微米級圖案廣泛應用於智能生物材料，透過圖案大小與形狀差異，可改變細胞之間的信號溝通，本論文目的為製備膠原蛋白的微米級島嶼與孔洞，以控制細胞貼附、生長與分化。而膠原蛋白是細胞外基質中的主要成分之一，因此本論文使用膠原蛋白模仿天然組織環境。
本論文第一部分為使用臭氧紫外光結合光罩以製備膠原蛋白島嶼，並利用trypsin (+EDTA) 移除被臭氧紫外光破壞的膠原蛋白，製備膠原蛋白島嶼於雲母片表面。將探討利用不同熱處理的溫度對於膠原蛋白型態的影響、以及熱處理後的膠原蛋白對於細胞生長行為之影響。當熱處理溫度自50度增加到90度，肺癌細胞 (LLC) 延展的比例自42.0 % 增加到 71.2 %。此外，培養間充質幹細胞於膠原蛋白微米級島嶼，肌動蛋白 (F-actin) 可依照島嶼形狀延展。但是因為製備膠原蛋白島嶼的結果不具良好重複性，因此調整膠原蛋白於雲母片上的沉積環境。
論文中第一部分所製備的微米級膠原蛋白島嶼的結果不具良好的重複性，可能因為是膠原蛋白與雲母片的附著力較弱，因此在製作膠原蛋白島嶼時候具有難度。本論文第二部分嘗試利用化學方法修飾基材，以增進膠原蛋白與基材的附著力。將氧氣電漿處理過的雲母片基材分別浸泡於APTES (3-aminopropyltriethoxysilane) GA與GA (glutaraldehyde) 兩種溶液，使用最適化的沉積條件，將膠原蛋白沉積於化學修飾的雲母片，利用水接觸角、原子力顯微鏡、以及甲基橙吸附等實驗進行討論，結果顯示在化學修飾過的雲母片表面，膠原蛋白沉積型態為亂序的絲狀結構且沒有D-banding。
因此，本論文進一步將嘗試利用轉移方式，將雲母片上的具D-banding的膠原蛋白，轉移至經化學修飾後的PDMS，原子力顯微鏡的結果顯示，轉移後的膠原蛋白型態形態為單一方向之絲狀結構，並具有D-banding特性。雲母片上的膠原蛋白島嶼之研究發現，使用化學移除膠原蛋白產生微米級圖案具有難度。因此本論文的第三部分，製備尺寸不同、圖案化的PEGDA在PVDF上，形成PEGDA微米牆圖案於PVDF上，再使用超音波震盪器移除孔洞中未交聯的PEGDA，製備由PEGDA微米牆圍繞的微米級孔洞。實驗發現控制超音波震盪時間是移除PEGDA重要參數，因為超音波震盪時間增加時，可有效移除孔洞中的PEGDA，但是如果超音波震盪過長，則PEGDA微米牆也會同時被移除，因此隨著目標PEGDA孔洞尺寸的不同，超音波震盪最適化時間也不同。一旦成功製備PEGDA微米牆在PVDF上，可將膠原蛋白沉積PVDF於微米級孔洞中，培養兩種不同的間充質幹細胞 (hMSC與WJ MSC)，以探討細胞的貼附、生長、與分化的情形。
本論文調整膠原蛋白沉積環境，使具有D-banding的絲狀膠原蛋白在雲母片上沉積成功率增加，利用臭氧紫外光與trypsin (+EDTA) 在雲母片上製備具有D-banding的膠原蛋白微米級島嶼的方法雖然可行，但是trypsin(+EDTA)能夠輕易移除膠原蛋白，使實驗失敗率高。因此藉由表面化學修飾，增加膠原蛋白與雲母片之附著力，而膠原蛋白成功沉積於膠過化學表面修飾的雲母片，但是不具有D-banding，因此發展膠原蛋白轉移之方法，將具有D-banding的絲狀膠原蛋白，轉移至表面經過化學修飾的PDMS，結果顯示轉移過後的膠原蛋白保有D-banding特色。因為研究結果發現，使用trypsin(+EDTA) 移除膠原蛋白而產生微米級圖案具有難度，因此本論文發展紫外光交聯PEGDA之方法，於PVDF表面製備膠原蛋白微米級孔洞，並且誘導間充質幹細胞進行貼附、生長與分化。
未來研究方向可以將具有D-banding的膠原蛋白，藉由表面化學修飾轉移至PDMS甚至是PVDF，再藉由紫外光交聯具有抗沾黏之材料，建立微米牆，圍繞具有D-banding的膠原蛋白，製備膠原蛋白微米級圖案。第二個方向為先將具有D-banding的膠原蛋白轉移至化學表面修飾的PDMS，再使用臭氧紫外光曝光膠原蛋白轉移後的PDMS，因為PDMS可受臭氧紫外光而受熱收縮，而產生凸式的膠原蛋白微米級圖案。

The goal of this thesis is to prepare collagen micro islands/wells by exposing UV light. Because of micro-patterns were widely used to apply in intelligent biomaterial and improve cell behavior such as cell attachment, cell proliferation, and cell differentiation in the micro environment. Additionally, Collagen I is one of abundant molecules in extracellular matrix (ECM). Thus, collagen was used in this thesis to mimic the natural environment.
The first part of thesis, the combination of UV ozone and photo mask was used to destruct collagen on col/mica and the destructed collagen was removed by trypsin (+EDTA). Therefore, collagen micro islands on col/mica were prepared. The effects of heat treatment on collagen fibers and the heat treated collagen fibers on cell behavior were described. With increasing temperature of heat treatment, from 50 oC to 90 oC, the fiber structure and D-banding were preserved and the percentage of elongated cell increased from 42.0 % to 71.2 %. The resulted collagen micro islands on col/mica were presented by fluorescence image and hMSC was cultivated on collagen micro islands. Moreover, the conformation of hMSC accorded to the island shape. However, the collagen micro islands on col/mica was not obtained repeatedly. Therefore, the combination of phosphate and KCl for collagen deposition was optimized and the characteristic of D-banding of collagen on mica was deposited repeatedly.
The second part of this thesis aims to promote the adhesion between collagen and substrate. Two additional substrates were used in this part: functionalized mica and PDMS. The functionalization was facilitated by immersing the substrates in APTES solution, followed by crosslinking using glutaraldehyde. The characterizations of the chemical functionalization were analyzed by WCA, AFM, and methyl orange. The formation of collagen on functionalized mica showed random direction without D-banding. On the other hand, the collagen with D-banding on col/mica was successfully transferred onto the functionalized PDMS. The AFM image showed the fiber structure and D-banding of collagen were preserved on the chemically functionalized PDMS.
The third part of this thesis presented the results of preparation collagen microwells on PVDF. PEGDA, an antifouling polymer, was used to create microwalls surrounding the microwells through UV crosslinking at the wavelength of 365 nm. Ultrasonic was applied to remove the PEGDA inside the microwells, therefore the time of ultrasonication was optimized for the patterns with different aspect ratios. Afterward, collagen was deposited onto the microwells for the modulation of cell proliferation and differentiation.
In conclusion, collagen micro islands on col/mica were fabricated by UV ozone and trypsin (+EDTA) treatment where hMSC proliferated to the island shape. However, the reproducibility was low because of trypsin (+EDTA) removed the collagen on col/mica easily. Thus, the adhesion between collagen and mica was increased by functionalizing mica. Although the deposition of collagen was achieved on functionalized mica but the resultant collagen showed no D-banding. On the other hand, the collagen with D-banding on col/mica can be successfully transferred onto the functionalized PDMS. In addition, collagen microwells were prepared on PVDF by 365 UV exposure to crosslink the PEGDA which formed the microwalls surrounding the PEGDA microwells. The results showed that the microwells decorated with collagen allowed to induce MSC attachment, proliferation, and differentiation.
This thesis discussed the methods to prepare collagen islands/wells for the induction of stem cell. According to the experimental results from this thesis, collagen transfer on functionalized substrates (PDMS, PVDF), followed by UV crosslinking to build the antifouling microwalls can be the future works. Additionally, because of the patterned PDMS could be fabricated by treating masked PDMS with UV ozone of 5 cm. Therefore, the collagen of characteristic D-banding was transferred on functionalized PDMS then treated by UV ozone such that convex collagen patterns could be fabricated.

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