協同金合歡醇/透明質酸預防暨治療手術誘導骨關節炎之研究

Synergistic Prevention and Repair of Farnesol/Hyaluronan on Surgical Resection-induced Osteoarthritis - A Rabbit Animal Model

陳俊宇(Chun-Yu Chen)

指導教授 ： 郭士民

共同指導教授 ： 黃蓮池(Lain-Chyr Hwang)

義守大學/智慧科技學院/電機工程學系/博士(2022年)

摘要

由於軟骨中的軟骨細胞佔比非常低（約1-5%），而且軟骨因為缺乏血液循環和淋巴循環，導致再生能力非常地差，導致軟骨無法在軟骨損傷或老化時進行自體修復。一旦到了晚期，單純的藥物和復健治療已經無法再減緩症狀時可能就需要考慮手術介入藉以治療。近幾十年來醫學的研究把重心放在軟骨再生和修復的組織工程。金合歡醇是自然界的產物，已知有許多作用對人體有幫助，例如抗發炎、抗氧化和促使腫瘤相關細胞凋亡。過去的研究證實金合歡醇可以減少發炎因子，同時也已證實金合歡醇可以調節細胞外基質及相關結締組織的合成，金合歡醇亦在體外研究被證實可恢复軟骨细胞的健康，並挽回膠原蛋白二型、多醣蛋白以及細胞外基質的生產能力。
藉由外側副韌带以及外側半月板的移除，本實驗在約8周大的紐西蘭兔中誘發關節退化。所有兔子隨機被分成對照组（健康兔子，A组，n=2）和實驗组。當中實驗組包括B组（關節退化誘導兔未經治療，n=3），C组（關節退化誘導兔接受0.4mM金合歡醇治療，n=3），D组（關節退化誘導兔接受0.8mM金合歡醇/透明質酸奈米微粒治療，n=3），E组（關節退化誘導兔接受單纯透明質酸奈米微粒治療，n=3）。接下来，被分配為治療組的患肢將在兩週後接受0.5毫升的0.4mM金合歡醇、0.8mM金合歡醇/透明質酸奈米微粒、純透明質酸奈米微粒的關節內藥物注射。在退化治療後的兩週和六週，將犧牲的兔子患肢髕骨肌腱後方的滑膜取出分離當中的蛋白比較MMP-1等等含量。同時取出整個股骨外髁關節軟骨，製成4毫米厚的切片進行組織切片、之後再加入免疫組織化學染色以及番紅0染色進一步研究分析。
用<0.4mM金合歡醇培養軟骨細胞24小時後，細胞的活力没有明顯下降，發現金合歡醇的最大半抑制濃度約為0.5mM。然而，在金合歡醇/透明質酸奈米微粒中培養則是加入到4mM金合歡醇後，才觀察到軟骨細胞的活力會顯著下降。在治療後的第六週，軟骨外觀照顯示為治療的外側髁軟骨有明顯而且廣泛的磨損。相比之下，0.4mM金合歡醇和0.8mM金合歡醇/透明質酸奈米微粒所治療的膝關節軟骨比起未治療組有更透亮健康的外觀。未治療兔子滑膜的MMP-1在兩週時有已經很快地高於其他三種治療的組別。金合歡醇在0.4mM和0.8mM时的MMP-1數值皆遠低於未治療组，而纯透明質酸奈米微粒治療组的MMP-1下降幅度則很小。在治療後第六周時， 0.4mM金合歡醇治療組的MMP-1已經上升，甚至高於未治療组，而0.8mM 金合歡醇/透明質酸奈米微粒治療組仍然能维持低水平。這時纯透明質酸奈米微粒治療组也表现出比兩週時更低的MMP-1。未治療组在治療兩週後即表現出明顯退化及表層軟骨破損，MMP-13免疫組織化學染色更是明顯反應及擴散到整个關節軟骨。此時的番紅0的染色只有未治療组有明顯軟骨基質流失，兩種不同濃度金合歡醇及純透明質酸奈米微粒治療組的整體軟骨表面外觀還是平順，組織切片沒有明顯差異，而番紅0的染色亦都顯示維持大量軟骨基質。在治療後6週，純透明質酸奈米微粒治療組和未治療组都出現嚴重的軟骨基質流失。又如膠原蛋白二型的免疫組織化學染色所示，在治療後6周，0.8 mM金合歡醇/透明質酸奈米微粒治療組的染色比0.4 mM金合歡醇的染色更明顯，顯示軟骨細胞製造膠原蛋白二型更活躍。
根據本篇研究成果顯示，將藥物封裝在透明質酸奈米微粒中，可以有效降低藥物對细胞活力抑制作用；另外，藥物在透明質酸奈米微粒中的承載濃度較大，透過減緩藥物的釋放速度及高濃度藥物乘載提高治療效果。在普遍被認為是關節退化起源的發炎反應部分，本篇研究的MMP-1水平，可以了解純透明質酸奈米微粒治療的作用較慢；0.8 mM金合歡醇/透明質酸奈米微粒治療組比0.4 mM金合歡醇能發揮更長久的作用。儘管我們發現純透明質酸奈米微粒治療也能保持軟骨表面相對地光滑，但進一步染色發現其實軟骨基質仍有大量流失。在損傷的初始階段弱能減少MMP反應可能對隨後的軟骨基質降解的程度有很大影響。本篇研究使用手術誘導退化的兔子動物模型，證實金合歡醇的早期介入可防止軟骨損傷造成的進一步退化。與透明質酸奈米微粒結合可以使金合歡醇的濃度更高、更安全，而且藥物的緩慢釋放可以使軟骨保護更持久。此外，依照0.8 mM金合歡醇/透明質酸奈米微粒在六週時的膠原蛋白二型表現，應該有更多的健康軟骨細胞恢復可用於協助軟骨自體修復。

關鍵字

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並列摘要

The articular cartilage is poorly regenerated due to its low density of chondrocytes, avascular nature, andabsence of lymphatics.Moreover, it may not be able to repair following cartilage injury or aging. Surgical intervention is required once the symptoms can no longer be controlled with medication and rehabilitation therapy at the advanced stage. To avoid the necessity for operation, tissue engineering for cartilage regeneration has been the focus of research in recent decades. An organic 15-carbon sesquiterpene compound, Farnesol, produced by Candida albicans, has exhibited antioxidant, antimicrobial, anti-inflammatory, and tumor apoptosis characteristics. Some investigatorshave demonstrated that farnesol inhibits the inflammatory cytokines, including IL-1β, IL-6, and tumor necrosis factor-alpha.Farnesol has also been shown to significantly restore ECM COL II and GAG production.
The animal osteoarthritis model was induced in 8-week-old male New Zealand rabbits through both lateral collateral ligament and meniscus resection. The rabbits were randomized into the control group (healthy rabbit, Group A, n=2) and the experimental groups: Group B (OA rabbits without treatment, n= 3), Group C (OA rabbits received 0.4 mM Farnesol treatment, n=3), Group D (OA rabbits received 0.8 mM Farn/HA nanoparticles treatment, n=3), and Group E (OA rabbits received pure HA nanoparticles, n=3). Next, the diseased limb assigned to treatment will receive intra-articular medicine injections with 0.5 mL of 0.4 mM Farnesol, 0.8 mM Farn/HA nanoparticles, or pure HA nanoparticles after two weeks. At two and six weeks following the OA treatment, upon sacrificing the rabbit, the synovium behind the patellar tendon was harvested, the total proteins were isolated from the synovium, and levels of MMP-1 were compared. The entire joint cartilage from the lateral femoral condyles is removed for histology investigation as 4 mm thick sections.
After incubation with 0.4 mM farnesol, the viability of the chondrocytes was not significantly decreased, and the IC50 of farnesol was approximately 0.5 mM.However, adding 4 mM Farn/HA nanoparticles significantly reduced the viability of chondrocytes. Six weeks after treatments, the gross photos revealed that the untreated lateral condyle has more evident and extensive wear. In contrast, the 0.4mM farnesol and 0.8mM Farn/HA treated knee cartilage showed a more transparent and brighter appearance than the untreated knees. MMP-1 levels in the untreated group were soon higher than the control group and other treatments at two weeks. The MMP-1 level of farnesol at 0.4 mM and 0.8 mM was lower than the untreated group, and the decrease in the HA group treatment was minimal. At six weeks, the low dose of farnesol 0.4mM started to rise even higher than untreated group, while farnesol 0.8mM could still be maintained at a lower level. At that time, the HA group also exhibited lower levels of MMP-1 than at two weeks. The untreated group showed significant degeneration two weeks after treatment, with MMP-13 IHC staining increasing significantly and spreading throughout the joint cartilage. The Safranin-0 stain at 2wk seems that only the untreated group had cartilage matrix loss, and there is no difference between HA and farnesol groups. Six weeks after treatment, severe cartilage matrix loss was observed in both the HA treated and untreated groups. As shown in the IHC staining for COL II, 0.8 mM at 6 weeks after treatment showed greater staining than 0.4 mM.
HA nanoparticles were found to effectively reduce the inhibitory effects of compounds and drugs on cell viability by encapsulating them; furthermore, a more significant proportion of drugs was encapsulated within HA nanoparticles, thus enhancing the therapeutic effect by slowing the release of the drug. In terms of inflammation, according to the MMP-1 level, HA has a slower action; 0.8mM Farn/HA can exert a more prolonged effect than 0.4mM farnesol. Despite the fact that HA can also keep the cartilage surface relatively smooth, there is still a significant loss of cartilage matrix. Reducing MMP at the initial stage of injury may have a significant impact on the extent of subsequent cartilage matrix degradation.This animal model of surgically induced degeneration has shown that farnesol treatment at an early stage could prevent degeneration. HA nanoparticles allow for more and safer concentrations of farnesol therapeutics, and the slow release of the drug allows for longer-lasting cartilage protection. In addition, COL II performance of 0.8 mM Farn/HA at six weeks suggests that more restored chondrocytes are available to aid cartilage repair.

並列關鍵字

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參考文獻

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