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研究生中文姓名:李毓雯
研究生英文姓名:Lee, Yu-Wen
中文論文名稱:探討不同比例混合白棘三列海膽刺殼粉與牡蠣殼粉做為生物醫學材料之表現
英文論文名稱:The Characteristics of Biomaterials Formed by Using Different Formula of Shell Powder from Sea Urchin Tripneustes gratilla and Oyster Crassostrea gigas
指導教授姓名:黃登福
口試委員中文姓名:教授︰鄭森雄
教授︰黃耀文
教授︰周薰修
教授︰陳石松
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:食品科學系
學號:10432018
請選擇論文為:學術型
畢業年度:106
畢業學年度:106
學期:
語文別:中文
論文頁數:103
中文關鍵詞:白棘三列海膽牡蠣廢棄物利用生物醫學材料氫氧機磷灰石β-磷酸三鈣
英文關鍵字:Tripneustes gratillaCrassostrea gigaswaste utilizingbiomateriahydroxyapatitebeta-tricalcium phosphate
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白棘三列海膽 (Tripneustes gratilla) 俗稱為馬糞海膽,為目前臺灣市面上最主要之食用海膽,然而海膽可食用部位僅約占其總重之 10%;牡蠣 (Crassostrea gigas) 也為台灣國人常見之貝類,因其味道鮮美,富含鎂、鋅、鐵等豐富營養價值,傳統上被認為有壯陽提振精神之作用,又有海中的牛奶之美稱。其可食用部位約占其總重之 30%,然而上述兩者不可食用部份,因其碳酸鈣組成不易被環境分解消化,造成台灣的環境危害。
臨床上病人於骨缺陷處進行骨移植手術,需要填補生醫骨質材料磷酸鈣鹽類等,使傷口能夠促進癒合。由於已知鈣磷酸鹽類於生物體中之降解速率為 α-三鈣磷酸鹽 (alpha-tricalcium phosphate, α-TCP) > β-三鈣磷酸鹽 (beta-tricalcium phosphate, β-TCP) (Chatterjea et al., 2014) > 氫氧基磷灰石 (hydroxyapatite, HA)。且因海膽刺殼中富含鎂離子,能使轉換之骨修復性生醫材料構型為鎂離子取代型 β- 三鈣磷酸鹽 (magnesium substituted tricalcium phosphate, (Ca,Mg)3(PO4)2, β-TCMP),已有研究指出 β-TCMP 培養人類骨母細胞 (MG-63) 增生及黏附情形較 β-TCP 佳。其次,牡蠣殼經水熱轉換方法 (hydrothermal reaction) 已知可形成 HA,其中由於 β-TCMP 之市場較大,但台灣白棘三列海膽產量較少,牡蠣殼產量較多,因此本研究之目的,為研究不同比例混合白棘三列海膽刺殼與牡蠣殼轉換成 β-TCMP 作為生醫材料之整體利用性。
本研究以混合不同比例之白棘三列海膽刺殼與牡犡殼透過添加 0.2 g/ml 之 diammonium hydrogen phosphate ((NH4)2HPO4 利用水熱轉換方法 (hydrothermal reaction) 在轉換條件 180℃、24 小時後即可得到富含鈣、磷之骨修復性生醫材料,再經過 X-ray 繞射分析、FTIR 圖譜分析、SEM-EDS 形態觀察和成分分析學材料鑑定後,確定白棘三列海膽刺殼成功從碳酸鈣轉換成骨修復性生醫材料 β-TCMP,其餘之混合比例材料與僅有牡蠣殼之組別則轉換成另一種骨修復性生醫材料氫氧基磷灰石 (hydroxyapatite; HA)。
由上述結果得知,減少白棘三列海膽刺殼的含量,則轉換後之骨修復性生醫材料會傾向於 HA 並不是 β-TCMP。進一步利用牡蠣殼添加不同劑量之 MgCl2·6H2O 與 SrCl2·6H2O 粉末,混合 0.2 g/ml 之 ((NH4)2HPO4 ,透過水熱轉換方法 (hydrothermal reaction) 在轉換條件 180℃、24 小時後即可得到骨修復性生醫材料,再經過 X-ray 繞射分析、FTIR 圖譜分析、SEM-EDS 形態觀察和成分分析學材料鑑定後,確定在牡蠣殼粉中額外添加 0.425 mg MgCl2·6H2O 與 0.015 mg SrCl2·6H2O 粉末,能將牡蠣殼從碳酸鈣成分轉換成骨修復性生醫材料 β-TCMP,與僅有牡蠣殼所轉換成的骨修復性生醫材料 HA 不同,且所需之 Mg2+、Sr2+ 離子比例能夠自由調控。
接著將樣品材料浸漬液及樣品本身利用與骨母細胞 (MG-63) 共培養來進行生物適應性評估,分別將兩組與骨母細胞 (MG-63) 進行體外培養,結果顯示所有樣品材料浸漬液皆無細胞毒性,所有樣品則於 800℃ 下處理之錠片對細胞不具毒性。在鹼性磷酸酶 (Alkaline phosphatase; ALP) 活性測試實驗與細胞礦化實驗 (Alizarin red staining assay; ARS) 中樣品浸漬液組與錠片組在第七天後兩組之 ALP 與 ARS 之偵測數值皆有明顯上升趨勢,結果說明兩組材料皆有促進骨母細胞生長和促進 ALP 和 ARS 表現提高之能力,尤其以牡蠣殼額外添加 2 倍 Mg2+、Sr2+ 離子的 DOSE-2 組別最佳,並且可以自由調配所需比例濃度。綜合上述結果說明由白棘三列海膽刺殼與牡蠣殼所混合製成轉換之骨修復性生醫材料與純牡蠣殼粉額外添加 Mg2+、Sr2+ 離子所製成之骨修復性生醫材料皆能達到降低環境汙染,具有充分利用廢棄物之目的,並能有效促進骨母細胞 MG-63 的生長。
Tripneustes gratilla is a common sea urchin in Taiwan but its edible part is only about 10% of the total weight. Crassostrea gigas is the most popular oyster in Taiwan but its edible part is only about 30% of the total weight. The purpose of this study was to use different formula of shell powder from sea urchin T. gratilla and oyster C. gigas for converting to magnesium substituted β-tricalcium phosphate (β-TCMP) as the feasibility of bone biomaterials.
After the assays of X-ray diffraction, fourier transmission infrared spectrometry, scanning electron microscopy, and energy dispersive X-ray spectrometry, all materials of different formula of shell powder from sea urchin T. gratilla and oyster C. gigas, are successfully converted to β-TCMP and hydroxyapatite (HA). Among them, only sea urchin T. gratilla group can be converted to the bone graft material β-TCMP. Furthermore, oyster shell C. gigas with different dose of MgCl2·6H2O / SrCl2·6H2O can be also converted to the bone graft material β-TCMP by hydrothermal reaction.
Then osteoblast MG-63 cells were cultured in soaking solution with bone graft biomaterials by using MTT assay to determine the cell viability. Otherwise, osteoblast MG-63 cells were cocultured with those bone graft biomaterial tablest, by using MTT assay to determine the cell viability. The result revealed that all bone graft biomaterials and those tablets were non-toxic. Therefore, the β-TCMP was found from using urchin shell only and oyster shell with higher dose of MgCl2·6H2O and SrCl2·6H2O than sea urchin shell. Those bone graft biomaterials formed from different formula of sea urchin and oyster shell, showed HA structure.
The bone graft biomaterials from sea urchin shell and oyster shell with MgCl2·6H2O / SrCl2·6H2O supplement exhibited to increase MG-63 cell growth, especially for 2 folds of dose of MgCl2·6H2O / SrCl2·6H2O in the oyster shell.
目錄
摘要 1
Abstract 3
壹、文獻整理 5
一、海膽簡介 6
二、牡蠣簡介 10
三、生醫材料簡介 12
貳、研究內容 19
第一章、探討由不同比例混合白棘三列海膽刺殼粉與牡蠣殼粉轉換之骨修復生醫材料之差異
一、前言 20
二、材料與方法 22
三、結果 28
四、討論 31
圖表………………………………………………………………………………32
第二章、探討添加不同比例鎂、鍶離子混合牡蠣殼粉轉換之骨修復生醫材料之差異
一、前言 48
二、材料與方法 49
三、結果 53
四、討論 55
圖表………………………………………………………………………………56
第三章、探討轉換後之骨修復性生醫材料與 MG-63 細胞共培養之表現
一、前言 68
二、材料與方法 69
三、結果 74
四、討論 77
圖表………………………………………………………………………………78
參考文獻 98
謝辭 103
附錄…………………………………………………………………………….........104

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全文檔開放日期:2019/07/03
 
 
 
 
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