本研究利用丙胺酸(L-alanine)和poly(ethylene glycol)(PEG)共聚合成一同時具有溫度敏感性和可進行光交聯的高分子。先在PEG以開環反應在兩末端接上poly(L-alanine)(L-PA)形成一三嵌段共聚物PA-PEG-PA(PEA)，並將其末端接上丙烯醯基(acryloyl group)，此過程稱為「diacrylation」。然後利用紫外光(UV light)對此材料進行照射，藉此引發聚合反應形成光交聯水膠。在本研究中，首先利用PEG與L-PA合成之高分子原本即具有溫度敏感之成膠特性，希望透過光交聯的步驟能增強其水膠的機械強度，此外由於alanine為一種生體具備的胺基酸，其作為共聚物的疏水鏈段相信比傳統聚酯類高分子(polyester)更有好的生物相容性(biocompatibility)。在本研究中證實，不論在水膠表面進行細胞培養，抑或是利用水膠包埋細胞，PEA水膠均相較PEG水膠有提升細胞相對存活率之功效；另外於PEA水膠中混摻人體骨骼主要成分磷酸鈣(β-TCP)，發現有助於細胞增生及骨分化能力。綜合以上之研究結果，PEA高分子材料對於未來組織工程上的應用有很大的發展空間，例如生物黏著劑和軟骨、硬骨修復等。

The synthesis of photocrosslinked and thermosensitive hydrogels are performed by copolymerization of poly(L-alanine)(L-PA) and poly(ethylene glycol)(PEG). Both ends of PEG were conjugated with L-PA to form a PA-PEG-PA(PEA) tri-block. Then, the terminal ends of PA-PEG-PA were modified by acroyloyl groups, which was called “diacrylation”. Exposing this material to UV-light, photo-crosslinking would begin. As photoinitiator attacked C=C double bond of vinyl groups and induced photopolymerized to form the gel. First, we want to use the thermosensitive property of the tri-block to form the hydrogel, and let it expose UV to begin photocrosslinking. This step we hope can increase mechanical strength of the hydrogel. Besides, alanine is an amino acid, it is more suitable to culture cell than polyester hydrogels. We think the hydrogel we made that can increase not only strength but with good biocompatibility. In this study, we prove that PEA hydrogel can enhance cell proliferation whether cell seeded on hydrogel surface or encapsulated into hydrogel. Comparing with PEG hydrogel, PEA hydrogel has better performance on cell viability than PEG hydrogel. In addition, we study that PEA incorporated with β-TCP which is main component of bone can promote cell proliferation and also induce osteogenic differentiation. In summary, this copolymer “PEA” has wide range of the development in the tissue engineering field, such as bioadhesive, cartilage or bone regeneration.

[1] Hoare, T. R., and Kohane, D. S. (2008) Hydrogels in drug delivery: Progress and challenges, Polymer 49, 1993-2007.
[2] Wichterle, O., and Lim, D. (1960) Hydrophilic Gels for Biological Use, Nature 185, 117-118.
[3] Dušek, K., and Patterson, D. (1968) Transition in swollen polymer networks induced by intramolecular condensation, Journal of Polymer Science Part A-2: Polymer Physics 6, 1209-1216.
[4] Nuttelman, C. R., Rice, M. A., Rydholm, A. E., Salinas, C. N., Shah, D. N., and Anseth, K. S. (2008) Macromolecular monomers for the synthesis of hydrogel niches and their application in cell encapsulation and tissue engineering, Progress in Polymer Science 33, 167-179.
[5] Yang, J., Han, C.-R., Duan, J.-F., Xu, F., and Sun, R.-C. (2013) Mechanical and Viscoelastic Properties of Cellulose Nanocrystals Reinforced Poly(ethylene glycol) Nanocomposite Hydrogels, ACS Applied Materials & Interfaces 5, 3199-3207.
[6] Ifkovits, J. L., and Burdick, J. A. (2007) Review: Photopolymerizable and degradable biomaterials for tissue engineering applications, Tissue Engineering 13, 2369-2385.
[7] Bryant, S. J., Nuttelman, C. R., and Anseth, K. S. (2000) Cytocompatibility of UV and visible light photoinitiating systems on cultured NIH/3T3 fibroblasts in vitro, Journal of biomaterials science. Polymer edition 11, 439-457.
[8] Chan, B. K., Wippich, C. C., Wu, C.-J., Sivasankar, P. M., and Schmidt, G. (2012) Robust and Semi-Interpenetrating Hydrogels from Poly(ethylene glycol) and Collagen for Elastomeric Tissue Scaffolds, Macromolecular Bioscience 12, 1490-1501.
[9] Cruise, G. M., Scharp, D. S., and Hubbell, J. A. (1998) Characterization of permeability and network structure of interfacially photopolymerized poly(ethylene glycol) diacrylate hydrogels, Biomaterials 19, 1287-1294.
[10] Yoshii, E. (1997) Cytotoxic effects of acrylates and methacrylates: Relationships of monomer structures and cytotoxicity, Journal of biomedical materials research 37, 517-524.
[11] Odian, G. (2004) Principles of Polymerization, 4th Edition, Wiley, New York.
[12] Bryant, S. J., and Anseth, K. S. (2001) The effects of scaffold thickness on tissue engineered cartilage in photocrosslinked poly(ethylene oxide) hydrogels, Biomaterials 22, 619-626.
[13] Burdick, J. A., and Anseth, K. S. (2002) Photoencapsulation of osteoblasts in injectable RGD-modified PEG hydrogels for bone tissue engineering, Biomaterials 23, 4315-4323.
[14] Nuttelman, C. R., Tripodi, M. C., and Anseth, K. S. (2004) In vitro osteogenic differentiation of human mesenchymal stem cells photoencapsulated in PEG hydrogels, Journal of Biomedical Materials Research Part A 68A, 773-782.
[15] Yu, L., and Ding, J. (2008) Injectable hydrogels as unique biomedical materials, Chemical Society Reviews 37, 1473-1481.
[16] Choi, Y. Y., Jang, J. H., Park, M. H., Choi, B. G., Chi, B., and Jeong, B. (2010) Block length affects secondary structure, nanoassembly and thermosensitivity of poly(ethylene glycol)-poly(l-alanine) block copolymers, Journal of Materials Chemistry 20, 3416-3421.
[17] Greenwald, R. B., Choe, Y. H., McGuire, J., and Conover, C. D. (2003) Effective drug delivery by PEGylated drug conjugates, Advanced Drug Delivery Reviews 55, 217-250.
[18] Yun, E. J., Yon, B., Joo, M. K., and Jeong, B. (2012) Cell Therapy for Skin Wound Using Fibroblast Encapsulated Poly(ethylene glycol)-poly(l-alanine) Thermogel, Biomacromolecules 13, 1106-1111.
[19] Yeon, B., Park, M. H., Moon, H. J., Kim, S. J., Cheon, Y. W., and Jeong, B. (2013) 3D Culture of Adipose-Tissue-Derived Stem Cells Mainly Leads to Chondrogenesis in Poly(ethylene glycol)-Poly(L-alanine) Diblock Copolymer Thermogel, Biomacromolecules 14, 3256-3266.
[20] Kweon, H., Yoo, M. K., Park, I. K., Kim, T. H., Lee, H. C., Lee, H.-S., Oh, J.-S., Akaike, T., and Cho, C.-S. (2003) A novel degradable polycaprolactone networks for tissue engineering, Biomaterials 24, 801-808.
[21] Li, S., Molina, I., Martinez, M., and Vert, M. (2002) Hydrolytic and enzymatic degradations of physically crosslinked hydrogels prepared from PLA/PEO/PLA triblock copolymers, Journal of Materials Science: Materials in Medicine 13, 81-86.
[22] Thi Hong Anh, N., and Van Cuong, N. (2010) Formation of nanoparticles in aqueous solution from poly(ε-caprolactone)–poly(ethylene glycol)–poly(ε-caprolactone), Advances in Natural Sciences: Nanoscience and Nanotechnology 1, 025012.
[23] Zhang, Y., Wu, X., Han, Y., Mo, F., Duan, Y., and Li, S. (2010) Novel thymopentin release systems prepared from bioresorbable PLA-PEG-PLA hydrogels, Int J Pharm 386, 15-22.
[24] Yu, L., Chang, G. T., Zhang, H., and Ding, J. D. (2008) Injectable block copolymer hydrogels for sustained release of a PEGylated drug, Int J Pharm 348, 95-106.
[25] Ko, C.-Y., Yang, C.-Y., Yang, S.-R., Ku, K.-L., Tsao, C.-K., Chwei-Chin Chuang, D., Chu, I. M., and Cheng, M.-H. (2013) Cartilage formation through alterations of amphiphilicity of poly(ethylene glycol)-poly(caprolactone) copolymer hydrogels, RSC Advances 3, 25769-25779.
[26] Vitale, A., Bongiovanni, R., and Ameduri, B. (2015) Fluorinated Oligomers and Polymers in Photopolymerization, Chemical Reviews 115, 8835-8866.
[27] Muggli, D. S., Burkoth, A. K., Keyser, S. A., Lee, H. R., and Anseth, K. S. (1998) Reaction Behavior of Biodegradable, Photo-Cross-Linkable Polyanhydrides, Macromolecules 31, 4120-4125.
[28] Anseth, K. S., Wang, C. M., and Bowman, C. N. (1994) REACTION BEHAVIOR AND KINETIC CONSTANTS FOR PHOTOPOLYMERIZATIONS OF MULTI(METH)ACRYLATE MONOMERS, Polymer 35, 3243-3250.
[29] Oh, H. J., Joo, M. K., Sohn, Y. S., and Jeong, B. (2008) Secondary Structure Effect of Polypeptide on Reverse Thermal Gelation and Degradation of l/dl-Poly(alanine)–Poloxamer–l/dl-Poly(alanine) Copolymers, Macromolecules 41, 8204-8209.
[30] Choi, Y. Y., Joo, M. K., Sohn, Y. S., and Jeong, B. (2008) Significance of secondary structure in nanostructure formation and thermosensitivity of polypeptide block copolymers, Soft Matter 4, 2383-2387.
[31] Blache, U., Metzger, S., Vallmajo-Martin, Q., Martin, I., Djonov, V., and Ehrbar, M. (2016) Dual Role of Mesenchymal Stem Cells Allows for Microvascularized Bone Tissue-Like Environments in PEG Hydrogels, Advanced Healthcare Materials 5, 489-498.
[32] Agrawal, V., and Sinha, M. (2016) A review on carrier systems for bone morphogenetic protein-2, Journal of Biomedical Materials Research Part B: Applied Biomaterials, n/a-n/a.
[33] Han, F., Yang, X., Zhao, J., Zhao, Y., and Yuan, X. (2015) Photocrosslinked layered gelatin-chitosan hydrogel with graded compositions for osteochondral defect repair, Journal of Materials Science: Materials in Medicine 26, 1-13.
[34] Ma, K., Titan, A. L., Stafford, M., Zheng, C. h., and Levenston, M. E. (2012) Variations in chondrogenesis of human bone marrow-derived mesenchymal stem cells in fibrin/alginate blended hydrogels, Acta Biomaterialia 8, 3754-3764.
[35] Goldshmid, R., Cohen, S., Shachaf, Y., Kupershmit, I., Sarig-Nadir, O., Seliktar, D., and Wechsler, R. (2015) Steric Interference of Adhesion Supports In-Vitro Chondrogenesis of Mesenchymal Stem Cells on Hydrogels for Cartilage Repair, Scientific reports 5, 12607.
[36] Jefferiss, C. D., Lee, A. J., and Ling, R. S. (1975) Thermal aspects of self-curing polymethylmethacrylate, The Journal of bone and joint surgery. British volume 57, 511-518.
[37] Dee, K. C., Andersen, T. T., and Bizios, R. (1999) Osteoblast population migration characteristics on substrates modified with immobilized adhesive peptides, Biomaterials 20, 221-227.
[38] Elluru, M., Ma, H., Hadjiargyrou, M., Hsiao, B. S., and Chu, B. (2013) Synthesis and characterization of biocompatible hydrogel using Pluronics-based block copolymers, Polymer 54, 2088-2095.