| Background and ObjectionFat grafting has emerged as a key technique in soft tissue reconstruction.Despite improved surgical techniques,especially with the advent of the BRAVA preconditioning,fat transplantation still results in donor site morbidity and is bedeviled by unpredictable fat retention.To achieve better engraftment survival,cell-assisted fat grafting has gained popularity in the field of plastic surgery.Recently,the addition of stromal vascular fraction(SVF)to the lipotransfer technique has been shown to improve results.These vascularized engraftments possess multiple cell types with different capacities and are available in sufficiently high cell numbers through liposuction.However,a particular drawback of cell-assisted lipotransfer in clinical practice is the need to harvest almost double the amount of fat intended for transplantation,which is not feasible in some patients,and the need to process the SVF,which adds a further period of 60-90 min to the existing operating time.Furthermore,the functionality of adipose stem cells(ASCs)within SVF varies according to patient profile.Previous studies have demonstrated that indomethacin,a known ligand for peroxisome proliferator-activated receptor-?(PPAR-?),promotes adipogenesis in stem cells in vitro,and found that the expression of adipogenic genes are greatly up-regulated.More importantly,some studies revealed that indomethacin,as the nonsteroidal anti-inflammatory drug(NSAID),protected the engrafted stem cells from the inflammatory insults,further regulating the local microenvironment and preventing pro-inflammatory cytokine-induced apoptosis.These data suggest that indomethacin influences the local inflammation,and co-treatment with lipoaspirate may improve final adipose retention.Thus,we hypothesize that indomethacin supplementation may enhance fat graft volume retention by triggering adipogenesis and protecting the viability of adipose progenitor cells in the grafted tissue.To test this hypothesis,human ASCs were cultured in different conditioned medium and supplemented with various concentrations of indomethacin and estimated by Oil-Red O and PCR for adipogenic genes.In vivo,fat grafts with or without indomethacin were compared with grafts with SVF.It would be great value since indomethacin's role as a pharmacological adjunct in fat grafting has hitherto been unexplored,despite its relatively non-toxic nature and FDA-approved status.However,its role as a pharmacological adjunct in fat grafting and culture medium for human ASCs have hitherto been unexplored despite its relatively non-toxic nature and FDA-approved status.Materials and Methods Preparation of human conditioned medium and Zuk mediumFresh human lipoaspirate were obtained with informed consent from patients in accordance with the Australian National Health and Medical Research Council guidelines and approval from the St.Vincent's Health Human Research Ethics Committee.Adipose tissue was harvested from the abdominal region that was discarded after surgical procedures.The harvested lipoaspirate was washed with phosphate-buffered saline to remove oil and other fluids and then maintained in complete medium(Dulbecco's modified Eagle's medium(DMEM),10%fetal calf serum,1%PS,1%glutamine)in proportion of 1:3,cultivating at 37?,5%carbon dioxide,and 95%humidity for 24 hrs.The cultured medium was then collected and passed through a 0.22um filter to remove debris.This extracted conditioned medium was then aliquoted and stored in-20 freezer,ready for use.In 2001,Zuk et al.,characterized multilineage differentiation potential of SVF-like cells,obtained from human lipoaspirates,and termed this stem cell population as processed lipoaspirate(PLA)cells.In his study,these PLA cells were induced towards adipogenic lineage using adipogenic induction factors,termed as Zuk medium,which was composed of DMEM supplemented with 10%Fetal bovine serum(FBS),0.5mM isobutyl-methylxanthine(IBMX),1 ?M dexamethasone.10?M insulin.200?M indomethacin and 1%antibiotic/antimycotic.11Isolation and culture of Human ASCsTissues were obtained with informed consent from five patients in accordance with the Australian National Health and Medical Research Council guidelines and approval from the St.Vincent's Health Human Research Ethics Committee.Adipose tissue was harvested from fat in the abdominal region that was discarded after surgical procedures.Isolation of primary ASCs was subsequently performed.Briefly,minced adipose tissue was digested with 0.1%typel collagenase(Worthington Biochemical)in phosphate-buffered saline(PBS)for 50 min.Upon centrifugation at 300 g for 5 min,cell pellets were resuspended in complete medium(DMEM)-high glucose containing 10%fetal calf serum and 1%antibiotic-antimycotic solution;(Invitrogen),filtered through a 100 ?m nylon mesh,and centrifuged at 700 g for 5 min.Cells were washed in PBS and then resuspended in complete DMEM and placed into tissue culture flasks for incubation overnight at 37? in a humidified atmosphere containing 5%C02.The next day,fresh medium was applied and ASCs were harvested when they reached 90%confluence,based on their adherence to plastic.The cells were then frozen in liquid nitrogen.Adipogenic differentiation of hASCsASCs in passage 2 were used.Healthy hASCs were plated at a density of 2.0 x 104 cells/well in 24-well plates and grown at 37? in 5%CO2.Cells were grown to 80%confluence in complete medium.One-days post-confluent cells were incubated in different culture medium including complete medium(negative control),Zuk medium(positive control),and adipogenesis-inducing medium.In the adipogenesis-inducing groups,indomethacin were applied in an incremental dosage to ascertain the best concentration for adipogenesis.Four groups of indomethacin-supplemented conditioned medium(Cd)were prepared at concentrations of 50,100,150,200?M.In addition,Zuk supplemented with 200?M indomethacin was prepared as double dose of 200?M indomethacin in conditioned medium.For the 2-week experiments,media was replaced every three days.Oil Red-O staining and quantitative analysisTo examine adipose-derived stem cells differentiation,cells were analyzed by Oil Red O staining for adipocyte detection after 2 weeks' culture.ASCs were fixed with 10%formalin and stained with Oil red O solution(in 60%iso-propanol)for 1 h following repeated washing with distilled water twice.1 ml of hematoxylin solution was added for 5 mins.Finally,hematoxylin was removed and the cells were then immersed in 1ml fresh water each well,visualized under light microscopy and photographed.To extract the incorporated oil red 0 from the stained cells,lml isopropanol was added to each well followed by 15 min of shaking at room temperature.The optical density(OD)of the solution was measured at 492 nm with Plate Readers.Quantitative Real-time Polymerase Chain ReactionTotal RNA was isolated and cDNA prepared using Superscript III Reverse Transcriptase(Life Technologies).Quantitative reverse transcription polymerase chain reaction was performed in triplicate using 1 ?l of cDNA and Quantifast SYBR Green(Qiagen,Toronto,Ontario,Canada).Data were collected on an RG-6000 Rotor-Gene(Corbett Research,Sydney,Australia)and analyzed using the 2-AACT relative quantification technique and expressed relative to an internal normalizing mRNA.High-stringency primer pairs were used for markers up-regulated during adipose differentiation,like peroxisome proliferator-activated receptor-y(PPARy),CCAAT-enhancer binding protein alpha(CEBP-a),fatty acid binding protein 4(FABP4),CCAAT-enhancer binding protein belta(CEBP-?)and lipoprotein lipase(LPL).(Invitrogen,Camarrillo,Calif.).18S primers served as an internal normalizing standard.Nonvascularised fat transplantation Animal ModelThis study was carried out in strict accordance with the recommendations of the Guide for the Care and health of Laboratory Animals of the Australian National Health and Medical Research Council.The animal protocol was approved by the St.Vincent's Hospital Animal Ethics Committee(#024/15).Eight-week-old female C57BL/6 mice(EMSU,O'Brien Institute Department,Victoria,Australia)were purchased from Animal Resources Centre,Perth,Western Australia.All efforts were made to minimize animal suffering.Fourty normal C57 mice were killed and the subcutaneous inguinal fat pads from both sides were harvested and gently dissected into extremely small pieces,similar to the size of aspirated fat tissue used for clinical fat injection in humans.We processed stromal vascular fraction(SVF)from a donor group of C57BL/6 mice according to a previous protocol by Stillaert et al.for cell-assisted lipotransfer12.The final pellet was resuspended in PBS to achieve a standardized concentration of 10,000 cells per injection in the cell-assisted lipotransfer group,which has been shown by Paik and colleagues to exert the maximum effect on fat graft retention13.200?l of isolated fat with/without SVF cells,as a bolus,was injected bilaterally beneath the dorsum of 45 recipient mice using a 1-ml syringe with a 19-gauge needle.The lower back was selected as recipient site,as the absence of subcutaneous fat and less compressing pressure on the grafts than the other area such as the scalp or nape.6 groups were employed for this study:(1)Lipoaspirate alone;(2)Lipoaspirate + SVF(Cell-assisted lipotransfer)(3)Lipoaspirate + 200?M Indomethacin(4)Lipoaspirate + 200?M Indomethacin + SVF;(5)Lipoaspirate + DMSO(Vehicle);and(6)Lipoaspirate + DMSO + SVF.At 2,4,12 weeks after transplantation(n = 5 per time point),the grafts were excised and analyzed as described below.The experimental design was shown in the summary drawing(Fig.1).Tissue harvesting and histologyAt 2,4 and 12 weeks after implantation,the mice were anesthetized(n=15 per time-point)Briefly,an incision was made in the midline of dorsal skin and the implanted regions were exposed.The transplanted fat were gently dissected away from the dermis and underlying muscle and they were evaluated based on the volume of fluid displacement.Harvested tissues were then fixed in 4%paraformaldehyde overnight before embedding for routine histology and immunohistochemistry.Five-micron serial sections were cut from paraffin blocks and were processed with hematoxylin and eosin(H&E)stain for analysis of fat graft morphology.ImmunohistochemistryTo distinguish live adipocytes from non-viable adipocytes,immunohistochemical staining was performed using antibodies against perilipin.Standard procedures recommended by the manufacturers were followed.The primary antibody(rabbit anti-rat Perilipin;Cell Signaling Technology,Danvers,MA,USA)was applied at a dilution ratio of 1:300(Dako,Glostrup,Denmark)for one hour at room temperature,followed by secondary antibody(Dako envision system anti-rabbit)for 30 minutes.3.3'-diaminobenzidene(DAB)chromagen was then applied to the slides for 2 minutes.CD31(platelet endothelial cell adhesion molecule 1)staining(rat anti-mouse;BD Pharmingen.)was also performed for analysis of graft vascularity,which was applied at 1:100 dilution for 1 hour,followed by a secondary antibody(Dako rabbit anti-rat biotinylated immunoglobulin)at 1:200 for 30 minutes.The avidin-biotin-horseradish peroxidase detection system(ABC Elite;Vector Laboratories)was then applied for 30 minutes followed by DAB chromagen.All sections were then counterstained in hematoxylin,dehydrated and coverslipped using DPX mounting medium.Cells that exhibited brown staining in the cytoplasm/nucleus were considered to be positive.Morphometric analysis of living adipocytes and vascularizationQuantification of living adipocytes and tissue vascularization were performed using video microscopy with a computer-assisted stereo investigator system(MBF Bioscience,VT,USA).14 For all the slides with perilipin and CD31 staining were counted with a 10X and 20x magnification objective,respectively.The edge of the grafts were traced and using systemic random sampling,16-point grids(400 ?m x 400 ?m)were superimposed on randomly selected fields representing 25%of the total area.Each point on the grid was recorded as positive or negative on the basis of perilipin staining live adipocytes or CD31 staining vessels.The percentage of adipose or vascular volume was determined by dividing the number of points in each of the selected fields that fell randomly on adipocytes or vessels by the total number of points counted for that tissue specimen,multiplied by 100.All counting was completed by two trained operators masked to the identity of the tissue samples.Statistical analysisData are presented as the mean ? standard error of the mean(SEM).The mean data were analysed with one-way analysis of the variance(ANOVA)with Bonferroni multiple comparison post-hoc test where appropriate.A P-value of<0.05 was considered statistically significant.ResultsIndomethacin increases differentiation of ASCs into mature adipocytes in a dose response mannerAdipogenesis of hASCs was induced by the addition of indomethacin at varying dosage.The cells were stained for oil red O to show presence of lipid droplets indicating the formation of adipocytes.Changes in cell morphology were evident 7 days after induction and cells started to accumulate lipids at 72h of the first treatment of differentiation medium.Two weeks treatment with adipogenesis-inducing conditioned medium resulted in increasing number of rounded,lipid-laden adipocytes.This was dose dependent as evidenced by increasing numbers of lipid containing cells with increases of the drug to 200 ?M,which showed a similar appearance to that of in Zuk medium(positive control).In contrast to a lower dose of indomethacin(200?M),Zuk combined with 200?M indomethacin(400?M)failed to further increase the number of lipid containing cells.A similar measurement of indomethacin treatment was observed when analyzing the absorbance of Oil Red-O solution.Indomethacin up-regulated the expression of adipogenic genes in the Presence of Conditioned mediumConditioned Imedium supplemented with indomethacin effectively induced adipogenesis at a dose of 200?M.significantly increasing the expression of adipogenic genes like PPAR-?,CEBP-?.FABP4 and LPL at 7 days;while failed to replicate this effect on CEBP-(3.It possibly due to that the upregulation of CEBP-?was more obvious at the later time point of adipogenesis rather than the early stage.Conditioned medium with indomethacin had an equivalent effect to Zuk to increase adipogenesis,caused a significantly greater increase in PPARy.This group also demonstrated higher increase for the rest of gene changes as compared with control and drug alone groups but lower expression when compared with Zuk.Generally,the highest expression in total genes that typically accompanies adipogenesis was accelerated in the presence of both indomethacin and conditioned medium,suggesting the drug-assisted adipogenic effect could only be amplified within an adipogenic microenvironment.In contrast,conditioned medium alone or drug alone(in complete medium)had much lower effect on stimulating those gene expression.These data indicate that indomethacin acts through PPAR-y to induce these genes.Macroscopic views on grafted tissue and volume assessmentA skin incision was made in the dorsal midline of the animal and the fat grafts appeared well encapsulated and well vascularized at earlier stage of lipotransfer.Macroscopic and microscopic images mainly focused on control(no indomethacin)and indomethacin-treated fat grafts at different time points post grafting.The fat grafts were dissected out and volumed at scheduled time points.Indomethacin and SVF-enriched groups exhibited significantly largest volumes at all time-points after engraftment when compared with control,cell-assisted groups and drug-assisted groups.After 12 weeks,there was a mean 85%reduction in control fat graft volumes from baseline,whereas indomethacin-treated fat grafts displayed a mean volume reduction of 63%.Furthermore,fat grafts retentions were calculated,suggesting the indomethacin-treated group illustrated significantly higher volume retention at 2 weeks and 4 weeks compared with control and vehicle groups,maintaining 75.6%and 65.7%respectively.However,no significance was found at 12 weeks,it possibly due to the one dose injection locally instead of continuously administration.Histological evaluation of grafted adipose tissueH&E demonstrated a less marked inflammatory response in the indomethacin-treated fat grafts,indicated by fewer inflammatory infiltration,less fibrous tissue,as well as a lesser degree of oil cysts and vacuoles as compared to the control group over time.Indomethacin Improves Fat Graft viability but Has No Effect on VascularityViable adipocytes can be observed in fat grafts from 2 weeks to 12 weeks,based on perilipin expression profiles.The morphology of viable adipocytes(perilipin-positive round cells)in indomethacin-treated group showed greater extent of integration of graft architecture than control group.The mean percentage of adipocyte coverage at 4 weeks was significantly larger in the indomethacin group(62%)than in controls(45± 16.2)(p = 0.016).suggesting that indomethacin may help to delay adipocyte death and/or promote the viability of adipose-derived stem/progenitor cells,which facilitates adipogenesis at later stages after transplantation.However,the adipose volume in indomethacin-treated groups(58.8%± 4.2%)was no difference as compared with SVF-assisted groups(p = 0.138).Similarly,no significant difference was found in CD31+ staining within all the groups.This is supported by quantifying the percentage of adipose volume and vascular volume within the fat grafts using histomorphometric analysis for perilipin and CD31 at 4 weeks respectively.This period has been validated by Youshimura's group to be the most indicative of regenerating adipocytes,when peak levels of perilipin-positive staining adipocytes were observed in his study.Adipogenesis and Proliferation in grafted tissueInterstingly,several a bunch of small round monolocular or multilocular cells(diameter of less than 30 ?m)strongly positive for perilipin were appeared in interstitial fibrous tissue or aside around vacuoles,suggesting ongoing adipogenesis,increased after fat grafting and peaked at 4 weeks in both control and indomethacin-treated grafts.In addition,double staining for Ki67 and CD34 was performed for comparison in both control and drug-assisted groups.In the drug-treated group,Ki67-positive cells were most frequently observed at week 2 and 4,but only occasionally observed in the control group during this period.Quantification of double positive cells for both Ki67 and CD34 revealed that the number of proliferating stem cells was much higher in the treatment group than that in the control group at week 2 and 4.Thereafter,the number of double positive cells in both groups declined to a low level at week 12,indicating that cell proliferation and differentiation stoppedDiscussionThe novel finding of our study is that treating fat grafts with indomethacin,without the need for SVF,produces comparable results to the cell-assisted lipotransfer group in terms of both volumes of the grafts and percentage of viable adipocytes.Volume retention for both indomethacin-treated groups(with and without SVF)was superior to that of the control.Several studies have shown SVF-assisted fat transplantation improves final retention.This may be due to increased host-derived adipogenesis and angiogenesis.18,19 However,controversy remains regarding the safety of this technique in a standard operative setting.In addition,the elusive SVF phenotype makes it difficult to standardize their use.20 Alternative,simpler ways to maximize final retention would have clinical appeal.Our in-vitro findings provided a more thorough understanding of the mechanism of drug interaction with ASCs.Firstly,we demonstrated that indomethacin has a stimulated role in inducing adipogenesis by up-regulating adipogenic genes.This is also supported by Styner et al.,8 who found that indomethacin stimulated adipogenesis in preadipocytes as well as bone marrow-derived MSCs by up-regulating PPAR?2 expression.Specifically,indomethacin served as the ligand for PPAR-?,enhanced transcription of critical adipogenic gene targets.8 In addition,indomethacin as a NSAID and a cyclooxygenase-2(COX-2)inhibitor,may target the Wnt pathway by inhibiting prostaglandin E2(PGE2).the product of COX-2,preventing the accumulation of ?-catenin and transcription of downstream effectors in the adipogenic process.21 Furthermore.Moshaverinia et al.11 found that the incorporation of indomethacin into the encapsulating scaffold could regulate the local microenvironment of the implants and prevent pro-inflammatory cytokine-induced apoptosis,thus improving the viability of engrafted MSCs.This is particularly important at the initial stage of the transplant where the engrafted fat is damaged by the immune system while awaiting neovascularization.It is noteworthy that local administration of indomethacin qualitatively reduced the inflammatory infiltration in the immediate microenvironment and it consistent with increasing the viability and adipogenic differentiation of preadipocytes within the grafts.Another beneficial effect of anti-inflammatory action of drugs,is their inhibitory effect on formation of fibrotic encapsulation which can compromise the viability and function of the adipose stem cells in a broad range of cell-based therapeutics.22 Therefore,local administration of anti-inflammatory drugs via fat injection could be a strategy to mitigate host immune response and to improve the stability of the whole graft.It has been proved that indomethacin promoted adipogenesis in a dose dependent manner,200?M was selected based on in vitro study.However,there is lack of published in vivo studies investigating subcutaneous injections with indomethacin,since most researchers report oral doses or focus on in vitro studies.The proposed clinical protocol in a single dose at the time of grafting is recommended.Continuously administered oral indomethacin is tolerated by most humans.23 The benefits and limitations of current drug-assisted lipotransfer as a compensation or supplement for cell-assisted lipotransfer in clinical use should be evaluated further.ConclusionOur results provide proof-of-concept support for using indomethacin as a pharmacological adjunct in fat grafting in view of its anti-inflammatory and pro-adipogenic properties.With further investigation,we believe this simple method may be used in clinical settings to improve free fat transplantation. |
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