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  • 學位論文

3D生物列印含脂肪幹細胞與透明質酸之雙交聯仿生水膠支架應用於軟骨組織修復

3D Bioprinting of human adipose stem cells (hADSCs) encapsulated hyaluronic acid (HA) based biomimetic double crosslinked hydrogel bioink for cartilage tissue engineering (CTE)

指導教授 : 王志光
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摘要


關節軟骨覆蓋骨骼的邊緣並提供耐磨承載能力,最終支持柔性關節運動。因此,一旦這些關節軟骨受損,它們就會限制患者的自由關節運動並導致嚴重的並發症。此外,關節軟骨本質上是無血管的,這也限制了其在損傷後自我修復的能力。為了解決與關節軟骨損傷相關的這些問題,近來科學家引入了軟骨組織工程(CTE)。 CTE通過使用細胞、生長因子和生物材料支架的組合策略幫助修復或再生受損的軟骨。由於水凝膠和天然軟骨之間的相似性,水凝膠具有吸收大量水的能力,被視為軟骨模擬支架的理想材料。此外,將幹細胞或軟骨細胞與水凝膠支架結合起來被認為是CTE的有前景的方法。因為該策略能夠支持高密度細胞群,細胞附著,細胞均勻分佈,且為細胞生長和分化提供理想的微環境。不幸的是,目前為止,開發結構性完整的水凝膠支架仍是限制水凝膠在CTE中應用的主要問題。因此,為解決現有CTE相關的問題,本論文主旨在利用3D生物列印技術,通過組合脂肪幹細胞(ADSCs)和透明質酸(HA)為基礎的水凝膠來列印軟骨支架,並分析軟骨支架的可行性後續將應用於軟骨組織再生。首先,為了開發新的軟骨細胞外基質模擬水凝膠系統,我們合成了生物素化(biotinylation)的透明質酸(HA-Biotin),並通過傅立葉變換紅外光譜分析證實了成功的接枝生物素。接下來,製備HA-Biotin-Avdin水凝膠,即通過生物素和鏈黴抗生物素蛋白(Streptavidin)之間的非共價鍵將Streptavidin linker與HA-Biotin水膠混合形成部分非共價交聯的HA基水凝膠。由於Streptavidin中存在細胞粘附位點,可使HA-Biotin-Streptavidin(HBS)水凝膠增加細胞的附著能力。隨後,將部分交聯的HBS水凝膠與海藻酸鈉(alginate)不同比例混合,再使用Rokit co./InVivo生物列印機進行可列印條件測試,於獲得較佳的列印參數後,將剛烈印的3D HBS支架浸沒在CaCl2溶液中,使其經由alginate的carboxyl group和CaCl2溶液的Ca2+螯合成離子交聯,即增強3D HBS水凝膠支架的機械強度。以上條件的參數可對纖維結構形成、支架支撐能力、列印解析度和交聯劑濃度等進行ADSCs細胞活性與分化能力探討,以冀望獲得最適合的3D列印軟骨再生支架。本研究結果顯示,用Streptavidin linker進行部分交聯HA-Biotin對可列印性具有顯著的影響,即由最佳3D列印HBS支架的形態分析顯示支架可具有優良的線性交錯幾何形狀和清晰的微孔洞。並在細胞活性分析的結果進一步證實,本研究雙交聯HA基質水凝膠具有3D生物列印軟骨組織工程支架的優異潛力。

並列摘要


Articular cartilage covers the edges of the bones and provides wear resistant load bearing capacity which ultimately supports the flexible joint movement. Therefore, once these articular cartilages get damaged, they limit the free joint movements in patients and cause severe complication. Also, articular cartilage is avascular in nature, which also restricts its ability to repair itself after any damage. To address these issues associated with articular cartilage damage, cartilage tissue engineering (CTE) has been introduced. CTE helps in repairing or regenerating damaged cartilages by using a combined strategy which involves cell, growth factors, and biomaterial scaffolds. Hydrogel with the ability to absorb a large amount of water viewed as an ideal material for cartilage mimetic scaffold owing to the similarity between the hydrogel and native cartilage. Combining stem cell or chondrocytes with hydrogel scaffold is regarded as a promising approach for CTE. This strategy is capable of supporting highly dense cell population, cell attachment, homogeneous cell distribution, and also offer an ideal microenvironment for cell growth and differentiation. Unfortunately, developing hydrogel scaffold with required structural integrity is a major issue that limits the application of hydrogel in CTE. Therefore, to address the problems associated with existing CTE, this thesis aimed to utilize 3D bioprinting to print cartilage constructs by combining adipose-derived stem cells (ADSCs) and hyaluronic acid (HA) based hydrogels. First, to develop a new cartilage extracellular matrix (ECM) mimetic hydrogel system, we synthesized biotinylated-hyaluronic acid (HA-Biotin) and confirmed the successful grafting of biotin with HA trough Fourier Transform Infrared Spectroscopy (FTIR) analysis. Next, HA-Bio hydrogel was prepared and Streptavidin was mixed with this hydrogel to form partially crosslinked HA-based hydrogel through non-covalent bonding between biotin and streptavidin. Addition of streptavidin also supports higher cell attachment due to the presence of cell adhesion sites in streptavidin. After that, partially crosslinked HA-Bio-Streptavidin (HBS) hydrogel was mixed with sodium alginate and subsequently printed using Rokit INVIVO bioprinter. After printing, 3D scaffolds were submerged into CaCl2 solution achieve ionic crosslinking through ion transfer between sodium alginate and CaCl2. Different parameters such as fiber formation, self-supporting ability, printing resolution, and crosslinker concentration were optimized to get desired 3D printed constructs. In vitro cell proliferation and live/dead staining assay were also performed on 3D cell-laden scaffolds. The result showed that partially crosslinking the biotinylated-HA based hydrogel with streptavidin has a significant effect on printability. Morphological analysis of optimal 3D printed scaffold showed clearly visible pores with desired shape and geometry. Favorable cell proliferation and growth was also observed in 3D HBSA based hydrogel scaffolds. These result further confirmed that double crosslinking HA-based hydrogel could be a good choice for 3D bioprinting based tissue engineering.

參考文獻


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