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論文名稱(外文):Development of Three Dimensional Polyethylene Glycol Based Hydrogel for Tissue Engineering Scaffold Application
指導教授(外文):Ming-Long Yeh
外文關鍵詞:Cartilage degenerationTwo-photon laser scanning microscopyPoly (ethylene glycol) diacrylateMicro-wellAdipose-derived stem cellTissue engineeringCo-culture
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Articular cartilage degeneration which is hard to repair by itself because of no nerves and blood vessels has become a public health issue since average of lifespan is increasing. Many repairing approaches did not restore the normal hyaline cartilage but fibrocartilage instead. Previous researches were limited by barrier of mass transfer, improper induction sequence, cell anchorage, or few amount of available cells. Buildup of the monitoring systems that simulate physiological conditions enables us to inspect the events of electrical and chemical communications between cells. In this study, we fabricated poly (ethylene glycol) diacrylate (PEGDA) into three kinds of devices by customized mold. PEGDA is hydrophilic, biocompatible and photo-crosslink-able. In addition, adipose-derived stem cells (ASC) were harvested as the standard cells to evaluate whether the systems were functional.
First product was thin film of PEGDA featuring micro-pattern created by two-photon laser scanning microscopy. Surface of the film was grafted RGD peptide so that cells could partially adhere to the surface of polymer. Second product was micro-well which hosted high quantity but small population of cells. The bonding between silane and PEGDA was competed by protein; thus, we were able to control the delamination speed of micro-well from culture stage. The last product was scaffold for cartilage tissue engineering with novel design to reduce barrier of mass transfer. ASC were encapsulated in PEGDA and induced by co-culture strategy to stimulate the protein express which are the characteristic of chondrogenesis. Compared with treatment of exogenous supplement of growth factors, co-culture strategy achieved the same results.
In general, different manufacturing process granted three devices which shared the same material with the abilities to monitor, house, and induce chondrogenesis. ASC is an ideal autologous cell source because it is abundant in quantity and has outstanding differential potential. Future work is to fully utilize these devices to acquire information of regulating chondrogenesis. Ultimate goal is to integrate three devices into tissue engineering scaffold with real-time surveillance on proliferation and differentiation status.

摘要 i
Abstract ii
Acknowledgements iv
Table of contents v
List of tables viii
List of figures ix
Chapter 1 Introduction 1
1.1 Background 1
1.1.1 Compositions and properties of cartilage 1
1.1.2 Cartilage degeneration and approaches of repair 2
1.2 Scaffolds 4
1.2.1 Design of scaffold 4
1.2.2 Natural biomaterials 5
1.2.3 Artificial biomaterials 6
1.3 Two-photon laser scanning microscopy 8
1.4 Micro-well array 10
1.5 Cell Source 10
1.5.1 Differentiated cells of normal tissue 10
1.5.2 Stem cells 11
1.5.3 Bone marrow stem cells 12
1.5.4 Adipose-derived stem cells 12
1.6 Environment 14
1.6.1 Mechanical stimulation 14
1.6.2 Biochemical stimulation 14
1.6.3 Co-culture system 15
1.7 Purpose and specific aims 16
1.7.1 Purpose 16
1.7.2 Aims 17
Chapter 2 Materials and methods 21
2.1 Materials and instruments 21
2.2 Cell culture 24
2.2.1 Harvest of ASC 24
2.2.2 Harvest of chondrocytes 24
2.2.3 Cell maintenance and passage 25
2.3 Gelation of PEGDA 25
2.3.1 Customized mold to load PEGDA solution 25 PEGDA film with micro-pattern created by TPLSM 25 Micro-well array with controllable delamination hosted cells 25 PEGDA encapsulated ASC to induce chondrogenesis by co-culturing with xenogeneic chondrocytes 26
2.3.2 Parameters to crosslink PEGDA 26
2.4 Mechanical property of PEGDA gel 26
2.5 Cell membrane fluorescent stain-Cell linker 27
2.6 Biochemical evaluation 27
2.6.1 Cell viability assay 27
2.6.2 Western blot 28
2.7 Histological section 29
Chapter 3 Results 30
3.1 PEGDA film with micro-pattern created by TPLSM 31
3.2 Micro-well array with controllable delamination hosted cells 33
3.3 PEGDA encapsulated ASC to induce chondrogenesis by co-culturing with xenogeneic chondrocytes 35
3.3.1 Geometric structure of scaffold 35
3.3.2 Young’s modulus of PEGDA hydrogel 35
3.3.3 Cell morphology within scaffold 38
3.3.4 Cell viability in hydrogel 39
3.3.5 Subcutaneous implant of hydrogel loaded with cell 41 Observation at the surface of PEGDA after implantation 41 Fluorescence image of cell within hydrogel 42
3.3.6 Sections of PEGDA hydrogel scaffold 43
3.3.7 Change of protein secretion of ASC upon treatment 47
Chapter 4 Discussion 48
4.1 PEGDA film with micro-pattern created by TPLSM 48
4.2 Micro-well array with controllable delamination hosted cells 48
4.3 PEGDA encapsulate ASC to induce chondrogenesis by co-culturing with xenogeneic chondrocytes 50
4.4 Integration of three tools 57
Chapter 5 Conclusion 59
References 60
Curriculum vitae 66

Asanbaeva, A., Masuda, K., Thonar, E.J., Klisch, S.M., and Sah, R.L. (2008a). Cartilage growth and remodeling: modulation of balance between proteoglycan and collagen network in vitro with beta-aminopropionitrile. Osteoarthritis Cartilage 16, 1-11.
Asanbaeva, A., Tam, J., Schumacher, B.L., Klisch, S.M., Masuda, K., and Sah, R.L. (2008b). Articular cartilage tensile integrity: modulation by matrix depletion is maturation-dependent. Arch Biochem Biophys 474, 175-182.
Aust, L., Devlin, B., Foster, S.J., Halvorsen, Y.D., Hicok, K., du Laney, T., Sen, A., Willingmyre, G.D., and Gimble, J.M. (2004). Yield of human adipose-derived adult stem cells from liposuction aspirates. Cytotherapy 6, 7-14.
Barron, V., Lyons, E., Stenson-Cox, C., McHugh, P.E., and Pandit, A. (2003). Bioreactors for cardiovascular cell and tissue growth: A review. Annals of Biomedical Engineering 31, 1017-1030.
Bongso, A., and Richards, M. (2004). History and perspective of stem cell research. Best Pract Res Cl Ob 18, 827-842.
Boquest, A.C., Shahdadfar, A., Brinchmann, J.E., and Collas, P. (2006). Isolation of stromal stem cells from human adipose tissue. Methods Mol Biol 325, 35-46.
Bruder, S.P., Fink, D.J., and Caplan, A.I. (1994). Mesenchymal stem cells in bone development, bone repair, and skeletal regeneration therapy. J Cell Biochem 56, 283-294.
Bunnell, B.A., Flaat, M., Gagliardi, C., Patel, B., and Ripoll, C. (2008). Adipose-derived stem cells: isolation, expansion and differentiation. Methods 45, 115-120.
Chang, N.J., Lin, C.C., Li, C.F., Wang, D.A., Issariyaku, N., and Yeh, M.L. (2012). The combined effects of continuous passive motion treatment and acellular PLGA implants on osteochondral regeneration in the rabbit. Biomaterials 33, 3153-3163.
Chou, P.J. (2010). Proliferation and Differentiation of Human Adipose-Derived Stem Cells by Suspension Culture. In Institute of Biomedical Engineering (Tainan, National Cheng Kung University), pp. 100.
Connelly, J.T., Garcia, A.J., and Levenston, M.E. (2007). Inhibition of in vitro chondrogenesis in RGD-modified three-dimensional alginate gels. Biomaterials 28, 1071-1083.
Fogler, H.S. (2006). Elements of chemical reaction engineering, 4th edn (Upper Saddle River, NJ, Prentice Hall PTR).
Gimble, J.M., and Nuttall, M.E. (2011). Adipose-derived stromal/stem cells (ASC) in regenerative medicine: pharmaceutical applications. Curr Pharm Des 17, 332-339.
Guo, A., Song, B., Reid, B., Gu, Y., Forrester, J.V., Jahoda, C.A., and Zhao, M. (2010). Effects of physiological electric fields on migration of human dermal fibroblasts. J Invest Dermatol 130, 2320-2327.
Hahn, M.S., Miller, J.S., and West, J.L. (2006). Three-dimensional biochemical and biomechanical patterning of hydrogels for guiding cell behavior. Advanced Materials 18, 2679-+.
Harris, J.M., Zalipsky, S., American Chemical Society. Division of Polymer Chemistry., and American Chemical Society. Meeting (1997). Poly(ethylene glycol) : chemistry and biological applications (Washington, DC, American Chemical Society).
Hern, D.L., and Hubbell, J.A. (1998). Incorporation of adhesion peptides into nonadhesive hydrogels useful for tissue resurfacing. Journal of Biomedical Materials Research 39, 266-276.
Hildner, F., Concaro, S., Peterbauer, A., Wolbank, S., Danzer, M., Lindahl, A., Gatenholm, P., Redl, H., and van Griensven, M. (2009). Human adipose-derived stem cells contribute to chondrogenesis in coculture with human articular chondrocytes. Tissue Eng Part A 15, 3961-3969.
Hume, P.S., and Anseth, K.S. (2010). Inducing local T cell apoptosis with anti-Fas-functionalized polymeric coatings fabricated via surface-initiated photopolymerizations. Biomaterials 31, 3166-3174.
Hunziker, E.B. (2002). Articular cartilage repair: basic science and clinical progress. A review of the current status and prospects. Osteoarthr Cartilage 10, 432-463.
Insall, J.N., Ranawat, C.S., Aglietti, P., and Shine, J. (1976). A comparison of four models of total knee-replacement prostheses. J Bone Joint Surg Am 58, 754-765.
Insall, J.N., Ranawat, C.S., Aglietti, P., and Shine, J. (1999). A comparison of four models of total knee-replacement prostheses. 1976. Clin Orthop Relat Res, 3-17; discussion 12.
Johnstone, B., Hering, T.M., Caplan, A.I., Goldberg, V.M., and Yoo, J.U. (1998). In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res 238, 265-272.
Kloxin, A.M., Benton, J.A., and Anseth, K.S. (2010a). In situ elasticity modulation with dynamic substrates to direct cell phenotype. Biomaterials 31, 1-8.
Kloxin, A.M., Kasko, A.M., Salinas, C.N., and Anseth, K.S. (2009). Photodegradable hydrogels for dynamic tuning of physical and chemical properties. Science 324, 59-63.
Kloxin, A.M., Kloxin, C.J., Bowman, C.N., and Anseth, K.S. (2010b). Mechanical Properties of Cellularly Responsive Hydrogels and Their Experimental Determination. Advanced Materials 22, 3484-3494.
Kutzner, I., Heinlein, B., Graichen, F., Bender, A., Rohlmann, A., Halder, A., Beier, A., and Bergmann, G. (2010). Loading of the knee joint during activities of daily living measured in vivo in five subjects. J Biomech 43, 2164-2173.
Lefebvre, V., and de Crombrugghe, B. (1998). Toward understanding SOX9 function in chondrocyte differentiation. Matrix Biol 16, 529-540.
Lefebvre, V., Huang, W.D., Harley, V.R., Goodfellow, P.N., and deCrombrugghe, B. (1997). SOX9 is a potent activator of the chondrocyte-specific enhancer of the pro alpha 1(II) collagen gene. Mol Cell Biol 17, 2336-2346.
Levangie, P.K., and Norkin, C.C. (2011). Joint structure and function : a comprehensive analysis, 5th edn (Philadelphia, F.A. Davis Co.).
Li, J., Tao, R., Wu, W., Cao, H., Xin, J., Guo, J., Jiang, L., Gao, C., Demetriou, A.A., Farkas, D.L., et al. (2010). 3D PLGA scaffolds improve differentiation and function of bone marrow mesenchymal stem cell-derived hepatocytes. Stem Cells Dev 19, 1427-1436.
Lin, C.C., and Anseth, K.S. (2011). Cell-cell communication mimicry with poly(ethylene glycol) hydrogels for enhancing beta-cell function. Proc Natl Acad Sci U S A 108, 6380-6385.
Loeser, R.F., Goldring, S.R., Scanzello, C.R., and Goldring, M.B. (2012). Osteoarthritis: A disease of the joint as an organ. Arthritis Rheum 64, 1697-1707.
Luo, Y., and Shoichet, M.S. (2004). A photolabile hydrogel for guided three-dimensional cell growth and migration. Nature Materials 3, 249-253.
Mankin, H.J., and Buckwalter, J.A. (1996). Restoration of the osteoarthrotic joint. J Bone Joint Surg Am 78, 1-2.
Mansour, J.M. (2008). Biomechanics of Cartilage. In Kinesiology: The Mechanics and Pathomechanics of Human Movement (Lippincott Williams & Wilkins), pp. 66-79.
Martin, U., and Amit, M. (2009). Engineering of stem cells (Berlin, Springer).
MeNickle, A.G., Provencher, M.T., and Cole, B.J. (2008). Overview of Existing Cartilage Repair Technology. Sports Med Arthrosc 16, 196-201.
Meyer, E.G., Buckley, C.T., Steward, A.J., and Kelly, D.J. (2011). The effect of cyclic hydrostatic pressure on the functional development of cartilaginous tissues engineered using bone marrow derived mesenchymal stem cells. J Mech Behav Biomed Mater 4, 1257-1265.
Mitalipov, S., and Wolf, D. (2009). Totipotency, pluripotency and nuclear reprogramming. Adv Biochem Eng Biotechnol 114, 185-199.
Moeller, H.C., Mian, M.K., Shrivastava, S., Chung, B.G., and Khademhosseini, A. (2008). A microwell array system for stem cell culture. Biomaterials 29, 752-763.
Nagy, L.J., Ding, L., Xu, L.S., Knox, W.H., and Huxlin, K.R. (2010). Potentiation of Femtosecond Laser Intratissue Refractive Index Shaping (IRIS) in the Living Cornea with Sodium Fluorescein. Investigative Ophthalmology & Visual Science 51, 850-856.
O'Cearbhaill, E.D., Murphy, M., Barry, F., McHugh, P.E., and Barron, V. (2010). Behavior of human mesenchymal stem cells in fibrin-based vascular tissue engineering constructs. Ann Biomed Eng 38, 649-657.
Oldershaw, R.A., Baxter, M.A., Lowe, E.T., Bates, N., Grady, L.M., Soncin, F., Brison, D.R., Hardingham, T.E., and Kimber, S.J. (2010). Directed differentiation of human embryonic stem cells toward chondrocytes. Nature Biotechnology 28, 1221-U1280.
Petersen, T.H., Calle, E.A., Zhao, L., Lee, E.J., Gui, L., Raredon, M.B., Gavrilov, K., Yi, T., Zhuang, Z.W., Breuer, C., et al. (2010). Tissue-engineered lungs for in vivo implantation. Science 329, 538-541.
Promega, C. (Revised 2009/06). CellTiter 96® AQueous One Solution Cell Proliferation Assay. In T e c h n i c a l B u l l e t i n, P. Corporation, ed. (Madison, Promega Corporation), pp. 12.
Ramos, T.V., Wang, T., Maki, C.B., Pascual, M., and Izadyar, F. (2009). Adipose stem cell side population in the mouse. J Tissue Eng Regen Med 3, 430-441.
Ratner, B.D. (2004). Biomaterials science : an introduction to materials in medicine, 2nd edn (Amsterdam ; Boston, Elsevier Academic Press).
Rettig, J.R., and Folch, A. (2005). Large-scale single-cell trapping and imaging using microwell arrays. Anal Chem 77, 5628-5634.
Rui, Y.F., Lui, P.P., Lee, Y.W., and Chan, K.M. (2012). Higher BMP receptor expression and BMP-2-induced osteogenic differentiation in tendon-derived stem cells compared with bone-marrow-derived mesenchymal stem cells. Int Orthop 36, 1099-1107.
Saadat, E., Lan, H., Majumdar, S., Rempel, D.M., and King, K.B. (2006). Long-term cyclical in vivo loading increases cartilage proteoglycan content in a spatially specific manner: an infrared microspectroscopic imaging and polarized light microscopy study. Arthritis Res Ther 8, R147.
Salinas, C.N., and Anseth, K.S. (2008a). The enhancement of chondrogenic differentiation of human mesenchymal stem cells by enzymatically regulated RGD functionalities. Biomaterials 29, 2370-2377.
Salinas, C.N., and Anseth, K.S. (2008b). The influence of the RGD peptide motif and its contextual presentation in PEG gels on human mesenchymal stem cell viability. Journal of Tissue Engineering and Regenerative Medicine 2, 296-304.
Salinas, C.N., Cole, B.B., Kasko, A.M., and Anseth, K.S. (2007). Chondrogenic differentiation potential of human mesenchymal stem cells photoencapsulated within poly(ethylene glycol)-arginine-glycine-aspartic acid-serine thiol-methacrylate mixed-mode networks. Tissue Engineering 13, 1025-1034.
Sekiya, I., Vuoristo, J.T., Larson, B.L., and Prockop, D.J. (2002). In vitro cartilage formation by human adult stem cells from bone marrow stroma defines the sequence of cellular and molecular events during chondrogenesis. Proc Natl Acad Sci U S A 99, 4397-4402.
Seo, S., and Na, K. (2011). Mesenchymal Stem Cell-Based Tissue Engineering for Chondrogenesis. J Biomed Biotechnol.
Shah, D.N., Recktenwall-Work, S.M., and Anseth, K.S. (2008). The effect of bioactive hydrogels on the secretion of extracellular matrix molecules by valvular interstitial cells. Biomaterials 29, 2060-2072.
Sharma, R.I., and Snedeker, J.G. (2012). Paracrine interactions between mesenchymal stem cells affect substrate driven differentiation toward tendon and bone phenotypes. PLoS One 7, e31504.
Shiers, L.G. (1954). Arthroplasty of the knee; preliminary report of new method. J Bone Joint Surg Br 36-B, 553-560.
Sommer, B., and Sattler, G. (2000). Current concepts of fat graft survival: histology of aspirated adipose tissue and review of the literature. Dermatol Surg 26, 1159-1166.
Takahashi, K., and Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663-676.
Tortelli, F., Tasso, R., Loiacono, F., and Cancedda, R. (2010). The development of tissue-engineered bone of different origin through endochondral and intramembranous ossification following the implantation of mesenchymal stem cells and osteoblasts in a murine model. Biomaterials 31, 242-249.
Tsuchiya, K., Chen, G.P., Ushida, T., Matsuno, T., and Tateishi, T. (2004). The effect of coculture of chondrocytes with mesenchymal stem cells on their cartilaginous phenotype in vitro. Mat Sci Eng C-Bio S 24, 391-396.
Ulloa-Montoya, F., Verfaillie, C.M., and Hu, W.S. (2005). Culture systems for pluripotent stem cells. J Biosci Bioeng 100, 12-27.
Villanueva, I., Weigel, C.A., and Bryant, S.J. (2009). Cell-matrix interactions and dynamic mechanical loading influence chondrocyte gene expression and bioactivity in PEG-RGD hydrogels. Acta Biomaterialia 5, 2832-2846.
Waldman, S.D., Couto, D.C., Grynpas, M.D., Pilliar, R.M., and Kandel, R.A. (2007). Multi-axial mechanical stimulation of tissue engineered cartilage: Review. Eur Cells Mater 13, 73-74.
Waldman, S.D., Spiteri, C.G., Grynpas, M.D., Pilliar, R.M., and Kandel, R.A. (2003). Long-term intermittent shear deformation improves the quality of cartilaginous tissue formed in vitro. J Orthop Res 21, 590-596.
Williams, C.G., Kim, T.K., Taboas, A., Malik, A., Manson, P., and Elisseeff, J. (2003). In vitro chondrogenesis of bone marrow-derived mesenchymal stem cells in a photopolymerizing hydrogel. Tissue Engineering 9, 679-688.
Wong, J.Y., Velasco, A., Rajagopalan, P., and Pham, Q. (2003). Directed movement of vascular smooth muscle cells on gradient-compliant hydrogels. Langmuir 19, 1908-1913.
Wylie, R.G., and Shoichet, M.S. (2008). Two-photon micropatterning of amines within an agarose hydrogel. Journal of Materials Chemistry 18, 2716-2721.
Zhao, Y.L., Zhang, S., Zhou, J.Y., Wang, J.L., Zhen, M.C., Liu, Y., Chen, J.B., and Qi, Z.Q. (2010). The development of a tissue-engineered artery using decellularized scaffold and autologous ovine mesenchymal stem cells. Biomaterials 31, 296-307.
Zuk, P.A., Zhu, M., Mizuno, H., Huang, J., Futrell, J.W., Katz, A.J., Benhaim, P., Lorenz, H.P., and Hedrick, M.H. (2001). Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 7, 211-228.

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