Effects Of Compressive Force On Expression Of Integrin ?5 And ?1 Of Human Gingival Fibroblasts Cultured On PLGA-Collagen Scaffold

Objective: To investigate the effects of compressive force on expression of Integrin ?5 and ?1 of human gingival fibroblasts(HGFs)cultured on PLGA-collagen scaffold,in order to better understand the role of Integrin on the early mechanotransduction of HGFs.Methods: HGFs were cultured by the method of tissue adhering and their histological origins were indentified.HGFs suspensions(1×105 cells/ml)were plated on the two PLGA scaffolds with or without collagen coated.After 6 hours,the seeding efficiency were examined.Fluorescence microscopy observation and confocal laser scanning microscopy observation were done after five days and CCK-8 assay was used to detect the cell proliferation activity of co-culture of HGFs.The flow cytometry method was used to detect the proliferation of cells cultured on PLGA-collagen scaffold after loading different magnitude of continuous static compressive force(0,5,15,25,35g/cm~2)for 24 hours.The optimal loading force was selected for the next experiment.The co-cultured cells were subjected to the optimal loading force for 0,1,3,6 12 hour,respectively.The m RNA and protein expression levels of Integrin ?5 and ?1 on different time were investigated by RT-q PCR and flow cytometry methods,respectively.Results:1.HGFs were successfully cultured in vitro.The cells at 4 to 6 passages with active proliferation speeds and stable shape were employed for the next experiments.2.The seeding efficiency on PLGA-collagen scaffold was significantly higher than that on PLGA scaffold.3.Fluorescence microscopy showed that there were large number of HGFs growing on these two scaffold.The number of cells on the PLGA-collagen scaffold was higher than that on the PLGA scaffold in the same view.Moreover,when focussing on different planes,there were various numbers of cells on different planes.4.Confocal laser scanning microscopy showed that HGFs grew intensively in all directions on these two scaffold.The cells associated with each other into a whole.5.The result of CCK-8 assay showed that HGFs presented the same growth patern on these two scaffold.The highest OD value were at the 5th day.The number of cells on the PLGA-collagen scaffold was significantly higher than that on the PLGA scaffold at every time point.6.Within proper range of magnitude(0-25g/cm~2),compressive force could promote the proliferation of HGFs,while excessive compressive force(35 g/cm~2)damaged HGFs partly.Therefore,the optimal loading force for our model was 25 g/cm~2.7.The stimulation of the optimal loading force(25 g/cm~2)could up-regulate the m RNA and protein expression of Integrin ?5 and ?1 over time.However,the peak value of Integrin ?5 was at the 3rd hour while that of Integrin ?1 was at the 6th hour.Moreover,the expression level of Integrin ?1 was significantly higher than Integrin ?5 at every time point.Conclusions: We successfully established the model of HGFs co-cultured with PLGA-collagen scaffold and the optimal loading force of compressive force for this model was 25 g/cm~2.The m RNA and protein expression leve of Integrin ?5and ?1 changed with time under the stimulation of the optimal loading force,indicating that they might play important roles in the early mechanotransduction of HGFs cultured in a 3D PLGA-collegan scaffold.