以功能性生醫材料開發人類眼角膜內皮細胞層片之組織工程及其再生醫學之應用

Functional Biomaterials for Human Corneal Endothelial Cell Sheet Engineering and Regenerative Medicine

賴瑞陽(Jui-Yang Lai)

指導教授 ： 薛敬和

國立清華大學/工學院/化學工程學系/博士(2006年)

https://doi.org/10.6843/NTHU.2006.00232

摘要

由於人類眼角膜內皮細胞在體內具有不會分化生長的特性，正常角膜內皮組織的細胞密度自出生後即不斷減少。當密度低於每平方毫米1000個細胞時，角膜內皮組織的排水生理功能將無法發揮，進而導致角膜水腫混濁及視覺喪失。在目前臨床上，全層角膜移植術為治療角膜內皮組織病變的主要方式。然而，捐贈角膜之來源不足與全層角膜移植的術後相關併發症仍是此法應用時必須克服之瓶頸。因此，以體外大量培養之人類眼角膜內皮細胞進行受損角膜內皮組織的單層置換乃一種極具潛力的替代方案。本研究之目的即是藉由製備與移植生醫工程的人類眼角膜內皮細胞層片，以開發一新穎性治療法進行眼組織重建。
研究首先根據電漿化學反應設計一套兩階段的材料表面改質方法，以製備感溫性聚異丙基丙烯醯胺高分子接枝表面，並應用於人類眼角膜內皮細胞之培養。進一步藉由外界溫度改變所產生的熱刺激達到有效控制細胞於培養界面上之貼覆與分離。經由能量分散式X射線光譜學、霍氏全反射紅外線光譜學、原子力顯微鏡學及靜態接觸角量測等表面鑑定分析，聚異丙基丙烯醯胺高分子於材料表面之最佳接枝量為每平方公分1.6微克。此外，導入丙烯酸高分子鏈段作為材料接枝的間距分子將可有效加速細胞於培養表面的分離，並防止細胞因過度低溫處理而衍生之不良效應。
接續探討以感溫性培養基材所製備之生醫工程人類眼角膜內皮細胞層片是否具有作為天然組織替代物之可行性。將來自眼庫捐贈組織的成體人類眼角膜內皮細胞在37°C下培養於感溫性高分子接枝表面。三週後，長滿之細胞可經由降溫至20°C而脫離培養表面，並獲得一層片狀組織。此生醫工程細胞層片的體外性質以存活率試驗、掃瞄式電子顯微鏡學、免疫組織化學及組織學進行評估。結果指出在與眼庫角膜內皮組織比較下，人類眼角膜內皮細胞層片於形態、結構、活性與功能等方面均有相似特徵，適合作為天然組織替代物。
由於細胞層片組織相當柔軟易碎，本研究亦設計一多功能水膠載體傳輸系統，以進行生醫工程人類眼角膜內皮細胞層片之眼內移植應用。實驗採用不同等電點(5.0或9.0)及分子量(3至100 kDa)的動物明膠為原料，製備水膠載體並接受γ射線照射消毒。藉由機械性質、含水率、分解率與細胞相容性等測試進行各種水膠載體之功能性評估。結果顯示具有等電點5.0及分子量100 kDa的動物明膠最適合作為細胞層片移植治療之水膠載體與發展穩定的眼內傳輸系統。
在體內試驗方面，本研究以兔子為動物實驗模式，進行生醫工程人類眼角膜內皮細胞層片之眼內移植治療可行性分析。經由臨床觀測與病理組織切片檢視，人類眼角膜內皮細胞層片在體內能夠順利貼覆結合於宿主病變組織上，並有效發揮其細胞特有生理功能。與僅製造眼角膜內皮傷口的對照組相較下，動物經植入細胞層片後，其受損角膜之病態水腫及其澄清度均可獲得大幅改善。此外，兔子眼角膜厚度幾乎恢復至原始狀態，也意謂著植入的細胞層片組織在體內確實能夠展現功能。這些實驗結果指出具有良好結構與功能之生醫工程細胞層片相當適合應用於眼角膜內皮組織修復。
基於上述研究發現，利用感溫性培養界面與多功能水膠載體，以製備及移植生醫工程人類眼角膜內皮細胞層片，能夠建立一套角膜內皮細胞治療之新策略。此外，功能性生醫材料在人類眼角膜內皮細胞層片組織工程及其再生醫學之開發極具潛力。最後，本研究希望此細胞層片新療法未來能有效改善角膜內皮相關病變，並應用於眼組織再生重建與臨床工程。

關鍵字

生醫材料 ； 細胞層片組織工程 ； 再生醫學 ； 感溫性聚異丙基丙烯醯胺接枝培養表面 ； 多功能性動物明膠傳輸載體 ； 人類眼角膜內皮層 ； 電漿化學 ； 眼科學 ； 移植

並列摘要

Human corneal endothelium in vivo demonstrates an age-related decrease in cell density and cannot be compensated due to its limited regenerative capacity. When the cell density is less than a critical level of 1000 cells/mm2, the endothelial monolayer no longer functions, causing corneal edema and loss of visual acuity. Penetrating keratoplasty (PK) is currently the common way to treat corneas that are opacified due to endothelial dysfunction. However, insufficient supplies of donor corneas and several complications associated with PK remain a worldwide problem. Therefore, transplantation of in vitro cultured human corneal endothelial cells (HCECs) to replace damaged corneal endothelium alone is a promising alternative to PK. In this study, we developed a novel therapy technique to fabricate and transplant cultured HCEC sheets for corneal endothelial reconstruction.
On the basis of plasma chemistry, we have designed a two-step method to prepare a thermo-responsive poly(N-isopropylacrylamide) (PNIPAAm)-grafted culture surface for controlling HCEC adhesion and detachment via a thermal stimulus. The results of surface characterization including energy-dispersive X-ray spectroscopy (EDX), attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), atomic force microscopy (AFM), and static contact angle measurements show that an optimal grafting amount of PNIPAAm is 1.6 μg/cm2. We have also demonstrated that the introduction of AAc segment as short spacers onto the culture support can accelerate the cell detachment, which is beneficial to protect these harvested cells from functional damage.
We further investigated whether the bioengineered HCEC sheets harvested from thermo-responsive culture supports could be used as biological tissue equivalents. Untransformed adult HCECs derived from eye bank corneas were cultivated on PNIPAAm-grafted surfaces for 3 weeks at 37°C. Confluent cell cultures were detached as a laminated sheet by lowering culture temperature to 20°C. In vitro characteristics of HCEC sheets were evaluated by viability, scanning electron microscopy, immunohistochemistry, and histology. Similar to the native corneal endothelium from eye bank donors, the fabricated HCEC monolayers having normal morphology, structure and viability are suitable to be used as tissue replacements for transplantation.
Because of the soft and fragile nature of bioengineered HCEC sheets, we have designed and developed a multi-functional hydrogel carrier system for intraocular delivery of these sheet grafts. The functionality of gamma-sterilized cell carriers made from raw gelatins with a different isoelectric point (IEP = 5.0 and 9.0) and a molecular weight (MW) range from 3 to 100 kDa, was investigated by the determination of mechanical properties, water content, dissolution degree, and cytocompatibility. The results of our study indicate that the gamma-sterilized hydrogel discs consisting of raw gelatins (IEP = 5.0, MW = 100 kDa) are promising candidates as cell sheet carriers for effective corneal endothelial cell transplantation and therapy.
In the in vivo tests, we evaluated the feasibility of HCEC transplantation by harvesting the cell sheets from the thermo-responsive culture supports and delivering with multi-functional gelatin hydrogel discs in a rabbit model. We have shown that the transplanted HCEC sheets could be integrated into rabbit corneas denuded of endothelium. Additionally, when endothelium alone was removed, the rabbit corneas became cloudy and remained opaque throughout the course of the experiment. Once receiving tissue-engineered HCEC sheets, the corneas have returned to a nearly normal thickness. These results imply the biological function of transplanted cell sheets. Our findings indicated that a well-organized and functional HCEC sheet is feasible to be used as tissue equivalents for replacing compromised endothelium.
In the present study, we have demonstrated that the bioengineered human corneal endothelium fabricated from thermo-responsive culture supports and delivered by multi-functional hydrogel carriers can potentially offer a new therapeutic strategy for corneal endothelial cell loss. In addition, functional biomaterials have great potential for development of HCEC sheet engineering and regenerative medicine in ophthalmology. We hope this work will lead to insights into cell sheet-based therapy for corneal endothelial dysfunction and will open an exciting new door to the future.

並列關鍵字

Biomaterials ； Cell sheet engineering ； Regenerative medicine ； Thermo-responsive poly(N-isopropylacrylamide)-grafted culture surface ； Multi-functional gelatin carrier ； Human corneal endothelium ； Plasma chemistry ； Ophthalmology ； Transplantation

參考文獻

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