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系統識別號 U0026-0812200910342801
論文名稱(中文) 氫氧基磷灰石複合骨水泥基本性質及植入結果研究
論文名稱(英文) Basic Properties and In Vivo Studies of Calcium Phosphate Composite Cement
校院名稱 成功大學
系所名稱(中) 材料科學及工程學系碩博士班
系所名稱(英) Department of Materials Science and Engineering
學年度 91
學期 1
出版年 92
研究生(中文) 陳文正
學號 n5885103
學位類別 博士
語文別 中文
口試日期 2002-09-16
論文頁數 182頁
口試委員 指導教授-朱建平
指導教授-陳瑾惠
召集委員-尹相姝
口試委員-林立民
口試委員-林瑞模
關鍵字(中) 生醫材料
生物可吸收性
磷酸鈣正鹽類
鈣磷系骨水泥
氫氧基磷灰石
關鍵字(英) bioresorbable
biomaterials
calcium othophosphate
calcium phosphate cement
hydroxyapatite
學科別分類
中文摘要   本研究內容主要是針對鈣磷系骨水泥機械性質、微結構、動物實驗及臨床反應做一系列的探討,本實驗室已發展出非常適合作為生醫植入材的骨水泥,從結果發現nd-CPC在相同的條件下尤其是在反應初期階段比起c-CPC顯示有較為緻密的型態,具較高的磷灰石生成速率,nd-CPC在硬化及浸泡於模擬人工體液過程表現較高的強度。
  反應過程中較後的階段大量Ca/P比接近HA的磷灰石晶體/鬚晶生成並有效的連接DCPA顆粒,藉著這些磷灰石晶體/鬚晶橋接大顆的TTCP顆粒並將其位置固定住,此時CPC達到硬化,顯微結構研究結果發現CPC通常在經過20到40分鐘反應後浸泡於模擬人工體液就不會溶解是因為大量鬚晶的生成將粉末互相連結。
  為了使nd-CPC更為實用,本實驗室以熱處理方式提供了一個熱處理方式(例如160-180℃處理1小時),此方式可以大幅度的改善以TTCP/DCPA為主要材料的鈣磷系骨水泥工作及硬化時間。n-CPC在動物植入方面具有相當高的吸收速率,植入兔子大腿骨24週的吸收率約50%,並且在牙科的臨床方面初步結果顯示並無不良反應有很好的組織相容性。
  總體而言,鈣磷系骨水泥nd-CPC是新一代生醫材料相當好的選擇。
英文摘要   The present work is a study of the mechanical properties, microstructure, animal and clinical study of a series of non-decay calcium phosphate cements. The author’s lab has developed a new calcium phosphate cement and showed excellent properties as a bone substitutes. From the results revealed that the apatite formation rate of nd-CPC was higher than that of c-CPC, especially at early stage. The nd-CPC showed a denser morphology, higher strength than c-CPC both during setting and after immersion in Hanks’ solution.
  During the later stages of reaction, the extensive growth of apatite crystals/whiskers, with a Ca/P ratio very close to that of HA, effectively linked particles together. It is suggested that, when the particles are locked in place by the bridging apatite crystals/whiskers, the CPC is set and would not dissolve when immersed in Hanks' solution after 20-40 min of reaction.
  For practical used, we provide a simple heat treatment method that can largely modify the working/setting time of TTCP/DCPA-derived CPC without using additives or sacrificing its strength. It can be seen that the nd-CPC cement can induce the growth of the natural bone, the resorb ratio was about 50% after 24 weeks implanted. From the result of nd-CPC clinical study applied in dental, no side reaction revealed nd-CPC has good biocompatibility. The nd-CPC has great potential for use as implant material.
論文目次 中文摘要…………………………………………………………………………5
Abstract……………………………………………………………………………6
研究動機…………………………………………………………………………7
總目錄……………………………………………………………………………8
圖目錄……………………………………………………………………………10
表目錄……………………………………………………………………………14
第一章 總緒論…………………………………………………………………15
  1-1 生醫材料沿革簡介……………………………………………………16
  1-2 生醫植入材料分類……………………………………………………22
  1-3 自然骨性質及基本簡介………………………………………………23
  1-4 骨科植入材料性質……………………………………………………24
  1-5 氫氧基磷灰石性質簡介………………………………………………33
  1-6 骨細胞型態……………………………………………………………42

第二章 鈣磷系骨水泥的發展與簡介………………………………………46
  2-1 鈣磷系列生醫陶瓷分類………………………………………………47
  2-2 鈣磷系列生醫陶瓷的發展……………………………………………47
  2-3 四鈣磷酸鹽的水解反應………………………………………………48
  2-4 鈣磷系骨水泥的分類-原料中只含磷酸鈣鹽類………………………50
  2-5 CPC 的崩解性及解決方法……………………………………………51
  2-6 CPC 的特性……………………………………………………………52
  2-7目前CPC 的主要應用…………………………………………………53
  2-8 CPC 的未來發展………………………………………………………54

第三章 非崩解性TTCP/DCPA為主的鈣磷系骨水泥浸泡在模擬
    人工體液(Hanks’ solution)後的結構及性質差異性研究…………59
  3-1 摘要……………………………………………………………………60
  3-2 前言……………………………………………………………………61
  3-3 實驗方法………………………………………………………………64
  3-4 結果與討論……………………………………………………………67
  3-5 結論……………………………………………………………………73

第四章 穿透式電子顯微鏡在TTCP/DCPA為主要材料
    的鈣磷系骨水泥硬化機制研究………………………………………97
  4-1 摘要……………………………………………………………………98
  4-2 前言……………………………………………………………………99
  4-3 實驗方法………………………………………………………………101
  4-4 結果與討論……………………………………………………………102
  4-5 結論……………………………………………………………………106

第五章 熱處理對於TTCP/DCPA為主要材料的鈣磷系骨
    水泥抗壓強度及硬化行為影響的研究………………………………119
  5-1 摘要……………………………………………………………………120
  5-2 前言……………………………………………………………………121
  5-3 實驗方法………………………………………………………………122
  5-4 結果與討論……………………………………………………………123
  5-5 結論……………………………………………………………………126

第六章 生物可吸收性TTCP/DCPA為主要材料的鈣磷系骨水泥在機械
    性質及與其它商用植入材料體內(in vivo)組織反應研究………135
  6-1 摘要……………………………………………………………………136
  6-2 前言……………………………………………………………………137
  6-3 實驗方法………………………………………………………………140
  6-4 結果與討論……………………………………………………………144
  6-5 結論……………………………………………………………………151

參考文獻…………………………………………………………………………167
誌謝………………………………………………………………………………180
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系統識別號 U0026-0812200910365154
論文名稱(中文) 生醫用鈦合金之滑動磨潤性質研究
論文名稱(英文) Wear properties research of biomedical titanium alloys
校院名稱 成功大學
系所名稱(中) 材料科學及工程學系碩博士班
系所名稱(英) Department of Materials Science and Engineering
學年度 91
學期 2
出版年 92
研究生(中文) 楊哲青
學號 n5690124
學位類別 碩士
語文別 中文
口試日期 2003-06-11
論文頁數 89頁
口試委員 指導教授-朱建平
指導教授-陳瑾惠
口試委員-田英俊
關鍵字(中) 生醫材料
鈦合金
磨耗
關鍵字(英) biomaterials
wear
titanium alloys
學科別分類
中文摘要 人體在長期的相對運動下造成磨損,跟著年齡增長,磨耗程度也更加嚴重,在嚴重磨損的情形下,導致嚴重疼痛,或是因為關節的病變發生時,便需要考慮人工關節的置換。
人工髖關節需要承受高應力且耐衝擊,故多選用金屬材料製作,本研究將以實驗室自行開發的高強度低彈性模數鈦合金(Ti-7.5Mo)與目前商用的Ti-6Al-4V與Ti-13Nb-13Zr兩種鈦合金作對陶瓷與金屬的滑動磨潤性質研究,希望能開發出不含毒性元素且具有更優良的機械性質之新型鈦合金。
對磨陶瓷的實驗中,Ti-13Nb-13Zr的耐磨性最差,Ti-6Al-4V有最佳的抗磨性﹔而在金屬對金屬的實驗中,Ti-6Al-4V有明顯優異的抗磨耗表現。實驗中發現,抗磨耗性質最優異的都是硬度最高的Ti-6Al-4V,所以單從磨耗行為來討論,Ti-6Al-4V會是適合作為人工關節元件的材料。
英文摘要 With the increase of age, the joint will get more serious damage. In some badly cases, they should consider total hip joint replacement.
Artificial hip joint require bearing high stress and shocks.Therefore the metal materials are wildly uesed. This research has used high strenghth low modulus titanium alloy(Ti-7.5Mo) and two other commercial titanium alloy Ti-6Al-4V and Ti-13Nb-13Zr to compare their wear properties in M/C and M/M test.
In M/C test Ti-13Nb-13Zr gets worse wear resistance.In M/M test Ti-6Al-4V has most good wear resistance. if only wear resistance was required,Ti-6Al-4V will be the suitable material of artificial joint devices.
論文目次 第一章 前言導論
1-1 研究背景……………………………………………………………1
1-2 研究目的……………………………………………………………3

第二章 理論基礎
2-1 植入物的條件簡介…………………………………………………5
2-2 生醫材料的分類……………………………………………………6
2-2-1 金屬類……………………………………………………………6
2-2-2 陶瓷類……………………………………………………………9
2-2-3高分子類…………………………………………………………10
2-2-4複合材料…………………………………………………………10
2-3鈦合金簡介…………………………………………………………11
2-3-1鈦的基本性質……………………………………………………11
2-3-2鈦合金的分類……………………………………………………13
2-3-3 Ti-Mo合金的基本介紹…………………………………………14
2-4人工髖關節之鬆脫機制……………………………………………16
2-4-1應力遮蔽效應(stress shielding)……………………………16
2-4-2 骨溶解效應(osteolysis) ……………………………………17
2-5磨潤機制……………………………………………………………19
2-5-1黏著(Adhesion wear)………………………………………20
2-5-2 研磨(Abrasion wear)…………………………………………22
2-5-3 轉移(transfer wear)…………………………………………23
2-5-4疲勞(fatigue wear)……………………………………………23
2-5-5第三顆粒(third body wear) …………………………………23
2-6潤滑(lubriction)條件的影響……………………………………24
2-7磨耗殘屑引起之生理機制…………………………………………26

第三章 實驗流程與方法
3-1實驗步驟……………………………………………………………28
3-2試片製備……………………………………………………………28
3-2-1鈦合金試片製備…………………………………………………28
3-2-2金屬試片的熔煉及鑄造…………………………………………30
3-2-3釔安定氧化鋯陶瓷片的製備……………………………………33
3-2-4試件機械加工成形………………………………………………36
3-3磨耗試驗……………………………………………………………38
3-3-1磨耗機簡介………………………………………………………38
3-3-2實驗條件…………………………………………………………41
3-3-3實驗步驟…………………………………………………………41
3-4顯微結構分析………………………………………………………43
3-4-1 掃描式電子顯微鏡(Scanning Electron Microscopy, SEM)43
3-4-2 X光晶體繞射儀(X-Ray Diffraction) ………………………43
3-5機械性質分析………………………………………………………44
3-5-1表面粗糙度分析…………………………………………………44
3-5-2微硬度分析………………………………………………………45
3-6表面化學分析………………………………………………………45
3-6-1電子能譜化學分析………………………………………………45

第四章 結果與討論
4-1金屬對陶瓷實驗結果………………………………………………47
4-1-1 X光繞射分析……………………………………………………47
4-1-2磨耗量與摩擦係數………………………………………………49
4-1-3磨耗面粗糙度測量………………………………………………53
4-1-4磨耗面硬度測量…………………………………………………56
4-1-5磨耗面SEM觀察 …………………………………………………56
4-1-6磨屑SEM觀察 ……………………………………………………58
4-1-7表面化學分析……………………………………………………61
4-1-8金屬對陶瓷實驗討論……………………………………………64
4-2金屬對金屬實驗結果………………………………………………65
4-2-1磨耗量與摩擦係數………………………………………………65
4-2-2磨耗面粗糙度測量………………………………………………68
4-2-3磨耗面硬度測量…………………………………………………72
4-2-4磨耗面SEM觀察…………………………………………………72
4-2-5磨屑SEM觀察……………………………………………………75
4-2-6表面化學分析……………………………………………………77
4-2-7金屬對金屬實驗討論……………………………………………79

第五章 結論
5-1金屬對陶瓷磨耗實驗結論…………………………………………81
5-2金屬對金屬磨耗實驗結論…………………………………………82

第六章 參考資料………………………………………………………83
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系統識別號 U0026-0812200914060344
論文名稱(中文) 神經元介面微電極陣列之製造與體外特性描述
論文名稱(英文) Fabrication and In-Vitro Characterization of Microelectrode Array for Neuronal Interfaces
校院名稱 成功大學
系所名稱(中) 醫學工程研究所碩博士班
系所名稱(英) Institute of Biomedical Engineering
學年度 96
學期 1
出版年 97
研究生(中文) 林淑萍
學號 p8892104
學位類別 博士
語文別 英文
口試日期 2008-01-17
論文頁數 93頁
口試委員 指導教授-陳家進
口試委員-婁世亮
口試委員-廖峻德
口試委員-曾淑芬
口試委員-曾清俊
口試委員-林克忠
關鍵字(中) 三維細胞培養基質
多點式神經元介面微電極
生物相容性
降解性材料
表面改質
阻抗量測
自我排列單分子膜
關鍵字(英) self-assembled monolayers
degradable biomaterials
biocompatibility
3D cell matrix
impedance measurement
surface modification
MEA
學科別分類
中文摘要 本研究主要是利用體外分析量測方法探討表面改質之神經元介面多點式微電極於未來體內植入為需求之應用。藉由微機電製程的技術(MEMS techniques)製造出兩種不同以聚亞醯胺(PI)為基底之多點式神經元介面的金微電極,包括培養皿式平面微電極(Petri-dishlike planar MEA)及軟性神經植入式電極(flexible neural implant)。利用MUA的自我排列單分子膜(SAMs)進行培養皿式微電極的表面改質處理。為了增加培養皿式微電極的生物相容性,選用具有生物活性的分子—PDL固定於經MUA自我排列單分子膜修飾過的電極上。紅外線光譜儀(FTIR)的頻譜顯示其表面處理後具有特殊的共價鍵結(covalent bonding)化學官能基,其分別為醯胺I鍵結(1,613 cm-1)及醯胺II鍵結(1,548 cm-1)。以阻抗量測儀測量未經表面改質與經表面改質過後培養皿式平面微電極的阻抗變化,發現改質過後的電極阻抗從原本的352.9 ± 34.4 kΩ稍微升高至524.6 ± 55.8 kΩ(p<0.05, N=20)。此外,經由七天連續量測隨時間變化之總阻抗(由實部電阻及虛部阻抗值組成)實驗中,可以進一步推得細胞株是否健康生長接觸於電極上。藉由細胞株及活體細胞培養於修飾過後電極上,證實有非常良好的生長結果。在另一方面,軟性神經植入式電極因為具有較小的硬度,所以在植入後可以順應組織的變化以減少不必要的組織傷害。然而,軟性神經植入式電極因為有彎曲效應以致於無法穿透軟性組織。因此,PDL混合於聚乙二醇凝膠(PEG)合成之PEGDL凝膠,可以應用於批覆於軟性神經植入式電極上用以增加電極的硬度。利用紅外線光譜儀及掃瞄式電子顯微鏡(SEM)分析凝膠的化學鍵結成分、降解反應及凝膠表面的觀察。這些體外分析顯示均質地披覆凝膠於軟性神經植入式電極除了可以增加電極的硬度,而且披覆的凝膠也會隨著時間產生降解。另外,所披覆的PEGDL凝膠於體外細胞測試結果顯示具有很良好的生物相容性。掃瞄式電子顯微鏡分析顯示額外的生物活性的分子—laminin於PEGDL凝膠中可以提供神經細胞更良好的生長環境。生化測試顯示神經細胞在軟性神經植入式電極以及披覆凝膠之軟性神經植入式電極具有神經細胞固有的功能特性。所以,PEGDL凝膠披覆可以提供軟性神經植入式電極足夠的硬度及增加其生物相容性。最後,本研究亦探討結合培養皿式平面微電極與PEGDL凝膠,進一步用阻抗量測探討細胞在三維空間下的動態特性描述。在連續八天之動態阻抗量測實驗中,可推估細胞在三維結構空間下生長的情形。另外,一系列的生物相容性測試驗證此一三維細胞培養基質不但可以提供一適合細胞生長的環境。亦可以在平面電極上架構一仿生性的系統以提供未來有效電生理刺激量測之計畫及診斷檢驗。
英文摘要 This study aims to investigate surface-modified microelectrodes on the microelectrode arrays (MEAs) for neuronal interfaces with in vitro cell culture. Two kinds of polyimide (PI) MEA, including Petri-dishlike planar MEA and flexible neural implant (NI), were fabricated by using micro-electro-mechanical systems (MEMS) techniques. Self-assembled monolayers (SAMs) of 11-mercaptoundecanoic acid (MUA) were utilized to modify the microelectrode surface of the planar MEA. To increase biocompatibility, the poly-D-lysine (PDL) was immobilized on the SAMs' modified microelectrodes. Spectra of the Fourier transform infrared reflection (FTIR) revealed that covalent amide bonding, amide I (at 1,613 cm-1) and amide II (at 1,548 cm-1), existed in PDL-MUA-SAMs modified surfaces. The impedance measurement of PDL-MUA-SAMs modified MEA only increased slightly to an average of 524.6 ± 55.8 kΩ from 352.9 ± 34.4 kΩ of bare gold microelectrode (p<0.05, N=20). In order to infer the growth of cell lines on the electrode contact of modified MEA, the time-course changes of total impedance resulting from cell sealing resistance and gap reactance were recorded for 7 days. The experiment of cell-line and primary-cell culture on the modified MEAs displayed a good adhesion rate.
On the other hand, flexible NIs with reduced stiffness are desirable for future implantation applications. Implantable NIs require the flexibility to conform to tissue movement, but they can easily affected by bending effect on mechanical properties and subsequently render them incapable of penetrating soft tissues. Poly(ethylene glycol) hydrogel supplemented with PDL (PEGDL) as a coat for flexible NIs was synthesized to enhance the stiffness of material surface. The PEGDL-coated and uncoated NIs were compared the mechanical properties by tensiometry. FTIR microscopy and scanning electron microscopy (SEM) were employed to characterize the chemical bonding, degradation, and surface topography of the PEGDL coat. These analyses indicated that a homogeneous coating increased the stiffness of NIs and the coating can be degraded over time in in-vitro tests. The ability of our coated implants to support the growth and differentiation of NIH3T3 fibroblasts and cortical neurons in vitro was examined as a measure of biocompatibility and found to be excellent. Furthermore, Western blot analysis of cell lysates from neurons treated with glutamate indicated proper neuronal function on our engineered material. Our observations indicate that PEGDL coating exhibits an increase in stiffness and improvement in biocompatibility.
Finally, the combination of PEGDL on surface-modified planar MEA was constructed to characterize the cell growth on 3D matrix MEA by using impedance measurement. The time-course changes of total impedance, cell sealing resistance, and gap reactance were recorded for 8 days for inferring the growth of cells in the 3D culture hydrogel system on the MEA. In addition, serial biocompatibility assays demonstrated that 3D matrix can not only provide a good environment for cell growth but also construct a biomimetic system on our planar MEA for effective electrophysiological stimulation/sensing schemes or diagnostic examinations in the future.
論文目次 Qualified certification of oral defense ..................I
Chinese abstract .......................................III
Abstract..................................................V
Acknowledgements in Chinese............................ VII
Contents ..............................................VIII
List of Tables.........................................XIII
List of Figures........................................ XIV

Chapter 1 Introduction................................... 1
1.1 Introduction of MEA.................................. 1
1.2 Cogitation of MEA.....................................3
1.3 Surface modification – self-assembled monolayers ....3
1.4 Biocompatibility .................................... 5
1.5 Impedance measurement................................ 6
1.6 Considerations of flexible interface................. 7
1.7 Aims of this study ................................. 10

Chapter 2 Materials and Methods......................... 12
2.1 Fabrication of MEA ................................. 12
2.2 Characterization of surface-modified planar MEA..... 14
2.2.1 Surface modification for planar MEA .............. 14
2.2.2 FTIR analysis .................................... 15
2.2.3 Surface topography for planar MEA................. 16
2.3 Impedance evaluation of surface-modified planar MEA..16
2.4 Assessments of biocompatibility of planar MEA ...... 18
2.4.1 Preparation of cell lines and primary cultures.... 18
2.4.2 Cell adhesion rate and growth assays ............. 19
2.5 PEGDL synthesis and flexible NI electrode coating... 20
2.6 Characterization of hydrogels ...................... 22
2.6.1 Mechanical test................................... 22
2.6.2 FTIR analysis .................................... 22
2.6.3 Evaluation of degradation......................... 22
2.7 Biocompatibility of hydrogel........................ 23
2.8 Western blot for neuronal function.................. 25
2.9 Application of hydrogel on planar MEA .............. 26
2.10 Characterization of 3D matrix on planar MEA ....... 27
2.10.1 Quantification of degradation of 3D matrix....... 27
2.10.2 FTIR analysis for degraded 3D matrix............. 28
2.10.3 Swelling test and degraded area evaluation of 3D matrix.................................................. 28
2.11 Cell growing on 3D matrix MEA ..................... 28
2.11.1 Cell culture..................................... 28
2.11.2 Cell viability assay............................. 29
2.11.3 Fluorescent observation.......................... 29
2.11.4 Impedance measurement for 3D matrix MEA.......... 30

Chapter 3 Results....................................... 32
3.1 Appearance of MEA .................................. 32
3.2 Quantification of chemical bonding on surface-modified Petri-dishlike MEA. .................................... 33
3.3 Quantifying the surface-modified Petri-dishlike MEA..36
3.4 Impedance evaluation for cell lines cultured on modified Petri-dishlike MEA. ........................... 38
3.5 Biocompatibility studies for Petri-dishlike MEA..... 41
3.6 Synthesis and characterization of hydrogel.......... 44
3.6.1 Synthesis of hydrogel ............................ 44
3.6.2 Mechanical property............................... 45
3.6.3 Characterization of chemical bonding on PEGDL-coated NI ..................................................... 47
3.6.4 Qualitative and quantitative analysis of degradation of hydrogel ............................................ 49
3.7 Assessments of biocompatibility for physical coating NI ..................................................... 51
3.8 Biochemical assay for neuronal function ............ 56
3.9 Characterization of a 3D cell culture hydrogel in in-vitro planar MEA........................................ 57
3.9.1 Evaluation of degradation of 3D matrix ........... 57
3.9.2 Characterization of chemical bonding of degraded 3D matrix ................................................. 58
3.9.3 Swelling test and degraded-area evaluation of 3D matrix ................................................. 59
3.10 Cell viability and fluorescent image on 3D matrix MEA .................................................... 61
3.11 Impedance evaluation for 3D matrix on MEA ......... 65

Chapter 4 Discussion ................................... 71

Chapter 5 Conclusions................................... 79

References.............................................. 81

Appendix................................................ 91
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系統識別號 U0026-1108201013204200
論文名稱(中文) 以力學觀點評估傷口注射膠原蛋白、透明質酸及明膠對皮膚癒合的影響
論文名稱(英文) Mechanical Evaluations of the Influence of Collagen, Hyaluronan and Gelatin Injections on Skin Wound Healing
校院名稱 成功大學
系所名稱(中) 醫學工程研究所碩博士班
系所名稱(英) Institute of Biomedical Engineering
學年度 98
學期 2
出版年 99
研究生(中文) 陳慶頤
學號 p8697421
學位類別 碩士
語文別 中文
口試日期 2010-07-15
論文頁數 72頁
口試委員 口試委員-黃玲惠
口試委員-林真福
口試委員-吳建一
指導教授-葉明龍
關鍵字(中) 傷口癒合
生物性材料
拉伸強度
膠原蛋白
明膠
透明質酸
關鍵字(英) Wound Healing
Biomaterials
Tensile Strength
Collagen
Hyaluronan
Gelatin
學科別分類
中文摘要 術後加速傷口癒合且減小疤痕大小一直是學者積極努力的目標。在過去,已有多種物理性刺激因子與敷傷材料被研究其對傷口癒合的影響;然而,鮮少研究以皮膚的力學性質作為傷口癒合指標。多種生物性材料已作為臨床上的使用,本研究使用大鼠傷口模型,將膠原蛋白(Collagen)、明膠(Gelatin)與透明質酸(HA)與其複合物等不同生物性材料注射於縫合的傷口,在1、2、4、6與8週分別以拉力測試測量皮膚拉伸強度作為皮膚癒合指標、使用組織切片染色了解癒合組織生長情況以及膠原蛋白定量觀察胞外基質的生成情況。
結果發現材料注射會有不同的疤痕外觀。癒合初期傷口在在單一材料注射會有縫線疤痕;複合材料則是有較大且向內微凹的傷疤。癒合晚期則複合材料有較相似於正常皮膚之外觀。力學性質恢復得知,HA較所有材料注射組有較高的力學性質恢復情況,其他組彼此間均無其差異性。組織切片觀察顯示,單一材料有較快的表皮恢復情況且真皮層組織內部結構較為緻密;複合材料則是具有較於相似正常皮膚真皮組織細部的構型。於組織內膠原蛋白生成量比例結果發現,除單一材料於癒合時期2週Gelatin與HA組有較高的分泌量之外,彼此間無明顯差異。
綜合以上結論發現,單一材料對於癒合初期表皮癒合與再上皮化程度提供顯著效果;癒合初期具有較佳的傷口密合,癒合晚期擁有較佳的皮膚拉伸強度與恢復情況,但存在隱約縫線的縫線疤痕。複合材料作用效果可能著重於癒合後期真皮結構的加速新生與重組。
英文摘要 Quick and smooth skin healing is the goal in wound healing researches. In the past, several physical stimulating factors and wound dressing materials have been studied for their influences on wound healing. However, only few studies investigated the mechanical recovery of healing wound. Several biological materials have been used in clinical practice. Natural materials such as Type I collagen, Hyaluranon (HA), gelatin and their composite are similar to the composition of natural skin and have beneficial potential in cell proliferation, differentiation and ECM synthesis at wound sites. So far, no research has reported the mechanical recovery of the healing skin by injecting those natural biomaterials directly into the wound site. The aim of this study was to investigate the mechanical recovery influences of injectable biomaterials such as collagen, HA, gelatin and their composite on incisional wound injury in rat model. Tensile strength of healing skin was measured at 1, 2, 4, 6 and 8 weeks after incision; than, the correlated recovery index was calculated. The architecture of skin-wound was observed with histological section and collagen synthesis was measured.
The results showed that different biomaterial injection provided different scar pattern. Mono biomaterial groups had suture-scar pattern and mixed biomaterial groups had sunken scar pattern. The HA group had the best recovery results of mechanical properties compared with other groups. Histological observations exhibited that mono biomaterial groups had faster epidermis recovery and more compact dermis architecture. The mixed group had similar dermis architecture to normal skin. Collagen synthesis ratio showed that Gelatin and HA group had a higher collagen content at 2 weeks of healing time. Overall, mono biomaterial had significant effects on epithelial wound healing and re-epithelialization during early stage. In the late stage, the skin had a better tensile strength and recovery index, but there were slight suture-scar at wound site. Effect of mixed biomaterials might focus on regeneration and remodeling of the architecture of skin-wound.
論文目次 中文摘要 I
Abstract II
致謝 IV
目錄 VI
圖目錄 VIII
表目錄 X
第一章 緒論 1
1.1皮膚的結構、組成與功能 1
1.1.1表皮層 (Epidermis layer) 2
1.1.2真皮層 (Dermis layer) 3
1.1.3皮下組織 (Hypodermis layer) 5
1.2實驗鼠與人類皮膚結構的差異性 7
1.3皮膚的傷口癒合過程 8
1.3.1發炎期 (Inflammatory phage) 8
1.3.2生長期 (Proliferation phase) 9
1.3.3成熟期 (Maturation phase) 10
1.4傷口修復之動物模型 10
1.4.1急性傷口模型(Acute wound models) 11
1.4.2慢性傷口模型(Chronic wound models) 11
1.4.3癒合能力受損模型(Impaired healing models) 11
1.5參與傷口癒合之細胞外基質-膠原蛋白、明膠與透明質酸 12
1.5.1膠原蛋白(Collagen) 12
1.5.2明膠(Gelatin) 14
1.5.3透明質酸(Hyaluronic acid or Hyaluronan) 15
1.6相關文獻探討 17
1.6.1實驗變數因子正規化與實驗流程標準化 17
1.6.2透明質酸對傷口癒合之影響 18
1.6.3膠原蛋白對傷口癒合之影響 19
1.6.4明膠對傷口癒合之影響 19
1.7研究動機 20
1.8研究目的 20
第二章 材料與方法 21
2.1實驗設計 21
2.2實驗材料與設備 23
2.2.1生物性材料 23
2.2.2試片裁切器與夾具 23
2.2.3拉力測試機台 24
2.2.4照相擷取系統 24
2.3實驗方法 25
2.3.1動物實驗 25
2.3.2標準試片準備 26
2.3.3抗拉強度試驗 28
2.3.4膠原蛋白成分分析 29
2.3.5組織切片染色 29
2.3.6統計分析 30
第三章 結果 31
3.1實驗一部分-單一材料 31
3.1.1不同癒合時間之傷口外觀 31
3.1.2不同單一生物性材料與不同癒合時間對癒合皮膚的影響 32
3.1.3組織切片觀察 38
3.1.4組織內膠原蛋白生合成變化量之觀察 41
3.2實驗第二部分-複合材料 42
3.2.1不同癒合時間之傷口外觀 42
3.2.2 不同複合生物性材料與不同癒合時間對癒合皮膚的影響 43
3.2.3組織切片觀察 49
3.2.4組織內膠原蛋白生合成變化量之觀察 52
3.3 單一生物性材料 與 複合生物性材料之結果 比較 53
3.3.1 不同材料對癒合傷口外觀之影響 53
3.3.2 不同癒合時間之癒合皮膚抗拉強度與癒合皮膚癒合指數比較 55
3.3.3 組織切片觀察 58
3.3.4組織內膠原蛋白生合成變化量之觀察 61
第四章 討論 63
4.1不同材料對癒合傷口外觀之觀察 63
4.2不同材料對皮膚力學性質恢復之影響 63
4.3不同材料對癒合皮膚組織內部之影響 64
4.4不同材料對組織內膠原蛋白生合成變化量之影響 65
4.5實驗限制 66
4.5.1實驗對象選擇與樣本標準化 66
4.5.2 動物模型與傷口模型的選擇 66
4.5.3 皮膚試片的處理 67
4.6未來研究 67
第五章 結論 69
參考文獻 70
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系統識別號 U0026-1502201109114700
論文名稱(中文) 不同缝線技術、矽凝膠、遠紅外線照射與生醫材料對大鼠皮膚癒合力學性質的影響
論文名稱(英文) Impact of Different Suturing Methods, Topical Application of Silicone Gel, Far-infrared Ray Exposure and Biomaterial Injections on the Mechanical Properties of Healing Skin in Rats
校院名稱 成功大學
系所名稱(中) 醫學工程研究所碩博士班
系所名稱(英) Institute of Biomedical Engineering
學年度 99
學期 1
出版年 100
研究生(中文) 楊正三
學號 p8896117
學位類別 博士
語文別 英文
口試日期 2011-01-21
論文頁數 71頁
口試委員 指導教授-葉明龍
口試委員-黃玲惠
口試委員-徐阿田
口試委員-謝式洲
口試委員-陳嘉炘
口試委員-林真福
口試委員-林鼎勝
關鍵字(中) 遠紅外光
可注射生物材料
恢復指數
矽凝膠
縫合方法
抗拉強度
傷口癒合
關鍵字(英) Far-infrared ray (FIR)
Injectable biomaterials
Recovery index (RI)
Silicone gel
Suture methods
Tensile strength (TS)
Wound healing
學科別分類
中文摘要 傷口癒合的過程是一個複雜的生物反應,包括發炎,增生,和重塑的階段。在過去研究中,許多物理和化學治療方法被大量研究,企圖找出影響傷口癒合的機制和改善癒合的關鍵因子。然而,大部分的研究以外觀和組織觀察,來評估治療方法的效用,卻少有研究,使用抗拉強度換算的癒合指數來量化傷口癒合的品質,其中癒合指數可用來正規化不同的生長條件的老鼠。因此,我們提出了一連串的研究,在大鼠切口模型來探討縫合方法、矽凝膠、遠紅外線照射、注射生物材料(膠原蛋白,透明質酸,明膠)的影響。在Sprague-Dawley大鼠背部分4個區域,其中1處作為控制組,其他3處切2公分的傷口並縫合,作為實驗組。經過不同的治療方法干預後,在1,2,4,6週將皮膚取下作拉伸測試和H&E染色。結果顯示,簡單的縫合方式、矽凝膠治療,對皮膚癒合過程有較佳的力學性質。而在前兩週的遠紅外線治療,可增加皮膚的抗拉強度。總而言之,其結果表明,縫線材料越穩固、縫合方法越簡單,癒合傷口的力學性能就越大。此外,矽凝膠治療組相較於較未治療組,其發炎細胞較少,表皮有更好的完整性在,以及較好的力學強度。遠紅外線能提供短期的力學強度,但其效果在較長時間則減弱。另外,玻尿酸注射組比膠原蛋白和明膠有較高的力學強度。
英文摘要 The wound healing process is a set of complicated and concurrent biological responses, which includes inflammation, proliferation, and remodeling phases. In the past, many physical and chemical treatments have been intensively studied in order to find the key factors that affect the mechanisms of wound healing and to improve healing. However, most of these studies used gross appearances and histology observations to evaluate the efficacy of the novel treatments. Only limited studies quantified the healing process using the mechanical recovery index (RI), which was introduced to normalize the different growth conditions of individual rats. Therefore, this study proposed a series of studies that investigated the mechanical recovery influences of suture methods, topical silicone gel, far-infrared ray (FIR) exposure, and injectable biomaterials (collagen, hyaluronic acid (HA), and gelatin) in rats used as models of incision injury. The dorsum of each the Sprague-Dawley rats was divided into four regions. The rats were randomly assigned, with one group of rats assigned to the normal group and the others to experimental groups with two centimeter long cuts and full-thickness skin suture closures. Following different treatment protocols, the skins were harvested for tensile testing and H&E stain at 1, 2, 4, and 6 weeks of healing time. The results showed that the simple suture method, the use of silicon gel, and HA injection groups provided better mechanical properties for the healing courses. FIR increased the tensile strength (TS) 2 weeks after injury. In conclusion, the results of suture methods indicated that the more supportive the suture materials are and the simpler the suture method is, the higher the mechanical property it will have. Besides, the wound treated with topical silicone gel had fewer inflammatory cells, higher integrity in the epidermis, and better mechanical strength than untreated groups. The FIR exposure enhances the RI of skin in the short term, but its effect diminished in the longer period. HA injection groups had higher mechanical strength than collagen and gelatin groups.
論文目次 ABSTRACT III
中文摘要 V
ACKNOWLEDGEMENTS VI
TABLE OF CONTENTS VII
LIST OF TABLES IX
LIST OF FIGURES X
1. BACKGROUNDS 1
1.1 WOUND HEALING PROCESS 1
1.1.1 Inflammatory phase 1
1.1.2 Proliferation phase 3
1.1.3 Remodeling phase 3
1.2 TYPES OF WOUND MODELS 3
1.3 LITERATURE REVIEWS 5
1.3.1 The Effect of Suture Methods on Wound Healing 5
1.3.2 The Effect of Silicone Gel on Wound Healing 7
1.3.3 The Effect of FIR on Wound Healing 9
1.3.4 The Effect of Biomaterial Injections on Wound Healing 10
1.4 PURPOSE OF STUDY 13
2. MATERIALS AND METHODS 15
2.1 ANIMAL EXPERIMENT 15
2.1.1 Different Suture Methods 15
2.1.2 Topical Silicone Gel 16
2.1.3 FIR 17
2.1.4 Biomaterial Injections 18
2.2 SOFT TISSUE TENSILE TEST 19
2.3 HISTOLOGY 21
2.4 STATISTICAL ANALYSIS 21
3. RESULTS 23
3.1 DIFFERENT SUTURE METHODS 23
3.2 TOPICAL SILICONE GEL 27
3.3 FIR EXPOSURE 33
3.3.1 Mechanical property of wound treated with FIR 33
3.3.2 Characteristics of Rats 36
3.3.3 TS of Unwound Skin treated with FIR 37
3.3.4 Histology 38
3.4 BIOMATERIAL INJECTIONS 41
3.5 RI FOR ALL TREATMENTS 45
4. DISCUSSION 47
4.1 DIFFERENT SUTURE METHODS 47
4.2 TOPICAL SILICONE GEL 48
4.3 FIR EXPOSURE 50
4.4 BIOMATERIAL INJECTIONS 54
4.5 LIMITATIONS 56
5. CONCLUSIONS 58
6. FUTURE WORKS 60
7. REFERENCES 61
8. BIOGRAPHY 67
9. PUBLICATION LIST 68
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18. Chan KY, Lau CL, Adeeb SM, Somasundaram S, Nasir-Zahari M. A randomized, placebo-controlled, double-blind, prospective clinical trial of silicone gel in prevention of hypertrophic scar development in median sternotomy wound. Plastic and reconstructive surgery. Sep 15 2005;116(4):1013-1020; discussion 1021-1012.
19. Murison M, James W. Preliminary evaluation of the efficacy of dermatix silicone gel in the reduction of scar elevation and pigmentation. J Plast Reconstr Aesthet Surg. 2006;59(4):437-439.
20. Chernoff WG, Cramer H, Su-Huang S. The efficacy of topical silicone gel elastomers in the treatment of hypertrophic scars, keloid scars, and post-laser exfoliation erythema. Aesthetic plastic surgery. Sep-Oct 2007;31(5):495-500.
21. Signorini M, Clementoni MT. Clinical evaluation of a new self-drying silicone gel in the treatment of scars: a preliminary report. Aesthetic plastic surgery. Mar-Apr 2007;31(2):183-187.
22. Lacarrubba F, Patania L, Perrotta R, Stracuzzi G, Nasca MR, Micali G. An open-label pilot study to evaluate the efficacy and tolerability of a silicone gel in the treatment of hypertrophic scars using clinical and ultrasound assessments. The Journal of dermatological treatment. 2008;19(1):50-53.
23. Mustoe TA. Evolution of silicone therapy and mechanism of action in scar management. Aesthetic plastic surgery. Jan 2008;32(1):82-92.
24. Mercer NS. Silicone gel in the treatment of keloid scars. British journal of plastic surgery. Jan 1989;42(1):83-87.
25. Ishibashi J, Yamashita K, Ishikawa T, et al. The effects inhibiting the proliferation of cancer cells by far-infrared radiation (FIR) are controlled by the basal expression level of heat shock protein (HSP) 70A. Med Oncol. 2008;25(2):229-237.
26. Udagawa Y, Nagasawa H. Effects of far-infrared ray on reproduction, growth, behaviour and some physiological parameters in mice. In Vivo. Mar-Apr 2000;14(2):321-326.
27. Inoue S, Kabaya M. Biological activities caused by far-infrared radiation. Int J Biometeorol. Oct 1989;33(3):145-150.
28. Toyokawa H, Matsui Y, Uhara J, et al. Promotive effects of far-infrared ray on full-thickness skin wound healing in rats. Exp Biol Med (Maywood). Jun 2003;228(6):724-729.
29. Al-Watban FA. Laser Therapy Converts Diabetic Wound Healing to Normal Healing. Photomed Laser Surg. Feb 4 2009;27(1):127-135.
30. Yu SY, Chiu JH, Yang SD, Hsu YC, Lui WY, Wu CW. Biological effect of far-infrared therapy on increasing skin microcirculation in rats. Photodermatol Photoimmunol Photomed. Apr 2006;22(2):78-86.
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33. Huang-Lee LL, Nimni ME. Crosslinked CNBr-activated hyaluronan-collagen matrices: effects on fibroblast contraction. Matrix Biol. Mar 1994;14(2):147-157.
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35. Singer AJ, Clark RA. Cutaneous wound healing. The New England journal of medicine. Sep 2 1999;341(10):738-746.
36. Lee SB, Jeon HW, Lee YW, et al. Bio-artificial skin composed of gelatin and (1-->3), (1-->6)-beta-glucan. Biomaterials. Jun 2003;24(14):2503-2511.
37. Courts FJ. Standardization and calibration in the evaluation of clinical performance. Journal of dental education. Dec 1997;61(12):947-950.
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系統識別號 U0026-1708201115374500
論文名稱(中文) 以力學觀點評估於術後不同時間點傷口注射透明質酸及明膠複合物對皮膚癒合的影響
論文名稱(英文) Mechanical Evaluation of the Influence of Hyaluronan and Gelatin Mixture Injections on Skin Wound Healing with Different Post Operation Time
校院名稱 成功大學
系所名稱(中) 醫學工程研究所碩博士班
系所名稱(英) Institute of Biomedical Engineering
學年度 99
學期 2
出版年 100
研究生(中文) 許哲維
學號 p86981203
學位類別 碩士
語文別 英文
口試日期 2011-07-27
論文頁數 58頁
口試委員 指導教授-葉明龍
口試委員-黃玲惠
口試委員-陳嘉炘
口試委員-粘譽薰
關鍵字(中) 傷口癒合
生物性材料
拉伸強度
明膠
透明質酸
關鍵字(英) Wound Healing
Biomaterials
Tensile Strength
Hyaluronic Acid
Gelatin
學科別分類
中文摘要 術後加速傷口癒合且減小疤痕大小一直是學者積極努力的目標。現今已有多種物理性刺激因素與敷傷材料被研究於動物實驗或臨床對傷口癒合的影響。然而,鮮少研究是以皮膚的力學性質恢復的客觀分析方法作為傷口癒合指標並。目前,多種生物性材料已在臨床上被廣泛使用,本研究使用Sprague Dawley大鼠手術切口模型,將明膠(Gelatin)與透明質酸(HA)兩種生物性材料之複合物,於手術後不同時間點(0, 7, 0&7天組)注射於縫合的傷口,在2、4、6、8與12週分別以力學拉伸測試量測皮膚拉伸強度作為傷口癒合指標,並使用組織切片染色(H&E staining, Alcian blue staining pH2.5)分析組織癒合情況。
由力學測試結果發現,0天組在6週後期力學性質恢復能力有趨緩的現象,而7天及0&7天組有隨癒合時間而逐漸增加之趨勢,癒合後期的恢復情況亦較0天組好。由組織切片觀察顯示,0&7天組在癒合時期12週有比較小的傷口範圍,較類似於正常皮膚的真皮結構與細部排列;7天組在癒合時期12週則有比較鬆散的透明質酸分布情形及較快的真皮組織恢復。
綜合以上實驗結果發現,適當延後施打複合材料的時間,對於癒合時期後期,具有比較好的皮膚拉伸強度及恢復情形,以及比較好的真皮層內部細部組織結構。
英文摘要 Quick and smooth skin healing is the goal in wound healing researches. In the past, several researches had focused on a variety of physical stimulation factors and the materials of wound dressing to promote wound healing. Nowadays, a variety of natural materials are used in clinical practice, such as collagen, gelatin and hyaluronic acid (HA) etc. which are close to the composition of these artificial skin to deal with the healing processes including cell differentiation, proliferation and secretion of extracellular matrix and other mechanisms to promote wound healing. The main purpose of this study was to evaluate the effects by different post-operation injection time (0day, 7day 0&7day groups) of HA and gelatin mixture biomaterials in skin healing at rat incision model. Mechanical tensile test and histological analysis (H&E staining and Alcian blue staining) of the healing skin were conducted at the healing time at 2, 4, 6, 8, and 12 weeks after the surgery.
The results for mechanical property showed that at the late stage of healing time, the 0day group’s mechanical property was gradually slow down after 6 weeks of healing time. The 7day and 0&7day groups continued to increase the mechanical property, and had better recovery situation with the healing time up to 12 weeks compared with the 0day group. The H&E stain analysis showed that the 0&7day group had a relatively smaller wound area, and a better dermis structure and arrangement. The Alcian blue stain analysis showed that the 7day group may have a loosely hyaluronic acid distribution and faster rate for dermis regeneration at the 12 weeks of healing time. Overall, delay the time of mixture biomaterial injection after the surgery, may have a better performance in mechanical property recovery, regeneration, reconstruction, and micro-integration to dermis structure recovery at later stage of wound healing, which compared to injection the same biomaterial instantly.
論文目次 中文摘要 I
Abstract II
致謝 IV
Content VI
Figure Contents IX
Table Contents X
Chapter 1 Introduction 1
1.1 The Structure of Skin 1
1.1.1 Epidermis Layer 2
1.1.2 Dermis Layer 4
1.1.3 Hypodermis Layer 6
1.2 The Difference of Skin Structure between Human and Rats 8
1.3 Processes of Skin Wound Healing 9
1.3.1 Inflammatory Phase 10
1.3.2 Proliferation Phase 11
1.3.3 Maturation Phase 12
1.4 Animal Models of Wound Healing 13
1.4.1 Acute Wound models 13
1.4.2 Chronic Wound Models 14
1.4.3 Impaired Healing Wound 14
1.5 Extracellular Matrix Participated in Wound Healing – Gelatin, Hyaluronic Acid 14
1.5.1 Gelatin 14
1.5.2 Hyaluronic Acid or Hyaluronan 15
1.6 Related Literature 17
1.6.1 Normalization of Experimental Variable Factors and Standardization of Experiment Procedure 17
1.6.2 The effects of HA to Wound Healing 19
1.6.3 The effects of Gelatin to Wound Healing 20
1.7 Motivation 21
1.8 Aim of Study 21
Chapter 2 Materials and Methods 22
2.1 Flow Chart of Experiment 22
2.2 Experiment Materials and Equipments 24
2.2.1 Biomaterials 24
2.2.2 Punchers and Clamps for Samples 24
2.2.3 Mechanical Testing System 25
2.2.4 Image Capture System 25
2.3 Experiment Methods 26
2.3.1 Animal Experiment 26
2.3.2 Preparation of Standard Specimen 27
2.3.3 Mechanical Tensile Test 29
2.3.4 Histological Staining 31
2.3.5 Statistical Analysis 32
Chapter 3 Results 33
3.1 Morphology of Healing Wounds 33
3.2 Influence of Biomaterial Injections on Skin Wound Healing with Different Post-Surgery Time 34
3.2.1 Mechanical Tensile Strength 34
3.2.2 Recovery Index 38
3.3 Observation of Histological Analysis 43
3.3.1 Hematoxylin & Eosin Staining 43
3.3.2 Alcian Blue staining, pH2.5 47
Chapter 4 Discussion 51
4.1 Influence of Biomaterial Injections on Wound Morphology with Different Post-Surgery Time 51
4.2 Influence of Biomaterial Injection Time on Recovery of Skin Mechanical Property 51
4.4 Experimental Limitations 54
4.4.1 Processing and normalization of skin specimens 54
4.4.2 Selection of animal model and wound model 55
4.5 Future Work 55
Chapter 5 Conclusion 56
References 57
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系統識別號 U0026-1807201113321600
論文名稱(中文) 利用自組裝胜肽奈米纖維進行梗塞後心血管再生
論文名稱(英文) Tissue Engineering Approaches to Post-infarction Cardiovascular Regeneration Using Self-assembling Peptide Nanofibers
校院名稱 成功大學
系所名稱(中) 醫學工程研究所碩博士班
系所名稱(英) Institute of Biomedical Engineering
學年度 99
學期 2
出版年 100
研究生(中文) 林意棟
學號 P8896110
學位類別 博士
語文別 英文
口試日期 2011-07-09
論文頁數 111頁
口試委員 口試委員-王水深
口試委員-蔡偉博
口試委員-張志涵
共同指導教授-葉明龍
指導教授-謝清河
關鍵字(中) 生醫材料
心肌組織工程
骨髓幹細胞
血管內皮生長因子
心肌梗塞
關鍵字(英) biomaterials
cardiac tissue engineering
bone marrow stem cell
vascular endothelial growth factor
myocardial infarction
學科別分類
中文摘要 缺血性心血管疾病之罹病率與所造成的死亡率在全球各國皆居高不下,在已開發國家當中尤甚。心肌梗塞即是最具威脅的缺血性心血管疾病之一,其常見於冠狀動脈因阻塞而造成下游的心肌缺血,進而在短時間內引發心肌細胞大量死亡,並進入病態的心室重塑,最後走向不可逆的心衰竭。當心肌梗塞發生之後,傳統的方式並無法有效改善與治療。因此,心血管組織工程與再生醫學是一門極待發展的新興技術。幹細胞移植即是一具有潛力的治療方式,然而,細胞移植的存留率與存活率不佳是一尚待克服的難關;另外一項有潛力的治療方式是打入生長因子誘導血管再生,然而,局部控制釋放不佳是此一方式的最大瓶頸。
在此,我們提出可以利用一自組裝胜肽奈米纖維(NFs)來克服上述瓶頸。本研究主要包含兩部份的研究,在第一個部份以迷你豬作為大動物實驗模型,我們假設自組裝胜肽奈米纖維(NFs)的材料充填可增加梗塞後的心壁厚度,同時提高自體骨髓單核細胞(MNCs)移植後的存留率,進而改善梗塞後有害的心肌重塑與功能異常。我們的結果顯示,胜肽奈米纖維注射即可改善梗塞後的心舒張功能,並減緩惡性心室重塑的效果;而注射自體骨髓細胞則以改善收縮功能為主。唯有將胜肽奈米纖維混合骨髓細胞作治療時,才能同時改善心臟收縮與舒張功能,提供較高的移植細胞存留率,進而達到較佳的血管新生與心肌保護之效果。
而在第二部份,我們以大鼠作為動物實驗模型,我們假設胜肽奈米纖維可以作血管內皮生長因子(VEGF)的局部與長效釋放,進而改善梗塞後血管新生。我們的結果證實胜肽奈米纖維混合內皮生長因子可顯著改善梗塞後的血管新生、動脈新生、與心收縮功能。重要的是,此部份的研究證實胜肽奈米纖維不只可以當作血管內皮生長因子控制釋放的載體,還可以建立心肌內合適組織再生的微環境,進而誘發內生性的動脈新生與潛在的心肌再生。
我們的結果顯示胜肽奈米纖維可以有效增強細胞與生長因子療法的功效。重要的是,在第一部份的研究中,我們以大動物實驗模型證實了本方法於臨床上使用的可行性與安全性;而在第二部份的研究中,我們藉由小動物實驗模型,進一步探討奈米纖維有利心肌組織工程的內在機制,並證實胜肽奈米纖維所建立的微環境可幫助新血管再生。我們相信本研究所提供兩種新穎治療技術與策略,不但能夠對基礎的組織工程研究提供珍貴的經驗與洞見,亦能在不遠的未來轉譯並應用於臨床上,嘉惠眾多受苦於心臟疾病的病患。
英文摘要 Ischemic cardiovascular disease (ICD) such as myocardial infarction (MI) is the leading cause of morbidity and mortality throughout the world. MI is often attribute to sudden occlusion of cornary arteries, which subsequently results in irreversible myocardium damage and heart failure. The current treatments, including drug prescription and surgical intervention, can only partially ameliorate the symptoms. Cardiac tissue engineering is thus an imperative need for vascular or myocardial regeneration. Accumulating studies have suggested that numerous adult progenitor / stem cells can contribute to de novo cardiovascular regeneration. Besides, angiogenesis can also be promoted by delivery of growth factors to induce vascular cell proliferation, differentiation, survival and migration. Nevertheless, the present clinical trials appear to be un-satisfactory and controversial. The major hurdle for cell therapy is low survival and retention rate of transplanted cells, while the problems associated with the growth factor administration is local-controlled and sustained delivery within the microenvironments.
Here, we provide a novel self-assembling peptide nanofibers (NFs) based therapy to overcome these difficulties. The nanofibrous hydrogel can not only function as scaffold to prolong cell retention but also control local delivery of proteins, and thereby enhancing angiogenic effects of delivered cells and proteins. Therefore, this thesis is divided into two parts. In the first part, we hypothesize that intramyocardial injection of NFs thickens the infarcted myocardium and increases transplanted autologous bone marrow mononuclear cell (MNC) retention to attenuate cardiac dysfunction. Our data revealed NF injection significantly improved diastolic function and reduced ventricular remodeling 28 days after treatment. Injection of MNCs alone ameliorated systolic function only, whereas injection of MNCs with NFs significantly improved both systolic and diastolic functions, increased transplanted cell retention, and promoted capillary density in the peri-infarct area.
In the second part, we hypothesize that intramyocardial injection of NFs along with vascular endothelial growth factor (VEGF) allows controlled local delivery with prolonged effect of VEGF and therapeutic angiogenesis. Our results demostrated intramyocardial injection of VEGF along with nanofibrous hydrogel has significantly improved post-infarction angiogenesis, arteriogenesis and thus cardiac performance. Importantly, this approach not only allowed controlled local delivery with prolonged effect of VEGF without systemic harmful effects, but also created a favorable microenvironment in vivo for recruitment and vascular integration of mural cells. Strikingly, the engineered vascularized microenvironment further attracted a population of cardiomyocyte-like small myocytes stained positive for cardiac troponin I, indicating the potential induction of cardiac regeneration.
Our results demonstrate that NF injection enhances the effcacy of cell and protein therapy. Importantly, we validate the feasibility and safety of this approach in the large animal study as a pre-clinical trial in the first part of thesis. Besides, we investigate the underlysing mechanism of beneficial NF injection using rodent model in the second part of thesis. We believe the unprecedented study holds not only valuable inspiration for basic biomedical research but also potency for translational application in the foreseeable future.
論文目次 Abstract I
摘要 III
誌謝 V
Index of Contents VII
Index of Figures XII
Index of Tables 1
Chapter1 Introduction 2
1.1 Cardiovascular disease is a leading cause of death 2
1.2 Current treatment to heart disease is inadequate 2
1.2.1 Medicine 2
1.2.2 Percutaneous coronary intervention 3
1.2.3 Surgery 3
1.3 Cardiac tissue engineering is a promising approach 4
1.3.1 Cell therapy 5
1.3.2 Growth factor therapy 8
1.3.3 Gene therapy 10
1.4 Challenges of cardiac regeneration 11
1.5 Novel delivery system leads revolutionary pharmaceutical treatment 12
1.6 Self-assembling peptide nanofibers 12
1.7 Overall approach 14
Chapter2 Intramyocardial NF injection improves post-MI ventricular remodeling and efficacy of MNC therapy in pigs 15
2.1 Background 15
2.2 Materials and methods 16
2.2.1 Experimental animals 16
2.2.2 Autologous bone marrow mononuclear cell isolation and DiI labeling 17
2.2.3 Myocardial infarction and treatment 18
2.2.4 Echocardiography 21
2.2.5 Hemodynamics 21
2.2.6 Ratio of necrotic tissue area, length, and thickness measurements 22
2.2.7 Collagen content measurement 22
2.2.8 Immunohistochemistry and fluorescence microscopy 22
2.2.9 Determination of NFs degradation after injection. 23
2.2.10 Blood chemistry 23
2.2.11 Quantitative evaluation of leukocytes 24
2.2.12 Statistical analysis 24
2.3 Results 24
2.3.1 Injection of NFs alone increases interventricular septum thickness, and prevents ventricular remodeling after infarction 24
2.3.2 Injection of NFs along with autologous MNCs improves both systolic and diastolic functions after infarction 32
2.3.3 Injection of NFs along with autologous MNCs decreases necrotic tissue and collagen content in the remote area after infarction 33
2.3.4 Injection of peptide nanofibers along with bone marrow mononuclear cells increases transplanted cell retention and differentiation as well as angiogenesis after infarction 33
2.3.5 Injection of NFs Reduce C-Reactive Protein Levels after Infarction 47
2.4 Discussion 49
2.4.1 Local repair by biomaterial injection preserves cardiac geometry and function 49
2.4.2 Cell retention determines cardiac functional improvement by cell therapy 50
2.4.3 MNC/NF injection has complementary effects for clinical application 50
2.4.4 Mechanisms of MNC Therapy remain to be determined 51
2.5 Summary and future direction 55
Chapter3 Nanofibrous Scaffold with VEGF Creates Intramyocardial Microenvironments for Arteriogenesis and Cardiac Benefits after Infarction 56
3.1 Background 56
3.2 Materials and methods 57
3.2.1 VEGF functional assay 57
3.2.2 NFs/VEGF binding capacity and in vitro releasing kinetics 58
3.2.3 Animals and experimental myocardial infarction 58
3.2.4 Preparation of treatments and in vivo releasing kinetics 58
3.2.5 Therapeutic study and echocardiography 59
3.2.6 Tissue processing and infarct size estimation 59
3.2.7 Fluorescence microscopy and analysis 60
3.2.8 Vascular permeability study 60
3.2.9 Bone marrow cell injection study 61
3.2.10 Cardiomyocyte-specific fate-mapping study 61
3.2.11 Statistical Analysis 62
3.3 Results 62
3.3.1 Intramyocardial NF/VEGF injection achieves sustained VEGF delivery and improves cardiac performance at 28 days after MI 62
3.3.2 NF/VEGF injection promotes both angiogenesis and arteriogenesis at 28 days after MI 70
3.3.3 Local delivery of VEGF along with NFs does not cause systemic harmful effects 75
3.3.4 NF injection recruits α-SMA+ cells and NF/VEGF injection achieves arteriogenesis 78
3.3.5 NF/VEGF injection recruits cardiomyocyte-like cTnI+ small myocytes after MI 84
3.4 Discussion 87
3.4.1 High dosage of VEGF achieves effective angiogenesis but not arteriogenesis and cardiac benefits 87
3.4.2 NFs/VEGF injection creates biomimic microenvironments to enhance therapeutic effects 88
3.4.3 NF/VEGF injection creates a vascular niche for cardiac regeneration 90
3.5 Summary and future direction 91
Chapter4 Conclusion 92
References 93
Curriculum Vitae 110
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