Treatment And Mechanism Of Autologous Chyle Fat Graft On Hypertrophic Scars

Objective:"Chyle Fat" is prepared by bolusing the autologous fat through a special transducer.To investigate the effect of Chyle Fat on the scar treatment of subcutaneously implanted human hypertrophic scar(hHS)nude mice model;Preliminary analysis of morphological and biological characteristics of Chyle Fat Derived Stromal/Stem Cells(CFSCs).Analysis of characteristics and biological characteristics;To observe the effects of CFSCs-conditioned medium(CFSCs-CM)on the proliferation,apoptosis,migration and invasion of hypertrophic scar fibroblasts(HSFbs),to provide theoretical basis for the research on the mechanism of action of Chyle Fat in the treatment of hypertrophic scar.Methods:1.Preparation of Chyle Fat:Emulsification was achieved by transferring the fat between two syringes connected to each other by a 0.8-mm nanometer transverter 30 times to ensure that predominantly chyle fat was retained,and finally filtered through a 0.5mm aperture filter.2.Preparation of Tissue Explants and HS Implantation Model:In total,36 specimens were subcutaneously implanted into the backs of 18 male BALB/c nude mice.The mice were randomly divided into 3 groups(n=6 per group):group control,group triamcinolone acetonide and group Chyle Fat.The scar weights,histology,and decorin staining was assessed to evaluate therapeutic efficacy.3.Preliminary study on biological characteristics of Chyle Fat.4.P3 CFSCs that were 80-90%confluent were starved with serum-free medium for 48 h,Then,the CFSC supernatant was collected as CFSCs-CM.DMEM/F12 mediumwithout serum was used as the control medium(CG-CM).5.Culture of HSFbs:Isolation and culture of HSFbs by tissue block attachment method,and passed to P3 generation for use.Conditioned medium treatment:P3 HSFbs were examined using Cell Counting Kit-8(CCK-8)for Cell Proliferation Assay,using an in vitro scratch wound assayfor Cell Migration Assay and using Annexin V-FITCApoptosis detection kit for Cell Apoptosis.6.HSFbs were harvested after 48-h treatment with CFSC-CM or control medium.Thereafter,the medium was sampled for ELISA analysis of type I collagen,type III collagen,and alpha smooth muscle actin(a-SMA)according to the kit manufacturer's protocol.HSFbs were harvested after 48-h treatment with CFSC-CM or control medium.The intensity of type I collagen,type III collagen,and alpha smooth muscle actin(a-SMA)protein expression on the membranes was analyzed using Western Blot.Results:1.Emulsification was achieved by transferring the fat between two syringes connected to each other by a 0.8-mm nanometer transverter 30 times to ensure that predominantly Chyle Fat was retained.The fat became an emulsified liquid with a whitish appearance.2.Injection of Chyle Fat in scar can improve histological features such as collagen accumulation and inhibit hypertrophic scar fibrosis.3.Experiment in the preparation of Chyle Fat,fat cells are destroyed during mechanical emulsification.However,in the later stage of cell culture and passage purification,ADSCs with good differentiation potential can be obtained.It is proved that although there are no fat cells in Chyle Fat,,there are a large number of ADSCs,which we call CFSCs.4.CFSCs can express mesenchymal stem cell surface markers with high proliferation ability and can differentiate into adipose tissue,bone tissue and cartilage tissue.5.CFSCs-CM can inhibit the growth and proliferation of HSFbs.CFSCs-CM can significantly inhibit the migration of HSFbs in vitro.Treatment of HSFbs with CFSCs-CM conditioned medium in vitro does induce apoptosis.6.CFSCs-CM conditioned medium inhibits the expression of fibrogenic protein factor type I collagen,type III collagen and a-SMA protein in HSFbs.CFSCs-CM conditioned medium exerts anti-fibrotic effect on HSFbs,and significantly inhibits the secretion of extracellular matrix proteins such as Col I.Conclusion:1.A nude mouse model of human hypertrophic scar transplantation was successfully constructed,and the injection of Chyle Fat in the scar can improve the histological features such as collagen accumulation and inhibit the process of hypertrophic scar fibrosis.2.The Chyle Fat is rich in CFSCs,which has a good proliferation abilityand can be differentiated into three lines.3.CFSCs conditioned medium can inhibit the cell division,proliferation and migration of HSFbs and affect cell apoptosis.4.CFSCs conditioned medium effectively inhibits the synthesis of extracellular matrix components of HSFbs.The conditioned medium of CFSCs has a significant inhibitory effect on HSFbs,suggesting that it may be achieved through the paracrine pathway.