指導教授：姜昱至臺灣大學：臨床牙醫學研究所黃菁菁Wong, Ching-ChingChing-ChingWong2014-11-302018-07-092014-11-302018-07-092014http://ntur.lib.ntu.edu.tw//handle/246246/264233活髓治療的目的在於治療任何可回復性的牙髓傷害，給予具生物活性與保護性之覆髓材料，並保持牙髓活性與誘導牙本質－牙髓修復再生。然而，活髓材料發展至今，在臨床上仍然沒有理想之活髓材料。目前在臨床上較常使用的活髓材料存在著操作性質差、硬化時間過長、機械性質不足或誘導牙髓–牙本質再生能力不足等缺點。為了改良並取代目前活髓材料的不足，本研究以奈米硫酸鈣和氫氧基磷灰石為基質，研發出可攜帶複方生長因子 (TGF-β1/VEGF) 之雙相生醫活髓材料。 本研究主要分成四大部分進行：第一部分為材料製成、物理性質評估與最佳化之研究。利用低溫真空法將二水硫酸鈣製成奈米級硫酸鈣，並利用化學共沉澱法合成氫氧基磷灰石，與醫療級半水硫酸鈣三種材料混合調整出最佳比例後，進行材料物理性質測試，包括以X光繞射分析材料結晶相、掃瞄式電子顯微鏡觀察材料表面結構、硬化時間、材料降解度、抗壓強度測試、以及生長因子釋放曲線分析等。第二部分為分析醫材體外細胞毒性與生物相容性；第三部分主要是分析材料誘導牙髓細胞之礦化能力，以評估其當活髓材料在誘導牙本質－牙髓組織再生中所扮演的角色。第四部分則以大鼠作為模型進行動物實驗、微電腦斷層(Micro-CT)非破壞性檢視，以及組織形態學分析。 結果顯示，雙相複合材料 nCS/HAp/CS 的初始硬化時間為 15 分鐘，符合臨床操作性質。浸泡在磷酸緩衝溶液中的複合材料 1 天的抗壓強度可維持在 7.0 MPa 左右，並在 1 週後有下降的趨勢。生長因子 (TGF-β1/VEGF) 在 14 天內可測得 60～70 %的釋放量。生物相容性測試結果顯示雙相複合材料並不會對人類牙髓細胞的增生或存活造成影響。在雙相材料中加入 6 ng/mL TGF-β1 和 0.5 ng/mL VEGF 亦可促進鹼性磷酸酶和細胞基質礦化小體的表現。而動物實驗方面，以攜帶 TGF- β1 和 VEGF 的雙相生醫材料活髓治療 4 週後之大鼠臼齒，藉由 Micro-CT 和組織切片觀察到明顯的修復性牙本質橋形成。 綜合以上結論，顯示以本實驗所研發之奈米硫酸鈣與氫氧基磷灰石雙相生醫材料攜帶 TGF- β1 和 VEGF，在操作性質、生物相容性、促進細胞礦化能力，以及誘導牙本質－牙髓修復再生之能力均不亞於市售產品之表現，故在臨床應用上對於活髓治療之發展應具有相當的潛力。To treat the reversible pulpal injury, vital pulp therapy aims to preserve pulp vitality as well as to induce dentin-pulp repair/regeneration by direct applying a bioactive material. However, after years of research and development of the capping agents, there is still a shortage of the ideal capping material for vital pulp therapy. Current pulp capping materials present several limitations, such as poor handling properties, prolonged setting time, insufficient mechanical properties, or difficulty to induce dentin-pulp complex regeneration, which in turn influences the prognosis of vital pulp therapy. Hence, the innovation of vital pulp therapy is important to overcome the limitations of current materials. The purpose of this study is to develop a biphasic biomaterial with inorganic materials, nano-calcium sulfate (nCS) and hydroxyapatite (HAp) as a bioactive pulp capping agent which is able to carry dual growth factors (TGF-β1/VEGF) for the induction of dentin-pulp complex regeneration. This study carried out in four parts-Part I: By using cryo–vacuum method, calcium sulfate dihydrate (CSD) was modified to nano-calcium sulfate hemihydrate. Hydroxyapatite was synthesized via chemical co-precipitation. We characterized crystalline phase via X-ray diffraction, and observed the microstructure by scanning electron microscope. The physical properties such as the setting time, compressive strength, in vitro degradation rate and the releasing profile of growth factors were determined while the ratio of nCS/HAp/CS was optimized. Part II: The cell-material interaction study, WST-1 and LDH assay was performed to evaluate the biocompatibility and cell cytotoxicity of nCS/HAp/CS/TGF-β1/VEGF cement. Part III: Evaluation of the cell mineralization ability of human dental pulp cell in response to the biomaterials so as to assess their roles in the pulp capping material in term of the induction of dentin-pulp complex regeneration. Part IV: An animal model of rat was established. In vivo evaluation was performed by non-destructive micro-CT determination and histological analysis. The results revealed that the initial setting time of the nCS/HAp/CS biphasic cement was 15 minutes, which exhibits substantial improvement compared with the commercial product. The compressive strength of the biphasic cement after immersion in phosphate buffered saline solution in 1 day was 7.0 MPa, and decreased after 1 week. Sustained release of TGF-β1/VEGF was achieved up to 60~70% through the biomaterial within 2 weeks. The excellent biocompatibility of the biphasic cement was also confirmed by WST-1 and LDH test, which indicates no significant difference between control group (DMEM) and experimental group (nCS/HAp/CS/TGF-β1/VEGF). Pulp cells treated with nCS/HAp/CS carrying 6 ng/mL TGF-β1 and 0.5 ng/mL VEGF showed increased level of ALP activity and formation of calcified nodules in vitro. In the animal study, the non-destructive determination with micro-CT evaluation showed hard tissue formation during 4-week measurements. Histological analysis demonstrated a prominent formation of reparative dentin bridge with nCS/HAp/CS/TGF-β1/VEGF cement after 4 weeks. Based on the present findings, in view of the advantages of the nCS/HAp/CS/TGF-β1/VEGF cement as the bioactive dressing material for vital pulp therapy, such novel compound is as competitive as the commercial products in minimally invasive treatment of dental pulp therapy and has promising prospects. We concluded that the developed nCS/HAp/CS cement could act as a potent carrier for sustained release of growth factors, and the nCS/HAp/CS/TGF-β1/VEGF cement is of great potential to promote dentin-pulp complex regeneration.謝誌……………………………………………………………………………………I 中文摘要…………………………………………………………………………….. II Abstract……………………………………………………………………………...... IV 目錄…………………………………………………………………………………VII 表目錄……………………………………………………………………………….XI 圖目錄………………………………………………………………………………XII 第一章 緒論.………………………………………………………………………….1 1.1 保存牙髓活性之重要性 ……………………………………………………1 1.1.1 牙髓之發育與構造………………………………………………….…...….1 1.1.2 保留牙髓於臨床治療的意義……………………………………….……..2 1.2 活髓治療 (Vital Pulp Therapy)………………………………………............3 1.3 現今活髓治療材料……………………………………………………………4 1.3.1 理想活髓材料之需求……………………………………………………..4 1.3.2 現今活髓材料之缺點與限制……………………………………………..5 1.3.2.1 氫氧化鈣……………………………………………………………..5 1.3.2.2 三氧礦化物…………………………………………………………..6 1.4 生長因子於活髓治療之應用潛力…………………………………………...7 1.4.1 TGF-β1 (Transforming Growth Factor-β1)………………………………..7 1.4.2 VEGF (Vascular Endothelial Growth Factor)……………………………..8 1.5 載體(Carrier)…………………………………………………………………9 1.6 雙相複合生醫陶瓷材料作為載體之選擇 …………………………………..11 1.6.1 雙相複合生醫陶瓷材料……………………………….............................11 1.6.2 氫氧基磷灰石(Hydroxyapatite)…………………………….....................12 1.6.3 半水硫酸鈣(Calcium sulfate hemihydrate; CaSO4 ‧ ½H2O)…................12 1.6.4 奈米級硫酸鈣 (Nano-calcium Sulfate)................................................... 14 1.7 雙相複合生醫材料攜帶生長因子於活髓治療之優勢..................................14 第二章 實驗動機與目的..............................................................................16 第三章 材料與方法..........................................................................................17 3.1實驗材料之製備...................................................................................17 3.1.1 奈米硫酸鈣之合成（Nano-Calcium Sulfate, nCS）................................17 3.1.2 氫氧基磷灰石顆粒之製備(Hydroxyapatite, HAp)...................................18 3.1.3 雙相生醫材料(nCS/HAp/CS)比例之最佳化.............................................19 3.2 材料性質分析..........................................................................................19 3.2.1 X光繞射分析 (X-ray diffraction, XRD).....................................................19 3.2.2 掃瞄式電子顯微鏡 (SEM) 觀察材料之顯微結構...................................19 3.2.3 硬化時間(Setting Time)..................................................................20 3.2.4 抗壓強度測試(Compressive Strength) .............................................20 3.2.5 材料降解率之測定( In Vitro Degradation Rate) ..............................21 3.2.6 生長因子之釋放測試(Releasing Profiles of Growth Factors)....................22 3.2.7 酸鹼值之評估(pH variation) .........................................................22 3.2.8 鈣離子釋放評估(Calcium Ions Releasing Profile).....................................23 3.3 體外細胞活性及生物相容性測試............................................................23 3.3.1 材料萃取液之製備..............................................................................23 3.3.2 人類牙髓細胞之初級培養..................................................................23 3.3.3 材料－細胞貼附行為之研究................................................................24 3.3.3.1 測試樣本之製備 ................................................................................24 3.3.3.2 細胞－材料共同培養 ........................................................................24 3.3.4 WST-1 細胞存活率測試(Cell Viability Assay) .................................25 3.3.5 LDH細胞毒性測試(Lactate Dehydrogenase Cytotoxicity Assay)............. 26 3.4 體外細胞礦化行為分析.........................................................................27 3.4.1 鹼性磷酸酶定性染色分析(ALP Staining Assay).......................................27 3.4.2 鹼性磷酸酶定量分析(ALP Activity Quantitative Assay)..........................29 3.4.2.1鹼性磷酸酶(ALP)活性定量 ...............................................................29 3.4.2.2 Total protein 定量 .......................................................................30 3.4.3 細胞基質礦化小體染色(Alizarin Red Staining, ARS)..............................30 3.5 動物實驗................................................................................................31 3.5.1 測試材料分組......................................................................................31 3.5.2 活髓治療之動物實驗模型................................................................32 3.5.3 微電腦斷層掃瞄(Micro-Computed Tomography, μ-CT)...................33 3.5.4 組織切片製備與分析......................................................................33 第四章 實驗結果 .....................................................................................35 4.1 材料性質分析......................................................................................35 4.1.1雙相生醫材料之 X 光繞射分析................................................................35 4.1.2 雙相生醫材料之顯微結構................................................................35 4.1.3 硬化時間測試.................................................................................36 4.1.4 抗壓強度測試
...................................................................................36 4.1.5 材料降解率之測定...........................................................................36 4.1.6 生長因子之釋放測試....................................................................37 4.1.7 酸鹼值之評估..................................................................................37 4.1.8 鈣離子釋放評估..............................................................................38 4.2 體外細胞活性及生物相容性測試..................................................................38 4.2.1 材料-細胞貼附行為之研究.......................................................................38 4.2.2 WST-1 細胞存活率測試....................................................................38 4.2.3 LDH 細胞毒性測試.............................................................................39 4.3 體外細胞礦化行為分析......................................................................39 4.3.1 鹼性磷酸酶定性染色分析..............................................................39 4.3.2 鹼性磷酸酶定量分析 ..................................................................40 4.3.3 細胞基質礦化小體染色分析.....................................................................40 4.4 動物實驗............................................................................................41 4.4.1 μ-CT 影像分析................................................................................41 4.4.2 組織切片觀察...................................................................................41 第五章 討論 ............................................................................................43 第六章 結論 .........................................................................................53 第七章 未來研究方向 ...................................................................................54 參考資料............................................................................................55 表目錄 表 4-1 材料之硬化時間測試結果............................................................70 圖目錄 圖 3-1 奈米級硫酸鈣之製備流程圖........................................................... 17 圖 3-2 氫氧基磷灰石之製備流程圖...........................................................18 圖 3-3 WST-1 檢測原理圖........................................................................ 25 圖 3-4 LDH 檢測原理圖...............................................................................26 圖 3-5 動物實驗說明............................................................................... 32 圖 4-1 奈米級硫酸鈣之X光繞射圖譜與JCPDS之半水硫酸鈣標準資料檔.....71 圖 4-2 氫氧基磷灰石之X光繞射圖譜與JCPDS之氫氧基磷灰石標準資料檔..71 圖 4-3 掃瞄式電子顯微鏡觀察奈米硫酸鈣(nCS)之顯微結構.............................72 圖 4-4 掃瞄式電子顯微鏡觀察氫氧基磷灰石(HAp)顆粒顯微結構........................73 圖 4-5 掃瞄式電子顯微鏡觀察nCS/HAp/CS試片之表面顯微結構..................74 圖 4-6 掃瞄式電子顯微鏡觀察nCS/HAp/CS試片之斷面顯微結構..................75 圖 4-7 不同時間點nCS/HAp/CS之抗壓強度及材料降解度測試結果..............76 圖 4-8 TGF-β1和VEGF於材料nCS/HAp/CS之累積釋放曲線.......................76 圖 4-9 材料浸泡於PBS中不同時間點之 pH 值變化..........................................77 圖 4-10 材料浸泡於PBS中之鈣離子累積釋放量..................................................77 圖 4-11 掃瞄式電子顯微鏡觀察細胞貼附於材料nCS/HAp/CS 表面之情形.....78 圖 4-12 掃瞄式電子顯微鏡觀察細胞貼附於材料nCS/HAp/CS/TGF-β1/VEGF表 面之情形...............................................................................79 圖 4-13 牙髓細胞於 1 天之 WST-1 測試結果.............................................80 圖 4-14 牙髓細胞於 3 天之 WST-1 測試結果.............................................80 圖 4-15 牙髓細胞於 1 天之 LDH 測試結果........................................................81 圖 4-16 牙髓細胞於 3 天之 LDH 測試結果........................................................81 圖 4-17 牙髓細胞培養 10 天後，鹼性磷酸酶定性染色結果................................82 圖 4-18 牙髓細胞於 5 天之 ALP 定量測試結果.................................................83 圖 4-19 牙髓細胞於 10 天之ALP 定量測試結果...............................................83 圖 4-20 牙髓細胞培養 18 天後，Alizarin red染色結果............................84 圖 4-21 經 CTVoxR處理μ-CT的影像分析結果...........................................85 圖 4-22 nCS/HAp/CS/TGF-β1/VEGF組於1週組織切片結果.............................86 圖 4-23 nCS/HAp/CS組於1週組織切片結果............................................87 圖 4-24 nCS/HAp/CS/TGF-β1/VEGF 組於2週組織切片結果............................88 圖 4-25 nCS/HAp/CS/TGF-β1/VEGF 組於2週組織切片結果............................89 圖 4-26 nCS/HAp/CS 組於2週及4週組織切片結果..........................................90 圖 4-27 nCS/HAp/CS/TGF-β1/VEGF組於4週組織切片結果.............................91 附圖 1 X光繞射分析儀.........................................................................92 附圖 2 臨界點乾燥機..................................................................................92 附圖 3 金屬離子覆膜機..........................................................................93 附圖 4 掃瞄式電子顯微鏡－Hitachi Scanning Electron Microscope S-2400...... 93 附圖 5 掃瞄式電子顯微鏡－Nova NanoSEM™ 30 Series, FEI, HK ...................94 附圖 6 Gilmore needle......................................................................... 94 附圖 7 萬用拉力測試機..............................................................................95 附圖 8 Multi-function water quality meter..................................................95 附圖 9 μ-CT Skyscan 1176.........................................................................96 附圖 10 光學顯微鏡－Zeiss Axiovert 200M..............................................964736035 bytesapplication/pdf論文使用權限：同意有償授權(權利金給回饋本人)活髓治療牙本質-牙髓再生奈米硫酸鈣氫氧基磷灰石生長因子thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/264233/1/ntu-103-R00422016-1.pdf