氧化透明質酸/己二酸二醯生物水膠對防止脊椎手術術後神經沾黏效能研究

In Situ Forming Oxidized Hyaluronic Acid/Adipic Acid Dihydrazide Hydrogel for Prevention of Epidural Fibrosis After Laminectomy

胡名孝(Ming-Hsiao Hu)

指導教授 ： 林峯輝

國立臺灣大學/工學院/醫學工程學研究所/博士(2018年)

https://doi.org/10.6342/NTU201800278

摘要

脊椎狹窄(spinal stenosis)指的是脊髓(spinal cord)及脊椎神經根(nerve root) 因各個不同的病理原因受到擠壓，而它往往導致病人臨床上出現腰背痛，肢體疼痛，感覺麻木或異常以及肌力喪失等症狀。而脊椎的退化性疾病是造成脊椎狹窄最常見的原因，當中包括面關節(facet joint)退化和黃韌帶增生(ligamentum flavum hypertrophy)等病理性因素。脊椎狹窄的致病學理說明神經的傷害主要來自骨刺(bone osteophyte)和韌帶增生(ligamentous hypertrophy)的物理性壓迫，以及因局部血液供應不足或靜脈血液鬱積(venous stasis)所造成的缺血性脊髓神經病變。在病程進行當中，大多數病患可藉由藥物或物理治療等保守性療法控制病情。當病患有無法承受的疼痛或持續性神經學缺失，而保守性治療失敗時，便需要施行手術來解除神經的壓迫，進而緩解臨床症狀。傳統上, 藉著手術移除椎弓(lamina)、面關節、黃韌帶和椎間盤(intervertebral disc)可以完成脊椎神經減壓(decompression)。一般在手術結束前，手術醫師會仔細完成止血以及傷口清洗，接著便進行傷口縫合，而暴露的脊髓神經組織便會直接與周圍肌肉組織及血腫(hematoma)接觸。這樣的脊椎減壓手段已經行之有年，並被證明是一種有效率且穩定性高的治療方式。
然而，神經減壓手術儘管成效顯著，手術後傷口復原過程中導致的神經硬脊膜上纖維化(epidural fibrosis)與硬脊膜沾黏(dura adhesion)是卻是無法避免的。而這樣的結果被證明和手術後的遺留的神經痛或背痛息息相關。神經沾黏的成因起於術後癒合的過程中，疤痕組織(scar tissue)取代了脊膜上的正常脂肪組織，進而造成脊髓及脊椎神經根和脊柱前後周圍組織相連。日常生活中，當我們的脊椎做前彎(bending forward),後彎(backward)以及側彎(sideway)動作時，同時會造成脊髓形狀的改變。這樣的改變在正常人身上並不會造成任何不適。然而，在一位發生脊髓神經沾黏的病患身上，這樣的活動會導致神經過度的拉扯，進而導致臨床症狀。除此之外，當我們施行二次手術時，容易發生包括如硬脊膜破裂(dura mater tear)及神經根受傷等等併發症，而使病人手術風險提高，導致手術效果不彰。現階段臨床上針對發生症狀的病人，施行硬膜上注射(epidural injection) 合併經皮沾黏解離(adhesiolysis)被證實對於緩解疼痛有一定助益。然而包括硬脊膜破裂，感染以及導管相關併發症機率卻也相對較高。因此如何減少術後神經發生沾黏，是決定椎弓切除減壓手術成效的重要議題。
目前許多證據證明神經管周圍沾黏是來自脊旁肌(paraspinal muscles)內纖維結締組織(fibrous connective tissue)進入手術位置血塊(hematoma)所導致。在先前文獻中，研究者已經應用許多材料，這當中包括生物性的與非生物性的，其目的都是用來阻擋神經組織和血塊的接觸，進而減少癒合過程中纖維結締組織的過度形成。然而，大部分的材料並未獲得進一步的驗證，而使得實際應用於臨床上的屈指可數。我們認為，一個防止脊髓神經沾黏的理想材料需要能抑制纖維母細胞(fibroblast)增長及疤痕組織的形成。除此之外，方便臨床使用者操作亦十分重要。可注射性的生物水膠近年來應用在組織工程，生物載體及藥物釋放的研究上，它具備許多適合用做防沾材料的材料特性。初始的液態條件使得它容易操作，可以快速有效率的覆蓋不規則神經露出的手術部位，迅速膠連(gelate)的特性可確保穩定性，不用擔心流動問題。在先前的研究中，我們成功發展出氧化透明質酸/己二酸二醯生物水膠 (oxidized hyaluronic acid/adipic acid dihydrazide (oxi-HA/ADH) hydrogel)，研究成果顯示此水膠注入體內後可於3分鐘內成膠，並可以維持膠態約5周左右，而且具有良好的生物相容性。這些特性說明此水膠適合用來測試其對於防止沾黏發生的成效。本實驗的目的希望藉著細胞及動物活體實驗來評估此水膠對於脊椎周圍組織的生物相容性及預防脊髓手術後發生神經沾黏的效果。實驗的第一步，針對神經細胞，肌肉細胞及纖維母細胞等在脊椎環境中會與材料接觸者來測試氧化透明質酸/己二酸二醯生物水膠的生物相容性。我們發現水膠對於神經細胞株PC-12並無不利影響，但對於纖維母細胞株(NIH/3T3)有活性抑制能力。然而對於肌肉細胞株(C2C12)和神經膜細胞株(Schwann cell, RSC96)，儘管細胞毒性測試證實乳酸脫氫酶釋放無差異，但水膠的濃度差異卻稍微影響了這兩種細胞的粒線體活性。此外，聚合酶連鎖反應(polymerase chain reaction)顯示纖維母細胞的基因表現受到抑制。本實驗第二階段，我們利用大鼠動物模型，成功測試此水膠在活體中防止脊椎神經沾黏的功效。核磁共振及組織切片結果顯示，實驗組(在大鼠椎弓切除減壓部位使用水膠)的疤痕組織和硬脊膜(dura)沾黏程度均較對照組(僅施行大鼠椎弓切除減壓)減輕。術後六個月的模擬再手術實驗中，使用水膠組硬膜和疤痕組織間沾黏少，較易被分離。
根據上述實驗發現，氧化透明質酸/己二酸二醯生物水膠具有不錯的脊椎環境生物相容性。良好的液態成膠轉換特性(sol-gel transformation property)使得它容易操作，大大增加將來臨床使用的可行性。而在大鼠實驗中，水膠確實能減少神經管周圍沾黏。然而，在小動物有限的手術視野中，影響暴露神經組織發生沾黏的因素變異程度相對較高，再者於實際臨床環境中，評估是否發生沾黏不易取得評估上的直接證據，因此使用大動物實驗，盡可能在模擬人體實際手術環境下測試此水膠成效是未來研究的可能方向。

關鍵字

神經沾黏 ； 注射型水膠 ； 椎弓切除 ； 氧化透明質酸 ； 纖維母細胞

並列摘要

Spinal stenosis is a condition in which the spinal cord and the nerve roots are compressed by a number of pathologic factors, leading to symptoms such as pain, numbness, and weakness. The most common type of spinal stenosis is caused by degenerative arthritis of the spine. It is most commonly localized at the facet joints and ligamentum flavum. Many theories regarding the pathophysiology of spinal stenosis suggest a number of confluent mechanisms. The spinal cord can be directly compressed by bone osteophyte and ligamentous hypertrophy. Also, compression of local vascular structures can lead to ischemia of the spinal cord from arterial insufficiency and venous stasis. Operative treatment is indicated for patients with severe pain and constant neurologic symptoms, and in patients where conservative treatment has failed. Decompression by laminectomy which can be done alone or with additional fusion is the treatment of choice for most of spinal stenosis. Traditionally, laminectomy is used to explore the spinal canal and decompress the neural elements by removing the lamina, facet joints, ligamentum flavum and intervertebral disc during spinal surgery. At the end of the surgical procedure, wound will be closed with the dura sac or spinal cord exposed and contact to paravertebral muscle. Spinal decompression surgery has been proven to be an effective method to relieve neurologic symptoms and improve short term patients’ outcomes.

Post-operative epidural fibrosis is a biological response after laminectomy that may lead to clinical symptoms, such as radicular pain. During post-operative healing process, replacement of epidural fat and fibrotic tissue occur and it can bind the dura mater and the nerve roots to the anterior and posterior structures. Epidural fibrosis can cause symptomatic problems, such as radicular pain. Principles of neuromechanics can explain the role of epidural fibrosis. Bending the spine forward, backward, and sideways causes the dura to deform. Limb lifting causes the nerve roots to move, and the spinal cord itself moves up and down with flexion-extension of the neck or back. In a person with peridural scarring, however, in whom the dura and nerve roots are bound by the scarring, this binding could very well result in pain. In addition, epidural adhesion leads to difficulties during a revision surgery. The rate of complications, such as iatrogenic nerve root injury or dura mater tear, remains unpredictably high in revision surgeries. For symptomatic epidural fibrosis, epidural injections with percutaneous adhesiolysis showed a promising results in pain relief. However, the procedure-related complications including dura tear, infection, arachnoiditis and catheter retention are reported. Therefore, prevention of epidural fibrosis and adhesion is an imperative for a successful laminectomy.

A variety of biological, pharmacological and synthetic materials have been tested in vitro or used in vivo as space-occupying agents with inconsistent results. An ideal material for prevention of epidural fibrosis should be able to inhibit fibroblast adhesions and reduce formation of scar tissue. An injectable hydrogel would be the material of choice for this purpose, since it could fill an irregular surgical defect completely, gelate in situ and be delivered in a minimally-invasive manner. We show development of an injectable oxidized hyaluronic acid/adipic acid dihydrazide (oxi-HA/ADH) hydrogel in our previous study and this hydrogel seems a good candidate for prevention of epidural fibrosis. The objective of this study was to evaluate, in vitro and in vivo, the cytocompatibility and anti-adhesive effect of an oxidized hyaluronic acid/adipic acid dihydrazide (oxiHA/ADH) hydrogel. First, different cell types present in the spine were used to test the cytocompatibility of the hydrogel. The hydrogel extraction medium had no deleterious effects on neural cells (PC-12), but reduced fibroblasts viability (NIH/3T3). Although the hydrogel did not change the release of lactate dehydrogenase from myoblasts (C2C12) and Schwann cells (RSC96), the extraction medium concentration slightly affected the mitochondrial activity of these two cell types. qPCR showed that the hydrogel down-regulated S100a and P4hb expression in NIH/3T3 cells, supporting the hypothesis that the hydrogel might inhibit fibroblast activity. In the second part of our study, we used rat laminectomy model to test the effect of this hydrogel for prevention of epidural adhesion. It showed a reduction of scar tissue formation as well as severity of adhesion between scar tissue and the dura mater in the experimental animal model. Superficially, the peel-off test showed significantly decreased tenacity.

In conclusion, based on the experimental findings, the hydrogel had reasonable biocompatibility for neural cells, Schwann cells, fibroblasts and myoblasts. In a rat study, it showed a good sol-gel transformation property, so that it could be handled composedly and easily in order to cover the exposed neural structure in the laminectomy site. Also, it showed good antiadhesive characteristics. The oxi-HA/ADH hydrogel is an effective and promising injectable and thermosensitive material for prevention of post-operative epidural fibrosis.

並列關鍵字

injectable hydrogel ； thermosensitive ； hyaluronic acid ； epidural fibrosis ； laminectomy

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