Effects of various factors produced by mechanically stressed human periodontal ligament fibroblasts (HPLF) on bone-related cells

各种体外研究表明，物理应激人牙周膜成纤维细胞(HPLF)在正畸牙齿移动引起的骨改建过程中可能起重要作用。为了确定机械应激条件培养液(S-CM)是否会影响成骨细胞的功能或活性，我们观察了在克隆小鼠成骨样细胞(MC_3T_3-E_1)中加入S-CM后，细胞的ALPase活性和cAMP的产生。在融合期，对HPLF施加1.0g/cm2的持续压缩应力，作用1、3、6、12、24 h，收集各条件培养液，加入MC3T3-E1。实验结果如下：1.S-CM诱导MC3T3-E1细胞ALPase活性和cAMP产生均呈时间依赖性增加。尤其是在S-CM存在下，MC3T3-E1细胞ALPase活性和cAMP产生分别在12h和6h显著增加(p&lt；3.S-CM在PGES抑制剂吲哚美辛的存在下，既不增加MC3T3-E1的ALPase活性，也不增加cAMP的产生。这些结果表明，HPLF在持续压力下产生的几个因素可能会影响MC3T3-E1的分化和/或细胞反应。此外，PGES似乎是这些因素之一，并作为MC3T3-E1细胞活动的调节器。

Various studies in vitro have shown that the physically stressed human periodontal ligament fibroblasts (HPLF) may have an important role in the regulation of bone remodeling evoked by orthodontic tooth movement. To determine whether conditioned medium (S-CM) obtained from the mechanically stressed HPLF would affect the osteoblastic function or activity, we investigated ALPase activity and cAMP production in clonal mouse osteoblast-like cells (MC3T3-E1), after an addition of S-CM.HPLF and MC3T3-E1 were routinely cultured in Dulbecco's Modified Eagle's Medium (D-MEM) containing 15% fetal bovine serum (FBS) at 37ﾟC under 5% CO_2 in air. At the confluent phase, continuously compressive stress of 1.0 g/cm^2 was applied to HPLF for 1,3,6,12 and 24 h. Each conditioned medium was gathered, and then added to MC3T3-E1. At the end of each experimental period, ALPase activity and cAMP production were measured by a modification of the method of Puzan and Brand, and the use of enzyme immunoassay kit for cAMP,respectively.The results obtained in this project were as follows :1.Addition of S-CM induced increases in both ALPase activity and cAMP production in MC3T3-E1, time-dependently, as observed in directly stressed MC3T3-E1.2.Especially in the presence of S-CM,significant increase in ALPase activity and cAMP production in MC3T3-E1 was recognized after 12h and 6h, respectively (p<0.01).3.S-CM in the presence of Indomethacin, an inhibitor of PGEs, increased neither ALPase activity nor cAMP production in MC3T3-E1.These findings suggest that several factors produced by HPLF subjected to continuously compressive stress might affect differentiation and/or cellular responses of MC3T3-E1. In addition, PGEs appear to be one of these factors and to act as a modulator of cellular activities of MC3T3-E1.