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研究生:陳葭菱
研究生(外文):CHEN, HSIA-LING
論文名稱:製備多層次軟骨支架並探討添加鈣磷系鹽類之生物相容性質
論文名稱(外文):Scaffolding Strategies for Tissue Engineering: Study on Preparation of Multi-Layered Cartilage Scaffold with Calcium Phosphate Additives and Investigate of Biocompatibility
指導教授:陳文正陳文正引用關係
指導教授(外文):CHEN, WEN-CHENG
口試委員:陳文正石啟仁胡晉嘉
口試委員(外文):CHEN, WEN-CHENGSHIH, CHI-JENHU, JIN-JIA
口試日期:2020-07-15
學位類別:碩士
校院名稱:逢甲大學
系所名稱:生醫資訊暨生醫工程碩士學位學程
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2020
畢業學年度:108
語文別:中文
論文頁數:87
中文關鍵詞:多層次軟骨支架鈣磷系鹽類體外細胞培養
外文關鍵詞:multi-layer cartilage scaffoldcalcium phosphatecell proliferation test
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隨著醫療設備的進步,老年人口比例逐漸增加,導致退化性關節炎逐漸普及,退化性關節炎常見的治療方式是使用藥物做短暫式的減緩退化治療,或是用顯微修補手術,將不平整處移除,甚者直接進行人工關節置換等;另一選擇為利用自體軟骨細胞移植,但是此方式受制於患者年齡,若無法培育有足夠的自體軟骨細胞移植,會造成手術後生成纖維軟骨而非透明軟骨產生之負面影響,所以對於軟骨的修復仍是相當大的挑戰。
本研究主要目的為製備具有連通孔洞、生物降解性、促進細胞貼附與增生之多層次軟骨支架,並添加鈣磷系鹽類與骨粉,模擬軟骨的鈣化區(calcified zone)與海綿骨,期望支架能提供細胞向內生長的良好環境,並伴隨著細胞的增生緩慢降解。
多層次支架主要分為三層,以明膠(gelatin)及玻尿酸(hyaluronic acid, HA)作為主要材料,第一層為調整氯化鈉的含量以控制孔徑的大小;第二層為添加不同含量的二鈣磷酸鹽及表面富含奈米鬚晶的二鈣磷酸鹽,並利用不同劑量碳化二亞胺進行交聯反應;第三層則以骨粉替代氯化鈉,再以不同劑量及濃度的碳化二亞胺進行交聯。
在SEM表觀結構的觀察得知,本實驗成功製備出具有多孔結構及連通孔特性的軟骨支架;在含水量及降解率的測試中,支架可於短時間內大量吸收水分,並在去離子水中降解;在胺基殘留率及表面化學性質分析中,可以證實支架有良好的交聯反應。
在體外生物相容性測試結果中,顯示多層次軟骨支架不具有細胞毒性,而在接觸性培養的細胞貼附觀察中,顯示軟骨細胞對支架的貼附狀況良好,且軟骨細胞隨著時間增加有明顯的細胞增生趨勢。
綜合上述的測試結果,代表本實驗所製備的多層次支架能提供給細胞良好的生長環境,期望未來可實際應用在臨床上,以加速軟骨的修復 。

With the advancement of medical technology, the proportion of the aging population is increasing. Degenerative osteoarthritis (OA) is a clas-sic age-related disorder, OA is the chronic disability in elder and leads to pain and stiffness in the joints. The more common treatment is to use medicines that could temporary relieve pain. Due to the medicines do not reduce inflammation, the surgeon maybe suggests a quick surgery by removing the uneven site through an arthroscope inside patient’s joint or even the surgeon would takes out the severely diseased parts and directly replaces them with an artificial joint. The other option is to use autologous chondrocyte implantation, but this method is subject to the patient's age and only has the significance for slowing down the progression or delaying knee replacement surgery. It would also result in negative influence of generating fibrocartilage rather than hyaline cartilage, so the current art of repairing OA still remains a considerable challenge
The main purpose of this study is to prepare a multi-layer cartilage scaffold with interconnecting pores, degradable and can promote cell attachment and proliferation. It is expected that the scaffold can slowly degrade with the proliferation of cells and provide a good environment for cell ingrowth, and that calcium-phosphorus polymers can induce and promote cell migration to stimulate the proliferation and differentiation of chondrocytes.
The multi-layer scaffold prepared in this experiment is divided into three layers, with gelatin (gelatin) and hyaluronic acid (HA) as the main materials. The first layer is to adjust the content of sodium chloride to control the pore size. The second layer is to add different content of dicalcium phosphate and dicalcium phosphate rich in nano whiskers on the surface, and use different doses of carbodiimide Perform cross-linking reaction. The third layer replaces sodium chloride with bone meal, and then crosslinks with different doses and concentrations of carbodiimide.
Observation of the apparent structure of SEM shows that this experiment successfully prepared a cartilage scaffold with porous structure and characteristics of interconnected pores. In the test of water content and degradation rate, the stent can absorb a large amount of water in a short time and degrade in de-ionized water. In the compressive strength test, it can be found that adding calcium phosphate salts can improve the mechanical properties of the stent. In the cross-linking degree test and FTIR, the formal scaffold can carry out a good cross-linking reaction. The results of the in vitro biocompatibility test showed that the multi-layer cartilage scaffold was not cytotoxic, and it was shown in the cell attachment observation of the contact culture that the Chond cells had a better attachment to the scaffold surface.
In the cell proliferation test, it can be observed that Chond cell has a tendency to increase with time. It is hoped that in the future, it can be practically applied in practical application of clinical treatment of OA, to accelerate the repair of cartilage.

誌  謝 1
摘  要 2
ABSTRACT4
第一章 緒論11
第二章 文獻探討13
2.1 退化性關節炎13
2.2 軟骨介紹14
2.3 軟骨受損16
2.4 組織工程18
2.5 組織工程支架的製備方法19
2.6 支架材料的選擇20
2.7 交聯劑22
2.8 明膠、玻尿酸與EDC的交聯反應23
2.9 二鈣磷酸鹽類24
2.10 氫氧基磷灰石25
2.11 研究目的26
第三章 實驗材料與方法27
3.1 實驗材料與儀器27
3.2 前驅實驗流程與測試30
3.2.1 DCP-rich DCPA製備30
3.2.2 骨粉製備31
3.2.3 支架製備32
3.3組別組成與測試35
3.4 軟骨支架之表面型態37
3.4.1 掃描式電子顯微鏡(scanning electron microscope, SEM)及孔徑分析37
3.5 軟骨支架之物化性質分析37
3.5.1 含水量分析 37
3.5.2 降解率分析 38
3.5.3 抗壓強度測試38
3.5.4 X-光繞射(X-ray diffraction analysis, XRD)分析38
3.5.5 交聯度測試39
3.5.6 傅立葉紅外線光譜儀 (FTIR)分析40
3.6 滅菌方式評估41
3.6.1無菌測試41
3.7 體外細胞培養相關測試42
3.7.1 細胞培養42
3.7.2萃取液的製備(Following ISO 10993-5)42
3.7.3 細胞毒性定量測試(XTT assay)43
3.7.4 細胞毒性定性測試43
3.7.5 細胞貼附型態觀察43
3.7.6 細胞增生44
3.7.7 Alcian blue stain44
3.7.8免疫螢光染色44
第四章 前驅結果與討論45
4.1 第一層氯化鈉支架45
4.1.1 SEM表觀結構45
4.1.2 含水量分析47
4.1.3 胺基殘留率分析48
4.2 第二層磷酸鹽支架49
4.2.1 SEM表觀結構49
4.2.2 含水量分析51
4.2.3 胺基殘留率分析53
4.3第三層骨粉支架55
4.3.1 SEM表觀結構55
4.3.2 含水量分析56
4.3.3 胺基殘留率分析58
第五章 前驅結論60
5.1 第一層氯化鈉支架60
5.2 第二層磷酸鹽支架60
5.3 第三層骨粉支架60
5.4 總結60
第六章 最佳參數結果與討論61
6.1最佳參數流程61
6.2 物化性質分析64
6.2.1 含水量分析64
6.2.2 降解率分析65
6.2.3 機械強度分析66
6.2.4 ATR-FTIR化學性質分析68
6.2.5 XRD71
6.2.6 滅菌確效72
6.3生物相容性73
6.3.1細胞毒性73
6.3.2 細胞貼附75
6.3.3 Alcian blue stain77
6.3.4 細胞增生79
6.3.5 免疫螢光染色81
第七章 結論82
參考文獻83

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