- 學位論文
原位成型氧化玻尿酸水膠於椎間盤髓核再生之應用
In Situ Forming Oxidized Hyaluronic Acid-Based Hydrogel for Nucleus Pulposus Regeneration
摘要
下背痛是病人求診骨科門診最常見的原因之一。儘管下背痛不會直接威脅到生命，但是該症狀會影響到個人的生活品質以及增加許多醫療及社會資源的支出。據統計，美國每年會有超過1000億美金的醫療支出花費在下背痛的治療上。大部的下背痛與末期的椎間盤退化(intervertebral disc degeneration)有關，老化、肥胖、抽菸、先天性基因變異或是從事體力勞動量較大的工作都是造成椎間盤退化的主要原因。服用止痛藥、進行椎間盤切除術或是脊椎融合手術是臨床上常見的治療方法，然而這些方法僅僅只能移除疼痛症狀而無法再生或是重建組織，因此如何有效地修復並重建椎間盤是目前醫學研究上一個重要的課題。 初期的椎間盤退化源於中心的髓核(nucleus pulposus)組織纖維化，而使得椎間盤失去減壓與吸震的能力。因此在本研究當中，我們嘗試開發出原位成型水膠以用於椎間盤髓核重建，實驗共分成三大部分。在第一部分的實驗，我們嘗試利用玻尿酸開發出原位成型水膠以用於椎間盤髓核之再生。玻尿酸先以高碘酸鈉進行氧化反應以獲得含有醛基的玻尿酸，稱之為氧化玻尿酸(oxidized hyaluronic acid)。該氧化玻尿酸可以進一步和已二酸二醯肼上的胺基反應交聯以形成具有黏彈特性的水膠。傅立葉轉換紅外線光譜分析儀和三硝基苯磺酸試劑用來分析氧化玻尿酸的結構以及氧化率。流變儀用來評估該水膠的操作性和黏彈特性。細胞的基因表現則是用即時聚合酶連鎖反應儀來分析。從實驗結果中可以發現該水膠注入體內後可於3分鐘內成膠，並可以維持膠態至少5周以上，而水膠的複變剪力模數數值較正常的髓核組織大一些。最重要的是該水膠有具良好的生物相容性，並且能協助椎間盤髓核細胞合成第二型膠原蛋白(COLII) 以及蛋白多醣 (AGG)。 第二部分的實驗，我們嘗試在先前的水膠系統內加入明膠以改善該水膠的細胞貼覆性以及黏彈特性。高分子量的玻尿酸(1900 kDa)先和不同濃度的明膠交聯，以形成玻尿酸-明膠高分子，之後再進行氧化反應以形成氧化玻尿酸-明膠高分子 (oxidized hyaluronic acid-gelatin)。最後，再將氧化玻尿酸-明膠高分子與已二酸二醯肼混和以形成氧化玻尿酸-明膠-已二酸二醯肼水膠。從流變實驗結果中可以發現該水膠的複變剪力模數(|G*|, complex shear modulus)數值(11~14 kPa)和正常椎間盤髓核組織(11.3 kPa)相似。螢光染色以及電子顯微鏡的結果中可以觀察到髓核細胞培養於不同明膠濃度的水膠內或是水膠上都具有貼附性，並且能以圓形的細胞型態存活。在含有高濃度明膠的水膠上，髓核細胞還能有顯著的增生表現。在細胞基質的修復上，我們發現了培養於水膠內的髓核細胞可以合成第二型膠原蛋白 (COLII) 以及蛋白多醣 (AGG)。此外，該細胞還能合成其他重要的信使核糖核酸像是SOX-9 以及缺氧誘導因子-1 (HIF-1α)。 為了要評估該水膠於臨床應用的可行性，實驗的最後一部分是動物實驗評估。大鼠的尾巴先以23號針頭於椎間盤中心處做穿刺以誘導椎間盤退化，並於誘導兩周後施打含有細胞之氧化玻尿酸水膠以及氧化玻尿酸-明膠水膠以重建退化的椎間盤。在給予水膠治療後的3周以及6周，利用核磁造影追蹤觀察椎間盤的退化及修復狀態，並於治療6周後犧牲動物以做後續的組織型態及染色分析。從核磁造影結果中可以發現有施打含細胞之水膠的椎間盤有較強的T2-weighted訊號，並呈現比較白的影像，而單純穿刺未給予任何治療的椎間盤則在穿刺8周後完全失去了訊號。從組織化學染色上也可以得到證實，單純穿刺的椎間盤在穿刺8周後完全退化，沒有任何的髓核組織存在，而原先外層整齊排列的纖維環嚴重坍塌並變得破碎。相較於退化的椎間盤，有施打含細胞之水膠的椎間盤，型態完整，中心髓核處具有許多細胞外基質以及髓核細胞，於髓核組織處亦能染到蛋白多醣(AGG)、第二型膠原蛋白 (COLII) 以及SOX-9。此外，在施打含細胞之氧化玻尿酸-明膠水膠的椎間盤髓核處還發現有均勻分布的緻密基質 (dense matrix)。從以上實驗結果中發現，含有細胞之氧化玻尿酸水膠以及氧化玻尿酸-明膠水膠具有再生椎間盤髓核的能力。 綜合以上所述，本研究結果顯示原位成型氧化玻尿酸水膠以及氧化玻尿酸-明膠水膠皆具有修復及再生椎間盤髓核之能力，因此該水膠應具備有相當的潛力應用在未來臨床治療上。
並列摘要
Low back pain (LBP) is one of the most common reasons for patients to visit or-thopedist. Although LBP is not life threatening, it affects the quality of life and contrib-utes to high medical expenditure. The total cost of LBP in the USA exceeds 100 billion dollars per year. It is believed most of the low back pain is associated with intervertebral disc degeneration. Aging, obesity, smoking, genetic or environmental factors are the major reason for intervertebral disc degeneration. Clinically, taking pain killer, discec-tomy and spinal fusion surgery are the most common treatments. These treatments can only relieve the symptoms, not repair the intervertebral disc. Therefore, how to repair the degenerated intervertebral disc is considered as an important issue. Early stage of the degeneration originates from the fibrosis of nucleus pulposus (NP) which is located in the center of the intervertebral disc. Hence, in this study, we try to develop an in situ forming hydrogel for NP regeneration. The whole experiment di-vided into three parts, in the first part, we try to develop an in situ forming hydrogel for NP regeneration by hyaluronic acid. Hyaluronic acid was first oxidized by sodium pe-riodate to form oxidized hyaluronic acid (oxi-HA), and then the oxi-HA can be further crosslinked with the amino groups of adipic acid dihydrazide to form gel matrix. Fourier transform infrared spectrometry and trinitrobenzene sulfonate assay are used to char-acterize the oxi-HA, and evaluate the oxidation degree. Rheometer is use to evaluate the working ability and the viscoelastic properties of the hydrogel. Real-time PCR is used for gene expression analysis. Results showed the hydrogel can transform from liquid form into a gel matrix under physiological conditions within 3 minutes, and maintain its gel state for at least 5 weeks. The complex shear modulus of the hydrogel is slightly higher than that of native nucleus pulposus. Importantly, the hydrogel can assist in NP cell synthesis of type II collagen and aggrecan mRNA gene expression, and shows good biocompatibility based on cell viability and cytotoxicity assays. In the second stage of the study, we try to introduce gelatin into previous system to improve the cell attachment ability and the viscoelastic properties of the hydrogel. First, high molecular weight (1900 kDa) hyaluronic acid was crosslinked with various con-centrations of gelatin to form hyaluronic acid-gelatin (HAG) polymers. Sodium perio-date was then oxidized HAG polymer to form oxidized HAG (oxi-HAG) polymer. The oxi-HAG polymer can be further crosslinked by adipic acid dihydrazide (ADH) to form oxi-HAG-ADH hydrogel. Results showed that the viscoelastic properties of the hydro-gels were similar to native tissue, as reflected in the complex shear modulus (11~14 kPa for hydrogels, 11.3 kPa for native NP). Cultured NP cells not only attached to the hy-drogels but also survived and maintained their round morphology. Besides, NP cells can also proliferate on the high gelatin content oxi-HAG-ADH hydrogel. In the aspect of matrix repair, we found that the hydrogels increased NP cell expression of several crucial genes, such as type II collagen, aggrecan, SOX-9, and HIF-1A. In order to assess the feasibility of the hydrogel for future application, the last part of the experiment is the animal study. Rat coccygeal intervertebral disc was stabbed by 23-gauge needle in the center of the disc to create the intervertebral disc degeneration. After two weeks, intervertebral discs were treated with NP cell contained oxi-HAG-ADH and oxi-HA-ADH hydrogels to regenerate the intervertebral disc. Mag-netic resonance imaging scan was used to monitor the degeneration and regeneration of the intervertebral disc after treatment for 3 and 6 weeks. Animals were sacrificed at 6 weeks after treatment for subsequent histological analysis including hematoxylin and eosin staining, Alcian blue staining and immunohistochemistry of aggrecan, type II col-lagen, SOX-9, and S-100. Results showed the successful repair of the degenerated in-tervertebral disc on T2-weighted signal intensity in the hydrogel treatment intervertebral discs. In the stab-only group, no T2-weighted signal intensity can be detected at 8 weeks after needle-stab. Similar results can be found in histochemical staining. No nucleus pulposus cells can be found in the stab-only intervertebral disc and the outer annulus ring collapsed. For hydrogel treated intervertebral discs, lots of extracellular matrix and cells can be found in the nucleus pulposus. Immunohistochemistry also showed positive findings for aggrecan, type II collagen, and SOX-9. Besides, there are some well-distributed dense matrix can be found in the oxi-HAG-ADH hydrogel treated intervertebral disc. From the results of animal study, we found the cell contained oxi-HA-ADH and oxi-HAG-ADH hydrogel can assist NP regeneration in vivo. In conclusion, the developed in situ forming oxidized hyaluronic acid-based hy-drogel (including oxidized hyaluronic acid hydrogel and oxidized hyaluronic ac-id-gelatin hydrogel) has the potential to perform as a suitable material for the repair of nucleus pulposus. It should be a promising hydrogel for nucleus pulposus regeneration, especially, for the treatment of early stage intervertebral disc degeneration.