- 學位論文
脈衝列電磁場刺激對骨母細胞生物活性的影響
EFFECTS OF PULSE BURST ELECTROMAGNETIC FIELD STIMULATION ON OSTEOBLASTS BIOACTIVITIES
摘要
摘 要 物理刺激在骨科疾病的治療應用範圍廣泛，包括加速骨折癒合、減緩骨質流失，迄今雖在臨床治療上，已獲致很好的療效，但是對於細胞層面之分子生物影響仍不清楚。本研究以小鼠為模式探討電磁場刺激骨母細胞在不同成熟階段之分子回應機制，瞭解電磁場刺激對骨形成作用，是導致細胞數的增加或加強細胞的分化機制。 本研究以初生三天之幼鼠培養骨母細胞，施以脈衝式電磁場刺激系統對骨母細胞之影響。其參數設定為頻寬 5 ms、頻待 61.6ms、 頻率15 Hz，磁場強度為1 高斯，每天刺激8小時並依序刺激1、3、5、12天。利用細胞活性、鹼性磷酸酶定量、鹼性磷酸酶染色、骨組織礦化染色、反轉錄聚合連鎖反應探討電磁場刺激之骨形成作用的分化機制之機轉。 結果發現，接受脈衝式電磁場刺激的骨母細胞較控制組的MTT吸光值高且具有統計上的意義；在刺激達第1，3，5天時，刺激組較控制組分別增加了34 %，11.5 %，13.3 %。但在第12天的時候，控制組的MTT吸光值幾乎接近刺激組，二者之間並沒有明顯的差異且無統計上的意義。所以接受脈衝式電磁場刺激的骨母細胞在細胞增生機制中的確有促進新生骨之形成作用。同時，其鹼性磷酸酶活性的濃度之於控制組，則分別減少了11.4 %，20.1 %，32.0 %。第12天的時候，一樣低於控制組的濃度達12.4 %。在本實驗初代骨母細胞培養模式下，經電磁場的刺激下可能轉變為一種延後調控分化的能力。但是，當這些細胞慢慢開始發展至成熟過程後，電磁場刺激效應就沒有很大的差別。因此，我們推測電磁場刺激效應在細胞分化機制中有可能使骨母細胞延緩分化的效果，進而維持及加強了前趨骨母細胞的活性。骨母細胞的成熟過程是一種複雜的機制且伴隨著特異性表徵的改變會有明顯的基因表現。接受脈衝式電磁場刺激的骨母細胞，其細胞內的OPG 之mRNA基因會提早表現；而PGL之mRNA基因則會延後表現。而這樣的結果似乎透露出電磁場效應可能會抑制蝕骨細胞的成熟作用，但是仍需進一步研究以證實此假說。 在本研究中我們發現，經脈衝式電磁場刺激後，可促進骨母細胞的增生能力，延遲細胞的分化及成熟作用，並降低礦化節點的生成機制。因此，在此模式下的影響對骨母細胞的骨形成作用，是比較傾向細胞數目的增加而不是加強骨母細胞的分化反應機制。
並列摘要
Abstract Electric stimulation has been used successfully to treat a wide range of bone disorders, including delayed and onounion fractures, fresh fracture healing, prevention and reverse of osteoporosis, and congenital pseudarthroses. However, further research is required to characterize specifically the molecular responses of osteoblasts exposed to electric stimulation in different stages of maturation. The purposes of this research were to assess the stimulatory effect of PEMF on bone cells and to validate that bone tissue-like formation was associated with the increase in the number of cells and/or with the enhancement of cellular differentiation. Newborn ICR mice calvarial osteoblasts model will be used in this study. A pulse electromagnetic field with a frequency15 Hz and magnetic filed strength of 1 gauss were applied. In order to assess the effects of PEMF stimulation on the cellular biology throughout the osteoblast maturation, a series of cultures was stimulated from 1 to 12 days with 8 hours per day. The temporal effects of PEMF on the osteoblast were evaluated by the status of proliferation, differentiation (ALP stain and titer measurement), mineralization (Von-Kossa stain), and gene expression (collagen type I, osteopontin, osteocalcin, osteopotegerin and osteopotegerin ligand) on the 1st, 3rd, 5th, and 12th days of stimulation. Our results demonstrated that PEMF stimulation significantly increased the osteoblast proliferation 34.0, 11.5, 13.3% over the control group on the 1, 3 and 5 days of stimulation; thereafter, there was no significantly difference in the optic density of MTT assay between the control and the PEMF stimulated groups until the end of the 12 days experiment. The PEMF stimulation significantly decreased the ALP activity in the neonatal calvarial osteoblast-like cell and this effect was partially blocked as the culture period increased. The addition of PEMF stimulation on the osteoblast culture did delayed the formation of mineralization nodules. The formation of mineralization nodules in the osteoblast cultures were significantly affected by the PEMF stimulation, especially during the first 5 days’ culture. In the present investigation, we also determined the effects of PEMF stimulation on the extracellular matrix synthesis. Under the PEMF stimulation, the OPG mRNA expression was up-regulated; the OPGL mRNA expression were down-regulated than that of the control. In conclusion, PEMF treatment on osteoblasts may accelerate cellular proliferation, delayed cellular differentiation, maturation and decreased mineralization nodules formation. The PEMF stimulated increase in the bone tissue-like formation was most likely associated with the increase in the number of cells, but not with the enhancement of the osteoblasts differentiation.