人類牙周韌帶細胞的分化、基因表現與對機械張力刺激反應之研究

Abstract

齒列矯正治療時，矯正力量所引起的牙周韌帶與牙周組織反應是促成牙齒移動的關鍵因素。牙周韌帶是厚度僅0.15-0.38公釐介於牙根與齒槽骨之間的特殊軟組織結構，它的主要功能是使牙齒固定在齒槽骨上維持正常的牙齒位置及牙周組織的結構形態，並且負責牙周組織的修復與再生。無論是咀嚼時的咬合力、傷害性咬合接觸力以及齒列矯正力量，在機械力量刺激所引起的牙周組織適應與重塑過程中，牙周韌帶細胞都扮演極關鍵角色。基礎細胞生物學的研究顯示牙周韌帶細胞具有造骨細胞表型特徵，許多研究也顯示機械力量刺激會影響牙周韌帶細胞的生長與分化，然而在細胞生長與檢測造骨細胞表型特徵上，不同研究的結果並不一致。本研究的目的在探討一般培養液與含有L-ascorbic acid 2-phosphate(0.05mM)、sodium β-glycerophosphate(10 mM)及dexamethasone(10 -7 M)的促進基質鈣化培養液下的牙周韌帶細胞，在不同培養時間(7天、14天、21天)、未受力與施予不同程度週期性張力刺激下(3%、10%；0.1Hz；24hours)，其細 胞分化、造骨細胞表型分化指標基因與其他相關基因的基因表現。 本研究結果發現：(1)牙周韌帶細胞在促進基質鈣化培養液下會表現細胞外基質鈣化並強化其成骨指標蛋白的基因表現，且隨著培養時間而增強。(2) 3%的週期性張力刺激會提升在正常培養液細胞的ALP、OC、Cbfa-1表現，10%則是抑制作用；3%與10%的週期性張力均抑制在促進基質鈣化培養液細胞的ALP、OC、Cbfa-1表現。(3) 3%與10%週期性張力會提升COL-I、COX-2的基因表現；3%週期性張力會提升COL-III mRNA表現，10%則是下降。因此本實驗推論牙周韌帶細胞在不同程度的分化時期，對於相同程度的機械力刺激反應並不相同。此外，細胞外基質也會受機械力 量所調控。

A mechanical force applied to a tooth is transmitted to the root surrounding tissues of the periodontium and initiates remodeling activities that allow for movement of the tooth through alveolar bone. The periodontal ligament (PDL) , in normal thickness from 0.15mm to 0.38mm, is a unique structure situated between the hard tissues of the alveolar bone and cementum. The PDL contains a large number of highly specialized fibroblasts that are believed to involve the normal maintenance, repair and regeneration of the ligament, the cementum and alveolar bone. PDL fibroblasts have been shown to possess the phenotypes of osteoblasts and to undergo osteoblast differentiation in response to various stimuli. The research results in the responses of PDL cells to mechanical strain varied due to different experiment models, different strain patterns and different strain levels. We hypothesize that the differentiation stage of PDL cells determines the gene expression of osteogenic markers (alkaline phosphatase, osteocalcin), transcription factor for osteoblastic differentiation (Cbfa-1/Runx2), extracellular matrix proteins (COL-I, COL-III) and proinflammatory gene (COX-2) when PDL cells responding to mechanical strain in vitro. In this study, the human PDL cells were cultured in standard medium or mineralizing medium with osteogenic factors, such as L-ascorbic acid 2-phosphate (0.05mM), sodium β- glycerophosphate (10 mM) and dexamethasone (10 -7 M). We examined the influences of culture periods (7 , 14, 21 days), addition of osteogenic supplements and the effects of different magnitudes of cyclic tensional force (3% v.s. 10% elongation, 0.1Hz, 6 cycles/ min ) for 24 hours on the responses of PDL cells. Our results demonstrated that the osteogenic supplements of mineralizing medium induced the extracellular matrix mineralization and enhanced the expression of osteoblastic phenotype in PDL cell culture, which increased in a time-dependent manner. The mRNA expression of ALP、OC、Cbfa-1 of PDL cells cultured with standard medium was up regulated by 3% cyclic tensional force, but down regulated by 10% cyclic tensional force. However, the mRNA expression of ALP、OC、Cbfa-1 in PDL cells cultured with mineralizing medium decreased by both levels of cyclic tensional force, and this regulation was dependent on the magnitude of the force. The mRNA expression of COL-1 and COX-2 was increased by both levels of mechanical stimulation. The mRNA expression of COL-III was up regulated by 3% cyclic tensional force, but down regulated by 10% tensional force. These results suggest that PDL cells when cultured with different culture medium receiving different mechanical loads, have the ability to induce a variety of biological responses including altering ECM protein synthesis.