Intact Adipocytes Research Articles

Adipose biopsy techniques for studies in human exercise physiology.

Adipose biopsy techniques are relatively undefined for exercise physiology research in individuals at or near normal weight. The purpose of this study was to compare the influence of two adipose biopsy techniques on tissue quality through measurements of adipocyte cell size, as well as mRNA and protein levels of select pro- and anti-inflammatory cytokines and adipokines. Thirteen participants (9 M, 4 W; 28 ± 4 yr; 27 ± 3 kg·m-2; V̇o2max: 3.3 ± 0.7 L·min-1) underwent subcutaneous adipose biopsies on either side of the umbilicus (incision: ∼8 cm lateral, sampling area: ∼5 cm lateral) using 1) a 6-mm Bergström biopsy needle and 2) a mini-liposuction approach with a 4-mm Mercedes biopsy needle that used prebiopsy tumescent delivery (∼30 mL 0.9% NaCl solution) into the sampling area (i.e., 'wet' technique). Tissue obtained was processed identically for analysis and both techniques returned high-quality tissue for histology (similar % intact adipocytes), mRNA (RNA integrity numbers >7.0), and protein. Adipocyte size was similar (P > 0.05) between both techniques (Bergström: 6,116 ± 1,652 μm2, 554-23,522 µm2; Mercedes: 6,517 ± 952 μm2, 926-21,969 µm2). There were also no differences (P > 0.05) between the two techniques for the measured cytokines (pro- and anti-inflammatory) and adipokines at the mRNA and protein levels. Adipocyte size was positively correlated with body mass index and body fat percentage, and negatively correlated with V̇o2max (P < 0.05). These results suggest both adipose biopsy techniques used in the current investigation are appropriate for histological, transcriptional, and translational level measurements in exercise physiology studies of nonobese women and men.NEW & NOTEWORTHY This study provides investigators with useful information related to adipose biopsy sampling approaches that can be used when planning studies that use measurements of adipose histology, as well as measurements at the mRNA and protein level. Adipose periumbilical sampling with the Bergström biopsy needle and the Mercedes wet mini-liposuction technique are both appropriate options for studies in exercise physiology and in nonobese individuals.

Chlamydia pneumoniae Lung Infection in Mice Induces Fatty Acid-Binding Protein 4-Dependent White Adipose Tissue Pathology.

Fatty acid-binding protein 4 (FABP4) is a critical immune-metabolic modulator, mainly expressed in adipocytes and macrophages, secreted from adipocytes in association with lipolysis, and plays essential pathogenic roles in cardiovascular and metabolic diseases. We previously reported Chlamydia pneumoniae infecting murine 3T3-L1 adipocytes and causing lipolysis and FABP4 secretion invitro. However, it is still unknown whether C. pneumoniae intranasal lung infection targets white adipose tissues (WATs), induces lipolysis, and causes FABP4 secretion invivo. In this study, we demonstrate that C. pneumoniae lung infection causes robust lipolysis in WAT. Infection-induced WAT lipolysis was diminished in FABP4-/- mice or FABP4 inhibitor-pretreated wild-type mice. Infection by C. pneumoniae in wild-type but not FABP4-/- mice induces the accumulation of TNF-α- and IL-6-producing M1-like adipose tissue macrophages in WAT. Infection-induced WAT pathology is augmented by endoplasmic reticulum (ER) stress/the unfolded protein response (UPR), which is abrogated by treatment with azoramide, a modulator of the UPR. C. pneumoniae lung infection is suggested to target WAT and induce lipolysis and FABP4 secretion invivo via ER stress/UPR. FABP4 released from infected adipocytes may be taken up by other neighboring intact adipocytes or adipose tissue macrophages. This process can further induce ER stress activation and trigger lipolysis and inflammation, followed by FABP4 secretion, leading to WAT pathology. A better understanding of the role of FABP4 in C. pneumoniae infection-induced WAT pathology will provide the basis for rational intervention measures directed at C. pneumoniae infection and metabolic syndrome, such as atherosclerosis, for which robust epidemiologic evidence exists.

A New Classification for Adipose-derived Stromal-cell Systems.

Obtaining regenerative cells from adipose tissue and their clinical use has become one of the most popular subjects of plastic surgery. However, there is no accepted classification in terms of methods. In this study, classification is proposed for the first time as a new idea. Accordingly, stromal cells can be obtained from adipose tissue by two approaches: direct methods for the bonds between parenchymal and stromal cells, and indirect methods, which target parenchymal cells rather than strong bonds, and increase the stromal cell ratio relatively. These methods can also be subclassified as fat (+), fat (-), fat (±) in terms of using the remaining fat in the final product as a graft. Direct methods include adinizing and enzymatic techniques; indirect methods include emulsification and micro-fragmentation/micronization techniques. In the enzymatic method, the fat tissue in the final product is considered dirty because it contains enzymes and must be discarded. That is why it is a fat (-) method. The adinizing method using ultra-sharp blades is fat (+) because the adipose tissue after the procedure can be used. Because the fat tissue is exposed to blunt pressure in emulsification techniques, it cannot be used as a graft. Thus, these are fat (-) methods. In micronization techniques using filter systems, there may still be intact adipocytes; therefore, it should be classified as fat (±). Adinizing provides both the highest efficiency and the full use of the end product. This classification will guide clinicians in terms of choosing the right product.

A high-content endogenous GLUT4 trafficking assay reveals new aspects of adipocyte biology.

Insulin-induced GLUT4 translocation to the plasma membrane in muscle and adipocytes is crucial for whole-body glucose homeostasis. Currently, GLUT4 trafficking assays rely on overexpression of tagged GLUT4. Here we describe a high-content imaging platform for studying endogenous GLUT4 translocation in intact adipocytes. This method enables high fidelity analysis of GLUT4 responses to specific perturbations, multiplexing of other trafficking proteins and other features including lipid droplet morphology. Using this multiplexed approach we showed that Vps45 and Rab14 are selective regulators of GLUT4, but Trarg1, Stx6, Stx16, Tbc1d4 and Rab10 knockdown affected both GLUT4 and TfR translocation. Thus, GLUT4 and TfR translocation machinery likely have some overlap upon insulin-stimulation. In addition, we identified Kif13A, a Rab10 binding molecular motor, as a novel regulator of GLUT4 traffic. Finally, comparison of endogenous to overexpressed GLUT4 highlights that the endogenous GLUT4 methodology has an enhanced sensitivity to genetic perturbations and emphasises the advantage of studying endogenous protein trafficking for drug discovery and genetic analysis of insulin action in relevant cell types.

Bcl-2 modified adipose-derived stem cells improve the retention of fat graft

ABSTRACT In cell-assisted lipotransfer, adipose-derived stem cells play a crucial role in enhancing fat graft retention. In vitro, human adipose-derived stem cells were modified with Bcl-2 gene. In vivo, aspirated fat was mixed with the Bcl-2-modified adipose-derived stem cells and then transplanted subcutaneously into nude mice. The retention of fat graft was evaluated. The surviving Bcl-2-modified adipose-derived stem cells were tracked after transplantation. Capillary density was quantified after transplantation. Transplantation with Bcl-2-modified adipose-derived stem cells enhanced fat graft retention by 49% and 114% at 6 weeks compared with the Fat + vector-modified adipose-derived stem cell group and Fat-only group, respectively. Transplants from the Fat + Bcl-2-modified adipose-derived stem cell group had significantly more intact adipocytes and lower levels of fat necrosis and fibrosis at 6 weeks. The survival of Bcl-2-modified adipose-derived stem cells increased by 33% at 3 weeks and 54% at 6 weeks, respectively, compared with vector-modified adipose-derived stem cells. The capillary density was 24% higher in Fat + Bcl-2-modified adipose-derived stem cell group than in Fat + vector-modified adipose-derived stem cell group or 60% higher than in Fat-only group at 3 weeks.

Oncostatin M Induces Lipolysis and Suppresses Insulin Response in 3T3-L1 Adipocytes.

Oncostatin M (OSM) is an immune cell-derived cytokine that is upregulated in adipose tissue in obesity. Upon binding its receptor (OSMR), OSM induces the phosphorylation of the p66 subunit of Src homology 2 domain-containing transforming protein 1 (SHC1), called p66Shc, and activates the extracellular signal-related kinase (ERK) pathway. Mice with adipocyte-specific OSMR deletion (OsmrFKO) are insulin resistant and exhibit adipose tissue inflammation, suggesting that intact adipocyte OSM–OSMR signaling is necessary for maintaining adipose tissue health. How OSM affects specific adipocyte functions is still unclear. Here, we examined the effects of OSM on adipocyte lipolysis. We treated 3T3-L1 adipocytes with OSM, insulin, and/or inhibitors of SHC1 and ERK and measured glycerol release. We also measured phosphorylation of p66Shc, ERK, and insulin receptor substrate-1 (IRS1) and the expression of lipolysis-associated genes in OSM-exposed 3T3-L1 adipocytes and primary adipocytes from control and OsmrFKO mice. We found that OSM induces adipocyte lipolysis via a p66Shc-ERK pathway and inhibits the suppression of lipolysis by insulin. Further, OSM induces phosphorylation of inhibitory IRS1 residues. We conclude that OSM is a stimulator of lipolysis and inhibits adipocyte insulin response. Future studies will determine how these roles of OSM affect adipose tissue function in health and disease.

Modification of emulsified fat (nanofat) obtaining procedures

Objective. To modify the procedure by reducing the number of filters and passages through them.&#x0D; Materials and Methods. 16 samples of fat, aspirated by a syringe from the abdominal region were examined. Fat filtration was carried out through anaerobic fat transfers with an inner diameter of 1.4 mm and 1.2 mm, as well as an emulsifying filter (nanofat filter). The content of destroyed adipocytes and fibroblast-like cells was assessed.&#x0D; Results. The 1.2 mm filter with 10 passages protocol provides determination of the minimal number of adipocytes while maintaining fibroblast-like cells. While filtrating through the 1.4 mm transfer, and then immediately through the nanofat filter, numerous connective tissue fibers with fibroblast-like cells are identified. When filtrating an adipose tissue using the 1.4 mm transfer 10 times, then using nanofat filter 5 times, there was obtained a homogeneous fat emulsion with a high content of morphologically intact adipocytes, the particle diameter of which allows injecting with a thin needle syringe.&#x0D; Conclusions. To obtain nanofat, it is possible to optimize the lipograft filtration protocol proposed by P. Tonnard (2013), depending on the purpose of using the obtained product.

ХАРАКТЕРИСТИКА ЖИЗНЕСПОСОБНОСТИ КЛЕТОК ЛИПОГРАФТА ПРИ РАЗЛИЧНЫХ МЕТОДИКАХ ЕГО ПОЛУЧЕНИЯ И ПОДГОТОВКИ

Objective. To determine the predictors of adipocytes and fibroblast-like cells survival in the lithographt using various techniques of lipoaspiration and adipose tissue preparation to autotraslantation. Methods. Cellular viability was analyzed in 57 adipose tissue samples prepared by various techniques of lipoaspiration, and analysis of damage to adipocytes and fibroblast-like cells - in 73 smears after passing of adipose tissue through filters of different diameters. Results. The average adipocyte viability in untreated lipoaspirate was 59%. Using water-jet technique, it was 65% (median 61%), syringe technique - 65% (median 74%), suction-assisted lipectomy - 55% (median 44%), p=0.18. The number of viable adipocytes depending on donor sites: thighs - 76%, waist - 67%, abdomen - 57%, buttocks - 50%, shoulders - 38%, knees - 35%. The number of intact adipocytes after 1.4 mm filter processing was 62-68%, fibroblast-like cells - 24-28%. The number of viable cells reduced with each passage. The number of intact adipocytes after 1.2 mm filter was 42-52%; of non-damaged fibroblast-like cells were 24-26% which located among connective tissue. The number of intact cells after emulsifying filter elevated up to 4-16% of adipocytes and 6-16% of fibroblast-like cells with a reduction of viable cells number with increasing of each passage. Single connective tissue fibers were determined, most content of the smear was represented by homogeneous fat. Conclusion. The viability of adipocytes is higher when using the syringe lipoaspiration technique with sampling from the inner and outer surfaces of the thighs and lower back. For regenerative purposes, it is preferable to use emulsified fat, characterized by the destruction of adipocytes and the elimination of connective tissue fibers, keeping intact up to 16% of fibroblast-like cells. The defects of soft tissue is better to fill with washed «macro-fat» without filtration, or use a 1.4 mm cell filter. For a combination of regenerative and volumizing purposes, it is advisable to use anaerobic cell filters. What this paper adds For the first time the viability of adipocytes using different lipoaspiration techniques and taken from different anatomical areas has been analyzed. An original technique for analyzing the viability of lipograft has been firstly applied. A reliable difference in the cellular composition of the lipograft when using different cell filters has been found. A reliable significant negative relationship between the number of viable cells and the number of passages through cell filters (D-1.4, 0.5 mm.) has been established. Recommendations for the preparation of various types of lipograft to obtain the maximum ratio of viable cells were substantiated.

Impact of Fat Graft Thickness and Harvesting Technique on Adipocyte Viability in a New Porcine Experimental Model: An Immunohistochemical Analysis.

Autologous fat grafting (AFG) has been employed in surgical practice as a filling method. However, controversies remain on the specifics of this technique. So far, few relevant experimental large animal studies have objectively assessed factors related to AFG integration. This study utilized an experimental, medium-sized animal model to compare the feasibility of AFG collected employing 2 different techniques with instruments of distinct thicknesses. Twenty minipigs (Sus scropha domesticus) were subjected to AFG harvesting via en bloc resection utilizing 3- (Group I) and 5-mm-diameter (Group II) round punch blades (PBs) and liposuction (LS) with 3- (Group III) and 5-mm-diameter cannulas (Group IV). Both samples were grafted intramuscularly (biceps femoralis). Hematoxylin and eosin staining was employed to identify intact adipocytes, fat necrosis, fibrosis, inflammation, and oil cysts. Immunohistochemical staining (perilipin-A, tumor necrosis factor alfa, and cluster of differentiation number 31) was utilized to quantify the feasibility of adipocytes, tissue necrosis, and neoangiogenesis, respectively. Hematoxylin and eosin analysis showed that fat necrosis and histiocyte presence were significantly lower in the AFG harvested utilizing a PB than in LS. For perilipin-A, a statistical difference was observed between subgroups I and III (P = 0.001) and I and IV (P = 0.004). Instrument diameter had no effect on graft integration in comparisons between groups II and III (P = 0.059) and II and IV (P = 0.132). In this experimental study, fat collected utilizing a PB demonstrated higher adipocyte viability than fat collected with LS. The diameter of the collection instruments, whether PB or LS, had no effect on graft integration.

Comparative study on the effect of adipose-derived stem cells versus alpha-lipoic acid in autologus fat graft survival in adult female albino rat: histological and immune histochemical study

Abstract Background Autologous fat transfer is a popular option for soft tissue augmentation, but has a low survival rate. So, enriching the transplanted fat with stem cells (cell assisted lipotransfer) or oral administration of alpha-lipoic acid (ALA) before and after graft injection, were thought to increase the survival rate of the transplanted fat. Aim of Study Is to compare between cell-assisted lipotransfer (CAL) and ALA therapy in improvement of fat graft survival. Materials and Methods Thirty adult female albino rats were used and divided into three groups. Group I: the control group. Group II: CAL group received autologous fat mixed with adipose derived stem cells (ASCs). Group III: ALA group in which animals received oral ALA four days before fat transplantation and continued throughout experiment. Fat grafts were collected from the sites of injection after twenty-eight days. Histological and immune histochemical studies were performed. Statistical analysis was also done. Results Histological evaluation of the fat grafts from animals of control group revealed atrophy and death of adipocytes with formation of fat cysts. Severe inflammatory infiltration with giant cells formation and fibrosis were detected. Significant decrease of VEGF reaction was seen. In CAL group significant increase in number of intact adipocytes together with decrease in inflammation and fibrosis occurred. Moreover, significant increase in VEGF reaction was observed. In ALA group, significant decrease in number of intact adipocytes and VEGF reaction was detected compared with CAL group. Also, significant increase in inflammatory infiltration and fibrosis were detected compared to CAL group. Conclusions Both ASCs and ALA showed improvement in all histological parameters compared with control group. However, enrichment of fat with ASCs showed the best results.

Abstract P6-15-07: Impact of increased mammary adiposity on DCIS progression

Abstract Background: The incidence of ductal carcinoma in situ (DCIS) has greatly increased since the introduction of mammographic screening. However, so far, it is still not possible to predict which patient with DCIS will further develop invasive breast cancer. Adipocyte hypertrophy and adipocyte inflammation has been associated with increased risk of developing breast cancer in postmenopausal women and with worse prognosis in breast cancer patients. In this study, we aimed 1) at evaluating the association between mammary adiposity, as assessed by digital pathology, with DCIS progression using a unique case-control series with long-term follow up and 2) study the interaction between DCIS cells and the surrounding adipocytes. Patients and methods: Mammary adipocyte size was measured in surgical specimens of 279 women with primary DCIS. These mammary adipocyte measurements were compared in 109 women with primary DCIS who subsequently developed ipsilateral invasive breast cancer (iIBC) (cases) and 170 women who did not (controls). Patients were matched for age and follow-up duration. Median follow-up time was 12.8 years. Post-menopausal patients were defined as &amp;gt;50 years at diagnosis and represented 220/279 (78,9%) of patients. All patients were treated with breast cancer-conserving surgery only. Intact adipocytes distant from the DCIS lesions were measured (largest diameter and area) using HALO™ image analysis software (Indica Labs, Corrales, NM) on H&amp;E-stained whole slide images of primary DCIS. Given the specific interest in the larger adipocytes, we systematically considered the 75th percentile as unique value for each patient. An average of 4259 adipocytes (min:70, max:21107) was measured per patient. Tissue segmentation was used to measure the adipose area. Adipose triglyceride lipase (ATGL clone 2138, Cell Signaling) immunohistochemistry (IHC) was applied to study ATGL expression in DCIS cells. Her2, ER and COX-2 IHC and RNAseq of microdissected pure DCIS was already available for this series (LL. Visser et al. Clin Can Res 24 (15) 2018). Conditional logistic regression was applied to investigate differences in adipocyte hypertrophy between groups of patients (e.g., cases and controls). Results: Adipocytes were larger and the proportion of adipose tissue was higher in post-menopausal patients (both p&amp;lt;0.001). Mean adipocyte maximum diameter and mean adipocyte area of the 75th percentile ranged from 46.9-115.9 µm and 2016-11336 µm2 respectively. For every 1000 µm2 increase in mean adipocyte area a DCIS patient had a 1.18 increased risk for iIBC (95% CI 1.02-1.36, p= 0.028). DCIS patients with large adipocytes (above the 75th percentile) had a 2.1 increased risk for developing iIBC (95% CI: 1.18-3.72, p=0.011). This observation remained when restricting the analysis to post-menopausal patients (OR 1.19 per 1000 µm2 95% CI 1.01-1.40, p=0.038 and adipocytes above 75th percentile OR 2.2, 95% CI 1.22-3.99, p= 0.009). To further explore the potential interactions between the epithelial cells from DCIS with the adjacent adipocytes, we compared the size of the DCIS-adjacent adipocytes to those away from DCIS lesions. DCIS-adjacent adipocytes showed a reduction in size, suggesting delipidation. Adipose triglyceride lipase (ATGL) is a rate limiting lipase that can release stored free fatty acids (FFA) in DCIS cells for fatty acid ß-oxidation. In a pilot series strong ATGL expression was observed in a subset of DCIS lesions (4 of 25). Conclusion and perspective: This study is the first to demonstrate that mammary adipocyte hypertrophy is associated with an increased risk of progression for patients with DCIS. Our findings might help to distinguish potentially hazardous from harmless DCIS, enabling overtreatment of indolent DCIS. Adipocyte size will be correlated to immunohistochemical expression of ATGL, Her2, ER and COX2 in DCIS, and RNAseq data of pure microdissected DCIS. Citation Format: Mathilde Matthea Machteld Almekinders, Michael Schaapveld, Bram Thijssen, Ingrid Hofland, Marjolijn Mertz, Dennis Peters, Annegien Broeks, Lodewijk Wessels, Wilbert Zwart, Jos Jonkers, Esther Lips, Christine Desmedt, Jelle Wesseling, on behalf of the PRECISION Team. Impact of increased mammary adiposity on DCIS progression [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-15-07.

Effects of Frozen Stromal Vascular Fraction on the Survival of Cryopreserved Fat Tissue

BackgroundNowadays, the use of cryopreserved fat tissue for soft tissue augmentation is common, except for its unpredictable fat graft absorption, and the toxicity of the cryoprotective agent remains a limitation. In this study, the effects of freezing stored fat tissue without a cryoprotector, and the addition of the stromal vascular fraction (SVF) on the survival of cryopreserved transplants was studied.MethodsLipoaspirates from six donors were processed and cryopreserved at − 20 °C, − 80 °C and − 196 °C, respectively. The authors evaluated the lipoaspirates in vitro, on the basis of fat tissue and SVF viability after cryopreservation. In vivo fat grafting was performed in nude mice. Six trenches were injected on the backs of mice. Cryopreserved tissues (− 20 °C, − 80 °C and − 196 °C) were injected on the right side, and the other side received the SVF combination. At 4 and 8 weeks after transplantation, the authors examined the weight, volume and morphology of the tissue and analyzed histochemical staining and immunohistochemistry (i.e., DIL, CD31 and VWF) to evaluate the survival of the fat grafts.ResultsAfter cryopreservation without the cryoprotective agent, adipose tissue maintained its morphology better in − 80 °C than − 20 °C and − 196 °C. SVF cells can retain their adhesive and proliferative properties after cryopreservation. Although cryopreservation caused damage to fat tissue, all explants showed intact adipocytes and vascular ingrowth. Most of all, the − 80 °C group had less graft resorption and fibrosis than the other temperature groups. There was increased survival of fat grafts in the SVF group compared with the control group.ConclusionIn this study, the authors demonstrated that the storage temperature of − 80 °C was promising for 3 months of adipose tissue cryopreservation without a cryoprotective agent, and SVF could increase the survival rate of cryopreserved fat tissue.No Level AssignedThis journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

Does non-activated platelet-rich plasma (PRP) enhance fat graft outcome? An assessment with 3D CT-scan in mice

The adjunction of autologous platelet-rich plasma (PRP) is emerging as a promising approach to enhance the long-term survival of fat grafting, but there are still insufficient data on its efficacy. The aim of this in vivo study was to assess the effect of the addition of non-activated PRP on fat graft outcome. Human adipose tissue mixed with 20% of non-activated PRP was injected under the scalp skin of nude Balb/cAnNRj mice and compared to grafted fat mixed with 20% of saline. The fat graft volume was analyzed by a computed tomography scan until day 90 and immunohistochemistry was then performed to assess adipocyte viability and graft revascularization. At day 90, the volume of fat graft was not enhanced by PRP compared to the saline control group. However, immunohistochemistry showed that PRP significantly increased the fat graft area occupied by intact adipocytes compared to the saline group (72.66% vs. 60.78%, respectively; p<0.05). Vascularity was also significantly higher in the PRP group compared to the control group (6695 vs. 4244 CD31+ cells/µm2, respectively; p<0.05). The adjunction of non-activated-PRP to fat grafts significantly increased adipocyte viability and tissue vascularity. However, in contrast to other studies adding activated-PRP, non-activated-PRP did not increase residual fat graft volume until day 90. Further studies are therefore needed to understand whether PRP has a positive effect on fat graft volume. As 3D computed tomography scan is a reproducible and precise technique, it should be used to analyze fat graft volume changes over time.

Adipose Tissue Dysfunction Occurs Independently of Obesity in Adipocyte-Specific Oncostatin Receptor Knockout Mice.

ObjectiveThis study examined the phenotypic effects of adipocyte-specific oncostatin M receptor (OSMR) loss in chow-fed mice.MethodsChow-fed adipocyte-specific OSMR knockout (FKO) mice and littermate OSMRfl/fl controls were studied. Tissue weights, insulin sensitivity, adipokine production, and stromal cell immunophenotypes were assessed in epididymal fat (eWAT); serum adipokine production was also assessed. In vitro, adipocytes were treated with oncostatin M (OSM) and adipokine gene expression assessed.ResultsBody weights, fasting blood glucose levels, and eWAT weights did not differ between genotypes. However, the eWAT of OSMRFKO mice was modestly less responsive to insulin stimulation than that of OSMRfl/fl mice. Notably, significant increases in adipokines including C-reactive protein, lipocalin 2, intercellular adhesion molecule-1 (ICAM-1), and insulin-like growth factor binding protein 6, were observed in the eWAT of OSMRFKO mice. In addition, significant increases in fetuin A and ICAM-1 were detected in OSMRFKO serum. Flow cytometry revealed a significant increase in leukocyte number and modest, but not statistically significant, increases in B and T cells in the eWAT of OSMRFKO mice.ConclusionsThe chow-fed OSMRFKO mouse exhibits adipose tissue dysfunction and increased pro-inflammatory adipokine production. These results suggest that intact adipocyte OSM-OSMR signaling is necessary for adipose tissue immune cell homeostasis.

Effects of Collagenase Digestion and Stromal Vascular Fraction Supplementation on Volume Retention of Fat Grafts.

The use of autologous fat as a soft tissue filler has increased over the past decade in both reconstructive and aesthetic surgeries. Enhancement of autologous fat grafts with the addition of the stromal vascular fraction (SVF) has been reported to improve long-term volume retention. Stromal vascular fraction is most commonly isolated using enzymatic digestion, but it is unknown what effect the digestion process has on the adipocytes and SVF cells that comprise the graft. Some clinicians have reported use of enzymatically digested fat grafts to alter the physical properties of the tissue in specialized applications. We have previously reported that increasing collagenase digestion duration adversely affects the viability of adipocytes and SVF cells. Here, we aimed to determine if collagenase digestion of adipocytes before grafting is detrimental to long-term graft retention and if SVF supplementation can abrogate these potential deleterious effects. We used a published xenograft model in which human lipoaspirate was implanted into the scalp of immunocompromised mice to study the effects of collagenase digestion on in vivo graft survival after 12 weeks. We used 4 experimental groups: grafts composed of collagenase-digested and nondigested adipocytes (50-minute digestion) and grafts with and without SVF supplementation. We used microcomputed tomography to serially and noninvasively quantify graft volume, in conjunction with hematoxylin-eosin staining of histological cross-sections of implanted and excised grafts to assess overall tissue viability. We found that adipocytes that were collagenase-digested before implantation had significantly lower retention rates at 12 weeks and poorer tissue health, which was assessed by quantifying the number of intact adipocytes, the number of cystic formations, and by scoring the degree of inflammation and fibrosis. Further, we found that SVF supplementation of the digested grafts improved graft survival, but not to the level observed in undigested grafts. We conclude that collagenase digestion adversely affects the long-term volume retention of fat grafts, but that graft retention is improved by SVF supplementation. These experimental results can serve as an initial framework to further elucidate the reported efficacy and safety of using collagenase-digested fat grafts and SVF in the clinical setting.

CL316243 induces phosphatidylinositol 3,4,5-triphosphate production in rat adipocytes in an adenosine deaminase-, pertussis toxin-, or wortmannin-sensitive manner.

The effect of beta(3)-adrenoceptor (beta(3)-AR) agonists on adipocytes treated or not treated with signaling modulators has not been sufficiently elucidated. Using rat epididymal adipocytes (adipocytes) labeled with [(32)P]orthophosphate, we found that treatment with the selective beta(3)-AR agonist CL316243 (CL; 1 microM) induces phosphatidylinositol (PI) 3,4,5-triphosphate (PI[3,4,5]P(3)) production and that this response is inhibited by adenosine deaminase (ADA, an adenosine-degrading enzyme; 2 U/ml), pertussis toxin (PTX, an inactivator of inhibitory guanine-nucleotide-binding protein; 1 microg/ml), or wortmannin (WT, a PI-kinase inhibitor; 3 microM). The results showed that CL induced PI(3,4,5)P(3) production in intact adipocytes and that this production was affected by signaling modulators. Taken together, our findings indicate that CL produces PI(3,4,5)P(3) in an ADA-sensitive, PTX-sensitive, or WT-sensitive manner and will advance understanding of the effect of beta(3)-AR agonists on adipocytes.

Identification of the Centrifuged Lipoaspirate Fractions Suitable for Postgrafting Survival.

The Coleman centrifugation procedure generates fractions with different adipocyte and progenitor cell densities. This study aimed to identify all fractions that are feasible for implantation. Human lipoaspirates were processed by Coleman centrifugation. The centrifugates were divided arbitrarily into upper, middle, and lower layers. Adipocyte viability, morphology, numbers of stromal vascular fraction cells, and adipose-derived mesenchymal stem cells of each layer were determined. The 12-week volume retention of subcutaneously implanted 0.3-ml lipoasperate of each layer was investigated in an athymic mice model. Most damaged adipocytes were located in the upper layers, whereas the intact adipocytes were distributed in the middle and lower layers. A gradient of stromal vascular fraction cell density was formed in the centrifugates. The implant volume retentions of samples from the upper, middle, and lower layers were 33.44 ± 5.9, 55.11 ± 4.4, and 71.2 ± 5.8 percent, respectively. Furthermore, the middle and lower layers contained significantly more adipose-derived stem cells than did the upper layer. The lower layer contains more viable adipocytes and stromal vascular fraction cells leading to the highest implant volume retention, whereas the most impaired cells are distributed in the upper layer, leading to the least volume retention. Although with a lower stromal vascular fraction content, the middle layer has a substantial number of intact adipocytes that are capable of retaining partial adipose tissue volume after implantation, suggesting that the middle layer may be an alternative fat source when large volumes of fat grafts are needed for transplantation.

Improved fat graft survival by different volume fractions of platelet-rich plasma and adipose-derived stem cells.

The success of soft-tissue augmentation is offset by the low survival rates of grafted fat tissue. Research shows that adipose-derived stem cells (ASCs) and platelet-rich plasma (PRP) are beneficial to tissue healing. To evaluate the long-term effects of different volume fractions of PRP combined with ASCs on fat graft. ASCs were isolated from human fat tissue, and PRP was obtained from human blood. Cell count kit-8 and real-time polymerase chain reaction (PCR) were used to evaluate the influence of PRP (0%, 10%, 20%, and 30%; volume/volume [v/v]) in medium on ASC proliferation and adipogenic differentiation, respectively. A novel lipoinjection consisting of granular fat, PRP, and ASCs was subcutaneously transplanted into nude mice. The grafts were volumetrically and histologically evaluated 10, 30, 60, and 90 days after transplantation. The addition of PRP improved ASC proliferation. Expression of adipogenic-related genes, peroxisome proliferator-activated receptor-γ, lipoprotein lipase, and adipophilin were up-regulated in PRP-induced ASCs. Compared with other groups, granular fat grafts formed with 20% (v/v) and 30% (v/v) PRP significantly improved residual volumes. More intact adipocytes and capillary formation, but less vacuolization, were observed in the 20% (v/v) and 30% (v/v) PRP groups at 30, 60, and 90 days. However, no significant difference was observed between the 20% (v/v) and 30% (v/v) PRP groups in retaining fat grafts and improving histology. Fat grafting with 20% (v/v) PRP and ASCs constitutes an appropriate transplantation strategy for improving graft survival and provides a potential approach for soft-tissue restoration in plastic and reconstructive surgery.

A quantitative analysis of the effects of different harvesting, preparation, and injection methods on the integrity of fat cells

The percentage of intact fat cells membrane obtained by even a well-known technique remains poorly studied. The purposes of this study were to evaluate and compare the structural effects of various harvesting, preparation, and injection methods on human fat cell integrity. We enrolled 20 healthy females in this study, ages ranged from 20 to 41 years with an average age of 31 ± 0.5 years. We harvested fat tissues from the abdomen and thigh of donors using two different methods: conventional and syringe liposuction; we treated the aspirated fat specimens within 30 min of harvesting by fine-mesh gauze or using centrifugation. We injected the prepared fat through small or large needle, and then obtained a specimen from each different method. We analyzed all fat graft samples by routine histological examination. There was a significant difference in the percentage of intact fat cells wall was observed in syringe aspiration (75.95 ± 0.31 %) versus vacuum liposuction (56.43 % ± 0.67). There was a significant difference in the percentage of intact fat cells when using centrifugation versus fine-mesh gauze preparation. Also there was a significant difference in the percentage of intact fat cells observed when using large versus small needle for injection. The highest intact adipocyte counts were in syringe suction, no centrifuge, and using large cannula, while the fewest intact adipocyte counts were in conventional liposuction, using centrifuge, and using small cannula. Level of Evidence: Level IV, prognostic study

Autologous Fat Processing Via the Revolve System: Quality and Quantity of Fat Retention Evaluated in an Animal Model

Currently, fat graft viability and retention cannot be reliably predicted. The reasons for this variability are not fully understood, although fat processing has been implicated. The authors compare the in vitro quantity and in vivo fat retention from lipoaspirate processed by the Revolve system (LifeCell, Bridgewater, New Jersey) compared with centrifugation and decantation. Ten patients were enrolled in this prospective study. Lipoaspirate from each patient was processed by each of 3 methods: decantation, centrifugation, and the Revolve system. Biochemical characteristics and free oil, adipose, and aqueous phases of the processed fats were determined. Fat grafts were implanted in nude mice; volume retention and quality of the fat grafts were evaluated after 28 days. Viability of retained fat was demonstrated by intact adipocytes and neovascularization on histology. Of the 10 patients, 9 were women and 1 was a man. Mean patient age was 40.7 ± 8.9 years (range, 30-55 years). Fat tissue obtained from all methods had good physiological properties with neutral pH and isotonic salt concentrations. The Revolve system yielded significantly less blood cell debris, a higher percentage of adipose tissue, and a lower percentage of free oil compared with the other 2 methods. Fat tissue retention from Revolve samples was significantly higher (73.2%) than that from decanted samples (37.5%) and similar to that from centrifuged samples (67.7%). The Revolve system produced physiologically compatible, preinjection fat with reduced contaminants and free oil in conjunction with high fat content. In an animal model, volume retention of Revolve-processed fat grafts was significantly greater than decanted samples. The Revolve system presents a fat-processing option that was less time-consuming, easier to use, and more efficient in this study than standard centrifugation or decantation.