Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/47868
標題: 以水平微振動平台控制細胞增生與幹細胞分化
Control Proliferation of Endothelia Cell and Differentiation of Stem Cell Using a Micro- Positioning Pzt Stage
作者: 王國禎
關鍵字: 醫學工程;應用研究
摘要: 
適當的剪應力刺激能夠影響細胞於生醫材料之增生與貼附以及影響幹細胞之分化,而如何控制剪應力之刺激強度以精確控制細胞增生以及幹細胞分化成特定組織細胞則是亟需進一步研究之課題。有鑑於此,本研究規劃進一步從物理方式著手,以壓電控制式水平往複微振動平台為工具,藉由其零至數百赫茲之可控頻率,以及微米尺度之可控振幅,探討不同細胞之最佳增生剪應力以及如何藉由剪應力控制幹細胞之分化,與傳統fluid shear flow 相較,此平台不僅是提供單向層流,而是藉由可精確控制之往覆式運動,對細胞產生擾動,提高剪應力之梯度。本研究之第一年將由奈微米技術著手,製備具不同奈米結構之PLGA 支架,以探討細胞在微米振幅與不同振動頻率下之增生情形,並設計製作介電泳力量測平台,以介電泳方式量測細胞於材料之貼附力,並利用螢光染色計算細胞之貼附面積,求出細胞在不同頻率下所承受之剪應力,繪製各種支架之最佳生長曲線圖並比較其關聯性,期望能找出不同細胞增生所需之最佳剪應力,則在組織工程之應用上,僅以簡單之水平往覆微振動平台即可有效控制細胞之增生,不需著力於耗時之生醫材料表面結構設計與材料合成。於第二年之研究中,本研究將利用黃光微影製程製備具表面奈米結構之微米柱狀PLGA 陣列支架,分別探討脂肪幹細胞於不同奈微米比例陣列結構以及不同剪應力變化下之生長特性與分化趨勢;藉免疫螢光染色及流式細胞分析儀鑑定分化後之脂肪幹細胞是否具內皮細胞表面蛋白expression of nitric oxidesynthase(eNOS)、CD31 與骨母細胞表面蛋白Bone-specific alkaline phosphatase(BAP)、Osteocalcin (OC),並利用ELISA 進行定量,以探討分化趨勢與分化能力,找出最佳分化能力趨勢表;接著延長分化後細胞之培養時間,並與由組織純化出之細胞比較其於不同奈米結構PLGA 支架下之生長特性。本研究之第三年,規劃將微振動應用於具表面奈米結構之微米柱狀PLGA陣列支架上,探討脂肪幹細胞是之生長特性與分化趨勢是否與其靜態培養時一致以及分化能力是否可因施加水平往覆微振動加以提升,以提出最佳分化能力趨勢表;並利用分化後之細胞與自組織純化後的細胞進行比較,求出兩者間之最佳生長曲線,量化描述細胞於PLGA 支架之最佳生長環境,探討以水平往覆微振動平台控制脂肪幹細胞分化成特定組織細胞之可行性,進而應用於組織工程中之組織器官損傷修復。

Appropriate excitation of shear stress can influence the proliferation andadhesion of cells and the differentiation of stem cells. How to precisely control theapplied shear stress such that the proliferation and adhesion of cells and thedifferentiation of stem cells can be achieved becomes an imperative issue in tissueengineering. This proposal proposes a simple method to precisely control the desiredshear stress using a micro-positioning PZT stage. Compared with the conventionalfluidic sheer stress, a micro- positioning PZT stage not only provides cultured cellswith laminar flow, but also is able to generate large shear stress gradient by precisereciprocating motions.In the first year of this research, we schedule to fabricate various nanostructuredPLGA scaffolds for further investigations of cell proliferation under differentvibrational excitations by a PZT stage. To efficiently measure the cell adhesion force,a MEMS based device for dielectricphoretic force detection will be designed andmanufactured. The fluorescence labeling will be adopted to estimate the adhesive areaof a cultured cell. Optimal growth curves of different scaffolds will then be drawn.Resultantly optimal shear stress for the proliferation of different kinds of cells can beestimated. It is hoped that a micro-positioning PZT stage can be used to efficientlyenhance the proliferation of cells.During the second year, the photolithographic fabrication method will be used toproduce PLGA scaffolds consisting of micro scale rod array with nanostructure on thetop surface of each rod. The micro/nano structure combined PLGA scaffolds will beused to investigate the differentiation tendency of adipose derived mesenchymal stemcells. The fluorescence activated cell sorting analysis method will be used to examinethe differentiation tendency for constructing the optimal differentiation ability table.The after differentiating culture period will be extended for further comparisons withthe tissue purified cells on their growth characters of being cultured on PLGAscaffolds of different nanostructures.In the last year, micro vibrational excitations will be applied to the cell culture onmicro/nano structure combined PLGA scaffolds. Comparisons with the withoutvibrational exciting culture on the growth characters and the differentiation tendencywill be carried out. The feasibility of the vibrational excitation on the differentiationenhancement will also be analyzed to make the optimal differentiation ability table.Comparisons with the tissue purified cells on their growth characters of being culturedon PLGA scaffolds of different nanostructures will then be conducted to obtain the optimal differentiation curves for both cells. The feasibility of controlling thedifferentiation of adipose derived mesenchymal stem cells into desired tissue cells bya micro-positioning PZT stage will be investigated. It is expected that the results ofthis research can be applied to the repair of wounded tissues.
URI: http://hdl.handle.net/11455/47868
其他識別: NSC100-2221-E005-014-MY3
Appears in Collections:機械工程學系所

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