利用白芨多醣體合併白藜蘆醇浸漬雙層海綿作為預防脊椎椎板切除術後硬脊膜外抗纖維化作用之效能研究

Antifibrotic Effect of Bletilla Striata Polysaccharide-Resveratrol-Impregnated Dual-Layer Carboxymethyl Cellulose-Based Sponge for the Prevention of Epidural Fibrosis After Laminectomy

陳宣佑(Hsuan-Yu Chen)

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(2021年)

https://doi.org/10.6342/NTU202102328

摘要

當病人患有嚴重背痛和持續神經痛經保守治療無效後，脊椎椎板切除術是一種常見且能有效治療例如椎間盤突出症、脊柱狹窄和腫瘤切除等脊椎疾病的手術方式。脊髓和神經根受壓會導致疼痛，麻木和無力等症狀。血管結構受阻會導致動脈供血不足和靜脈淤滯導致脊髓缺血。脊椎椎板減壓手術過程需要去除椎板，小關節，黃韌帶和椎間盤來減壓神經脊隨，手術後暴露的脊髓神經組織便會直接與周圍肌肉組織及血腫接觸。儘管手術效果顯著且穩定性高，且能有效減輕神經壓迫所造成的痛楚，但仍有8％–40％的患者在脊椎椎板切除術後開始有脊椎手術後疼痛症候群，其中4％–9％脊椎手術後疼痛症候群的患者甚至嚴重到需要再次接受脊椎翻修手術。
疤痕組織形成過程是創傷反應造成誇大和失調的“癒合”機制，最終導致一系列局部急性炎症，細胞外基質沉積，纖維化和新血管生成。硬脊膜外纖維化的發病機制中涉及多個因素，例如個體差異，傷口癒合過程，血腫形成，軟組織和骨頭創傷多寡。復原過程中，疤痕組織取代了硬脊膜上的正常組織，導致硬脊膜與周遭軟組織纖維化，當背部彎曲或伸展的動作時會造成周遭軟組織收縮進而刺激或壓迫神經，而這樣的結果被證明和手術後遺留的神經痛或背痛息息相關，上述纖維化可能佔所報告的脊椎手術後疼痛症候群病例約20％–36％，因為手術切除纖維組織不易，會增加傷口癒合不良, 感染和硬脊膜破裂等相關手術併發症的風險，且翻修手術後的症狀改善度難以預測。因此預防硬膜外纖維化的成形，是決定椎弓切除減壓手術成效的重要議題。
已有各種臨床策略來減輕硬脊膜外纖維化生成，包括微創手術，放射療法，藥物治療和生醫材料放置。新型生物可降解的聚合物材料已被用作佔據術後空間屏障，可以透過限制纖維母細胞的過度增殖和減輕硬膜外纖維化的形成來減少脊椎手術後疼痛症候群的發生機率。因此，本研究欲藉由製成可生物降解的雙層海綿概念，在接觸硬脊膜側以含有羧甲基纖維素-白芨多醣體-白藜蘆醇（CMC-BSP-RES）來抑制椎板切除術後的硬膜外纖維化的形成，在接觸周遭軟組織側以羧甲基纖維素為基底維持正常癒合機制。體外實驗首先針對纖維母細胞，肌母細胞，神經細胞和許旺細胞等在模擬脊椎環境中會接觸的細胞來測試與CMC-BSP-RES雙層海綿的生物相容性。在不同濃度萃取的CMC-BSP-RES雙層海綿培養皿中四個細胞系均顯示出良好的生存力，而細胞毒性沒有顯著差異。在CMC-BSP-RES雙層海綿中培養的纖維母細胞NIH / 3T3中，S100a4和P4hb的mRNA表現下調，這顯示該海綿可能抑製纖維母細胞的活性。動物實驗中對大鼠進行脊椎椎板切除術並且在隨機分配下指派有或沒有使用CMC-BSP-RES雙層海綿覆蓋硬脊膜。在術後8周執行核磁共振成像分析，剝離手術和組織病理學切片對硬脊膜外和疤痕組織之間的纖維化進行定性評分。在核磁共振影像上未看到手術部位的硬脊膜有不正常的術後收縮或擴張；剝離試驗中硬脊膜和纖維化組織之間沾黏少且相較於未使用CMC-BSP-RES雙層海綿組較易被分離；組織病理切片結果進一步證實，CMC-BSP-RES雙層海綿組的平均成纖維母細胞數量明顯減少。
根據上述實驗結果，在神經側含有羧甲基纖維素-白芨多醣體-白藜蘆醇（CMC-BSP-RES）且可生物降解的雙層海綿設計，在體外脊椎細胞環境下有良好的生物相容性，在大鼠實驗中能減少纖維母細胞數量，CMC-BSP-RES雙層海綿可有效減輕脊椎椎板切除術後硬脊膜外纖維化的成形。

關鍵字

硬脊膜外纖維化 ； 椎板切除術 ； 白芨多醣體 ； 羧甲基纖維素 ； 白藜蘆醇

並列摘要

Laminectomy, indicated for patients whose conservative treatment has failed with severe back pain and constant neurologic symptoms, is a surgical procedure typically applied in the treatment of numerous spinal disorders, such as lumbar disk herniation, spinal stenosis, and tumor excision. Compression of the spinal cord and nerve roots will lead to symptoms such as pain, numbness, and weakness. Compression of vascular structures can lead to ischemia of the spinal cord from arterial insufficiency and venous stasis. Except removing the pathology, laminectomy needs to decompress the neural elements by removing the lamina, facet joints, ligamentum flavum and intervertebral disc. Dura sac or spinal cord will be exposed contacting to paravertebral muscle after operation. Although this procedure is generally effective and relieves neural compression, 8%–40% of patients suffer from failed back surgery syndrome (FBSS) post-laminectomy, with 4%–9% FBSS patients receiving revision surgery.
The scar tissue formation process exaggerated and dysregulated “healing” mechanisms, as a response to trauma, it culminates to a series of local acute inflammations, extracellular matrix deposition, fibrosis, and neo-angiogenesis. Several factors, such as individual variability, the wound healing process, hematoma, the amount of soft tissue and bone traumatization, are involved and have important roles in the pathogenesis of epidural fibrosis. The epidural fibrosis potentially contributes to 20%–36% of the reported FBSS cases, by inducing nerve irritation, dura sac compression, and back or radicular pain (especially with movements including the flexion and/or extension of the back) and increases the technical difficulties and risks associated with revision surgery. The goal of treatment is to prevent epidural fibrosis formation because surgical removal of fibrous tissue is often associated with poor results.
Various clinical strategies have been introduced to attenuate epidural fibrosis, including minimal invasive surgery, radiotherapy, drug treatment, and local material placement. Novel biodegradable polymeric materials have been used as space-occupying barrier to inhibit fibroblast adhesions and reduce formation of scar tissue.
An ideal barrier to prevent or reduce adhesions between dura sac and paravertebral soft tissue should mimic the physiologic features of original epidural fat. Barrier materials are meant to avoid the formation of scar tissue between them and decrease the dead space. The application of antifibrotic materials can alleviate epidural fibrosis by restricting the exaggerated proliferation of fibroblasts and by mitigating scar tissue formation. A dual-layer biodegradable carboxymethyl cellulose (CMC)-Bletilla striata polysaccharide (BSP)-resveratrol (RES) sponge was fabricated to inhibit scar tissue formation after post-laminectomy surgery in this study. Fibroblast cell line NIH/3T3, myoblasts C2C12, neural cells PC-12, and Schwann cells RSC96 were first used to evaluate the in vitro cytocompatibility of dual-layer CMC-BSP-RES sponge. Laminectomy on Sprague Dawley rats randomized with/without the application of dual-layer CMC-BSP-RES sponge were performed. The severity of fibrosis between the dura mater and the formed scar tissue was qualitatively scored by magnetic resonance imaging (MRI) analysis, peel-off surgery, and histopathological examination. All four cell lines exhibited good viability with no significant difference in cytotoxicity when cultured with variable extractions of dual-layer CMC-BSP-RES sponge. The mRNA expressions of S100a4 and P4hb were down-regulated in fibroblast NIH/3T3 cultured in the CMC-BSP-RES sponge. Thereby implying that this sponge potentially inhibits fibroblast activity. No post-operative shrinkage or expansion of the dura mater along the surgical site were detected through MRI. The peel-off tests revealed that the tenacity of adhesion between the dura mater and scar tissue was decreased. Histopathological examinations further verified that the average number of fibroblasts in the dual-layer CMC-BSP-RES group was considerably decreased.
In conclusion, dual-layer CMC-BSP-RES sponge is a biocompatible and effective material for alleviating the development of post-operative epidural fibrosis and mitigating fibroblast expression following laminectomy.

並列關鍵字

epidural fibrosis ； laminectomy ； Bletilla striata ； carboxymethyl cellulose ； resveratrol

參考文獻

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