可注射式氧化透明質酸/己二酸二醯肼水膠做為人工玻璃體之應用

Abstract

眼球玻璃體(vitreous body)是一種眼球內容物，為無色透明的膠狀物質，主要成份由膠原蛋白纖維、透明質酸、葡萄氨聚糖及水所組成，人類玻璃體體積約4ml，重量約4克，佔眼球內腔體積的二分之三。玻璃體會因老化、變性退化或其它相關眼球病變而受損，包括透明質酸變性或膠原蛋白纖維析出而產生玻璃體基質液化，進而阻礙視線或產生視網膜剝離現象，嚴重時對於這部份的病變往往必須用玻璃體切除手術才能治療。

玻璃體切除術(vitrectomy) 是臨床上常用來處理眼球相關疾病的手術之一，包括了糖尿病視網膜病變(diabetic retinopathy)，視網膜剝離(complex retinal detachment)或黃斑裂孔(macular hole)。患者接受玻璃體切除術，大部份的玻璃體會被移除，而人工玻璃體替代物主要是用於填充手術切除玻璃體後所留下之眼球空腔，避免眼球塌陷及幫助視網膜貼附。目前臨床使用上主要是以氣體或矽油當成人工玻璃體替代物，以氣體做為玻璃體替代物時，由於氣體的密度低於人體體液，因此患者必須在手術後保持臉部朝下的姿勢長達數天至數週之久，對患者的生活品質造成相當大的影響。為克服此一缺點，自1960年代以來，矽油(silicone oil)開始做為人工玻璃體替代物，用於某些無法維持特定姿勢的患者，例如小孩。然而許多研究指出，矽油長期植入後若未移除則可能造成長期併發症，也可能會對鄰近組織產生細胞毒性。也有相當多的研究嘗試開發出各種材料以作為人工玻璃體替代物。然而，到目前為止尚未有適合的人工玻璃體材料可以完全符合手術的需求。 在本研究中，我們使用氧化後的透明質酸及己二酸二醯肼為材料，開發出一個透明無色且可注射式的水膠做為人工玻璃體替代物。透明質酸是眼球玻璃體的主要成份之一，研究中我們利用高碘酸鈉(NaIO4)來氧化透明質酸，使透明質酸的乙醯氨基葡糖(N-acetyl-D-glucosamine)以及葡糖醛酸(D-glucuronic acid)開環而形成醛基(aldehyde group)，並藉由傅立葉轉換紅外線光譜分析儀進一步確定醛基之形成。透明質酸開環產生的醛基可與己二酸二醯肼上的胺基產生交聯反應而形成透明無色且具彈性之水膠。氧化透明質酸/己二酸二醯肼水膠具有良好的光學性質，其折射率(refractive index, RI)介於1.3420 and 1.3442之間，與人體眼球玻璃體之折射率1.3348近似，可避免眼球屈光參數改變，對患者視力的影響較小，並且有利於術後的眼底檢查和治療。此外，經流變分析結果証實，氧化透明質酸與己二酸二醯肼混合後可在低溫環境下維持液態長達8分鐘以上，有利於手術過程之注射。在注射入人體之後，由於溫度上升的影響，氧化透明質酸與己二酸二醯肼混合物可迅速形成固態水膠，避免滲漏情形發生，並緊密填充於眼球內腔。從體外生物降解測試結果顯示氧化透明質酸/己二酸二醯肼水膠可在含有濃度10,000 unit/ml溶解酵素(lysozyme)之生理食鹽水中維持至少5週以上。並且在體外生物相容性評估，以視網膜色素上皮細胞(retinal pigment epithelial cells)測試結果也証實此水膠有良好的生物相容性，且無明顯細胞毒性產生。綜合以上結果顯示，透明質酸/己二酸二醯肼水膠外觀為透明無色並具有適當的折射率，且能以注射方式進行手術，在未來應用於人工玻璃體替代物應具有良好的發展潛力。在動物實驗評估中，注射氧化透明質酸/己二酸二醯肼水膠的眼球，藉由眼球內壓力量測、角膜厚度量測、裂隙燈觀察、視網膜電圖及組織切片評估，在術後四週的結果顯示與正常眼球比較並無明顯異常情形。 綜合以上所述，本研究結果顯示氧化透明質酸/己二酸二醯肼水膠應為具有相當潛力的人工玻璃替代材料。

The vitreous body is a clear, transparent gelatinous substance in the vitreous cavity of the eye that is posterior to the lens and anterior to the retina. It occupies two-thirds of the ocular volume, with a weight of approx. 4 g and a volume of about 4 ml. The main components of vitreous body include water (98%), collagen fibrils, glycosaminoglycans, hyaluronic acid (HA) and other rest solutes. Specific diseases, age-related degeneration or trauma can lead to pathological changes in the vitreous body, including HA degeneration and collagen precipitation, which result in liquefaction of the matrix [5]. A degenerated or liquefied vitreous body will lead to floater formation and eventually result in posterior vitreous detachment and possible retinal detachment. Among clinical treatments, pars plana vitrectomy (PPV) is one of the most important surgeries for treating a number of ocular-related diseases, including diabetic retinopathy, complex retinal detachment (for example, due to trauma) and macular hole, during PPV, the vitreous body is cut and aspirated, and then is typically replaced with a vitreous substitute, such as gas (air, perfluropropane or sulfur hexafluoride) or silicone oil. Vitreous substitutes are used to fill vitreous cavity and help reattach the retina after vitrectomy surgery. Postoperatively, a vitreous substitute can keep the retina in position while the adhesion between the retina and the retinal pigment epithelium (RPE) cells are formed. Gases, which are lighter than water, are useful for flattening a detached retina and keeping it attached while healing occurs. However, it is frequently necessary to maintain a face-down position following surgery for a week or more when gas is used. Since the 1960s silicone oil is sometimes used instead of gases to keep retina attached postoperatively in complicated retinal detachments, or in patients unable to position postoperatively (e.g., children), but long-term complications can occur if the silicone oil is not removed later. Besides, silicone oil also seems to be cytotoxic to ocular tissues, such as corneal endothelial cells In the present study, we developed a colorless, transparent and injectable hydrogel with appropriate refractive index as a vitreous substitute. The hydrogel is formed by oxidated hyaluronic acid (oxi-HA) cross-linked with adipic acid dihydrazide (ADH). Hyaluronic acid (HA) was oxidized by sodium periodate to create aldehyde functional groups, which could be cross-linked by ADH. The refractive index of this hydrogel ranged between 1.3420 and 1.3442, which is quite similar to human vitreous humor (1.3345). Rheological properties were measured to evaluate the working ability of the hydrogel for further clinical application. The oxi-HA/ADH in situ forming hydrogel can transform from liquid form into a gel-like matrix within 3–8 min, depending on the operational temperature. The degradation tests demonstrated that the hydrogel could maintain the gel matrix over 35 days, depending on the ADH concentration. In addition, the cytotoxicity was evaluated on retina pigmented epithelium (RPE) cells cultivated following the ISO standard (tests for in vitro cytotoxicity), and the hydrogel was found to be non-toxic. In the animal study, the oxi-HA/ADH hydrogel was injected into the vitreous cavity of rabbit eyes. The evaluations of slit-lamp observation, intraocular pressure, cornea thickness, electroretinography (ERG) and histological examination showed no significant abnormal biological reactions for 4 weeks. This study suggests that the injectable oxi-HA/ADH hydrogel should be a potential vitreous substitute.