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研究生: 吳柏廷
Wu, Po-Ting
論文名稱: 以阿基里斯肌腱疾病動物模式探討肌腱病變的評估和玻尿酸治療的可行性
The Evaluation and Hyaluronate Treatment Strategies in Tendinopathy: An Achilles Tendon Model
指導教授: 蘇芳慶
Su, Fong-Chin
共同指導教授: 周一鳴
Jou, I-Ming
學位類別: 博士
Doctor
系所名稱: 工學院 - 生物醫學工程學系
Department of BioMedical Engineering
論文出版年: 2016
畢業學年度: 104
語文別: 英文
論文頁數: 89
中文關鍵詞: 肌腱病變玻尿酸動物模式玻尿酸動物模式發炎動態負重基質金屬蛋白酶
外文關鍵詞: tendinopathy, hyaluronate, animal model, inflammation, dynamic weight bearing, MMP
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  • 肌腱病變是一種常見疾病,據統計,30-50%的運動相關傷害及約半數的職業疾病為肌腱病變。然而如此高盛行率的疾病,目前對其病理機轉仍未十分明瞭。動物模式有助於研究疾病機轉及評估療效,目前對於肌腱病變小動物模式的功能性評估多僅限於動作分析,缺乏對於步態的力學分析。玻尿酸為肌腱病變眾多可能治療方式之一,無論在臨床或動物實驗上,玻尿酸皆顯示對肌腱病變有其療效,然而對於不同分子量與濃度的效果並未著墨。而臨床上施打玻尿酸時,皆不採取肌腱內注射,但尚未有研究顯示肌腱內注射玻尿酸是否會產生傷害。。因此,本研究將以阿基里斯肌腱模式為主軸探討肌腱病變的評估與玻尿酸的治療方式:(1) 以自製力板軌道系統探討以膠原脢(collagenase)注射引起之肌腱病變模式的步態力學特徵,並對照組織學和超音波特徵;(2) 探討肌腱內注射玻尿酸引起的急性發炎反應; (3) 探討不同分子量及濃度的玻尿酸在肌腱病變中對於基質金屬蛋白酶1和3的表現。
    本研究發現超音波引導大鼠阿基里斯腱肌腱內膠原脢注射確實為一有效的肌腱病變動物模式,除了比對照組有顯著對應的組織病理變化和超音波特徵外,注射後足較對側後足有顯著較小的動態負重,而對側前足則有代償性增加的動態負重。此一顯著步態力學變化從第一週持續至第八週,而靜態後肢負重比例則無法反應此一變化。對比於肌腱戳針(Sham group),肌腱內注射玻尿酸或磷酸緩衝生理食鹽水皆會引起明顯的急性肌腱傷害和發炎反應-病理組織變化、ED1+和ED2+巨噬細胞聚集、IL-1β表現和血管新生。然而在上述各項指標裡,玻尿酸注射皆比磷酸緩衝生理食鹽水表現較為嚴重,因此應該避免肌腱內注射玻尿酸。而在IL-1β刺激的肌腱細胞中,高分子量玻尿酸 (HA3000)明顯的減弱基質金屬蛋白酶1和3的mRNA和蛋白質的表現,而此一效果與玻尿酸濃度成正比,且藉著先行處理OX-50來抑制CD44表現可明顯反轉此一較果。臨床上,高分子量玻尿酸的治療可明顯的降低患者的VAS分數和減弱基質金屬蛋白酶1和3的表現。因此,高分子量玻尿酸藉著降低基質金屬蛋白酶1和3的表現,可能是一個有效的肌腱病變治療方式。本研究結果希望可以較為完備小動物肌腱病變模式及玻尿酸於肌腱病變治療的立論基礎。

    Tendinopathy is a high-prevalence common disease that accounts for 30-50% of sports injuries and around half of occupation-related diseases. However, the pathogenetic mechanism of tendinopathy is largely unknown. Animal models are thus essential to evaluate the disease mechanism and treatment efficacy. To date, only the results of motion analysis has been reported in the small-animal pain assessment of tendinopathy, and kinetic changes in gait cycle have received much less attention. Hyaluronate (HA), one of possible treatment modalities, has been reported to mitigate tendinopathy effectively in both clinical series and animal models. However, the impacts of molecular weight and concentration of HA are still unknown. Moreover, intratendinous injection of HA is anecdotally avoided, despite a lack of evidence that direct intratendinous injection leads to an acute injury. Therefore, this dissertation will assess the evaluation and HA treatment strategies in tendinopathy using a rat Achilles tendon model. The specifics of this work are as follows: (1) to elucidate the kinetic features in a collagenase-induced tendinopathy model using a customized corridor with multi-directional force sensors, and compare the features with the histopathological and ultrasonographical findings; (2) to elucidate the possible detrimental inflammation induced by intratendinous injection of HA; and (3) to examine the effects of HA with different molecular weights and different concentrations on matrix metalloproteinase (MMP)-1 and -3 expression.
    Our results showed ultrasound (US)-guided intratendinous injection of collagenase into rat Achilles tendons is a validated animal model with corresponding higher histopathological scores and higher US features scores than the control tendons. Our customized corridor clearly revealed significantly less dynamic weight bearing in the injected hind limbs than in the contralateral hind limbs, and compensatorily higher dynamic weight bearing in the contralateral fore limbs from week 1 to week 8. The static weight bearing ratio could not reflect the tendinopathic changes. Compared with intratendinous needle puncture, intratendinous injection of either HA or phosphate buffered saline (PBS) induces acute tendon injury and inflammatory reactions: histopathological changes, ED1+ and ED2+ macrophages accumulation, IL-1β expression, and neovascularization. The injury induced by intratendinous injection of HA was more severe than that induced by PBS. Therefore, intratendinous injection of HA should be avoided. Furthermore, high-molecular-weight (HMW) HA (HA3000) significantly attenuated the mRNA and protein expression of MMP-1 and -3 in IL-1β stimulated tenocytes. These effects were dose-dependent and significantly reversed by pretreating tenocytes with OX-50 to inhibit CD44 expression. Clinically, HMW-HA treatment significantly reduced VAS scores and attenuated MMP-1 and -3 expression in patients with LHB tendinopathy. These findings indicate that HMW-HA might be an effective treatment for tendinopathy by attenuating MMP-1 and -3 expression. It is hoped that, this dissertation will contribute to pain-related assessments in the small-animal tendinopathy model and to further research into HA treatment with regard to tendinopathy.

    摘要 I Abstract III 致謝 V Content VI List of Tables X List of Figures XI Chapter 1 Introduction 1 1.1 Research Background 2 1.1.1 Tendinopathy 2 1.1.2 Treatment of Tendinopathy 2 1.1.3 Mechanism of Tendinopathy 3 1.1.4 Animal Models in Tendinopathy 3 1.2 Motivations 6 1.3 Specific Aims 7 1.3.1 Specific Aim I 7 1.3.2 Specific Aim II 7 1.3.3 Specific Aim III 7 1.4 Dissertation Outline 9 Chapter 2 Dynamic weight bearing analysis in a collagenase-induced tendinopathy rat model : a customized corridor with multi-directional force sensors 10 2.1 Introduction 11 2.2 Materials and Methods 13 2.2.1 Ethics Statement 13 2.2.2 Animal Model 13 2.2.3 US guidance procedures and scoring of US features 14 2.2.4 Histopathological grade 15 2.2.5 Customized corridor design 15 2.2.6 Validation of customized corridor data 18 2.2.7 Kinetic analysis in customized corridor 19 2.2.8 Static weight bearing paradigm 20 2.2.9 Statistical Analysis 20 2.3 Results 22 2.3.1 Histopathological grading 22 2.3.2 US features scores 24 2.3.3 Validity test and reliability test of the customized corridor 26 2.3.4 Kinetic parameters and static weight bearing ratio 26 2.4 Discussion 30 2.5 Conclusion 35 Chapter 3 Intratendinous Injection of Hyaluronate Induces Acute Inflammation: A Possible Detrimental Effect 36 3.1 Introduction 37 3.2 Materials and Methods 39 3.2.1 Ethics statement 39 3.2.2 Animal Model 39 3.2.3 Ultrasonographic guidance procedures 40 3.2.4 Histopathological grade 41 3.2.5 Immunohistochemistry 42 3.2.6 Statistical analysis 43 3.3 Results 44 3.3.1 Histopathological grade 44 3.3.2 ED1+ and ED2+ macrophage density and IL-1β expression 44 3.3.3 Neovascularization 45 3.4 Discussion 53 3.5 Conclusion 57 Chapter 4 High-molecular-weight hyaluronic acid attenuates matrix metalloproteinase-1 and -3 expression via CD44in tendinopathy 58 4.1 Introduction 59 4.2 Material an Methods 61 4.2.1 Ethics statement 61 4.2.2 Tenocyte culturing and identification 61 4.2.3 HA treatment in patients with long head of biceps tendinopathy 62 4.2.4 IL-1β stimulation and HA treatment of various molecular weights 63 4.2.5 Tenocyte proliferation and viability 63 4.2.6 Measurement of MMP-1 and -3 mRNAexpression by reverse-transcription and real-time PCR 64 4.2.7 Measurement of MMP-1 and -3 protein using ELISA 66 4.2.8 HA preparations of various concentrations 66 4.2.9 Statistical Analysis 66 4.3 Results 67 4.3.1 Identification of Tenocytes form Primary Culture 67 4.3.2 Effects of HA on tenocyte proliferation and viability 67 4.3.3 Effects of HA of various molecular weights on the mRNA and protein expression of MMP-1 and -3 68 4.3.4 Effects of various concentrations of HA on the MMP-1 and-3 mRNA expression 71 4.3.5 OX-50 treatment inhibited CD44 expression and blocked the attenuating effects of HA 72 4.3.6 The dynamic changes of MMPs and VAS in patients with long head of biceps tendinopathy after high-molecular-weight HA treatment 74 4.4 Discussion 75 Chapter 5 Summary and Future Work 79 5.1 Summary of the Present Work 80 5.2 Limitations and the Future Work 81 References 82

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