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標題: | 釋放控制洛伐他汀之聚乳酸聚甘醇酸奈米顆粒於覆髓治療之應用 Controlled Release of Lovastatin in Poly(lactic-coglycolic acid) Nanoparticles for Application on Direct Pulp Capping |
作者: | Han-Ping Tu 涂瀚平 |
指導教授: | 李伯訓(Bor-Shiunn Lee) |
關鍵字: | 活髓治療,覆髓材料,聚乳酸聚甘醇酸,洛伐他汀,骨生成,生物活性材料, vital pulp therapy,pulp capping materials,PLGA,lovastatin,osteogenesis,bioactive materials, |
出版年 : | 2017 |
學位: | 碩士 |
摘要: | 活髓治療中使用具生物相容性的材料覆蓋於暴露牙髓尖端,透過材料特性給予患部治療、修復,避免牙髓進一步的感染與破壞,保存其活性。適當的修復材料可促使細胞修復、和緩軟組織發炎情況、協助硬組織形成。MTA 具良好生物相容性,能有效刺激修復型牙本質生成,封閉性能佳且生物活性強。然而其硬化時間長、操作性質差的缺點卻會影響牙本質橋再生能力下降。本研究以生物可降解性材料- 聚乳酸聚甘醇酸[Poly(lactic-co-glycolc acid), PLGA]做為包覆奈米載體,攜帶洛伐他汀(Lovastatin)進行緩慢穩定藥物釋放,大幅降低藥物細胞毒性,提升生物相容強度。先前實驗已證實此奈米顆粒具有良好的釋放效率和促使細胞骨分化的能力,本研究進一步將此載體系統試驗於人類初代牙髓細胞。
本研究分為三部分:(一)材料製備:使用雙乳化製備法,參考先前研究團隊尋找出的最佳PLGA 和洛伐他汀配置比例製成。以動態粒徑分析儀(Dynamic Light Scattering)檢測粒徑大小,並使用穿透式電子顯微鏡(TEM)觀察顆粒大小一致性、型態與分散性。在0 至150 天時,使用可見光紫外光分光光譜儀(UVvis)定量洛伐他汀包覆釋放效率。(二)體外生物相容性分析與骨分化能力試驗:此系列實驗使用初代培養之人類牙髓細胞,將實驗分為六個組別,觀察藥物包覆前和包覆後,以及不同濃度下的細胞反應。經過細胞活性檢測(MTT assay),了解細胞對此奈米顆粒的適應性。另外,由鹼性磷酸酶(ALPase)活性測試評估該藥物促進骨分化進行的能力。於分子生物面向,使用定量即時聚合酶鏈鎖反應(Realtime PCR)觀察其對骨分化過程相關基因-DSPP、DMP1、OCN 表現量之影響; 西方墨點法(Western blot)和酵素結合免疫吸附分析法(ELISA)分別用以分析DSPP、DMP1 和OCN 蛋白質生成量。(三)動物模型建立:以大鼠為試驗樣本進行實驗,透過組織解剖學分析硬組織和軟組織的形態變化。 研究結果顯示本PLGA 包覆洛伐他汀奈米聚合物平均粒徑約為174.8 nm。藥物(1 mg/ml)控制釋放可穩定緩速釋放,於第44 天時釋放量達22.54 μg/mg,並持續釋放至第150 天。從細胞活性試驗及骨分化能力試驗中皆發現該奈米顆粒在濃度100 μg/ml 情況下具有最佳生物相容性與促進骨分化進行的潛能,並且鹼性磷酸酶於第9 天和第13 天時明顯上升,骨分化過程參與之基因與蛋白質表現量亦於第14、21 天時呈現顯著成長趨勢。動物實驗組織切片染色觀察以PLGA 製成藥物緩釋洛伐他汀奈米顆粒作為覆髓材料,在第二週與第四週皆出現明顯修復性牙本質生成。 綜合以上結論,本研究研發聚乳酸聚甘醇酸攜帶洛伐他汀之奈米釋放系統應用於牙髓細胞與活髓治療上,在生物相容性、促進細胞鈣化能力,及誘導牙本質橋修復之能力均有良好表現,作為改善臨床應用之材料具有其相當之發展性與潛力。 An ideal pulp capping material with function of maintaining pulp vitality has yet to be developed. The material has to be a biocompatible material that can provide dental tissue repair and recovery without further infection and damage. In addition, it is expected to stimulate cellular repair and to promote formation of stable reparative dentin bridge. MTA has good biocompatibility, does not have the microleakage problems of calcium hydroxide, and can promote generation of reparative dentin. However, drawbacks such as long setting time and poor operating convenience could influence the ability of dentin bridge regeneration. Our previous study has developed a delivery system by encapsulating lovastatin into poly(latic-co-glycolic acid) (PLGA) nanoparticles. The PLGA nanoparticles exhibit high release efficiency and ability to promote osteogenesis. The aim of the present research is to investigate the effects of lovastatin carried in PLGA nanoparticles on human dental pulp cells. This study was carried out in three parts. Part I: PLGA-lovastatin nanoparticles were prepared using double emulsion method. The nanoparticle size and characterization were observed by dynamic light scattering and TEM. A UV-Vis spectrophotometer was used to quantify the lovastatin release from 0 to 150 days at 248 nm. Part II: Six groups were designed for in vitro assay of cell reaction. The control group received no treatment. Three concentrations (50, 100, and 200 μg/ml) of PLGA-lovastatin nanoparticles and two concentration (1, 2 μg/ml) of free lovastatin were used to evaluate the cytotoxicity to primary culture of human dental pulp cells (permission from the Ethical Committee of National Taiwan University Hospital with case no. 201105080RC) by 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Alkaline phosphatase (ALPase) activity was measured using a Micro BCA protein assay kit. The osteogenesis gene and protein expression- DMP1 (Dentin matrix acidic phosphoprotein 1), OCN (Osteocalcin) and DSPP (Dentin Sialophosphoprotein) were examined in realtime PCR assay, western, and ELISA. Part III: An animal model was established. In vivo study was determined by histological analysis. The results demonstrated that PLGA-lovastatin nanoparticles have the median diameter approximately in 174.8 nm. Nanoparticles with 1 mg/ml concentration could control the release of 22.54 μg/mg lovastatin within 44 days, and continue released toward 150 days. In cell reaction assay revealed that PLGA-lovastatin nanoparticles (100 μg/ml) had the ability of inducing osteogenesis. Histological analysis illustrated reparative dentin formation with controlled-release of lovastatin by PLGA nanoparticles after 2 and 4 weeks. The PLGA-lovastatin nanoparticles with concentration of 100 μg/ml demonstrated the best biocompatibility, ALPase activity, well ability to promote human dental pulp cells osteogenesis and reparative dentin formation. These results suggest that PLGA-lovastatin nanoparticles have great potential for clinical application on direct pulp capping. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/59331 |
DOI: | 10.6342/NTU201701110 |
全文授權: | 有償授權 |
顯示於系所單位: | 口腔生物科學研究所 |
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