Enzyme-Crosslinked Gelatin Hydrogel with Human Adiposed-Derived Stem Cells Applied in Tissue Engineering

• Main Authors: Ching-Cheng Tsai, 蔡景丞
• Other Authors: 游佳欣
• Format: Others
• Language: en_US
• Published: 2019
• Online Access: http://ndltd.ncl.edu.tw/handle/72x3vv

碩士 === 國立臺灣大學 === 化學工程學研究所 === 107 === 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.