臺灣博碩士論文加值系統

人體骨骼缺損修補，常需進行骨骼移植，由於國人對器官移植觀念尚未建立，人體骨骼移植材料取得不易，另外異體骨骼在移植中常有感染及排斥等風險。因此，開發人造硬骨植入物以突破上述自體、異體移植骨骼容易發生感染、排斥等問題，並確保人造骨骼細胞增生、分化能力，已成為組織工程重要之研究課題。另外高分子水膠是一種立體網狀結構具親水性之材料，可包覆活體細胞、提供細胞體外增生環境，並產生修補組織缺陷植入物之功能，然而高分子水膠也是一種高含水率之溫感性液/固相變化材料，雖然有良好之細胞增生條件，但缺乏結構支撐強度，無法適用可承受力量之硬骨缺損修補，造成臨床應用受限。爰此，本研究係利用本團隊所自行開發之雷射燒結快速成型系統，設計、製造生物可吸收聚己內酯(PCL)立體孔洞支架，並灌注高分子水膠與骨母細胞，透過體外實驗培養硬骨活體組織，並進行物、化性測試與細胞分化及增生品質驗證，以期發展一種結合高分子水膠與聚己內酯(PCL)孔洞支架，使能經由體外細胞培養成兼具結構支撐強度與細胞增生能力之生物可降解人造活體硬骨植入物。另外，為了使本研究所開發之硬骨組織增生技術能符合臨床需求，也進行了豬下顎骨缺損植入活體支架修補之動物實驗，並初步觀察活體支架在動物體內被吸收及硬骨組織生長情形。
本研究工作係分三部份進行，第一部份主要是進行所選定生物可降解聚己內酯(PCL)粉末材料之測試分析，首先係將PCL分散於水或有機溶劑中之超微細樣品，在未經稀釋之條件下，利用動態光散射儀進行粒徑分析，接著利用SEM觀察PCL粉末材料燒結前、後微觀結構，最後再量測其熔點溫度(Tm)，做為設定雷射燒結製程材料預熱溫度之依據；接著，利用本團隊所自行開發之雷射燒結快速成型機，製作三種不同支架結構（0°/45°/0°/45°，0°/90°/ 0°/90°及0°/45°/90°/135°）之立體孔洞支架，透過巨觀、微觀結構分析實驗，觀察不同孔洞支架之孔洞形狀、大小、分布均勻性及孔洞間的交聯性，並測定支架之抗拉、抗壓強度和孔隙率，以了解多孔性支架是否有足夠抗拉、抗壓強度及具備細胞植入後貼附與生長的孔洞結構；第二部份先透過高分子水膠灌注孔洞支架實驗，以三種不同之灌注方法即浸泡法(immersion)、針孔注入法(injection)、重力垂降(gravitational dropping)將水膠灌注入孔洞支架，以評估水膠與孔洞支架結合之成效；接著，進行兔子骨髓分離間葉幹細胞培養實驗，透過鈣染色來確認間葉幹細胞是否能分化為骨母細胞，再進行體外細胞培養實驗分析。體外細胞培養實驗之設計係分為三個組別進行實驗分析：控制組(水膠加細胞)、實驗A組（支架加細胞）及實驗B組（水膠加細胞加支架）；實驗係以28天為週期，每七天三組各取出11個樣本進行系列實驗分析以瞭解細胞生長情况，至於每七天所進行之系列實驗為鹼性磷酸酶染色法(ALP)、VonKossa’s 染色實驗及MTS 色度分析實驗等；第三部份則是將PCL孔洞支架結合骨母細胞，進行豬大塊下顎骨缺陷修補實驗規劃與先期評估；首先製作30mm X 30mm X 10mm之PCL孔洞支架，配合臨床醫師進行動物實驗，將具有骨母細胞之PCL孔洞支架植入大蘭嶼花斑豬下顎骨缺陷部位，以觀察動物植入手術後是否發生感染，並評估PCL孔洞支架與動物組織之生物相容性以及所植入之骨母細胞於孔洞支架內是否能持續增生及分化。
本研究已完成使用雷射燒結快速成型技術製作PCL孔洞支架，並灌注高分子水膠，進行硬骨細胞體外增生及分化實驗與臨床評估；在研究第一部份生物支架設計製作方面，已完成了PCL粉末材料分析，並透過微觀結構分析實驗，證實PCL孔洞支架內具有300~500m之孔洞，符合文獻中所述適合骨母細胞之生長環境；接著，也透過抗壓實驗證明PCL孔洞支架之彈性模式3.75MPa，但根據文獻指出人體受力區之彈性模式為100~500MPa，因此本研究所製作之PCL生物支架機械強度，較適合應用於人體非受力區骨組織修復；本研究第二部份生物支架結合高分子水膠包覆骨母細胞實驗，經過14天鈣染色分析，以光學顯微觀察幹細胞，觀察出幹細胞顏色為暗紅色，表示幹細胞慢慢分化為骨母細胞；另外所製作之PCL孔洞支架與水膠結合後，利用MTS 色度分析、鹼性磷酸酶染色法及VonKossa’s 染色進行體外實驗，並使用SPSS統計分析軟體，透過ANOVA分析各組間統計上差異。由MTS 色度分析實驗結果得知細胞培養第7天時，對照組、實驗A組、實驗B組等三組之OD值分別為0.12±0.094、0.029±0.033、0.092±0.058，實驗A組與實驗B組比較P<0.05，實驗A組與對照組比較P<0.05，培養第14天時對照組、實驗A組OD值分別0.29±0.083、0.285±0.029比較p>0.05，培養第21天實驗A組、實驗B組OD值分別為0.491±0.161、0.0585±0.210 經統計分析比較p>0.05，第7天與第21天，實驗A組OD值明顯比對照組高，可能原因是在加入養份時，對照組養份要從水膠外進入到裡面較不容易，而實驗A組細胞可直接吸收養份；另外，由鹼性磷酸酶染色法(ALP)實驗分析結果得知第7天OD值量三組皆有明顯上昇趨勢，對照組、實驗A組與實驗B組等三組之OD值分別為0.0063±0.0004、0.0073±0.0007、0.0070±0.0006，對照組與實驗A組比較P=0.168、對照組與實驗B組比較P=0.365、實驗A組與實驗B組比較P=0.808；以上三組經 ANOVA分析比較後P=0.177 ，培養第14天時對照組、實驗A組與實驗B組等三組之OD值分別為0.0076±0.0008、0.0070±0.0005、0.0063±0.0002，對照組與實驗A組 比較P=0.436、對照組與實驗B組 比較P=0.05、 實驗A組與實驗B組比較P=0.339，以上三組經ANOVA分析比較後 P=0.76，培養第21天時對照組、實驗A組與實驗B組等三組之OD值分別為0.0093±0.0006、0.0056±0.0007、0.0061±0.0002對照組與實驗A組 比較P=0.0001，對照組與實驗B組 比較P=0.001、 實驗A組與實驗B組比較P=0.535 ；以上三組經ANOVA分析比較後P=0.001<0.05 有顯著差異，培養第28天時對照組、實驗A組與實驗B組等三組之OD值分別為0.0026±0.0.002、0.0016±0.0002、0.0053±0.0006，對照組與實驗A組比較 P=0.051、對照組與實驗B組比較 P=0.585 、實驗A組與實驗B組比較P=0.201；以上三組經ANOVA分析比較後 P=0.57，代表ALP 活性在21天後細胞開始分泌出其他如OPN等其他物質，經由統計分析結果顯示，開始培養骨母細胞1 週後ALP 的活性表現最活躍，接著數據呈現先上昇再下降的趨勢；至於VonKossa’s 染色實驗，結果發現實驗初期時細胞分散，沒有礦化物質產生，培養1 週時細胞已開始形成群落，但並沒有暗褐色礦物質化結節形成，培養2 週時細胞明顯形成群落，有暗褐色礦物質化結節開始形成，結果顯示PCL孔洞支架水膠包覆下能提供細胞生長環境，進而提高細胞增生與分化效果。
本研究第三部份在硬骨組織活體支架臨床評估方面，完成修復豬下顎骨缺陷實驗流程設計，並製作30mm X 30mm X 10mm之PCL孔洞支架結合骨母細胞，並將孔洞支架植入豬下顎進行骨缺損修補，植入28天後取出支架術後28天評估並無感染情況，驗證本研究所設計之多孔性PCL支架與動物組織相容性良好，為可應用於組織工程以供組織培養的支架。
本研究證實了結合高分子水膠包覆硬骨細胞與雷射燒結成形之聚己內酯(PCL)孔洞支架，可經由體外培養成兼具結構支撐強度與細胞增生能力之生物可降解人造活體硬骨植入物。各項物化性實驗分析驗證硬骨細胞增生與分化成效。除此，本研究所開發之雷射燒結快速成型技術結合高分子水膠於硬骨組織支架設計方法，將可成為組織工程培養人造活體硬骨植入物之新策略。


關鍵詞：組織工程、支架、雷射燒結、快速成型、高分子水膠、幹細胞

英文摘要
Bone defect, and often the need for bone transplants, people on the organ transplant as a result of the concept has not yet been established, the body is not easy to obtain bone graft material, while in the transplantation of allograft bone are often the risk of infection and rejection. Therefore, the development of artificial bone implants hard to break through the above-mentioned autologous, allograft bone prone to infection, rejection and so on, and to ensure that the artificial bone cell proliferation, differentiation, tissue engineering has become an important research topic. Another water-binder polymer is a three-dimensional network structure of a hydrophilic material, living cells can be coated to provide in vitro proliferation of the environment, and organizations have a defective repair of functional implants, however, water-binder polymer is a high moisture content emotional temperature of liquid / solid phase change materials, although there are good conditions for cell proliferation, but the lack of structural support strength, not the application of force can withstand the bony defect, resulting in limited clinical application. Yuan, the purpose of this study was to use the team's self-developed laser sintering rapid prototyping systems, the design, manufacture bioabsorbable polycaprolactone (PCL) three-dimensional scaffold holes and perfusion with water-binder polymer Osteoblastoma through in vitro Experimental cultivation bony tissues and structures, resistance testing with cell differentiation and proliferation of quality certification, with a view to developing a water-binder combination of polymer and polycaprolactone (PCL) stent holes so that they can by in vitro cell culture as both the structure of support the strength and the ability of cells biodegradable in vivo bony man-made implants. Work of this study is divided into three sections, the first part of the principal is selected biodegradable polycaprolactone (PCL) powder materials test and analysis, first of all, the Department of the PCL dispersed in water or organic solvents in samples of ultra-fine without dilution of the conditions, the use of dynamic light scattering particle size analysis instrument, and then the use of SEM observation of sintered powder material PCL before and after the micro-structure, and then measuring the temperature of its melting point (Tm), as a set of laser sintering process based on material pre-heating temperature; the second part is using the team's self-developed laser sintering rapid prototyping machine, the production of three different stent structure (0 ° / 45 ° / 0 ° / 45 °, 0 ° / 90 ° / 0 ° / 90 ° and 0 ° / 45 ° / 90 ° / 135 °) of three-dimensional scaffold holes through macro and micro structural analysis of the experiment to observe the different holes of the holes stent shape, size, and pore distribution crosslinking between, and support the determination of tensile, compressive strength and porosity, porous scaffold in order to understand whether there is sufficient tensile strength, compressive strength and post-implantation with cells attached to the pore structure and growth; Finally, The study also through water-binder polymer stent holes perfusion experiments, three different methods of reperfusion immersion (immersion), pinhole injection (injection), reduced vertical gravity (gravitational dropping) irrigation water into the holes of plastic stents to assessment of water combined with plastic stent and the effectiveness of hole; then, the separation of rabbit bone marrow mesenchymal stem cell culture experiments, through the calcium staining to confirm whether the mesenchymal stem cells can differentiate into bone cells, further analysis of in vitro cell culture experiments. In vitro cell culture experiments Design of experiments is divided into three groups analysis: control group (water-binder plus cells), the experimental A (scaffold plus cells) and experimental B (water-binder plus cells plus stent); experiment based on 28-day cycle, every seven days out three groups of 11 samples for analysis of a series of experiments to understand the cell growth, as each of the seven-day series of experiments carried out by staining for alkaline phosphatase (ALP), VonKossa's staining experiments and MTS Colorimetric analysis of experiments; third part of combination type of PCL scaffold holes Osteoblastoma for pig large jaw bone defects repair planning and pre-assessment test; first of all, the production of 30mm X 30mm X 10mm holes on the PCL scaffold, with the clinical physicians to carry out animal experiments, will have the PCL Osteoblastoma stent holes big Orchid Flower mandible defect site, in order to observe the animals implanted in the occurrence of post-operative infection, and to assess the PCL holes of stent biocompatibility and animal tissue and the implanted cells in the bone cavity whether stent proliferation and differentiation can be sustained.
This study has been completed using the laser sintering rapid prototyping technique PCL stent openings and water-binder polymer perfusion, the bony proliferation and differentiation in vitro experiments and clinical evaluation; the first part in the study of biological stent design has been completed PCL powder materials analysis and experiment through the micro-structural analysis confirmed that PCL stent with holes 300 ~ 500  m of the hole, in line with the literature mentioned in Osteoblastoma of suitable habitat; then also proved through the compression holes PCL scaffold flexible mode of 3.75MPa, but according to the literature that the human body by the force of the elastic model of the District 100 ~ 500MPa, therefore the production of the Institute of PCL scaffolds mechanical strength, is more suitable for non-human force applied to areas of bone tissue repair; of this study was the first Second, some combination of polymer scaffolds coated plastic water Osteoblastoma experiment, after 14 days of calcium staining analysis to optical microscopy observation of stem cells, stem cells observed for the dark red color, that stem cells gradually differentiate into bone cells; other PCL holes produced by the plastic stent combined with water, the use of MTS color analysis, staining and alkaline phosphatase staining VonKossa's in vitro experiments, and the use of SPSS statistical analysis software, by ANOVA analysis of statistical differences between groups. Color analysis by the MTS cell culture results indicate that the first 7 days, the control group, experimental group A, group B and other experiments on the OD value of the three groups were 0.12 ± 0.094,0.029 ± 0.033,0.092 ± 0.058, the experimental group and A Experimental B group P <0.05, experiment A group with the control group P <0.05, cultured 14 days in control group, experimental A group OD values were 0.29 ± 0.083,0.285 ± 0.029 p> 0.05, the first 21 days of culture A group of experiments, OD value of experimental group B, respectively, 0.491 ± 0.161,0.0585 ± 0.210 p> 0.05, the first 7 days, with the first 21 days, experimental A group of OD was significantly high than the control group, the possible reasons for adding nutrients in when nutrients to the control group apart from the plastic into the water than there is not easy, and the experimental A group of cells to absorb nutrients directly; In addition, staining by the alkaline phosphatase (ALP) analysis of the results of experiments that the OD value of the first 7 days, the volume of the three groups have clear upward trend in the control group, experimental group A and group B and other experiments on the OD value of the three groups were 0.0063 ± 0.0004,0.0073 ± 0.0007,0.0070 ± 0.0006, control group A and experimental group P = 0.168, control group and experimental group B P = 0.365, experiment A Group B Group and experimental P = 0.808, ANOVA analysis, P = 0.177, cultured 14 days in control group, experimental group A and group B and other experiments on the OD value of the three groups were 0.0076 ± 0.0008,0.0070 ± 0.0005,0.0063 ± 0.0002, control group A and experimental group P = 0.436, control group B and experimental group P = 0.05, experiment A Group B Group and experimental P = 0.339, ANOVA analysis, P = 0.76, cultured 21 days the control group, experimental group A and group B and other experiments on the OD value of the three groups were 0.0093 ± 0.0006,0.0056 ± 0.0007,0.0061 ± 0.0002 in control group A and experimental group P = 0.0001, control group B and experimental group P = 0.001, experiment A group B group and the experimental P = 0.535, ANOVA analysis, P = 0.001 <0.05 there were significant differences in training the first 28 days in control group, experimental group A and group B and other experiments on the OD value of the three groups were 0.0026 ± 0.0.002 , 0.0016 ± 0.0002,0.0053 ± 0.0006, control group A and experimental group P = 0.051, control group B and experimental group P = 0.585, experiment A Group B Group and experimental P = 0.201, ANOVA analysis, P = 0.57, on behalf of ALP activity 21 days after the beginning of the secretion of other cells, such as OPN and other substances, through the statistical analysis showed that began training 1 week Osteoblastoma ALP activity after the performance of the most active, rising at first and then a downward trend, VonKossa's staining experiments, through the cell dyeing observation, the beginning of culture, the cells dispersed, there is no mineralized material have, when the cell culture 1 week community has begun to take shape, but no dark brown nodules of the formation of minerals, cultured cells in two weeks when the formation of communities, there are dark brown mineral of the beginning of the formation of nodules showed that PCL stent water holes under the plastic-coated cells can provide the environment, to improve the effect of cell proliferation and differentiation.
The third part of this study in the hard bone tissue scaffold in vivo clinical assessment, complete the repair of bone defects in experimental pig jaw design process and the production of 30mm X 30mm X 10mm holes on the PCL scaffold Osteoblastoma combination, and stent implantation of pig jaw holes for bone defect, 28 days after implantation of stents removed 28 days after infection to assess the situation does not, verify the design of the Institute of PCL porous scaffold with a good animal tissue compatibility for tissue engineering can be applied to the scaffold for tissue culture .
This study confirmed the combination of polymer plastic coated water bony cells and Laser Sintering of Polycaprolactone (PCL) stent hole via in vitro into both structural strength to support the ability of cell proliferation and biodegradable in vivo bony man-made implants. Physico-chemical analysis of the experimental verification of bony effectiveness of cell proliferation and differentiation. In addition, the development of the Institute of Laser Sintering polymer technology in hard plastic water bone tissue scaffold design, tissue engineering will become in vivo culture bony man-made implants the new strategy.
Key words: tissue engineering, stent, laser sintering, rapid prototyping, polymer water-binder, stem cell

目 錄
指導教授推薦書………………………………………………………i
口試委員會審定書……………………………………………………ii
授權書…………………………………………………………………iii
誌謝……………………………………………………………………iv
中文摘要………………………………………………………………x
目錄……………………………………………………………………xv
圖目錄…………………………………………………………………xix表目錄…..……………………………………………………………xxiii
第一章 研究背景、動機與目的………………………………………...1
1.1研究背景 ………………..1
1.2研究動機 5
1.3研究目的 5
1.3.1 PCL生醫粉末材料分析及生物支架設計製作及分析 6
1.3.2生物支架結合高分子水膠包覆硬骨細胞體外實驗...........6
1.3.3硬骨組織活體支架臨床評估 7
1.4論文架構 7
第二章 文獻回顧 8
2.1組織工程支架結構設計與分析技術相關文獻...........................8
2.2傳統組織工程支架製作技術相關文獻 12
2.2.1 纖維鍵結法 12
2.2.2溶劑鑄造/鹽析法 13
2.2.3冷凍乾燥法 14
2.2.4相分離法 15
2.2.5氣體發泡法 16
2.3快速成型組織支架製作技術相關文獻 16
2.3.1 精密擠出製造法 17
2.3.2 溶解沈積法 18
2.3.3 低溫沉積法 20
2.3.4 多噴嘴沉積.......................................................................20
2.3.5 立體生醫噴塗機 22
2.3.6 快速成型自動沉積系統 23
2.3.7 立體纖維沉積技術 24
2.3.8 精密擠出沉積技術 25
2.3.9 立體印刷技術 26
2.3.10 美國TheriFormeTM公司所提出之製造技術 26
2.3.11 雷射燒結技術 27
2.4 支架材料簡介 29
2.4.1 氫氧基磷灰石 29
2.4.2 聚乳酸 30
2.4.3 聚己內酯 30
2.4.4 聚乳酸-聚甘醇酸 30
2.4.5 聚甘醇酸 31
2.5組織工程孔洞支架分析相關文獻 31 2.6高分子水膠相關文獻 33 2.7文獻總結 34
第三章 PCL生醫粉末材料分析及生物支架設計製作及分析 ……36
3.1 PCL生醫粉末材料分析…………………………….………….36
3.1.1粒徑分……………………………………...…………......38
3.1.2粉末材料微觀觀測實驗…………..………………….…..36
3.1.3熔點溫度測定實驗…………………..………….....……..37
3.2立體孔洞支架設計製作…………………..……………......37
3.2.1立體支架不同角度設計……………………..…………...37
3.2.2立體支架不同角度製作……………………..…………...38
3.3立體孔洞支架特性分析..…………………………..…………...39
3.3.1巨觀結構分析……………………………..……………...40
3.3.2微觀觀結構分析………………………..………………...40
3.3.3拉伸實驗………………………………..………………...40
3.3.4抗壓實驗..………………………………………………...41
3.3.5孔隙率測定實驗..………………………………………...42
第四章 生物支架結合高分子水膠包覆硬骨細胞體外實驗..………...44
4.1兔子骨髓分離間葉幹細胞培養實驗.………………………....45
4.2高分子水膠灌注生物支架實驗.………………………………45
4.3生物孔洞支架包覆硬骨細胞實驗....…………………..……....49
4.3.1 MTS比色分析實驗…….……………………….……………....49
4.3.2 Von Kossa’s 組織染色實驗…….………………………………50
4.3.3 ALP 活性測定實驗….……………………….………………...50
第五章 硬骨組織活體支架臨床評估………………………………….52
5.1 實驗流程設計……………………….…………………….......52
5.2 生物支架設計製作及結合骨母細胞實驗.…………..…….…53
5.3生物支架結合硬骨細胞修復豬下顎骨缺陷應用實驗………..53
第六章 結果與討論…………………………………………………….55
6.1 PCL生醫粉末材料分析及生物支架設計製作及分析……… 55
6.2 生物支架結合高分子水膠包覆硬骨細胞體外實驗…………65
6.3 硬骨組織活體支架臨床評估…………………………………76
第七章 未來研究方向…………………………………………. .…….77
參考文獻 ………………………………………………………………79

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