The Effect Of CGF Fibrin Membrane On The Adhesion And Proliferation Of Human Gingival Fibroblasts

Objective:Autologous Concentrated Growth Fators (CGF) fibrin which is obtainedby differental centrifugation of autologous, as a new generation of palateletconcentrate has been used for soft tissue and bone regeneration. To investigatethe effect of the membrane on the proliferation and adhesion of humangingival fibroblasts by using scanning electron microscopy and HE staining invitro, and provide theoretical basis for periodontal reconstruction and clinicalapplication of CGF.Methods:1The culture and identification of human gingival fibroblastsNormal human gingival tissue was obtained from patients with ages from18to25years in Department of Oral&Maxillofacial Surgery, Hospital ofStomatology, Hebei Medical University. The fibroblasts were cultured in lowglucose DMEM contained20%fetal bovine serum by using traditionalmethod of primary culture in vitro. The gingival fibroblasts were digested by0.25%trypsin when fibroblasts overgrowed the80%of bottom of the bottle.The vimentin and cytokeratin antibodies were applied to identify the secondgeneration fibroblasts by Immunohistochemistry.2The experimental groups and experimental materialsThe experimental groups included group CGF, group Bio-gide and groupcontrol.CGF preparation: Venous blood was obtained from the patients and wascentrifuged in9ml sterile Vacuette tubes for13minutes. After centrifuging,the venous blood was divided into three layers. The upper layer was discardedwhile the junction of the middle layer and the bottom layer was cut and waspressed into CGF membrane. The CGF membrane was cut into5mm square tissue and then preserved in sterile normal saline.The Bio-gide membrane was cut into5mm square tissue and thenpreserved in sterile normal saline.3The culture of human gingival fibroblastsThe third generation of human gingival fibroblasts were dispensed intosingle cell suspension with the density of5×104/ml and the cell suspensionwere respectively inoculated on CGF membrane and Bio-gide membrane,andthe cells of group control were inoculated in the bottle without any membrane.Added in low glucose DMEM contained20%fetal bovine serum, at37℃and5%CO2culture saturated humidity incubator, once every2days in liquid.4Histological observationsAfter5days of culture, the cells of group CGF membrane and groupBio-gide membrane were fixed in10%formalin, embedded in paraffinsections, and then stained with HE.5SEM observationsAfter5days of culture, the cells of group CGF membrane and groupBio-gide membrane were fixed in2.5%glutaraldehyde, dehydrated, sprayedcoating, Scanning electron microscopy (scanning, electron microscopy, SEM)was used to observe the morphology of human gingival fibroblasts on twodifferent membrane.6The proliferation of gingival fibroblasts by MTT assayThe mean absorbance values (x±s) of there groups(N=8) were obtainedby MTT assay after2days and5days of culture at490nm wavelengths.7Statistical analysesStatistical analyses of the data were performed with SPSS13.0. One-wayANOVA test was applied to compare the data of the three groups.P<0.05wasconsidered as the level of statistical siginificance.Results:1The cell culture and identificationAfter6days of primary cell culture, cells emigrated from the tissueblocks and growed, long spindle with2~3nucleolus, spherical or elliptic nuclei, uniform cytoplasm, After12~14days the cells began to converge withspiral radial growth. Immunocytochemical assay was used to observe theexpression of vimentin protein and cytokeratin protein.2Histological observationsThe morphology of human gingival fibroblasts in CGF membrane wereclear with full body, nucleolar staining migrating into the film. The fibroblastsin Bio-gide membrane were wizened with small nuclear locating on thesurface of membrane.3SEM observationsThe fibroblasts on the surface of CGF membrane were in good condition,and more intensive. The cell body was plump, star shape, extending and thinpseudopodia connecting with each other and growing into lamellar.There were a few Human gingival fibroblasts on the surface of Bio-gidemembrane. Most cells showed long fusiform and some the cells were flat,small pseudopod adhesion to fibers, and connection can be seen in a few cells.4The cell growth and proliferation of human gingival fibroblastsdetected by MTT assayAfter2days of culture, the absorption values of three groups were0.596±0.049(group CGF),0.601±0.035(group Bio-gide),0.575±0.014(groupcontrol), respectively.There were no significant difference among three groups(P>0.05).After5days of culture, the absorption values of three groups were0.853±0.031(group CGF),0.733±0.027(group Bio-gide),0.634±0.013(groupcontrol), There were significant difference among three groups (P<0.05).Conclusions1CGF membrane can obviously promote the proliferation of Humangingival fibroblasts.2CGF membrane has advantage to the adhesion of Human gingivalfibroblasts. And make gingival fibroblasts to grow well. 3CGF membrane is expected to become a new scaffold material inreconstruction of periodontal tissue engineering.