| Background:Progressive Hemifacial Atrophy(PHA)is a rare craniomaxillofacial disease,which involves the hemifacial soft tissues,including skin,subcutaneous tissue,muscle,and cartilage,especially subcutaneous and connective tissues.Those who develop the disease in childhood also suffer from dysplasia of the zygomatic temporal bone,maxillary and mandibular.Some patients also develop neurological and ocular complications,alopecia areata,and hair color changes.The etiology of this disease is not clear at present,so it cannot be treated according to its etiology.At present,plastic surgery treatment is aimed at the sunken deformity of the hemifacial left by the disease to restore and rebuild the morphology and fullness of the face and strive to make the affected side and the normal side symmetrical.Vascularized free flap transplantation and orthognathic surgery or bone filling are the most commonly used methods for repairing facial asymmetry.However,due to the enormous trauma of free flap transplantation and craniomaxillofacial orthognathic surgery and the uncertainty of the survival of the transplanted flap,patients have been rejected to a certain extent.Autologous fat transplantation has been promoted more and more for the treatment of mild to moderate PHA in recent years due to its advantages,such as the relatively simple operation and the wide range of sources of adipose tissue,no immunogenicity,and fast recovery.However,whether the fat survival rate of patients with hemifacial atrophy who underwent autologous fat transplantation was significantly different from that of regular patients,whether adipose tissue cells and cell derivatives are significantly different from those of healthy patients,and whether cell-assisted lipotransfer(CAL)and exosome-assisted lipotransfer(EAL)is valuable for these patients are still lacking in detailed experimental studies and data support at home and abroad.Objectives:1.To compare the in vitro biological characteristics of adipose-derived adipogenic stem cells and exosomes in liposuction area between patients with progressive hemifacial atrophy and healthy patients,including the differentiation of ADSCs between the two groups,the phenotypes,the growth increment curve of CCK-8,the increment under OGD condition,and the gene expression differences in autophagy,apoptosis,adipogenesis,and senescence.The morphology of exosomes by electron microscopy,NTA particle size,and the expression of surface proteins by WB were detected.2.To establish granular fat transplantation model in nude mice.To compare the differences in fat graft volume and mass retention at 2,4,8.and 12 weeks after transplantation among two groups of cell-assisted fat transplantation(PHA-ADSC-assisted group and NORM-ADSCs-assisted group),one experimental group of exosome-assisted fat transplantation and the control group(granulated fat transplantation alone).3.To perform hematoxylin-eosin(H&E)and immunohistochemical staining,including CD31 staining for angiogenesis,CD68 staining for macrophage infiltration,and Perilipin staining for adipogenesis,on the four groups of fat grafts removed from the subcutaneous skin of nude mice.RT-qPCR was used to analyze and compare the similarities and differences of autophagy,apoptosis,lipid senescence,and angiogenesis.This experiment aims to provide theoretical support for the treatment of PHA patients with adipogenic stem cells and exosomes assisted fat transplantation.Methods:Part I:This part was the in vitro cytological experiments part.ADSCs were extracted from abdominal fat aspirated from healthy young patients and patients with hemifacial atrophy according to the standard procedure of collagenase digestion and labeled PHA-ADSCs and Norm-ADSCs,respectively.Proliferation ability(CCK-8),stem cell phenotype(flow cytometry+BD kit),Triallel differentiation ability(oil red O staining,alizarin red staining,and alizarin blue staining after lipid,osteogenic and chondrogenic induction),migration ability(Transwell),apoptosis of ADSCs under OGD conditions,self-repair ability,apoptosis and autophagy(RT-qPCR for related gene expression)were detected respectively.The exosomes(PHA-ADSCs-Exos and Norm-ADSCs-Exos)were extracted from the supernatant of ADSCs in the two groups.NTA particle size,TEM,and WB were used to detect the similarities and differences of CD63 and TSG101.Part Ⅱ:This part is the establishment of the BALB/C nude mouse model.Fat grafts were divided into four groups.Two groups were cell-assisted fat transfer(CAL)group,including PHA-ADSCs(about 105 cells/100pL)mixed with 400p L of granular fat from corresponding PHA patients and NORM-ADSCs(about 105 cells/100μL)mixed with 400μL of granular fat from corresponding healthy patients.The third group was the exosome-assisted fat transfer group consisting of 100μL granular fat from PHA patients with exosomes(about 50μg/100μL)mixed with 400μl granular fat from PHA patients.The fourth group was the control group,including 400μl granular fat from PHA patients and 100μL phosphate buffer salt(PBS).Four different groups of fat grafts were transplanted into nude mice(bilateral shoulders and back).Each of the four injection sites in nude mice was injected clockwise to eliminate possible differences due to different injection sites.Four different groups of fat grafts were transplanted into nude mice(bilateral shoulders and back).Each of the four injection sites in nude mice was injected clockwise to eliminate possible differences due to different injection sites.Part Ⅲ:This part is the in vivo animal experiments part.APRC5,ATG5,ATG7 ATG12,BAX,PPARG,CDKN1A,and CDKN2A gene and protein expression levels were detected on the fat grafts removed at different time points by real-time(RT)-qPCR to compare the gene and protein expression differences of fat grafts between the PHA-ADSCs-assisted group and NORM-ADSCs-assisted group.H&E staining,CD31,CD68,and Perilipin-1 immunohistochemical staining were used to evaluate the specific histological changes of fat grafts in each group.Results:Part I:CCK-8 showed a statistically significant increase in cell proliferation on day 6.and the cell proliferation ability of NORM-ADSCs was significantly better than that of PHA-ADSCs from day six after transplantation.Both groups of ADSCs had typical adipogenic stem cell phenotype and differentiation ability.PHA-ADSCs showed weaker droplet formation ability than NORM-ADSCs.Transwell showed cell migration ability in PHA-ADSCs was weaker than that in NORM-ADSCs.After Calcein/PI double staining,ImageJ calculated the alive/dead ratio of PHA-ADSCs and NORM-ADSCs after OGD treatment was 46.11%and 54.21%,respectively.ATG7 and ATG12 were significantly down-regulated in PHA-ADSCs,and the apoptosis rate was higher in PHA-ADSCs.BAX expression was significantly up-regulated in PHA-ADSCs.In PHA-ADSCs,the expression of ARPC5 was significantly down-regulated.The expression of CDKN1A and CDKN2A was significantly up-regulated in PHA-ADSCs.WB analysis confirmed that CD63 and TSG101 were the positive expressions of ADSCs exosome surface markers in the two groups,but the difference in expression intensity was significant.Part Ⅱ:The fat grafts of the four groups all showed a yellow adipose tissue appearance.Compared with the control group,PHA-ADSCs,NORM-ADSCs and exosome groups had larger fat grafts in volume and weight.At 12 weeks,the volume retention rate of fat grafts was 40.66%in the PHA-ADSCs group.48%in the NORM-ADSCs group,and 38.24%in the exosome group.The volume retention rate in the control group was 27.44%.In addition,the weight retention rate was similar to that at 12 weeks.The weight retention rate was 24.67%in the control group,35.50%in the PHA-ADSCs group,42.47%in the NORM-ADSCs group,and 31.08%in the exosome group.Part III:HE staining panoramas showed better preservation of new fat cells in the peripheral and central areas of fat grafts,especially in the NORM-ADSCs group,among the four groups of fat grafts except the control group at 2 and 12 weeks after transplantation.In addition,there was no significant difference in adipocyte formation between the PHA-ADSCs and exosome groups.Quantitative analysis of the number of adipocytes showed that both cell-assisted and exosomal assistance increased the number of adipocytes in the central and peripheral regions from 2 weeks to 12 weeks.CD31 staining showed that adipose grafts in the ADSCs and exosomes group had better vascular network reconstruction at 12 weeks than the control group.The mRNA expression analysis of VEGF confirmed that the adipocyte activity and vascular remodeling ability of the norm-Adscs group were better than that of the PHA-ADSCs group.At week 12,the perilipin-1 staining showed that the number of perilipin-1 adipose-positive adipocytes in the ADSCs group and exosome group was significantly higher than that in the control group,and the NORM-ADSCs group was higher than the PHA-ADSCs group,which was entirely consistent with the expression result of adipogenesis gene PPARG at 12 weeks.CD68 staining showed that at 12 weeks,macrophage infiltration in the other three groups was significantly reduced compared with the control group.RT-PCR results showed that autophagy was down-regulated,apoptosis was up-regulated,and cell regeneration ability was down-regulated in the PHA-ADSCs group.Conclusions:Compared with NORM-ADSCs,PHA-ADSCs have relatively weak proliferation ability,higher apoptosis rate,less lipid droplet formation,weak cell migration ability,poor tolerance to OGD,easy aging,and weak self-renewal and repairability.PHA-ADSCs-assisted fat transplantation and exosome-assisted fat transplantation can significantly improve fat retention after fat transplantation in hemifacial atrophy patients.Cell-assisted and exosome-assisted fat transfer is an effective method to treat progressive hemifacial atrophy. |
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