請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/49717
標題: | 以PDMS薄膜及微孔洞始去細胞之真皮支架上形成真皮乳頭微球陣列應用在毛囊重建工程之研究 Formation of dermal papilla spheroid array on decellurized dermis scaffold by PDMS film and microwell for hair follicle regeneration engineering |
作者: | Chia-Hsiem Tsai 蔡佳憲 |
指導教授: | 黃義侑 |
關鍵字: | 真皮乳頭細胞,聚二甲基矽氧烷,微孔陣列,去細胞化,毛囊重建, Dermal papilla,PDMS,microwell array,Decellularization,Hair follicle regeneration, |
出版年 : | 2016 |
學位: | 碩士 |
摘要: | 禿髮的發生不僅會失去毛髮的功能,更會對患者產生巨大的心理負擔,而在組織工程上製作支架並使毛囊再生的概念也成為一個可行的方法。成功的毛囊新生,需要有誘導力的真皮細胞及具有分化力的表皮細胞進行表皮-間葉組織的交互作用才能發生。在毛囊重建工程上,屬於特化間葉組織的真皮乳頭細胞十分廣泛的被應用,然而在體外培養數代後誘導能力會快速的消退。相比於傳統的平面培養,三維空間的培養更能模擬細胞真實生長的環境,也能加強細胞之間與胞外基質的交互作用。
因此本研究的目的希望可以培養出真皮乳頭微球組織,並有高產量、大小均一等特點。由於聚二甲基矽氧烷(PDMS)本身具有良好的生物相容性及細胞的低貼附性,藉由直接將PDMS塗層在96孔盤,並施加微弱搖晃使中央部分形成高細胞密度並聚縮成微組織。我們發現當控制細胞密度在3000/well下,不論大鼠或人類真皮乳頭細胞皆可以產生與活體大小相近的微組織,在短時間的培養甚至是十天以上,大部分的真皮乳頭細胞皆能保有其生長活性。為了確認在此培養方法下的微組織保有誘導毛囊新生的能力,我們對此進行免疫螢光染色進行真皮乳頭細胞標定物的分析,像是α-SMA、Versican、Vimentin等都有明顯表現。 同時我們以凍融、洗滌劑及生物酶的綜合去細胞處理程序來製作真皮的去細胞支架,經過DNA及GAG含量測定分析後,證實我們確實去除了絕大多數的細胞並保有大部分的胞外基質。此外結合以雷射雕刻的PDMS微孔陣列,藉由事先的設計我們便能讓真皮乳頭微組織在此支架上有特定的排列。因此本實驗的設計及後續的培養方式,確實能夠產生大量的真皮乳頭微組織,保有其誘導毛囊新生能力,並能在之後應用於毛囊重建工程上。 Hair loss, also called alopecia not only loses function of hair but also cause physiological impacts on the patient. Fabrication of scaffold for regeneration of hair follicle by tissue engineering provides a promising alternative. Successful hair follicle (HF) neogenesis depends on the existence of both capable dermal cells and competent epidermal keratinocytes through epithelial–mesenchymal interaction. Dermal papilla (DP) is a highly specialized mesenchymal cell population and widely applied for hair follicle regeneration engineering. However, DP cells tend to lose their inductivity in vitro. Three-dimensional (3D) culture system mimics real microenvironment and cell−extracellular matrix (ECM) interactions are enhanced compared to two-dimensional (2D) monolayers culture. The aim of this study was to construct a strategy for DP microtissues which are high-throughput and can be produced in uniform-sized manner. Due to high biocompatibility and low cell attachment of Poly(dimethylsiloxane) (PDMS), local high cell density is observed and cellular aggregates are formed by direct PDMS coating on 96-well plate and weak shear force application. We found that both rat and human dermal papilla microtissues grow as in vivo size under 3000/well operation condition. Most cells still represented viable within the microtissues for short-term culture. To confirm the preservation of inductivity of DP microtissues, we conducted immune-fluorescent staining and found that specific markers, such as α-SMA, Versican and Vimentin are expressed. Furthermore, acellular dermal scaffold was fabricated by serial steps of decellularization. By DNA and GAGs content analysis, we ensured native cells are almost removed but keeps ECM intact. Besides, laser-fabricated PDMS micro-array makes microtissues grow in specific arrangement. Hence we can generate large-scale DP microtissues by PDMS low attachment 3D culture and have high potential for hair follicle regeneration engineering. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/49717 |
DOI: | 10.6342/NTU201602545 |
全文授權: | 有償授權 |
顯示於系所單位: | 醫學工程學研究所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-105-1.pdf 目前未授權公開取用 | 3.43 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。