不同矽含量生物活性玻璃對細胞毒性與生物活性影響之研究

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研究生：	藺莛恩 Ting-An Lin
論文名稱：	不同矽含量生物活性玻璃對細胞毒性與生物活性影響之研究 Influence of Si concentration on cytotoxicity and bioactivity of spray dried bioactive glass
指導教授：	施劭儒 Shao-Ju Shih
口試委員:	王丞浩 Chen-Hao Wang 鍾仁傑 Ren-Jie Chung 吳孟晃 Meng-Huang Wu
學位類別：	碩士 Master
系所名稱：	工程學院 - 材料科學與工程系 Department of Materials Science and Engineering
論文出版年：	2018
畢業學年度：	106
語文別：	中文
論文頁數：	95
中文關鍵詞：	生物活性玻璃、噴霧乾燥法、生物活性、細胞毒性
外文關鍵詞：	bioactive glass, spray dry, bioactivity, cytotoxicity
相關次數：	點閱：236 下載：10
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上一筆

隨著高齡化社會的來臨，老年人普遍有骨質疏鬆的問題，一旦發生骨折意外將不易修復，因此生物活性玻璃(Bioactive glass, BG) 因為具有良好的生物活性與骨傳導性，而受到許多關注。BG的製程有許多種，例如:傳統玻璃製程、溶膠-凝膠法、噴霧熱解法等等，但是傳統玻璃製程的純度不高，溶膠-凝膠法製程時間過長，而噴霧熱解法製程的粒徑不易控制。噴霧乾燥法(Spray dry, SD)法式一個較為新穎的生物活性玻璃製程，此法提供了一個純度高且能夠量產(i.e. 1kg/h)的方法。實驗中成功製備Si-Ca-P系統的生物活性玻璃，依照SiO2的含量分為58S、68S與76S的粉體及燒結後的塊材。
利用X光繞射技術(XRD)鑑定其相結構，使用電子顯微鏡(SEM)觀察其表面形貌。在體外測試中，將BG浸泡於人體模擬體液十二小時後，測量其pH值變化，並利用XRD與SEM測量其生物活性，最後進行MTT測試其細胞毒性。由實驗結果發現，對於生物活性(相同時間的HA生成量)而言，58S最佳，68S其次，而76S生物活性最差；但是對於細胞毒性而言，卻是76S細胞毒性最低，58S細胞毒性最高的結果。細胞毒性主要是因為過量的鈣離子釋放，造成細胞死亡，因此若將粉體燒結成塊材，鈣離子釋放較慢，使其細胞毒性降低。

The problem of the aging society is becoming more and more obvious. The most serious problem is the issue of osteoporosis associated with population aging. Once the bone fracture accident happens, it will not be easy to repair for the elderly people. Thus, bioactive glass (BG) has attracted much attention due to its excellent bioactivity and osteoconductivity properties. There are many kinds of processes to synthesize the BG, such as traditional glass process, sol-gel method, and spray pyrolysis. However, the BG powder in the traditional glass process has the problem of the low purity; in the sol-gel method need a long time to compose the BG powder; it is difficult to control the particle size using spray pyrolysis. For the Spray Dry (SD) method, which is a newly developed bioactive glass process that can provide a high purity and mass production (i.e. 1kg/h).

In this research, the powders and bulks of the Si-Ca-P system bioactive glass, 58S, 68S and 76S were successfully prepared by SD. The phase structure was characterized by X-ray diffraction (XRD). The surface morphology was observed using an electron microscope (SEM). The in-vitro bioactive tests were measured by XRD and SEM, and finally, the cytotoxicity was tested by MTT. The results indicated that 58S is the best sample for the bioactivity test (HA growth in the same soaking time) followed by 68S and 76S. However, the results of the cytotoxicity test are different from the bioactivity test, it is shown that 76S is the least cytotoxic in all the sample. In addition, 58S is the most cytotoxic sample due to a lot of the Ca2+ ions content in the 58S. The excess of Ca2+ ions release leads to the cell apoptosis. In order to overcome this problem to decrease the cytotoxicity, sintering the powder into a bulk can reduce the release rate of Ca2+ ions.

摘要    I
Abstract    II
致謝    II
目錄    V
圖目錄    IX
表目錄    XI
第一章、緒論    1
1.1研究背景    1
1.2研究動機    2
第二章 文獻回顧    3
2.1 骨骼    3
2.1.1 骨骼的成分    3
2.1.2 骨骼的構造    4
2.1.3 骨骼組織內細胞    6
2.1.4 骨折    7
2.1.5 骨折癒合    9
2.2 生醫材料    11
2.2.1 生醫材料簡介    11
2.2.2 生醫材料檢測    13
2.2.3 生醫陶瓷材料    15
2.2.4 生物活性玻璃    18
2.2.5 生物活性    20
2.3 細胞毒性    22
2.3.1 ATP染色法    22
2.3.2 LDH法    23
2.3.3 MTT法    24
2.4 噴霧乾燥法介紹    25
2.4.1 生物活性玻璃製程概述    25
2.4.2 噴霧乾燥法簡介    26
2.4.3 前驅物溶液的配製    27
2.4.4 影響粉體形貌與大小之機制    29
第三章、實驗方法    33
3.1實驗設計    33
3.2實驗藥品與儀器    37
3.2.1 X光繞射儀    39
3.2.2 聚焦型離子束顯微鏡    40
3.2.3 酸鹼度與體外生物活性測試    42
3.2.4 體外生物相容性測試    42
3.2.5 熱機械分析    44
第四章、實驗結果    45
4.1 市售商品Nova Bone分析    45
4.1.1 Nova Bone之相結構分析    45
4.1.2 Nova Bone之SEM圖    46
4.1.3 Nova Bone之pH變化圖    47
4.1.4 Nova Bone之生物活性測試    48
4.1.5 Nova Bone之細胞毒性測試    49
4.2 生物活性玻璃粉體分析    50
4.2.1 未酸洗生物活性玻璃之相結構分析    50
4.2.2 酸洗後生物活性玻璃之相結構分析    51
4.2.3 未酸洗生物活性玻璃之SEM圖    52
4.2.4 酸洗後生物活性玻璃之SEM圖    53
4.2.5 未酸洗與未酸洗生物活性玻璃之酸鹼度測試    54
4.2.6 未酸洗與酸洗後生物活性玻璃之細胞毒性比較    56
4.2.7 生物活性玻璃之比表面積量測    57
4.2.8 生物活性玻璃之生物活性試驗    58
4.2.9 生物活性玻璃之細胞毒性試驗    60
4.3生物活性玻璃塊材之分析    62
4.3.1 生物活性玻璃之熱機械分析    62
4.3.2 生物活性玻璃塊材之相結構圖    64
4.3.3 生物活性玻璃塊材之SEM圖    65
4.3.4 生物活性玻璃塊材之生物活性    66
4.3.5 生物活性玻璃塊材之細胞毒性    68
第五章、實驗討論    69
5.1 Nova Bone產品細胞毒性與生物活性之探討    69
5.2 pH值與生物活性玻璃之體外測試結果的關聯    70
5.2.1 pH值與生物活性和細胞毒性之關聯    70
5.2.2 生物活性玻璃之細胞毒性成因    72
5.3 生物活性玻璃粉體與塊材之細胞毒性比較    74
第六章、結論    76
第七章、未來工作    77
第八章、參考文獻    78

 
圖目錄
Figure 1.1 The diagram of hip fracture    1
Figure 2.2 The mechanism of absorption by osteoclasts    7
Figure 2.3 The different tissues involved in bone regeneration are shown above with the two important revascularization steps during tissue development. In the lower line the 5 consecutive phases of bone healing are depicted    10
Figure 2.4 Raman spectra of PLGA/Bioglass1 composite samples (100/50) as function of incubation time in PBS for (a) 0 days, (b) 7 days, (c)35 days. The formation of HA is indicated by the P-O peaks at 962cm-1    21
Figure 2.5 General spray-drying equipment configuration    26
Figure 2.6 Dependence of water concentration on reaction time during hydrolysis of TEOS catalysed by strong acids.    28
Figure 2.7 Dependence of water concentration on reaction time and pH’ during TEOS hydrolysis.    28
Figure 2.8 Various particle morphologies prepared using the spray-drying method    29
Figure 2.9 The effect of flow rate on the stability of droplets    31
Figure 2.10 Proposed particle formation process for high Peclet numbers    32
Figure 3.1 The experimental flow chart of Nova Bone analysis    34
Figure 3.2 The experimental flow chart of 58S,68S and 76S bioactive glass powder    35
Figure 3.3 The experimental flow chart of 58S,68S and 76S bioactive glass bulk    36
Figure 3.4 Diffraction by an array of point scatterers may be considered as Bragg reflection from planar arrays    40
Figure 3.5 The Schematic of TMA    44
Figure 4.1 XRD pattern of Nova Bone    45
Figure 4.2 SEM image of Nova Bone    46
Figure 4.3 The pH values as the function of soaking time of Nova Bone    47
Figure 4.4 The XRD patterns of Nova Bone soaked in SBF for 24h    48
Figure 4.5 The SEM image of Nova Bone soaked in SBF for 24h    48
Figure 4.6 The cell viability as a function of extract concentration of Nova bone powder and bulk    49
Figure 4.7 XRD patterns of 58S, 68S, and 76S as-received bioactive glass powders    50
Figure 4.8 XRD patterns of 58S, 68S, and 76S bioactive  glass powders after HNO3- washing    51
Figure 4.9 SEM images of (a) 58S, (b) 68S, and (c)76S as-received bioactive glass powders calcined at 600oC for 1h.    52
Figure 4.10 SEM images of nitric acid washed (a) 58S, (b) 68S, and (c) 76S bioactive glass powders calcined at 600oC for 1h.    53
Figure 4.11 The pH values as the function of soaking time of    54
(a)    as-received and (b) HNO3- washed bioactive glass powders    54
Figure 4.12 The cell viability as a function of SiO2 concentration for as-received and HNO3- washed bioactive glass powders    56
Figure 4.13 Specific surface areas as a function of SiO2 concentration of bioactive glasses    57
Figure 4.14 XRD patterns of 58S, 68S and 76S bioactive glass powders soaked in SBF for 24h    58
Figure 4.15 SEM images of (a) 58S, (b) 68S, and (c) 76S bioactive glass powders soaked in SBF for 24h    59
Figure 4.16 The cell viability as a function of extract concentration of bioactive glass powders    60
Figure 4.17 Percent shrinkage as a function of temperature of 58S, 68S and 76S bioactive glass powders    62
Figure 4.18 Percent shrinkage rate a function of temperature of 58S, 68S and 76S bioactive glass powders    63
Figure 4.19 XRD patterns of 58S, 68S, and 76S bioactive glass bulks sintered at 800oC for 1h    64
Figure 4.20 SEM images of (a) 58, (b) 68, and (c) 76S bioactive glass bulks sintered at 800oC for 1h    65
Figure 4.21 XRD patterns of 58S, 68S and 76S bioactive glass bulks soaked in SBF for 24h    66
Figure 4.22 SEM images of (a) 58S, (b) 68S, and (c) 76S bioactive glass bulks soaked in SBF for 24h    66
Figure 4.23 The cell viability as a function of extract concentration of bioactive glass bulks    68
Figure 5.1 HA integrate intensity and cell viability as a function of SiO2 concentration of bioactive glass powders    70
Figure 5.2 The cell viability of bioactive glasses added with the Ca2+ inhibitor    73
Figure 5.3 The cell viability as a function of SiO2 concentration of bioactive glass powders and bulks    75

表目錄
Table 2.1 Description of the Gustilo Classification System of Open Fracture    8
Table 2.2 The table of each kind of biomaterials    12
Table 2.3 Characteristic Features of Ceramic Biomaterials    17
Table 2.4 Sequence of Interfacial Reactions Involved in Forming a    19
Bond between Tissue and Bioactive Ceramics    19
Table 3.1 Information of experimental chemicals    37
Table 3.2 Details of experimental instruments    38


                                

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