本研究主要探討成球細胞與非成球細胞於不同纖維尺度之聚胺酯(polyurethane, PU)電紡膜上的細胞表現差異，之後進一步比較在相同尺度下之不同材料(聚胺酯及聚己內酯)和不同之薄膜加工方式(電紡膜及相轉換薄膜)，對於成球細胞分化能力之影響。其中三種不同纖維直徑(200-400 nm、600-800 nm及1.4-1.6 μm)之聚胺酯電紡膜的製備方式為結合高低沸點溶劑二甲基乙醯胺及三氟乙醇，於調整高分子溶液濃度、流速、電壓及接收距離下所得，而選用之細胞以人類骨髓間葉幹細胞(human bone marrow mesenchymal stem cells, hBMSC)為研究模式。由貼附增生及基因表現之結果可得知，於不同纖維尺度及不同親疏水性之聚胺酯電紡膜上，成球細胞不僅能穩定地維持貼附率，同時比非成球細胞具有更佳之軟硬骨分化能力，特別是在與細胞外基質結構相似之聚胺酯奈米電紡絲上(200-400 nm)。至於比較成球細胞在相同纖維尺度之聚胺酯及聚己內酯電紡膜上之分化能力，基因表現結果得知幹細胞於聚胺酯電紡膜上具有較佳之軟硬骨化能力，其中又以硬骨化之表現程度更為顯著。而在聚胺酯電紡膜及相轉換薄膜對於成球細胞硬骨化能力影響之部分，基因和蛋白質表現顯示幹細胞於聚胺酯相轉換薄膜上之硬骨化表現為最佳，不過由於其延展性過低而應用相對受到限制。因此，將hBMSC成球細胞結合聚胺酯電紡絲能具有較佳之軟硬骨組織工程上的應用潛力。

The behavior of human bone marrow derived mesenchymal stem cells (hBMSC) was evaluated on biodegradable polyurethane electrospun fibers with different diameter ranges (200-400 nm, 600-800 nm, and 1.4-1.6 μm). Cells were seeded on the fibrous membranes in the form of single dispersed cells or self-assembled spheroids. The effect of material elasticity was further elucidated by comparing cell behavior on polyurethane and poly(ε-caprolactone). Fibers were electrospun from polymer solutions in N,N-dimethylacetamide and 2,2,2-trifluoroethanol. Differentiation experiments showed that hBMSC spheroids seeded had greater differentiation capacities than single cells. Gene expression revealed that nanofibers of 200-400 nm diameters significantly promoted the osteogenic and chondrogenic differentitation of hBMSC spheroids than fibers of the other diameters. hBMSC also demonstrated significantly higher osteochondrogenic differentiation potential on polyurethane vs. poly(ε-caprolactone) electrospun fibers. hBMSC on polyurethane membranes fabricated by wet phase separation (WPS) showed more bone-related marker gene expression and matrix mineralization than electropsun fibers, but WPS membranes had limited elongation. We suggested that hBMSC spheroids seeded on electrospun fibers may be advantageous for cartilage and bone tissue engineering.

誌謝..................... ................................ I
摘要..................................................... II
Abstract .............................................. III
目錄 ................................................... V
圖目錄 ..................... ......................... IX
表目錄 ................... ......................... XI
第一章 文獻回顧 ....................................... - 1 -
1-1. 聚胺酯 ...................................... - 1 -
1-2. 生物可降解聚胺酯 .......................... - 3 -
1-3. 電紡絲 .......................... ...... - 5 -
1-4. 組織工程上的電紡絲 ....... .............. - 8 -
1-4-1. 電紡絲纖維的方向性對細胞影響........................ - 8 -
1-4-2. 電紡絲纖維直徑對細胞的影響 ....................... - 9 -
1-5. 多細胞成球之三維培養技術 .......................... - 11 -
1-6. 研究目的 ....... ................................ - 13 -
第二章研究方法 ................................ .... - 15 -
2-1. 研究架構 .. ................................ - 15 -
2-2. 生物可降解聚胺酯之合成 ...................... .... - 17 -
2-3. 電紡纖維膜之製備................ - 19 -
2-3-1. 四種變因對電紡纖維膜的影響..... ................. - 19 -
2-3-2. 以共溶劑系統製備聚胺酯奈米及微米電紡纖維膜 ......... - 21 -
2-3-3. 製備聚己內酯奈米電紡纖維膜 ..... ................. - 22 -
2-4. 電紡纖維膜之物性分析 ..................... ........ - 23 -
2-4-1. 掃描式電子顯微鏡分析 ............................ - 23 -
2-4-2. 纖維密度及孔隙度分析 .......................... - 23 -
2-4-3. 接觸角分析 .......... ................. - 23 -
2-4-4. 拉伸試驗分析 ........ ............. - 23 -
2-5. 三維細胞成球之技術.... ............ - 24 -
2-6. 成球與非成細胞於不同尺度之聚胺酯電紡膜上之細胞試驗 ...... - 24 -
2-6-1. 細胞植覆率測試 .... ......... - 24 -
2-6-2. 細胞增生測試 ............ - 25 -
2-6-3. 細胞軟硬骨化之基因表現 ......................... - 26 -
2-7. 成球細胞於聚胺酯及聚己內酯電紡膜上之軟硬骨化能力........ - 27 -
2-7-1. 細胞數分析 ......... ................. - 27 -
2-7-2. 基因表現分析.......... ............. - 28 -
2-8. 比較成球細胞於聚胺酯電紡薄膜及相轉換薄膜上之硬骨化能力.... - 29 -
2-8-1. 細胞數分析 ........ ................. - 29 -
2-8-2. 基因表現分析 .......... ............. - 29 -
2-8-3. 鈣含量及膠原蛋白含量分析 ........................ - 30 -
2-9. 統計分析 .................. - 31 -
第三章實驗結果 ............................... .... - 32 -
3-1. 電紡纖維膜之物性分析 ............. ........ - 32 -
3-1-1. 四種變因對於電紡纖維膜的影響 ... ............. - 32 -
3-1-2. 共溶劑系統對於電紡纖維膜的影響 .......... ......... - 32 -
3-1-3. 聚己內酯電紡纖維膜之表面形態 ........ ............. - 33 -
3-1-4. 電紡纖維膜之纖維密度及孔隙度.... ............. - 33 -
3-1-5. 接觸角分析 ............ ................. - 34 -
3-1-6. 拉伸試驗分析 ........ ............. - 34 -
3-2. 成球與非成球細胞於不同尺度之聚胺酯電紡膜上之細胞試驗 .... - 35 -
3-2-1. 細胞植覆率與增生情形 ........................... - 35 -
3-2-2. 細胞軟硬骨化之基因表現 ........................ - 35 -
3-3. 成球細胞於聚胺酯及聚己內酯電紡膜上之軟硬骨化能力 ........ - 36 -
3-3-1. 基材上之細胞形態 ........................ ..... - 36 -
3-3-2. 細胞數分析 ....... ................. - 37 -
3-3-3. 基因表現分析 ...... ............. - 37 -
3-4. 比較成球細胞於聚胺酯電紡薄膜及相轉換薄膜上之硬骨化能力 ... - 38 -
3-4-1. 細胞數分析 .......... ................. - 38 -
3-4-2. 基因表現分析 ......... ............. - 38 -
3-4-3. 鈣含量及膠原蛋白含量分析 ...................... - 38 -
第四章 討論 ................ .......... - 40 -
4-1. 電紡纖維膜之物性分析 ....... ........ - 40 -
4-1-1. 四種變因對於電紡纖維的影響 ...... ............. - 40 -
4-1-2. 共溶劑系統對於電紡纖維膜的影響 ....... ......... - 41 -
4-1-3. 接觸角分析 ............ ................. - 41 -
4-1-4. 拉伸試驗分析 ............. ............. - 42 -
4-2. 成球與非成球細胞於不同尺度之聚胺酯電紡膜上之細胞試驗 .... - 43 -
4-2-1. 細胞植覆率與增生情形 .... - 43 -
4-2-2. 細胞軟硬骨化之基因表現 .................. - 43 -
4-3. 成球細胞於聚胺酯及聚己內酯電紡膜上之軟硬骨化能力 ....... - 45 -
4-4. 比較成球細胞於聚胺酯電紡薄膜及相轉換薄膜上之硬骨化能力.... - 46 -
4-5. 未來展望 ................................ - 46 -
第五章 結論 .................... .......... - 48 -
參考文獻 ....................... ................. - 49 -
圖........................... - 68 -
表.......................................... - 84 -
附錄 ................................... - 86 -

1.Gunatillake PA, Meijs GF. Polyurethanes in Biomedical Engineering. Encyclopedia of Materials: Science and Technology. 2001. 7746-7752.
2.Ratner BD. Polymeric Implants. Polymer Science: A Comprehensive Reference. 2012. 397-411.
3.Borkenhagen M, Stoll RC, Neuenschwander P, Suter UW, Aebischer P. In vivo performance of a new biodegradable polyester urethane system used as a nerve guidance channel. Biomaterials. 1998. 19: 2155-2165.
4.Fujimoto KL, Tobita K, Merryman WD, Guan JJ, Momoi N, Stolz DB, Sacks MS, Keller BB, Wagner WR. An elastic, biodegradable car-diac patch induces contractile smooth muscle and improves cardiac remodeling and function in subacute myocardial infarction. Journal of the American College of Cardiology. 2007. 49: 2292-2300.
5.McDevitt TC, Woodhouse KA, Hauschka SD, Murry CE, Stayton PS. Spatially organized layers of cardiomyocytes on biodegradable polyurethane films for myocardial repair. Journal of Biomedical Materials Research Part A. 2003. 66: 586-595.
6.Alperin C, Zandstra PW, Woodhouse KA. Polyurethane films seeded with embryonic stem cell-derived cardiomyocytes for use in cardiac tissue engineering applications. Biomaterials. 2005. 26: 7377-7386.
7.Saad B, Hirt TD, Welti M, Uhlschmid GK, Neuenschwander P, Suter UW. Development of degradable polyesterurethanes for medical applications: in vitro and in vivo evaluations. Journal of Biomedical Materials Research. 1997. 36: 65-74.
8.Chen Q, Liang S, Thouas GA. Elastomeric biomaterials for tissue engineering. Progress in Polymer Science. 2013. 38: 584–671.
9.Martina M, Hutmacher DW. Biodegradable polymers applied in tissue engineering research: a review. Polymer International. 2007. 56: 145-157.
10.Guelcher SA. Biodegradable Polyurethanes: Synthesis and Applications in Regenerative Medicine. Tissue Engineering: Part B. 2008. 14: 3-17.
11.Skarja GA, Woodhouse KA. Synthesis and characterization of degradable polyurethane elastomers containing an aminoacid based chain extender. Journal of Biomaterials Science, Polymer Edition. 1998. 9: 271-295.
12.Elliott S, Fromstein JD, Santerre JP, Woodhouse KA. Identifica-tion of biodegradation products formed by l-phenylalanine based segmented polyurethaneureas. Journal of Biomaterials Science Polymer Edition. 2002. 13: 691-711.
13.Guan JJ, Wagner WR. Synthesis, characterization and cytocompatibility of polyurethaneurea elastomers with designed elastase sensitivity. Biomacromolecules. 2005. 6: 2833-2842.
14.Liang D, Hsiao BS, Chu B. Functional electrospun nanofibrous scaffolds for biomedical applications. Advanced Drug Delivery Reviews. 2007. 59: 1392-1412.
15.Bhardwaj N, Kundu SC. Electrospinning: A fascinating fiber fabrication technique. Biotechnology Advances. 2010. 28: 325-347.
16.Sill TJ, von Recum HA. Electrospinning: Applications in drug delivery and tissue engineering. Biomaterials. 2008. 29: 1989-2006.
17.Xu CY, Inai R, Kotaki M, Ramakrishna S. Aligned biodegradable nanofibrous structure: a potential scaffold for blood vessel engineering. Biomaterials. 2004. 25: 877-886.
18.Deitzel JM, Kleinmeyer J, Harris D, Tan NCB. The effect of processing variables on the morphology of electrospun nanofibers and textiles. Polymer. 2001. 42: 261-272.
19.Zhang Y, Qian J, Ke Z, Zhu X, Bi H, Nie K. Viscometric study of poly (vinyl chloride)/poly (vinyl acetate) blends in various solvents. European Polymer Journal. 2002. 38: 333-337.
20.Luu YK, Kim K, Hsiao BS, Chu B, Hadjiargyrou M. Development of a nanostructured DNA delivery scaffold via electrospinning of PLGA and PLA-PEG block copolymers. Journal of Controlled Release. 2003. 89: 341-353.
21.Subbiah T, Bhat GS, Tock RW, Parameswaran S, Ramkumar SS. Electrospinning of nanofibers. Journal of Applied Polymer Science. 2005. 96: 557-569.
22.Ramakrishna S, Fujihara K, Teo WE, Yong T, Ma Z, Ramaseshan R. Electrospun nanofibers: solving global issues. Materials Today. 2006. 9: 40-50.
23.Cui W, Zhou S, Li X, Weng J. Drug-loaded biodegradable polymeric nanofibers prepared by electrospinning. Tissue engineering. 2006. 12: 1070.
24.Wu Y, He JH, Xu L, Yu JY. Electrospinning drug-loaded poly (Butylenes Succinate-cobytylene Terephthalate) (PBST) with acetylsalicylic acid (aspirin). International Journal of Electrospun Nanofibers and Applications. 2007. 1: 1-6.
25.Barnes CP, Sell SA, Knapp DC, Walpoth BH, Brand DD, Bowlin GL. Preliminary investigation of electrospun collagen and polydioxanone for vascular tissue engineering applications. International Journal of Electrospun Nanofibers and Applications. 2007. 1: 73-87.
26.Welle A, Kroger M, Doring M, Niederer K, Pindel E, Chronakis S. Electrospun aliphatic polycarbonates as tailored tissue scaffold materials. Biomaterials. 2007. 28: 2211-2219.
27.Nishida T, Yasumoto K. The network structure of corneal fibroblasts in the rat as revealed by scanning electron microscopy. Investigative Ophthalmology and Visual Science. 1988. 29: 1887–1890.
28.Kadler KE, Holmes DF. Collagen fibril formation. Biochemical Journal. 1996. 316: 1–11.
29.Laurencin C, Ambrosio A. Tissue engineering: orthopedic applications. Annual Review of Biomedical Engineering. 1999. 1: 19–46.
30.Stitzel J, Liu J, Lee SJ, Komura M, Berry J, Soker S. Controlled fabrication of a biological vascular substitute. Biomaterials. 2006. 27(7): 1088-1094.
31.Lee SJ, Yoo JJ, Lim GJ, Atala A, Stitzel J. In vitro evaluation of electrospun nanofiber scaffolds for vascular graft application. Journal of Biomedical Materials Research Part A. 2007. 83(4): 999-1008.
32.Thomas V, Jose MV, Chowdhury S, Sullivan JF, Dean DR, Vohra YK. Mechano-morphological studies of aligned nanofibrous scaffolds of polycaprolactone fabricated by electrospinning. Journal of Biomaterials Science, Polymer Edition. 2006. 17(9): 969-984.
33.Sui G, Yang X, Mei F, Hu X, Chen G, Deng X. Poly-L-lactic acid/hydroxyapatite hybrid membrane for bone tissue regeneration. Journal of Biomedical Materials Research Part A. 2007. 82(2): 445-454.
34.Mohammadi Y, Soleimani M, Fallahi-Sichani M, Gazme A, Haddadi-Asl V, Arefian E. Nanofibrous poly(epsilon-caprolactone)/poly(vinyl alcohol)/chitosan hybrid scaffolds for bone tissue engineering using mesenchymal stem cells. The International Journal of Artificial Organs. 2007. 30(3): 204-211.
35.Schnell E, Klinkhammer K, Balzer S, Brook G, Klee D, Dalton P. Guidance of glial cell migration and axonal growth on electrospun nanofibers of poly-epsilon-caprolactone and a collagen/poly-epsilon-caprolactone blend. Biomaterials. 2007. 28(19): 3012-3025.
36.Yang F, Murugan R, Wang S, Ramakrishna S. Electrospinning of nano/microscale poly(L-lactic acid) aligned fibers and their potential in neural tissue engineering. Biomaterials. 2005. 26(15): 2603-2610.
37.Fertala A, Han WB, Ko FK. Mapping critical sites in collagen II for rational design of geneengineered proteins for cell-supporting materials. Journal of Biomedical Materials Research. 2001. 57: 48-58.
38.Li WJ, Laurencin CT, Caterson EJ, Tuan RS, Ko FK. Electrospun nanofibrous structure: a novel scaffold for tissue engineering. Journal of Biomedical Materials Research. 2002. 60: 613-621.
39.Li WJ, Danielson KG, Alexander PG, Tuan RS. Biological response of chondrocytes cultured in three-dimensional nanofibrous poly (epsilon-caprolactone) scaffolds. Journal of Biomedical Materials Research Part A. 2003. 67: 1105-1114.
40.Rho KS, Jeong L, Lee G, Seo BM, Park YJ, Hong SD. Electrospinning of collagen nanofibers: effects on the behavior of normal human keratinocytes and early-stage wound healing. Biomaterials. 2006. 27: 1452-1461.
41.Venugopal J, Ramakrishna S. Biocompatible nanofiber matrices for the engineering of a dermal substitute for skin regeneration. Tissue Engineering. 2005. 11: 847-854.
42.Zong X, Ran S, Fang D, Hsiao BS, Chu B. Control of structure, morphology and property in electrospun poly (glycolide-co-lactide) non-woven membranes via post-draw treatments. Polymer. 2003. 44: 4959-4967.
43.Zong XH, Bien H, Chung CY, Yin LH, Fang DF, Hsiao BS. Electrospun fine-textured scaffolds for heart tissue constructs. Biomaterials. 2005. 26: 5330-5338.
44.Sahoo S, Ouyang H, Goh JC, Tay TE, Toh SL. Characterization of a novel polymeric scaffold for potential application in tendon/ligament tissue engineering. Tissue Engineering. 2006. 12(1): 91-99.
45.Lee CH, Shin HJ, Cho IH, Kang YM, Kim IA, Park KD. Nanofiber alignment and direction of mechanical strain affect the ECM production of human ACL fibroblast. Biomaterials. 2005. 26(11): 1261-1270.
46.Li WJ, Mauck RL, Cooper JA, Yuan X, Tuan RS. Engineering controllable anisotropy in electrospun biodegradable nanofibrous scaffolds for musculoskeletal tissue engineering. Journal of Biomechanics. 2007. 40(8): 1686-1693.
47.Baker BM, Mauck RL. The effect of nanofiber alignment on the maturation of engineered meniscus constructs. Biomaterials. 2007. 28: 1967-1977.
48.Wise JK, Yarin AL, Megaridis CM, Cho M. Chondrogenic differentiation of human mesenchymal stem cells on oriented nanofibrous scaffolds: engineering the superficial zone of articular cartilage. Tissue Engineering Part A. 2009. 15: 913-921.
49.Lim SH, Mao HQ. Electrospun scaffolds for stem cell engineering. Advanced Drug Delivery Reviews. 2009. 61: 1084-1096.
50.Yim EK, Pang SW, Leong KW. Synthetic nanostructures inducing differentiation of human mesenchymal stem cells into neuronal lineage. Experimental Cell Research. 2007. 313: 1820-1829.
51.McBeath R, Pirone DM, Nelson CM, Bhadriraju K, Chen CS. Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. Developmental Cell. 2004. 6: 483-495.
52.Chen M, Patra PK, Warner SB, Bhowmick S. Optimization of electrospinning process parameters for tissue engineering scaffolds. Biophysical Reviews and Letters. 2006. 1: 153-178.
53.Soliman S, Pagliari S, Rinaldi A, Forte G, Fiaccavento R, Pagliari F, Franzese O,Minieri M, Nardo PD, Licoccia S, Traversa E. Multiscale three-dimensional scaffolds for soft tissue engineering via multimodal electrospinning. Acta Biomaterialia. 2010. 6: 1227-1237.
54.Lowery JL, Datta N, Rutledge GC. Effect of fiber diameter, pore size and seeding method on growth of human derman fibroblasts in electrospun poly(ε-caprolactone) fibrous mats. Biomaterials. 2010. 31: 491-504.
55.Chen M, Patra PK, Lovett ML, Kaplan DL, Bhowmick S. Role of electrospun fibre diameter and corresponding specific surface area (SSA) on cell attachment. Journal of Tissue Engineering and Regenerative Medicine. 2009. 3: 269-279.
56.Bashur CA, Dahlgren LA, Goldstein AS. Effect of fiber diameter and orientation on fibroblast morphology and proliferation on electrospun poly(D,L-lactic-co-glycolic acid) meshes. Biomaterials. 2006. 27: 5681-5688.
57.Badami AS, Kreke MR, Thompson MS, Riffle JS, Goldstein AS. Effect of fiber diameter on spreading, proliferation, and differentiation of osteoblastic cells on electrospun poly(lactic acid) substrates. Biomaterials. 2006. 27: 596-606.
58.Sisson K, Zhang C, Farach-Carson MC, Chase DB, Rabolt JF. Fiber diameters control osteoblastic cell migration and differentiation in electrospun gelatin. Journal of Biomedical Materials Research Part A. 2010. 94: 1312-1320.
59.Szentivanyi A, Chakradeo T, Zernetsch H, Glasmacher B. Electrospun cellular microenvironments: Understanding controlled release and scaffold structure. Advanced Drug Delivery Reviews. 2011. 63: 209-220.
60.Cukierman E, Pankov R, Stevens DR, Yamada KM. Taking cell-matrix adhesions to the third dimension. Science. 2001. 294: 1708-1712.
61.Grayson WL, Zhao F, Izadpanah R, Bunnell B, Ma T. Effects of hypoxia on human mesenchymal stem cell expansion and plasticity in 3D constructs. Journal of Cellular Physiology. 2006. 207: 331-339.
62.Cukierman E, Pankov R, Yamada KM. Cell interactions with three-dimensional matrices. Current Opinion in Cell Biology. 2002. 14: 633-639.
63.Griffith LG, Swartz MA. Capturing complex 3D tissue physiology in vitro. Nature Reviews Molecular Cell Biology. 2006. 7: 211-224.
64.Bartosh TJ, Ylostalo JH, Mohammadipoor A, Bazhanov N, Coble K, Claypool K. Aggregation of human mesenchymal stromal cells (MSCs) into 3D spheroids enhances their antiinflammatory properties. Proceedings of the National Academy of Sciences of the United States of America. 2010. 107: 13724-13729.
65.Wang CC, Chen CH, Hwang SM, Lin WW, Huang CH, Lee WY. Spherically symmetric mesenchymal stromal cell bodies inherent with endogenous extracellular matrices for cellular cardiomyoplasty. Stem Cells. 2009. 27: 724-732.
66.Dittmer A, Hohlfeld K, Ltzkendorf J, Mller L, Dittmer J. Human mesenchymal stem cells induce E-cadherin degradation in breast carcinoma spheroids by activating ADAM10. Cellular and Molecular Life Sciences. 2009. 66: 3053-3065.
67.Frith JE, Thomson B, Genever PG. Dynamic three-dimensional culture methods enhance mesenchymal stem cell properties and increase therapeutic potential. Tissue Engineering Part C: Methods. 2010. 16: 735-749.
68.Lutolf MP, Gilbert PM, Blau HM. Designing materials to direct stem-cell fate. Nature. 2009. 462: 433-441.
69.Watt FM, Hogan BL, Eden O. Stem cells and their niches. Science. 2000. 287: 1427-1430.
70.Gerecht-Nir S, Cohen S, Itskovitz-Eldor J. Bioreactor cultivation enhances the efficiency of human embryoid body (hEB) formation and differentiation. Biotechnology and Bioengineering. 2004. 86: 493-502.
71.Otsuka H, Hirano A, Nagasaki Y, Okano Y, Horiike Y, Kataoka K. Two-Dimensional multiarray formation of hepatocyte spheroids on a microfabricated PEG-Brush surface. ChemBioChem. 2004. 5: 850-855.
72.Wang W, Itaka K, Ohba S, Nishiyama N, Chung UI, Yamasaki Y, Kataoka K. 3D spheroid culture system on micropatterned substrates for improved differentiation efficiency of multipotent mesenchymal stem cells. Biomaterials. 2009. 30: 2705-2715.
73.Liu H, Lin J, Roy K. Effect of 3D scaffold and dynamic culture condition on the global gene expression profile of mouse embryonic stem cells. Biomaterials. 2006. 27: 5978-5989.
74.Casciari JJ, Sotirchos SV, Sutherland RM. Mathematical modelling of microenvironment and growth in EMT6/Ro multicellular tumour spheroids. Cell Proliferation. 1992. 25: 1-22.
75.Sutherland RM, Inch WR, McCredie JA, Kruuv J. A multi-component radiation survival curve using an in vitro tumour model. International journal of radiation biology and related studies in physics, chemistry, and medicine. 1970. 18: 491-495.
76.Jessup JM, Frantz M, Sonmez-Alpan E, Locker J, Skena K, Waller H, Battle P, Nachman A, Bhatti Weber ME, Thomas DA, Curbeam RL Jr, Baker TL, Goodwin TJ. Microgravity culture reduces apoptosis and increases the differentiation of a human colorectal carcinoma cell line. In Vitro Cellular & Developmental Biology - Animal. 2000. 36: 367-373.
77.Ingram M, Techy GB, Saroufeem R, Yazan O, Narayan KS, Goodwin TJ, Spaulding GF. Three-dimensional growth patterns of various human tumor cell lines in simulated microgravity of a NASA bioreactor. In Vitro Cellular & Developmental Biology - Animal. 1997. 33: 459-466.
78.Goodwin TJ, Prewett TL, Wolf DA, Spaulding GF. Reduced shear stress: A major component in the ability of mammalian tissues to form three-dimensional assemblies in simulated microgravity. Journal of Cellular Biochemistry. 1993. 51: 301-311.
79.Schwarz RP, Goodwin TJ, Wolf DA. Cell culture for three-dimensional modeling in rotating-wall vessels: An application of simulated microgravity. Journal of Tissue Culture Methods. 1992. 14: 51-57.
80.Kelm JM, Timmins NE, Brown CJ, Fussenegger M, Nielsen LK. Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types. Biotechnology and Bioengineering. 2003. 83: 173-180.
81.Emfietzoglou D, Kostarelos K, Papakostas A, Yang WH, Ballangrud A, Song H, Sgouros G. Liposome-mediated radiotherapeutics within avascular tumor spheroids: Comparative dosimetry study for various radionuclides, liposome systems, and a targeting antibody. Journal of Nuclear Medicine. 2005. 46: 89-97.
82.Foty R. A simple hanging drop cell culture protocol for generation of 3D spheroids. Journal of Visualized Experiments : JoVE. 2011. (51).
83.Banerjee M, Bhonde RR. Application of hanging drop technique for stem cell differentiation and cytotoxicity studies. Cytotechnology. 2006. 51(1): 1-5.
84.Huang GS, Dai LG, Yen BL, Hsu SH. Spheroid formation of mesenchymal stem cells on chitosan and chitosan-hyaluronan membranes. Biomaterials. 2011. 32(29): 6929-6945.
85.Yeh HY, Liu BH, Hsu SH. The calcium-dependent regulation of spheroid formation and cardiomyogenic differentiation for MSCs on chitosan membranes. Biomaterials. 2012. 33(35): 8943-8954.
86.Heldin CH, Rubin K, Pietras K, Ostman A. High interstitial fluid pressure - an obstacle in cancer therapy. Nature Reviews Cancer. 2004. 4: 806-813.
87.Kim JB, Stein R, O''Hare MJ. Three-dimensional in vitro tissue culture models of breast cancer - a review. Breast Cancer Research and Treatment. 2004. 85: 281-291.
88.Bhatia SN, Balis UJ, Yarmush ML, Toner M. Microfabrication of hepatocyte/fibroblast co-cultures: Role of homotypic cell interactions. Biotechnology Progress. 1998. 14: 378-387.
89.Cukierman E, Pankov R, Stevens DR, Yamada KM. Taking cell-matrix adhesions to the third dimension. Science. 2001. 294: 1708-1712.
90.Albrecht DR, Underhill GH, Wassermann TB, Sah RL, Bhatia SN. Probing the role of multicellular organization in three-dimensional microenvironments. Nature Methods. 2006. 3: 369-375.
91.Liu VA, Bhatia SN. Three-dimensional photopatterning of hydrogels containing living cells. Biomedical Microdevices. 2002. 4: 257-266.
92.Griffith LG, Swartz MA. Capturing complex 3D tissue physiology in vitro. Nature Reviews Molecular Cell Biology. 2006. 7: 211-224.
93.Yuan XY, Zhang YY, Dong CH, Sheng J. Morphology of ultrafine polysulfone fibers prepared by electrospinning. Polymer International. 2004. 53: 1704-1710.
94.Megelski S, Stephens JS, Chase DB, Rabolt JF. Micro- and nanostructured surface morphology on electrospun polymer fibers. Macromolecules. 2002. 35(22): 8456-8466.
95.Doshi J, Reneker DH. Electrospinning process and applications of electrospun fibers. Journal of Electrostatics. 1995. 35(2-3): 151-160.
96.Reneker DH, Yarin AL. Electrospinning jets and polymer nanofibers. Polymer. 2008. 49: 2387-2425.
97.Srivastava Y, Marquez M, Thorsen T. Multijet electrospinning of conducting nanofibers from microfluidic manifolds. Journal of Applied Polymer Science. 2007. 106: 3171-3178.
98.Reneker DH, Chun L. Nanometre diameters of polymer, produced by electrospinning. Nanotechnology. 1996. 7: 216-223.
99.Demir MM, Yilgor I, Yilgor E, Erman B. Electrospinning of polyurethane fibers. Polymer. 2002. 43: 3303-3309.
100.Rutledge GC, Warner SB, Ugbolue SC. Electrostatic Spinning and Properties of Ultrafine fibers. National Textile Center Annual Report M01-MD22. 2004.
101.Liu Y, Wu N, Wei Q, Cai Y, Wei A. Wetting behavior of electrospun poly (L-lactic acid)/poly(vinyl alcohol) composite nonwovens. Journal of Applied Polymer Science. 2008. 110: 3172-3177.
102.Wang N, Burugapalli K, Song W, Halls J, Moussy F, Zheng Y, Ma Y, Wu Z, Li K. Tailored fibro-porous structure of electrospun polyurethane membranes, their size-dependent properties and trans-membrane glucose diffusion. Journal of Membrane Science. 2013. 427: 207-217.
103.Pai CL, Boyce MC, Rutledge GC. Mechanical properties of individual electrospun PA 6(3)T fibers and their variation with fiber diameter. Polymer. 2011. 52: 2295-2301.
104.Arinstein A, Burman M, Gendelman O, Zussman E. Effect of supramolecular structure on polymer nanofibre elasticity. Nature Nanotechnology. 2007. 2: 59-62.
105.Santini MT, Rainaldi G, Indovina PL. Apoptosis, cell adhesion and the extracellular matrix in the three-dimensional growth of multicellular tumor spheroids. Critical Reviews in Oncology/Hematology. 2000. 36: 75-87.
106.Li CL, Tian T, Nan KJ, Zhao N, Guo YH, Cui J, Wang J, Zhang WG. Survival advantages of multicellular spheroids vs. monolayers of HepG2 cells in vitro. Oncology Reports. 2008. 20: 1465-1471.
107.Takagi M, Umetsu Y, Fujiwara M, Wakitani S. High Inoculation Cell Density Could Accelerate the Differentiation of Human Bone Marrow Mesenchymal Stem Cells to Chondrocyte Cells. Journal of Bioscience and Bioengineering. 2007. 103(1): 98-100.
108.Park JS, Chu JS, Tsou AD, Diop R, Tang Z, Wang A, Li S. The effect of matrix stiffness on the differentiation of mesenchymal stem cells in response to TGF-β. Biomaterials. 2011. 32: 3921-3930.
109.Hsu SH, Huang GS, Lin SY, Feng F, Ho TT, Liao YC. Enhanced Chondrogenic Differentiation Potential of Human Gingival Fibroblasts by Spheroid Formation on Chitosan Membranes. Tissue Engineering Part A. 2012. 18: 67-79.
110.Ritchie RO, Buehler MJ, Hansma P. Plasticity and toughness in bone. Physics Today. 2009. 62: 41.
111.Burstein AH, Zika JM, Heiple KG, Klein L. Contribution of collagen and mineral to the elastic-plastic properties of bone. The Journal of Bone and Joint Surgery. 1975. 57(7): 956-961.
112.Mehta SS, Oz OK, Antich PP. Bone Elasticity and Ultrasound Velocity Are Affected by Subtle Changes in the Organic Matrix. Journal of Bone and Mineral Research. 1998. 13(1): 114-121.
113.DeLise AM, Fischer L, Tuan RS. Cellular interactions and signaling in cartilage development. Osteoarthritis and Cartilage. 2000. 8(5): 309-334.
114.Zhang L, Su P, Xu C, Yang J, Yu W, Huang D. Chondrogenic differentiation of human mesenchymal stem cells: a comparison between micromass and pellet culture systems. Biotechnology Letters. 2010. 32: 1339-1346.
115.Li B, Wang Y, Jia D, Zhou Y. Gradient structural bone-like apatite induced by chitosan hydrogel via ion assembly. Journal of Biomaterials Science. 2011. 22: 505-517.
116.Markovic D, Zivojinovic V, Jokanovic V, Krstic V. Biocompatibility of nanostructured carbonated calcium hydroxyapatite obtained by hydrothermal method. 2006. 56: 541-551.