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，物理应力的人牙周膜成纤维细胞（HPLF）可能在正畸牙齿移动引起的骨重建的调节中发挥重要作用。为了确定从机械应激的HPLF获得的条件培养基（S-CM）是否会影响成骨细胞的功能或活性，我们研究了克隆小鼠成骨样细胞中的ALP活性和cAMP产生（MC3T3-E1），加入S-CM后，HPLF和MC 3 T3-E1常规培养在含有15%胎牛血清（FBS）的Dulbecco改良Eagle培养基（D-MEM）中。在37 ℃，5%CO_2的空气中。在融合期，对HPLF施加1.0 g/cm^2的持续压应力1、3、6、12和24 h。收集每种条件培养基，然后加入到MC 3 T3-E1中。在每个实验期结束时，分别通过改进的Puzan和Brand的方法和使用cAMP的酶免疫测定试剂盒来测量ALP活性和cAMP产生。1. S-CM可诱导MC 3 T3-E1细胞内ALP活性和cAMP生成量增加，并呈时间依赖性，与直接应激的MC 3 T3-E1细胞相似。3.S-CM在PGEs抑制剂吲哚美辛存在下，既不增加MC 3 T3-E1的ALP活性，也不增加cAMP的产生。此外，PGEs似乎是这些因子之一，并作为MC 3 T3-E1细胞活性的调节剂。

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.