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系統識別號 U0026-2107201611355500
論文名稱(中文) 光反應核黃素應用於牙本質黏著中抑制基質金屬蛋白酶
論文名稱(英文) Application of photoreactive riboflavin in dentin bonding to inhibit matrix metalloproteinase
校院名稱 成功大學
系所名稱(中) 口腔醫學研究所
系所名稱(英) Institute of Oral Medicine
學年度 104
學期 2
出版年 105
研究生(中文) 謝蓉珮
研究生(英文) Jung-Pei Hsieh
學號 T46034036
學位類別 碩士
語文別 英文
論文頁數 61頁
口試委員 指導教授-莊淑芬
指導教授-陳玉玲
口試委員-林睿哲
口試委員-張志涵
中文關鍵字 核黃素  膠原蛋白交聯  牙本質黏著  基質金屬蛋白酶 
英文關鍵字 riboflavin  collagen crosslinking  dentin binding  matrix metalloproteinases 
學科別分類
中文摘要 牙本質與複合樹脂間建立良好的鍵結是復形治療中的重要基礎。近年來發現一種光反應劑─核黃素,搭配紫外光或藍光可增加膠原蛋白的交聯,並抑制基質金屬蛋白酶 (MMP)。本篇的研究目的是評估使用核黃素搭配藍光,是否可增加牙本質黏著層內膠原蛋白交聯,並抑制基質金屬蛋白酶,以改善牙本質黏著。
四個實驗組分別為0.1% 與1% 的核黃素照射藍光1或2分鐘,以2% 氯己定做為抑制基質金屬蛋白酶的對照組。首先以膠體電泳檢測膠原蛋白交聯的效果。為了檢測抑制基質金屬蛋白酶的反應,於四種蛋白酶選擇近似牙本質的基質金屬蛋白酶。以酶譜分析測試核黃素等處理對近似牙本質基質金屬蛋白酶的影響,並以膠體電泳檢視交聯處理後膠原蛋白抵抗基質金屬蛋白酶降解效果。另將牙本質與樹脂黏著的試片,再以微拉伸黏著強度測試、掃描式電子顯微鏡觀察銀離子微滲漏的現象、共軛焦顯微鏡觀察牙本質黏著試片中基質金屬蛋白酶表現的位置。
膠體電泳的結果顯示第一型膠原蛋白特有型態呈現三聚體、雙體及單體的位置;經核黃素處理後三聚體表現明顯增加;且0.1% 核黃素表現比1% 核黃素顯著。重組人類基質金屬蛋白酶 (recombinant MMP 2)與牙本質中的膠原蛋白酶相似因此應用於後續實驗;經核黃素處理後,其基質金屬蛋白酶表現強度降低。經基質金屬蛋白酶處理後的膠體電泳結果,1% 核黃素處理的組別降解較0.1% 明顯,顯示抑制基質金屬蛋白酶效果較低。初期的黏著強度測試中,0.1% 核黃素兩組強度最高;1% 核黃素、氯已定與控制組無明顯的差異;經降解後,所有組別的強度都降低;0.1% 核黃素比其他組強度較高;氯己定組則保持鍵結強度。掃描式電子顯微鏡的結果顯示,微滲漏發生於黏著層靠近牙本質位置,0.1% 核黃素處理組呈現較少微滲漏。共軛焦顯微鏡的結果顯示,基質金屬蛋白酶會出現在牙本質與樹脂的交界。根據這些研究結果,基質金屬蛋白酶活性出現於經黏著劑處理後的牙本質表面,光反應核黃素可增加膠原蛋白的交聯且抑制基質金屬蛋白酶。且0.1% 核黃素交聯效果與抵抗基質金屬蛋白酶降解的能力比1% 核黃素佳,並可增加黏著強度及降低黏著層的微滲漏。核黃素搭配藍光應用在牙本質黏著過程可有效改善膠原蛋白交聯與抑制蛋白酶活性,可預期於牙本質黏著劑臨床應用增強黏著強度並維持長期成功率。
英文摘要 A good bonding between composite resin and dentin is important in restorative dentistry. It has been reported that riboflavin (RF), a cross-linker reactive to ultraviolet A (UVA) or blue light (BL), could increase collagen crosslinking and inhibit matrix metalloproteinases (MMP). The purpose of this study was to use RF under BL irradiation to enhance the degree of dentinal collagen crosslinking and inhibit MMP, and thus to promote dentin bonding.
Four experimental groups were 0.1%, 1% RF combined with BL 1- and 2- mins irradiation. 2% chlorhexidine (CHX) was a control as MMP inhibition. First, degrees of collagen crosslinking were examined by gel electrophoresis. Four collagenases were examined to choose a dentinal MMP mimetic. Effect of RF treatment in inhibiting MMP was examined by gelatin zymography. The resistance of crosslinked collagen to MMP degradation was tested by gel electrophoresis. Additionally, dentin was prepared for resin adhesion. Bond strengths and stability were checked by a microtensile bond strength (μTBS) test. Nanoleakage was observed by scanning electron microscopy (SEM). In situ zymography assay in the dentin specimens was analyzed by confocal microscopy.
In the gel electrophoresis, the pattern of type I collagen showed at γ chain, β chain, α1, and α2 chain. The density of γ chain increased in all RF treatment groups, while 0.1% RF groups were more effective than 1% RF groups. The recombinant human MMP 2 was chosen as a dentinal MMP mimetic due to their similar MMP compositions. After RF treatment, MMP activity decreased. Collagen treated by 0.1% RF showed less enzymatic degradation than 1% RF did. In initial stage, 0.1% RF groups had the highest bond strength. 1% RF, CHX and control groups were not significantly different. After enzymatic degradation, bond strength of all groups decreased. 0.1% RF maintained the highest bond strength among groups. CHX maintained bond strength. Nanoleakage expression of dentin-resin interface was observed near dentin site. 0.1% RF groups showed less nanoleakage. Under in situ zymography, the MMP activity presented at dentin-resin interface. According to these findings, photoreactive RF increased collagen crosslinking, improved adhesion strength and decreased enzymatic degradation. 0.1% RF showed better crosslinking and resistance MMP degradation than 1% RF did. Application of RF-BL in bonding procedure might be practical and effective to improve collagen crosslinking and enzyme inhibition, and thus to enhance bond strength and long-term success rate.
論文目次 中文摘要 I
Abstract III
致謝 V
Content VI
List of tables IX
List of figures X
Chapter 1 Introduction 1
1.1 Principle of dentin bonding 1
1.2 Degradation of hybrid layer 2
1.3 Matrix metalloproteinases (MMPs) 5
1.3.1 MMPs degradation in hybrid layer 7
1.3.2 Application of MMP inhibitor in preserving dentin bonding 8
1.4 Dentinal collagen 10
1.4.1 Intrinsic cross-links in type I collagen 11
1.4.2 Application of extrinsic collagen crosslinkers in promoting dentin bonding 13
1.5 Motivation and objective 17
Chapter 2 Materials and Methods 18
2.1 Effect of RF-BL on collagen crosslinking (CXL) 21
2.2 Selection of dentinal MMP mimetics 24
2.2.1 Identification of dentinal MMP by gelatin zymography 25
2.2.2 Comparisons of different enzymes by gelatin zymography 26
2.3 Inhibition of MMP by RF-BL treatments 28
2.3.1 Effect of RF-BL treatment on EnzR activity 28
2.3.2 Resistance to enzymatic degradation in RF-BL treated collagen 30
2.4 Resin-dentin bond stability 32
2.4.1 Initial microtensile bond strengths test 33
2.4.2 After EnzR degradation microtensile bond strengths test 34
2.5 Effect of nanoleakage expression on bond interface 34
2.6 Observation MMP activity in hybrid layer 35
Chapter 3 Results 37
3.1 Effect of RF-BL on collagen crosslinking 37
3.2 Selection of dentinal MMP mimetics 38
3.2.1 Identification of dentinal MMP 38
3.2.2 Zymography of different enzymes 39
3.3 Inhibition of MMP by RF-BL treatments 41
3.3.1 Effect of RF-BL treatment on EnzR activity 41
3.3.2 Resistance to enzymatic degradation in RF-BL treated collagen 42
3.4 Resin-dentin bond stability 44
3.4.1 Initial microtensile bond strengths 44
3.4.2 Post-degradation microtensile bond strengths 45
3.5 Nanoleakage expression on bond interface 46
3.6 MMP activity in hybrid layer 47
Chapter 4 Discussion 49
Chapter 5 Conclusion 54
References 55
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