水性3D列印墨水之開發與軟骨組織工程之應用研究

Abstract

本研究探討材料物理化學特性對細胞行為之影響，並發展多成份水性三維(three-dimensional, 3D)列印墨水以作為製造可促進組織修復之3D支架用。第一部分為製備一系列不同親水基團比例及薄膜厚度之陰離子型水性生物可降解聚胺酯(polyurethane, PU)以了解高分子於水溶液環境下表面鏈段重整情形對細胞行為的影響。研究中發現聚胺酯硬段中的羧基與胺基會與水溶液中的鈣離子作用，並進入到間葉幹細胞中造成細胞快速移動與形成自組裝團聚，研究中也發現此細胞團聚的形成與否和團聚大小分別與NF-kB 路徑和Hippo 路徑相關，此聚胺酯薄膜上所形成的細胞團聚相較於貼附的細胞具有較高的幹性基因(Oct4、Nanog和Sox2)表現和多分化能力。本部分顯示高分子於細胞培養環境中的鏈段重整及表面官能基螯合鈣質的能力將可促進間葉幹細胞自我團聚的形成，並提升幹細胞的分化能力，此有助於設計出合適的材料化學結構以影響幹細胞行為並促進組織修復。第二部分為利用水性3D列印方式並輔以聚氧乙烯作為增黏劑，將水性生物可降解聚胺酯進行組織工程支架成形，此製程相較於其他的3D列印方式無需使用有毒有機溶劑、交聯劑及光起始劑。所形成後的水性3D列印聚胺酯支架具有良好的彈性及細胞相容性，並發現軟骨細胞於支架中可形成細胞團聚，此可促進軟骨細胞增生及分泌細胞外基質，透過此部分研究我們發展出水性3D列印製程與彈性3D支架，並證實此支架適用於軟骨組織工程中。第三部分中我們利用水性3D列印技術發展出了可促幹細胞自動軟骨化的支架，當中利用水性生物可降解聚胺酯、玻尿酸(hyaluronan, HA)與生長因子TGFB3或小分子藥物Y27632進行列印。當幹細胞種植於水性三成份支架中可形成細胞團聚，而釋放出的TGFB3或Y27632可促進其軟骨分化，且研究也發現相較於使用TGFB3，若使用Y27632可避免MSC往肥大化軟骨分化，經由兔子關節軟骨實驗也證實此含Y27632支架具有良好的修復效果，本部分證實所發展之三成份水性3D列印墨水所形成的3D列印支架於客製化軟骨組織工程上具有極高的應用性。本論文第四部份為探討材料碎形維度對細胞行為的影響。由研究中可發現較高碎形維度的水凝膠中纖維母細胞與間葉幹細胞皆有較高的增生速率，而幹細胞分化方面當細胞分別培養於碎形維度≥ 1.8、≥ 1.6或 ≤ 1.4的水凝膠中時可分別促進幹細胞的軟骨分化、硬骨分化與神經分化，此顯示當幹細胞培養於具有和目標組織相同之碎形維度的水凝膠中時可促進其往目標組織分化，此部分有助於未來促進幹細胞分化之3D列印水凝膠墨水的設計。透過本論文之四個部分探討材料物化性質對細胞行為的影響及水性3D列印的可行性，期望幫助未來可設計出更合適的材料化學結構和物理構型，並搭配水性3D列印與多成份列印墨水配方的優點，達到更優異的組織修復效果。

This study investigated the effect physo-chemical properties of materials on cell behavior and developed the water-based multi-component three-dimensional (3D) printing inks for use in tissue engineering. In the fist section, a series of biodegradable anionic polyurethane (PU) was synthesized with different extents of surface functional group rearrangement in response to aqueous environment. The recruitment of carboxyl and amino groups from the bulk material to the surface can interact with calcium ion. The surface-bound calcium was observed to enter mesenchymal stem cells (MSCs), which prompted MSC migration and assembly. The MSC aggregate formation was associated with the NF-kB pathway while the aggregate size was connected to the Hippo pathway. The MSC aggregates had greater expressions of Oct4, Nanog, and Sox2 as well as multi-differentiation capacities than attached MSCs. This part of study suggested that the critical importance of surface functional group and its calcium binding capacity on the self-assembly of MSCs, which may help define and design the appropriate MSC-substrate interaction for tissue engineering applications. In the second section, scaffolds were fabricated from the biodegradable PU dispersion by water-based 3D printing using polyethylene oxide as a viscosity enhancer. Not any toxic organic solvent, crosslinker, or initiator was used. The green process generated a highly elastic scaffold with good affinity to cells. In the 3D-printed PU scaffolds, cells tended to aggregate in clusters. Chondrocytes in 3D-printed PU scaffolds have excellent proliferation and matrix production. In this part of study, we developed a green water-based 3D printing platform to fabricate biodegradable/elastic scaffolds for cartilage tissue engineering applications. In the third section, we developed a 3D-printed scaffold to promote the spontaneous chondrogenesis of MSCs. The scaffolds were printed from the water-based ink containing PU, hyaluronan (HA), and Y27632 (or TGFB3). MSCs seeded in the scaffolds were self-assembled into MSC aggregates and underwent chondrogenesis effectively. The use of Y27632 could prevent the expression of hypertrophic marker. Transplantation of the MSC-seeded PU/HA/Y scaffold in rabbit chondral defects significantly improved the cartilage regeneration. This part of study suggested that the water-based 3D printed PU/HA/Y scaffolds may have potential applications in customized cartilage tissue engineering. In the fourth section, we evaluated the effect of fractal dimension (Df) of hydrogels on cell proliferation and stem cell differentiation. Fibroblasts and mesenchymal stem cells grow faster in hydrogels with a higher Df. Hydrogels with the Df matched to that of a specific tissue favor the tissue-specific differentiation. Chondrogenesis, osteogenesis, and neurogenesis are each preferred in hydrogels with Df ≥ 1.8, ≥ 1.6, and ≤ 1.4, respectively. This part of study suggested that the fractal structure of gel can modulate cell proliferation and fate, which supply a new design rationale to design the appropriate fractal and molecular structure of hydrogels for applications of 3D printing.