研發促進骨生成的SIMVASTATIN骨材與探討藥物促進骨生成之分子機制

Development of simvastatin osteo-inductive biomaterial and study of the osteogenesis molecular mechanism of simvastatin

戴宜均(I-Chun Tai)

指導教授 ： 何美泠

共同指導教授 ： 張瑞根(Je-ken Chang)

高雄醫學大學/醫學院/醫學研究所博士班/博士(2015年)

摘要

Statins藥物是HMG-CoA reductase之抑制劑(3-hydroxy-3-methyl- glutaryl-coenzyme A reductase inhibitor)，抑制膽固醇合成的初步速率限制步驟，可以降低肝細胞內膽固醇的生合成，為臨床上常用之降血脂劑。前人研究指出statins可以在活體外促進骨生成蛋白的基因表現，包括：BMP-2、Osteocalcin、Runx-2，以及增加骨生成細胞的鹼性磷酸酶(alkaline phosphatase, ALP)活性、礦質化(mineralization)，進而促進細胞走向骨分化。然而在活體內以及臨床研究的結果仍具有爭議性。原因在於口服給予simvastatin (SIM)後首先會在肝臟進行初始代謝，以致於在身體週邊血液中的藥物濃度過低，而無法達到促進骨生成的劑量。若給予高劑量口服或是持續注射simvastatin雖能夠促進骨癒合，卻可能會造成橫紋肌溶解症或是肝臟疾病等的副作用產生。為了避免肝臟的代謝而降低藥物的作用，過去曾有些研究使用局部性給予的方式促進骨生成。然而藥物若無法由材料中控制釋出，過量的simvastatin則會造成週邊組織的傷害，而降低了simvastatin促進骨癒合的效率。因此在本論文第一部分的研究【Part I】是為了能更有效的促進無法自行癒合及延遲癒合的骨修復作用，我們使用脛骨缺損及移植壞死骨之小鼠動物模式將控放型之徐放生物高分子材料包覆之simvastatin於骨缺損處，觀察其是否能有效促進骨生成作用。實驗結果顯示其持續釋放的simvastatin (0.03~1.6 µg/day and 0.05~2.6 µg/day)不只能夠啟動骨癒合，也能夠增加血管新生以及細胞生長入移植骨的能力。在第二部分的研究【Part II】中更進一步利用小鼠的骨髓間質幹細胞來探討simvastatin促進幹細胞骨分化與細胞骨架相關之分子機轉。近年來的研究指出，活化RhoA訊息傳遞路徑能增強間質幹細胞之細胞骨架和細胞內張力，此作用在骨分化過程中扮演重要的角色。因此，本研究旨在探討simvastatin是否參與RhoA 訊息傳遞路徑以及相關之細胞骨架、細胞內張力及促進小鼠骨髓間質幹細胞走向骨分化的過程。結果顯示，simvastatin雖減少了RhoA在細胞膜上的量卻仍能增加活化態RhoA量並使其訊號傳遞更進一步活化下游分子ROCK substrate phospho-MYPT 以及 phospho-myosin light chain (MLC)，進而增強了細胞骨架(actin cytoskeleton) ，focal adhesion，以及增加細胞內張力(cell rigidity)。更進一步地， 給予抑制劑以破壞細胞骨架或減少細胞內張力則能降低simvastatin所誘導之老鼠骨髓間質幹細胞之骨化能力。活體內研究則利用異位性骨化之動物模式進一步證實細胞骨架以及細胞內張力對於simvastatin所促進之骨生成的重要性。我們的結果首次證實了simvastatin增強細胞骨架，進而增加細胞內張力，其所促進之骨髓間質幹細胞的骨分化作用中扮演重要的角色。根據我們第一部份以及第二部分的研究顯示simvastatin可作為骨誘導因子，能夠促進骨髓幹細胞之細胞內骨架重新排列以及增加細胞內張力而增促進骨分化，和骨引導性材料結合可應用於以細胞為基礎的骨再生醫學。

關鍵字

辛伐他订骨生成 ； 控制徐放骨材 ； 幹細胞 ； 細胞骨架 ； 細胞張力

並列摘要

Statins, 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, reduce cholesterol synthesis and thus prevent cardiovascular disease. Previous in vitro studies have shown that statins increase osteogenic marker genes, such as Runt-related transcription factor 2 (Runx-2), bone morphogenetic protein 2 (BMP-2), osteocalcin (OC), osteopontin, and alkaline phosphatase (ALP), in cultured osteoblastic cells and bone marrow-derived cells. However, the effects of statins on bone formation are still controversial based on animal and clinical studies because orally administered statins may be degraded during the first pass metabolism in the liver. Systemic administration of higher doses of statins may lead to rhabdomyolysis or liver failure. Local administration of statins can bypass the hepatic degradation of statins to achieve therapeutic concentrations in bone and avoid using high dose and side effects. Recent studies have shown that inflammation or delayed bone union is caused by a high dose or burst release of locally applied statin. Accordingly, Part I of this thesis was to develop a novel controlled-release drug carrier for simvastatin, (SIM)/poly(lactic-co-glycolic acid) (PLGA)/hydroxyapatite (HAp) microspheres, for local implantation around the bridged necrotic bone graft in mice. The results showed that SIM was constitutively released (0.03-1.6 µg/d and 0.05-2.6 µg/d) from SIM/PLGA/HAp that not only enhanced the callus formation at the initiation stage but also increased neovascularization and cell ingrowth in the grafted bone. Part II of this thesis was to investigate the cytoskeletal-related signaling pathway that involved in SIM-enhanced osteogenic differentiation in mouse bone marrow mesenchymal stem cells (BMSCs) (D1 cells). Activation of Ras homolog gene family member A (RhoA) signaling has been reported to increase cytoskeleton tension, which is crucial in the osteogenic differentiation of mesenchymal stem cells (MSCs). Accordingly, we hypothesized that RhoA signaling is involved in SIM-enhanced osteogenesis in BMSCs. In our experimental mouse BMSCs (D1 cells) culture system, we found that, although SIM treatment shifts the RhoA protein localization from the membrane to the cytosol, it still increases the level of active RhoA dose-dependently. SIM also enhanced RhoA signaling pathway downstream molecules including Rho-associated kinase substrate phosphomyosin phosphatase target protein and phosphomyosin light chain as well as increased actin filament density, focal adhesion composition and enhanced cellular tension. Furthermore, disrupting actin cytoskeleton or reducing cell rigidity by chemical agents reduced SIM-induced osteogenic differentiation. The results of an in vivo ectopic bone formation study also confirmed that actin filament density is increased in SIM-induced ectopic bone formation. Our study is the first to demonstrate that maintaining intact actin cytoskeleton and enhancing cell rigidity are crucial in SIM-induced osteogenesis. According to our Part I and Part II studies, we found that SIM is an osteoinductive agent and acts by increasing actin filament organization and cell rigidity, thus stimulating osteogenesis. Our results suggest that combination use of controlled-release PLGA/SIM with osteoconductive biomaterials may be benefit for stem cell-based bone regeneration.

並列關鍵字

simvastatin ； bone formation ； controlled release carrier ； stem cell ； cytoskeleton ； cell rigidity

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