奈米表面應用於造骨細胞的生長調控和人工植入物的設計

Abstract

仿造細胞外基質[extracellular matrix(ECM)]作成的微米基材促進細胞生長跟分化，但是對於奈米結構是否促進細胞生長分化卻少有人說明，為了指出奈米結構表面是否控制細胞的生長情形，我們將 MG63 成骨細胞培養在奈米點陣列上，而這些奈米點的分布範圍由10-nm到200-nm。奈米表面對於細胞的生長、型態、貼附、細胞骨架以及礦化能力將被明確的探討，在所有的實驗數據中，50-nm的表面最適合MG63細胞生長。我們將四種不同型態的牙釘：Uncoated、TPS、HA、Nano Tite，以電子顯微鏡 (electron microscope) 作細部結構的探討，並且應用於生物體外以及臨床實驗上的研究。此篇研究中指出約50-nm的奈米結構的確可以有效的在生物體外調控成骨細胞的生長，而在臨床研究上擁有與50-nm的奈米點結構類似的HA牙釘也可使成骨細胞生長得比同樣擁有奈米結構的Uncoated和TPS牙釘以及結構定義不明確的Nano Tite牙釘好。 接著我們製作出一個包含有九片奈米點表面結構片段的裝置，此表面結構有Flat、10-nm、50-nm、100-nm和200-nm。接著將HELA、C33A、ES2、PA-1、TOV-112D、TOV-21G、MG63和NIH-3T3細胞培養於我們做出的裝置上三天，計算細胞的個數來當作其細胞成長的程度，而SEM可觀察出細胞型態變化的程度，再利用vinculin以及F-actin螢光染色來觀察細胞在不同表面上的貼附能力以及細胞骨架分布情形，並且將以上四種關於細胞的不同特性以數據的方式呈現。我們可以利用此裝置來分辨出HELA和C33A兩種癌細胞之間向外侵略能力的差異，不僅如此，我們的裝置對於分處於不同階段的ES2、PA-1、TOV-112D、TOV-21G四種癌細胞其生長也有不同的表現，我們還更進一步的探討特定奈米點表面對於MG63細胞的生長影響。 我們架設了一個簡單且可分析表面結構對於細胞特性影響的平台，這簡單的製造流程可以大量的生產並且降低生產成本，更甚的，這個裝置可以用來分別出癌細胞的級別與侵蝕能力，並且帶給現今人工植入物質一個參考的指標，此裝置提供組織工程以及癌症治療一個方便且快速的檢測平台。

Microstructure that mimics extracellular substratum promotes cell growth and differentiation while cellular reaction to nanostructure is poorly defined. To evaluate modulating ability of nano-scaled surface, MG63 osteoblasts were grown onto nanodot arrays with dot diameters ranging from 10 to 200 nm. Cell proliferation, morphology, adhesion, cytoskeleton, and mineralization were evaluated. Nanodot with 50-nm in diameter behaved the best in all evaluation. Four different types of dental implants, Uncoated, TPS, HA, and Nano Tite, were characterized by electron microscope and subjected to in vitro and clinical test. Here we show that nanostructure is capable of modulating the in vitro growth of osteoblasts at approximately 50-nm in diameter. Best clinical outcome for dental implants with nanostructure of similar dimension (HA) behaves the best compared to nanoscaled structure (Uncoated and TPS) and undefined structure (Nano Tite). We have fabricated a nanodevice composed of a matrix of 9 nanodot arrays with various dot sizes ranging from flat surface, 10-nm, 50-nm, 100-nm, and 200-nm. HELA, C33A, ES2, PA-1, TOV-112D, TOV-21G, MG63, and NIH-3T3 cells were seeded on the device and cultured for 3 days. Cell density was counted to examine the proliferation of cells. Scanning electron microscopy (SEM) was performed to assess the morphological change of cells. To evaluate cell adhesion and cytoskeleton reorganization, immunostaining specific to vinculin and actin filaments was performed. The scores of cell proliferation, morphology, distribution of focal adhesions, and cytoskeleton organization were obtained. We were able to distinguish the invasive ability of HELA versus later-staged C33A. Ovarian cancer cell lines (ES2, PA-1, TOV-112D, and TOV-21G) also exhibited differential growth parameters which are associated with the cell type, grade, and stage. Modulation for the growth of MG63 was also achieved. We have established a platform which can assess basic parameters for cell growth. The simplified fabrication process ensured mass production and cost down. Apparently, the device was capable of distinguishing cancer cell line of various stages and also provided basic designing parameters for artificial implants. Our device will serve as a convenient and fast tool for tissue engineering and cancer treatment.