藍藻蛋白（cyanophycin granule polypepite, CGP）是一種非核糖體合成的胺基酸聚合物，基因重組菌株生產的藍藻蛋白在組成結構有所改變，除了天然藍藻蛋白含有的aspartic acid（Asp，天門冬氨酸）和arginine（Arg，精氨酸）之外，還多了lysine（Lys，離氨酸）穿插在側鏈上；透明質酸由葡萄糖醛酸（D-glucuronic acid）和N-乙醯氨基葡萄糖（N-acety-glucosamine）以β-1,3與β-1,4醣苷鍵的形式重複連接形成的直鏈生物高分子，與多數糖胺聚糖不同，透明質酸屬未硫酸化的糖胺聚醣，組成中不含硫，其相對分子量可達106量級。
藍藻蛋白與透明質酸均具備良好生物相容性以及生物降解性，兩者在組織工程的研究中各有其優勢與劣勢。藍藻蛋白在中性環境下帶正電，有助於細胞貼附與增生，卻受限於對水溶解度與機械強度，生醫材料方面的應用尚未普及；透明質酸彈性佳、親水性好、接近組織基質組成，但不利於細胞貼附，是在組織工程應用上急需克服的缺陷。故本研究利用戊二醛（glutaraldehyde）交聯反應結合藍藻蛋白與透明質酸，製備出不同比例之藍藻蛋白-透明質酸接枝支架，希望能從中找到適合的比例，綜合兩項材料的優勢，彌補劣勢，擴大應用範圍，並期待能成為組織工程研究中另一項值得深入研究的基材。
本研究先利用EDC/NHS結合藍藻蛋白與透明質酸，以TNBSA測定法推定接枝程度後，在細胞培養過程中於培養基加入藍藻蛋白-透明質酸接枝產物進行生物相容性測試。後續利用戊二醛進行交聯反應，製備出不同比例之藍藻蛋白-透明質酸接枝支架，再以掃描式電子顯微鏡（scanning electron microscope, SEM）觀察藍藻蛋白-透明質酸接枝支架之表面以及切面結構，並進行螢光染色支架測試與膨潤度測試。
實驗結果顯示，在生物相容性測試部分，相較於控制組，透明質酸可提升CHO cell增生速率達2.26倍，藍藻蛋白僅可達0.77倍，藍藻蛋白-透明質酸在HA-CGP40%的組別增生速率達到1.17倍，在不同接枝比例中有最好的增生效果。藍藻蛋白-透明質酸接枝支架在SEM觀察下表面以及切面結構並無顯著差異，但兩者皆隨著藍藻蛋白比例增加而有較大之孔洞大小和較廣之孔洞範圍分布；螢光染色結果為螢光強度隨著藍藻蛋白比例增加而有上升的趨勢；四組接枝比例在浸泡24小時後膨潤度顯著提高。
由上述結果可總結出，藍藻蛋白-透明質酸接枝支架孔洞直徑分布範圍有助於營養物質及生長因素的傳輸。同時擁有良好的保水性質，並能夠在培養基中持續維持支架型態。

Cyanophycin (CGP) and hyaluronic acid (HA) are capable of good biocompatibility and biodegradability, and there are pros and cons of both biomaterials. The structure of CGP is positive charged, favorable for cell-adhesion, under neutral environment, but the limitations of water solubility and mechanical strength result in the applications are yet widely available. HA is flexible, hydrophilic and similar to the structure of extracellular matrix, while the shortage of cell-adhesion for polysaccharide needs to be improved in tissue engineering. Therefore, we crosslinked CGP and HA with glutaraldehyde in different ratio in this study to form HA-CGP grafted scaffolds. We looked forward to combining the advantages of them to expand the applications.
In this study, we used EDC/NHS as crosslinker to produce HA-CGP conjugates in different ratio, estimating the grafted level by TNBSA assay, and the biocapabilities was determined by MTT assay. Then, the HA-CGP scaffolds were crosslinked with glutaraldehyde, observing the surface and cross-section of scaffolds with SEM, using fluorescent method and fluorescence microscopy to observe the crosslinking results, and test the swelling ratio in different points.
According to the results, the relative viability of HA and CGP are 2.26 and 0.77 times higher than control relatively, and there was the highest relative viability of HA-CGP40%, 1.17 times higher than control, of different ratio HA-CGP conjugates. In other words, compared the other ratio HA-CGP conjugates, HA-CGP40% could supply the most suitable environment for cells' proliferation. The structures of HA-CGP grafted scaffolds on surface yet differed from those on cross section significantly, and the pore size and diameter range of HA-CGP grafted scaffolds got larger with the CGP content increased. The fluorescent intensity also went up with the higher CGP content. After being immersed in 1×PBS for 24 hours, the swelling ratio of HA-CGP grafted scaffolds increased significantly.
Based on the above results, we concluded that the range of pore size and diameter of the HA-CGP grafted scaffolds is helpful for nutrients and growth factors transition, and good water retention which made the structure of scaffolds complete in medium.

1.M.M. Allen, F. Hutchison, and P.J. Weathers, Cyanophycin granule polypeptide formation and degradation in the cyanobacterium Aphanocapsa 6308. Journal of Bacteriology, 1980. 141: p. 687-693.
2.M. Obst, et al., Isolation and Characterization of Gram-Positive Cyanophycin-Degrading BacteriaKinetic Studies on Cyanophycin Depolymerase Activity in Aerobic Bacteria. Biomacromolecules, 2004. 5: p. 153-161.
3.R.D. Simon and P. Weathers, Determination of the structure of the novel polypeptide containing aspartic acid and arginine which is found in cyanobacteria. Biochimica et Biophysica Acta, 1976. 420: p. 165-176.
4.H. Mooibroek, et al., Assessment of technological options and economical feasibility for cyanophycin biopolymer and high-value amino acid production. Applied microbiology and biotechnology, 2007. 77: p. 257-267.
5.A.H. Mackerras, N.M. de Chazal, and G.D. Smith, Transient accumulations of cyanophycin in Anabaena cylindrica and Synechocystis 6308. Journal of General Microbiology, 1990. 136: p. 2057-2065.
6.M. Schwamborn, Chemical synthesis of polyaspartates: a biodegradable alternative to currently used polycarboxylate homo- and copolymers. Polymer Degradation and Stability, 1998. 59: p. 39-45.
7.K. Ziegler, et al., Molecular characterization of cyanophycin synthetase, the enzyme catalyzing the biosynthesis of the cyanobacterial reserve material multi-L-arginyl-poly-L-aspartate (cyanophycin). European Journal of Biochemistry, 1998. 254: p. 154-159.
8.G. Hannig and S.C. Makrides, Strategies for optimizing heterologous protein expression in Escherichia coli. Trends Biotechnology, 1998. 16: p. 54-60.
9.J. Kroll, S. Klinter, and A. Steinbuchel, A novel plasmid addiction system for large-scale production of cyanophycin in Escherichia coli using mineral salts medium. Applied microbiology and biotechnology, 2011. 89: p. 593-604.
10.K.M. Frey, et al., Technical-Scale Production of Cyanophycin with Recombinant Strains of Escherichia coli. Applied and Environmental Microbiology, 2002. 68: p. 3377-3384.
11.A. Steinle, K. Bergander, and A. Steinbuchel, Metabolic engineering of Saccharomyces cerevisiae for production of novel cyanophycins with an extended range of constituent amino acids. Applied and Environmental Microbiology, 2009. 75: p. 3437-3446.
12.Tseng, W.C., et al., Assessments of growth conditions on the production of cyanophycin by recombinant Escherichia coli strains expressing cyanophycin synthetase gene. Biotechnology Progress, 2012. 28: p. 358-363.
13.M. Obst and A. Steinbuchel, Microbial degradation of poly(amino acid)s. Biomacromolecules, 2004. 5: p. 1166-1176.
14.K. Neubauer, et al., Isolation of cyanophycin from tobacco and potato plants with constitutive plastidic cphATe gene expression. Journal of Biotechnology, 2012. 158: p. 50-58.
15.M. Krehenbrink, F.-B. Oppermann-Sanio, and A. Steinbüchel, Evaluation of non-cyanobacterial genome sequences for occurrence of genes encoding proteins homologous to cyanophycin synthetase and cloning of an active cyanophycin synthetase from Acinetobacter sp. strain DSM 587. Archives of Microbiology, 2002. 177: p. 371-380.
16.C.L. Yeh, et al., Effect of arginine on cellular adhesion molecule expression and leukocyte transmigration in endothelial cells stimulated by biological fluid from surgical patients. Shock, 2007. 28: p. 39-44.
17.D. Mazia, G. Schatten, and W. Sale, Adhesion of cells to surfaces coated with polylysine. Applications to electron microscopy. Journal of Cell Biology, 1975. 66: p. 198-200.
18.陳柏劭, 以藍藻蛋白修飾細菌纖維素進行細胞生長之探討, in 化學工程系. 2011, 國立臺灣科技大學: 台北市. p. 113.
19.陳陞陽, 豬軟骨細胞於藍藻蛋白-葡聚糖接枝薄膜生長之探討, in 化學工程系. 2014, 國立臺灣科技大學: 台北市. p. 214.
20.M.N. Collins and C. Birkinshaw, Hyaluronic acid based scaffolds for tissue engineering—A review. Carbohydrate Polymers, 2013. 92: p. 1262-1279.
21.J. Baier Leach, et al., Photocrosslinked hyaluronic acid hydrogels: natural, biodegradable tissue engineering scaffolds. Biotechnology and Bioengineering, 2003. 82: p. 578-589.
22.C. Zhu, D. Fan, and Y. Wang, Human-like collagen/hyaluronic acid 3D scaffolds for vascular tissue engineering. Materials Science and Engineering: C, 2014. 34: p. 393-401.
23.N. Davidenko, et al., Collagen–hyaluronic acid scaffolds for adipose tissue engineering. Acta Biomaterialia, 2010. 6: p. 3957-3968.
24.S. Yan, et al., Silk fibroin/chondroitin sulfate/hyaluronic acid ternary scaffolds for dermal tissue reconstruction. Acta Biomaterialia, 2013. 9: p. 6771-6782.
25.C. Li, et al., Structural properties of pepsin-solubilized collagen acylated by lauroyl chloride along with succinic anhydride. Materials Science and Engineering: C, 2015. 55: p. 327-334.
26.L.H.H. Olde Damink, et al., Cross-linking of dermal sheep collagen using a water-soluble carbodiimide. Biomaterials, 1996. 17: p. 765-773.
27.Z. Grabarek and J. Gergely, Zero-length crosslinking procedure with the use of active esters. Analytical Biochemistry, 1990. 185: p. 131-135.
28.J.E. Coligan, J.P. Tam, and J. Shao, Production of antipeptide antisera. Current Protocols in Neuroscience, 2001. 5: p. 5.6.1-5.6.21
29.L. Peng, B. Wang, and P. Ren, Reduction of MTT by flavonoids in the absence of cells. Colloids Surf B Biointerfaces, 2005. 45: p. 108-111.
30.T. Mosmann, Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. Journal of Immunological Methods, 1983. 65: p. 55-63.
31.J.C. Stockert, et al., MTT assay for cell viability: Intracellular localization of the formazan product is in lipid droplets. Acta Histochem, 2012. 114: p. 785-96.
32.K. Satake, et al., The spectrophotometric determination of amine, amino acid and peptide with 2,4,6-trinitrobenzene 1-sulfonic acid. Journal of Biochemistry, 1960. 47: p. 654-660.
33.K. Satake, et al., THE SPECTROPHOTOMETRIC DETERMINATION OF AMINE, AMINO ACID AND PEPTIDE WITH 2, 4, 6-TRINITROBENZENE 1-SULFONIC ACID. The Journal of Biochemistry, 1960. 47: p. 654-660.
34.R. Imani, M. Rafienia, and S.H. Emami, Synthesis and characterization of glutaraldehyde-based crosslinked gelatin as a local hemostat sponge in surgery: an in vitro study. Biomed Mater Eng, 2013. 23: p. 211-224.
35.B.E. Magun and J.W. Kelly, A new fluorescent method with phenanthrenequininone for the histochemical demonstration of arginine residue in tissues. Journal of Histochemistry & Cytochemistry. Journal of Histochemistry & Cytochemistry, 1969. 17: p. 821-827.
36.H.A. Itano, et al., Mechanism and specificity of the phenanthrenequinone test for monosubstituted guanidines. Analytical Biochemistry, 1976. 76: p. 134-141.
37.B.E. Magun and J.W. Kelly, A NEW FLUORESCENT METHOD WITH PHENANTHRENEQUINONE FOR THE HISTOCHEMICAL DEMONSTRATION OF ARGININE RESIDUES IN TISSUES. Journal of Histochemistry & Cytochemistry, 1969. 17: p. 821-827.
38.Y. Ni, et al., Tough and elastic hydrogel of hyaluronic acid and chondroitin sulfate as potential cell scaffold materials. International Journal of Biological Macromolecules, 2015. 74: p. 367-375.
39.H.S. Yoo, et al., Hyaluronic acid modified biodegradable scaffolds for cartilage tissue engineering. Biomaterials, 2005. 26: p. 1925-1933.
40.M.M. Caron, et al., Redifferentiation of dedifferentiated human articular chondrocytes: comparison of 2D and 3D cultures. Osteoarthritis Cartilage, 2012. 20: p. 1170-1178.