本研究運用雙交聯法來製備海藻酸/多巴胺/明膠(Alginate/Dopamine/Gelatin)雙網絡支架，首先利用EDC/NHS的方式將Alginate接上Dopamine，此化合物（簡稱AD）易溶於水，利用AD及Gelatin（簡稱G）皆易溶於水的特性，使兩種材料於水中均勻混合，並進行冷凍乾燥來固定形貌，再利用鈣交聯反應及Genipin交聯反應，製備出三維多孔隙支架。而我們將探討AD/G雙交聯支架不同比例的形貌，並透過傅立葉轉換紅外線光譜儀(FT-IR)探討兩種高分子雙交聯狀態與AD/G支架分子間氫鍵的變化。再從熱性質分析AD/G與鈣交聯的比例，進而討論支架的差異，並觀察AD/G雙交聯支架的機械性質與降解率，使支架的機械強度與延展性提高一倍以上與30%，同時改善明膠降解性過高的特性，將降解率降低40%。
而為了進一步了解AD/G雙交聯支架在骨組織工程的應用，我們將支架進行人類成骨肉瘤MG-63細胞培養後的生物活性分析、成骨礦化分析及細胞在支架上的形貌。藉由這些分析驗證AD/G雙交聯支架對於骨細胞的增殖與分化有明顯的幫助。最後，我們執行週期性動態培養測試，讓細胞在生長時接受外在力量刺激，並利用雙交聯支架的機械穩定性讓整體支架所受的應力相同，使應變均勻地對組織之結構、形態和功能產生影響，使其在提升細胞活性及分化能力表現更為顯著。

In this study, the double cross-linking method was used to prepare the Alginate/Dopamine/Gelatin double network scaffold. First of all, Dopamine was connected to Alginate by EDC/NHS. This compound (AD) is easily soluble in water. Secondly, using AD and Gelatin (abbreviated as G) that is also easily soluble in water to mix the two materials uniformly in water, and freeze-dried to fix the morphology, and then crosslinked AD with calcium and crosslinked Gelatin with Genipin to prepare three-dimensional porous polymers scaffold.
We observed the morphology of the AD/G double cross-linked scaffold in different proportions, and use the Fourier Transform Infrared Spectroscopy (FT-IR) to survey the changes in the hydrogen bond between the two polymer. Then analyzing the ratio of AD/G and calcium cross-linked in the scaffold by using Thermogravimetric analysis (TGA) to discuss the difference between the samples. We observed the mechanical properties and degradation rate of the AD/G double cross-linked scaffold to reach the goal of increasing the mechanical strength and ductility of the scaffold to be over double and 30%. At the same time, AD could improve the high degradation rate characteristics of Gelatin, and decrease the degradation rate to 40%.
In order to understand the application of AD/G double cross-linked scaffold in bone tissue engineering, we did the research of the biological activity analysis of human osteosarcoma MG-63 cell culture, bone mineralization analysis and the morphology of the cells on the scaffold. Through these analyses, it is verified that the AD/G double cross-linked scaffold is obviously helpful for the proliferation and differentiation of bone cells. Finally, we perform a cyclic stretching cell culture to give the cells external force stimulation during growth, and use the mechanical stability of the double cross-linked scaffold to make the overall scaffold suffer the same stress, so that the strain can evenly affect the structure and morphology of the tissue to make it more significant in enhancing cell viability and differentiation ability.

Cieza, A., Causey, K., Kamenov, K., Hanson, S. W., Chatterji, S., & Vos, T. (2020). Global estimates of the need for rehabilitation based on the Global Burden of Disease study 2019: a systematic analysis for the Global Burden of Disease Study 2019. The Lancet, 396(10267), p.2006-2017.
[2] Brennan-Olsen, S.L., et al.（2017）, Prevalence of arthritis according to age, sex and socioeconomic status in six low and middle income countries: analysis of data from the World Health Organization study on global AGEing and adult health (SAGE) Wave 1. BMC Musculoskelet Disord, 18(1): p. 271.
[3] XIAOHUA LIU, PETER X. MA(2004), Polymeric Scaffolds for Bone Tissue Engineering. Annals of Biomedical Engineering, Vol. 32, No. 3, March 2004 (©2004) pp. 477–486
[4] Muhammad Iqbal Sabir, Xiaoxue Xu, Li Li (2009) . A review on biodegradable polymeric materials for bone tissue engineering applications. J Mater Sci 44:5713–5724 DOI 10.1007/s10853-009-3770-7
[5] Jean-Yves Reginster , Nansa Burlet (2006) , Osteoporosis: A still increasing prevalence. Bone 38 S4–S9
[6] Jiali Shen, Dongjian Shi, Liangliang Dong, Zhuying Zhang, Xiaojie Li, Mingqing Chen(2018). Fabrication of polydopamine nanoparticles knotted alginate scaffolds and their properties. J Biomed Mater Res Part A：106A：3255–3266，2018
[7] Sruthi Ranganathan, Kalimuthu Balagangadharan, Nagarajan Selvamurugan(2019). Chitosan and gelatin-based electrospun fibers for bone tissue engineering. International Journal of Biological Macromolecules 133 354–364
[8] Dirk Schultheiss (2004). Regenerative Medicine in Andrology:Tissue Engineering and GeneTherapy as PotentialTreatment Options for Penile Deformations and Erectile Dysfunction. European Urology 46 162–169
[9] Akter, F., Chapter 1 - What is Tissue Engineering?, in Tissue Engineering Made Easy, F. Akter, Editor. 2016, Academic Press. p. 1-2.
[10] Suh, H., Tissue restoration, tissue engineering and regenerative medicine. Yonsei Med J, 2000. 41(6): p. 681-4.
[11] Vinatier C, Mrugala D, Jorgensen C, Guicheux J, Noël D（2009）.Cartilage engineering: a crucial combination of cells, biomaterials and biofactors.Trends Biotechnol. 27(5):307-14. doi: 10.1016/j.tibtech.2009.02.005. Epub 2009 Mar 28.PMID: 19329205
[12] Akter, F., Chapter 2 - Principles of Tissue Engineering, in Tissue Engineering Made Easy, F. Akter, Editor. 2016, Academic Press. p. 3-16.
[13] Olson, J.L., A. Atala, and J.J. Yoo, Tissue engineering: current strategies and future directions. Chonnam medical journal, 2011. 47(1): p. 1-13.
[14] Fergal J. O’Brien（2011）. Biomaterials & scaffolds for tissue engineering. Materials Today.Volume 14, Issue 3, March 2011, Pages 88-95
[15] Steve KW Oh and Andre BH Choo. Frontiers in Research Review: Cutting-Edge Molecular Approaches to Therapeutics,HUMAN EMBRYONIC STEM CELLS: TECHNOLOGICAL CHALLENGES TOWARDS THERAPY.Clinical and Experimental Pharmacology and Physiology (2006) 33, 489–495
[16] Pau Atienza-RocaXiaolin CuiGary J. HooperTim B. F. WoodfieldKhoon S. Lim（2018）. Growth Factor Delivery Systems for Tissue Engineering and Regenerative Medicine. Cutting-Edge Enabling Technologies for Regenerative Medicine pp 245-269
[17] Badylak, S.F., The extracellular matrix as a biologic scaffold material. Biomaterials, 2007. 28(25): p. 3587-3593.
[18] Blais, M., et al., Concise review: tissue‐engineered skin and nerve regeneration in burn treatment. Stem cells translational medicine, 2013. 2(7): p. 545-551.
[19] Amini, A.R., C.T. Laurencin, and S.P. Nukavarapu, Bone tissue engineering: recent advances and challenges. Critical Reviews™ in Biomedical Engineering, 2012. 40(5).
[20] Chung, C. and J.A. Burdick, Engineering cartilage tissue. Advanced drug delivery reviews, 2008. 60(2): p. 243-262.
[21] Kasten, P., et al., Porosity and pore size of β-tricalcium phosphate scaffold can influence protein production and osteogenic differentiation of human mesenchymal stem cells: an in vitro and in vivo study. Acta biomaterialia, 2008. 4(6): p. 1904-1915.
[22] Blunk, T., et al., Differential effects of growth factors on tissue-engineered cartilage. Tissue engineering, 2002. 8(1): p. 73-84.
[23] Akter, F. and J. Ibanez, Chapter 8 - Bone and Cartilage Tissue Engineering, in Tissue Engineering Made Easy, F. Akter, Editor. 2016, Academic Press. p. 77-97.
[24] Clarke, B., Normal bone anatomy and physiology. Clinical journal of the American Society of Nephrology, 2008. 3(Supplement 3): p. S131-S139.
[25] Gilbert, S.F., J.M. Opitz, and R.A. Raff, Resynthesizing evolutionary and developmental biology. Developmental biology, 1996. 173(2): p. 357-372.
[26] Kahn, A. and D. Simmons, Investigation of cell lineage in bone using a chimaera of chick and quail embryonic tissue. Nature, 1975. 258(5533): p. 325-327.
[27] In Vitro Osteogenic Differentiation of Human ES Cells. Cloning and Stem Cells, 2003. 5(2): p. 149-155.
[28] Huang, Z., et al., The sequential expression profiles of growth factors from osteroprogenitors to osteoblasts in vitro. Tissue engineering, 2007. 13(9): p. 2311-2320.
[29] Jaiswal, N., et al., Osteogenic differentiation of purified, culture‐expanded human mesenchymal stem cells in vitro. Journal of cellular biochemistry, 1997. 64(2): p. 295-312.
[30] Robert Proulx Heaney , G.D.W. Bone: Remodeling,growth and development,Bone remodeling. Encyclopædia Britannica,2019 March 19; Available from: https://www.britannica.com/science/bone-remodeling.
[31] Rucci, N., Molecular biology of bone remodelling. Clinical cases in mineral and bone metabolism : the official journal of the Italian Society of Osteoporosis, Mineral Metabolism, and Skeletal Diseases, 2008. 5(1): p. 49-56.
[32] The Sequential Expression Profiles of Growth Factors from Osteroprogenitors to Osteoblasts In Vitro. Tissue Engineering, 2007. 13(9): p. 2311-2320.
[33] Taisuke Masuda, Ichiro Takahashi, Takahisa Anada, Fumihito Arai, Toshio Fukuda, Teruko Takano-Yamamoto, Osamu Suzuki,Development of a cell culture system loading cyclic mechanical strain to chondrogenic cells. Journal of Biotechnology 133 (2008) 231–238
[34] JAMES H.-C. WANG, GUOGUANG YANG, and ZHAOZHU LI. Controlling Cell Responses to Cyclic Mechanical Stretching. Annals of Biomedical Engineering, Vol. 33, No. 3, March 2005 (© 2005) pp. 337–342 DOI: 10.1007/s10439-005-1736-8
[35] A.O. Stucki, J.D. Stucki, S.R. Hall, M. Felder, Y. Mermoud, R.A. Schmid, T. Geiser, O.T. Guenat. A lung-on-a-chip array with an integrated bio-inspired respiration mechanism. Lab Chip, 15 (2015), pp. 1302-1310
[36] S. Kazunori, S. Atsushi, M. Kenichi, H. Mitsuru, K. Satoshi. Development of a biochip with serially connected pneumatic balloons for cell-stretching culture Sens. Actuators B Chem., 156 (2011), pp. 486-493
[37] Y. Katanosaka, J.H. Bao, T. Komatsu, T. Suemori, A. Yamada, S. Mohri, K. Naruse. Analysis of cyclic-stretching responses using cell-adhesion-patterned cells. J. Biotechnol., 133 (2008), pp. 82-89
[38] Shen-Jui Tseng, Chi-Hui Cheng, Tzer-Min Lee, Jui-Che Lin. Studies of osteoblast-like MG-63 cellular proliferation and differentiation with cyclic stretching cell culture system on biomimetic hydrophilic layers modified polydimethylsiloxane substrate. Biochemical Engineering Journal Volume 168, April 2021, 107946
[39] Chu, J., et al., Biphasic regulation of myosin light chain phosphorylation by p21-activated kinase modulates intestinal smooth muscle contractility. J Biol Chem, 2013. 288(2): p. 1200-13.
[40] Boerckel, J.D., et al., In Vivo Model for Evaluating the Effects of Mechanical Stimulation on Tissue-Engineered Bone Repair. Journal of Biomechanical Engineering, 2009. 131(8).
[41] Neidlinger‐Wilke, C., H.J. Wilke, and L. Claes, Cyclic stretching of human osteoblasts affects proliferation and metabolism: a new experimental method and its application. Journal of Orthopaedic Research, 1994. 12(1): p. 70-78.
[42] Stanford, C., J. Morcuende, and R. Brand, Proliferative and phenotypic responses of bone‐like cells to mechanical deformation. Journal of Orthopaedic Research, 1995. 13(5): p. 664-670.
[43] Matsuda, N., et al., Proliferation and differentiation of human osteoblastic cells associated with differential activation of MAP kinases in response to epidermal growth factor, hypoxia, and mechanical stressin vitro. Biochemical and biophysical research communications, 1998. 249(2): p. 350-354.
[44] Tanaka, S.M., et al., Effects of broad frequency vibration on cultured osteoblasts. Journal of biomechanics, 2003. 36(1): p. 73-80.
[45] You, L., et al., A model for strain amplification in the actin cytoskeleton of osteocytes due to fluid drag on pericellular matrix. Journal of biomechanics, 2001. 34(11): p. 1375-1386.
[46] A. Tondon, R. Kaunas. The direction of stretch-induced cell and stress fiber orientation depends on collagen matrix stress. PLOS One, 9 (2014), pp. 1-10
[47] Chen, Y.-J., et al., Mechanoregulation of osteoblast-like MG-63 cell activities by cyclic stretching. Journal of the Formosan Medical Association, 2014. 113(7): p. 447-453.
[48] Javier Aragón, Simona Salerno, Loredana De Bartolo, Silvia Irusta, Gracia Mendoza. Polymeric electrospun scaffolds for bone morphogenetic protein 2 delivery in bone tissue engineering. Journal of Colloid and Interface Science 531 (2018) 126–137
[49] Maryam Hatamzadeh, Peyman Najafi-Moghadam, Ali Baradar-Khoshfetrat, Mehdi Jaymand, Bakhshali Massoumi. Novel nanofibrous electrically conductive scaffolds based on poly(ethylene glycol)s-modified polythiophene and poly(ε- caprolactone) for tissue engineering applications. Polymer 107 (2016) 177-190
[50] Albuquerque, P., et al., Approaches in biotechnological applications of natural polymers. 2016.
[51] Asadi, N., et al., Common Biocompatible Polymeric Materials for Tissue Engineering and Regenerative Medicine. Materials Chemistry and Physics, 2019: p. 122528.
[52] Seal, B., T. Otero, and A. Panitch, Polymeric biomaterials for tissue and organ regeneration. Materials Science and Engineering: R: Reports, 2001. 34(4-5): p. 147-230.
[53] Markstedt, K., et al., 3D bioprinting human chondrocytes with nanocellulose–alginate bioink for cartilage tissue engineering applications. Biomacromolecules, 2015. 16(5): p. 1489-1496.
[54] Levite, M., Dopamine and T cells: dopamine receptors and potent effects on T cells, dopamine production in T cells, and abnormalities in the dopaminergic system in T cells in autoimmune, neurological and psychiatric diseases. Acta Physiol (Oxf), 2016. 216(1): p. 42-89.
[55] Cao, X. and A. Aballay, Neural Inhibition of Dopaminergic Signaling Enhances Immunity in a Cell-Non-autonomous Manner. Curr Biol, 2016. 26(17): p. 2329-34.
[56] Ku SH, Park CB. Human endothelial cell growth on mussel-inspired nanofiber scaffold for vascular tissue engineering. Biomaterials 2010;31(36):9431–9437.
[57] Shi D, Zhang L, Shen J, Li X, Chen M, Akashi M. Fabrication of rod-like nanocapsules based on polylactide and 3, 4-dihydroxyphenylalanine for a drug delivery system. RSC Adv 2015;5(125):103414–103420.
[58] Wang R, Li J, Chen W, Xu T, Yun S, Xu Z, Xu Z, Sato T, Chi B, Xu H. A biomimetic mussel-inspired ε-poly-L-lysine hydrogel with robust tissue-anchor and anti-infection capacity. Adv Funct Mater 2017;27(8):1604894.
[59] Ma H, Luo J, Sun Z, Xia L, Shi M, Liu M, Chang J, Wu C. 3D printing of biomaterials with mussel-inspired nanostructures for tumor therapy and tissue regeneration. Biomaterials 2016;111:138–148.
[60] J. Maitra, V.K. Shukla. Cross-linking in hydrogels-a review. Am. J. Polym. Sci, 4 (2) (2014), pp. 25-31
[61] Zoran S.Petrović, JamesFerguson. Polyurethane elastomers. Progress in Polymer Science.Volume 16, Issue 5, October 1991, Pages 695-836
[62] Qiang Chen, Hong Chen, Lin Zhu, Jie Zheng. Fundamentals of double network hydrogels. Journal of Materials Chemistry B DOI: 10.1039
[63] J. P. Gong, Y. Katsuyama, T. Kurokawa and Y. Osada, Adv. Mater., 2003, 15, 1155–1158.
[64] Q. Chen, L. Zhu, C. Zhao, Q. Wang and J. Zheng, Adv. Mater., 2013, 25, 4171–4176.
[65] Suesca, E., et al., Multifactor analysis on the effect of collagen concentration, cross-linking and fiber/pore orientation on chemical, microstructural, mechanical and biological properties of collagen type I scaffolds. Materials Science and Engineering: C, 2017. 77: p. 333-341.
[66] Ruchi Mishra, Bikramjit Basu, Ashok Kumar. Physical and cytocompatibility properties of bioactive glass–polyvinyl alcohol–sodium alginate biocomposite foams prepared via sol–gel processing for trabecular bone regeneration. J Mater Sci: Mater Med (2009) 20:2493–2500 DOI 10.1007/s10856-009-3814-1
[67] Sema Ekici, Dursun Saraydin. Interpenetrating polymeric network hydrogels for potential gastrointestinal drug release. Polymer International 56:1371–1377 (2007)
[68] Yu-Shu Lai, Wen-Chuan Chen, Chang-Hung Huang, Cheng-Kung Cheng, Kam-Kong Chan, and Ting-Kuo Chang. The Effect of Graft Strength on Knee Laxity and Graft In-Situ Forces after Posterior Cruciate Ligament Reconstruction. PLoS One. 2015; 10(5): e0127293.