添加中藥杜仲之多孔性骨科材料對骨細胞再生之研究

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题名:	添加中藥杜仲之多孔性骨科材料對骨細胞再生之研究 A Study of porous bone composite containing Chinese Medicine Eucommia Ulmoides on regeneration of bone cells
作者:	劉耀中
贡献者:	中醫學系碩士班
关键词:	杜仲;骨細胞再生;骨母細胞;蝕骨細胞;細胞培養 Du-Zhong;Eucommia Ulmoides;bone regeneration;osteoblast;osteoclast;cell culture
日期:	2011-08-08
上传时间:	2011-10-17 16:05:46 (UTC+8)
出版者:	中國醫藥大學
摘要:	意外事故的發生與老年人口的增加讓骨缺陷的修復與治療成為臨床上一個重要的議題。雖然目前臨床上有各種的修補技術，但卻還沒有一種既快速又便宜的治療方式。傳統的治療方式除了對骨骼可能造成二次的傷害外，也消耗太多的時間成本。基因療法則因治療費用太高而無法普及。骨科複合材料的應用是目前少數既有效又便宜的治療方式。除了可以提供骨細胞一個良好的骨誘導及骨傳導的環境以利生長外，更重要的是，材料還可添加各種促進骨骼生長之物質，並於體內降解以避免對人體組織細胞造成二度傷害。杜仲自古以來就是一種受到廣泛使用的中藥材，具有補肝腎，強筋骨的功效。在現代的研究中，其被證實對心血管疾病擁有降壓及血管擴張之效果。但對於骨細胞再生的能力則尚在研究及探討之階段。由於依據傳統中藥之書籍記載，杜仲對於治療骨疾患者擁有良好之療效，而藥材在經過炮製後，有效成分較易煎出；因此，我們嘗試將生杜仲及鹽製杜仲分別進行測試以瞭解它們對骨細胞再生的效果。本研究將中藥杜仲分別於體外進行各種骨細胞生化分析，以評估其對骨母細胞的增生、分化及礦化能力，以及對蝕骨細胞的增生是否具有促進還是抑制的效果。針對實驗結果發現，鹽製杜仲相對於生杜仲擁有更好的骨母細胞增生的促進能力，尤其在125μg/ml的濃度中。而在骨母細胞的鹼性磷酸酵素活性的分析中，鹽製杜仲相對於生杜仲的促進效果也是略勝一籌，尤其在250μg/ml的濃度中。不過，在細胞的礦化能力上，生杜仲則表現出比鹽製杜仲更好效果，尤其是在250μg/ml的濃度中。至於針對蝕骨細胞所進行的TRAP實驗顯示，不論是生杜仲或是鹽製杜仲，對於蝕骨細胞的促進或是抑制在低濃度中均無統計上的意義。反觀，在高濃度下則呈現促進的效果。在材料的實驗部分，添加中藥杜仲的GGT 骨科材料在各項測試中，均與控制組的GGT材料無明顯差異。細胞在植入材料後，使用電子掃描式顯微鏡 (SEM) 觀察其型態，發現細胞均可良好的生長，並完整貼附在材料表面。這些結果顯示，添加杜仲後的 GGT 材料並不會影響材料原有之特性，且可以提共骨母細胞一個良好之生長環境。 The injuries and traumas caused by the accidents and the increased of the population among elderly have caused the treatment and healing of the bone defect become a serious clinical issue. Although there are a variety of clinical repair techniques, none of them can provide an inexpensive and fast treatment. Conventional treatments, in addition caused a secondary injury to the skeleton, it too consumed too much on the time cost. Gene therapy, however, can’t be popularized due to the expensive cost. The application of orthopedic composite material is among the minority which provided both effective and inexpensive method of treatment. Besides providing an osteoconductive and osteoinductive environment, more importantly, the drugs can be added to the material to enhance the growth of the bone cell, and degraded in the body to avoid the adding of insult to injury. The medicinal use of Du-Zhong (Eucommia ulmoides) dates back to ancient Traditional Medicinal Chinese, possesses the effects on tonifying the liver and kidney, and strengthens the bones and sinews. In modern studies, it has been proved to have antihypertensive and vasodilatory effects. However, its ability on promoting the regeneration of bone cells is still under investigate and study. As mentioned in the ancient medical books of TCM, Eucommia ulmoides has a good effect in the treatment of bone disease, and the active ingredients are more easily to be decocted by using the processed herb, therefore, raw and salt roasted Eucommia ulmoides were used in this study to explore their effects on the regeneration of bone cells. In this study, in vitro test was performed to evaluate the effects of Eucommia ulmoides in the proliferation, differentiation and mineralization of osteoblast cell, and its effect in the proliferation of the osteoclast cells. As the result, salt roasted Eucommia ulmoides (SEU) showed a better proliferation effect in the osteoblast cells compared to the raw Eucommia ulmoides (REU), especially in the concentration of 125μg/ml. In the analysis of alkaline phosphatase assay (ALP assay), SEU too showed a higher promotion effect rather than REU, especially in the concentration of 250μg/ml. However, REU was performed better than SEU in the Von Kossa staining especially in the concentration of 250μg/ml. For the TRAP assay, both REU and SEU showed no statistically significant in the low concentration, but displayed a promoting effect in high concentration of 500μg/ml and 1mg/ml. In the material test, GGT porous composite material added with Eucommia ulmoides showed no significant difference compare to the control group. The MG63 cells were seeded into the GGT porous composite and observed under scanning electron microscope (SEM). Cells were grown well and attached to the surface of GGT porous composite. These results showed that GGT added with Eucommia ulmoides will not affect the original properties of the material, yet provided a good environment for the growth of osteoblast cells.
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