The Effect Of Different Dynamic Mechanical Strain On The Expression Of Integrinsα₅,α₆,β₁ And The Proliferation, Synthetic Function In Human Periodontal Ligament Fibroblasts In Vitro

Periodontal tissues, especially the periodontal ligament, are exposed different mechanical forces generated by occlusion in normal or trauma condition, orthodontical treatment and dental prosthetic restoration. Periodontal ligament fibroblasts (PDLF), act as main receptive cells and effector cells, can play a key role in the remodeling of periodontal ligament, and the change of its proliferation and synthetic function directly reflect the remodeling condition of periodontal ligament. Therefore, it is important to study the biological response and mechanism of periodontal ligament fibroblasts to mechanical strain.Objectives:The objective of the present research was to study the change of proliferation, synthetic function and expression of integrinsas, α₆, β₁ in human periodontal ligament fibroblasts (HPDLF) in response to dynamic mechanical strain of different mode, magnitude and duration. Accordingly the change of biological behaviors of HPDLF to dynamic mechanical strain and the relationship between mechanical strain and signal transductionmediated by integrins were initially investigated.Methods:1. Periodontal ligament tissue attached to the mid-third of the root of premolars extracted immediately was removed with a surgical scalpel. HPDLF were cultured by the method of digesting by I -type collagenase combining with tissue adhering spontaneously. The cultured cells were passaged successfully and their histological origins were identified. The alkaline phosphatase (ALP) staining also carried out. The morphologic characteristics of HPDLF were observed and the growth curve was plotted.2. HPDLF were cultured on special culture plates and placed on a four point bending system. Cells were loaded at 0.5Hz frequency, at 3 levels of strain-force (1000, 2000, 4000ustrain, respectively equal to 0.1, 0.2, 0.4% deformation of cells), and carried out for different periods in 12hours. The effect of dynamic mechanical strain of different mode, magnitude and duration on the activity of proliferation in HPDLF was investigated through analyzing the change of cell cycles by flow cytometry (FCM).3. HPDLF were load by four point bending system. The effect of dynamic mechanical strain of different mode, magnitude and duration on the total protein level and the activity of ALP in HPDLF was investigated through quantitative analysis.4. HPDLF were load by four point bending system. The effect of dynamic mechanical strain of different mode, magnitude and duration on the expression of integrinsots, a^, Pi on the membrane of HPDLF was investigated through analyzing by FCM.Results:1. The cultured cells were typical HPDLF with steady proliferation speeds, and were suitable for mechanical loading experiment.2. Cell percent of G0/G1, S phase and proliferation index (PI) of HPDLF were significantly influenced by dynamic mechanical micro-strain, produced by our loading system. The decrease of cell percent of G0/G1 phase, the increase of percent of S phase and PI, suggested the transformation from Gl to S phase and increase of the activity of proliferation in HPDLF. These changes closely correlated with mode, magnitude and duration of strain.3. Dynamic mechanical micro-strain significantly increased total protein level and activity of ALP in HPDLF, which closely correlated with mode, magnitude and duration of strain. The change tendency of total protein level and the activity of ALP were a little different.4. The result showed that there were actual expression of integrinsas, ot6, Pi on the membrane of HPDLF without mechanical strain, and that the expression of integrinspi is highest, intergrins(X6 is lowest. Dynamic mechanical micro-strain up-regulated the expression of integrinsas, a.^, Pi on the membrane of HPDLF, which closely correlated with mode, magnitude and duration of strain. Up-regulation of expression of integrinsas, a6, Pi correlated with each other, but varied to some extent.Conclusion:Above-mentioned results suggested that the changes of expression of integrinsas, ct6, Pi closely correlated with the changes of proliferation and synthetic function and with cell differentiation in HPDLF, which were caused by dynamic mechanical strain. The changes of expression of integrinsas, ci6, Pi were relatively early cell response, which happened before the changes of proliferation and synthetic function. This suggested that there were possible causal relation between these changes. Up-regulation of expression of integrinsas, ct6, Pi caused by dynamic mechanical strain of suitable magnitude and duration possibly initated aserial of signal transduction pathways, which increased activity of proliferation and synthetic function of HPDLF, and which facilitated cell differentiation. Accordingly it indicated that integrinsPi subtribe possibly plays an important role in regulation of mechanotransduction and proliferation and synthetic function in HPDLF.To sum up, our results to some extent reveal the remodeling of periodontal ligament as a result of mechanical force, from the point of view of cellular level and molecular biology. Our results have actual significance in maintenance and rebuilding of periodontal ligament in clinic, and provide some experiment evidences for the principle of controlling forces during design of prosthetic restoration.