成椎間環損傷，使勁度變得比健康組椎間盤大。而高程度疲勞負載組的椎間盤，其吸震能力與黏滯力比健康組來得差。低力量疲勞負載組其吸震能力與黏滯力比健康組高，但並不顯著。疲勞負載造成的椎間盤退化，使椎間盤變硬、吸震能力變差。高程度疲勞負載組會使椎間盤的硬度、吸震能力、彈力比低程度疲勞負載組來的小。

Objective: To find the effect of nucleus pulposus denaturation and anulus fibrosus fatigue loading on the disc viscoelastic properties. Background: Intervertebral disc is a viscoelastic organ composed of collagen fibers, proteoglycan and water. The major mechanical functions of discs are to resist external loading and absorb shock energy. Resistance to external loading is majorly provided by collagen fiber network and water content. Shock energy absorption is resulted from the friction force between water flow and solid tissue. The bonds between water and proteoglycan contribute to this friction force. Declination of disc function makes discs vulnerable to external stress, which in turn accelerates disc degeneration. The aging and excessive fatigue loading are recognized as major triggers of disc degeneration. Two major methods, i.e. the enzymatic denaturation and mechanical fatigue loading, are used to create disc degeneration models. The hydrolysis enzymes, such as matrix metalloproteinases (MMPs), are activated to cleave collagen fibers and proteoglycan in the aging process. Highly repeated activities or staying in vibrating environment accumulate mechanical micro-injuries within discs. Disc functions can be quantitatively represented by disc viscoelastic properties. Understanding the alteration of disc viscoelastic properties of injured discs improves the knowledge on degeneration etiology. Methods and Materials: Thirty-two fresh porcine thoracic discs were prepared by cutting off the adjacent vertebral bodies. The soft tissue and posterior element was removed carefully. Disc specimens were equally assigned to 4 groups, i.e., intact, denatured, low-level fatigue loading, high-level fatigue loading groups. The initial disc height and dimensions were measured. The disc of intact group did not receive any forms of injury. The discs of denatured group were injected with trypsin solution (0.5 ml, 0.5 %). The discs of low- and high-level fatigue loading groups were respectively applied with a 2 hour fatigue loading at 2 magnitudes, i.e. 0.1-0.8 MPa, 0.8-1.6 MPa. The frequency of fatigue loading was 5 Hz. All specimens were soaked in the phosphate buffer saline (PBS), wrapped with gauze and then stored at -20 degree Celsius before tests. Disc viscoelastic properties were measured by dynamic mechanical analysis (DMA) techniques using a material testing apparatus (Bose ElectroForce 3510). The discs were placed in the center of a chamber mounted on the material testing apparatus. The chamber was filled with the PBS. A Teflon plate was attached to the actuator of the material testing machine to transfer cyclic loadings to the specimen. All discs were applied with 0.1-0.8 MPa sinusoidal loading at frequencies sweeping from 0.01 to 10 Hz. Stiffness, damping coefficient, storage modulus (E’), loss modulus (E’’), phase angle (δ) of discs at frequency of 0.01, 0.063, 0.1, 0.63, 1.0, 6.3, 10 Hz were analyzed according to the resultant force and deformation. One-way ANOVA was respectively performed to evaluate the effect of enzymatic denaturation and fatigue loading on the stiffness, damping coefficient, storage modulus, loss modulus and phase angle. Statistical significance was set at p<0.05. Results: The stiffness, storage modulus and loss modulus of denatured discs were significantly lower than of intact discs. The damping coefficient and phase angle of denatured discs didn’t change significantly with intact groups when frequency was over 0.63 Hz. The stiffness of denatured discs significantly increased with increasing frequency, but the damping coefficient and phase angle were decreased. The storage modulus of denatured discs don’t increase significantly when frequency was over 0.1 Hz. However, loss modulus of denatured discs did not changed significantly with variation of frequencies. The damping coefficient, loss modulus and phase angle trends of low level fatigue discs were lower than intact discs at frequency lower than 0.01 Hz. On the other hand, the damping coefficient, loss modulus and phase angle increased with frequency when compared to the intact discs. The trend of stiffness of high level fatigue discs were higher than of intact discs, but the trend of damping coefficient, phase angle, storage modulus and loss modulus were lower than of intact one. In the low-level fatigue discs, the stiffness increased but the damping coefficient decreased with frequency, respectively. However, no significant changes for storage modulus and phase angle were found when frequency was over 0.1 Hz. The loss modulus did not change significantly with frequency. In the high-level fatigue discs, the stiffness was not significantly increased when frequency was over 0.63 Hz. The damping coefficient and phase angle significantly decreased with frequency. The storage modulus significantly increased with frequency. The loss modulus did not change significantly with frequency. The stiffness, damping coefficient, storage modulus, loss modulus and phase angle of high level fatigue loading discs were lower than low level fatigue loading discs. Conclusion: (1) The denatured disc shows softer, weaker shock absorption characteristics, elastic modulus and viscosity in comparison with intact disc. However, the ability of shock absorption of the denatured disc did not change significantly in comparison with intact disc when frequency was over 0.63 Hz. (2) The fatigue loading caused injury in anulus fibrosus. The disc after fatigue loading was stiffer than intact one. The high level fatigue loading showed weaker shock absorber and viscosity than intact disc. The ability of shock absorption and viscosity subjected to low level fatigue loading were higher than intact, but the differences were not significant. Degenerated disc with fatigue loading showed stiffer, weaker shock absorption characteristics. The hardness, ability of absorbing shock and elasticity of high level fatigue loading were lower than of low level fatigue loading.