人體由軟組織和硬組織組成。 遺傳和外源因素，如衰老、疾病和損傷等，對人體造成的的影響非常迫切需要通過臨床修復來恢復受損組織或器官的功能和形態。 組織工程是一個新興的多學科領域，涉及生物學、醫學和工程學，旨在通過方法的現代化來恢復、維持或增強組織和器官功能，從而改善病人的健康和生活質量。 成功的支架構造需要在生理學和解剖學方面來模仿天然組織。 3D 列印是公認可行的技術，因為它具有可控的支架建構、再現性且更緊密地模仿在解剖學上感興趣的形狀。 然而，即使以該技術目前的最新水平亦無法產生可模擬生理學的微觀結構，即人體組織的天然細胞外基質 (ECM)。
另一方面，靜電紡絲是從各種可生物降解的聚合物和具有不同形態的生物材料構建 ECM 模擬纖維支架中最成熟的技術之一。 然而，由於複雜的射流-電場相互作用，導致射流路徑的可控性差，而產生細胞無法滲入的片狀二維支架。 在這篇論文中，我們的第一項工作裡，報告了一種“新型自導向/自駕式聚合物單射流 (AJ) 靜電紡絲技術”，由於已沉積纖維上的電荷餘留和耗散過程，致使射流在懸臂式運動 (M1) 和鞭打運動 (M2) 之間自行切換。這種獨特的自噴射切換工藝將纖維沉積在收集器上，隨著此過程的持續，可產生具有不同屈曲程度的纖維形態及堆積密度的差異，並因此而共同引入了孔隙大小梯度和力學強度。 因此，只要使用簡單且成本低廉的自製常規靜電紡絲裝置，經單一步驟即可成功地構建多孔但力學強度堅韌的微纖維支架 (MFS)。各項物理表徵顯示，其與傳統的隨機多噴射流 (RJ) 生產的奈米纖維支架 (NFS) 相比，AJ 生產的微纖維支架具有近10 倍的多孔性和 8 倍的力學可拉伸性。 通過 M1 模式用高階屈曲構造的基礎層提供了出色的形狀記憶和力學魯棒性，可見於補充視頻資料。 對 3T3 小鼠胚胎纖維母細胞進行的體外研究結果表明，與通過多噴射流靜電紡絲的傳統奈米纖維支架相比，AJ 生產的支架有助於更高的細胞附著、滲透和增殖到支架的內層。 在這項研究中，我們還展示了 AJ 靜電紡絲具有可重複性、穩健性和可調性，並且史上首次為靜電紡絲技術生產的支架帶來了一致性。
雖然我們的第一個工作成功地構建了模擬生理的支架，但我們在第二個工作中展示了 AJ 在各種三維模板上的自我導向建構過程，試圖在符合解剖學的原則下製造模擬人體器官的三維支架時。 AJ 採用各種三維彎曲路徑，這些路徑遵循最佳場力線，將纖維沉積在三維收集器的不同側面，從而實現共形纖維沉積，產生精確的三維複製品。當採用了新穎的書寫建構策略，即可避免具有複雜三維幾何形狀模板而致的場力線競爭，例如本研究中的人臉模板。 模擬（COMSOL-Multiphysics 軟件）和實驗結果均表明，AJ 顯示出高目標鑑識率和解析度，圖案特徵可小至100微米(µm) 大至數十公分，並且能夠直接寫入極具挑戰性的圖案化二維及三維支架，包括人體器官尺寸的三維臉部、女性乳房和乳頭以及血管支架。 物理和生物學特性亦表明，紡成的支架呈海綿狀，並顯示出通過 AJ 靜電紡絲的 M1/M2 切換所實現的特殊形狀記憶性，且具有多向 3T3 細胞之附著、增殖和遷移能力，且細胞穿透深度超過 250 µm。 活細胞和死細胞的熒光染色表明，AJ 紡絲支架可以成功地將三維細胞網絡形成到支架中； 而膠原蛋白和細胞核的天狼星紅染色顯示細胞外基質的分泌和擴散以及細胞的共定位，並有掃描式電鏡影像佐證。我們的方法第一次展示將具有百年歷史的靜電紡絲技術引入可重複性的研究，並能將具有良好形狀記憶的三維支架直接寫出，從而為將該技術添加到3D支架製造技術中開啟了新路。

Human body is made up of both soft and hard tissues. Genetics and exogenous factors, such as aging, diseases and injury, affecting the human body highly demand clinical repairs to restore the functions and morphologies of the damaged tissues or organs. Tissue engineering is an emerging multidisciplinary field involving biology, medicine, and engineering to modernize the ways, which can improve the health and quality of life of affected people, by restoring, maintaining or enhancing tissue and organ functions. A successful scaffold construction requires to mimic the native tissue in terms of both physiologically and anatomically. 3D printing is the well-recognized technique for its controllable scaffold writing, reproducibility, and imitating shapes of interest more closely, in other words anatomically. However, the current state-of-art of this technique is unable to produce its microstructures mimicking physiologically i.e. the native extracellular matrix (ECM) of human tissues.
On the other hand, electro(static) spinning is one of the most established techniques for constructing ECM mimicking fibrous scaffolds from various biodegradable polymers and biomaterials with diverse morphologies. However, the poor controllability over its jet path due to complex jet-field interactions produce cell-impermeable sheet-like 2D scaffolds. In this thesis, in our first work, we report a “novel self-directing/autopilot polymer single jet (AJ) electrospinning” that self-switched between cantilever-like armed jet motion (M1) and whipping motion (M2) due to charge-retention and dissipation of pre-deposited fibers. This unique self-jet switching process deposited fibers on the collector with distinct fiber morphologies of high-order and low-order buckling and difference in packing density together introduced gradient porosity and mechanical strength as the process continued. Thereby, it successfully constructed porous yet mechanically robust microfibrous scaffolds (MFS) using the simple and cost-effective, homemade conventional electrospinning setup in a single-step. The physical characterizations revealed that the AJ-produced MFS showed nearly 10X porous and 8X mechanically stretchable as compared to the conventional random jet (RJ) produced nanofibrous scaffolds (NFS). The base layer constructed with high-order buckling by M1 mode provided excellent shape memory and mechanical robustness, bestowed with supporting videos. The in-vitro study carried out with 3T3 mouse embryonic fibroblasts results show AJ-produced scaffolds allow higher cell attachment, penetration and proliferation into inner layers of scaffold compared to the conventional nanofibrous scaffolds by multi-jets electrospinning. In this study, we also show the AJ-electrospinning is reproducible, robust and tunable, and that first time brings consistency to electrospinning produced scaffolds.
Whilst our first work successfully constructed scaffolds mimicking physiologically, we demonstrate the self-searched writing of AJ on various 3D templates in an attempt of fabricating 3D scaffolds mimicking human organs anatomically in our second work. The AJ took various 3D bending paths which followed optimal field lines to deposit fibers on the different sides of the 3D collectors, thereby achieving conformal fiber deposition that produced exact 3D replicas. Novel writing strategies were adopted thus avoiding the competitive field lines to templates having complex 3D geometries, for example human face in this study. Both the simulation (COMSOL-Multiphysics software) and experimental results show the AJ displayed high target recognition/specificity and resolution with pattern features as small as 100 µm to 10’s cm and is capable of direct writing challenging patterned-2D and 3D scaffolds, including human-organ-scale 3D face, female breast & nipple and vascular graft. The physical and biological characteristics revealed as-spun scaffolds were spongy and showed exceptional shape memory achieved through M1/M2 switching of AJ-electrospinning with multi-directional 3T3 cells attachment, proliferation and migration with deeper cell penetration over 250 µm. The live and dead cell fluorescent staining showed the AJ-spun scaffolds allowed successful formation of 3D cell networks into the scaffold; whereas, Sirius red staining of collagen and cell nucleus showed the secretion and spreading of ECM and co-localization of cells along with SEM imaging. By doing so, this is the first study to introduce reproducibility, capable of direct-writing 3D scaffolds with good shape memory to the century-old electrospinning technique thus paving the path to add this technique to the new list of 3D scaffolds fabrication techniques.

Abstract…………………………………………………………………………………………....i
Acknowledgements………………………………………………………………………………vi
Chapter 1: Motivation…………………………………………………………………………...1
1.1 Introduction………………………………………………………………………1
1.2 Motivation………………………………………………………………………...2
1.2.1 Novel Self-directing/autopilot Polymer Jet Developing Layered-like 3D Buckled Microfibrous Scaffolds for Tissue Engineering Applications…...2
1.2.2 Self-searched Writing of Human-organ-scale 3D Topographic Scaffolds with Shape Memory by Silkworm-like Electrospun Autopilot Jet………..6
1.3 Outline of the thesis……...………….…………………………………………...7
Chapter 2: Literature review……………………………………………………………………8
2.1 Background…………………………………………………………………………..8
2.1.1 Extracellular matrix…………………………………………………………8
2.1.2 Tissue Engineering…………………………………………………………10
2.1.3 Need for the fabrication of 3D porous scaffolds…………………………...12
2.2 Introduction to the electrospinning technique……………………………………14
2.2.1 Working principle………………………………………………………….14
2.2.2 Factors influencing electrospinning process……………………………….16
2.2.2.1 Solution properties……………………………………………….16
2.2.2.2 Processing parameters……………………………………………17
2.3 Complications in the conventional electrospinning process……….…………….20
2.3.1. Poor jet path controllability……………………………………………….21
2.3.2 Fiber diameter: dual role in porosity and mechanical properties…………..23
2.3.3 Fiber packing density: dual role in porosity and mechanical properties…..28
2.3.4 Fiber orientation: dual role in porosity and mechanical properties………..37
2.4 Post-processing electrospun scaffolds……………………………………………..41
2.5 Near-field electrospinning………………………………………………………….43
2.6 Melt-electrowriting…………………………………………………………………47
2.7 Summary…………………………………………………………………………….53
Chapter 3: Novel Self-directing/autopilot Polymer Jet Developing Layered-like 3D Buckled Microfibrous Scaffolds for Tissue Engineering Applications………………………………..57
3.1 Introduction…………………………………………………………………………57
3.2 Experimental section……………………………………………………………….58
3.2.1 Materials and Fabrication of fiber constructs and reproducibility…………58
3.2.2 Characterization of the fibers………………………………………………59
3.2.3 Analysis of water-uptake…………………………………………………..60
3.2.4 Preparation of scaffolds to mimic ECM…………………………………...60
3.2.5. Proliferation assay and morphological studies of cells on fibers………….61
3.2.6 Statistical analysis………………………………………………………….61
3.3 Results and Discussion……………………………………………………………..61
3.3.1 High-speed camera observation of unique self-directing/autopilot (AJ) jet electrospinning process…………………………………………………………..61
3.3.2 Reproducibility and tunability of the autopilot jet…………………………66
3.3.3 Characterizations of the fibrous scaffolds………………………………….69
3.3.4 Gradient porosity of the microfibrous scaffolds…………………………...72
3.3.5 Gradient mechanical strength of the microfibrous scaffolds………………75
3.3.6 Cell proliferation on the 3D microfibrous scaffolds……………………….77
Chapter 4: Self-searched Writing of Human-organ-scale 3D Topographic Scaffolds with Shape Memory by Silkworm-like Electrospun Autopilot Jet………………………………..82
4.1 Introduction…………………………………………………………………………82
4.2 Experimental section……………………………………………………………….83
4.2.1 Materials…………………………………………………………………...83
4.2.2 Solution preparation and electrospinning………………………………….83
4.2.3 Electric field simulation……………………………………………………84
4.2.4 Physical characterization of the scaffolds………………………………….85
4.2.5 Scaffold preparation for in-vitro cell culture………………………………85
4.2.6 In-vitro study……………………………………………………………….86
4.2.7 Green CMFDA (5-chloromethylfluorescein diacetate) imaging study....…86
4.2.8 Immunostaining study……………………………………………….……..87
4.2.9 Sirius Red staining…………………………………………………………87
4.2.10 FESEM analysis of cell-scaffold morphology……………………………88
4.3 Results and Discussion……………………………………………………………...88
4.3.1 2D pattern writing………………………………………………………….91
4.3.2 3D scaffolds construction………………………………………………….94
4.3.3 Characteristics of the AJ-produced scaffolds………………..…………...106
Chapter 5: Conclusions and future work………………..…………………………………..114
BIBLIOGRAPHY…………………….……………………………………………………….118
Appendix A.................................................................................................................................135

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