External Volume Expansion Induces Adipose Tissue Regeneration By Regulating Cell Proliferation And Differentiation Via Self-synthesis Collagen

BackgroundAt present,repairing soft tissue defect is a major problem in plastic surgery.Fat transplantation and skin flap transplantation are the main methods to fill the defect,but these methods cannot obtain satisfactory results due to the problems of high retention rate and donor defect.An external volume expansion(EVE)based on tissue engineering theory provides us with a new idea.Roger Khouri K,1999 invention soft tissue expansion Brava successfully induce breast adipose tissue regeneration,and its mechanism of action of Brava conjecture,puts forward the Brava for 3 weeks to attract highly vascularized autogenous tissue engineering scaffolds can be induced,followed by adipose tissue stem cells as seed cells migration on it and eventually differentiate into fat cells,and induced fat tissue regeneration.Subsequently,basic experiments based on EVE confirmed that edema,inflammation and mechanical force were considered as the three major mechanisms of fat regeneration.In our previous animal experiments in EVE,the experimental group found that the release of cell-cell contact could promote the proliferation of adipose tissue-derived stem cells,and that the expansion could cause the increase of chemotactic factor CXCL12 and collect hematology-derived mesenchymal stem cells.All the experiments confirmed that EVE could provide seed cells for fat regeneration.Meanwhile,in previous experiments,we found that fat regeneration in EVE showed a regular pattern of strong early stage cell proliferation and late stage cell adipogenic differentiation.However,the mechanism of adipose tissue regeneration in EVE in this rule is not clear,and it requires further exploration.In previous experiments,we also found that fat regeneration in EVE was accompanied by extracellular matrix(ECM)exudation.In tissue engineering,ECM can be used as a scaffold to provide a growing environment for seed cells.ECM plays an important role in embryonic development,tissue regeneration and tissue homeostasis regulation.In recent years,studies on adipose tissue engineering have reported and used different ECM collagen components to regulate the directional functions of cells,such as migration,proliferation and differentiation.Therefore,we hypothesized that the external volume expansion model could induce the exudation of autologous collagen matrix and thus regulate adipose tissue regeneration.Methods1.Animal modelFirst,the weight of SD rats was measured and configured.Animals were anesthetized with pentobarbital.After anesthesia,SD rats were fixed in the animal operation table for hair removal and disinfection of the operation area with iodide.SD rats were followed by a 3cm longitudinal incision in the right inguinal region to fully expose the fat flap in the inguinal region.Hemostatic forceps were used to separate the inguinal fat flap and retain the axial vessels,and then the distal side was fixed to the alba line.Finally,the incision was sutured and the location and contour of the fat flap were marked with marker on the skin.SD rats were given lweek postoperative recovery period,and wound healing was examined one by one during wound recovery.One week after the operation,negative pressure suction was performed after the wound was completely healed.Firstly,a 3cm diameter hemispherical plastic cover was connected with a negative pressure suction device,and the suction pressure was adjusted to-25mmhg.Then,the cover was placed in the marked area of the fat flap for negative pressure suction.A total of 48 SD rats with body weight of 350±20g were divided into experimental groups.Among them,24 were subjected to negative pressure external attraction for 10 hours every day,and only 24 were used for the control group of simple surgical flap transfer.At week 1,4,8 and 12,SD rats in the experimental group and the control group were sacrificed and adipose tissue was collected.Fat flap along the bar is divided into three parts,one part with paraformaldehyde fixed overnight,subsequent to wax block embedding,sectioning followed by a part in-80 ℃storage and used for subsequent protein and RNA analysis.2.HE staining,Masson staining and immunohistochemical analysisThe sample was embedded in paraffin and cut into 2 mm.Hematoxylin,eosin and masson staining were carried out according to the reported standard procedures.Immunohistochemical sections were used for antigen thermal repair and primary antibody overnight.A fight for the experiments of Fibronectin,Laminin,and Collagen I,Collagen IV antibody,rtwo resistance for horseradish peroxidase labeled goat rabbit antibody.3.Fluorescence stainingAfter negative pressure suction for 1,4,8 and 12 weeks,the samples were embedded in paraffin and sliced into 2mm,followed by immunofluorescence staining,and the primary antibody was overnight.The primary antibodies used in the experiment were Ki67 and Perilipin antibodies.The nucleophilic staining was 4’,6-diamidine-2-phenyl indole staining.After the primary antibody was incubated,488nm goat anti-rabbit fluorescence secondary antibody was added.After staining,the film was photographed by Zeiss laser confocal microscope.4.Isolation and culture of adipose stem cellsSubcutaneous adipose tissue in the groin area was taken from 3-week-old SD rats.Firstly,the fat tissue granules were physically cut into 10 mm and digested with collagenase type I for 30 minutes.After complete digestion,complete medium was added to terminate digestion,centrifugation,supernatant was taken and precipitation was obtained.After resuspension,cells were inoculated in DMEM medium,10%fetal bovine serum,and 1%penicillin.After 48 hours,the unadhered cells were removed and the new culture medium was added.The culture medium was changed every 3 days,and the adherent spindle cells were defined as adipose stem cells after passage for 3 times.5.Decellularization of Adipose tissueAdipose tissue acellular pattern was treated according to the standard pattern reported in the literature.Briefly,go through the 5-day detergent decellularization process.Experience physical homogenate of adipose tissue fully broken,followed by an hour-80 ℃ to 37 ℃ repeated freezing and thawing,using the polarity extractant isopropyl alcohol extraction,then use of trypsin digestion and nuclease adequately remove cellular components.Will eventually deal with a good fat tissue to take off the cell substrate is placed in a sterile PBS in 4 ℃ storage,stay after inspection and cell cultivation.6.Reseed ASCs on decellularized adipose tissue5 mg of decellularized adipose tissue(DAT)was placed on a 24-well plate and washed with PBS for 3 times.PBS was then removed and added into the complete culture medium and placed in a cell culture dish for pretreatment before cell implantation.Will stay one day,after pretreatment,5× 10 ^ 5 of the third generation fat source of stem cells to grow on decellularized adipose tissue(DAT).After replanting for 48 hours,the unadhered cells were removed and the new medium was added.The medium was changed every 3 days.After 3 days of culture,the proliferation and lipogenic differentiation of the cells on DAT were detected.7.Oil red O stainingThe cells were cultured in adipoacellular matrix(DAT)with lipogenic induction culture(DMEM,10%fetal bovine serum,1%penicillin,rosiglitazone,insulin,IBMX and indomethacin)for 7 days,fixed with 4%paraformaldehyde,and stained with oil red o dye at room temperature.8.Statistical analysisStatistical data were expressed as mean standard deviation,and statistical software SPSS 21.0 was used for statistical data processing.Independent t test was used to compare the data between groups at a single time point.P<0.05 was considered statistically significant.Results1.Histology and morphology of adipose tissueCompared with the control group,the morphological structure of adipose tissue remained unchanged.In the suction group,obvious fibrous connective tissue-like structures were observed in adipose tissue at 40x magnification,and collagenous fibrous exudation was observed at 200x magnification.No significant fibrous exudation was observed in the control group.At 200x magnification,only a relatively tight intercellular space was observed,and no significant fibrous exudation was observed.2.Masson stainingMasson’s staining results showed that in the negative pressure suction group at 1,4,8 and 12 weeks,obvious collagen exudation was observed in the interstitial space,while only a small number of positive staining was observed in the control group.Further observation showed that there were a large number of new small fat cells in the exudation collagen in the positive area.3.Collagen types ImmunohistochemistryIn order to explore the exudation of collagen,immunohistochemistry was used to detect four main collagen components:fibronectin,laminin,type I collagen and type iv collagen in the attraction group.Immunohistochemical results showed that compared with collagen type I and collagen type iv,the expression trends of f:ibronectin and laminin were significantly different.Fibronectin increased gradually at week 1 and 4,peaked at week 4,and then decreased sharply.Laminin expression increased slowly from week 4 to week 12.4.Cellular proliferation and adipogenesisIn order to explore adipose tissue regeneration,cell proliferation-Ki67 and small adipose cell regeneration-perilipin were detected by immunofluorescence.Statistical results showed that cell proliferation increased significantly at 1 and 4 weeks,followed by a gradual decline at 8 and 12 weeks.The number of small fat cells increased gradually with the attraction,and reached the peak at 8 and 12 weeks.At the same time,we detected the expression trend of the peroxisomal proliferator-activated receptor(PPAR gamma)of the expression gene of the adipogenic differentiation,which gradually increased with the increase of the suction time.5.ASCs reseed on decellularized adipose tissue(DAT)Immunofluorescence results of acellular adipose tissue showed that fibronectin was highly expressed in DAT of the 1-week aspiration group,and laminin was highly expressed in DAT of the 12-week aspiration group.Cell proliferation and lipogenesis were detected after cell replantation.The results showed that DAT promoted cell proliferation in week 1 group,and DAT promoted cell proliferation and lipogenic differentiation in week 12 group.ConclusionEVE device enhanced collagen deposition in EPAT.High fibronectin expression during early stage collagen deposition may enhance ASC proliferation,whereas high laminin expression during later stages may promote adipogenesis.