本實驗在聚乙二醇(polyethylene glycol, PEG)兩端共聚合上己內酯(ε-caprolactone)，並於其末端進行烯基化(acrylation)，使其分子量2500的高分子鏈上具有碳-碳雙鍵成為PCL-PEG-PCL-DA (PEC-DA)。此共聚物溶於水後經UV照射可形成光交聯水膠。成品和烯基化的聚乙二醇-聚乳酸(PLA-PEG-PLA-DA, PEL-DA)、烯基化的聚乙二醇(PEG-DA)兩種光交聯水膠比較。將應用這些水膠包埋臍帶血間葉幹細胞，同時誘導其分化成骨母細胞，以期達到骨生成之目的。使用三種材料10%(w/v)、20%(w/v)濃度的水膠做比較，結果顯示水膠的膨潤率隨水膠濃度增加而降低、交聯度和壓縮應力隨水膠濃度增加而增加。透過熱性質分析證實其交聯程度隨水膠濃度增加而增加。PEG-DA與PEC-DA水膠在磷酸緩衝液中的降解情形類似，28天後最多降解30%，pH最低降到6.5；PEL-DA水膠28天後最多降解40%，pH最低降到3.7。使用300 ppm的光起始劑在細胞包埋過程中能使細胞受最少影響。三種材料各濃度包埋人類間葉幹細胞的包埋率在90%以上、一天後的細胞存活率在90%以上。28天的誘導培養期間，首次觀察到細胞在PEL-DA 20%水膠中能伸展，具最佳細胞形態；細胞在PEC-DA水膠中也可伸展但較不明顯；在PEG-DA水膠中則無。包埋細胞後PEL-DA水膠降解最快，無法維持28天。各材料20%水膠內的細胞到28天後細胞存活率較10%水膠低。膠原蛋白一型、鹼性磷酸酶基因表現和細胞內鹼性磷酸酶活性的測定都可確認在各水膠中的間葉幹細胞誘導成骨母細胞。在PEC-DA 10%水膠內的細胞誘導成骨母細胞的表現最好。

In this research, polyethylene glycol (PEG) was copolymerized with ε-caprolactone and acrylated to obtain C-C double bond at both ends. The product PEC-PEG-PEC-DA(PEC-DA) was able to form hydrogel upon UV irradiation. This hydrogel was compared with those formed by PEG and PEG-PLA(PEL) as scaffolds to encapsulate umbilical mesenchymal stem cells and to induce toward osteogenesis. In the study, 10%(w/v) and 20%(w/v) hydrogels of three materials were prepared. Swelling ratio decreased as hydrogel concentration increased. Meanwhile, thermodiagrams showed that crosslinking density increased as hydrogel concentration increased. Monitoring scaffold degradation in phosphate buffer showed that PEG-DA and PEC-DA have similar weight loss about 30% on day 28 and the pH decreased to 6.5. PEL-DA hydrogel maximum weight loss was about 40% and the pH decreased to 3.7. The photoinitiator used at 300 ppm showed little impact on encapsulated cell and encapsulation rate of cell was above 90%. The relative viabilities of cell after 1 day encapsulation in hydrogels of three scaffold materials were above 90%. During 28 days induction culture, we observed cell spread within PEL-DA 20% hydrogel. Cells also spread within PEC-DA hydrogels to a less degree, but not in PEG-DA hydrogels. After cell encapsulation, PEL-DA hydrogels degrade fastest and could not last for 28 days. Cell within 20% hydrogels has less viability then 10% hydrogels of three materials. Assays for collagen type I gene expression, alkaline phosphatase (ALP) gene expression and ALP activity confirm osteogenesis. PEC-DA 10% hydrogel is the best scaffold material for cell encapsulation in this study.

1 楊婷琪. 組織工程相關產品發展及重要公司分析. (工研院IEK生醫與生活組, 2002).
2 Salgado, A. J., Coutinho, O. P. & Reis, R. L. Bone Tissue Engineering: State of the Art and Future Trends. Macromolecular Bioscience 4, 743-765 (2004).
3 Langer, R. & Vacant, J. P. Tissue engineering Science 260, 920 - 926 (1993).
4 Canalis, E., Giustina, A. & Bilezikian, J. P. Mechanisms of Anabolic Therapies for Osteoporosis. N Engl J Med 357, 905-916 (2007).
5 Kanczler, J. M. Osteogenesis and angiogenesis: the potential for engineering bone. European Cells and Materials 15, 100-114 (2008).
6 Sommerfeldt, D. W., Rubin, C. T. . Biology of bone and how it orchestrates the form and function of the skeleton. Eur Spine J 10, S86-95. (2001).
7 Pittenger, M. F. et al. Multilineage potential of adult human mesenchymal stem cells. Science 284, 143-147 (1999).
8 Aggarwal, S. & Pittenger, M. F. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood 105, 1815-1822 (2005).
9 Mack, G. S. Osiris seals billion-dollar deal with Genzyme for cell therapy. Nat Biotech 27, 106-107 (2009).
10 Tuan, R. S., Boland, G. & Tuli, R. Adult mesenchymal stem cells and cell-based tissue engineering. Arthritis Research & Therapy 5, 32-45 (2003).
11 Drury, J. L. & Mooney, D. J. Hydrogels for tissue engineering: scaffold design variables and applications. Biomaterials 24, 4337-4351 (2003).
12 Fedorovich, N. E. et al. Hydrogels as extracellular matrices for skeletal tissue engineering: state-of-the-art and novel application in organ printing. Tissue Engineering 13, 1905-1925 (2007).
13 Mano, J. F. Stimuli-Responsive Polymeric Systems for Biomedical Applications. Advanced Engineering Materials 10 (2008).
14 Sawhney, A. S., Pathak, C. P. & Hubbell, J. A. Bioerodible Hydrogels Based on Photopolymerized Poly(Ethylene Glycol)-Co-Poly(Alpha-Hydroxy Acid) Diacrylate Macromers. Macromolecules 26, 581-587 (1993).
15 Jinku Kim, T. E. H., Michael J. Yaszemski, Lichun Lu. Potential of Hydrogels Based on Poly(Ethylene Glycol) and Sebacic Acid as Orthopedic Tissue Engineering Scaffolds. Tissue Engineering Part A 15, 2299-2307 (2009).

16 Poon Y.F., C. Y., Zhu Y., Judeh Z.M., Chan-Park MB. Adition of beta-malic acid-containing poly(ethylene glycol) dimethacrylate to form biodegradable and biocompatible hydrogels. Biomacromolecules 10, 2043-2052 (2009).
17 Peppas, N. A., Hilt, J. Z., Khademhosseini, A. & Langer, R. Hydrogels in biology and medicine: From molecular principles to bionanotechnology. Advanced Materials 18, 1345-1360 (2006).
18 Benoit, D. S., Durney, A. R. & Anseth, K. S. Manipulations in hydrogel degradation behavior enhance osteoblast function and mineralized tissue formation. Tissue Engineering 12, 1663-1673 (2006).
19 Jaiswal, N., Haynesworth, S. E., Caplan, A. I. & Bruder, S. P. Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro. Journal of Cellular Biochemistry 64, 295-312 (1997).
20 Nicodemus, G. D. & Bryant, S. J. Cell Encapsulation in Biodegradable Hydrogels for Tissue Engineering Applications. Tissue Engineering Part B: Reviews 14, 149-165 (2008).
21 Bryant, S. J. & Anseth, K. S. Hydrogel properties influence ECM production by chondrocytes photoencapsulated in poly(ethylene glycol) hydrogels. Journal of Biomedical Materials Research Part A 59, 63-72 (2001).
22 Lu, S. & Anseth, K. S. Release Behavior of High Molecular Weight Solutes from Poly(ethylene glycol)-Based Degradable Networks. Macromolecules 33, 2509-2515 (2000).
23 Weber, L. M., He, J., Bradley, B., Haskins, K. & Anseth, K. S. PEG-based hydrogels as an in vitro encapsulation platform for testing controlled beta-cell microenvironments. Acta Biomaterialia 2, 1-8 (2006).
24 Bryant, S. J., Anseth, K. S., Lee, D. A. & Bader, D. L. Crosslinking density influences the morphology of chondrocytes photoencapsulated in PEG hydrogels during the application of compressive strain. Journal of Orthopaedic Research 22, 1143-1149 (2004).
25 Rice, M. A. & Anseth, K. S. Controlling cartilaginous matrix evolution in hydrogels with degradation triggered by exogenous addition of an enzyme. Tissue Engineering 13, 683-691 (2007).
26 Burdick, J. A. & Anseth, K. S. Photoencapsulation of osteoblasts in injectable RGD-modified PEG hydrogels for bone tissue engineering. Biomaterials 23, 4315-4323 (2002).
27 Gonen-Wadmany, M., Oss-Ronen, L. & Seliktar, D. Protein-polymer conjugates for forming photopolymerizable biomimetic hydrogels for tissue engineering. Biomaterials 28, 3876-3886 (2007).

28 Lieb, E. et al. Poly(D,L-lactic acid)-poly(ethylene glycol)-monomethyl ether diblock copolymers control adhesion and osteoblastic differentiation of marrow stromal cells.
29 Lieb E Fau - Tessmar, J. et al. Poly(D,L-lactic acid)-poly(ethylene glycol)-monomethyl ether diblock copolymers control adhesion and osteoblastic differentiation of marrow stromal cells.
30 Davis, K. A., Burdick, J. A. & Anseth, K. S. Photoinitiated crosslinked degradable copolymer networks for tissue engineering applications. Biomaterials 24, 2485-2495 (2003).
31 Miyasako, H., Yamamoto, K., Nakao, A. & Aoyagi, T. Preparation of Cross-Linked Poly[(e-caprolactone)-co-lactide] and Biocompatibility Studies for Tissue Engineering Materials. Macromolecular Bioscience 7, 76-83 (2007).
32 Lutolf, M. P., Raeber, G. P., Zisch, A. H., Tirelli, N. & Hubbell, J. A. Cell-Responsive Synthetic Hydrogels. Advanced Materials 15, 888-892 (2003).
33 Benoit, D. S. W., Schwartz, M. P., Durney, A. R. & Anseth, K. S. Small functional groups for controlled differentiation of hydrogel-encapsulated human mesenchymal stem cells. Nat Mater 7, 816-823 (2008).
34 Nuttelman, C. R., Tripodi, M. C. & Anseth, K. S. In vitro osteogenic differentiation of human mesenchymal stem cells photoencapsulated in PEG hydrogels. Journal of Biomedical Materials Research Part A 68A, 773-782 (2004).
35 Dikovsky, D., Bianco-Peled, H. & Seliktar, D. The effect of structural alterations of PEG-fibrinogen hydrogel scaffolds on 3-D cellular morphology and cellular migration. Biomaterials 27, 1496-1506 (2006).
36 Kloxin, A. M., Kasko, A. M., Salinas, C. N. & Anseth, K. S. Photodegradable Hydrogels for Dynamic Tuning of Physical and Chemical Properties. Science 324, 59-63 (2009).
37 Yeung, T. et al. Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion. Cell Motility and the Cytoskeleton 60, 24-34 (2005).
38 Engler, A. J., Sen, S., Sweeney, H. L. & Discher, D. E. Matrix Elasticity Directs Stem Cell Lineage Specification. Cell 126, 677-689 (2006).
39 Stein, G. S., Lian, J. B., Stein, J. L., Van Wijnen, A. J. & Montecino, M. Transcriptional control of osteoblast growth and differentiation. Physiol. Rev. 76, 593-629 (1996).

40 Lian, J. B. & Stein, G. S. Development of the osteoblast phenotype: molecular mechanisms mediating osteoblast growth and differentiation. The Iowa orthopaedic journal, 118-140 (1995).
41 Kulterer, B. et al. Gene expression profiling of human mesenchymal stem cells derived from bone marrow during expansion and osteoblast differentiation. BMC Genomics 8, 70 (2007).