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研究生:林立群
研究生(外文):Lin, Lichun
論文名稱:透明質酸/第二型膠原蛋白/軟骨細胞微粒載體於軟骨修復之研究
論文名稱(外文):The Study Of Chondrocyte-Seeded Hyaluronan/Collagen II Microspheres On Cartilage Repair
指導教授:郭士民郭士民引用關係莊景文莊景文引用關係
指導教授(外文):Kuo, ShyhmingChuang, Chinwen
口試委員:郭士民莊景文姚俊旭黃志賢李明偉
口試委員(外文):Kuo, ShyhmingChuang, ChinwenYao, ChunhsuWong, ChiyinLee, Mingwei
口試日期:2012-06-15
學位類別:博士
校院名稱:義守大學
系所名稱:電機工程學系
學門:工程學門
學類:電資工程學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:英文
論文頁數:92
中文關鍵詞:透明質酸第二型膠原蛋白軟骨細胞微粒載體軟骨修復
外文關鍵詞:HyaluronanCollagen IIMicrospheresChondrocyte-seededCartilage Repair
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第二型膠原蛋白及透明質酸為關節軟骨細胞外基質中最主要的成分,不僅提供軟骨組織機械強度,同時亦是細胞貼附及生長之基板。本研究利用實驗室所建立的高壓電場微粒製備系統,於水相環境中製備透明質酸/第二型膠原蛋白 ( HA/Col II microspheres) 微粒,以二階段交聯反應(氯化鐵及EDC),最後以回溫37℃強化微粒機械強度。研究結果顯示本系統能於水相環境中製備出透明質酸/第二型膠原蛋白微粒,其微粒粒徑大小約介於486  43~ 679  24 m之間,且微粒之表面微結構、機械強度、含水量之基本性質影響,可依處理溶劑(氯化鐵及EDC)條件的改變而加以調整。將軟骨細胞培養於HA / COL II微粒7天後,再植入兔子軟骨缺損處, 4周後取出觀察顯示,未植入微粒的缺陷內呈現半透明的結締組織;不論是植入HA / COL II微粒或軟骨細胞/HA / COL II微粒之缺陷,其癒合缺陷有新生組織融入周圍的軟骨組織。GAG染色組織切片觀察,植入HA / COL II微粒之缺陷處,其GAG含量較低,顯示大部分修復組織是纖維軟骨;而植入軟骨細胞/HA / COL II微粒之缺陷,呈現較高含量的GAG,顯示新生組織為透明軟骨。綜合上述之結果得知,軟骨細胞/HA / COL II微粒對於軟骨修補上有所裨益,顯示軟骨細胞/HA / COL II微粒應用於軟骨組織工程具有其發展潛能。
Collagen II and hyaluronan are the two major components of the native extracellular matrix (ECM). Both biopolymers are responsible for providing the associated tissues with tensile strength, and also serve as a structural scaffold for cell adhesion and growth. Over the years, many researchers have focused on the preparation and evaluation of man-made ECM that comprises of the two polymers in form of membrane shape for chondrocyte culture applications. Here, a simple and in-situ method, by injecting the hyaluronan/collagen II (HA/Col II) mixture solution through a pair of hollow-centered parallel disc electrodes (HCPDE) of a high-voltage electrostatic field system, was developed and employed to prepare HA/Col II microspheres in watery phase. The HA/Col II microspheres were firmed up by a two-step cross-linking treatment (first by FeCl3 and then by 1-ethyl-3-(3-dimethyl aminopropyl) carbodimide, EDC) to secure the spherical structure shape. Then, at 37 C, reconstitution treatment of the collagen II molecules was conducted to further strengthen the microspheres. Depending on treatment conditions, the resulting series of HA/Col II microspheres all exhibited good sphericity in the range of 486  43 to 679  24 m in diameter. Furthermore, the ratio and amount of HA/Col II in the mixture solutions would affect the morphological structure and basic characteristics, including mechanical strength, thermo property and water content. The animal model study evaluated the effects on a 4-week healing process of chondral defects by the implantation of allogenous chondrocyte-seeded HA/Col II microspheres that had been cultured in vitro for 7 days prior to implantation compared with unseeded HA/Col II microspheres or an untreated wound. Four weeks post surgery, the untreated group’s defect was filled with translucent soft tissue. At the same time, the edges and demarcation lines of the healing defects that were implanted with either HA/Col II microspheres or chondrocyte-seeded HA/Col II microspheres were infused yet recognizable. Furthermore, the new tissues were well integrated into the surrounding articular cartilage. Less GAG staining was observed in the defects implanted with HA/Col II microspheres, which indicated that most of the repair tissues were derived from fibrocartilage formation. Conversely, more GAG staining appeared in the defect implanted with chondrocyte-seeded HA/Col II microspheres, which demonstrated a higher level of hyaline cartilage regeneration.
Abstract I
List of Contents III
List of Figures V
Chapter 1-Introduction 1
1.1 Background 1
1.2 Extracellular Matrix 2
1.3 Microspheres 6
1.4 The Research Purpose 8
Chapter 2-Materials and Equipments 9
2.1 Materials 9
2.2 Equipments 11
Chapter 3-Methods 12
3.1 Purification of type II collagen 12
3.2 Construction of the hollow-centered parallel disc electrodes (HCPDE) system and preparation of HA/Col II microspheres 12
3.3 Characterization of HA/Col II microspheres 16
3.3.1 Size and morphology of the microspheres 16
3.3.2 Mechanical durability of HA/Col II microspheres 16
3.3.3 Water content of HA/Col II microspheres 17
3.3.4 Thermal analysis of HA/Col II microspheres 17
3.3.5 Two-photon fluorescence microscopy 17
3.4 Chondrocyte isolation 18
3.5 Cell culture 18
3.6 Transplantation of chondrocyte-seeded HA/Col II microspheres 19
3.7 Histological and histochemical analysis 19
Chapter 4-Results 23
4.1 Type II collagen isolation and identification 23
4.2 The preparation system of HA/Col II microspheres 24
4.2.1 HCPDE system 24
4.2.2 Effect of Fe 3+ concentration on the diameters of HA/Col II microspheres 25
4.2.3 Effect of HA and Collagen II Concentration on the diameters of HA/Col II microspheres 27
4.2.4 Effect of EDC crosslinking reaction on the diameters of HA/Col II microspheres 30
4.3 The characterization of the HA/Col II microspheres 32
4.3.1 Morphology of the HA/Col II microspheres 32
4.3.2 Mechanical durability of HA/Col II microspheres 43
4.3.3 Water content and swelling profile of HA/Col II microspheres 48
4.3.4 Thermal property analysis 50
4.3.5 Two-photo fluorescence microscopy 52
4.4 Characteristics of chondrocyte-seeded HA/Col II microspheres 56
4.5 Animal implantation 58
4.5.1 Macroscopic observation 58
4.5.2 Histological Observations 62
Chapter 5-Discussions 73
Chapter 6-Conclusions 78
References 80
List of Figures
3.1 Schematic presentation of the HA/Col II microsphere generation system and preparation procedures 14
3.2 The hollow-centered parallel disc electrodes (HCPDE) system 15
3.3 Surgical procedures in this present study: man-made 6-mm defect 20
3.4 Surgical procedures in this present study: filled with HA/Col II microspheres 21
3.5 Surgical procedures in this present study: covered with periosteum and sutured 22
4.1 SDS-polyacrylamide gel electrophoresis of type I collagen from Vitrogen (A) and purified type II collagen (B) 23
4.2 Simulation of the electric field distribution between two parallel disc electrodes 24
4.3 Mechanism of the formation of HA/Col II microspheres by Fe3+ and EDC reactions and thermal reconstitution 26
4.4 Effects of Fe3+ concentration on the yield and size of the prepared HA/Col II microspheres 27
4.5 Effects of Col II concentrations on the yield and size of the prepared HA/Col II microspheres 29
4.6 Effects of EDC treatment on the size of the prepared HA/Col II microspheres 31
4.7 Photomicrographs of HA microspheres treated with Fe3+, EDC for 1 h and 37℃ reconstitution for 24 h 33
4.8 Photomicrographs of HA/Col II-7/3.5 microspheres treated with Fe3+, EDC for 1 h and 37℃ reconstitution for 24 h 33
4.9 Photomicrographs of HA/Col II-7/5 microspheres treated with Fe3+, EDC for 1 h and 37℃ reconstitution for 24 h 34
4.10 Photomicrographs of HA/Col II-7/7 microspheres treated with Fe3+, EDC for 1 h and 37℃ reconstitution for 24 h 34
4.11 SEM micrographs of the surface and cross-section of the HA microspheres after treatment with Fe3+, EDC for 1 h and 37℃ reconstitution for 24 h 35
4.12 SEM micrographs of the surface and cross-section of the HA/Col II-7/3.5 microspheres after treatment with Fe3+, EDC for 1 h and 37℃ reconstitution for 24 h 36
4.13 SEM micrographs of the surface and cross-section of the HA/Col II-7/5 microspheres after treatment with Fe3+, EDC for 1 h and 37℃ reconstitution for 24 h 37
4.14 SEM micrographs of the surface and cross-section of the HA/Col II-7/7 microspheres after treatment with Fe3+, EDC for 1 h and 37℃ reconstitution for 24 h 38
4.15 SEM micrographs of the surface and cross-section of the HA microspheres after treatment with Fe3+ and EDC for 1 h without 37 ℃ reconstitution for 24 h 39
4.16 SEM micrographs of the surface and cross-section of the HA/Col II-7/3.5 microspheres after treatment with Fe3+ and EDC for 1 h without 37 ℃ reconstitution for 24 h 40
4.17 SEM micrographs of the surface and cross-section of the HA/Col II-7/5 microspheres after treatment with Fe3+ and EDC for 1 h without 37 ℃ reconstitution for 24 h 41
4.18 SEM micrographs of the surface and cross-section of the HA/Col II-7/7 microspheres after treatment with Fe3+ and EDC for 1 h without 37 ℃ reconstitution for 24 h 42
4.19 Mechanical strength of pure HA microspheres by various treatment procedures 44
4.20 Mechanical strength of HA/Col II-7/3.5 microspheres by various treatment procedures 45
4.21 Mechanical strength of HA/Col II-7/5 microspheres by various treatment procedures 46
4.22 Mechanical strength of HA/Col II-7/7 microspheres by various treatment procedures 47
4.23 Water contents for pure HA, HA/Col II-7/3.5, HA/Col II-7/5 and HA/Col II-7/7 microspheres 49
4.24 DSC thermograms of pure HA, HA/Col II-7/3.5, HA/Col II7/-5 and HA/Col II-7/7 microspheres 51
4.25 Two-photon fluorescence micrograph of pure HA microspheres before and the excitation 53
4.26 Two-photon fluorescence micrograph of pure HA microspheres after the excitation 53
4.27 Two-photon fluorescence micrograph of HA/Col II-7/7 microspheres before after the excitation 54
4.28 Two-photon fluorescence micrograph of HA/Col II-7/7 microspheres after the excitation 54
4.29 Two-photon fluorescence micrograph of HA/Col II-7/7 microspheres after the excitation 55
4.30 Chondrocytes cultured on HA/Col II microspheres for 7 days 57
4.31 Gross observations of chondral defects without treatment (control group) after 4 weeks implantation period 59
4.32 Gross observations of chondral defect implanted with HA/Col II microspheres alone after 4 weeks implantation period 60
4.33 Gross observations of chondral defects implanted with chondrocytes-seeded HA/Col II microspheres after 4 weeks implantation period 61
4.34 Typicl histological sections after 4 weeks implantation period were shown in control group 64
4.35 Typical histological sections of the defect implanted with HA/Col II microspheres alone after 4 weeks implantation period 65
4.36 Typical histological sections of the defect implanted with chondrocytes-seeded HA/Col II microspheres after 4 weeks implantation period 67
4.37 Histochemical Alcian blue-stained sections of the defect implanted with HA/Col II microspheres alone after 4 weeks implantation period 69
4.38 Histochemical Alcian blue-stained sections of the defect implanted with chondrocytes-seeded HA/Col II microspheres after 4 weeks implantation period 71
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