黃義侑臺灣大學:醫學工程學研究所王得耀Wang, De-YaoDe-YaoWang2010-06-022018-06-292010-06-022018-06-292009U0001-2001200912222500http://ntur.lib.ntu.edu.tw//handle/246246/184659本研究將精密加工中的雷射雕刻技術 (Laser micromachining)、半導體製程衍生出的軟蝕刻技術 (Soft lithography) 以及奈米科技中的電紡織技術 (Electrospinning) 應用於神經再生領域，用來評估誘導的細胞、製備多功能性的支架、發展新式的導管加工方式以及提供排列的訊息。了提供神經再生研究中新的細胞來源途徑，我們分別利用生物性和物理性的方式，將間葉幹細胞 (Mesenchymal stem cell) 誘導分化成類神經細胞。首先，應用雷射雕刻技術去製作用來排列細胞的微流道系統 (Microfluidic system)。再利用微流道系統將綠色螢光基因轉殖小鼠的間葉幹細胞和紅色螢光蛋白基因轉殖小鼠的神經細胞 (Neuron cell) 區隔排列在玻片上，進行共同培養，來探討單純神經細胞的旁分泌（Paracrine）對於間葉幹細胞轉分化 Transdifferentiation) 的影響。發現利用該系統可以提供不具有擴散障礙的共同培養平台，讓培養其中的間葉幹細胞有較高的神經相關蛋白表現，並且在區隔排列之下，兩種細胞的融合現象 (Cell fusion) 更容易被觀察。此外，利用電紡織技術來製備奈米尺寸的聚己內酯 (Polyε-Caprolactone) 細絲，以物理結構來誘導分化間葉幹細胞，使該細胞具有類神經細胞的型態表現，以及表現神經相關蛋白和增加神經相關基因的表現量。者，為了提供誘導細胞具有孔洞性的仿生基材，我們改良傳統的電紡織技術，製備具有微米尺寸孔洞的電紡織支架，用來模擬相似於生理微環境 (Microenvironment) 的結構。首先，應用改良式電紡織技術結合顆粒蝕刻技術 (Particle leaching)，同時製備並且均勻混合電紡織明膠 (Gelatin) 細絲以及聚己內酯顆粒，當扮演成孔劑（Pore generator）的聚己內酯溶解後，留下孔洞提高了材料的孔隙度以及細胞對材料的穿透性。此外，利用幾丁聚醣 (Chitosan) 溶液收集具有物理性誘導分化潛力的電紡織聚己內酯細絲，在冷凍乾燥(Lyophilizing) 之後形成同時具有微米尺寸孔洞以及奈米尺寸紋理的電紡織支架。後，我們提供創新的加工方式將多孔性的仿生基材製備成神經導管，以及使用排列生物分子來引導細胞生長的方向。首先，應用雷射雕刻技術並且結合電腦輔助設計以及電腦輔助製造，可以快速、精確、客製化、批次量造地製造神經導管 (Nerve conduit)，使得多孔性的高分子材料不會在導管加工過程中坍塌變形。並且應用軟蝕刻技術去製作具有微米尺寸結構的次級神經導管模具，再利用高分子灌模、成型、脫模之後，以共軸方式將次級神經導管堆疊成神經導管。以及應用軟蝕刻技術製作軟性結構將生物分子層黏連蛋白 (Laminin) 以微接觸式印刷技術 (Microcontact printing) 拓印在細胞培養之基材上，可以引導許旺氏細胞 (Schwann cell) 依特定方向性生長，用來探討訊息和細胞間的交互作用。This work demonstrates how micro/nanotechniques, laser micromachining, soft lithography, and electrospining define material properties and applications at the nanoscale or microscale for broad capabilities in the development of processes of nerve regeneration such as needs of induced cell, engineered scaffold, and patterned signal.o obtain alter neuron cell resources, induced cues of cocultured neuron and physical structure were used to transdifferentiate mesenchymal stem cells (MSCs) into euron-like cells. Green fluorescent protein expressing (GFP+) mMSCs and red fluorescent protein expressing (RFP+) neuron cells were microfluidic patterned separately on the same cover glass. When cocultured with neuron cells, more mMSCs expressed neural markers, Beta tubulin III and Glial fibrillary acidic protein (GFAP) in the microfluidic patterned coculture system than cells in a transwell system. Also, two reporters, GFP and RFP, provide us a way to assay that a very few case of fused cell happened. The microfluidic patterned coculture system facilitates to valuate the plasticity and behavior of cells and dynamic cross-talks between cells. Besides, aligned and random collected electrospun polycaprolactone (PCL) fibers ere fabricated to provide not only contact guidance but also nanometric cues to affect cell fate. Compared to mMSCs cultured on cover glass, cells expressed protein evel of Beta tubulin III and GFAP, and even higher mRNA level of Nestin, Beta tubulin III, TH, Synapsin, GFAP, and MBP. Nanometric topographies used to change cell functions are a way to evaluate cell plasticity and cell-biomaterial interactions.urthermore, to provide the induced cell with physical supporting and potential transdifferentiated cues, particle leaching and lyophilizing were introduced into lectrospinning to fabricate engineered scaffold with electrospun fibers and microstructured pores. We used the rotating multichannel electrospinning (RM-ELSP) o produce gelatin electrospun scaffolds with controllable porosity. Gelatin electrospun fibers and PCL microparticles were formed and blended simultaneously using the RM-ELSP. The composites were turned into the porous electrospun caffolds with the use of acetone to leach out PCL microparticles and leave space for cell ingrowth to improve its poor porosity. Besides, a chitosan solution as a collector of electrospun PCL fibers is used to support the fibers after changed it to be a porous sponge using lyophilizing. A chitosan/PCL composute, a porous chitosan sponge distributed electrospun PCL fibers within its microstructure, provided topographical cues on its surface to not only improve GFP+ mMSCs infiltration within the lectrospun scaffolds but also increase higher mRNA level of Nestin, TH, Synapsin, GFAP and MBP. It implied that nano-topographical cues in engineered scaffold have great potential to make mMSCs transdifferentiated into neuron-like cells.oreover, turning engineered scaffold to nerve conduits employed multiple channels and microstructure in their lumen surface and providing pattern signal to orient the ell growth were considered also. We fabricated porous chitosan conduits employed designed patterns of engraved channels using the direct-write CO2 laser icromachining. Laser micromachining allows us to shape various selected materials in the regions engraved with the designed patterns. Besides, we presented a new way o fabricate nerve conduits using soft lithography and molding process. Afterwards the conduit subunit microfabrication, the conduit subunits were stacked coaxially to form a nerve conduit. Due to the precise capability and cost-effective of soft lithography, it s a well-suited way for us to fabricate nerve conduits having complex designs. Finally, we demonstrated the efficacy of microcontact printed laminin to align and edirect Schwann cells growth; and therefore, microcontact printing is able to pattern cell-recognition molecules on scaffolds for guided cell growth in tissue regeneration. hese micro- and nanotechniques and approaches, laser micromachining, soft lithography, and electrospinning, are useful in advanced material and biological studies in tissue engineering such as change of functions and behaviors of cells to be a new resource of induced neuron cells; development of engineered scaffolds with properties of scaffold size, network interconnectivity, and geometrical designs; and establishment of artificial microenvironment composed of biochemical, physical, and topographical cues used for regenerated cell adherence, viability, proliferation, and differentiation to integrate the nerve regeneration processes.Acknowledgement Ibstract (Chinese) IIbstract IVable of Contents VIIIist of Figures XIIIist of Tables XVist of Publications XVIist of Patents XVIIhapter 1: Background and literature review 1.1. Tissue engineering triad 1.1.1. Cell 2.1.2. Signal 3.1.3. Scaffold 5.2. Nerve regeneration 9.2.1. Nervous system 9.2.2. Nerve injury 11.2.3. Nerve repair using tabulation 13.3. Challenge in nerve regeneration 14.4. Micro/nanotechniques and approaches in nerve regeneration 15.5. References 21ART I: INDUCED CELL 25hapter 2: Evaluation of transdifferentiated from mesenchymal stem cells to euron-like cells using microfluidic patterned coculture system……...26.1. Introduction 27.1.1. Mesenchymal stem cell 29.1.2. Neuron stem cell 31.1.3. Mesenchymal stem cell and neuron cell interactions 33.1.3.1. MSCs transdifferentiated into neuron-like cells in vivo 34.1.3.2. MSCs transdifferentiated into neuron-like cells in vitro 34.1.3.3. Concerns of transdifferentiation 35.1.3.4. MSCs rescued dying neuron related cells 37.1.4. Microfluidic patterning 38.2. Materials and methods 40.2.1. Microfluidic patterning device fabrication 40.2.2. Primary culture of GFP+ mMSCs and RFP+ neuron cells 41.2.3. Microfluidic patterning 43.2.4. Immuno-cytochemistry analysis 44.2.5. Stereological cell count 45.3. Results 46.3.1. Microfluidic device 46.3.2. Differentiated neurons from neuron sphere 47.3.3. Cocultured MSCs and neuron cells 50.3.4. Neural markers expressed in mMSCs with and without ell-cell contact induction 52.3.5. Cell fusion 56.4. Discussion 57.5. Conclusions 60.6. References 61hapter 3: Electrospun polycaprolactone fibers facilitated mesenchymal stem cells to transdifferentiate into neuron-like cells 65.1. Introduction 66.2. Materials and methods 68.2.1. Electrospinning 68.2.2. MSCs isolation and culture 69.2.3. Morphology observations 70.2.4. Immuno-cytochemistry analysis 70.2.5. Quantitative real-time polymerase chain reaction 71.3. Results 73.3.1. PCL electrospun fibers and membrane 73.3.2. Induced elongation of GFP+ mMSCs along electrospun fibers 74.3.3. Living cell observation 76.3.4. Neural marker expressed GFP+ mMSCs 78.3.5. Quantitative real-time polymerase chain reaction 81.4. Discussion 83.5. Conclusions 85.6. References 86ART II: ENGINEERED SCAFFOLD 90hapter 4: Fabricating microparticles/nanofibers composite and nanofiber scaffold ith controllable pore size via the rotating multichannel lectrospinning 91.1. Introduction 92.1.1. Electrospinning 94.1.2. Gelatin 98.1.3. Polycaprolactone 98.2. Materials and methods 99.2.1. Electrospinning 99.2.2. Glutaraldehyde vapor crosslinking 101.2.3. Pore formation 102.2.4. Degradation test 102.2.5. Fiber and cell morphology observations 103.2.6. Cell seeding and culture 103.2.7. Cell proliferation and cell toxicity assay 104.3. Results 105.3.1. Electrospinning of PCL and the PCL/gelatin composites 105.3.2. Glutaraldehyde vapor crosslinking and PCL leaching 109.3.3. Cell morphologies 111.3.4. Cell proliferation and total cell number assay 113.4. Discussion 114.5. Conclusion 117.6. References 117hapter 5: Microstructure of chitosan sponge blended with electrospun olycaprolactone fibers facilitated neural differentiation of esenchymal stem cells 120.1. Introduction 121.2. Materials and methods 123.2.1. Electrospinning 123.2.2. Lyophilizing and neutralization of chitosan/PCL composites 124.2.3. Mesenchymal stem cell culture 125.2.4. Srtucture morphology 126.2.5. Quantitative real-time polymerase chain reaction 127.3. Results 128.3.1. Electrospinning of PCL 128.3.3. The chitosan/PCL composite 129.3.4. Observations of seeded cells within the chitosan sponge 132.3.5. Quantitative real-time polymerase chain reaction 133.4. Discussion 134.5. Conclusion 136.6. References 137ART III: NERVE CONDUIT AND SIGNAL PATTERNING 139hapter 6: Manufacture of nerve conduits via the direct-write CO2 laser icromachining 140.1. Introduction 141.1.1. Chitosan 144.1.2. Laser micromachining 145.2. Materials and methods 147.2.1. Preparation of chitosan sponge 147.2.2. Processes of laser micromachined conduits 147.2.3. Wire-heated conduit process 149.2.4. Conduit structures 149.2.5. Pore structure comparisons 150.3. Results 150.3.1. Laser micromachined conduit structure 150 .3.2. Comparisons of pore structures 151.4. Discussion 153.5. Conclusion 156.6. References 157hapter 7: Fabricate coaxial stacked nerve conduits through soft lithography nd molding processes 160.1. Introduction 161.1.1. Soft lithography 163.2. Materials and methods 164.2.1. Lithographic processes 164.2.2. PDMS mold production 165.2.3. Conduit subunit production 165.2.4. Nerve conduit production 166.3. Results 166.3.1. PDMS mold production 166.3.2. Conduit subunit production 168.3.3. Coaxial sacking by rational symmetry 169.4. Discussion 171.5. Conclusion 172.6. References 173hapter 8: Microcontant printing (μCP) of laminin on oxygen plasma activated substrates for alignment and morphology of Schwann cells 175.1. Introduction 176.1.1. Schwann cell 178.1.2. Plasma activation 180.2. Materials and methods 182.2.1. Elastomeric stamp preparation 182.2.2 Substrate preparation 185.2.3. Contact angle measurement before and after xygen plasma treatment 186.2.4. Microcontact printing 187.2.5. Primary culture of Schwann cell 187.3. Results 189.3.1. Effect of oxygen plasma treatment 189.3.2. Validation of the microcontact pattern 190.3.3. Orientation and morphology of Schwann cellson micropatterned substrates 190.4. Discussion 192.5. Conclusion 194.6. References 195hapter 9: Conclusions 197.1. Conclusions 197application/pdf31846672 bytesapplication/pdfen-US組織工程神經再生軟蝕刻技術電紡織技術間葉幹細胞微流道系統轉分化聚己內酯微環境神經導管微接觸式印刷技術許旺氏細胞Laser micromachiningSoft lithographyElectrospinningMesenchymal stem cellMicrofluidic systemTransdifferentiationPolycaprolactoneMicroenvironmentNerve conduitMicrocontact printingSchwann cellthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/184659/1/ntu-98-F93548019-1.pdf