Effect Of Twinlight Laser Treatment On The Attachment Of Human Gingival Fibroblasts To The Root Surface

Objective: To compare the clinical efficacy and the changes of IL-1? and MMP-9 levels in gingival crevicular fluid of the treatment of moderate and severe chronic periodontitis,which treated with hand instruments,Er: YAG laser and twinlight laser.Prepare cementum slices and observe the root surface morphology after different treatments.Human gingival fibroblasts were seeded on them to detect the initial adhesion,proliferation and cell growth morphology of human gingival fibroblasts on different root surfaces.To experiment the effect and mechanism of HGFs attachment on the root surface treated with Twinlight laser,in comprasion to hand instruments and Er: YAG laser through Enzyme-linked Immunosorbent Assay(ELISA),CCK-8 method,Scanning election microscopy(SEM)and other experimental methods.Aim to provide theoretical basis and experimental data for the promotion and the formation of new periodontal attachment of Twinlight laser-assisted periodontal therapy.Method:1.Thirty-five patients with chronic periodontitis were selected and randomly allocated into three groups after supra-gingival scaling: Group SRP(mechanical SRP therapy),Group ERL(Er:YAG laser-assisted SRP therapy),Group TPT(Twinlight laser-assisted SRP therapy).Probing depth(PD),clinical attachment level(CAL)and bleeding index(BI)were recorded and gingival crevicular fluid was collected at baseline,6 weeks,12 weeks after treatment.The gingival crevicular fluid levels of IL-1? and MMP-9 were analyzed by enzyme-linked immunosorbent assay.SPSS software version 21.0 was used for analysis.2.Single-rooted teeth extracted due to severe periodontal disease were used.Calculus deposits were removed,and the teeth were divided into three groups according to the applied treatment: Group SRP(root planing with Gracey curet);Group ERL(irradiations with Er:YAG laser);Group TPT(irradiations with combined Er:YAG laser and Nd:YAG laser).Fragments(4mm×4mm×1mm)were obtained from the experimental surfaces and observed by scanning electron microscopy.HGFs were cultured in vitro using tissue block method and anti-vimentin and keratin immunohistochemical staining were used to identify the source of the cells.The cell growth curve was determined by the CCK-8 method.HGFs were seeded on the surface of each fragment.the adhesion and proliferation of HGFs were measured by the CCK-8 method,and the cell morphology of HGFs on each fragment was observed by scanning electron microscopy.Result:1.Clinical indicators:A total of 2832 sites with 472 teeth were selected in clinical observation.After treatment,gingival swelling almost disappeared,appearance changed from dark red to pink,texture changed from soft to tough,and the amount of bleeding was significantly reduced.Repeated measure analysis of variance showed that there were interaction between groups and time points(P<0.05).At the baseline(T1),there was no statistically significant difference in clinical indicators among the three groups(P>0.05).The clinical indicators detected in the three groups were statistically different at different time points(P<0.05).The clinical indicators of each group were lower than T1 at T2 and T3(P<0.05).At T2 and T3,there were statistically significant differences in PD,CAL,and BI detection values among the three groups(P<0.05).All indicators in the TPT group were lower than those in the other two groups(P<0.05),while the indicators in the ERL group were no signficant difference from those in the SRP group(P>0.05).2.IL-1??MMP-9 content in gingival crevicular fluid:Repeated measure analysis of variance showed that there were interaction between groups and time points(P<0.05).Further analysis of the individual effects,there was no significant difference in the levels of IL-1? and MMP-9 in the gingival crevicular fluid of the three groups(P>0.05)at the baseline.The levels of IL-1? and MMP-9 in the gingival crevicular fluid of the three groups were different at T2 and T3(P<0.05).The levels of IL-1? and MMP-9 in the gingival crevicular fluid of the TPT group were significantly lower than those of the ERL group and SRP group(P<0.05).The levels of IL-1? and MMP-9 in the ERL group were significantly lower than the SRP group(P<0.05).3.Scanning electron microscopy of sample surfaceGroup SRP:The root surface was smooth and flat,with scratches,defiled layer,and scattered small calculus.There were traces of cementum ablation and no dentin tubules were exposed.Group ERL:The root surface showed a relatively uniform rough morphology,no obvious fusion,carbonization,scratches,fissure,and defiled layer were found.The surface showed obvious granular protrusions,the cementum part was ablated in some areas and no molten minerals were deposited.Group TPT:The root surface presents an unevenly rough morphology.It can be seen that the hard tissue of the tooth is ablated to varying degrees.The dentin tubules are partially fused or closed,no carbonization,.There is a small amount of defiled layer and molten mineral deposits are present.4.Adhesion and proliferation of HGFs on different treated surface HGFs cultured in vitro consistent with the appearance of fiber cell.Immunocytochemical staining showed that vimentin expressed positive and cytokeratin expressed negative.Comparisons of mean value of relative adhesion rate(sx ±)of HGFs cultured on three group for 2 hours showed statistical significance(P<0.05),the mean value of ERL group was 35.10 ± 1.08,which was significantly higher than the other two groups(P<0.05).The overall proliferation of HGFs on three groups showed an upward trend and then a gradual decline.The OD value of the SRP group on the first day was significantly lower than that of the ERL and TPT groups(P<0.05),while there was no significant difference between the ERL and TPT groups(P>0.05),the results of the other three groups were statistically different(P<0.05).The proliferation ability of HGFs in the ERL group was significantly better than that of the TPT group,and the proliferation ability of HGFs in the TPT group was significantly better than that of the SRP group(P<0.05).Adhesion microscopy of HGFs on the sample surfaces were observed by SEM.The cells on group SRP were sparse and poorly stretched,flat and similar to round.The pseudopodia were few and short.And the extracellular matrix was less.The cells on group ERL adhere well and densely packed.The cells extended out and spread the pseudopodia uniformly around the cells.The pseudopodia were intertwined into a network and tightly adhere to the surface of the sample.The body of the cells were full and well stretched.The surface of the rough sample was covered with extracellular matrix in the distant pore.The number of HGFs cells in each field of the TPT group was less than that in the ERL group.Pseudopodia that fewer and weaker than group ERL extended into the surrounding space and fixed firmly.The cell extension range was smaller.And the shape of the cells was narrow triangular or polygonal in the distant pore.Conclusions:1.Both SRP and laser treatment can effectively control and eliminate inflammation and reduce the content of IL-1? and MMP-9 in gingival crevicular fluid.2.Compared with SRP treatment,twinlight laser treatment effectively improved root surface biocompatibility.Moreover,Er:YAG laser was more suitable for processing root surface hard tissue.3.Twinlight laser-assisted periodontal treatment promoted the formation of new periodontal adhesionscan help by improving the adhesion and proliferation of HGFs on the root surface.But the damage of root surface caused by the direction of laser irradiation should be avoided.