| 摘要: | 本研究主要是利用發光二極體（Light-Emitting Diodes, LED）應用於骨折之骨再生。先前實驗室已建立利用血小板濃厚液（Platelet Rich Plasma, PRP）促進骨前驅細胞走向骨分化，進而預防骨質疏鬆症之發生，該研究已發表於2011年與2012年之國際期刊（Biomaterial, 2011&2012）。為了進一步了解LED是否可達到骨再生的效果，本研究首先利用細胞增生實驗，證實紅光LED（波長630 nm，劑量2 J/cm2）對骨前驅細胞（MC3T3-E1）具增加細胞增生之能力；細胞骨分化實驗，分別以骨分化基因（OPN及OCN）及骨分化染色（Alizarin Red S）結果證實紅光LED可促進細胞骨分化及骨礦化之作用；同時於細胞划痕實驗（Wound Healing Assay）與遷移小室實驗（Transwell Assay），證實紅光LED具促進細胞遷移之能力。接著將利用上述結果之發現，紅光LED不僅可促進細胞增生及促進骨分化之作用，同時也具促進細胞遷移之能力，進而應用至老鼠骨折之骨再生。動物實驗設計，將利用紅光LED治療具骨折之老鼠，並於第14天，取下老鼠腿骨，由X-ray、免疫化學組織染色（Immunohistochemistry）及微型電腦斷層掃描（ Micro computed tomography ），評估老鼠之骨癒合程度與骨生長情形（骨質密度、骨體積及骨小樑數目），並利用發炎相關基因（L-1s、IL-6，及TNF-α）、噬骨（RANK、RANKL， 及OPG）及造骨相關基因（RUNX2、OPN，及OCN）探其作用之機制。結果證實，紅光LED於老鼠骨折上，具增加骨癒合之速度及促進骨折部位之骨質密度、骨體積及骨小樑數目，進而修復老鼠之骨折，且於骨環境中證實LED可降低發炎因子的表現，同時調控噬骨細胞之分化，並增加骨細胞之分化，以上結果證實LED具有骨再生之能力，預期能作為後續以骨折研究為基礎之臨床前發展。
The aim of this study is to evaluate the effect of light-emitting diodes (LED) on bone regeneration and the therapeutic potential of bone fracture. In our previous study, we established the use of Platelet Rich Plasma (PRP) to induce osteogenesis in preosteoblast cells for the prevention and treatment of osteoporosis (Biomaterials, 2011 and 2012). Thus, in vitro analysis of the effect of LED on cell growth, osteogenic differentiation, and cell migration on preosteoblast cells (MC3T3-E1) were performed. The results demonstrated that LED with a wavelength of 630 nm and at the dose of 2 J/cm2 showed significantly increasing the capacity of cell proliferation and colony formation of MC3T3-E1, and meanwhile improving the gene expression level of OPN and OCN, and also enhancing the matrix mineralization. Additionally, the irradiated-cells also represented the advancing of the ability of migration by transwell assay and wound healing assays. For in vivo animal study, an animal model of bone fracture were established and used the Hematoxylin and Eosin stain (H&E stain), micro-computed tomography (Micro-CT), and X-ray analysis to evaluate the progress of bone fracture healing and the condition of bone regeneration. To further investigate the mechanism of LED on bone regeneration, the gene expression of inflammation, osteoclastogenesis, and osteogenesis were used in the experiments. The results ex vivo performed that LED could control the balance in micro-environment between inflammation and osteogenesis during fracture healing by reducing the expression of inflammatory genes (IL-6, IL-s, and TNF-α), and meanwhile control the osteoclastic differentiation (RANK, RANKL, OPG) that could reduce the absorption of bone and increase the osteogenesis (RUNX2, OPN, and OCN). These findings of LED performed that LED could potentially be applied in bone fracture therapy. |
