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  • 學位論文

電磁場與超音波刺激對骨母細胞生長影響之比較

Comparison of Electromagnetic Field and Ultrasound Stimulation on Osteoblast Growth

指導教授 : 張恆雄
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摘要


縮短骨折癒合時間不但可減輕病人身心壓力,且能降低社會經濟成本與整體負擔。雖然許多學者皆已證實機械或電性環境是影響骨折癒合快慢的重要因子,且在臨床與動物實驗中超音波與脈衝式電磁場能有效的促進骨折癒合﹐但是未來若欲找出較佳的骨折癒合促進模式,勢必要對其作用機轉有所了解。探討骨折癒合機轉時,骨重塑作用是重要的一環。為對超音波與電磁場刺激促進骨折癒合機轉有進一步的了解,我們以骨重塑過程中重要的骨母細胞增生現象來評估其訊息傳導路徑的異同。實驗分成三階段,首先找出超音波或電磁場刺激促進體外培養骨母細胞增生的最佳參數;再以此最佳參數刺激骨母細胞﹐分析其生化因子的改變量;最後以不同的訊息傳導抑制劑檢測超音波及電磁場刺激促進骨母細胞增生之可能路徑。 實驗結果顯示,最佳的單脈衝電磁場刺激條件為頻率7.5 Hz、時間常數432 酨、誘導電場強度2 mV/cm、刺激時間2小時、骨母細胞分盤密度5e104 cells/cm2、刺激24小時後分析。在相同的細胞密度及分析時間下,最佳的超音波刺激條件為頻率1 MHz、重複頻率100 Hz、脈衝形式1:19、刺激強度600 mW/cm2、刺激時間15分鐘。生化因子分析方面,超音波及電磁場刺激有相同的趨勢,前列腺素E2 (PGE2)及轉換生長因子(TGF1)濃度都會在兩種刺激後高於控制組。鹼性磷酸酵素(ALP)活性值僅會在刺激第三天後才有顯著的增加。超音波刺激還會使介白質(IL6)及腫瘤壞死因子(TNF)的濃度低於控制組。這表示超音波與電磁場刺激可能是藉由促進培養皿內前列腺素E2及轉換生長因子濃度的增加而使骨母細胞增生,且於骨母細胞生長初期的作用主要為促進細胞增生而非分化。另外,超音波刺激抑制骨母細胞分泌介白質及腫瘤壞死因子可能會間接影響破骨細胞的形成,有待將來更進一步的研究。在兩種刺激促進骨母細胞增生的訊息傳導機轉探討方面,相同的路徑為促進骨母細胞內鈣離子的增加,活化鈣調蛋白,進而造成骨母細胞增生,且這些現象可能部份經由蛋白質活化酵素(PKA, PKC或PKG)來調控。超音波刺激促進骨母細胞增生與電磁場不同處為,其主要的訊息傳導機制可能是經由一氧化氮(NO)來調控而電磁場卻不是。本研究首次提出超音波及單脈衝電磁場刺激促進骨母細胞增生機轉之可能路徑,非但可對此種刺激促進骨折癒合的機制有所了解,且可作為將來應用於骨疾病治療的理論基礎。

並列摘要


The ability of fracture patients to return early to work or to recreational activities would not only have a substantial economic impact on society, but also improve the overall physical and mental well-being of the patients. It is well known that the mechanical or electrical factors can play an important role in fracture healing. In order to develop new methods for promoting fracture healing by mechanical, electrical, or other means, it is fundamental to understand the way in which stimulus is transduced into cellular and molecular events. The conversion of an extracellular signal, such as a mechanical or electrical stimulus, into an intracelluar response, such as cellular proliferation, is termed signal transduction. The biochemical pathway mechanism of Wolff’s law mediating the proliferative response of bone cells to a mechanical stimulus had been postulated in 1996. They hypothesized that the inositol phosphate cascade-cytosolic Ca2+-cytoskeletal calmodulin system plays a dominant role in the signal transduction of a mechanical stimulus into increased proliferation of osteoblasts. Other studies found that both mechanical and electrical stimulation of osteoblasts increased the level of prostaglandin E2 (PGE2), and transforming growth factor beta 1 (TGFβ1). These changes are probably mediated by the inositol phosphate cascade-calmodulin pathway. However, the detail mechanism of action at the cellular level in osteogenesis induced by pulsed electromagnetic field (PEMF) and ultrasound stimulus (especially in ultrasound stimulation) remains unknown. Pulsed electromagnetic field and ultrasound stimulation on osteoblasts are examined and compared in the following three aspects. (1) Determining the optimum parameters of PEMF or ultrasound stimulation on osteoblast proliferation in vitro. (2) Checking the cytokine secretion of osteoblasts after PEMF or ultrasound stimulation. (3) Investigating the mechanism of PEMF and ultrasound stimulation on osteoblasts proliferation. The results showed that the optimum parameters of specific 7.5 Hz single pulsed PEMF were exposure time 2 hours, time constant 432 酨, induced electric field intensity 2 mV/cm, exposure duration 1 day with osteoblast seeding density of 5 e 104 cells/cm2. With the same seeding density, the optimum parameters of ultrasound were exposure time 15 minutes, 1:19 pulsed, 1 MHz, 100 Hz repetition, and at the intensity of 600 mW/cm2. The important finding of osteoblast growth after treatment with optimum PEMF and ultrasound stimulations on osteoblasts was that PGE2 and TGF1 concentrations in the supernatants of the culture wells can be changed significantly by exposure. The change in alkaline phosphatase (ALP) activity is not obvious at the early stage of osteoblast culture stage, but it will occur apparently after three days of exposure. TGF1 concentration varies with the number of cells and increases day by day, while PGE2 concentration decreases day by day and is regulated by treatments. Ultrasound exposure can decrease the concentration of interleukin-6 (IL6) and tumor necrosis factor alpha (TNF) in the culture medium. There is now solid evidence that there are different transduction pathways for PEMF (partially through the PKA, PKC or PKG pathway) and ultrasound stimulation (mainly through the NO pathway, partially though the PKA, PKC or PKG pathway) which enhance osteoblast proliferation. Moreover, the pathways are complementary in that they all lead to an increase in cytocolic Ca2+ and activation of calmodulin. PEMF and ultrasound stimulation are finally moving beyond the long-held “black box” image, and these studies provide a theory of cellular mechanisms to support the clinical efficacy of PEMF and ultrasound stimulation.

參考文獻


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被引用紀錄


Fang, T. (2008). 高壓靜電場對人類骨母細胞生長之影響 [master's thesis, National Taipei University of Technology]. Airiti Library. https://doi.org/10.6841%2fNTUT.2008.00174
陳泊汎(2003)。不同低頻率之脈衝式電磁場刺激對骨母細胞成長之研究〔碩士論文,中原大學〕。華藝線上圖書館。https://doi.org/10.6840%2fcycu200300561
戴岑芳(2006)。TGF- ß 2對人類牙髓細胞的影響及其訊息傳導機制〔碩士論文,國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342%2fNTU.2006.01999
李詩苗(2008)。超音波刺激對植牙後骨整合的影響〔碩士論文,中臺科技大學〕。華藝線上圖書館。https://www.airitilibrary.com/Article/Detail?DocID=U0099-0311200815301029

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