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研究生:葉佐儀
研究生(外文):TSO-I YEH
論文名稱:微細纖維狀仿生薄膜及立體生物性支架之製作及其做為全層皮膚應用之探討
論文名稱(外文):Fabrication and application of biomimic fine-fibrous membranes and three-dimensional biodegradable scaffold in skin tissue engineering
指導教授:曾厚
指導教授(外文):Tseng How
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:醫學科學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:109
中文關鍵詞:人工皮膚電氣紡絲多孔質結構物
外文關鍵詞:artificial skinelectrospinningKeratinocyte3D porous scaffold
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組織工程中「人工全層皮膚」的發展被認為可替代傷口敷料之治療方式,以創造無疤痕組織的皮膚再生。本研究將人工皮膚分成表皮層及真皮層進行模擬。研究主要利用電氣紡絲技術,以生物可降解性高分子PLLA、PBSA為原料,製備粗細差異近十倍之纖維狀薄膜,經表面被覆玻尿酸(Hyaluronic acid)、膠原蛋白(Collagen)及聚葡萄胺糖(Chitosan)等不同物質後,首先進行毒性測試,再進行HaCaT、人類角質細胞(Keratinocyte)貼附及增生試驗;Keratinocyte並進行多層化試驗。由毒性測試結果確認經電紡技術製備之纖維薄膜可有效提供細胞貼附與生長;再由HaCaT及Keratinocyte結果顯示10w/v % PLLA對於細胞之貼附及增生均具較佳;於薄膜表面被覆HA、Collagen則可有效增加細胞貼附及增生效果,其表現甚至優於商品化之細胞培養皿;同時,結果亦顯示:播植細胞前將纖維薄膜浸潤於細胞培養液中,對於未被覆之纖維薄膜於細胞生長表現可大幅提升至與被覆HA或Collagen相近。本研究同時利用Paraffin及PLLA製備多孔質結構物作為真皮層基質之用;製備之轉速與Paraffin spheres的大小成反比關係;而PVA溶液濃度對Paraffin spheres粒徑的影響則不明顯,但會造成不規則狀之Paraffin spheres。大粒徑之Paraffin spheres所形成之多孔成型物愈脆弱。多孔質結構物經細胞毒性測試亦確認可適於細胞生長。總結,本研究結果可推論10w/v% PLLA為較相似於細胞間質之結構,故細胞生長結果最佳;而多孔質結構體推論亦將提供細胞生長甚至產生血管化之良好環境。
It’s well known that in the living system, the extracellular matrix (ECM) plays an important role in controlling cell behavior. The ECM is composed of a basement membrane (BM) and complex cross-linked network of proteins and glycosaminoglycans(GAGs), and those non-woven fibrous architecture are already reach nano-sized. It is therefore important to know how to fabricate an artificial nano-sized fibrous structure for use in organ repair and tissue regeneration.
In present study, the major goal is to mimic the epidermal and dermal-like structure as a artificial skin. Biodegradable polymer Poly L-lactic acid (PLLA) and Poly butylenes succinate-co-adipate(PBSA) were used to fabricated various three-dimensional fibrous scaffolds via electrospinning process. Culture HaCaT and epithelial cell (keratinocyte) to determine whether the fibrous scaffolds are perfect for cell growth. The results show that 10w/v% PLLA and coated with HA、collagen respectively on the surface performed the better environment for cells growth. The second section was to create a dermal-like structure. A novel processing technique has been developed to create three-dimensional biodegradable polymer scaffolds with well-controlled interconnected spherical pores. Paraffin spheres were fabricated with a dispersion method, and were bonded together through a heat treatment to form a three-dimensional assembly in a mold. The inter-pore connectivity could be controlled by varying the heat treatment time of the paraffin spheres and the rotation rate , and mechanical properties could be controlled by varying the porosity of the scaffolds. Then the PLLA solution was casted onto the scaffold to form the framework. With an interconnected macroporous structure that promotes cell seeding throughout the interstices of the 3D porous scaffold, these novel matrices may be an excellent scaffold for vascularization in tissue engineering.
誌謝--------------------------------------------------------------------------------II
中文摘要-------------------------------------------------------------------------III
Abstract---------------------------------------------------------------------------V
縮寫表---------------------------------------------------------------------------VII
目錄-------------------------------------------------------------------------------IX
第一章 緒 論----------------------------------------------------------------------1
1.1 前言--------------------------------------------------------------------------1
1.2 研究動機-------------------------------------------------------------------2
1.3 研究目的-------------------------------------------------------------------3
第二章 文獻回顧----------------------------------------------------------------5
2.1組織工程--------------------------------------------------------------------5
2.2 皮膚組織工程原理-------------------------------------------------------6
2.3 人工皮膚發展及挑戰----------------------------------------------------8
2.4電氣紡絲------------------------------------------------------------------13
2.5 人工真皮-----------------------------------------------------------------14
2.6血管新生------------------------------------------------------------------16
2.7 實驗室製備立體組織工程皮膚--------------------------------------19
2.8立體孔洞性生物降解性支架------------------------------------------22
第三章 研究材料與方法-----------------------------------------------------24
3.1材料與試劑---------------------------------------------------------------24
3.2儀器設備------------------------------------------------------------------25
3.3研究方法------------------------------------------------------------------26
3.4實驗步驟------------------------------------------------------------------29
3.5 材料物性分析---------------------------------------------------------------31
3.6角質細胞培養與觀察---------------------------------------------------33
3.7多孔質結構支架製備---------------------------------------------------38
第四章 結果與討論------------------------------------------------------------40
纖維薄膜型態及其物理特性---------------------------------------------41
細胞貼附---------------------------------------------------------------------47
細胞增生---------------------------------------------------------------------57
立體結構---------------------------------------------------------------------65
第五章 結論--------------------------------------------------------------------71
第六章 未來研究方向---------------------------------------------------------73
6.1 表皮層--------------------------------------------------------------------73
6.2 真皮層--------------------------------------------------------------------74
6.3 皮膚組織工程的未來挑戰--------------------------------------------74
參考文獻-------------------------------------------------------------------------76


圖 索 引
第二章 文獻回顧
Fig. 2.1 The structure of human skin. ---------------------------------------7
Fig. 2.2 Some examples of commercialized artificial skin products. -10
第三章 研究材料與方法
Fig. 3.1 The flow chart of the study. ---------------------------------------28
Fig. 3.2The morphology of the cells. --------------------------------------35
第四章 結果與討論
Fig. 4.1 The different conditions of the membranes. --------------------83
Fig. 4.2 Contact angle of four various membranes. ---------------------84
Fig. 4.3 NIH 3T3 fibroblast adhesion in four various membranes. ---85
Fig. 4.4 HaCaT adhesion in four different membranes. -----------------85
Fig. 4.5 HaCaT adhesion in various coating materials. -----------------86
Fig. 4.6 HaCaT adhesion in four different membranes. The non-coating membranes had been rinsed before seeding cells. -----------------------86
Fig. 4.7 HaCaT adhesion in various coating materials. -----------------87
Fig. 4.8 HaCaT adhesion in four various filament membranes. -------88
Fig. 4.9 HaCaT adhesion in various coating materials. -----------------89
Fig. 4.10 Keratinocyte adhesion in four different membranes. --------90
Fig. 4.11 Keratinocyte adhesion in various coating materials. ---------90
Fig. 4.12 Keratinocyte adhesion in four different membranes. The non-coating membranes had been rinsed before seeding cells. --------91
Fig. 4.13 Keratinocyte adhesion in various coating materials. ---------92
Fig. 4.14 Keratinocyte adhesion in four various filament membranes. -----------------------------------------------------------------------------------92
Fig. 4.15 Keratinocyte adhesion in various coating materials. ---------93
Fig. 4.16 HaCaT proliferation in four different membranes. -----------94
Fig. 4.17 HaCaT proliferation in various coating materials. -----------94
Fig. 4.18 HaCaT proliferation in four different membranes. The non-coating membranes had been rinsed before seeding cells. --------95
Fig. 4.19 HaCaT proliferation in various coating materials. -----------96
Fig. 4.20 HaCaT proliferation in four various filament membranes. -----------------------------------------------------------------------------------96
Fig. 4.21 HaCaT proliferation in various coating materials. -----------97
Fig. 4.22 Keratinocyte proliferation in four different membranes. ----98
Fig. 4.23 Keratinocyte proliferation in various coating materials. ----99
Fig. 4.24 Keratinocyte proliferation in four different membranes. The non-coating membranes had been rinsed before seeding cells. --------99
Fig. 4.25 Keratinocyte proliferation in various coating materials. ---100
Fig. 4.26 Keratinocyte proliferation in four various filament membranes. ------------------------------------------------------------------101
Fig 4.27 Keratinocyte proliferation in various coating materials. ----101
Fig. 4.28 Keratinocytes air-lifting in the fine and coarse filament membranes. ------------------------------------------------------------------102
Fig. 4.29 The relationship between RPM and the diameter of paraffin spheres. -----------------------------------------------------------------------103
Fig. 4.30 The paraffin spheres. --------------------------------------------103
Fig. 4.31 Size distribution of the paraffin spheres ---------------------104
Fig. 4.32 Paraffin spheres made from various concentration of PVA solution. ----------------------------------------------------------------------105
Fig. 4.33 The asymmetry paraffin spheres-------------------------------106
Fig. 4.34 The heated paraffin spheres------------------------------------107
Fig. 4.35 Paraffin spheres with sizes of (a) 104-350μm. (b) > 500μm ---------------------------------------------------------------------------------108
Fig. 4.36 Heated paraffin disc. --------------------------------------------108
Fig. 4.37 3D porous scaffold immersed in the hexane for different duration. ----------------------------------------------------------------------108
Fig. 4.38 3D porous scaffold--------------------------------------------109
Figure 4.39 Toxicity trial of 3D porous scaffold. ----------------------109

表 索 引
第四章 結果與討論
Table 4.1 The formula and electrospinning condition of four membranes. ------------------------------------------------------------------82
Table 4.2 The viscosity of PBSA. ----------------------------------------82
Table 4.3 Contact angle of the various membranes -----------------84
Table 4.4 The relationship between RPM and the diameter of paraffin spheres -----------------------------------------------------------102
Table 4.5 The yield of paraffin spheres from various range of sieve. ---------------------------------------------------------------------------------104
Table 4.6 Different concentration of PVA solution. ----------------105
Table 4.7 Heat treatment for the paraffin spheres------------------109
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