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研究生:林均霈
研究生(外文):Chun-Pei Lin
論文名稱:探討藥物遞送機制於牙本質小管暴露相關疾病之治療
論文名稱(外文):Investigate drug delivery mechanism in the treatment of exposed dentinal tubules related diseases
指導教授:林俊彬林俊彬引用關係
指導教授(外文):Chun-Pin Lin
口試委員:林弘萍王姻麟謝明佑
口試日期:2018-06-02
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:臨床牙醫學研究所
學門:醫藥衛生學門
學類:牙醫學類
論文種類:學術論文
論文出版年:2018
畢業學年度:106
語文別:中文
論文頁數:111
中文關鍵詞:牙本質小管抗菌藥物抗發炎藥物牙本質小管暴露相關疾病動物實驗
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牙本質小管暴露相關疾病,源自於牙齦萎縮、牙齒磨耗、深度齲齒、牙冠斷裂、牙根整平或牙冠修型等因素而使牙本質小管暴露,後續引發牙齒知覺敏感症、牙髓炎,更甚者可能因細菌入侵造成牙髓壞死,進而引發根尖膿腫發炎等疾病。若能在牙本質小管暴露初期,給予一局部藥物塗佈,包含抗菌及抗發炎之功效,深入牙本質小管進入牙髓腔,進行殺菌以及減少因為牙本質小管暴露或細菌所引發之牙髓炎,並且將暴露之牙本質小管有效地封閉與外界隔絕,或許將可以有效地解決因牙本質小管暴露所引發之相關問題,並減少後續需要根管治療的機會,亦可以降低因為根管治療失誤所衍生之牙齒穿孔、器械斷裂等相關問題。

本研究的目的為探討抗菌及抗發炎藥物滲透牙本質之機制,第一部分先藉由transwell dentin disc model體外實驗觀察並分析抗菌及抗發炎藥物滲透牙本質小管的能力,篩選出滲透能力較理想之藥物組合,進一步評估結合中孔洞鈣氧化矽材料(CCMS)後藥物滲透的情形。第二部分則是利用人類牙髓幹細胞進行各組藥物體外生物相容性測試和對牙髓幹細胞硬骨分化能力之影響,包括以alamar blue測細胞存活率、LDH測細胞毒性以及進行ALP鹼性磷酸酶和鈣沉澱定性染色,並以濾紙擴散法驗證各組藥物對S. aureus、E. faecalis、E. coli的抗菌能力。第三部分則藉由LPS和LTA誘導人類牙髓幹細胞進行發炎模式分析,偵測細胞激素TNF-α、PGE2、IL-8和IL-6釋放與基因表現量,並進一步評估抗菌及抗發炎藥物對細胞發炎模式之效用。最後以米格魯犬作為模型進行動物實驗,並以根尖x光影像及組織學切片觀察分析,評量藥物在活體牙髓發炎模式之成效。

第一部分體外實驗結果顯示:Clindamycin phosphate、Ampicillin sodium、Betamethasone sodium phosphate、Lysine acetylsalicylate、Diclofenac sodium及Ginsenoside Rg1都能有效通過牙本質試片。Hydrocortisone sodium succinate和Piroxicam僅能有少量通過牙本質試片,而Hinokitiol則是完全無法通過牙本質試片。篩選滲透較佳的藥物組合與CCMS混合使用,發現CCMS會在牙本質小管形成的結晶,反而會阻礙藥物穿透牙本質小管。因此在使用上,應先讓藥物滲透牙本質小管後再以CCMS封閉之。

第二部分以篩選出的藥物:Ampicillin sodium、Betamethasone sodium phosphate、Lysine acetylsalicylate、Diclofenac sodium及Ginsenoside Rg1進行細胞生物相容性測試,以人類牙髓幹細胞進行藥物直接接觸與間接接觸模式之alamar blue 與 LDH 生物相容性測試,結果顯示Betamethasone sodium phosphate、Lysine acetylsalicylate、Ginsenoside Rg1和Ampicillin sodium皆無明顯生物毒性,生物相容性良好,而Diclofenac sodium的生物毒性較高,生物相容性較差。此外,各組藥物對誘導牙髓幹細胞(DPSCs)硬骨分化之影響,不論是ALP鹼性磷酸酶或是鈣沉澱染色結果,Ampicillin sodium、Lysine acetylsalicylate和Ginsenoside Rg1組與控制組較無差異,但Diclofenac sodium組所染到的ALP和鈣沉澱皆較少,顯示Diclofenac sodium會影響牙髓幹細胞硬骨分化之能力。而藥物通過transwell dentin disc tube model後的抗菌測試發現:Ampicillin sodium對E. faecalis和E. coli屬高敏感型,對S. aureus屬極敏感型。Ampicillin sodium單獨使用與混合抗發炎藥物同時使用,抑菌能力無顯著差異,代表抑菌效果主要來自Ampicillin sodium而非抗發炎藥。

第三部分以LPS和LTA誘導牙髓幹細胞發炎,以LPS誘導效果較LTA為佳,在發炎激素TNF-α、IL-8、IL-6的釋放量和基因表現量都較顯著,而在PGE2的釋放量和基因表現量相較於控制組並未有顯著差異。以LPS誘導牙髓幹細胞發炎添加抗菌抗發炎藥物,Betamethasone sodium phosphate及Betamethasone sodium phosphate/Ampicilin sodium的組別能有效降低TNF-α、IL-8、IL-6的釋放量,而Ginsenoside Rg1也能降低TNF-α的釋放。動物實驗結果發現不論是控制組或實驗組,皆無法有效阻止牙髓組織的壞死,x光影像上能觀察到根尖病灶產生,組織切片中除了觀察到牙髓壞死還伴隨著細菌存在,而各組別皆能觀察到修復性牙本質的生成,但是在Betamethasone sodium phosphate/Ampicilin sodium組別能觀察到較多修復性牙本質的生成。

綜合以上結果,本研究發現能夠滲透牙本質試片並最具潛力的抗菌抗發炎藥物組合為Betamethasone sodium phosphate/Ampicilin sodium,能有效在細胞發炎實驗降低發炎激素的釋放,儘管受限於動物實驗結果,但仍具有潛力能應用在牙本質小管暴露相關疾病之治療。
Exposed dentintal tubules may cause many problems in clinical aspect, such as dentin hypersensitivity, pulp inflammation and pulp necrosis or apical abscess due to bacteria infection. In general condition, dentinal tubules were covered and protected by enamel or cementum. However, gingival recession, tooth attrition, deep caries, crown fracture, root planing and tooth preparation would let dentinal tubules exposed to oral cavity. The simuli from oral cavity would affect the pulp and evoke pulp inflammatory response which may lead to pulp necrosis.

Anti-bacterial and anti-inflammatory drugs applied to exposed dentinal tubules and infiltrated through the tubules may give a chance for pulp recovery and preserve vital pulp tissue. Thus, there is no need of root canal treatment and more tooth structure will be preserved. Besides, the risk of vertical root fracture and procedure accidents such as perforation, instrument separation will be reduced. Therefore, the purpose of this study is to investigate anti-bacterial and anti-inflammatory drugs delivery mechinism in the treatment of exposed dentinal tubules related diseases. This research comprised of three parts:

Part I:
To investigate the permeability of different anti-bacterial and anti- inflammation drugs by using the transwell dentin disc model. Drugs with better permeability, Ampicillin sodium, Betamethasone sodium phosphate (BSP), Lysine acetylsalicylate, Diclofenac sodium and Ginsenoside Rg1, were selected. However, Hinokitiol cannot pass the dentin disc. Only a little amount of Hydrocortisone sodium succinate and Piroxicam can be detected through the disc. Besides, to investigate the permeability and crystallization of the previously developed CaCO3@mesoporous silica(CCMS) with different anti-bacterial and anti-inflammation drugs through UV spectrophotometer detection. The results suggested that CCMS may block the dentinal tubules and affect the ability of penetrating dentin tubules of drugs. Therefore, to optimize the effect of anti-bacterial and anti-inflammation drugs, CCMS should be applied after the drugs penetrated through dentinal tubules.

Part II:
To investigate the cell viability and cytotoxicity of Ampicillin sodium, Betamethasone sodium phosphate, Lysine acetylsalicylate, Diclofenac sodium and Ginsenoside Rg1 through in vitro model. The cell viability of Diclofenac sodium group was extremely lower than other drugs. Moreover, Diclofenac sodium showed higher cytotoxicity than other drugs. The influence of drugs on the osteogenesis differentiation of human dental pulp cells (HDPSCs) was evaluated. The secretion of alkaline phosphatase in day 7 and day 14, only Diclofenac sodium and Diclofenac sodium/Ampicilin sodium group showed less dotted and less calcium compared to other groups. The results suggested that Diclofenac sodium may inhibit the osteogenesis differentiation of human dental pulp cells (hDPSCs). To evaluate the antibacterial ability of anti-bacterial and anti-inflammatory drugs against S. aureus, E. faecalis and E. coli.. The results showed the inhibition zone of Ampicillin sodium against S. aureus belongs to extremely sensitive type and the inhibition zone against E. faecalis and E. coli both belongs to highly sensitive type. Furthermore, there was no significant difference of antibacterial effect between Ampicillin sodium single use and Ampicillin sodium combined with anti-inflammation drugs.

Part III:
To induce inflammation, lipopolysaccharides (LPS) and lipoteichoic acid (LTA) were applied to human dental pulp cells (hDPSCs). The releasing of inflammatory cytokines and gene expression of TNF-α, PGE2, IL-8, IL-6 were detected. LPS-induced groups revealed higher secretion and gene expression of TNF-α, IL-8 and IL-6 than LTA-induced groups. However, PGE2 secretion and gene expression showd no siganificant difference in all groups. The anti-inflammatory effect of Betamethasone sodium phosphate, Lysine acetylsalicylate, Ginsenoside Rg1 and Ampicilin sodium were evaluated through cell inflamtion model. The results showed Betamethasone sodium phosphate and Betamethasone sodium phosphate/Ampicilin sodium groups with reducing secretion of TNF-α, IL-8 and IL-6. Ginsenoside Rg1 group showed lower secrtion of TNF-αcompared to the control group. Betamethasone sodium phosphate, Lysine acetylsalicylate, Ginsenoside Rg1 and Ampicilin sodium were applied to animal study. Beagle dog animal model with bacteria-induced pulpitis treated with anti-bacterial and anti-inflmmatory drugs in combination. However, the post-operative periapical x-rays showed apical lesion around the teeth in all groups. Pulp necrosis with bacteria invasion was observed in all the H&E stain slides. Reparative dentin was found in all groups under H&E stain, and more reparative dentin formation was observed in Betamethasone sodium phosphate/Ampicilin sodium group.

According to the results of this study, Betamethasone sodium phosphate/Ampicilin sodium not only can pass through dentin disc, but also can reduce the secretion of TNF-α, IL-8 and IL-6 from cell inflammatory model. Although with the restriction of animal study, Betamethasone sodium phosphate/Ampicilin sodium is still with great potentional for treating exposed dentinal tubules related disease.
誌謝 II
中文摘要 III
ABSTRACT VI
目錄 IX
圖目錄 XIII
表目錄 XVIII
第一章前言 1
第二章文獻回顧 3
2.1 保存活髓(vital pulp)於臨床牙科治療之重要性 3
2.2 活髓保存材料於活髓治療的重要性 4
2.3 現今活髓治療材料遭遇的困難與限制 5
2.4 暴露性牙本質小管相關疾病 6
2.5 牙髓牙本質複合體(pulp-dentin complex) 7
2.6 牙本質結構 8
2.7 牙本質滲透性(dentin permiablility) 9
2.7.1 牙本質滲透原理 9
2.7.2 影響牙本質通透性的因素 11
2.8 牙本質小管之離子通透性及選擇性 12
2.8.1 擴散傳送(diffusion transport) 12
2.8.2 對流傳送(convection transport) 13
2.8.3 牙齒硬組織的離子通透性 14
2.9 牙本質小管帶電情形 15
2.10抗發炎及抗菌藥物於保存活髓之重要性 15
2.11抗菌及抗發炎藥物 16
2.11.1 抗菌藥物 17
2.11.2 抗發炎藥物 18
2.11.2 具抗發炎效果之天然萃取物 19
2.12 中孔洞鈣氧化矽材料 20
第三章動機與目的 21
第四章材料與方法 22
4.1 牙本質試片滲透模型之製備 22
4.1.1 牙本質試片之製備 22
4.1.2 Transwell dentin disc tube model 之製作 22
4.1.3 Transwell dentin disc model 之製作 22
4.2 抗菌抗發炎藥物之製備 23
4.2.1 抗發炎藥物Betamethasone sodium phosphate (BSP) 之製備 23
4.2.2 抗發炎藥物Hydrocortisone sodium succinate之製備 23
4.2.3 抗發炎藥物Piroxicam之製備 23
4.2.4 抗發炎藥物Lysine acetylsalicylate之製備 24
4.2.5 抗發炎藥物Diclofenac sodium之製備 24
4.2.6 抗菌藥物Clindamycin phosphate之製備 24
4.2.7 抗菌藥物Ampicillin sodium之製備 24
4.2.8 天然萃取物Ginsenoside Rg1(GRg1)之製備 24
4.2.9 天然萃取物Hinokitiol之製備 25
4.3 抗菌及抗發炎藥物之牙本質滲透檢測 25
4.4 CCMS製劑混合抗菌抗發炎藥物之牙本質滲透檢測 26
4.4.1 CCMS製劑混合抗菌抗發炎藥物滲透測試 26
4.4.2 掃描式電子顯微鏡觀察 26
4.5 抗菌抗發炎藥物生物相容性測試 27
4.5.1 人類牙髓幹細胞(human dental pulp stem cell, hDPSCs)之初級培養(primary culture) 27
4.5.2 細胞解凍 28
4.5.3 細胞計數 28
4.5.4 細胞存活率實驗(alamar blue® cell viability assaay) 29
4.5.5 細胞毒性測試(lactate dehydrogenase, LDH) 30
4.5.6 細胞骨整合能力測試- ALP鹼性磷酸酶定性染色 31
4.5.7 細胞骨整合能力測試-鈣沉澱染色 31
4.6 抗菌性測試(antimicrobial test) -濾紙擴散法 32
4.7 抗菌抗發炎藥物細胞發炎模式測試 32
4.7.1 LPS和LTA細菌代謝產物誘導細胞發炎模式之建立 32
4.7.2 LPS和LTA對細胞活性之影響 33
4.7.3 LPS和LTA對細胞之生物毒性 33
4.7.4 LPS和LTA誘導細胞發炎偵測細胞激素的釋放量 34
4.7.5 LPS和LTA誘導細胞發炎偵測細胞激素的基因表現量 36
4.7.6 評估抗菌及抗發炎藥物對細胞發炎之治療效果 38
4.8 動物實驗 41
4.8.1 實驗動物之選擇與照顧 41
4.8.2 實驗步驟 41
4.8.3 動物灌流 43
4.8.4 標本製備 43
4.8.5 組織學切片標本製備 43
4.8.6 組織學切片評分標準 44
第五章結果 45
5.1 抗菌及抗發炎藥物之牙本質滲透檢測 45
5.1.1 抗菌藥物 45
5.1.2 抗發炎藥物 45
5.1.3 具抗發炎效果之天然萃取物 46
5.1.4 抗菌抗發炎藥物滲透牙本質試片之篩選 46
5.2 CCMS製劑混合抗菌抗發炎藥物之牙本質滲透檢測 47
5.2.1 CCMS製劑混合抗菌抗發炎藥物之牙本質滲透檢測 47
5.2.2 掃描式電子顯微鏡觀察 47
5.3 抗菌抗發炎藥物生物相容性測試 48
5.3.1 細胞存活率實驗(alamar blue® cell viability assaay) 48
5.3.2 細胞毒性測試(lactate dehydrogenase, LDH) 49
5.3.3 細胞骨整合能力測試 49
5.4 抗菌性測試(antimicrobial test) -濾紙擴散法 50
5.5 抗菌抗發炎藥物細胞發炎模式測試 51
5.5.1 LPS和LTA對細胞活性之影響 51
5.5.2 LPS和LTA之細胞毒性 52
5.5.3 LPS和LTA誘導細胞發炎偵測細胞激素的釋放量 52
5.5.4 LPS和LTA誘導細胞發炎偵測細胞激素之基因表現量 53
5.5.5 評估抗菌及抗發炎藥物對細胞發炎之治療效果 54
5.6 動物實驗 55
5.6.1 術後根尖片x光觀察 55
5.6.2 組織學切片觀察 55
第六章討論 57
6.1 抗菌抗發炎藥物滲透牙本質之探討 57
6.2 混合抗菌抗發炎藥物與CCMS對牙本質滲透之影響 58
6.3 以LPS與LTA誘導牙髓幹細胞發炎模式之影響 58
6.4 抗菌抗發炎藥物對牙髓幹細胞發炎模式之影響 59
6.5 動物實驗牙髓發炎模式及使用抗菌抗發炎藥物之探討 60
第七章結論 62
第八章未來研究方向 64
參考文獻 65
附圖 69
附表 109
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顏煒慶 (2014). 鈣磷生醫材料於牙本質小管內的離子滲透與再結晶機制之研究, 台灣大學臨床牙醫學研究所碩士論文.
陳嬿伊 (2016). 研發根管治療用生醫活性材料:結晶機制與封閉側根管及分支, 台灣大學臨床牙醫學研究所碩士論文
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