應用奈米結構表面探討細胞介面的貼附行為與功能性表現

Application of nanostructured interface to estimate the cell behaviors and functional expression for biomedical engineering

潘叙安(Hsu-An Pan)

指導教授 ： 黃國華

國立交通大學/工學院/奈米科技研究所/博士(2012年)

https://doi.org/10.6842/NCTU.2012.00317

摘要

在細胞生長的過程中，細胞本身具有感知外在各式各樣的化學或物理信號並整合與分析這些外在環境訊息的能力，進而改變其細胞形態及生長行為模式。此外大多數細胞於偵測環境表面適於生長後，則開始進行貼附行為並表現該細胞的生長模式與型態變化，因此對於細胞貼附基質的特性研究將是發展細胞工程中重要的一環。目前在仿生基材的研究成果已廣泛應用於醫學領域，而最近的研究報告指出，微米結構化的表面具有模仿天然細胞外基質的特性，能夠促進細胞生長和分化，但細胞外基質是由微米與奈米結構所交織組成的天然結構表面，而目前細胞對奈米結構的反應與行為機制仍處於摸索階段。為了進一步研究微米級細胞對於奈米級結構表面的感知與行為反應，本研究利用直徑從10到200奈米的奈米點陣列探討各式不同類型的細胞型態與行為，以評估細胞對奈米尺度的反應。奈米點陣列以陽極氧化鋁為模板，製造於鍍上TaN的矽晶圓上，並濺鍍約 5奈米厚的白金層以提供一個同質性與良好的生物相容性表面。
為了探討微米級細胞與奈米級基材之間的作用反應，我們將觀察細胞如何透過貼附奈米結構基材，並展現其生長型態的變化與胞內的功能機轉，本研究中分為四個部分:
本論文研究第一部分主要探討利用系統化的奈米尺度點陣列，範圍從10奈米到200奈米的奈米點陣列，探討3T3纖維母細胞貼附行為與生長條件。我們發現以奈米點陣列的基材表面可能會誘導3T3纖維母細胞異常的凋亡。異常的細胞形態在培養於200奈米的點陣列24小時候被觸發，細胞自本體延展的偽足數目明顯的減少。在利用纖維連接蛋白與第一型膠原蛋白預塗層處理後，則可以有效預防奈米拓樸結構所引發的程序性細胞死亡，因此奈米結構觸發細胞異常凋亡的調控程序主要是透過Focal adhesion的形成。
在本論文研究的第二部分，利用Cardiomyoblast (H9c2)培養於直徑介於10和200 nm的奈米點陣列上進行觀察。我們發現H9c2細胞在50奈米的奈米點陣列上具有廣泛的偽足延展與面積分佈，並具有極佳的增生能力。相對於大尺寸奈米點陣列，100奈米與200奈米則減少了53.7％和72.6％的細胞增生數目，並生成較少的貼附蛋白與細胞骨架。而細胞纖維化和心肌肥大相關基因在培養於100奈米的細胞基因表現分析中則具有顯著的提升，此外，在胞內蛋白質分析中，培養於50奈米的細胞則具有大量Vinculin與PAI-1蛋白表現。根據這些分析結果我們可以藉由調整奈米點的直徑，調節Cardiomyoblast的增生與相關功能性基因及蛋白質的表現。
在本論文的第三部分，我們運用分析系列尺度的奈米點結構對於成骨細胞在體外培養的反應，以提供牙根植體表面優化的策略與設計依據。在體外培養分析中，利用直徑範圍從10奈米到200奈米的點陣列進行對類成骨細胞(MG63)的細胞活性，自體凋亡，貼附行為以及細胞骨架組成進行評估。我們發現50奈米點陣列相對於平面結構增加了44％的細胞活性，減少2.7％細胞凋亡程度，並促進30％的肌動蛋白纖維束增長，同時增加了73％的細胞貼附程度，此外也加強了約50%的細胞礦化程度。在本研究中，我們應用類成骨細胞模型系統可以評估出最佳的奈米點結構表面，有利於改善牙根植體表面的設計以達到最佳的生物相容性。
在本論文的最後一部分，我們製備了一個具有快速分析並調控癌細胞的增生，凋亡，侵襲能力以及胞內骨架重組的奈米表面平台。透過從一個平面至10奈米，50奈米，100奈米和200奈米點陣列所組成的奈米點矩陣，我們能夠有效區分出子宮頸癌細胞HeLa與較後期的C33A的細胞侵襲能力。在各類型的卵巢癌細胞株（ES2, PA-1, TOV-112D, TOV-21G）體外培養分析中，癌細胞本身的類型與相關期數也表現出相對差異性的生長模式。因此透過簡單與低成本的製造過程，我們所建製的奈米點矩陣平台可有效用於評估各類型細胞生長的基本特徵，並能夠區分不同期數與類型的癌細胞，同時也可以提供人工植入物的基本設計參數，因此本研究中的奈米點陣列平台將可作為一個方便且快速的癌症檢測工具。

關鍵字

奈米結構 ； 細胞貼附 ； 奈米點 ； 生醫工程

並列摘要

Cells sense and respond to a wide range of external signals, both chemical and physical. Extra-cellular information penetrates cell membrane, transmits through cytoskeleton, affects muscle genes expression, and alters the cytoskeleton organization. Eventually, cell morphology is changed leading to better growth or death. The modification of biomimetic substratum has been wildly used to study the effects of bio-nano interface on cell adhesion and subsequent growth and function. Micro-structures direct cell migration; however the cellular response to nanostructures is yet to be explored. In the current study, nanotopology is defined by nanodot arrays with dot diameters ranging from 10 to 200 nm. The nanodot arrays were fabricated by AAO processing on TaN-coated wafers. A thin layer of platinum, 5 nm in thickness, was sputtered onto the structure to provide a well biocompatible and homogeneous surface.
The defined nanostructures utilized in this study significantly facilitate our understanding of cell-nanosubstratum interactions. Our results were separated into four sections in this dissertation.
In the first part of this dissertation, nanodot arrays ranging from 10 nm to 200 nm were utilized to study the cellular adhesion behavior and growth condition in 3T3 fibroblasts. We found nanotopography, in the form of nanodot arrays, induced an apoptosis-like abnormality for cultured 3T3 fibroblast cells. Abnormality was triggered after as few as 24 hours of incubation on a 200 nm dot array. The number of filopodia extended from the cell bodies was lower for the abnormal cells. Pre-coatings of fibronectin or collagen type I promoted cellular anchorage and prevented the nanotopography-induced programmed cell death. The occurrence of the abnormality was mediated by the formation of focal adhesions.
In the second part of this dissertation, the cardiomyoblasts H9c2 were cultured on nanodot arrays. On the 50 nm nanodot arrays H9c2 showed maximum attachment and proliferation with largest cell area and extended lamellipodia. In contrast, 53.7% and 72.6% reductions of growth were observed on the 100- and 200 nm nanodot arrays. Immunostaining indicated that nanodots smaller than 50 nm induced cell adhesion and cytoskeleton organization. Expression of genes associated with fibrosis and hypertrophy was up-regulated in cells grown on 100 nm nanodots. The analysis of protein expression showed high levels of expression for vinculin and plasminogen activator inhibitor-1 for cells cultured on 50 nm nanodots. By adjusting the diameter of the nanodots, we could modulate the growth and expression of function-related genes and proteins in cardiomyoblasts.
In the third part of this dissertation, a strategy was proposed for the topologic design of dental implants based on the in vitro survey of optimized nanodot structures. An in vitro survey was performed using nanodot arrays with dot diameters ranging from 10 nm to 200 nm. Cell viability, apoptosis, cell adhesion, and cytoskeletal organization of MG63 osteoblasts were evaluated. Nanodots with a diameter of approximately 50 nm enhanced 44 % cell viability, minimized apoptosis to 2.7 %, promoted 30 % increase in actin filament bundles, and maximized cell adhesion with a 73 % increase in focal adhesions. Enhancement of ~50 % in mineralization was observed. We showed optimization for the biocompatibility of dental implants using nano-structures/MG 63 osteoblasts model system, providing a topologic approach beneficial for the design of dental implants.
In the last part of this dissertation, different sizes of nanodot arrays were integrated into a nanodevice for rapid modulation of proliferation, apoptosis, invasive ability, and cytoskeletal reorganization for cancer cells. The nanodevice composed of a matrix of nine nanodot arrays ranging from a flat surface to 10 nm, 50 nm, 100 nm, and 200 nm arrays. The invasive ability of HELA versus later-staged C33A cells was distinguished. Ovarian cancer cell lines (ES2, PA-1, TOV-112D, and TOV-21G) exhibited differential growth parameters that are associated with cell type, grade, and stage. We have established a platform that can be used to assess basic parameters of cell growth. The device is capable of distinguishing among cancer cell lines at various stages and also provides basic design parameters for artificial implants. Our device will serve as a convenient and fast tool for tissue engineering and cancer treatment.

並列關鍵字

nanostructure ； cell adhesion ； nanodot ； biomedical engineering

參考文獻

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