臺灣博碩士論文加值系統

　　牙周病是一種細菌誘發宿主免疫反應造成牙齒周圍支持齒槽骨破壞的一種疾病。如何恢復已喪失的牙周支持組織，一直是許多學者研究的目標。已經有許多研究證實使用引導組織再生術在牙周垂直性骨內缺損可以得到臨床上的牙周附連增加、新骨生成以及有效的囊袋降低效果。引導組織再生術的關鍵在於使用再生膜隔絕上皮與纖維組織，使牙周韌帶與骨骼組織能在穩定的缺損空間內生長。再生膜從過去的不可吸收到如今的可吸收再生膜，大大減少了使病人需要二次手術的痛苦；然而可吸收性再生膜中主要的膠原蛋白膜，常因為需要延長在體內被降解的時間而使用化學交聯，導致一定程度的細胞毒性。最近有一種新式仿生膠原薄膜，此種去細胞化組織間質薄膜由於保留原組織之結構，避開了化學交聯的步驟，具有極佳之生物相容性與操作性質。本研究旨在透過動物實驗測試此種新式仿生膠原薄膜於引導組織再生術之功效，並與市售產品進行相關之比較。
　　本實驗使用三隻混種犬，首先將犬隻的下顎雙側第三小臼齒與第一大臼齒拔除，經過10週的癒合期後，以手術的方式在實驗動物的下顎雙側第四小臼齒近心與遠心側各製備一個高5mm寬4mm的單壁骨缺損，接著將12個骨缺損平均分配到三個組別：實驗組使用新式仿生膠原薄膜（SIS）＋BioOssR；陽性對照組使用“吉士德”百歐吉（Geistlich BioGideR）可吸收性再生膜+ BioOssR；陰性對照組則不使用任何再生膜，只用BioOssR。手術當天、術後四周及八周各以皮下注射不同骨螢光標定染劑。三隻實驗動物於術後第12週犧牲，取下之標本於福馬林固定後進行後續的斷層掃描分析、組織切片判讀與測量、以及骨頭螢光標定觀察等方式分析各組材料促進牙周組織再生的程度。
　　結果的部份，三個組別所使用之材料在術後癒合皆無不良反應。組織形態測量的結果顯示，新生骨在牙根表面的高度（new bone height）在實驗組（SIS+BioOssR）和陽性對照組（BioGideR+BioOssR）相近，比陰性對照組（BioOssR only）高，但尚未達統計顯著（依序為4.80±0.98mm , 4.43±1.29mm, 3.29±0.83mm）。新生骨面積百分比（New bone area ％）的數值在三組排序由多至少分別為陽性對照組（25.90±12.22%）、實驗組（23.77±9.94％）、陰性對照組（20.32±8.59％），然而彼此間並無統計顯著差異。斷層掃瞄之結果顯示，新生骨體積百分比（new bone volume %）在實驗組和陽性對照組非常接近（分別為37.34±9.56% 與38.07±16.15％），高於陰性對照組（24.70±7.97％）較多，然而亦無統計顯著。螢光顯微鏡的部份，骨生長的方向大致上是從缺損外圍往中心生長為主，然而在各組中骨生長的方式還是略有不同，實驗組似乎在術後癒合的前期有加速骨生長的傾向。
　　整體來說，此仿生膠原薄膜合併骨移植材料之使用看似具有促進牙周再生的效果的潛力，然而因樣本數較小，統計上不易達到顯著差異，後續還需更多研究證實。另外透過骨螢光標定技術，除了呼應本實驗其他結果，也使我們進一步觀察到不同組別骨缺損中新骨生成的模式。

Background: Periodontitis is a kind of disease caused by the interaction of bacterial biofilm and host immune response, which usually leads to the breakdown of periodontal apparatus. How to regenerate the lost periodontal supporting tissue has been the primary goal of many researchers for several years. There have been many studies confirmed the use of guided tissue regeneration (GTR) in periodontal intrabony defects increases clinical attachment level, new bone formation and effectively reduces periodontal pocket. The key for guided tissue regeneration is to use a barrier membrane to exclude unwanted tissue type in the protected area. The evolution of barrier membrane from non-resorbable membranes in the past to resorbable membranes used today significantly reduced the need and pain of the second surgery after GTR. However, collagen membrane, the most used and biocompatible resorbable membrane, usually need chemical crosslinking for longer degradation time in vivo, that inevitably cause some extent of cytotoxicity. Recently, a new biomimetic membrane without chemical crosslinking has been proposed. This membrane made of decellularized extracellular matrix shows excellent biocompatibility and physical property. The primary object of this animal study was to test the efficacy of this biomimetic membrane in guided tissue regeneration. The second object is to compare that with a standard collagen membrane in the market.

Materials and Methods: The third mandibular premolars and first molars of three mongrel dogs were extracted bilaterally, and the extraction sites were allowed to heal for 10 weeks. One-wall intrabony defects were prepared bilaterally on the mesial and distal side of the fourth mandibular premolars. Twelve intrabony defects were assigned to three treatment groups: test group (SIS + BioOssR); positive control (BioGideR + BioOssR); negative control (BioOssR only). Fluorescence bone labeling was administrated subcutaneously on the day of GTR surgery, four weeks and eight weeks post-operatively. The animals were sacrificed 12 weeks after surgery. For histometric analysis, defect height, junctional epithelium migration, new cementum, new bone height, and new bone area were measured. New bone volume was measured using microcomputed tomography. Fluorescence microscopic observation was done to figure out the possible sequence of new bone formation in each group.

Result: No adverse effect was found in all three groups after GTR surgery. For new bone height, test group (4.80 ± 0.98mm) and positive control group (4.43 ± 1.29mm) showed superior results compared to negative control group (3.29 ± 0.83mm), but there is no statistical significance. New bone area (%) of the three groups was 23.77 ± 9.94% for the test group, 25.90 ± 12.22% for the positive control group, and 20.32 ± 8.59% for the negative control group. The differences between each group were not statistically significant. For new bone volume (%), analyzed from micro-CT, the result of the test group and the positive control group is very close (respectively 37.34 ± 9.56% and 38.07 ± 16.15%), higher than that of the negative control group (24.70 ± 7.97%), nevertheless, there is still no statistical significant. For fluorescence microscopy, the pattern of new bone formation usually begins from the border area of the defect toward the central part of the defect. However, the timing and the specific area of new bone formation are different in all three groups. It seems that the test group has a tendency of accelerating bone formation in the early phase of healing.

Conclusion: The combination of the biomimetic membrane with bone grafting materials seems to have the potential to promote periodontal regeneration. However, due to limited sample size in this study, there is less possibility that the result between groups achieving statistic significance. More research is needed on the regeneration potential of this new biomimetic membrane. In addition, with the fluorescence bone labeling technique, not only the results of histology and micro-CT were supported, but also can we understand the mode of new bone formation in different groups.

口試委員會審定書 I
誌謝 II
中文摘要 III
英文摘要 V
目次 VII
圖目錄 X
表目錄 XII
第一章　緒論 1
1.1前言 1
1.2研究動機與目的 2
1.3論文架構 3
第二章　文獻回顧 4
2.1組織再生的理論基礎與臨床應用 4
2.2再生膜的種類 5
2.2.1歷史沿革與分類 5
2.2.2膠原蛋白膜之來源與製備方式 6
2.3取自哺乳類動物小腸黏膜下層之去細胞化組織膜結構 8
2.3.1先前技術 8
2.3.2本實驗之新式去細胞化組織膜之製備方法與性質 9
2.4動物實驗模型之選擇 14
2.5組織切片製備與組織形態量測 16
2.6斷層掃描（Micro-CT） 16
2.7骨螢光標定 17
第三章　材料與方法 19
3.1實驗動物的選擇 19
3.2實驗目標 19
3.3實驗設計 20
3.4手術過程及術後照顧 22
3.4.1實驗動物的麻醉 22
3.4.2第一階段的手術步驟 23
3.4.3第二階段的手術步驟 24
3.4.4術後觀察及照顧 28
3.5骨螢光標定 28
3.5.1標定方法 28
3.5.2本實驗所用之螢光染劑 28
3.5.3標定步驟 29
3.6動物犧牲及標本取得 29
3.6.1動物犧牲 29
3.6.2標本之取得 30
3.7標本製作與染色 31
3.7.1標本的初步切割 31
3.7.2脫鈣標本製備 32
3.7.3磨片標本製備 34
3.8脫鈣標本觀察 35
3.8.1 組織玻片掃描、觀察與分析 35
3.8.2 新生骨所佔面積的計算方法 38
3.9放射線影像與斷層掃描 38
3.9.1放射線影像 38
3.9.2斷層掃瞄 39
3.9.3新生骨所佔體積之計算方法 39
3.10螢光顯微鏡觀察 41
第四章　實驗結果 44
4.1臨床觀察 44
4.2脫鈣組織切片分析 44
4.2.1組織學描述分析 44
4.2.2組織形態測量分析 48
4.3電腦斷層分析 51
4.4骨螢光標定 54
第五章　討論 59
5.1手術癒合情形之探討 59
5.2牙周齒槽骨再生率之探討 59
5.2.1脫鈣組織切片分析 59
5.2.2斷層掃瞄分析 61
5.2.3 組織形態分析與斷層掃瞄之相關性 62
5.3骨螢光標定之探討 62
5.4實驗設計限制之探討 68
第六章　結論 70
參考文獻 71

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