周期性張力刺激對人類牙根尖乳突細胞生長及分化之影響

Abstract

隨著生物組織工程研究的發展，研究者已經可以從胚胎或成體組織中分離取得幹細胞；含有幹細胞的牙齒組織包括齒槽骨、牙周韌帶、牙髓、牙濾泡、牙胚等等，由牙齒組織培養幹細胞，相對於胚胎或其它器官而言，則是較不具爭議性、較易取得且傷害較小。體外研究已證實在適當礦化誘導刺激下，齒源幹細胞具有細胞外基質礦化能力，可做為硬組織工程研究發展所需的細胞來源，未來也可能應用在牙齒或齒槽骨缺失的修復。由牙根尖乳突組織分離出來的牙根尖乳突細胞擁有間葉細胞及幹細胞的特性，亦被證實在適當刺激下，具有與骨髓間葉細胞相當的成骨分化能力。回顧文獻中許多研究證實機械力量刺激會促進骨髓間葉幹細胞的成骨分化，但是機械力量刺激對牙根尖乳突幹細胞的生長及成骨/成牙本質分化有何影響，則至今尚未被探討。 牙齒矯正移動時，矯正力對牙周組織造成張力刺激，當牙齒的牙根發育未完全時，則牙根尖乳突組織也須承受張力刺激。本實驗藉由體外實驗模式，以Flexercell system施予週期性張力刺激（3%、0.5 Hz，48小時）， 探討張力刺激對牙根尖乳突細胞的形態及排列之影響，以及成骨/成牙本質標記基因或蛋白質表現之變化。 本實驗對於所培養的人類牙根尖乳突細胞進行初步的細胞表面抗原鑑定，結果顯示部份細胞對STRO-1及CD146抗體呈現陽性反應，表示此群細胞內含有間葉細胞及幹細胞的組成。受週期性張力刺激的細胞在形態及細胞骨架排列較具規則性，可知細胞接收外力刺激，將訊息傳入細胞核內進行一連串的基因及蛋白質製造，並表現於形態及排列上，以應對外界環境變化。此外，即時聚合酶鏈鎖反應分析發現受力後6小時及24小時，Cbfa1/Run2（Core-binding factor alpha-1/Runt-related transcription factor-2）、ALP（Alkaline phosphatase）及DSPP（Dentin sialophosphoprotein）基因表現都隨時間增加而上升，但皆未達到統計上的顯著性，週期性張力刺激作用48小時後，Cbfa1、ALP及DSPP基因顯著被向上調控，顯示張力刺激促使牙根尖乳突細胞往成骨/成牙本質分化的方向發展。 本實驗另以西方墨點法觀察Cbfa1/Runx2蛋白質及磷酸化的表現，Cba1/Runx2是成骨分化的重要轉錄因子，也與牙齒硬組織的成熟有關。本實驗結果發現牙根尖乳突細胞受力6小時後，無論是Cbfa1/Runx2總量或其磷酸化之比例，受力組及對照組之間皆無明顯的差異，但在24小時及48小時，受力組的Cbfa1/Runx2總量及磷酸化的程度都較明顯，且在24小時組別達到統計上的顯著性。 本研究首次發現周期性張力刺激可促進人類牙根尖乳突細胞在成骨/成牙本質分化標記基因的向上調控，也說明了受張力的人類牙根尖乳突細胞會往成骨/成牙本質分化的方向發展，但成骨與成牙本質分化的標記往往有所重疊而難以單獨討論，故期許以此研究為基礎，日後能繼續對成骨/成牙本質分化標記及成牙本質分化機制有更多探討。

Recent advancements in tissue engineering enable us to discover stem cells from human embryonic and somatic origins. However, because of ethical consideration and risks of operation approaching vital organs, dental tissues such as alveolar bone, periodontal ligaments, dental pulp, dental follicle, and dental papilla, are more easily accessible sources without life-threatened surgical intervention. It was reported that the cells derived from apical papilla (APCs) of developing teeth were capable of osteogenic differentiation as bone marrow stromal cells did while cultured in osteogenic induction. Previous studies showed that mechanical force drive the differentiation of marrow stromal cells into osteogenic lineage. However, there is little understanding of how does the mechanical force affect on osteogenic/dentinogenic differentiation of APCs. The orthodontic forces applied to an immature tooth might produce the tensional stress to the apical papilla tissue. The aim of this study was to investigate the effects of cyclic mechanical tensional force on the shape, alignment, or the gene or protein expressions of osteogenic/dentinogenic differentiation of the human APCs. By using Flexercell strain unit, we applied the mechanical stimulation with the protocol of 3% elongation at 0.5Hz for 48 hours.. The cultured human APCs were characterized by anti-STRO-1 antibody and anti-CD146 antibody. The stretched APCs were well-oriented in the alignment of cytoskeleton compared to unstretched control. The expressions of Cbfa1/Runx2 (Core-binding factor alpha-1/Runt-related transcription factor-2), ALP (alkaline phosphatase) and DSPP (dentin sialophosphoprotein) genes were slightly, but not significantly elevated in the APCs subjected to cyclic mechanical tensional force for 6-hr and 24-hr. However, the mRNA levels of Cbfa1/Runx2, ALP and DSPP significantly iuncreased in APCs subjected to mechanical stimulation for 48-hr. This results implied that the stretched APCs exhibited the potential to differentiate into osteoblastic/odontoblastic cells. Furthermore, we investigated the protein expression and phosphorylation of Cbfa1/Runx2. Cbfa1/Runx2 is a key transcriptional factor in osteogenic differentiation, and also plays lots role in maturation and formation of dental hard tissue. In the groups with short-term mechanical stretching (6-hr), there was no obvious difference in Cbfa1/Runx2 expression between stretched group and control group. However, the expression of total protein and phosphorylation of Cbfa1/Runx2 significantly increased in the APCs subjected to cyclic mechanical tensional force for 24-hr and 48-hr. Our study was the first to report the upregulation of osteogenic/dentinogenic genes in human APCs by cyclic tensional force. It implied that appropriate mechanical stimulation could promote the osteogenic/dentinogenic differentiation of human APCs. Further studies are needed to discover more the underlying mechanism for controlling the osteogenic/dentinogenic differentiation of human dental apical papilla cells.