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標題: | 動物明膠/維他命C/幾丁聚醣複合材料
對於骨髓及牙髓幹細胞骨分化潛能之效應 Effects of Gelatin/Ascorbic acid/Chitosan Composite Materials on the Osteogenic Differentiation of Bone Marrow Stromal Cells and Stem Cells from Human Exfoliated Deciduous Teeth |
作者: | Ya-Hsi Hwang 黃雅希 |
指導教授: | 謝學真 |
關鍵字: | 幾丁聚醣,動物明膠,維他命C,人類乳牙牙髓幹細胞,骨髓間葉幹細胞株,骨分化,生醫材料, chitosan,gelatin,ascorbic acid,stem cells from human exfoliated deciduous teeth,bone marrow stromal cells,osteogenic differentiation,biomaterials, |
出版年 : | 2009 |
學位: | 碩士 |
摘要: | 適當的細胞來源和擴增技術,以及良好的生醫材料對於組織工程的應用而言極為重要。本研究以骨組織工程為目標,針對細胞的來源,以及促骨組織工程生醫材料進行深入研究和探討。首先,以人類骨髓間葉幹細胞株 (bone marrow mesenchymal stem cell line,BMSC) 及人類乳牙牙髓幹細胞 (stem cells from human exfoliated deciduous teeth,SHED) 探討其在體外擴增之可行性;繼而開發一種能提供細胞生長、並引導及誘導骨質新生之生醫材料,以期能使用於骨骼重建。
本研究使用幾丁聚醣作為主體之生醫材料,部份材料另外添加動物明膠及維他命C (縮寫分別為:C (幾丁聚醣)、CG (幾丁聚醣/動物明膠)、C-A (幾丁聚醣/維他命C)、CG-A (幾丁聚醣/動物明膠/維他命C) ),再進行間葉幹細胞之培養。另外,由光學顯微鏡觀察細胞形態及測定細胞DNA總量,可發現添加動物明膠於生醫材料中可以改善細胞的貼附情形,並促進細胞增生。 其次,利用即時定量聚合酶連鎖反應 (real-time polymerase chain reaction,real-time PCR) 及流式細胞分析技術 (flow cytometry) 探討骨鈣素 (osteocalcin,OCN) 基因及蛋白質表現。在不更換骨分化誘導培養基 (osteogenic medium,OM) 之情況下,在不同生醫材料表面上培養BMSC之OCN基因表現量並沒有顯著的改變。但若更換為OM,並在CG-A上培養甚至會降低OCN基因表現。而在不更換為OM時,在C-A表面上培養SHED,其OCN的基因表現與更換為OM的結果相當。在OCN之蛋白質表現方面,在更換為OM的情況下,BMSC在TCPS表面上培養其OCN-positive 細胞約佔50 %,在C表面上佔1.8 %,在CG表面上佔9.8 %,在CG-A表面上佔11.0 %。而未更換為OM時,BMSC在CG-A表面上培養其OCN-positive細胞佔20.5 %,甚至比更換為OM時更多。當更換為OM時,SHED在TCPS表面上培養其OCN-positive細胞約佔7.6 %,在CG表面上佔10.0 %。 除了OCN的表現之外,本研究另外探討鹼性磷酸酶 (alkaline phosphatase,ALP) 之活性。結果顯示,BMSC在生醫材料上培養並分化後第三天ALP之活性達到極大值,而SHED則無明顯的趨勢。 在分化後骨細胞之功能性表現方面,利用Alizarin red S染色以及測定 鈣含量。培養於CG-A表面上分化21天的BMSC鈣含量較在TCPS表面上提高1.56倍,而SHED的鈣含量則是提高了50倍。在不更換為OM的條件下,培養在C-A表面上的SHED的鈣含量與更換為OM相當。 由實驗結果來看,動物明膠能促進細胞貼附。另外,將本來存在於骨分化誘導培養基 (OM) 中之維他命C固定於生醫材料上,與維他命C存在於培養基中的效應相似。當不更換為OM的情況下,在CG-A表面上培養細胞其OCN-positive細胞佔20.5 %,甚至比更換為OM並培養於CG-A表面上之細胞比例為高。當材料中存在維他命C (C-A及CG-A表面),BMSC及SHED之ALP活性均會提高,鈣含量亦會有顯著的提升。結果顯示,本研究所開發之生醫材料,對於骨組織工程應用上,十分具有潛力。 Suitable cell sources with good expansion technique, as well as excellent biomaterials are important for tissue engineering applications. In this study, we aimed at the application of bone tissue engineering and the feasibility of in vitro expansion of bone marrow mesenchymal stem cell line (BMSC) and stem cells from human exfoliated deciduous teeth (SHED), and the development of biomaterials with osteoinductive characteristics. First of all, chitosan-based biomaterials were prepared namely, C (chitosan), CG (chitosan/gelatin), C-A (chitosan/ascorbic acid), CG-A (chitosan /gelatin/ascorbic acid)). By using inverted microscopy and measuring the DNA quantity, we found that gelatin could improve cell adhesion, and thus improve the proliferation rate. In this study, we also used real-time PCR and flow cytometry to detect the gene and protein expression of osteocalcin (OCN). Adding ascorbic acid and gelatin to chitosan biomaterials had little effect on the gene expression of OCN in BMSC. When BMSC exposed to osteogenic stimulus (OS), the gene expression of OCN in BMSC cultured on CG-A would decrease. When there was no OS, the expression of OCN in SHED cultured on C-A approximated that cultured in osteogenic medium. When exposed to OS, BMSC cultured on TCPS were 50 % OCN-positive, on C 1.8 %, on CG 9.8 %, on CG-A 11.0 %; SHED cultured on TCPS were 7.6 % OCN-positive, on CG 10.0 %. When there was no OM, the OCN-positive BMSC cultured on CG-A was 20.5 %. In addition to the expression of OCN, we also measure the activity of alkaline phosphatase (ALP). We found that the activity of ALP peaking at the 3rd day after osteogenic differentiation, while SHED didn’t have this intendency. In addition to the gene and protein expression, we also measured the function of cultured cells by using Alizarin red S staining and the content of calcium of BMSC or SHED. When BMSC cultured on CG-A under OS, the content of calcium increased to 1.56 folds of BMSC cultured on TCPS. For the case of SHED, the increment was up to 50 folds. Even if there was NS, SHED cultured on C-A still had similar amounts of calcium content. We demonstrated that the adsorption of ascorbic acid onto biomaterials had effect similar to the presence of ascorbic acid in medium. When there was NS, the OCN-positive BMSC cultured on CG-A was 20.5 %. These biomaterials (C-A and CG-A) enhanced the activity of ALP, and increased the content of calcium. Therefore, these biomaterials could be good candidates for bone-related tissue engineering applications. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/45573 |
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