【摘要】：背景和目的 隨著我國(guó)國(guó)民對(duì)品質(zhì)生活和健康體魄需求的提升,運(yùn)動(dòng)健身已逐漸成為日常生活中的重要組成部分,國(guó)人參加體育鍛煉和競(jìng)技運(yùn)動(dòng)的數(shù)量明顯增加,運(yùn)動(dòng)損傷也隨之增加。近十年來(lái),作為關(guān)節(jié)腔內(nèi)維持膝關(guān)節(jié)穩(wěn)定和功能運(yùn)動(dòng)的重要韌帶,前交叉韌帶(Anterior cruciate ligament, ACL)損傷后的組織學(xué)重建和生物力學(xué)功能恢復(fù)越來(lái)越受到骨科醫(yī)生的極大關(guān)注。由于相對(duì)缺乏血供,種子細(xì)胞不足以及關(guān)節(jié)腔惡劣的液態(tài)環(huán)境,ACL再生能力非常有限,因此其損傷后的功能性修復(fù)在臨床治療中仍然面臨巨大挑戰(zhàn)。當(dāng)前,自體移植、同種異體移植一直是修復(fù)ACL的金標(biāo)準(zhǔn)。然而,這些手術(shù)重建再生技術(shù)有不同的弊端,例如供體部位并發(fā)癥、疾病傳播、免疫排斥、較差整合性和較高的復(fù)發(fā)率。近年來(lái),人工韌帶的出現(xiàn)為提高ACL等軟組織的修復(fù)質(zhì)量帶來(lái)了全新的希望。 已經(jīng)面市的商業(yè)化產(chǎn)品大多為不可降解的合成材料。他們雖然克服自體和異體移植的弊端,在短期內(nèi)產(chǎn)生令人滿意的效果,然而長(zhǎng)期隨訪發(fā)現(xiàn)依然會(huì)引起包括移植物破裂,骨關(guān)節(jié)炎發(fā)生,慢性滑膜炎和異物反應(yīng)等不良事件。 近來(lái),基于蠶絲的生物材料由于其良好的生物相容性和較大的力學(xué)強(qiáng)度被廣泛應(yīng)用于各種軟組織的修復(fù)。有研究顯示單純捆綁型蠶絲支架用于ACL修復(fù)取得了一定效果,我們先前運(yùn)用“內(nèi)部空間預(yù)占”理論原創(chuàng)性的設(shè)計(jì)了一種編織狀蠶絲-膠原海綿支架用于皮下肌腱韌帶再生取得了滿意效果。本研究將進(jìn)一步探索其用于關(guān)節(jié)腔惡劣液態(tài)環(huán)境中的前交叉韌帶重建的長(zhǎng)期效果,并評(píng)估其對(duì)骨關(guān)節(jié)炎的預(yù)防效果。 方法和結(jié)果 1、編織狀蠶絲-膠原海綿材料的制備和性能檢測(cè)：復(fù)合蠶絲膠原材料的制備是通過(guò)在模具中加入10ml10%膠原溶液和編織狀蠶絲,經(jīng)過(guò)冷凍真空干燥法和真空干熱交聯(lián)法。最后制得的復(fù)合材料通過(guò)力學(xué)測(cè)試儀檢測(cè)其力學(xué)性能。 2、運(yùn)用兔間充質(zhì)干細(xì)胞(MSC)進(jìn)行體外相容性實(shí)驗(yàn)：將蠶絲-膠原復(fù)合材料與MSC共培養(yǎng),3、7、14天之后,運(yùn)用掃描電鏡(SEM)觀察材料表面細(xì)胞形態(tài),1、3、7、10、14天之后,通過(guò)細(xì)胞計(jì)數(shù)試劑盒-8(CCK-8)方法檢測(cè)細(xì)胞的增殖情況并評(píng)估材料的體外相容性。 3、單純蠶絲組和蠶絲膠原組分別與MSC共培養(yǎng),3、7天之后,通過(guò)轉(zhuǎn)錄因子(mRNA)檢測(cè)I型膠原(COL Ⅰ)、黏蛋白(TNC)、雙糖鏈蛋白多糖(BGN)、核心蛋白多糖(DCN)并完成統(tǒng)計(jì)分析,評(píng)估實(shí)驗(yàn)組對(duì)成腱分化的影響。 4、前交叉韌帶(ACL)重建模型復(fù)合支架體內(nèi)移植評(píng)估ACL修復(fù)能力：將實(shí)驗(yàn)組蠶絲膠原復(fù)合支架和對(duì)照組單純蠶絲支架分別植入建立的兔前交叉韌帶重建模型。術(shù)后2個(gè)月、6個(gè)月、18個(gè)月分別進(jìn)行組織學(xué)染色及評(píng)分、三色染色、腱系因子表達(dá)檢測(cè)、I型膠原(COL Ⅰ)、III型膠原(COL Ⅲ)免疫組化以及偏正光評(píng)估腱性部位修復(fù)情況。并對(duì)2個(gè)月和18個(gè)月進(jìn)行膠原含量檢測(cè)。同時(shí)運(yùn)用SO染色和micro-CT對(duì)腱骨交界處進(jìn)行評(píng)估。 5、骨關(guān)節(jié)炎(OA)預(yù)防的療效評(píng)估：通過(guò)對(duì)關(guān)節(jié)面進(jìn)行墨汁染色、SO染色組織學(xué)評(píng)分、半月板HE染色以及關(guān)節(jié)X線評(píng)估OA進(jìn)程。 結(jié)果顯示蠶絲膠原支架相比單純蠶絲支架擁有更好的力學(xué)性能,體外細(xì)胞共培養(yǎng)有效促進(jìn)了MSC的粘附、增殖和腱性因子的表達(dá),體內(nèi)移植2個(gè)月蠶絲膠原支架組顯著提高了遷移和粘附的梭形細(xì)胞,并于術(shù)后6個(gè)月發(fā)現(xiàn)再生組織可表達(dá)更多的腱系因子和表現(xiàn)出更自然的韌帶微結(jié)構(gòu)表型,進(jìn)一步在術(shù)后18個(gè)月,編織狀蠶絲膠原海綿支架呈現(xiàn)出具有更成熟的韌帶結(jié)構(gòu)和相對(duì)較好的骨腱愈合。同時(shí)關(guān)節(jié)腔內(nèi)蠶絲膠原支架相比皮下降解更慢,而且在術(shù)后18個(gè)月可以看到蠶絲膠原支架組有效得保護(hù)了關(guān)節(jié)軟骨面以及關(guān)節(jié)間隙維持在較理想寬度。 結(jié)論 本研究顯示編織狀蠶絲一膠原海綿支架可以促進(jìn)ACL功能再生并在遠(yuǎn)期有效預(yù)防骨關(guān)節(jié)炎的發(fā)生,提示其可以作為一種功能性生物支架用于ACL的重建,為其臨床轉(zhuǎn)化奠定了實(shí)驗(yàn)基礎(chǔ)。
[Abstract]:Background and Purpose With the improvement of the people's demand for quality and health, the sports body-building has gradually become an important part of daily life, and the number of people participating in the physical exercise and the competitive sports is obviously increased, and the sports injury also In the last 10 years, the histological and biomechanical function of the anterior cruciate ligament (ACL), which is an important ligament for maintaining the stability and functional movement of the knee joint in the joint cavity, is getting more and more affected by the orthopaedic surgeon. Concerned that the ability to regenerate the ACL is very limited due to the relatively short supply of blood, insufficient seed cells, and the poor liquid environment of the articular cavity, so that the functional restoration after injury still faces great challenges in the clinical treatment Challenge. Current, autograft, allografts have been the gold to repair the ACL Standards. However, these surgical reconstruction and regeneration techniques have different disadvantages, such as donor site complications, disease spread, immune rejection, poor heterozygosity, and higher complex In recent years, the appearance of the artificial ligament has brought a brand-new appearance to improve the quality of the soft tissue such as ACL Hope. The commercial products in the market are mostly non-degradable. Synthetic materials. They have a satisfactory effect in the short term, although they overcome the drawbacks of both autograft and allograft. However, long-term follow-up found that graft rupture, osteoarthritis, chronic synovitis, and foreign body reactions can still be caused. Recently, silk-based biological materials have been widely used for their good biocompatibility and high mechanical strength The repair of various soft tissues has shown that the simple binding type silk stent is used for ACL repair and has a certain effect. We have previously applied the 鈥渋nternal space preoccupation鈥，

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