Effects Of Compressive Pressure On The Expression Of RhoA/ROCK And F-actin In Human Gingival Fibroblasts On PLGA Scaffolds

Objective:To investigate the effect of compressive pressure on the Rho A/ROCK signaling pathway and F-actin expression in gingival fibroblasts on PLGA scaffolds,and further understand the roles of Rho A/ROCK signaling pathway and cytoskeleton in mechanical response of HGFs.Methods:1.HGFs were cultured by tissue piece method,and cell source identification were performed.HGFs lines were established to provide experimental cells for subsequent experiments.2.HGFs suspension with the concentration of 1×10~5/ml from the 4th to 6th generation was inoculated on the PLGA scaffold to construct a three-dimensional culture model of HGFs.3.Using heavy load model to applied 25g/cm~2 compressive pressure to the HGFs-PLGA.The mRNA and protein expression of Rho A and ROCK in HGFs were detected by RT-q PCR and Western blot after 0,6,24,48,72 hours.4.Stainning of F-actin with rhodamine-phalloidin was performed,and the laser scanning confocal microscope was used to observe the changes of F-actin in HGFs.Results:1.The cells cultured with the tissue piece method were confirmed as HGFs by morphological observation and immunogenic identification.2.The character of HGFs was stable in the 4th to 6th generations.After inoculated on PLGA scaffolds,the cells were observed to grow densely in three dimensions on the PLGA scaffolds and the morphology of cells were normal.3.When 25g/cm~2compressive pressure was applied to HGFs-PLGA,the mRNA expression of Rho A was up-regulated within 24 hours,and the mRNA expression of ROCK was up-regulated within 48 hours,both of which showed highest expression at 6hours.Besides,The mRNA expression of ROCK was higher than that of Rho A within 48 hours.With the prolongation of the force exertion time,the mRNA expression gradually returned to the level of 0 hour group.4.After 25g/cm~2compressive pressure was applied to HGFs-PLGA,the protein expression of Rho A increased within 48 hours,and the protein expression of ROCK increased within 72 hours.The highest level of protein expression of Rho A and ROCK were both achieved at 24 hours.With the prolonging of the exertion time,the protein expression of Rho A and ROCK gradually recovered to the 0 hour group level at 72 hours.5.After 25g/cm~2 compressive pressure was applied to HGFs-PLGA for 0 hour,the HGFs arranged disorderly,and F-actin fibers arranged along the long axis of the cells.The morphology of F-actin fibers was more slender.The F-actin fibers accumulated in cells edge but less around the nucleus.After 6 hours of pressurization,the arrangement of HGFs became neatly and F-actin fibers remodeled.The average fluorescence intens ity of F-actin increased,and F-actin became thickened to form stress fibers.They began to accumulate around the cell pole and nucleus.After 24 hours of pressurization,the cells arranged more neatly,and the F-actin fibers arranged around the nucleus further increased,and the fibers in the cytoplasm became denser.After 48 and 72 hours of pressurization,the orientation of the cells remained directional,and the average fluorescence intensity of F-actin began to decrease,but it was still higher than that of the 0 hour group.F-actin fibers further decrease and became thinner,and the arrangement of the F-actin fibers was recovered to the unforced level.Conclusions:1.HGFs are showed good biosafety and compatibility after co-culture with PLGA.The three-dimensional culture model of HGFs-PLGA can be successfully constructed.2.The genes and protein expression of Rho A and ROCK in HGFs up-regulate and the Rho A/ROCK signaling pathway is active under mechanical force.3.Cytoskeletal is remodeled and stress fibers are formed under mechanical force.