冠狀動脈硬化為心血管系統最常見的疾病，臨床上有數種不同的治療方法，其中最有效的就屬經皮冠狀動脈擴張術(PTCA)。但是大約有30~50﹪的病患，在PTCA手術後3至6個月內，會發生血管再狹窄的病變。血管再狹窄的原因，主要是由於PTCA手術後血管回彈(recoil)與血管內膜的過度增生(hyper-proliferation)所致。為了防止擴張後血管的回彈，PTCA搭配血管支架(stent)的置放可達到良好的效果。至於血管內膜的過度增生方面，本研究擬以生物相容性佳的膠原蛋白(collagen)，包覆抗血管內膜增生的藥物(sirolimus)並塗佈至血管支架上，進行藥物的控制釋放，以有效的抑制血管內膜的過度增生、降低血管再狹窄的機率。
本研究主要分為三部分。第一部分為膠原蛋白塗佈實驗，主要目的為尋找膠原蛋白均勻塗佈於支架上的最佳製程，並分析塗佈後其表面形態、均勻度、塗佈厚度與對支架之附著力。確定塗佈膠原蛋白之製程後，我們以天然交聯劑genipin來交聯膠原蛋白，並尋找其最適當的交聯條件，使交聯後的膠原蛋白仍能維持一定的完整性與交聯度。最後，將交聯後的collagen-coated stent進行膨脹（stent expansion）測試。第二部分為包藥釋放實驗，採多層式噴塗法（multi-layer spray coating，膠原蛋白層與sirolimus層交替噴塗），主要目的為以膠原蛋白高分子包覆抗血管內膜增生藥sirolimus，再以genipin進行交聯，製備一新型的藥物釋放型血管支架（drug-eluting stent）。最後於PBS中進行in vitro的藥物釋放實驗，探討不同包藥量（low dose ~30 mg/stent；high dose ~65 mg/stent）及包藥模式（有無阻礙層, with or without topcoat）之藥物釋放動力學情形。第三部分為血液相容性與抗酵素分解實驗，前者之目的為探討不同交聯程度及接枝肝素（heparin）的膠原蛋白之血液相容性；後者為探討不同交聯程度的膠原蛋白，其抗酵素分解的能力。
第一部分的塗佈結果顯示，於室溫下噴塗（spray coating）弱酸性之膠原蛋白溶液（pH~4），可達到最佳的膠原蛋白附著率、均勻度與塗佈後的平整性。在定量分析方面，發現膠原蛋白的噴塗量與塗佈厚度間，呈現一線性關係。此外，由原子力顯微鏡（AFM）的結果顯示，噴塗的膠原蛋白與支架表面之附著力，至少大於1500 nN（已達AFM探針的偵測極限）。為了使交聯後的膠原蛋白能維持一定的完整性與交聯度，又要避免交聯時膠原蛋白因過度吸水膨潤而變形，我們選擇在含有5﹪genipin之70﹪酒精中進行交聯。最後，我們以氣球導管擴張術將交聯後的collagen-coated stent由直徑1.5 mm膨脹至直徑2.6 mm，結果並沒有發生膠原蛋白剝落或分離的情形，進一步證實膠原蛋白與支架表面有相當好的附著力。
在第二部分的實驗裡，我們利用多層式噴塗法，以少量多層的方式分別將膠原蛋白及sirolimus噴塗至支架上，再噴塗含有5% genipin之70% 酒精溶液進行交聯。由in vitro的藥物釋放結果顯示，高劑量且沒有添加阻礙層的藥物釋放型血管支架可持續地釋放sirolimus至少達14天。此外，由低劑量支架的釋放結果來看，with topcoat的包藥模式能延緩藥物的釋放速率，降低burst release的情形發生。
第三部分血液相容性的實驗結果顯示，經肝素接枝及交聯程度達65%以上的膠原蛋白，具有較佳的血液相容性。由抗酵素分解的實驗結果顯示，隨著交聯程度的增加，膠原蛋白抗酵素分解的能力也越好。綜合以上的結果顯示，經genipin交聯處理的collagen-coated stent，很有潛力發展成一良好的藥物釋放型血管支架。

Background: Many clinical trails have shown that drug-eluting stents are effective in reducing restenosis. These stents are commonly coated with nondegradable polymers as a drug reservoir. However, as the drug is completely eluted, the permanent presence of the nondegradable polymer may induce unfavorable effects. The study was to develop a novel drug eluting stent with a biodegradable collagen coating. Preparation of this stent and its in vitro characteristics and drug release profiles are reported.
Methods: A spray coating process was developed to prepare the stent coated with collagen. The force required to remove the collagen coating from the metal stent was investigated by AFM (atomic force microscopy) and a balloon-expansion device. Collagen and sirolimus were sprayed layer-by-layer alternatively onto the external surface of the stent and subsequently crosslinked by genipin, a naturally occurring crosslinking agent. The in vitro hemocompatibility and degradability of the collagen coatings with distinct degrees of crosslinking were evaluated (n = 5). Four forms of the sirolimus-loaded stents were examined in an in vitro elution experiment: a low dose of ~30 mg per stent (0.78 mg/mm2) with or without topcoat; and a high dose of ~65 mg per stent (1.70 mg/mm2) with or without topcoat (n = 3).
Results: The results obtained in the AFM examination and the balloon expansion test demonstrated that the collagen coating adhered tightly to the stent surface. The hemocompatibility and the resistance against enzymatic degradation of the genipin-crosslinked collagen increased significantly as its degree of crosslinking increased. The results of the in vitro drug elution study showed that release of sirolimus from the stent without topcoat was able to maintain more than 14 days. The drug release study for the stent with topcoat is currently underway.
Conclusions: The aforementioned results indicated that the drug eluting stent developed in the study has a tightly adhered multi-layer collagen coating and can be used as a drug reservoir to sustain release of sirolimus.

1. Liu, M.W., Roubin, G.S., King 3d, S.B., “Restenosis after coronary angioplasty. Potential biologic determinants and role of intimal hyperplasia,” Circulation, 79, 1374-1387, 1989.
2. 許朝添, “病理學,” 第二版, 藝軒圖書出版社, 台北巿, 175-177, 2002.
3. 王宗道, 王宗德, “簡明病理學,” 合記圖書出版社, 台北市, 125-129,1990.
4.http://www.heartcenteronline.com/myheartdr/common /articles.cfm
5. 陳永泰, 陳建中, 林昇鋒等, “病理學,” 合記圖書出版社, 台北市, 362-373, 1991.
6. 王宗道, 王宗德, “簡明病理學,” 合記圖書出版社, 台北市, 148, 1990.
7. Grossman, W., “Diastolic dysfunction in congestive heart failure,” New England Journal of Medicine, 325(242), 1557-1564, 1991.
8. 張根源, “新世代冠狀動脈支架Drug-eluting stent,” 化工科技與商情, 第42期, 2003.
9. Lewis, A.L., Tolhurst, L.A., Stratford P.W., “Analysis of a phorsphorlcholine-based polymer coating on a coronary stent pre- and post-implantation,” Biomaterials, 23, 1697-1706, 2002.
10. Kuntz, R.E., Gibson, C.M., Nobuyoshi, M., Baim, D.S., “Generalized model of restenosis after conventional balloon angioplasty, stenting and directional atherectomy,” Journal of the American College of Cardiology, 21, 15-25, 1993.
11. Mintz, G.S., Popma, J.J., Pichard, A.D., “Arterial remodeling after coronary angioplasty. A serial intravascular ultrasound study,” Circulation, 94, 35-43, 1996.
12. Serruys, P.W., de Jaegere, P., Kiemeneij, F., “A comparison of balloon-expandable stent implantation with balloon angioplasty in patients with coronary artery disease,” New England Journal of Medicine, 331, 489-495, 1994.
13. Fischman, D.L., Leon, M.B., Baim, D.S., “A randomized comparison of coronary-stent placement and balloon angioplasty in the treatment of coronary artery disease,” New England Journal of Medicine, 331, 496-501, 1994.
14. Nakayama, Y., Ji-Youn, K., Nishi, S., Ueno, H., Matsuda, T., “Development of high-performance stent: gelatinous photogeo-coated stent that permits drug delivery and gene transfer,” Journal of Biomedical Materials Research, 57, 559-566, 2001.
15. Kereiakes, D.J., “How drug-eluting stents will impact the treatment of acute coronary syndromes, use of GP Iib/IIIa inhibitors, and U.S. hospitals,” Cath Lab Digest, 2, 1, 6-10, 2002.
16. http://www.biocompatibles.co.uk
17. Monhan, N., Babapulle, M.D., Mark, J., Eisenberg, M.D., “Coated stent for the prevention of restenosis: part I,” Circulation, 106, 2734-2740, 2002.
18. 葉明熙, 張原嘉, “淺談生醫材料－膠原蛋白,” 化工科技與商情, 第39期, 2002.
19. Djabourov, M., Lechaire, J.P., Gaill, C., “Structures and rheology of gelatin and collagen gels,” Biorheology, 30, 191-205, 1993.
20. Murayama, Y., Satoh, S., Oka, T., Imanishi, J., Noishiki, Y., “Reduction of the antigenicity and immunogenicity of xenografts by a new cross-linking reagent,” Trans. Am. Soc. Artif. Intern. Organss, 34, 546-549, 1988.
21. Nimni, M.E., Cheung, D., Strates, B., Odama, M.K., Sheikh, K., “Bioprosthesis derived form cross-linked and chemically modified collagenous tissues,” Nimni, M.E. ed. Collagen, Biotechnology, CRC Press, Inc., Fllorida, 1-37, 1988.
22. Tu, R., Lu, C.L., Thyagarajan, K., Wang, E., Nguyen, H., Shen, S., Hata, C., Quijano, R.C., “Kinetic study of collagen with polyepoxy fixatives,” J. Biomed. Mater. Res., 27, 3-9, 1993.
23. Trowbridge, E.A., Lawford, P.V., Crofts, C.E., “Pericardial heterografts－Why do these valves fail?” J. Thorac. Cardiovasc. Surg., 95, 577-585, 1988.
24. Okoshi, T., Noishiki, Y., Tomizawa, Y., “Development of an antithrombogenic cardiac wall substitute which can be reconstructed by infliltration of host cells,” Trans. Am. Soc. Artif. Intern. Organs., 34, 532-537, 1988.
25. Sung, H.W., Huang, R.N., Huang, L.L.H., Tsai, C.C., “In vitro evaluation of cytotoxicity of a naturally occurring crosslinking reagent for biological tissue fixation,” J. Biomater. Sci. Polymer Edn., 10, 63-78, 1999.
26. Sung, H.W., Tu, R., Shen, S.H., “Can cross-linking with a polyepoxy compound improve performance of porcine heart valves?” Asian. Cardio. & Thorac. Annals., 1, 104-109, 1993.
27. Fujikawa, S., Yokota, T., Koga, K., Kumada, S.I., “The continuous hydrolysis of geniposide to genpin using immobilized β-glucosidase on calcium alginate gel,” Biotech. Lett., 9, 697-702, 1987.
28. Sung, H.W., Huang, R.N., Huang, L.L.H., Tsai, C.C., Chiu, C.T., “Feasibility study of a natural crosslinking reagent for biological tissue fixation,” J. Biomed. Mater. Res., 42, 560-567, 1998.
29. Chang, Y., Tsai, C.C., Liang, H.C., Sung, H.W., “Reconstruction of the right ventricular outflow tract with a bovine jugular vein graft fixed with a naturally occurring agent (genipin) in a canine model,” Journal of Thoracic and Cardiovascular Surgery, 122, 1208-1218, 2001.
30. Sehgal, S.N., “Sirolimus: Its discovery, biological properties, and mechanism of action,” Transplantation Proceedings, 35（Suppl 3A）, 7S-14S, 2003.
31. Bhargava, B., Karthikeyan, G., Abizaid, A.S., Mehran, R., “New approaches to preventing restenosis,” British Medical Journal, 327, 274-279, 2003.
32. “組合式醫療器材範例－藥物釋放型血管支架（上）,” 醫療器材報導月刊, 37-40, 2003.
33. Martin, O., Christian, H., Andreas, B., Karl, R.K., “Stent-based antirestenotic coatings（Sirolimus / Paclitaxel）,” Catheterization and Cardiovascular Interventions, 55, 404-408, 2002.
34. Drachman, D.E., Edelman, E.R., Kamath, K.R., Palasis, M., Yang, D., Nott, S.H., Rogers, C., “Sustained stent-based delivery of paclitaxel arrests neointimal thickening and cell proliferation,” Circulation, 98 (suppl I), I-740, 1998.
35. Murphy, E.F., Joseph, J.L., Lu, J.R., Brewer, J., Russell, J., “The reduced adsorption of lysozyme at the phosphorylcholine incorporated polymer/aqueous solution interface studied by spectroscopic ellipsometry,” Biomaterials, 20, 1501-1511, 1999.
36. Spatz, J.P., Sheiko, S., Möller, M., “Substrate induced lateral microphase separation of a diblock copolymer,” Advanced Materials, 8, 513-517, 1996.
37. 范慧雯, “減切力誘導PS-PLLA團聯共聚合物奈米為結構定向－奈米圖案成行模板之製備,” 國立中興大學化學工程系碩士論文, 59-74, 2003.
38. Lewis, A.L., Cumming, Z.L., Goreish, H.H., Kirkwood, L.C., “Crosslinkable coatings from phosphorylcholine-based polymers,” Biomaterials, 22, 99-111, 2001.
39. Lewis, A.L., Tolhurst, L.A., Stratford, P.W., “Analysis of a phosphorylcholine-based polymer coating on a coronary stent pre- and post-implantation,” Biomaterials, 23, 1697-1706, 2002.
40. Meus, P.J., Wernly, J.A., Campbell, C.D., Takanashi, Y., Pick, R.L., Qui, Z.K., Replogle, R.L., “Long-term evaluation of pericardial substitutes,” J. Thorac. Cardiovasc. Surg., 85, 54-58, 1983.
41. Dorogosz, W. J., Lindgren, S.E., U.S. Patent 5413960, 1995.
42. Silvestro, L., Viano, I., Macario, M., Colangelo, D., Montrucchio, G., Panico, S., Fantozzi, R., “Effects of heparin and its desulfated derivatives on leukocyte-endothelial adhesion,” Seminar in Thrombosis and Hemostasis, 20, 254-258, 1994.
43. Lincoff, A.M., Furst, J.G., Ellis, S.G., Tuch, R.J., Topol, E.J., “Sustained local delivery of dexamethasone by a novel intravascular eluting stent to prevent restenosis in the porcine coronary injury model,” Journal of the American College of Cardiology, 29, 808-816, 1997.
44. Maleki, S., Graves, S., Becker, S., Horwatt, R., Hicks, D., Stroshane, R. M., Kincaid, H., “Therapeutic monitoring of sirolimus in human whole-blood samples by high-performance liquid chromatography,” Clinical Therapeutics, 22(suppl. B), B25-B37, 2000.
45. Schampaert, E., Cohen, E.A., Schluter, M., Reeves, F., Traboulsi, M., Title, L.M., Kuntz, R.E., Popma, J.J., “The Canadian study of the sirolimus-eluting stent in the treatment of patients with long de novo lesions in small native coronary arteries（C-SIRIUS）,” Journal of the American College of Cardiology, 43(6), 1-6, 2004.
46. 許哲誠, “以天然交聯劑(Genipin)交聯的生物組織材料表面性質探討,” 國立中央大學碩士論文, 46-49, 1999.
47. White, M.J., Kohno, I., Rubin, A.L., Stenzel, K.H., “Collagen films; effect of crosslinking on physical and biological properties,” Biomat. Med. Dev. Org., 1, 703-715, 1973.