| Abstract: | 矯正治療技術與觀念之發展非常迅速，當前治療上除要求矯正裝置之品質精良外，更要求患者治療之時間能縮短，加速矯正治療牙齒移動速度，因此治療上有使用藥物注射、電刺激或是超音波之應用等方法加速骨頭之變化。然而這些方法應用上有其缺點如藥物之處理為侵入性，電刺激會造成患者不適等。過去矯正應用雷射之範圍為減低牙齒移動之疼痛或做局部牙齦之切除與漂白為主。本研究室之先導試驗發現，低能雷射對於L929 細胞具有促進癒合之反應。近年低能量雷射之發展迅速，應用於牙科之範圍越來越廣泛，由於矯正牙齒時，相對之骨細胞也會有發炎反應，究竟雷射處理是否有幫助牙齒移動，則令人好奇。先導實驗之另一結果也發現，低能量雷射對於MG63 骨細胞也有類似反應。過去矯正治療上有報告指出，雷射可以影響人牙齒之移動，但文獻上並未有雷射加速牙齒移動之相關機轉研究作更詳細之報告。本研究目的：實驗將於體外與體內進行一系列之低能量雷射對於矯正牙齒移動之相關系列研究，即以低能量雷射處理培養之牙周韌帶細胞(PDL)與骨細胞(SaOS2) 後，觀察其細胞之生物學上訊息反應(signal expression )；並將體外試驗之結果應用於動物試驗上，以了解雷射加速牙齒移動之相關機轉。本計畫預計以三年時間，完成下面之系列研究：第一年研究目的，以低能量雷射於不同條件下，比較不同劑量雷射處理時間下，探討對於牙周細胞(牙周韌帶細胞與骨細胞)之細胞生物學上訊息之表現，作為研究加速牙齒矯正移動之基礎。第二年研究目的，於體外模擬活體矯正之狀況，將牙周細胞(牙周韌帶細胞與骨細胞)分別培養在具矯正力的環境下，依第一年研究得到之雷射劑量參數照射作用後，比較牙周韌帶細胞株與骨細胞株之骨訊息因子變化。第三年研究目的，以動物試驗，模擬牙齒之矯正模式下，比較有、無低能量雷射照射後，被矯正牙齒之移動速度變化與動物之骨細胞反應。研究方法：第一年以牙周細胞(牙周韌帶細和胞骨細胞)為試驗研究對象，分為實驗組與對照組，實驗組以不同劑量的二極體雷射照射細胞，對照組則無雷射照射。細胞處理後以mitochondrial colorimetry (MTT)分析其細胞之生長存活率；以西方墨點分析方法和RTPCR方法，比較ERKinase，Alkalain phosphatase, osteopotinin, osteocalcin, iNOS, MMPs (MMP3)和Type I collagenase 之表現。結果以ANOVA 統計比較。第二年，由第一年得到之參數值，將骨細胞和牙周韌帶細胞之細胞培養置於受壓力（模擬牙齒移動時之骨細胞環境）的狀況下， 實驗組以不同劑量的二極體雷射照射細胞，對照組則無雷射照射，比較二組細胞處理後之反應。以mitochondrial colorimetry (MTT)分析其細胞之生長存活率；顯微鏡下官察細胞之形態；以 RTPCR 分析發炎iNOS 之變化； 以西方墨點分析方法和RTPCR 方法比較 ERKinase ， RANKL,Alkalain phosphatase, osteopotinin, osteocalcin 和Type I collagenase 之表現。結果以ANOVA 統計比較。第三年，以大鼠為試驗對象，於口內裝上彈簧矯正裝置，施力後，同隻動物之口腔一側為實驗組，分別接受低能量雷射照射，另一側口腔作為對照組，無接受雷射照射。以測距儀比較於14, 21, 28, 42 天之牙齒移動量變化；以HE stain 染色觀察取下之牙齒與骨頭組織變化；以西方墨點分析方法和RTPCR 方法比較 ERKinase，RANKL,Alkalain phosphatase, osteopotinin, osteocalcin, iNOS, MMPs 和Type I collagenase 之表現，結果以ANOVA 統計比較。預期由本研究之結果了解低能雷射對於骨細胞和牙周韌帶細胞之變化，以及應用於活體動物實驗下，牙齒移動速率是否會因雷射之處理而有差異，作為日後應用於臨床上之參考。
To achieve faster teeth movement in orthodontic treatment is desired by orthodontists and patients. In the past, many efforts have been applied on it. Such as medicine injection, electrostimulation, ultrasonic stimulation etc. have been tested in animals. Recently, cortication on periodontal alveolar bone to increase bone turnover rate was introduced too. But all these methods were make patient uncomfortable or scare. Laser applied on orthodontic field was common seen in relief pain control. According to studies, low level laser can increase tissue healing. Also in our previous pilot study, similar findings was shown that low level laser can increase L929 cells healing. Because orthodontic tooth movement can induce the bone inflammation. It is interesting to discover the mechanism that when low level laser applied on the orthodontic tooth movement. This study will be a three years design. The purpose of this study in first and second years is to doing the in vitro study. The third year is to doing the in vivo study. In first year, we like to detect the bone survival rate on low level laser treated peridontoal cell [periodontal ligament cell (PDL) line and bone cell line (human clonal osteoblastic cell line SaOS-2)] and to discover its bone markers expression. The second year is to study the PDL cell and bone cell’s bone and inflammatory markers expression with low laser energy treatment under orthodontic treatment simulated negative pressure culture environment. The third year is in vivo study on rat. It is to discovering the rat teeth movement changes under orthodontic and laser treatment and evaluating its bone markers expression after low level laser treatment. Materials and methods The first year, PDL and bone cells will be tested with different low level laser. MTT assay will be used to analysis cells survival rate. Western blot assay and RT-PCR assay will be to discover bone markers expression. The second year, both PDL and bone cells will be cultured in negative pressure oven and applied with different laser dose. MTT assay will be to analysis cells survival rate. Western blot assay and RT-PCR assay will be to compare bone and inflammatory markers expression. The third year, it is in vivo study on rat. The rat mouth will be fixed with an orthodontic spring appliance. In same animal mouth, one side achieves laser treatment, the other does not. The distance of teeth movement will be recorded. The sacrified animal tissue will be done histology observation. The tissue will be analysed the bone marker by RT-PCR assay. From present study design, we may understand the low energy laser biomechanism on the bone physiology changes and its effects on orthodontic tooth movement. |
