Application And Mechanism Research On Keloid Treatment Using Nanofat And Its Adipose-derived Stem Cells

Objective:Keloid,a common disease of colored race,is a great challenge in clinical settings characterized by excessive proliferation and contracture.Adiposederived(mesenchymal)stem cells(Adipose-derived Stem Cells/Adipose tissuederived Mesenchymal Stem Cells,ADSCs/ASCs)can regulate skin fibrosis through an incompletely clear mechanism and have the potential to treat keloids.Nanofat,also known as Chylous Fat,contains a large amount of ADSCs,and can be considered as a carrier of ADSCs for clinical treatment.This study intends to investigate the clinical application prospects of nanofat in the treatment of keloids through mixed procedures.First,we examined the effect of nanofat injection combined with ADSCs into the human-derived keloid model on the back of nude mice;Second,we conducted clinical observation of the autologous nanofat transplantation in the treatment of multiple keloids;Moreover,cytological experiments were also adopted to understand the specific mechanism of ADSCs in the treatment of keloids,and to testify whether mi R-106b-5p can activate the TGF-β1/Smads pathway through targeting inhibition of Smad7,and therefore suppress keloid fibroblasts(KFs).Materials and Methods:We obtained human-derived nanofat and established a model of human-derived keloids on both sides of mice back(6 mice and 12 locations)and divided them into4 groups.After successful modeling,nanofat,triamcinolone acetonide and normal saline were respectively injected into the scar tissue of the mice in each group,and the control group was left untreated.Four weeks later,the nude mice were sacrificed and the general view,weight and volume of the keloid were recorded.Data were also collected including HE staining,Masson staining,decorin detection through immunehistochemical staining,α-SMA,collagen I and collagen III expression,Western Blot was used to detect the expression of Smad7,TGF-β1,p-Smad2/3,Smad2/3 and other indicators.Keloid fibroblasts(KFs)were cultured with ADSCs conditioned medium and divided into ADSCs group and control group.We tested cell viability of KFs by CCK8 and cell apoptosis by flow cytometry,and used scratch test and Transwell to detect cell migration and invasion.Western Blot was also adopted to detect the expression of Smad7,TGF-β1,p-Smad2/3,and Smad2/3.Smad7 was predicted to be the site of mi R-106b-5p by Targetscan,and the KFs cell line was divided into two groups.Group 1: NC mimic+Smad7wt;mi R-106b-5p mimic+Smad7wt.The fluorescence intensity was detected by dual luciferase gene reporter assay,and Smad7 expression was tested by q PCR;Group 2: According to the results of Group 1,we used conditioned medium to culture KFs,and tested the expression level of mi R-106b-5p in KFs by q PCR.We studied the impact of mi R-106b-5p overexpression in the KFs-inhibition process of ADSCs.Four groups were divided,namely,ADSCs-CM group;mi R-106b-5p mimic group;ADSCs-CM+mi R-106b-5p mimic group,and control group.We tested cell viability of ADSCs by CCK8 and cell apoptosis by flow cytometry,and used scratch test and Transwell to detect cell migration and invasion.Western Blot was also adopted to detect the expression of Smad7,TGF-β1,p-Smad2/3,and Smad2/3.Ten patients with multiple keloids(32 skin lesions in total)were enrolled.We obtained nanofat from the adipose tissue near the incision of a chosen keloid,and then injected them into other untreated keloids.The Vancouver Scar Scale Score(VSS)and Ultrasonic Shear Wave Elastography(SWE)were used to evaluate the change of keloids before the operation and 3 months later.Results:1.After 4 weeks of nanofat injection into the keloid of mice model,the following changes were observed:(1)Compared with the control group,the weight percentage of scar tissue mass after/before transplantation in the nanofat group and triamcinolone acetonide group were significantly reduced.(2)As shown by H&E and Masson staining,the accumulation of collagen fibers in both nanofat group and triamcinolone acetonide group were improved.(3)Immunohistochemical results indicated that,compared with the control group,the expression of decorin in both nanofat group and triamcinolone acetonide group were significantly increased and the expression of α-SMA,collagen I and collagen III were decreased.(4)Western blot results showed that,compared with the control group,the expression of Smad7 in nanofat group and triamcinolone acetonide group were significantly increased,and the expression of Smad7 in the triamcinolone acetonide group was higher than that of the nanofat group.In addition,the expression of TGF-β1 and the activation of Smad2 and Smad3 in the nanofat group and triamcinolone acetonide group were significantly lower than those in the control group,while the expression of TGF-β1,Smad2 and Smad3 activation levels in the triamcinolone acetonide group were significantly lower than the nanofat group.2.The results of cytological experiments were as follows:(1)As shown by CCK8 test,ADSCs-CM culture reduces the proliferation activity of KFs.(2)Scratch test showed that the scratch spacing of the two groups of KFs were shortened at 24 h compared to 0 h.The migration distance of the ADSCs-CM group was significantly smaller than that of the control group.(3)The number of cell invasion in the ADSCsCM group was significantly lower compared with the control group.(4)Flow cytometry indicated that the apoptosis rate of the ADSCs-CM group was significantly higher than that of the control group.(5)It was shown by Western blot that ADSCsCM treatment significantly increased the expression of Smad7,decreased the expression of TGF-β1,and inhibited the activation of Smad2 and Smad3.(6)The double-luciferase gene reporter experiment suggested that there were direct binding between mi R-106b-5p and Smad7.(7)Overexpression of mi R-106b-5p increased the activity of KFs and reversed the inhibitory effect of ADSCs-CM culture on KFs cell activity.(8)Overexpression of mi R-106b-5p increased the migration ability of KFs and reversed the inhibition of ADSCs-CM culture on the migration ability of KFs cells.(9)Overexpression of mi R-106b-5p increased the invasion ability of KFs and reversed the inhibitory effect of ADSCs-CM culture on the invasion of KFs cells.(10)Overexpression of mi R-106b-5p partially prevented the apoptosis of KFs cells induced by ADSCs-CM.(11)Overexpression of mi R-106b-5p reversed the inhibitory effect of ADSCs-CM on the TGF-β1/Smads pathway by targeting Smad7.3.Clinical observation results of autologous nanofat transplantation in the treatment of multiple keloids: 10 patients(with 32 keloid skin lesions)were examined three months later after nanofat injection treatment.The pruritus and tingling feeling of the scar was self-reported improved(32/32),and the following changes were observed,lighten of the scar color(21/32),decreased hardness(25/32),discontinuation of scar increase(32/32),and reduced scar size(18/32).The normality test(Shapiro-Wilk)indicated that the VSS scores before and after the treatment were both non-normally distributed(P=0.02).The Wilcoxon rank-sum test of paired data indicated the VSS decreased after the treatment(Z=-4.842,P<0.05,with statistical difference).The maximum Young’s modulus of the hardest part of keloids detected by SWE was 340.6±102.7k Pa,and 230.5±68.3k Pa three months later after treatment(t=5.048,P<0.001,with statistical difference).Conclusion:Smad7 is likely to be the target site of mi R-106b-5p,and ADSCs can suppress KFs from the mi R-106b-5p/Smad7 axis through the paracrine pathway,and thereby improve keloids.The injection of nanofat(riched in ADSCs)into keloids may suppress its hyperplasia and reduce the size and hardness of keloids,and therefore alleviate itching,tingling and other discomforts.This could be a promising alternative procedure for patients who are not suitable for surgery,drug and laser treatment,given its minimally invasive and relatively safe characteristics.Ultrasound SWE can objectively and accurately assist in the assessment of changes of keloids.