| Objective:to observe the rabbit ears hypertrophic scar morphology and histology change by the stromal vascular fraction cells(SVFs)joint fractional CO2 laser in the treatment of rabbit ears hypertrophic scar model,analyzing and comparing different treatment in the case of scar collagen type I,III ratio change and apoptosis of the fibroblast,investigate whether the stromal vascular fraction cells(SVFs)can successfully add hypertrophic scar stem cells as well as whether can more effectively scar hyperplasia and realize the functional repair of the skin combined with the fractional CO2 laser.Methods:1.New Zealand Big Eared rabbits 10,totally make rabbit ear hypertrophic scar model 80.be randomized:scar control group(group A),SVFs injection group(group B),SVFs injection + fractional CO2 laser group(group C),fractional CO2 laser group(group D),each group of 20.2.SVFs extraction and injection:after 28 days of modeling,Removal of the rabbit's double-side inguinal fat pad was washed repeatedly with PBS solution,the blood vessels were removed and scissors.PBS solution repeatedly wash to remove blood,Equal volume 0.25%type I collagen enzyme digest 30 min 37 ? constant temperature water bath liquid,during vortex generator oscillation 2-3 times,join in centrifuge tube such as PBS solution volume fully mixed termination of digestion,200 mesh sieve filter undigested fat mass Filtrate,1200 r/min centrifugation for 5 min,abandon the supernatant,PBS solution resuspended cells,Add 6 times volume of red blood cell lysate.,incubation 8 to 10 min at room temperature,1200 r/min centrifugation for 5 min,abandon the supernatant,get fresh separation SVFs cell suspension.group B and group C were injected with SVFs cell suspension,group A and group D were not treated.3.CO2 laser therapy(DeepFx:energy:15 mj,frequency:300 hz,Shape:2,Size:8,Pulse:1,the Density:5%):after injection,C and D were immediately treated with CO2 laser therapy,and A and B were not treated.4.specimen collection:to observe the morphological changes of the hypertrophic scar of rabbits in each group,cut lesion tissues in 7 days,14 days 21 days,28 days,2 months after treatment,paraffin embedding preservation.5.Evaluation of the effect of each group:HE staining observed histological changes,TUNEL detected the changes of apoptosis rate of fibroblasts and the changes in the ratio of type I/III collagen after picric acid staining.6.Data analysis:with SPSS21.0 software to analyze data,shown with mean +/-standard deviation(x + s),using the single factor analysis of variance(One-way ANOVA)to compare mean differences between groups,with LSD posthoc test to compare the difference between the two groups.P<0.05 was considered statistically significantResults:1.overall observation:28 days after modelling,each rabbit ear wound healing completely and form hypertrophic scar of red,hard and protruding.C,D group after treatment of hypertrophic scar thickness gradually slow thinning,soft texture,color close to normal skin with time.In group B,the hypertrophic scar texture was soft and pale,but the thickness was thicker than group C and D,and thinner than group A.In group A,the hyperplasia was maintained.2.Regular histopathological observation:microscopically observed after HE staining showed thickening of hypertrophic scar the dermis,which is rich in fibroblasts and capillary hyperplasia,part of the inflammatory cells infiltration,collagen density,disordered arrangement,nodular or vortex pattern distribution,the skin adnexal disappear.the hypertrophic scar in group B,C and D had different degrees transformate to normal skin after treatment,The transformation of group A was not obvious.3.TUNEL detected the apoptosis rate of fibroblasts:in the early stage of scar formation,only a small number of apoptotic cells were found in group A,gradually increased over time.B,C,D three groups after treatment for 7 days,14 days,21 days,28 days,2 months fibroblast apoptosis rate increased,apoptosis rate in each period was higher than that in the control group A,the difference was statistically significant(P<0 05).C?D group after treatment fibroblast apoptosis rate is higher than group B in the same period and after treatment for 7 days,14 days,21 days,28 days,2 months,the difference was statistically significant(P<0.05),C,D two groups after treatment for 7 days,14 days,21 days fibroblast cell apoptosis rate were similar,there was no statistically significant difference(P>0.05),and further to 28 days,2 months after treatment,significant difference was statistically significant(P<0.05).4.Special dyeing type I/III ratio:B,C,D three groups after treatment for 7 days,14 days,21 days,28 days,2 months collagen type I/III ratio is significantly lower than the same period of scar in the control group A(P<0.05,the difference was statistically significant),and with the rapid growth of collagen deposition of group A,the ratio is higher,reaching peak and stabilizing.The ratio of type I/III collagen in group D was lower than that in group B,and the difference was statistically significant(P<0.05).in Group C,the type I/III collagen ratio is lower than that of group D after treatment at 7 days,14 days,21 days,but the difference was statistically significant(P>0.05),and further to 28 days,2 months after treatment,comparing C,D two groups,significant difference was statistically significant(P<0.05).Conclusion:1.stromal vascular fraction cells(SVFs)joint fractional CO2 laser treatment of scars can promote the apoptosis of fibroblasts.Improve collagen morphology,density,arrangement,reduce and collagen type I/III ratio and vascular proliferation and inflammatory cells infiltration.The curative effect was superior to the simple matrix CO2 laser and the simple matrix vascular component cell(SVFs)injection.2.After injection of scar tissue by stromal vascular fraction cells(SVFs)may successfully add stem cells to scar tissue,using fractional CO2 laser therapy may be more effective in stimulating the proliferation and differentiation of adipose-derived stemc ells and promoting the restoration and repair of the skin in situ. |
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