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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 游佳欣(Jiashing Yu) | |
| dc.contributor.author | Hsin-Yu Chen | en |
| dc.contributor.author | 陳信宇 | zh_TW |
| dc.date.accessioned | 2021-06-15T11:29:07Z | - |
| dc.date.available | 2021-08-25 | |
| dc.date.copyright | 2016-08-25 | |
| dc.date.issued | 2016 | |
| dc.date.submitted | 2016-08-17 | |
| dc.identifier.citation | [1] B.J. Huang, J.C. Hu, K.A. Athanasiou. Cell-based tissue engineering strategies used in the clinical repair of articular cartilage, Biomaterials 98 (2016) 1-22.
[2] J.C. Bernhard, G. Vunjak-Novakovic. Should we use cells, biomaterials, or tissue engineering for cartilage regeneration, Stem cell research & therapy 7 (2016) 56. [3] C. Vinatier, J. Guicheux. Cartilage tissue engineering: From biomaterials and stem cells to osteoarthritis treatments, Annals of physical and rehabilitation medicine 59 (2016) 139-144. [4] I.M. El-Sherbiny, M.H. Yacoub. Hydrogel scaffolds for tissue engineering: Progress and challenges, Global cardiology science & practice 2013 (2013) 316-342. [5] B.A. Lindborg, J.H. Brekke, C.M. Scott, Y.W. Chai, C. Ulrich, L. Sandquist, E. Kokkoli, T.D. O'Brien. A chitosan-hyaluronan-based hydrogel-hydrocolloid supports in vitro culture and differentiation of human mesenchymal stem/stromal cells, Tissue engineering. Part A 21 (2015) 1952-1962. [6] C. Arakawa, R. Ng, S. Tan, S. Kim, B. Wu, M. Lee. Photopolymerizable chitosan-collagen hydrogels for bone tissue engineering, Journal of tissue engineering and regenerative medicine (2014). [7] Z. Chen, M. Zhao, K. Liu, Y. Wan, X. Li, G. Feng. Novel chitosan hydrogel formed by ethylene glycol chitosan, 1,6-diisocyanatohexan and polyethylene glycol-400 for tissue engineering scaffold: in vitro and in vivo evaluation, Journal of materials science. Materials in medicine 25 (2014) 1903-1913. [8] Y.H. Cheng, K.H. Hung, T.H. Tsai, C.J. Lee, R.Y. Ku, A.W. Chiu, S.H. Chiou, C.J. Liu. Sustained delivery of latanoprost by thermosensitive chitosan-gelatin-based hydrogel for controlling ocular hypertension, Acta biomaterialia 10 (2014) 4360-4366. [9] S. Khetan, M. Guvendiren, W.R. Legant, D.M. Cohen, C.S. Chen, J.A. Burdick. Degradation-mediated cellular traction directs stem cell fate in covalently crosslinked three-dimensional hydrogels, Nature materials 12 (2013) 458-465. [10] F. Han, F. Zhou, X. Yang, J. Zhao, Y. Zhao, X. Yuan. A pilot study of conically graded chitosan-gelatin hydrogel/PLGA scaffold with dual-delivery of TGF-b1 and BMP-2 for regeneration of cartilage-bone interface, Journal of Biomedical Materials Research Part B: Applied Biomaterials 103 (2015) 1344-1353. [11] H. Naderi-Meshkin, K. Andreas, M.M. Matin, M. Sittinger, H.R. Bidkhori, N. Ahmadiankia, A.R. Bahrami, J. Ringe. Chitosan-based injectable hydrogel as a promising in situ forming scaffold for cartilage tissue engineering, Cell biology international 38 (2014) 72-84. [12] A. Sukarto, C. Yu, L.E. Flynn, B.G. Amsden. Co-delivery of adipose-derived stem cells and growth factor-loaded microspheres in RGD-grafted N-methacrylate glycol chitosan gels for focal chondral repair, Biomacromolecules 13 (2012) 2490-2502. [13] F. Han, X. Yang, J. Zhao, Y. Zhao, X. Yuan. Photocrosslinked layered gelatin-chitosan hydrogel with graded compositions for osteochondral defect repair, Journal of materials science. Materials in medicine 26 (2015) 160. [14] N.J. Steinmetz, E.A. Aisenbrey, K.K. Westbrook, H.J. Qi, S.J. Bryant. Mechanical loading regulates human MSC differentiation in a multi-layer hydrogel for osteochondral tissue engineering, Acta biomaterialia 21 (2015) 142-153. [15] I.Y. Kim, S.J. Seo, H.S. Moon, M.K. Yoo, I.Y. Park, B.C. Kim, C.S. Cho. Chitosan and its derivatives for tissue engineering applications, Biotechnology advances 26 (2008) 1-21. [16] M.M. Masatoshi Sugimoto, Hitoshi Sashiwab, Hiroyuki Saimotob, Yoshihiro Shigemasabl. Preparation and characterization of water-soluble chitin and chitosan derivatives, Carbohydrate Polymers 36 (1998) 49-59. [17] D.K. Knight, S.N. Shapka, B.G. Amsden. Structure, depolymerization, and cytocompatibility evaluation of glycol chitosan, Journal of biomedical materials research. Part A 83 (2007) 787-798. [18] H.W.T.M. J.-K. Francis Suh. Application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering: a review, Biomaterials 21 (2000) 2589-2598. [19] A.S. Ashkan Lahiji, David S. Hungerford, Carmelita G. Frondoza. Chitosan supports the expression of extracellular matrix proteins in human osteoblasts and chondrocytes, Journal of biomedical Materials Research 51 (2000) 586-595. [20] A.S. Brian G. Amsden, Darryl K. Knight, and Stephen N. Shapka. Methacrylated glycol chitosan as a photopolymerizable biomaterial, Biomacromolecules 8 (2007) 3758–3766. [21] T.V.C. Xia Lou. Swelling behavior and mechanical properties of chemically cross-linked gelatin gels for biomedical use, Biomaterials Applications 14 (1999) 184-191. [22] D.I. Jalindar Totre, and Abraham J. Domb. Properies and hemostatic application of gelatin, Biodegradable Polymers in Clinical Use and Clinical Development (2011) 91-109. [23] I.R. Serra, R. Fradique, M.C. Vallejo, T.R. Correia, S.P. Miguel, I.J. Correia. Production and characterization of chitosan/gelatin/beta-TCP scaffolds for improved bone tissue regeneration, Materials science & engineering. C, Materials for biological applications 55 (2015) 592-604. [24] W. Xia, W. Liu, L. Cui, Y. Liu, W. Zhong, D. Liu, J. Wu, K. Chua, Y. Cao. Tissue engineering of cartilage with the use of chitosan-gelatin complex scaffolds, Journal of biomedical materials research. Part B, Applied biomaterials 71 (2004) 373-380. [25] A. Gupta, S. Bhat, P.R. Jagdale, B.P. Chaudhari, L. Lidgren, K.C. Gupta, A. Kumar. Evaluation of three-dimensional chitosan-agarose-gelatin cryogel scaffold for the repair of subchondral cartilage defects: an in vivo study in a rabbit model, Tissue engineering. Part A 20 (2014) 3101-3111. [26] B.S. Jerry Huang, J. William Futrell, M.D., Adam J. Katz, M.D.,, M.D. Prosper Benhaim, H. Peter Lorenz, M.D.,, M.D. Marc H. Hedrick, P.D. Patricia A. Zuk, Min Zhu, M.D., Hiroshi Mizuno, M.D. Multilineage cells from human adipose tissue: Implications for cell-based therapies, Tissue Engineeing 7 (2001) 211-228. [27] P.A. Zuk, M. Zhu, P. Ashjian, D.A. De Ugarte, J.I. Huang, H. Mizuno, Z.C. Alfonso, J.K. Fraser, P. Benhaim, M.H. Hedrick. Human adipose tissue is a source of multipotent stem cells, Molecular biology of the cell 13 (2002) 4279-4295. [28] A.M. Rodriguez, C. Elabd, E.Z. Amri, G. Ailhaud, C. Dani. The human adipose tissue is a source of multipotent stem cells, Biochimie 87 (2005) 125-128. [29] N.C. Cheng, S. Wang, T.H. Young. The influence of spheroid formation of human adipose-derived stem cells on chitosan films on stemness and differentiation capabilities, Biomaterials 33 (2012) 1748-1758. [30] F.T. Xu, H.M. Li, C.Y. Zhao, Z.J. Liang, M.H. Huang, Q. Li, Y.C. Chen, G.Y. Chi. Characterization of chondrogenic gene expression and cartilage phenotype differentiation in human beast adipose-derived stem cells promoted by ginsenoside Rg1 in vitro, Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology 37 (2015) 1890-1902. [31] J.S. Lee, G.I. Im. Influence of chondrocytes on the chondrogenic differentiation of adipose stem cells, Tissue engineering. Part A 16 (2010) 3569-3577. [32] T. Debnath, S. Ghosh, U.S. Potlapuvu, L. Kona, S.R. Kamaraju, S. Sarkar, S. Gaddam, L.K. Chelluri. Proliferation and differentiation potential of human adipose-derived stem cells grown on chitosan hydrogel, PloS one 10 (2015) e0120803. [33] Z. Cai, B. Pan, H. Jiang, L. Zhang. Chondrogenesis of human adipose-derived stem cells by in vivo co-graft with auricular chondrocytes from microtia, Aesthetic plastic surgery 39 (2015) 431-439. [34] C.-L.Tang. Y. Zhang, W.-J. Chen, Q. Zhang, S.-L. Wang. Dynamic compression combined with exogenous SOX-9 promotes chondrogenesis of adipose-derived mesenchymal stem cells in PLGA scaffold, European Review for Medical and Pharmacological Sciences 19 (2015) 2671-2678. [35] J.H. Lee, Y.S. Han, S.H. Lee. Long-duration three-dimensional spheroid culture promotes angiogenic activities of adipose-derived mesenchymal stem cells, Biomolecules & therapeutics 24 (2016) 260-267. [36] Z. Cesarz, K. Tamama. Spheroid culture of mesenchymal stem cells, Stem cells international 2016 (2016) 9176357. [37] L. Xie, M. Mao, L. Zhou, B. Jiang. Spheroid mesenchymal stem cells and mesenchymal stem cell-derived microvesicles: Two potential therapeutic strategies, Stem cells and development 25 (2016) 203-213. [38] G.S. Huang, L.G. Dai, B.L. Yen, S.H. Hsu. Spheroid formation of mesenchymal stem cells on chitosan and chitosan-hyaluronan membranes, Biomaterials 32 (2011) 6929-6945. [39] T. Mosmann. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays, Journal oflmmunologicalMethods, 65 (1983) 55-63. [40] D.G.a.N. Thomasset. Use of MTT colorimetric assay to measure cell activation, Journal of Immunological Methods 94 (1986) 57-63. [41] S.L. Waters, J. Alastruey, D.A. Beard, P.H. Bovendeerd, P.F. Davies, G. Jayaraman, O.E. Jensen, J. Lee, K.H. Parker, A.S. Popel, T.W. Secomb, M. Siebes, S.J. Sherwin, R.J. Shipley, N.P. Smith, F.N. van de Vosse. Theoretical models for coronary vascular biomechanics: progress & challenges, Progress in biophysics and molecular biology 104 (2011) 49-76. [42] E. Senses, P. Akcora. Mechanistic model for deformation of polymer nanocomposite melts under large amplitude shear, Journal of Polymer Science Part B: Polymer Physics 51 (2013) 764-771. [43] H. Shafiee, M.B. Sano, E.A. Henslee, J.L. Caldwell, R.V. Davalos. Selective isolation of live/dead cells using contactless dielectrophoresis (cDEP), Lab on a chip 10 (2010) 438-445. [44] B.H. Liu, H.Y. Yeh, Y.C. Lin, M.H. Wang, D.C. Chen, B.H. Lee, S.H. Hsu. Spheroid formation and enhanced cardiomyogenic potential of adipose-derived stem cells grown on chitosan, BioResearch open access 2 (2013) 28-39. [45] H.Y. Yeh, T.Y. Lin, C.H. Lin, B.L. Yen, C.L. Tsai, S.H. Hsu. Neocartilage formation from mesenchymal stem cells grown in type II collagen-hyaluronan composite scaffolds, Differentiation; research in biological diversity 86 (2013) 171-183. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/49447 | - |
| dc.description.abstract | 軟骨組織由於缺乏血管和神經系統，遭受損傷後極難修復，且由於其多於關節等骨頭相鄰處作為緩衝，受損未經修復容易惡化致使整個關節失去功能，因此修復軟骨組織為組織工程中一重要課題。人類脂肪幹細胞可發現於成人脂肪組織中，具有多種諸如脂肪、硬骨和軟骨等分化潛力，易於取得且可用於自體移植避免免疫反應之發生，已成為組織工程研究中一重要之細胞來源。乙二醇幾丁聚醣擁有和幾丁聚醣相似的特性且可溶於中性的水中，其結構和軟骨組織中的糖胺聚醣相似。此研究將其和明膠混合形成之水凝膠作為人類脂肪幹細胞之載體，觀察細胞生長情形和型態，並分析其軟骨分化之潛力。
明膠的存在可增進水凝膠的細胞貼附性質，然而明膠會隨著時間從膠中釋出。經觀察，幹細胞會逐漸在水凝膠表面聚集最後形成球狀，推測其原因可能為水凝膠之貼附性質降低，且幾丁聚醣的表面有助於幹細胞球的生成，即有研究是以幾丁聚醣形成之薄膜使幹細胞於其表面成球。細胞球的結構已被證實可以增進幹細胞的分化能力，傳統形成細胞球的技術諸如液滴、模具和幾丁聚醣膜等，其中細胞的聚集和避免細胞的攤平貼附為重要之因素。 細胞球之外觀和性質由顯微鏡和螢光染色等觀察之。而後以軟骨分化培養液對細胞球進行軟骨分化的刺激，在一定的天數以螢光染色和測量基因表現來分析軟骨分化的傾向。在水凝膠上以一定的細胞密度培養，可在一至兩天形成細胞聚集進而成球，然而由於明膠的脫離，水凝膠難以支持長時間的培養。 在軟骨分化方面，細胞的聚集成球對於幹細胞在SOX9和COL II兩者的基因表現有所助益，顯示對於軟骨分化的潛力。 | zh_TW |
| dc.description.abstract | The repair of damaged cartilage is a challenge due to its hard self-repair in cartilage tissue engineering. Human adipose stem cells are proved that have the ability to differentiate into different linage including adipogenic, osteogenic and chondrogenic lineage. They are considered to be the cell sources in cartilage tissue engineering.
Glycol chitosan, which is a soluble derivative of chitosan, is studied for cartilage repairing due to its similar structure to glycosaminoglycans in cartilage and keeps the properties of biocompatibility and degradability as chitosan. In this study, the hydrogel was combined by glycol chitosan and gelatin which made cells able to attach on and its performance in cell culture was researched. This hydrogel could make cells adhere on its surface and make the hASCs form a sphere structure after culturing for several days. The formation of spheroids was attributed to the releasing of gelatin and the main material, glycol chitosan. These spheroids were observed by SEM and fluorescence and tested their properties and gene expression of differentiation. There were studies showing that the stem cells in spheroids were viable mostly and had better differentiation ability. Moreover, the material we used was usually studied for cartilage tissue engineering. Therefor the chondrogenic differentiation potential of cells in spheroids was researched. We hope the GAGs-like glycol chitosan in hydrogel and formation of spheroids are able to promote the chondrogenic differentiation. The results showed the chondrogenic differentiation performance appeared in spheroids. Moreover, the spheroids formed with induction medium were larger and had more complete shape of sphere. They also had higher performance in chondrogenic differentiation. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T11:29:07Z (GMT). No. of bitstreams: 1 ntu-105-R03524037-1.pdf: 1748755 bytes, checksum: 16f3f52eddcad622507375a23c89d4de (MD5) Previous issue date: 2016 | en |
| dc.description.tableofcontents | 口試委員會審定書 #
致謝 i 摘要 ii ABSTRACT iii CONTENT v LIST OF FIGURES xi LIST OF TABLES xiii Chapter 1 Introduction 1 1.1 Tissue Engineering 1 1.1.1 Purposes of Tissue Engineering 1 1.1.2 Scaffolds 2 1.1.3 Cells 3 1.1.4 Signaling Molecules 4 1.1.5 Cartilage Tissue Engineering 5 1.2 Hydrogel 6 1.2.1 Glycol Chitosan 8 1.2.2 Gelatin 9 1.3 Cell Culture 10 1.3.1 Human Adipose Stem Cells 10 1.4 Cell Spheroid 12 1.4.1 Spheroid Structure 12 1.4.2 Formation of Spheroids 13 1.4.3 Spheroids in Tissue Engineering 13 1.5 Motive and Aims 14 1.6 Research Framework 15 Chapter 2 Materials and Methods 17 2.1 Materials 17 2.2 Experimental Equipment 21 2.3 Solution Formula 23 2.3.1 Phosphate Buffered Saline Solution (PBS) 23 2.3.2 DMEM/F-12 Culture Medium 23 2.3.3 MTT Assay Working Solution 23 2.3.4 DMEM – High Glucose Culture Medium (DMEM-HG) 24 2.3.5 Chodrogenic Differentiation Medium 24 2.3.6 Bicinchoninic Acid (BCA) Assay Working Reagent 25 2.3.7 Phosphate Buffered Saline Solution with Tween 20 (PBST) 26 2.4 Methods 26 2.4.1 Synthesis of Methacrylate Glycol Chitosan and Gelatin 26 2.4.2 Synthesis of Glycol Chitosan Hydrogel Combined with Gelatin 27 2.4.3 Preparation of Hydrogel Surface 28 2.4.4 HASCs Isolation and Culture 28 2.4.5 MTT Assay 29 2.4.6 Gelatin Release 31 2.4.7 Mechanical Property Test 31 2.4.8 Scanning Electron Microscope 33 2.4.9 Live/Dead Staining 33 2.4.10 DAPI and Phalloidin Staining 34 2.4.11 Fluorescence Staining 35 2.4.12 Alcian Blue Staining 36 2.4.13 RNA Extraction 36 2.4.14 Reverse Transcription – Polymerase Chain Reaction (RT-PCR) 37 2.4.15 Electrophoresis 40 2.4.16 Real Time – Polymerase Chain Reaction (qPCR) 40 2.4.17 Statistical Analysis 42 Chapter 3 Results and Discussions 43 3.1 Hydrogel 43 3.1.1 Mechanical Property 43 3.1.2 Gelatin Releasing 44 3.2 Cell Culture on Hydrogel 45 3.2.1 Cell Adhesion on Hydrogel 45 3.2.2 Cell Seeding Density 47 3.3 Viability of Cells on Hydrogel 48 3.3.1 MTT Assay 48 3.3.2 Live/Dead Staining 49 3.4 Morphology of Spheroid 50 3.4.1 Spheroid Observation 50 3.4.2 Scanning Electron Microscope Image of Spheroid 51 3.4.3 Spheroid Size 53 3.4.4 DAPI and Phalloidin Staining 54 3.5 Chondrogenic Differentiation 55 3.5.1 Alcian Blue Staining 55 3.5.2 Fluorescence Staining 57 3.5.3 Gene Expression 59 3.6 Discussion 61 Chapter 4 Conclusion 63 Appendices 64 References 66 | |
| dc.language.iso | en | |
| dc.subject | 細胞球 | zh_TW |
| dc.subject | 軟骨分化 | zh_TW |
| dc.subject | 水凝膠 | zh_TW |
| dc.subject | 乙二醇幾丁聚醣 | zh_TW |
| dc.subject | Hydrogel | en |
| dc.subject | Chondrogenic differentiation | en |
| dc.subject | Glycol chitosan | en |
| dc.subject | Spheroid | en |
| dc.title | 乙二醇幾丁聚醣結合明膠之水凝膠上形成的人類脂肪幹細胞球其軟骨分化傾向 | zh_TW |
| dc.title | Chondrogenic Differentiation Potential of Human Adipose Stem Cell Spheroid Forming on Glycol Chitosan and Gelatin Hydrogel | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 104-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 蔡偉博(Wei-Bor Tsai),陳賢燁(Hsien-Yeh Chen),楊凱強(Kai-Chiang Yang) | |
| dc.subject.keyword | 細胞球,乙二醇幾丁聚醣,水凝膠,軟骨分化, | zh_TW |
| dc.subject.keyword | Spheroid,Glycol chitosan,Hydrogel,Chondrogenic differentiation, | en |
| dc.relation.page | 73 | |
| dc.identifier.doi | 10.6342/NTU201602735 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2016-08-17 | |
| dc.contributor.author-college | 工學院 | zh_TW |
| dc.contributor.author-dept | 化學工程學研究所 | zh_TW |
| 顯示於系所單位： | 化學工程學系 | |
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