Effect of Molecular Weight and External Loading on Particle Diffusion within Intervertebral Disc: An in Vitro Porcine Model

中文摘要 背景簡介：椎間盤為人體最大之無血管軟骨組織。椎間盤的退化會導致椎終板鈣化與椎間核纖維化，兩者皆會影響椎間盤內的分子運輸，溶質運輸的減少將引致間盤退化的惡性循環。研究椎間盤營養交換於擴散作用(Diffusion)與日常動作對椎間盤產生的對流作用(Convection)方式，將有助於了解與治療退化性椎間盤。 目的：階段一、建立一個可用以研究椎間盤分子運輸的螢光攝影系統。階段二、找出椎間盤內分子量對擴散效能的影響。 材料與方法：發展出一個合乎成本效益的螢光巨觀攝影儀器用以偵測椎間盤內的多醣分子。此儀器包含了一台移除紅外線截止鏡的數位相機(Canon 450D)，以及兩組用以偵測紅色與綠色螢光試劑的光源與濾鏡。並將所有元件組裝於31.5cm x 26.5cm x 55.5cm的暗房內。實驗用到的三組注射液使用了螢光素鈉(FS, 0.4 kDa, F6377, Sigma-Aldrich, Saint Louis, Missouri, USA)、四甲基異硫氰酸羅丹明-多醣共軛物(TRITC-dextran, 4.4 kDa, T1037, Sigma-Aldrich)、異硫氰酸螢光素-多醣共軛物(FITC-dextran, 40 kDa, FD40S, Sigma-Aldrich)三種螢光試劑。激發光源以及偵測用的濾鏡可適度調整用來偵測此三種螢光試劑的放射光。 實驗中調配了三種注射液。 A. FS (M=0.4 kDa, 100μM) B. FS (M=0.4 kDa, 100μM) + TRITC (M=4.4 kDa, 100μM) C. TRITC (M=4.4 kDa, 100μM) + FITC-Dextran (M=40 kDa, 100μM) 以六月齡豬隻的椎間盤單元72個，依上述注射溶液分為三大組別(8個，32個，32個)，組別一為Intact組，在組別二與組別三中則依外加載荷細分成四小組(Intact組、1小時420 N靜態載荷組、0.5小時190 N~590 N頻率5Hz循環載荷組、1小時190 N~590 N頻率5Hz循環載荷組)，各小組數量皆為8個，試樣照分類之組別以22號針經其腹側各打入0.25 ml注射液並施加各類載荷。完成之試樣以-20 ℃冷凍固定溶液的溶質分子，之後將試樣包埋作縱向切片，並以自製之螢光攝影平台拍攝相對應之螢光影像。後續以擴散面積DIArea、橫向擴散長度Lx,r.m.s、縱向擴散長度Ly,r.m.s、椎終板穿透指標DIEP等四項指標分析各個螢光影像。 結果：階段一中成功的建立一個合乎成本效益的螢光影像系統。在高感光度(ISO-1600)長時間曝光下，高溫將導致影像的熱燥訊。在組裝冷卻系統後，可將CMOS的工作溫度從 30 ℃降至5℃，並成功的減少熱燥訊(雜訊比率從5.5%降至0.2%)。本系統可以藉由更換濾鏡與相機鏡頭適度的調整光波段與對焦距離。階段二中，無載荷下的擴散面積(diffusion area)與靜態載荷、循環載荷引致的對流面積(convection area)在FS中有類似結果。FS的對流面積不受中分子大小溶質(如:TRITC)的存在影響。然而，TRITC的對流面積則會受到大分子溶質(如:FITC)的鉗制。1時靜態載荷與0.5小時循環載荷使FITC的運輸增加而TRITC亦如此。1小時靜態載荷與0.5小時循環載荷對大分子運輸有相似的效果。1小時循環載荷的運輸面積則比0.5小時的循環載荷來的小。 結論：本實驗中發現小分子(0.4 kDa)與中分子(4.4 kDa)溶質並不受外力引致的對流所影響。然而外加載荷引致的對流卻會影響大分子(40 kDa)的運輸。大分子對椎間盤內部孔洞(包含椎終板與椎間環)引致的阻塞效應鉗制住了中分子，然而並不確定是否對小分子溶質有影響。更多的現象，如螢光試劑於內部的對流機制以及更長時間靜態或循環載荷試驗的量值影響，則可再做進一步探討。Abstract Background: Intervertebral Disc (IVD) is the biggest avascular cartilage in human body. The degeneration of disc would results in the calcification of endplate and fiborsis of nucleus pulposus; which both affect the molecular transportation within disc. The degradation of solute transportation would induce a vicious circle of disc degeneration. The study of diffusion of nutrition exchange in IVD and convection due to daily activities can be helpful for the understanding of disc degeneration and treatment. Objective: Step I: Set up a fluorescent imaging system for the study of molecular transport within disc. Step II: Find the effect of molecular weight on the diffusion capability within the disc. Methods: A cost effective fluorescence macroscopic photographic apparatus to detect the dextran molecule within the disc matrix was developed. This apparatus includes one digital camera (Canon 450D) with removal of IR-Cut filter, and two paired light sources and filters to detect “red” and “green” fluorescent reagent. All these components are fitted into a box of 31.5cm x 26.5cm x 55.5cm. Three fluorescent reagents, Fluorescein sodium salt (FS, 0.4 kDa, F6377, Sigma-Aldrich, Saint Louis, Missouri, USA), Tetramethylrhodamine isothiocyanate–dextran (TRITC-dextran, 4.4 kDa, T1037, Sigma-Aldrich), Fluorescein isothiocyanate–dextran (FITC-dextran, 40 kDa, FD40S, Sigma-Aldrich) were used for three groups of solution. The light source for excitation and filter for detection are fine tuned for the detection of emission of these three fluorescent reagents. Three groups of solutions were formulated. The solution A includes the FS (0.4 kDa, 100μM) only. The solution B includes both FS (0.4 kDa, 100μM) and TRITC-dextran (4.4 kDa, 100μM), and the solution C includes both TRITC-dextran (4.4 kDa, 100μM) and FITC-dextran (40 kDa, 100μM). A 0.25 ml solute was injected into the center of disc before the loading. Four types of loading, which include no load, 1 hr 420 N creep loading, 0.5 hr, 5 Hz, 190 N to 590 N, and 1 hr, 5 Hz, 190 N to 590 N peak-to-peak fatigue loading were applied for these three groups of solutions. After the loading, the discs were cryopreserved. All specimens were sagittally cut in half using a diamond blade saw. The fluorescent images of specimens were photographed using the developed fluorescent photographic system. For the solution A, green light and filter is used to detect FS. For the solution B and C, the “green” and “red” lights and filters were used to differentiate the FS from TRITC, and FITC from TRICT. After the subtraction of disc and vertebrae images, the fluorophore was identified by the gray scale above 20. The area covered by the fluorophore was calculated to represent the penetration of solutions. Results: Step I. We successfully build a cost effective fluorescent imaging system. The noises due to high temperature are found under long-time exposure when using high ISO mode (ISO-1600). After assembling the cooling system, the working temperature of CMOS decreases from 30 ℃ to 5℃, and the hot noise is reduced (noise ratio: reduced from 5.5% to 0.2%). This system is capable for adjusting band wavelengths and focal length by changing the filters and lens. Step II. The diffusion area (no load) and convention area (creep and fatigue) of FS were similar. The convection area of FS is not affected by the existence of medium size solute i.e., the TRITC. However, the convection area of TRITC was entangled by the existence of larger solute, i.e., the FITC. The 1 hr creep and 0.5 hr fatigue loading increased the transport of FITC, hence the TRITC as well. The effect of 1 hr creep and 0.5 hr fatigue on the large solute transport was similar. The transport area of solute of 1 hr fatigue loading is smaller than the one of 0.5 hr fatigue loading. Conclusion: In this study, we found that the small (0.4 kDa) and medium (4.4 kDa) solute is not affected by the load induced convection. However, the external loading induced convention does affect the transport of large solute (40 kDa). The large molecule induces a steric hindrance within the disc space (including both endplate and anulus fibrosus) hence entangles the medium molecule. However, it is not sure if the large molecule would affect the transport of small one. Few more phenomena should be studied in the near future, for example, the inward flow mechanism of the fluorescent solute and the quantitative effect of longer creep or fatigue loading