椎間核與椎間環間之粒子大小對擴散效能的影響

Abstract

背景介紹：擴散作用是椎間盤內部養分與廢物代謝的主要方式之一，因此擴散作用與受傷椎間盤的自我修復能力有著重要的關聯性，而這些養分和廢物的擴散能力亦會受到椎間盤的退化程度與重組結構影響，但是目前對於這方面的研究尚不明朗。在退化性的椎間盤模型方面可注射生化藥劑進行模擬，例如使用胰蛋白酶改變椎間盤內的生物分子組成。另外，近年來許多藥劑被使用於恢復椎間盤的組成與功能，其中胜肽交聯劑的梔子素已被證實可使肽結構間彼此產生交聯反應，並且恢復椎間盤的力學性質。因此研究變性及交聯後的椎間環與椎間核的擴散效能，將有助於了解胜肽交聯劑在椎間盤再生治療方面的粒子傳輸效能。 研究目的：本研究的目的在於使用螢光影像分析技術評估經由變性及交聯後的椎間環與椎間核之粒子擴散效能變化。 材料與方法：本實驗所使用的椎間環與椎間核皆取自六個月大的豬隻腰椎。在實驗上，會使用自製手術刀將椎間環切成立方體形狀，另一方面則使用1c.c.的針筒來收集椎間核，每次實驗會將椎間核(約0.09克)擠入自製夾具中。試樣上，使用72組環向椎間環、72組徑向椎間環與72組椎間核，試樣總數為216組。而這三種形式的試樣又將進一步區分為三種狀態，分別為健康組，降解組與交聯組。為了模擬退化的試樣，將胰蛋白酶(0.25%, 0.5c.c.)注入椎間盤中並且於4℃與室溫環境下各反應12小時，接著將椎間盤剖開取出椎間核。椎間環則是於取下後，浸泡於胰蛋白酶溶液中並且於4℃與室溫環境下各反應12小時，接下來椎間環的準備步驟則是與健康組相同。交聯組方面，所有的試樣將先經由降解組的流程反應，接著將梔子素(0.33%，0.5c.c.)注入椎間盤中於室溫下反應24小時之後剖開取出椎間核，椎間環則是浸泡於梔子素中於室溫下反應24小時。本實驗使用三種不同分子量的螢光染劑進行測試，分別為螢光素鈉(FS，MW=376 Da)、四甲基異硫氰酸羅丹明-多醣共軛物(TRITC-Dextran，MW=4400 Da)、異硫氰酸螢光素-多醣共軛物(FITC-Dextran，MW=40000 Da)。試樣將被置於兩端設有水槽的夾具中，右側的上游槽將注入螢光染劑(100μL，100 μM)，左側的下游槽則注入相同體積的生理食鹽水。實驗中會使用自製的螢光攝影系統來測試60分鐘內每隔5分鐘上、下游槽螢光訊號的變化，再經由Fick’s 第一定律便可計算出擴散係數。

實驗結果：小分子量染劑(FS)的擴散係數都比中分子量(TRITC)與大分子量的染劑(FITC)來得高。環向椎間環的擴散效能比徑向椎間環與椎間核來得好。健康組的椎間環與椎間核之擴散係數都比降解組與交聯組的擴散係數來的高，然而降解組與交聯組之間的擴散係數並無顯著性差異。 結論：(1)染劑的分子量大小與擴散係數成反比關係，即分子量越大的染劑，其擴散係數將越小。(2)由環向椎間環的擴散係數大於徑向椎間環可知試樣的方向性會影響其擴散效能。(3)經過胰蛋白酶降解與梔子素交聯的試樣，兩者的擴散性會比健康組來得低。

Background：Diffusion is one of the nutrient and waste particles transportation mechanisms within intervertebral discs, and thus could be crucial for disc self-recovery from destructive injury. Disc diffusion capacity could be affected by the degeneration change and structural remodeling. However, the underlying mechanism is not well investigated yet. Disc degeneration can be modeled by introducing the biochemical stresses, e.g., trypsin, which may denature the biomolecular compositions within disc. Recently, many reagents have been studied in recovering the disc function. The genipin, a peptide crosslinker, is well proved to restore disc mechanical properties by forming crosslinking between the peptide structures. Determining the diffusion capacity of anulus fibrosus and nucleus fibrosus tissue after denaturation and crosslinking will help to verify the feasibility of peptide crosslinkers for disc regeneration therapy from the aspect of particle transportation function. Purpose：The purpose of this study is to evaluate the diffusion capacity of anulus fibrosus (AF) and nucleus pulposus (NP) after denaturation and crosslinking with fluorescent image analysis techniques Material and method：Specimens of AF and NP were harvested from healthy 6-month-old juvenile pigs. The AF was dissected by a home-made scalpel into a cubic shape and NP were store in a inject syringe (1cc.), and 0.09 g of NP was measured and squeezed into the home-made cubic slots. Seventy-two AF along the circumferential direction, Seventy-two AF along the radial direction, and Seventy-two NP (total two hundred and sixteen groups) specimens were prepared. The three types of specimens were further assigned into 3 groups; i.e., intact group, denatured group and crosslinked group. To simulate the denaturation, trypsin (0.25%, 0.5cc.) was injected into the disc and stored at 4℃ for 12 hours and at room temperature for 12 hours, and then the NP was obtained by dissecting the disc. The AF were first dissected and immerged in trypsin solution at 4℃ for 12 hours and at room temperature for 12 hours, then the AF were prepared using the same procedure as intact group. For the crosslinked group, all the specimen were prepared first using the denatured group protocol, and then the specimen were treated with genipin for 24 hours at room-temperature. The Fluorescein Sodium Salt (FS), Tetramethylrhodamine Isothiocyanate-Dextran (TRITC-Dextran) and Fluorescein Isothiocyanate-dextran (FITC-Dextran) were used in this study. Specimens were set in a channel with two sink at both end. The source sink at right hand side was filled with 100μL fluorescence dye at 100 μM intensity and the drain sink at left hand side was filled with same amount of saline. An in-house fluorescent photographic system was used to find the signal changes of both source and drain sink every 5 minutes for 60 minutes. The diffusion coefficient can be calculated from these images using Fick’s first law. Result：Smaller molecule (FS) always has higher diffusion coefficient than the medium (TRITC) and large (FITC) molecule. The diffusion coefficient of AF along the circumferential direction is higher than the AF along radial direction and NP. The diffusion coefficient of intact AF and NP is higher than the coefficient of denatured and crosslinked one. However, the difference between the denatured and crosslinked disc is not significant. Conclusion：(1) Increasing the particle size lead to decrease the diffusion coefficient. (2) Higher diffusion coefficient through circumferential specimen in comparison with radial one shows the effect of direction. (3) The diffusion coefficient decreased significantly after denaturation and crosslinking.