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系統識別號 U0007-2707201013155700
論文名稱(中文) 磁性冷凍對牙髓解剖構造和機械特性之影響
論文名稱(英文) Effects of Magnetic Cryopreservation on the Anatomical Structures and Mechanical Properties of Dental Pulp
校院名稱 臺北醫學大學
系所名稱(中) 牙醫學系碩博士班
系所名稱(英) School of Dentistry
學年度 98
學期 2
出版年 99
研究生(中文) 林宴莊
研究生(英文) Yen-Chuang Lin
學號 M204095010
學位類別 碩士
語文別 中文
口試日期 2010-07-06
論文頁數 84頁
口試委員 委員-鄭景暉
委員-郭宗甫
指導教授-李勝揚
共同指導教授-黃豪銘
委員-黃彥華
中文關鍵字 冷凍  磁性程式降溫儀  細胞數目 
英文關鍵字 cryopreservation  magnetic program freezer  cell number 
學科別分類
中文摘要 隨著牙醫學的進步,牙齒疾患的處理趨向保守性地保留牙齒,同時修復與再生成為牙醫領域愈發重視的課題。近年來,冷凍醫學的發展愈發成熟,利用玻璃化冷凍技術來完整保存細胞組織的活性已漸廣泛應用。由於牙齒組織中富含間質幹細胞 (mesenchymal stem cell, MSC),擁有自我更新與多元分化能力,具再生醫療應用價值,所以牙齒與相關組織的保存實具學術與臨床雙重重要意義。臨床上,智齒或因齒顎矯正需求而拔除的小臼齒等,往往成為醫療廢棄物,若能突破冷凍技術門檻而將牙齒完好保存,不僅能廢物利用而於未來解凍後再植 (autotransplantation),也有機會利用富含MSC的牙髓組織,成為組織再生工程元素。本研究比較牙齒拔下後,於不同時間點,在冷凍過程中分別利用新式磁性冷凍法以及 -200C傳統冷凍法來保存整顆老鼠門齒及人類小臼齒,而以另一側未冷凍之牙齒為對照組。牙齒的選擇,為28天齡健康大公鼠的上下門齒,其冷凍時間點為拔下後0、12、24小時;而人類則為10-35歲健康無病變且因齒顎矯正所需而拔除的小臼齒,其冷凍時間點則為0及24小時。解凍且經脫鈣處理後,將牙髓組織進行切片並以HE染色觀察,比較牙髓之解剖構造變化,同時在光學顯微鏡下計算牙本質母細胞 (odontoblast),及富含細胞層 (cell rich zone) 細胞數目的差異,另外藉由拉力測試機來分別測量牙髓組織的楊氏模數,以瞭解冷凍對牙髓結構的影響,利用t-test及 Two-Way ANOVA 作統計。結果顯示,不同冷凍法間所呈現的牙髓解剖形態變化差異不大,但在細胞數量變化上,不同冷凍法間卻有明顯不同。老鼠門齒拔下後立即處理比較,牙髓之單位面積牙本質母細胞數在不冷凍組、磁性冷凍組、傳統冷凍組分別為:130.4 ± 6.8 cells/mm2、93.1 ± 16.0 cells/mm2、74.5 ± 7.9 cells/mm2 (p<0.05 );而富含細胞層之單位面積細胞數分別為 95.2 ± 16.4 cells/mm2、74.7 ± 18.8 cells/mm2、63.7 ± 16.0 cells/mm2 (p<0.05)。在門齒拔下後12小時後實驗部分,牙本質母細胞數在不冷凍組、磁性冷凍組、傳統冷凍組分別為:86.9 ± 0 cells/mm2、71.0 ± 13.8 cells/mm2、68.3 ± 6.8 cells/mm2;而富含細胞層之單位面積細胞數分別為:75.9 ± 15.0 cells/mm2、 54.8 ± 15.9 cells/mm2、47.4 ± 13.0 cells/mm2 ( p<0.05 )。在門齒拔下後24小時後實驗部分,牙本質母細胞數在不冷凍組、磁性冷凍組、傳統冷凍組分別為:70.4 ± 6.4 cells/mm2、68.3 ± 21.5 cells/mm2、66.2 ± 7.2 cells/mm2;而富含細胞層之單位面積細胞數分別為:59.6 ± 13.4 cells/mm2、52.3 ± 19.4 cells/mm2、39.9 ± 12.0 cells/mm2。上述結果均顯示,牙本質母細胞數及富含細胞層細胞數,都是不冷凍多於磁性冷凍,而前兩者則均多於傳統冷凍;在時間方面,則是0小時多於12小時,而前兩者均多於24小時。經由二因子變異數分析 (Two-Way ANOVA),亦呈現不同冷凍方法、不同時間冷凍都有顯著差異。在人類小臼齒部份,將冷凍實驗組除以不冷凍對照組之標準化數據進行比較:牙齒拔下後立刻實驗,牙本質母細胞數之實驗組/對照組比值在磁性冷凍部分為0.97,傳統冷凍法為0.59 ± 0.32;富含細胞層部分,磁性冷凍與傳統冷凍法比值分別為0.88及0.67 ± 0.30。在牙齒拔下後24小時實驗上,牙本質母細胞數部分,磁性冷凍組為0.96 ± 0.02,傳統冷凍組為0.57 ± 0.17 ( p<0.05 );而在富含細胞層部分,磁性冷凍與傳統冷凍比值分別為0.82 ± 0.14與 0.45 ± 0.01 (p<0.05 )。在力學性質部分,人類小臼齒牙髓經拉力測試機實驗所計算出的楊氏模數,牙齒拔下0小時實驗組部分:不冷凍組為 3.1 ± 1.7 MPa,磁性冷凍組為 3.6 ± 1.9 MPa,傳統冷凍組為 3.4 ± 1.7 MPa,三組並無顯著差異。而牙齒在拔下後放在00C~40C的運送液中,經過24小時後實驗部分:不冷凍組為 3.3 ± 1.0 MPa,磁性冷凍組為 3.5 ± 1.5 MPa,傳統冷凍組為 4.7 ± 1.9 MPa,亦無顯著差異。本研究結果顯示,磁性冷凍與傳統冷凍法,對牙髓支持結構的影響並無顯著差異,亦即牙髓在硬組織的保護下經冷凍及解凍,其機械性質仍能被保護住。但細胞數的差異則顯示,磁性冷凍可以獲得較傳統冷凍更好的組織保存效益。
英文摘要 Along with the tooth medicine's progress, dental treatment tends to be conservative and tooth preservative. The repair and regeneration become topics which the dental domain increasingly takes seriously. In recent years, the development of freezing medicine was getting more and more mature. The complete preservation of cell and tissue activeness by using the vitrification technology has gradually widely applied. The mesenchymal stem cell (MSC) of pulp, having the self-renewal and multiple differential abilities, has the value of regenerative application. Therefore, the preservation of tooth and relative structures has solid academic and clinical important meanings. On clinical, the wisdom tooth and the bicuspids extracted due to orthodontic demand often become the medical rejects. If teeth can be well preserved with breaking through the freezing technology limitation, tooth which was completely preserved can be an autotransplantation, MSC uasage, or element of regeneration engineering. We preserve the rat incisors and the human bicuspids in program freezer and traditional refrigeration. Control group, not being frozen, was the opposite side . The choices of teeth are from healthy rats of age of 28 days. One of the independent variables is the period from extraction to freezing of 0, 12, and 24 hours. The choices of healthy humans of 10 to 35 years old which do not have any systemic diseases and pathological problems. And the period from extraction to freezing is 0 and 24 hours. After decalcify processing, slicing and HE dyeing, we observed the dental pulp structure changes and calculates the numbers of the odontoblastic and cell rich zone cells simultaneously under the optical
microscope. Another experiment design is elastic modules testing of pulp. Theresults were made the statistical computation by t-test and Two-Way ANOVA. Theresult showed that the difference of dental pulp structure is not great between frozen and non-frozen ones. But in the cell quantities, the difference are actually obvious. In the 0 hour experiment of rat incisors, the odontoblast numbers of non-frozen, cryopreserved by program freezer, traditional cryopreservation are 130.4 ± 6.8 , 93.1 ± 16.0 and 74.5 ± 7.9 cells/mm2 (p<0.05 ). The cell rich zone cells are 95.2 ± 16.4、74.7 ± 18.8、63.7 ± 16.0 cells/mm2 (p<0.05). In the 12 hours experiment of rat incisors, the odontoblast numbers of non-frozen, cryopreserved by program freezer, traditional cryopreservation are 86.91 ± 0、71.0 ± 13.8、68.3 ± 6.8 cells/mm2. The cell rich zone cells are 75.9 ± 15.0、54.8 ± 15.9、47.4 ± 13.0 cells/mm2 ( p<0.05 ). In the 24 hours experiment, the odontoblast numbers are 70.4 ± 6.4、68.3 ± 21.5、66.2 ± 7.2 cells/mm2. The cell rich zone cells are 59.6 ± 13.4、52.3 ± 19.4、39.9 ± 12.0 cells/mm2. It reveals that cell numbers of nonfrozen teeth are the most, followed by program freezing ones, and traditional ones. Similar results were observed in 0 hour, 12 hours, and 24 hours designs. Also, there were significant difference during the two factors of time and freezing analyzed by Two-Way ANOVA. In the experiment of human bicuspids, the difference was presented by the ratio of freezn and non-frozen teeth. In dontoblastic cells area of 0 hour group, the ratio of program freezer/control is 0.97, and traditional one is 0.59 ± 0.32. In cell rich zone of 0 hour group, the program freezing one is 0.88 compare to traditional group of 0.67 ± 0.30. In the 24 hours experiments of odontoblastic cells , the program freezer group is 0.96 ± 0.02 compare to traditional group of 0.57 ± 0.17 (p<0.05). In cell rich zone of 24 hours group , the magnetic freezing ratio is 0.82 ± 0.14 compare to traditional group of 0.45 ± 0.01 (p < 0.05).In the mechanical properties experience of human dental pulp, the module of elasticity was measure. In the 0 hour
experimental group part: Non-frozen group is 3.1 ± 1.7 MPa. The program freezer group is 3.6 ± 1.9 MPa. The traditional freezing group is 3.4 ± 1.7 MPa. The 24 hours parts are as follows. Non-frozen group is 3.3 ± 1.0 MPa. The program freezer group is 3.5 ± 1.5 MPa. The traditional freezing group is 4.7 ± 1.9 MPa. There are no difference in elastic test. Therefore, this research revealed that there are no significant difference of the elasticity of pulp. But the difference of cell numbers revealed that magnetic program freezer can be more effective than traditional one.
論文目次 致謝…………………………………………………………………Ⅰ
中文摘要……………………………………………………………Ⅲ
英文摘要……………………………………………………………Ⅶ
目錄…………………………………………………………………ⅩⅠ

第一章、 緒論
第一節 研究動機與重要性……………………………………1
第二節 研究目的………………………………………………3
第三節 研究假設………………………………………………4
第四節 名詞界定………………………………………………5

第二章、 文獻回顧與查證
第一節 冷凍醫學的演進及發展……………………………11
第二節 牙髓組織之解剖構造與機械特性…………………13
第三節 牙髓幹細胞之探討與研究…………………………14

第三章、材料與方法
第一節 廣島大學程式降溫儀………………………………17
第二節 牙齒樣本的選擇與實驗設計………………………18
第三節 牙齒切片之製作……………………………………19
第四節 牙髓組織之切片觀察及細胞計算…………………21
第五節 人類小臼齒牙髓組織之機械性質測試……………22

第四章、研究結果………………………………………………24

第五章、研究討論………………………………………………27

第六章、結論與未來展望………………………………………32

第七章、參考文獻………………………………………………33

附錄………………………………………………………………37
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