結合生物陶瓷及控制釋放之骨引導藥物應用於大鼠骨修復之研究

The study for combination of bioceramic and controlled release osteoinductive agents for bone repair

傅尹志(Yin-Chih Fu)

指導教授 ： 何美泠

高雄醫學大學/醫學院/醫學研究所博士班/博士(2015年)

https://doi.org/10.6832/KMU.2015.00117

摘要

骨塑型蛋白 (BMPs) 是一種低分子量糖蛋白生長因子，其功能從細胞外和骨骼器官形成到骨形成與再生。 通常，在沒有載體的關係之下，生長因子的短半衰期和使用途徑限制其臨床上的應用。找出非蛋白質小分子以促進骨癒合則是另一種方法。 Simvastatin，一種3-hydroxy-3-methylglutaryl輔酵素A還原酶抑制劑，臨床用於減少血液膽固醇。 Mundy等說明出statins可刺激骨母細胞BMP-2的基因表現。 我們的早先研究顯示，具控制徐放的simvastatin微小球不僅促進骨折早期骨痂生成，而且增加移植骨的血管新生和細胞在內成長。 一個理想的骨移植替代物應該具備骨傳導、骨引導、生物可吸收性及易使用的特性。 已經有很多市售骨移植材料可供骨科使用，但多數只具備骨傳導並且很少甚至沒有骨引導能力。 所以，如何包覆骨引導蛋白質或藥物在骨移植替代物，且使其具備控制徐放作用將是一個大挑戰。
針對上述本論文第I部分利用新多孔陶瓷和結合我們早先研究具徐放型rhBMP-2微小球載體，評估使用這些具有骨傳導及骨引導能力之骨材於萎縮型骨癒合不良動物模型。動物組織學研究結果顯示 rhBMP-2載體組， 多孔陶瓷組和多孔陶瓷合併rhBMP-2載體組顯示比控制組在1 mm股骨缺損處有更好的骨痂形成。其中多孔陶瓷及rhBMP-2微小球載體骨材更能促進骨頭形成。
另外，第II部分研究則是要確效合併具徐放型 simvastatin 微小球(SIM/PLGA) 及能迅速吸收具藥物-運載特性的硫酸鈣複合骨材對重大骨缺損能提高骨頭癒合。研究的結果顯示此複合骨材對骨髓幹細胞沒有細胞毒性。跟控制組比較，複合骨材組提高細胞貼附能力。動物實驗顯示合併使用之骨材不只促進頭蓋骨大缺損之骨癒合，而且BMP-2和新微血管生成量也增加。 總之，結合simvastatin 微小球(SIM/PLGA) 及硫酸鈣之骨材具有骨傳導及骨引導能力，可以加速骨再生及骨癒合不良之骨折。
根據第I部分 及 第II部分 研究，我們建議合併具徐放型 rhBMP-2 或simvastatin 微小球及陶瓷具有臨床之潛力，可應用於骨生成及骨癒合不良。

關鍵字

負溫感水膠 ； 多孔性生物陶瓷 ； 骨塑型蛋白 ； 硫酸鈣複合骨材 ； 骨缺損

並列摘要

Bone morphogenetic proteins (BMPs) are low-molecular-weight glycoproteins that have pleomorphic functions ranging from extracellular and skeletal organogenesis to bone generation and regeneration. Generally, the clinical application of native growth factors without carriers is mainly limited by their short half-life and delivery way. Therefore, identification of non-protein small molecules to stimulate fracture healing is an alternative method. Simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, has been used clinically to reduce blood cholesterol levels. Mundy et al demonstrated that statins stimulate BMP-2 gene expression in osteoblasts. Our previous study showed that the controlled-release properties of a simvastatin microsphere not only enhance callus formation during the initiation of fracture healing but also increase neovascularization and cell in-growth in the grafted bone. An ideal bone graft substitute should be osteoconductive, osteoinductive, bioresorbable, and easy to use. A large numbers of bone-graft alternatives are commercially available for orthopedic use, but most are osteoconductive and have little or no osteoinductive capability. Therefore, encapsulating osteoinductive proteins or drugs in bone substitutes with controlled-release properties is a major challenge.
Accordingly, Part I of this thesis was to evaluate the new porous bioceramic in vivo and combined our previous controlled release of rhBMP-2 microsphere carriers to determine that these materials can be used as both osteoconductive and osteoinductive functional bone scaffolds in an atrophic non-union animal model. The results of animal study showed histological micrographs of the 1-mm defect in the femurs, with the rhBMP-2 carrier group, the bioceramic spacer group and the bioceramic spacer with rhBMP-2 carriers group showing better callus formation around the femur defect site than the control group. The optimal dual effects of the bone growth factors from osteoconductive bioceramics and osteoinductive rhBMP-2 carriers produced better bone formation.
Part II of this thesis was to verify that the combined use of a controlled-release simvastatin-encaptured microsphere (SIM/PLGA) and rapidly absorbable CS with drug-carrying properties to produce osteoinductive and osteoconductive cues can enhance bone healing in critical bone defects. The results of the combination treatment with a SIM/PLGA and calcium sulfate bone substitute showed no cytotoxic effect on bone marrow stem cells. Compared with controls, cell adhesion in rats was substantially enhanced following combination treatment with a SIM/PLGA and calcium sulfate bone substitute. In vivo, the combination bone substitute implantation also promoted fracture healing of critical-sized calvarial defects in rats, and bone morphogenetic protein-2 production and neovasculization were also enhanced in the defect area. In summary, the SIM/PLGA microspheres combined with a calcium sulfate bone substitute have osteoconductive and osteoinductive properties, indicating that these can be clinically applied for bone regeneration of bone defects.
According to our Part I and Part II studies, we suggested that the use of bioceramic scaffolds with controlled release rhBMP-2 or Simva carriers to support bone growth shows a wealth of potential clinical applications for the treatment of non-unions and fracture repair.

並列關鍵字

reverse thermo-responsive ； biphasic calcium phosphates ； bioceramic ； Recombinant human bone morphogenetic protein-2 carriers ； non-union defect ； calcium sulfate (CS) ； simvastatin ； calvarial bone defects ； osteoconductive ； osteoinductive

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