An Vitro Study Of The Cytoskeletal And Apoptosis Of Human Gingival Fibroblasts Under Compressive Resistance

Objective: The investigation of periodontal tissue remodeling underorthodontic force has been becoming a hotspot, and promotion of gingivalreconstruction is beneficial to the stability of the treatment outcomes. At present,how gingival fibroblasts react to the orthodontic force and translate it intobiological signals and how various gingival components play a role in the signaltransduction has still remained to be explored. With the advancement ofexperiment methods and the deepening of the research about human gingivalfibroblasts(HGFs), the perception and response to orthodontic force havebecome the orientation to further research. Cytoskeleton determines cellmorphology. cytoskeleton is composed of microfilament, microtubule andintermediate filaments, fibrous actin(F-actin) play a significant role in themaintenance and reconstruction of the cytoskeleton of human gingivalfibroblasts. This experiment focused on common problems about the patterns ofgingival response to tooth movement in the process of orthodontic treatment. Bycompressive force for human gingival fibroblasts, to explore biological variationof F-actin, further to investigate the relationship between the cytoskeletonchanges and cell apoptosis. Subjects to cultivate human gingival fibroblasts invitro, study of cytoskeleton time effect change in the role of mechanicalcompressive force.Methods: First, HGFs were cultured in vitro, purification and identification, provide the material basis for the subsequent mechanical testing; changing thenumber of the container is placed inside the lead bead stress is applied to adjustthe size of1,2,3,4,5g/cm2, the observation time is8hours,24hours and48hours. Special fluorescent antibody specificity experiments, the choice ofRhodamine Phalloidin to mark F-actin，HGFs projected areas and shapes as wellas the structure of F-actin were tested by use of laser scanning confocalmicroscope(LSCM).Then the relation ship among compressive force, the time,the shape and the structure of F-actin of HGFs was detected. By compressiveforce for24h, TUNEL method is used to detect the apoptosis of HGFs.Results:(1) At the first stage, HGFs were successfully cultured withclassic tissue-explant method and the cell source was confirmed. Fibroblast-likecell swimmed out from the edge of tissue block after7-12days’ cultivation. The3-6generations’ grew fast and had stable biological character.(2) Mechanical compressive force, short-term load,1,2,3g/cm2loadedgroup actin fibers disorganized, thinner filaments shorter fluorescence intensitydecreased, there are statistically significant between1,2,3g/cm2loaded groupand no loaded group in the average projected area and the mean fluorescenceintensity of HGFs. With prolonged load cell morphology is close to no loadedgroup, and the average increase in the average fluorescence intensity of theprojected area of HGFs, but there are still significant differences than no loaded.4,5g/cm2loaded group structure fracture, disorder, fluorescence intensitydecreased, the average projected area of the cell and the mean fluorescenceintensity loaded with prolonged declines linearly, there are significantdifferences with the group is not loaded.(3) Compressive force acting on the HGFs for24h, the apoptosis index (AI)with a strength size change is on the rise,1,2g/cm2loaded group with no statistical difference compared with no loaded group,3,4,5g/cm2loaded groupstatistically significant compared with no loaded group.1,2g/cm2load groupaction under the fiber F-actin cytoskeleton depolymerization and rearrangementof a small number of cells undergoing apoptosis; with increasing force values,F-actin fracture stimulation can not adapt to the mechanical environment, andmore cell apoptosis. Showed that F-actin is an early mediator of apoptosis, andthe change of cytoskeleton earlier than cell apoptosis.Conclusions: Comprehensive show that the compressive force can makethe HGFs cytoskeleton microfilament structure change, under the lightercompressive force, the structure of cytoskeleton microfilament depolymerizationis not aggravated with prolonged, F-actin rearrangement, produce adaptivealterations, but this kind of adaptive renovation is limited; As the loading forcevalue, HGF depolymerization of cytoskeleton microfilament structure also isaggravating, adaptive alterations will not happen, fiber rupture, cell apoptosis.