臺灣博碩士論文加值系統

摘要
組織工程的目的為：利用多孔性的支架（scaffold）來模擬人體原有的extracellular matrix，使細胞附著、增生、遷移，然後產生功能，以重建缺損的組織或器官。氫氧基磷灰石（HA）具有良好的生物相容性與生物活性，植入人體後不會引起毒性，不刺激組織，具有引導骨骼相內生長，及在骨骼外組織誘導骨細胞分化生長的能力。利用磷酸鈣鹽泥(Calcium phosphate ceramic slurry)製成的多孔性HA磷酸鈣鹽具有適當的強度，能控制其孔洞大小、孔隙度，可消毒，也具有生物可吸收性，磷酸鈣鹽泥的可塑性並能製出3-D不規則的立體結構，是一種適合作為組織工程支架的材料。
本研究將磷酸鈣鹽泥混合NaCl粉末填模加壓成型，利用salt leaching方式製成多孔性HA磷酸鈣鹽，並改變不同製程條件來同時提高材料的抗壓強度與孔隙度，同時以SEM觀察材料表面形態，並以XRD分析不同製程條件與不同添加物含量對磷酸鈣鹽組成的影響，最後進行PH值量測及細胞測試，期望能獲得一個適合應用於組織工程的scaffold。
實驗發現磷酸鈣鹽泥中NaCl的含量增加，能提高磷酸鈣鹽的孔隙度，但卻使抗壓強度降低。加長磷酸鈣鹽泥的浸泡時間，或利用適當條件的熱處理能將磷酸鈣鹽的強度提高，同時不影響材料孔隙度與結晶性差的apatite相。細胞測試發現，當磷酸鈣鹽孔隙度高，細胞容易附著，細胞毒性也低。因此利用磷酸鈣鹽泥混合水溶性鹽類，浸泡後加上適當熱處理，利用磷酸鈣鹽泥中原本就有的微孔洞加上salt leaching產成的巨孔洞，形成一巨孔微孔同時存在的高孔隙度結構，加速磷酸鈣鹽吸收與新骨生長並具有一定的強度，非常有潛力成為軟骨/硬骨重建的組織工程支架。

總目錄

致謝……………………………………………………………………3
摘要……………………………………………………………………4
總目錄…………………………………………………………………5
表目錄…………………………………………………………………8
圖目錄…………………………………………………………………9
第一章 前言與研究目的………………………………………………12
1-1 簡介……………………………………………………………...12
1-2 研究目的……………………………………………...…………13
第二章 理論基礎與文獻回顧…………………………………………15
2-1 生醫陶瓷的種類………………………………………………...15
2-2 氫氧基磷灰石（hydroxyapatite；HA）[Ca10(PO4)6(OH)2]……15
2-2-1 氫氧基磷灰石（HA）的特性……………………………..15
2-2-2 氫氧基磷灰石結構中的離子取代現象…………………...16
2-3 塊狀氫氧基磷灰石燒結製程與性質…………………………..16
2-4 多孔性磷酸鈣陶瓷……………………………………………...18
2-5 組織工程………………………………………………………...19
2-5-1 組織工程簡介……………………………………………...19
2-5-2 軟骨/硬骨組織工程支架的特性與需求…………………..20
2-5-3 組織工程支架材料………………………………………...21
2-5-4 組織工程支架的做法……………………………………...22
2-6 磷酸鈣鹽PH值…………………………………………………23
2-7 細胞毒性………………………………………………………...24
第三章 實驗方法與步驟………………………………………………31
3-1 試片準備………………………………………………………..31
3-1-1 多孔性試片製備…………………………………………...31
3-1-2 抗壓強度與孔隙度試片製備……………………………..31
3-1-3 試片熱處理………………………………………………..31
3-2 材料分析與測試………………………………………………..32
3-2-1 抗壓強度測試（Compressive strength）…………………32
3-2-2 孔隙度測試（Porosity）…………………………………..32
3-2-3 X光繞射（XRD）…………………………………………32
3-2-4 掃描式電子顯微鏡分析（SEM）…………………………33
3-3 PH值測試………………………………………………………..33
3-4 細胞測試………………………………………………………...33
3-4-1 細胞培養（Cell culture）……………………………….…33
3-4-1-1 實驗前預備……………………………….…………...34
3-4-1-2 細胞冷凍管解凍………………………………………34
3-4-1-3 細胞培養………………………………………………34
3-4-1-4 細胞冷凍（Cell freezing）……………………………35
3-4-2 細胞毒性測試（Cytotoxicity）……………………………35
3-4-3 細胞附著性測試（Bioaffinity）……………………………36
第四章 結果與討論……………………………………………………..47
4-1 浸泡處理後的多孔性磷酸鈣鹽………………………………...47
4-1-1 抗壓強度測試……………………………………………...47
4-1-2 孔隙度測試…………………………………………….…..50
4-2 熱處理後的多孔性磷酸鈣鹽…………………………………...73
4-2-1 抗壓強度………………………………….………………..73
4-2-2 熱處理後的孔隙度……………………….………………..74
4-3 PH值測試……………………….………………………………87
4-4 細胞測試………………………………………………………...94
4-4-1 細胞毒性測試……………………………………………...94
4-4-2 細胞附著性測試…………………………………………...95
第五章 結論……………………………………………………….…...100
第六章 參考文獻………………………………………………………102
表目錄

表1-1 氫氧基磷灰石（HA）植入材的發展………………………….14
表2-1 氫氧基磷灰石燒結方法………………………………………..25
表2-2 生物吸收性高分子聚合物……………………………………..26
表2-3 多孔性磷酸鈣鹽的製法、孔隙度與孔洞尺寸…………………27
表3-1 多孔性試片製備的溫度與時間參數…………………………..37
表3-2 Hank’s solution成份表………………………………………….39
表3-3 細胞培養測試所需溶液與器材列表…………………………..40
表3-4 食品工業發展研究所菌種中心/國家衛生研究院細胞庫細胞株資料單……………………………………………………………41
表4-2-1 不同NaCl含量試片，熱處理前後的孔隙度（%）……….76
表4-3-1 不同製程條件下，不同NaCl含量試片的PH值………….89
圖目錄

圖2-1 商用氫氧基磷灰石粉末與不同形態、尺寸的多孔質塊狀氫氧基磷灰石示意圖……………………………………………………29
圖2-2 常用於組織工程支架的生物降解性高分子聚合物…………..30
圖2-3 Cleavage of the tetrazolium salt WST-1 to formazan……….…..30
圖3-1 多孔性磷酸鈣鹽製程與測試分析流程………………………..42
圖3-2 Cell culture更換細胞培養基溶液步驟流程……………………43
圖3-3 Cell culture細胞繼代步驟流程…………………………………44
圖3-4 細胞冷凍步驟流程……………………………………………..45
圖3-5 細胞毒性測試步驟流程………………………………………..46
圖4-1-1 不同含量NaCl（未過篩），T2浸泡t4的抗壓強度…………53
圖4-1-2 不同含量NaCl（未過篩），T2浸泡t5的抗壓強度…………54
圖4-1-3 不同NaCl含量試片，T2去離子水浸泡的強度比較………55
圖4-1-4 不同含量NaCl，T2浸泡t4的抗壓強度比較……………….56
圖4-1-5 不同含量NaCl（未過篩），T3浸泡t2的抗壓強度…………57
圖4-1-6 不同含量NaCl（未過篩），T3浸泡t4的抗壓強度…………58
圖4-1-7 NaCl含量W3的試片，T3浸泡不同天數的抗壓強度………59
圖4-1-8 不同NaCl含量，不同浸泡溫度的抗壓強度比較………….60
圖4-1-9 不同NaCl含量，T3浸泡t2的抗壓強度…………………….61
圖4-1-10 NaCl含量W3的試片，T1浸泡的抗壓強度………………..62
圖4-1-11 NaCl含量W3的試片，不同溫度下浸泡的抗壓強度變化…63
圖4-1-12 NaCl含量W3的磷酸鈣鹽試片，不同溫度浸泡t4的SEM照片……………………………………………………………64
圖4-1-13 不同含量NaCl（未過篩），T2浸泡不同天數的孔隙度….65
圖4-1-14 不同NaCl含量（未過篩）試片，T2浸泡的孔隙度比較..67
圖4-1-15 不同NaCl含量（未過篩）試片，T2溫度浸泡t2時間與含NaCl未浸泡的SEM照片………………………………….68
圖4-1-16 不同NaCl含量（未過篩）試片，T2溫度浸泡t4時間的SEM照片…………………………………………………………69
圖4-1-17 NaCl含量W6（未過篩）試片，T2溫度浸泡t4時間，試片巨孔洞與微孔洞的SEM照片………………………………..70
圖4-1-18 不同含量NaCl（未過篩），T3浸泡不同天數的孔隙度….71
圖4-1-19 不同NaCl含量試片，T3浸泡的孔隙度變化……………..72
圖4-2-1 NaCl含量W2（未過篩）試片，T2浸泡t4後，不同溫度Ht5熱處理的抗壓強度比較……………………………………..76
圖4-2-2 NaCl含量W3（未過篩）試片，T2浸泡t4後，不同溫度Ht5熱處理的抗壓強度比較……………………………………..77
圖4-2-3 NaCl 含量W2/W3的試片，T2浸泡t4，不同溫度熱處理Ht5的SEM照片…………………………………………………….78
圖4-2-4 HT4熱處理Ht5對T2浸泡t4試片抗壓強度的影響…………79
圖4-2-5 HT4熱處理Ht5對T3浸泡t2試片抗壓強度的影響…………80
圖4-2-6 NaCl含量W3的試片，T2浸泡t4，HT4熱處理不同時間的抗壓強度比較…………………………………………………..81
圖4-2-7 含W3 NaCl試片，T2浸泡t4，HT4熱處理不同時間的SEM照片…. ……………………………………………………….82
圖4-2-8 NaCl含量W3的試片，T2浸泡t4，HT1熱處理不同時間的抗壓強度………………………………………………………...85
圖4-2-9 HT9熱處理不同時間對T2浸泡t4試片的抗壓強度的影響…86
圖4-3-1 不同NaCl含量試片，T2浸泡t4，HT4熱處理Ht5的PH值變化……………………………………………………….……..89
圖4-3-2 不同NaCl含量試片，T2浸泡t5的PH值變化……………90
圖4-3-3 不同NaCl含量試片，T3浸泡t2的PH值變化……………91
圖4-3-4 不同NaCl含量試片，T3浸泡t2，HT4熱處理Ht5的PH值變化……………………………………………………….…….92
圖4-4-1 淬取液的細胞毒性測試……………………………………...97
圖4-4-2 細胞在不同材料淬取液中培養24hrs後的細胞形態………98
圖4-4-3 不同製程條件的磷酸鈣鹽試片的生物附著性……………...99
圖4-4-4熱處理前後試片表面形態…………………………………..100

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