牵拉应力对三维培养的成骨样细胞的生物学效应

Biological responses of osteoblast-like cells in three-demensional culture under mechanical stretch

目的研究三维培养的克隆的鼠颅骨成骨样细胞(colonalmurinecalvarialosteoblast-likecellline,MC3T3-E1)在特定的周期性牵拉应力刺激下的增殖能力、碱性磷酸酶(alkalinephosphatase,ALP)活性及骨桥蛋白(osteopontin,OPN)mRNA表达的变化,探讨牵拉应力对成骨细胞的生物学效应。方法以明胶海绵(2.00cm×2.00cm×0.25cm)作为MC3T3-E1的三维培养支架,每块明胶海绵种植细胞悬液100μl,细胞数目为1.25×105个。明胶海绵拉伸度为5%,频率60cycles/min,15min/h,牵拉后2、4、6、8、10d分别从牵拉组和对照组中各取3个样本,行细胞计数、细胞及培养液的ALP活性测定、细胞的OPNmRNA测定。结果牵拉后第2d牵拉组的细胞数目即多于对照组(P<0.05),细胞倍增时间从71h提前至55h。除第2d外,牵拉组细胞的ALP活性低于对照组(P<0.05),两组培养液中ALP活性均处于低水平,差异无统计学意义。各时相牵拉组OPNmRNA的表达整体水平高于对照组,对照组第4dOPNmRNA的表达接近牵拉组,其余各时相均低于牵拉组(P<0.05)。结论周期性牵拉应力促进三维培养的MC3T3-E1细胞增殖,抑制细胞的ALP活性,促进OPN的合成,增强细胞的分化能力。

Objective In order to investigate the effectiveness of unique cyclic biomechanical stretch on proliferation, alkaline phosphatase (ALP) activity and osteopontin (OPN) mRNA of colonal murine osteoblast-like cell line MC3T3-E1 in three-demensional culture. Methods Gelatin sponges with a size of 2.00 cm×2.00 cm×0.25 cm were used as the three-demensional culture vector of MC3T3-E1, 100 μl cell suspension was applied to the surface of sponge, the cell number was 1.25×105. The gelatin sponges could be stretched to 5% with 60 cycles/min and 15 min/h on computerized bio-stretch controller. At 2, 4, 6, 8 and 10 d after stretch stimulation, three samples were collected at each time point from the stretch and con-trol group respectively. Cell counting, activity assay of ALP both of cells and medium as well as OPN mRNA measurement were carried out. Results The cell number of the stretch group was more than that of control group from 2 d (P<0.05), and reached the peak at 8 d, and cell diploid time of the stretch group was reduced from 71 h to 55 h. The ALP activity of cells and medium was in low level both in the stretch and control group at all time points, however, ALP activity of the stretch group was lower than that of control group (P<0.05) except at 2 d. In the stretch group, OPN mRNA maintained a high level at all the time points, and it was higher than in control group (P<0.05). In control group, OPN mRNA reached the peak at 4 d, and then decreased gradually to the lowest level at 8 d. Conclusion Cyclic stretch stress is a biome-chanical stimulation and can be converted into a biological signal in cell culture. Cyclic stretch can promote proliferation of MC3T3-E1 in three-dimensional culture, and inhibit ALP activity and promote synthesis of OPN as well as the differentiation of MC3T3-E1.