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Development of trachea and biphasic cartilage-bone tissue engineering
|關鍵字:||組織工程氣管;Tissue engineering trachea;聚己內酯;第二型膠原蛋白;旋轉式生物反應器;幾丁聚醣;低溫層積成形法;大氣電漿;明膠;聚麩胺酸鈉;透明質酸;poly(ε-caprolactone) (PCL);type II collagen (CII);rotational bioreactor;chitosan;Liquid-frozen deposition manufacturing (LFDM);air plasma (AP);gelatin;sodium poly (;hyaluronic acid (HA)||出版社:||化學工程學系所||引用:||Kojima K, Bonassar LJ, Roy AK, Vacanti CA, Cortiella J. Autologous tissue-engineered trachea with sheep nasal chondrocytes. J Thorac Cardiovasc Surg 2002;123:1177.  Ruszymah BH, Chua K, Latif MA, Hussein FN, Saim AB. Formation of in vivo tissue engineered human hyaline cartilage in the shape of a trachea with internal support. Int J Pediatr Otorhinolaryngol 2005;69:1489.  Wu W, Feng X, Mao T, Feng X, Ouyang HW, Zhao G., Chen F. Engineering of human tracheal tissue with collagen-enforced poly-lactic-glycolic acid non-woven mesh: A preliminary study in nude mice. Br J Oral Maxillofacial Surg 2007;45:272.  Moroni L, Curti M, Welti M, Korom S, Weder W, de Wijn JR, van Blitterswijk CA. 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本論文之第一部份為探討聚幾內酯結合第二型膠原蛋白氣管支架於體內與體外之研究，第一階段以聚己內酯結合第二型膠原蛋白所開發之新穎氣管支架，接著將軟骨細胞種入氣管支架後於裸鼠皮下進行培養，結果顯示，經8週培養可看見類似軟骨組織形成，經由組織學分析顯示，軟骨陷窩(lacunae)均勻的分布於基質中與分泌大量的基質。第二階段探討生物反應器流場對組織工程氣管軟骨生長之影響，反應器內的流體在一個5到20 rpm轉速下運作，反應器流場經由模擬軟體分析剪應力最大值與最小值分別為0.189-0.752 dyne/cm2與30.3×10-5-104×10-5 dyne/cm2，實驗結果顯示在轉速15 rpm下培養8週，軟骨細胞數增加兩倍，GAG增加170%與膠原蛋白增加240%，接著將此轉數下所培養的軟骨組織移植到兔子氣管缺陷處，兔子平均存活達52天。第三階段為增加生物相容性，PCL的內腔以明膠經梔質素交聯的水膠來進行改質，結果顯示，以梔質素交聯的明膠能抑制肉芽組織的過度生長，進而延長動物的存活率。
第二部份設計一種兩相支架(軟骨與硬骨)作為全關節的支架。在軟骨部分，探討第二型膠原蛋白混摻不同濃度之透明質酸複合支架的生物相容性，結果顯示，透明質酸可以明顯增加膠原蛋白支架的壓縮強度，並促進軟骨細胞向支架內部生長。在硬骨部分探討幾丁聚醣的薄膜與支架以大氣電漿進行改質，結果顯示經電漿處理的薄膜接觸角明顯降低與zeta電位趨向於正電，整體而言，經大氣電漿處理過的幾丁聚醣支架促進MC3T3-E1細胞向內部生長與礦物的形成可能是經由ALP與OCN的基因向上調節。此外，在生物膠部份，探討不同分子量的poly (gamma-glutamic acid)與明膠類型(type A or B)的對生物膠特性的影響，結果顯示在10%明膠(type A, 300 Bloom)與 2%gamma-PGA (880 kDa)有最強的粘著強度與最短的凝膠時間。生物膠在含有不同濃度的交聯劑下對纖維母細胞沒有毒性反應產生，在老鼠皮下植入也沒有明顯的發炎反應。
The first part of study was to develop a tissue engineering-trachea scaffold for in vitro and in vivo studies. A novel composite scaffold comprising a poly(epsilon-caprolactone) (PCL) stent and a type II collagen sponge for tissue engineering trachea was first fabricated. The chondrocytes-scaffold constructs were implanted subcutaneously in the dorsum of nude mice. The results indicated that gross appearance of the constructs revealed cartilage-like tissue at 8 weeks. Histological and biochemical analyses of the tissue engineering tracheal cartilage revealed evenly spaced lacunae embedded in the matrix, with abundant proteoglycans and type II collagen. Then a scaffold-bioreactor system was further developed for growing tissue-engineered trachea, and the effect of fluid flow on producing trachea-like neotissue was investigated. The bioreactor operated under continuous flow at a rotational speed from 5 to 20 rpm. Flow analysis showed that the maximal and minimal shear stress in the bioreactor was 0.189-0.752 dyne/cm2 and 30.3×10-5-104×10-5 dyne/cm2, respectively. Especially at 15 rpm, a two-fold increase in cell number, 170% increase in GAG, and 240% increase in collagen were found compared to static culture at 8 weeks. The constructs grown under 15 rpm was selected for implantation into tracheal defects of rabbits. The mean survival of six animals was 52 days. As a separate endeavor, the lumen of the PCL stent was modified by gelatin hydrogel crosslinked with genipin. The results showed that PCL modified by genipin crosslinked gelatin suppressed granulation tissue growth and prolonged animal survival time in comparison with the original PCL tube.
The second part of study was to develop materials for a biphasic scaffold (cartilage and bone) for the use as a osteochondral graft. In the cartilage portion, hyaluronic acid (HA) was added into collagen type II (CII) to prepare the composite scaffolds. The effect of HA on the biocompatibility of CII-HA composite scaffolds was investinged. The incorporation of HA into a CII scaffold was found to increase the compression strength. In vitro culture suggested that CII-HA scaffolds may promote the ingrowth of chondrocytes into the scaffolds due to the presence of HA. In the bony portion, chitosan precision scaffolds were fabricated. To further improve the surface properties of chitosan, chitosan films and scaffolds were treated with air plasma (AP) in this study. The results showed that the water contact angle of AP-treated films was significantly reduced and the zeta potential tended toward a more positive charge after AP treatment. Overall, the results showed that changes in surface chemistry and surface charge may account for the better cell proliferation on the treated chitosan films. AP treatment enabled the penetration of MC3T3-E1 cells into scaffolds, facilitated their proliferation and promoted the mineral deposition probably through ALP and OCN gene upregulation. Finally, the bioadhesive to integrate cartilage and bone scaffolds was developed based on poly (gamma-glutamic acid) (gamma-PGA) and gelatin. The influences of the molecular weight and the type (A or B) of gelatin, as well as the molecular weight of gamma-PGA, on the properties of gelatin/gamma-PGA mixed bioadhesives were studied. The mixture of 10% type A gelatin 300 Bloom and 2% gamma-PGA of 880 kDa showed the shortest gelation time and the greatest bonding strength. The mixed glues crosslinked with various concentrations of EDC (1.7-2.5%) showed no cytotoxicity to fibroblasts. In addition, no significant inflammatory response was observed in the rat subcutaneous implantation.
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