評估表面固定生物分子之支架應用於骨組織工程的應用性

The Evaluation of Bio-molecules Immobilized on Scaffolds Applied Bone Tissue Engineering

賴宜慧(Yi-hui Lai)

指導教授 ： 樓靜文

中臺科技大學/健康科學院/醫學工程暨材料研究所/碩士(2010年)

摘要

組織工程之構成要素包含支架、細胞以及調控細胞生長之訊息，相關研究多致力於如何使細胞容易在支架上貼附、遷移及分化？其中，支架的表面性質將扮演十分重要的角色。本研究以明膠及氫氧基磷灰石製作成骨支架材料，並固定骨生長因子於材料表面，藉此促進幹細胞在材料上生長，進一步有效分化成骨母細胞。
本研究透過化學法與生物結合法進行骨生長因子之固定。首先，使用EDC/NHS將卵白素(avidin)共價鍵結在材料表面，再利用卵白素與生物素(biotin)的生物結合作用，使骨生長因子第二型骨形成成蛋白(type II bone morphogenetic protein,BMP-2)在不暴露在化學環境之下，穩定的固定在材料表面，保存原有的生物活性。並進行骨髓間質幹細胞種植於材料中的體外評估，使用掃描式電子顯微鏡(scanning electron microscope, SEM)評估細胞的貼附生長情況，以鹼性磷酸酉每活性分析(ALP activity assay)和骨鈣素(osteocalcin)表現量評估幹細胞分化情況。最後，以兔體頭顱骨植入實驗，體內評估固定BMP-2之支架對骨缺陷處的修復效果。
實驗結果顯示以卵白素與生物素之結合系統可有效將BMP-2固定在材料上，體外培養結果也顯示固定化之BMP-2可促進幹細胞分化為骨細胞，在體內測試也顯示將BMP-2固定在材料上可促進骨組織之修復。

關鍵字

組織工程 ； 間質幹細胞 ； 卵白素

並列摘要

Three elements of Tissue Engineering is cells, scaffolds and signals. Surface characteristizations of scaffolds play an important role to facilitate cell adhesion, migration and differentiation. In this study, we prepared gelatin and surface-modified hydroxyapatite to make bone scaffolds. The surface modification is performed by the methods of covalence and non-covalence to immobilize biomolecules on scaffolds. Firstly, avidin, as a medium, is immobilized on scaffolds surface by a chemical reaction of EDC/NHS. Then, biotin-conjugated growth factor is added and combined with immobilized avidin. BMP-2 is not only effectively immobilized on scaffolds surface, but also maintained bioreactive while it is excluded from the exposure of chemical environment.
Otherwise, an experimental evaluation was adopted for seeding mesenchymal stem cells on scaffolds. We examined the growth and attachment of the cell by SEM, and observed the differentiation of the cell by ALP assay and osteocalcin expression.We took the surgery for rabbit cranium to appraise the outcome of repair on a bone defect. The research confirms that Avidin-Biotin system can effectively immobilize BMP-2 on the scaffold; In vitro study supports that immobilized BMP-2 can promote stem cell to differentiate into bone cell; Last but not least, In vivo shows that BMP-2 immobilize on the scaffold can enhance bone repair.

並列關鍵字

tissue engineerin ； avidin ； mesenchymal stem cell

參考文獻

[4] 黃惠玲、陳坤鴻、黃昭誠：組織工程發展現況與未來趨勢。台灣醫學2001；5:375-382

[5] 董國忠：化學固定骨生長因子於磷酸鈣陶瓷作為生物活性骨替代材料之研究。私立中原大學化學系，博士論文，2002。

[1] Huether SE, McCance KL. Understanding Pathophysiology. Second edition, 2000.

[7] Leary Swan EE, Popat KC, Desai TA.. Peptide-immobilized nanoporous alumina membranes for enhanced osteoblast adhesion. Biomaterials. 2005; 26: 1969–1976.

[8] Ho MH, Wang DM, Hsieha HJ, Liu HC, Hsienc TY, Lai JY, Hou LT. Preparation and characterization of RGD-immobilized chitosan scaffolds. Biomaterials. 2005; 26: 3197–3206.