Experimental Study Of The Effect Of Composite Laser And Pingyangmycin On Rabbit Ear Scars

Objective:Explore whether the composite laser combined with pingyangmycin injection can effectively interve the hyperplasia of blood vessels and inhibit hyperplasia of scars.To find a more effective method to interve the hyperplasia of blood vessels of scar.1.Methods:Fifteen New Zealand white rabbit with big ears were used to make hypertrophic scar models on the ventral side of ear,four models in each ear.Then got 150 hypertrophic scar models in total and randomly divided into five groups:control group（group A）,pulsed dye laser group（group B）,fractional CO₂laser and pulsed dye laser group（Composite laser group,group C）,pingyangmycin injection group（injection group,group D）and composite laser and pingyangmycin injection group（combined group,group E）.30 scar models in each group,and normal skin group F.2.Treatment parameters:Fractional CO2 laser therapy（Deep FX:energy30 m J,frequency 300 HZ,shape:2,size:8,Density:5%,1 pass）,pulsed dye laser（energy:10J/cm2,light spot size:7 mm,pulse width:3 ms,dynamic cold spray time:20 ms）.There is no treatment for Group A;Group B is treated with pulsed dye laser;Group C:use fractional CO2laser treat immediately after the treatment of pulsed dye laser,Group D:About 0.1 ml pingyangmycin（2mg/ml）is injected into the inner of the scar,Group E:after the treatment of composite laser,and then inject pingyangmycin into scar just like Group D.3.Collect Specimen:Dynamically Observe the general morphological changes of the rabbit ear hypertrophic scar model after treatment,and cut off part of the scar tissue immediately after treatment and at the 3rd,7th,14th and 28th days after treatment.Then store the scar tissue in paraffin.4.Evaluate the effect of each group:HE staining was used to observe the histological changes in scar;CD31 immunohistochemical staining was used to observe the changes of the number of microvessels and the morphology of vascular endothelial cells in the scar,and then counte the micro-vessel density（MVD）.VEGF immunohistochemical staining was used to observe the improvement of blood vessels.Masson staining was used to observe collagen expression and vascular distribution in scar,and then measure collagen volume fraction.5.statistical analysis:SPSS 22.0 statistical software was used for statistical analysis of the experimental data.The data were described as the mean standard deviation（（?）±S）.One-way analysis of variance was used to compare the differences in the mean of each group.LSD Posthoc test was used to compare the difference between two groups.The difference was statistically significant when P<0.05.Result:1.General morphological observation:about 28 days after ear modeling,all the rabbit ear wounds healed completely,and formed pale red,hard and hyperplastic scars protruding on the skin plane.After the rabbit ears in each group were treated,the scar of group A remained red and hard with the passage of time,and the scar thickness gradually increased.The thickness of scar in Group C and Group E was significantly thinner,the texture was softened,and the scar color was gradually similar to the surrounding normal skin,but the color change in Group E was more obvious than that in Group C.The main manifestations of scar in group B and D were obvious changes in scar color,with the red gradually becoming lighter and the thickness of scar slightly thinner,but the texture and thickness of scar were not as obvious as those in group C and group E.On the 28th day after treatment,among the five groups,group E,namely the combined laser combined with Pingyangmycin group,had the thinest and softest scar thickness,and the overall color and thickness of scar were the closest to normal skin.2.Regular pathological histology observation:Immediately,3,7 and 14days after treatment,in group A,the dermal layer of scar was significantly thicker than that in the other four groups.Collagenous fibers were thick and dense and disorderly arranged,and some fibers were arranged in nodules.A large number of proliferative fibroblasts could be seen,inflammatory cells infiltrated,neovascularization was obvious,and no obvious skin accessory organs were observed.However,the other four groups of scar collagen fibers were sparse to varying degrees,and the infiltration of fibroblasts was also improved.After 28 days of treatment,the scar of group A still showed hyperplasia of collagen fibers.The scar collagen fibers of group B,C,D and E were reduced,sparse and orderly.The number of fibroblasts was reduced,the density of vessels was reduced,and some vascular lumens were occlusion.Among them,the collagen density of group E was closest to that of normal skin,and no hyperplastic vessels were observed under the microscope.3.CD31 immunohistochemical staining observation:vascular hyperplasia was obvious in the hypertrophic scar in group A,and the expression of CD31（light yellow or brownish-yellow particles）was increased.On the3rd,7th and 14th day,CD31 positive particles in group A gradually increased under cicatoscopy,and the positive particles began to decrease significantly after 28 days of treatment.On the third day after treatment,the capillary density in scar of group B,C,D and E began to decrease,and the expression of CD31 positive particles decreased.On the 7th and14th day,the microvascular decrease of the four groups of scars was more obvious.After treatment,the capillary density and the expression of CD31 positive particles in group B,C,D and E decreased.After CD31labeling vessels,and then counted the micro-vessel Density（MVD）values in each group.After 28 days of treatment,we compared the microvessel density of the four groups of scars with that of group A.The results showed that P_A-B<0.05,P_A-C<0.05,P_A-D<0.05,P_A-E<0.05,and the differences were statistically significant.Then,we compared the MVD values between groups B,C,D and E,and the results showed that:Group E<Group C<Group D<Group B,the difference between Group B and D was statistically significant（P<0.05）,while there was no statistically significant difference in the microvessel density between Group C and D（P>0.05）,while there was statistically significant difference in the microvessel density between Group C and E（P<0.05）.4.VEGF Immunohistochemical staining:At 3,7,14 and 28 days after treatment,VEGF values in the group A showed a slow downward trend.ANOVA was performed on VEGF values at each time point,and the results showed no statistically significant change（P>0.05）.On the 3rd day after treatment,VEGF positive particles in group B,C,D and E were significantly decreased compared with group A,and the decrease was more obvious on the 7th and 14th day.After 28 days of treatment,the VEGF values of these four groups were compared with those of group A,and the difference was statistically significant（P<0.05）.At the same time,the values of B,C,D and E were pairwise compared,and the results showed that:Group E<Group C<Group D<Group B,the difference between Group B and D was statistically significant（P<0.05）,while there was no statistically significant difference in the microvessel density between Group C and D（P>0.05）,while there was statistically significant difference in the microvessel density between Group C and E.5.Masson staining observation:Collagen fibers in the scar were blue staining after Masson staining.In group A,Collagen was thick,dense and disordered,the fibroblast aggregation could be seen between the collagens.After treatment,the blue-stained collagen fibers gradually decreased in group B,C,D and E,and the fibers became thin and sparsely arranged.Throuh counting the CVF of each group,and then found that the CVF value of group E was the smallest.Conclusion:1.Composite laser and Pingyangmycin can effectively soften rabbit ear scar,inhibit the proliferation of blood vessels and collagen;2.The inhibit effect of composite laser and pingyangmycin on blood vessels was in rabbit ear scar were better than that of composite laser,pingyangmycin and pulsed dye laser alone;3.Pingyangmycin injection can effectively inhibit the hyperplasia of scar blood vessels in rabbit ears,and the effect was better than that of PDL alone.4.Pingyangmycin could be used to assist the composite laser to inhibit angiogenesis.