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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 楊宗霖(TSUNG-LIN YANG) | |
dc.contributor.author | CHIN-CHEAN WONG | en |
dc.contributor.author | 黃錦前 | zh_TW |
dc.date.accessioned | 2021-06-17T02:12:38Z | - |
dc.date.available | 2023-02-22 | |
dc.date.copyright | 2018-02-22 | |
dc.date.issued | 2017 | |
dc.date.submitted | 2017-12-22 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/68099 | - |
dc.description.abstract | 背景
軟骨損傷是骨科臨床常遇到的問題,可能因意外受傷、過度運動、肌力不足及老化等原因所造成,然而有效的治療卻非常有限。由於軟骨是屬於無血管的組織,故而其自我修復能力有限。因此在關節損傷後,軟骨損傷的程度會隨著時間而變得越來越嚴重,最後導致骨關節炎 (Osteoarthritis, OA) 的產生。為了防止關節軟骨的進一步退化,在過去幾十年中已經開發了許多治療的方式,如微裂縫骨髓刺激治療法 (Microfracture)、骨軟骨組織自體移植法 (Osteochondral autograft transplantation) 和自體同源軟骨細胞移植法 (Autologous chondrocyte implantation)。雖然利用微裂縫的骨髓刺激方法甚為簡單,但其修復的結果通常無法預測也難以複製。此外,經由此法修復的軟骨組織為纖維軟骨,其組織型態遠遜於關節的玻璃軟骨。自體骨軟骨移植為單一步驟即可達成的修補方法,其使用患者本身的骨軟骨移植填充至缺損處,但提供軟骨的部位容易產生病變、移植的骨軟骨要符合患處的外型輪廓較困難、缺損部位的大小限制以及會有軟骨及骨塌陷的風險。九零年代開始發展的自體軟骨細胞移植一開始被視為一優良且合適的技術。報告顯示相較於微裂縫骨髓刺激方法,自體軟骨細胞移植法可以產生玻璃軟骨般的再生組織。然而美中不足的是,自體軟骨細胞移植法需要多次的手術且體外的軟骨細胞培養過程非常昂貴且曠日廢時。另外,軟骨細胞在體外培養擴增過程中容易發生細胞 ‘去分化’的現象,從而不再表現軟骨細胞正常的基因及表現型特性,應用的效果因而大打折扣。 為解決臨床上軟骨修復的問題,作者提出從兩個層面切入問題的核心。第一,從技術層面上發展簡易有效的一階段軟骨修復技術,從而降低軟骨修復在臨床上的應用門檻。第二則是著眼於解決軟骨細胞來源不足的問題。此一部分的研究為利用異位性軟骨細胞嵌入第二型膠原蛋白基質後形成之'組織工程性軟骨組織'應用於動物關節軟骨修復之可行性。本研究的出發點在於解決關節軟骨細胞來源不足之問題。目前軟骨組織工程面臨之難題在於能夠用於修復軟骨缺損之關節軟骨細胞來源非常有限,且必須犧牲挖取關節內較不承重之軟骨部位以獲得細胞。由此衍生的問題包括所謂的捐贈來源位點組織損傷(donor site morbidity)及所獲之細胞數目遠遠不足,實難以用做組織培養。而骨髓幹細胞之取得較為不易且須較長時間之分化導引,修復結果也遠不如利用自體之軟骨細胞來得妥善。 體外研究 I.富含血小板血纖維蛋白(PRF)是由自體血液產生的第二代血小板濃縮物。PRF為具有高濃度生長因子的纖維蛋白生物材料,不需經過額外的活化步驟且生長因子可以穩定而持續的釋放。本研究首先分析了從兔子血液離心分離提取得到的PRF作性質分析。包含在電子顯微鏡下的結構解離分析。另外也利用ELISA 方法分析了各種生長因子的濃度。本研究利用合成所謂 ‘經PRF處理過之培養基’(conditioned medium)探索PRF對於軟骨細胞的生物相容性、遷移能力、分化能力、細胞外間質的分泌能力以及基因表現的影響。 II.。根據本研究團隊先前之實驗證明,第二型膠原蛋白能有效阻止關節軟骨細胞於體外大量培養時之'去分化'現象及引導骨髓幹細胞分化成軟骨組織。因此,本研究將觀察培養於第二型膠原蛋白被覆表面之耳朵軟骨細胞,評估第二型膠原蛋白是否能調控耳朵軟骨細胞分化為具有關節軟骨細胞特性。評估方式包含其葡萄糖胺聚糖(glycosaminoglycan)沉積之能力,關節軟骨特異mRNA之表現。為進一步提升其未來臨床應用之可能性,亦將利用耳朵軟骨細胞嵌入第二型膠原蛋白基質後形成立體'組織工程軟骨組織'。經過適當時日之培養後,進行組織切片免疫染色分析和物理特性分析(如彈性模數)。 動物體內研究 本研究利用紐西蘭大白兔兔子及豬隻軟骨缺損模式發展測試新式一階段軟骨修復法來避免多次的手術和體外細胞培養技術來克服軟骨修補技術之缺點。此法可以利用PRF的趨化效應使得軟骨細胞經由切碎的軟骨組織遷移及附著至受損區域,以達到軟骨的修復。同時也可以透過PRF來提供生長因子增進細胞增長,進一步增進受損處的癒合。另外,在兔子動物模式下,我們將經轉化的耳朵軟骨細胞(Converted AU Chondrocytes)和第二型膠原蛋白組成的'組織工程軟骨組織'植入膝關節軟骨缺損並且評估其修復情形。 結果 體外研究 經高倍率的電子顯微鏡分析PRF具有特殊的網狀結構及孔洞有利於細胞的長入。生長因子分析顯示PRF含有不同濃度的轉化生長因子β(TGF-β),鹼性成纖維細胞生長因子(bFGF),胰島素樣生長因子I(IGF-1),血管內皮生長因子(VEGF)和血小板衍生生長因子(PDGF)。 另外,PRF有明顯優越的效應在於提升軟骨細胞的遷移、增生及分化。另一方面,由第二型膠原蛋白組成的細胞微環境的改變確實能引導耳朵軟骨細胞轉化成類關節軟骨細胞並用於組織修復。 動物體內研究 動物軟骨缺損模型顯示PRF合併自體軟骨碎塊能夠有效地修復外科手術製造的軟骨缺損。利用國際軟骨修復協會制訂之評分指標分析得知PRF合併軟骨碎塊相較於控制組及單純給予PRF組別、能顯著提昇軟骨修復之結果並達到統計學上的差異。由轉化的耳朵軟骨細胞包覆第二型膠原蛋白組合成的3D軟骨結構成功修復兔子關節軟骨缺損。 結論 本研究經由抽取血液並製備出PRF,並量測生長因子的含量及觀察其結構來了解PRF的基本性質;除此之外,PRF對於軟骨細胞的生物相容性、遷移能力、分化能力、細胞外間質的分泌能力以及基因表現都被有系統的分析;最後,利用兔子動物模型,以PRF合併軟骨碎塊來填補人為創造的軟骨缺陷,從而證實了PRF的體內生物相容性以及對軟骨細胞的修復表現。 | zh_TW |
dc.description.abstract | Background
Cartilage injuries are common problem in orthopedic clinical practice with an estimated 60% of prevalence as revealed by routine knee arthroscopic procedures. Since cartilage is an avascular tissue with limited capacity of self-repair, these chondral lesions are likely to continuously progress that results in osteoarthritis. To prevent further degeneration of articular surface, many treatments have been developed to promote cartilage healing by applying cells or tissues, such as bone marrow stimulation therapy, osteochondral autograft transplantation (OAT), and autologous chondrocyte implantation (ACI). Although bone marrow stimulation through microfractures is a simple approach, the outcome of repair is often unpredictable. Moreover, the tissue generated for repair is fibrocartilage. OAT is a single-stage approach, in which defects are filled with autologous osteochondral plugs. The disadvantages of this technique include donor-site morbidity, technical difficulties in matching the lesion contour, limitation by the size of defect, and the risk of cartilage and bone collapse. ACI can yield hyaline-like tissues more similar to native cartilage in histological, mechanical and clinical aspects than that produced by microfractures. However, it requires multiple surgical procedures and a costly process of in vitro chondrocyte expansion, which may cause chondrocytes dedifferentiation. To avoid complicated procedures required in in vitro chondrocytes expansion for cartilage repair, development of a culture-free, one-stage approach combining Platelet-rich fibrin (PRF) and autologous cartilage graft may be the solution. Secondly, to solve the cell source problem in cartilage tissue repair, it is important to explore the feasibility of using heterotopic chondrocyte embedded in type II collagen (Col II) matrix in articular (AR) cartilage defect regeneration. Methods: The chemotactic effects of PRF on chondrocytes harvested from the primary culture of rabbit cartilage were evaluated both in vitro and ex vivo. The rabbit chondrocytes were cultured with different concentrations of PRF medium, and were evaluated for their ability of cell proliferation, chondrogenic gene expression, cell viability, and extracellular matrix syntheses. In second part of study, auricular chondrocytes (AU) were cultured with Col II supplement and analyzed for their articular chondrogenesis. The regulatory effects of Col II on differentiation of AU chondrocytes as having the characteristics of articular cartilage will be carefully determined. Methods of assessment includes glycosaminoglycan deposition and the expression of articular cartilage-specific mRNA (such as type II collagen mRNA; aggrecan, Lubricin, and SOX 9). To further enhance the possibility of its future clinical applications, the auricular chondrocytes will be embedded in Col II matrix to form a three-dimensional ‘tissue-engineered cartilage’. Histological analysis and physical characteristics analysis (such as elastic modulus) of this tissue will also be checked. For in vivo study, the chondral defects were created on the established animal models of rabbit and porcine. The results were evaluated by gross anatomy, histology, and objective scores to validate the treatment results. Results: PRF improved the chemotaxis, proliferation, and viability of cultured chondrocytes. Gene expression of chondrogenic markers including type II collagen and aggrecan revealed that PRF induced the chondrogenic differentiation of cultured chondrocytes. The efficacy of PRF on cell viability was comparable with fetal bovine serum. In the animal disease models, the morphological, histological, and objectively quantitative evaluation demonstrated that PRF combined with cartilage granules is feasible in facilitating chondral repair. In AU chondrocytes conversion study, the AU chondrocytes fabricated collagen constructs were implanted into rabbit osteochondral defect and their repair was evaluated by histological analysis. Conclusions: PRF enhances migration, proliferation, viability, and differentiation of chondrocytes, thus showing appealing capacity for cartilage repair. The data altogether provide evidences to confirm the feasibility of the one-stage, culture-free method of combining PRF and autologous cartilage for repairing articular chondral defects. On the other hand, chondrocytes harvested from AU cartilage can be converted to become similar to cells of AR cartilage exclusively by extracellular matrices. The results confirmed the feasibility of engineering AU chondrocytes to mitigate the drawbacks of applying heterotopic chondrocytes for cartilage repair. In this study, we propose a feasible and efficient methodology for the application of autologous cartilage transplantation using heterotopic chondrocytes, which is expected to expedite the potential clinical application of cartilage repair. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T02:12:38Z (GMT). No. of bitstreams: 1 ntu-106-D99421011-1.pdf: 10969850 bytes, checksum: aea280a508d5184f56e300fa2ea89c6a (MD5) Previous issue date: 2017 | en |
dc.description.tableofcontents | I. Verification Letter from the Oral Examination Committee
II. Acknowledgment III. Chinese Abstract IV. English Abstract V. Abbreviation Table VI. Main Text of the Thesis Chapter 1:.Introduction 1.1 Basic Science of Articular Cartilage: Structure, Composition and Function-------1 1.2 Articular Cartilage Injury and its Clinical Implications-------------------------------5 1.3 Evolution of Cartilage Repair Techniques----------------------------------------------7 1.4 Biological Strategy for Cartilage Repair---------------------------------------------- 13 1.5 Application of PRF for Cartilage Repair and Regeneration------------------------18 1.6 Research Hypothesis & Aims-PRF in Cartilage Repair-----------------------------23 1.7 Heterotopic Source of Chondrocytes for Articular Repair -------------------------26 1.8 Research Hypothesis & Aims-Col II-mediated AU chondrocytes Cell Conversion---------------------------------------------------------------------------------29 Chapter 2. Materials and Methods 2.1 Preparation & Characterization of PRF-------------------------------------------------32 2.2 Isolation of Rabbit Articular Chondrocytes, Auricular Chondrocytes & Meniscocytes-------------------------------------------------------------------------------34 2.3 Chemotactic Effects of PRF on Chondrocytes and Meniscocytes, in vitro and ex vivo-----------------------------------------------------------------------------------------36 2.4 Biological Effects of PRF on Chondrocytes and Meniscocytes---------------------39 2.5 Effects of PRF in Promoting Chondrocytes Viability---------------------------------43 2.6 Facilitating Cell Conversion of Auricular Chondrocytes towards Articular Chondrogenesis via Microenvironment Modulation----------------------------------44 2.7 Fabrication of Three-Dimensional Cartilaginous Constructs------------------------48 2.8 Rabbit Osteochondral Defect Repair Model--------------------------------------------50 2.9 Porcine Chondral Defect Repair Model-------------------------------------------------52 2.10 Rabbit Meniscal Defect Repair Model-------------------------------------------------58 Chapter 3. Results A. PRF Studies in rabbit chondrocytes & Meniscocytes 3.1 Microstructure of PRF & Its Cytokine Content----------------------------------------60 3.2 PRF Chemotactic Effects on Rabbit Chondrocytes &Meniscocytes----------------61 3.3 Effects of PRF on Rabbit Chondrocytes & Meniscocytes Morphological Alteration & Cell Proliferation ----------------------------------------------------------65 3.4 PRF Promotes Rabbit Chondrocytes & Meniscocytes Chondrogenic Differentiation------------------------------------------------------------------------------67 3.5 PRF Promotes Rabbit Chondrocytes Viability-----------------------------------------71 3.6 Successful Rabbit Osteochondral Repair by PRF Associated Cartilaginous Graft------------------------------------------------------------------------------------------73 3.7 Successful Porcine Chondral Repair by PRF Associated Cartilaginous Graft------------------------------------------------------------------------------------------74 3.8 PRF Augments Rabbit Meniscal Repair------------------------------------------------77 B. Type II Collagen Modulating Cell Conversion of Auricular Chondrocytes 3.9 Type II Collagen Modulated Heterotopic Chondrocytes Conversion---------------79 3.10 Characterization of Three-Dimensional AU-Col II Constructs--------------------83 3.11 Repair of Rabbit Knee Joint Osteochondral Defect by AU-Col II Constructs---85 Chapter 4. Discussion 4.1 Current Status of Cartilage Repair Strategy--------------------------------------------87 4.2 Limitations of Current Treatment Strategies-------------------------------------------88 4.3 Rationales of PRF on Cartilage Repair--------------------------------------------------91 4.4 Anabolic Effects of PRF on Chondrocytes-proliferation, differentiation & migration------------------------------------------------------------------------------------96 4.5 Pre-clinical Studies using PRF Augmented Tissue Healing------------------------102 4.6 Heterotopic Chondrocytes for Articular Cartilage Repair--------------------------105 4.7 Conflicting Outcomes of Heterotopic Chondrocytes Applied in Cartilage Tissue Engineering--------------------------------------------------------------------------------110 4.8 Type II Collagen-mediated Cellular Conversion of AU chondrocytes------------111 4.9 Facilitating in vivo Cartilage Repair using Converted AU Chondrocytes--------116 4.10 Study Limitations-----------------------------------------------------------------------118 Chapter 5. Perspective 5.1 Cartilage Repair, From Bench work to Clinical Application-----------------------120 5.2 Novel 1-stage Cartilage Repair Technique in Clinical Application----------------121 5.3 Future Prospect of PRF in Human Clinical Trial-------------------------------------125 5.4 Individual Variation of PRF-Clinical Impact-----------------------------------------125 5.5 New Concept Emerged-Cellular Conversion of Heterotopic Chondrocytes for Articular Repair---------------------------------------------------------------------------127 5.6 Converted AU Chondrocytes in Cartilage Tissue Engineering, its Clinical Implications-------------------------------------------------------------------------------131 5.7 Heterotopic Chondrocytes for Articular Repair-Future Clinical Barrier----------132 References----------------------------------------------------------------------------------134 Appendix -----------------------------------------------------------------------------------238 | |
dc.language.iso | en | |
dc.title | 關節軟骨修補與再生之轉譯醫學研究與應用 | zh_TW |
dc.title | Translational Approach for Cartilage Repair and Regeneration | en |
dc.type | Thesis | |
dc.date.schoolyear | 106-1 | |
dc.description.degree | 博士 | |
dc.contributor.coadvisor | 陳佑宗(You-Tzung Chen) | |
dc.contributor.oralexamcommittee | 楊偉勛(WEI-SHIUNG YANG),陳晉興(JIN-SHING CHEN),陳志華(CHIH-HWA CHEN),曾永輝(YANG-HWEI TSUANG) | |
dc.subject.keyword | 一階段式修復,富含血小板血纖維蛋白,關節軟骨,軟骨修復,異位性軟骨, | zh_TW |
dc.subject.keyword | Chondrocytes,One-stage cartilage repair,Platelet-rich fibrin,articular cartilage,chondral repair,heterotopic chondrocytes, | en |
dc.relation.page | 238 | |
dc.identifier.doi | 10.6342/NTU201704458 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2017-12-25 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 臨床醫學研究所 | zh_TW |
顯示於系所單位: | 臨床醫學研究所 |
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