- 學位論文
酵素交聯之明膠水凝膠裝載人類脂肪幹細胞於組織工程之應用
Enzyme-Crosslinked Gelatin Hydrogel with Human Adiposed-Derived Stem Cells Applied in Tissue Engineering
摘要
Part 1:人類脂肪幹細胞是間充質幹細胞的主要來源之一，其不但在適當的培養下能夠分化成多種分支，像是脂肪、軟骨和硬骨細胞，同時也具有跨胚層分化的潛力，例如內胚層的肝細胞和外胚層的神經細胞。然而，對於人類脂肪幹細胞來說重要的再生和分化能力的多能基因，其表現在二維的細胞培養方式下會減弱，越來越多的實驗結果顯示，細胞在三維培養方式聚集成細胞球，不但更能模擬細胞在體內的生長環境，也能夠提高人類脂肪幹細胞在體外培養時的幹性表現。在此研究中，我們藉由將細胞種在洋菜膠微孔盤來形成均一性的人類脂肪幹細胞球，且細胞球的大小可以被調整，最重要的是，細胞球的多能基因有顯著的提升。此外，我們使用微生物轉谷氨酰胺酶使明膠交聯，此酵素在廣泛的溫度和酸鹼值下都具有高活性的表現，相較於使用化學交聯的方式可能會導致細胞死亡，酵素交聯有較溫和的交聯反應。我們仔細地評估水凝膠的性質，並成功地使用水凝膠將細胞球包覆，研究結果顯示水凝膠對於細胞幾乎沒有毒性，且細胞展現良好的生長能力，不僅如此，相對於散細胞組別，細胞球組別擁有更佳的分化潛力尤其是脂肪和軟骨分化，老鼠皮下注射實驗結果也證明此水凝膠擁有不錯的可注射性和生物相容性。 Part 2:透明軟骨的再生一直是臨床上的挑戰。在此研究中，我們使用微生物轉谷氨酰胺酶使明膠交聯。此外，我們將能夠幫助軟骨分化，主要含有膠原蛋白和醣胺聚醣的關節軟骨細胞外間質包覆於此明膠水凝膠中，並仔細地檢驗此水凝膠的性質。將人類脂肪幹細胞包覆在這富有軟骨細胞外間質的水凝膠後，實驗結果顯示此酵素交聯方式對於細胞幾乎沒有毒性，更多的是，細胞在軟骨細胞外間質的幫助下有不錯的生長表現，最重要的是，細胞在這含有軟骨細胞外間質的水凝膠中展現更佳的軟骨分化潛力。我們更近一步的評估富含軟骨細胞外間質和人類脂肪幹細胞之水凝膠來修復全層骨軟骨缺陷的治療效果。巨觀上來說，GCC組別(水凝膠包覆細胞跟軟骨細胞外間質)手術經過八週後有光滑的關節表面帶有透徹像新透明組織的再生。在微米級電腦斷層掃描評定中，在手術四週後骨體積比例和平均骨小樑厚度在GCC組別有顯著的提升。組織學上，比起GC(水凝膠只包覆細胞)組別，於術後八週時，GCC組別展現出低程度的發炎反應且有更多的軟骨細胞在新生成的軟骨層中。相對於D組別(只有缺陷)，GC組別和GGC組別表現出顯著的醣胺聚醣含量。此外，在術後四週和八週之間，GCC組別的醣胺聚醣含量有明顯的增加。而且，從阿新藍染色的圖片中，說明GCC組別在術後八週有發現新生成的透明組織。總結來說，將富有關節軟骨細胞外間質和人類脂肪幹細胞的明膠水凝膠移植至兔子膝蓋關節缺陷模型中，能顯著的提升透明軟骨再生。
並列摘要
Part 1:Human adipose-derived stem cells (hASCs), an abundant source of mesenchymal stem cells (MSCs), not only can differentiate into multiple lineages when cultivated in appropriate conditions, including adipogenic, chondrogenic and osteogenic lineages but also have the potential of transdifferentiation such as hepatocyte of endoderm origin and neuron of ectoderm origin. However, the expression of pluripotency markers which is important for the renewal and differentiation capabilities of hASCs decreases during monolayer culture. Increasing evidence has proven that cells aggregated to form cell spheroids in 3D cell cultures better mimic the in vivo microenvironment and can enhance the expression of stemness markers. In this study, uniform hASC spheroids were formed by seeding cells in agarose microwell plates, and the size of the spheroids could be adjusted. Most importantly, the stemness expression of the spheroids increased significantly. Additionally, we utilized microbial transglutaminase (mTG), which is an enzyme that exhibits highly specific activity over a wide range of temperature and pH, to crosslink gelatin. The enzymatic crosslinking reaction is milder than physical and chemical methods, which may lead to cell death. The properties of the gelatin/mTG hydrogel were evaluated in detail. In addition, the spheroids were encapsulated in the 3D hydrogel successfully. The results showed that the hydrogel has low toxicity to the cells, which significantly proliferated in the 3D hydrogel. Moreover, the analysis of the differentiation potential indicated that the cell spheroids in the 3D hydrogel exhibited good activity, especially adipogenesis and chondrogenesis, compared to the cell suspension group. Furthermore, the in vivo data of subcutaneous injection in ICR mice confirmed the excellent injectability and biocompatibility of the 3D hydrogel. Part 2:The regeneration of hyaline cartilage remains clinically challenging. In this study, we applied microbial transglutaminase (mTG) to crosslink gelatin. Besides, the articular cartilage extracellular matrix (cECM) mainly comprising collagen and GAGs which can support chondrogenesis was encapsulated in the gelatin/mTG hydrogel. The properties of the cECM-encapsulated hydrogel were examined in detail. After human adipose-derived stem cells (hASCs) were encapsulated in the hydrogel enriched with cECM, the results demonstrated the enzymatic crosslinking reaction has little toxicity to the cells. Moreover, the cells in the hydrogel performed great proliferation with the assistance of cECM. Most importantly, the cells in cECM-encapsulated hydrogel exhibited better chondrogenic differentiation potential. Furthermore, we evaluated the therapeutic effects of using hydrogel enriched with cECM and hASCs to repair a full-thickness osteochondral defect. Macroscopically, the GCC group (hydrogel encapsulated cells and cECM) regenerated a smooth articular surface with transparent new hyaline-like tissue at 8 weeks after surgery. In the micro-CT assessment, the bone volume fraction (BV/TV) and trabecular thickness (Tb.Th) values were significantly higher in the GCC group at week 4. Histologically, the GCC group displayed the low level of inflammatory response and more chondrocytes in the newly formed chondral layer compared to the GC (hydrogel encapsulated cells only) group at week 8. Both GC and GCC groups exhibited significantly higher glycosaminoglycan (GAG) content at 4 and 8 weeks after surgery compared to the D (defect only) group. Moreover, the GAG content of the GCC group improved significantly between week 4 and 8. Also, the images of Alcian blue stain illustrated there were newly formed hyaline-like tissues in the GCC group at week 8. In summary, the implantation of gelatin hydrogel enriched with cECM and hASCs can significantly promote the hyaline cartilage regeneration in rabbit knee joint models.