Adipose Tissue Extracts Research Articles

Adipocyte‑rich microenvironment promotes chemoresistance via upregulation of peroxisome proliferator‑activated receptor gamma/ABCG2 in epithelial ovarian cancer.

The effects of adipocyte‑rich microenvironment (ARM) on chemoresistance have garnered increasing interest. Ovarian cancer (OVCA) is a representative adipocyte‑rich associated cancer. In the present study, epithelial OVCA (EOC) was used to investigate the influence of ARM on chemoresistance with the aim of identifying novel targets and developing novel strategies to reduce chemoresistance. Bioinformatics analysis was used to explore the effects of ARM‑associated mechanisms contributing to chemoresistance and treated EOC cells, primarily OVCAR3 cells, with human adipose tissue extracts (HATES) from the peritumoral adipose tissue of patients were used to mimic ARM invitro. Specifically, the peroxisome proliferator‑activated receptorγ (PPARγ) antagonist GW9662 and the ABC transporter G family member 2 (ABCG2) inhibitor KO143, were used to determine the underlying mechanisms. Next, the effect of HATES on the expression of PPARγ and ABCG2 in OVCAR3 cells treated with cisplatin (DDP) and paclitaxel (PTX) was determined. Additionally, the association between PPARγ, ABCG2 and chemoresistance in EOC specimens was assessed. To evaluate the effect of inhibiting PPARγ, using DDP, a nude mouse model injected with OVCAR3‑shPPARγ cells and a C57BL/6 model injected with ID8 cells treated with GW9662 were established. Finally, the factors within ARM that contributed to the mechanism were determined. It was found that HATES promoted chemoresistance by increasing ABCG2 expression via PPARγ. Expression of PPARγ/ABCG2 was related to chemoresistance in EOC clinical specimens. GW9662 or knockdown of PPARγ improved the efficacy of chemotherapy in mice. Finally, angiogenin and oleic acid played key roles in HATES in the upregulation of PPARγ. The present study showed that the introduction of ARM‑educated PPARγ attenuated chemoresistance in EOC, highlighting a potentially novel therapeutic adjuvant to chemotherapy and shedding light on a means of improving the efficacy of chemotherapy from the perspective of ARM.

A novel combined technology for treating hypertrophic scars: adipose tissue extract combined with fractional CO2 laser.

Introduction: Owing to the need for liposuction and its unsuitability for allogeneic transplantation, the clinical application of stromal vascular fraction gel (SVF-gel) combined with fractional CO2 laser for scar treatment is limited. Adipose tissue extract (ATE), rich in cytokines and growth factors, offers a more convenient option for clinical practice as it can be easily prepared using purely physical methods and has low immunogenicity. We aimed to evaluate the effectiveness of ATE combined with fractional CO2 laser in the treatment of hypertrophic scars. Methods: ATE was prepared using discarded liposuction fluid from patients undergoing liposuction. A rabbit ear hypertrophic scar model was established and treated with ATE, fractional CO2 laser, or a combination. PBS was used as a control. The scar appearance and histological changes were observed. The immunohistochemistry method was used to evaluate the expression of α-SMA, while perilipin was detected using immunofluorescence. Additionally, the level of adipogenic signal C/EBPα and PPARγ mRNA was studied. Results: Following treatment, the volume of hypertrophic scar decreased, resulting in a softer texture and thinner dermis. Additionally, there was a decrease in the infiltration of inflammatory cells, and the collagen arrangement became looser and more regular, and the expression of α-SMA also decreased, with the combination of ATE and fractional laser showing the most significant improvement. Moreover, the combination group was found to promote subcutaneous fat regeneration and increase the expression of adipogenic signals C/EBPα and PPARγ. Conclusion: The combination of ATE and fractional CO2 laser treatment has been shown to inhibit the development of hypertrophic scars. This effect may be attributed to the enhancement of adipogenesis and decrease in collagen deposition.

Current Situation of Adipose-Derived Stem Cells Treatments for Knee Ostheoarthritis and Hypothesis for a New Protocol Based on Cryoconservation and Multiple Injections of Mechanically Fragmented Adipose Tissue Extracts

Osteoarthritis (OA) is a widespread degenerative joint disease. The knee arthroplasty has represented for about 40 years the main treatment option, when pain and functional decrease became unbearable for the patient. The knee arthroplasty represents a challenging surgical operation, in which, based on current data available, about 20% of the patients are left totally or partially unsatisfied with the clinical result. A recent therapeutic alternative for mild-to-moderate grade OA (Kellgren-Lawrence grade II to III) in order to delay (or ultimately avoid) the need for prosthetic surgery, is a treatment with Adipose-Derived Stem Cells (or ADSCs). The safety and efficacy in terms of functional improvement, cartilage repair and inflammation reduction were already reported for OA treatment through a single intra-articular injection of Micro-Fragmented Autologous Fat Tissue (M-FAT), which is known to be rich in ADSCs. This efficacy period appears to be ranging from several months to a few years. Research has shown that the effect of single-dose M-FAT treatment, despite being evident in most cases, is not statistically uniform among the studies now available. This is also due to different treatment protocols, that leave to every research group the ultimate choice of preparation methods, rules and exclusion criteria. It is possible, because of this, to hypothesize a new kind of treatment protocol based on a single surgical harvesting operation. The extract would then need to be cryopreserved and administered multiple times over a several-years period. We hope, thanks to the subsequent injections of purified extract, to prolong and boost the effect of the treatment. We tried to elaborate, based on our experience in the field, a new protocol for this multi-injection treatment. This protocol is going to be applied to a small number of patients and evaluated over time, to elaborate the actual benefit from a treatment like this.

Fish Fingerprinting: Identifying Crude Oil Pollutants using Bicyclic Sesquiterpanes (Bicyclanes) in the Tissues of Exposed Fish.

In the present study, we investigated the possibility of identifying the source oils of exposed fish using ratios of bicyclic sesquiterpane (bicyclane) chemical biomarkers. In the event of an oil spill, identification of source oil(s) for assessment, or for litigation purposes, typically uses diagnostic ratios of chemical biomarkers to produce characteristic oil "fingerprints." Although this has been applied in identifying oil residues in sediments, water, and sessile filtering organisms, so far as we are aware this has never been successfully demonstrated for oil-exposed fish. In a 35-day laboratory trial, juvenile Lates calcarifer (barramundi or Asian seabass) were exposed, via the diet (1% w/w), to either a heavy fuel oil or to Montara, an Australian medium crude oil. Two-dimensional gas chromatography with high-resolution mass spectrometry and gas chromatography-mass spectrometry were then used to measure selected ratios of the bicyclanes to examine whether the ratios were statistically reproducibly conserved in the fish tissues. Six diagnostic bicyclane ratios showed high correlation (r2 > 0.98) with those of each of the two source oils. A linear discriminatory analysis model showed that nine different petroleum products could be reproducibly discriminated using these bicyclane ratios. The model was then used to correctly identify the bicyclane profiles of each of the two exposure oils in the adipose tissue extracts of each of the 18 fish fed oil-enriched diets. From our initial study, bicyclane biomarkers appear to show good potential for providing reliable forensic fingerprints of the sources of oil contamination of exposed fish. Further research is needed to investigate the minimum exposure times required for bicyclane bioaccumulation to achieve detectable concentrations in fish adiposetissues and to determine bicyclane depuration rates once exposure to oil has ceased. Environ Toxicol Chem 2023;42:7-18. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

A multilayered nanofibrous patch functionalized with adipose tissue extract for the treatment of bladder regeneration

Bladder tissue engineering is a promising approach to generating new tissues to replace damaged or diseased organs, breaking the limitations of current therapies using reconfigured autogenous bowel segments. Adipose tissue extract (ATE) is a non-cellular three-dimensional macromolecular network that provides an appropriate local microenvironment for cells and affects cellular metabolism, proliferation, differentiation, or organoid formation. This study integrated electrospun nanofibers mats and hyaluronic acid colloidal solution to constitute a multilayered nanofibrous patch, where ATE was added as a biological additive. In vitro study, we observed an increase in the pluripotent marker proteins (Oct-4, Sox-2, and Nanog) of adipose-derived stem cells (ADSCs) and bone mesenchymal stem cells (BMSCs) when cultured with ATE. The CCK-8 test, scratch assay, transwell migration assay, and tube formation assay demonstrated the positive effect of ATE on endothelial cells (ECs) proliferation, migration, and angiogenesis. The feasibility and effectiveness of the patch were investigated in a rat bladder defect model. The primary ideal results revealed the patches' great potential for tissue engineering repair and regenerative medicine.

An Improved Genetically Encoded Fluorescent cAMP Indicator for Sensitive cAMP Imaging and Fast Drug Screening.

Cyclic adenosine 3′,5′-monophosphate (cAMP) is an important intracellular second messenger molecule downstream of many G protein-coupled receptors (GPCRs). Fluorescence imaging with bright and sensitive cAMP indicators allows not only dissecting the spatiotemporal dynamics of intracellular cAMP, but also high-content screening of compounds against GPCRs. We previously reported the high-performance circularly permuted GFP (cpGFP)-based cAMP indicator G-Flamp1. Here, we developed improved G-Flamp1 variants G-Flamp2 and G-Flamp2b. Compared to G-Flamp1, G-Flamp2 exhibited increased baseline fluorescence (1.6-fold) and larger fluorescence change (ΔF/F0) (1,300% vs. 1,100%) in HEK293T cells, while G-Flamp2b showed increased baseline fluorescence (3.1-fold) and smaller ΔF/F0 (400% vs. 1,100%). Furthermore, live cell imaging of mitochondrial matrix–targeted G-Flamp2 confirmed cytosolic cAMP was able to enter the mitochondrial matrix. G-Flamp2 imaging also showed that adipose tissue extract activated the Gi protein-coupled orphan GPCR GPR50 in HEK293T cells. Taken together, our results showed that the high-performance of G-Flamp2 would facilitate sensitive intracellular cAMP imaging and activity measurement of compounds targeting GPCR-cAMP signaling pathway during early drug development.

Adipose tissue as a source of growth factors to promote wound healing: a human study of skin graft donor sites.

In the microenvironment of wound sites, naturally occurring growth factors are crucial for cell migration, opsonisation, chemotaxis, differentiation and angiogenesis. Exogenous growth factors, such as platelet-rich plasma (PRP) and adipose tissue, also improve healing. In the present within-subject study, we described the effects of PRP and adipose tissue extract (ATE) on skin graft donor site wound healing in patients requiring split-thickness skin grafts. Each patient, having at least two donor sites, received both control (no growth factor) and experimental (PRP or ATE) treatments. Wounds were evaluated on days 5, 7, 10, 15, 30 and 60. Digital photography and spectral images were used to analyse haemoglobin and melanin content, and re-epithelialisation area. Pain was assessed by visual analogue scale. Scar characteristics were scored on days 30 and 60. Biomaterial samples were analysed for growth factor and protein content. The study included 24 patients (18 male and six female; mean age: 59.1 years). PRP was topically applied to wounds in 11 patients (13 donor sites) and ATE in 13 patients (15 sites). ATE-treated donor sites exhibited significantly accelerated wound re-epithelialisation on days 5 and 7 compared with control sites (p=0.003 and 0.04, respectively). PRP accelerated healing on day 7 compared with control sites (p=0.001). Additionally, the application of ATE improved scar quality on days 30 and 60 (p=0.0005 and 0.02, respectively). Pain scores did not differ significantly between treatments. In this study, both growth factor sources stimulated wound healing. ATE is an alternative source of growth factors that promote early wound healing and improve scar quality.

Stem Cell Harvest From Adipose Tissue

Background: Differentiation, self-renewal, immunomodulation, and angiogenesis are all capabilities of stem cells that can help improve tissue structure (Bacakova et al, 2018). In adipose tissue, there is a stromal vascular fraction (SVF) that contains a variety of cell types that can transdifferentiate between endothelial and adipogenic cell lineages. (Schreml et al., 2009). The procedure used to harvest adipose tissue, such as liposuction or excision, has an impact on the quality of SVF and ADSC recovered from it. Barzelay et al., 2015). Literature review: The two most common locations for stem cell harvest are bone marrow and fat tissue. Taking stem cells can be done in a variety of ways, depending on the source of the cells. Isolating stem cells from preimplantation embryos is how embryonic stem cell retrieval is done. Liposuction is the removal of subcutaneous tissue with the help of a suction pump and an aspiration cannula introduced through a skin incision. Suction-assisted lipectomy, suction lipoplasty, and liposculpture are other synonyms for liposuction. The advantage of liposuction over resection for obtaining stem cells is that it is less intrusive and can preserve neurovascular structures. In addition, liposuction preserves fluid balance and reduces patient discomfort. A high-speed cannula is used in liposuction to disrupt adipocytes. This procedure has the potential to cause cellular harm. Adipocyte viability and transplant survival will be improved by using less stressful adipose tissue extraction procedures. (Simonacci et al., 2017). Under general anaesthesia, the resected tissue was removed whole. In order to separate ADSC, fibrous tissue and visible blood vessels were removed from the retrieved adipose tissue. Resection of adipose tissue produced more SVF and ADSC cells than liposuction, according to Iyyanki et al and Faustini et al. This differs from the findings of a prior study by Schreml et al. Conclusion: Liposuction and resection both have advantages and downsides when it comes to extracting stem cells from fat tissue. There was no discernible difference in the removal of stem cells from adipose tissue by liposuction versus excision.

Comparison of two cell-free therapeutics derived from adipose tissue: small extracellular vesicles versus conditioned medium

BackgroundCell-free therapy has been inspired as a promising approach to overcome the limitations of traditional stem cell therapy. However, the therapeutic effect between extracellular vesicles and conditioned medium with the same source had not been compared. Our previous studies have shown that both the conditioned medium of adipose tissue (adipose tissue extract, ATE) and its further purification product small extracellular vesicles (sEV-AT) contributed to adipose tissue regeneration. In this study, we aimed to compare the ATE and sEV-AT in composition, inductivity on cells and de novo adipose regenerative potential.MethodsThe characteristics of sEV-AT and ATE were compared through protein and particle yield, particle size distribution and composition. The inductivity of sEV-AT and ATE on cells were compared through co-culture of sEV-AT or ATE with ASC, HUVEC and RAW264.7 in vitro. The capacity of promoting de novo adipogenesis was compared by implanting the silicone tube containing sEV-AT or ATE subcutaneously in vivo.ResultsMore particles and concentrated particle size distribution were detected in sEV-AT. In turn, more soluble factors and multiple peaks in particle size distribution were detected in ATE. In 1662 common proteins of sEV-AT and ATE, there were 984 (59.2%) proteins enriched twice more in sEV-AT than in ATE. With the prerequisite of equivalent protein concentration, sEV-AT outperformed ATE in promoting proliferation, migration and regeneration potential of cells those contributing adipose tissue regeneration in vitro. Furthermore, sEV-AT expedited the de novo adipose tissue regeneration and angiogenesis at the early stage than ATE in vivo, but sEV-AT and ATE group formed similar neoadipose tissue and new vessels at week 12.ConclusionsOur results provided a direct comparison between EV and conditioned medium as cell-free therapeutic strategy. Both sEV and ATE had specific biological signature to facilitate tissue repair. Considering the convenience of extraction and acceptable effect, ATE represented a feasible product of cell-free therapy, providing another option for different situations in clinical application. Furthermore, the complex contents of both sEV-AT and ATE should be studied comprehensively to avoid possible negative effects and to ensure sufficient safety for clinical applications.

Point-of-care treatment of geometrically complex midfacial critical-sized bone defects with 3D-Printed scaffolds and autologous stromal vascular fraction

Critical-sized midfacial bone defects present a unique clinical challenge due to their complex three-dimensional shapes and intimate associations with sensory organs. To address this challenge, a point-of-care treatment strategy for functional, long-term regeneration of 2 cm full-thickness segmental defects in the zygomatic arches of Yucatan minipigs is evaluated. A digital workflow is used to 3D-print anatomically precise, porous, biodegradable scaffolds from clinical-grade poly-ε-caprolactone and decellularized bone composites. The autologous stromal vascular fraction of cells (SVF) is isolated from adipose tissue extracts and infused into the scaffolds that are implanted into the zygomatic ostectomies. Bone regeneration is assessed up to 52 weeks post-operatively in acellular (AC) and SVF groups (BV/DV = 0.64 ± 0.10 and 0.65 ± 0.10 respectively). In both treated groups, bone grows from the adjacent tissues and restores the native anatomy. Significantly higher torque is required to fracture the bone-scaffold interface in the SVF (7.11 ± 2.31 N m) compared to AC groups (2.83 ± 0.23 N m). Three-dimensional microcomputed tomography analysis reveals two distinct regenerative patterns: osteoconduction along the periphery of scaffolds to form dense lamellar bone and small islands of woven bone deposits growing along the struts in the scaffold interior. Overall, this study validates the efficacy of using 3D-printed bioactive scaffolds with autologous SVF to restore geometrically complex midfacial bone defects of clinically relevant sizes while also highlighting remaining challenges to be addressed prior to clinical translation.

Treatment of faecal incontinence with autologous expanded mesenchymal stem cells: results of a pilot study.

Management of faecal incontinence (FI) remains challenging because no definitive optimal treatment for this condition has yet been determined. Regenerative medicine could be an attractive therapeutic alternative for treating FI. Here, we aimed to determine the safety and feasibility of autologous expanded mesenchymal stem cells derived from adipose tissue (AdMSCs) in the treatment of patients diagnosed with structural FI. This was a randomized, multicentre, triple-blinded, placebo-controlled pilot study conducted at four sites in Spain with 16 adults with FI and a sphincter defect. Autologous AdMSCs were obtained from patients from surgically excised adipose tissue. These patients were intralesionally infused with a single dose of 4×107 AdMSCs or a placebo while under anaesthesia. We assessed the safety and feasibility of the treatment as the cumulative incidence of adverse events and the treatment efficacy using the Cleveland Clinic Faecal Incontinence Score, Faecal Incontinence Quality of Life score and Starck criteria to classify sphincter defects and anorectal physiology outcomes. Adipose tissue extraction, cell isolation and intralesional infusion procedures were successful in all the patients. There was only one adverse event connected to adipose tissue extraction (a haematoma), and none was associated with the injection procedure. There were no significant differences in any of the assessed clinical, manometric or ultrasonographic parameters. This study indicates that this infusion procedure in the anal sphincter is feasible and safe. However, it failed to demonstrate efficacy to treat patients with structural FI.

FABP4 promotes invasion and metastasis of colon cancer by regulating fatty acid transport

BackgroundThe prognosis of colon cancer is poor for metastasis, while the mechanism, especially adipocytes related, is not yet clear. The purpose of this study is to determine the effects of fatty acid binding protein 4 (FABP4), a transporter for lipids, on colon cancer progression.MethodsThe distribution of lipids and FABP4 was tested in the colon cancer tissues and adjacent normal tissues, and their relationship was also verified in vitro. Experiments about cellular invasion, migration and proliferation were performed to detect the impacts of FABP4 on the biological behaviors of colon cancer, and the positive results were checked in vivo. Meanwhile, the regulatory role of FABP4 in the energy and lipid metabolism was evaluated by the levels of triglyceride, ATP, LDH, glycerol and NEFA. At last, GO and KEGG analysis based on FABP4 overexpressed cells was performed, and the AKT pathway and epithelial–mesenchymal transition (EMT)-related proteins were determined by Western blot.ResultsHigher accumulation of lipids and stronger FABP4 transcription were observed in colon cancer tissues. Having been incubated with adipose tissue extract and overexpressed FABP4, colon cancer cells demonstrated enhanced lipid accumulation. In functional experiments, co-culture with adipose tissue extract significantly enhanced the invasion and migration of colon cancer cells, as well as the energy and lipid metabolism, and all these processes were reversed by FABP4 inhibitor. In addition, the metastasis of FABP4-overexpressed colon cancer cells was also significantly enhanced in vitro and in vivo. In terms of mechanism, the bioinformatics analysis showed that FABP4 was enriched in 11 pathways related to metabolic processes in FABP4 overexpressed cells. Finally, FABP4 overexpression improved EMT progression of colon cancer, as evidenced by the upregulation of Snail, MMP-2 and MMP-9, the downregulation of E-cadherin. The expression of p-Akt was also elevated.ConclusionFABP4 overexpression could increase FAs transport to enhance energy and lipid metabolism, and activate AKT pathway and EMT to promote the migration and invasion of colon cancer cells.

Mature Adipocytes and FGF2 Influence Angiogenic Processes

The process of wound healing requires the transportation of substrates needed for the rebuilding of tissue through appropriate vessel formation; angiogenesis. Adipose tissue extract is often used to facilitate the wound healing process, which is attributed to its potent angiogenic properties. Most angiogenic process are regulated through growth factors such as vascular endothelial growth factor (VEGF), angiopoietin, and fibroblast growth factor (FGF). Fibroblast growth factor has been utilized to facilitate wound healing in the biomedical community, which is one of the growth factors synthesized and secreted by adipocytes. Therefore, it was hypothesized that mature adipocytes augment angiogenic processes in dispersed adipose tissue in the presence of FGF2 (basic). Adipose tissue was collected from 4 prepubertal female pigs (gilts) and enzymatically dispersed (collagenase type II). Cells were separated into 2 groups 1) mature lipid‐filled adipocytes (MA) and 2) all other cell types (OC) i.e., vascular endothelial cells (VEC), preadipocytes, and fibroblasts. The OC were incubated for 24 hr in cell culture media with 10% fetal bovine serum (FBS) to promote attachment and angiogenic processes. After the first 24 hr incubation period, media was removed and replaced with 2% FBS and MA were added with or without FGF2 (10−11 M) or rabbit polyclonal anti‐FGF2 (1:100 dilution) and incubated for an additional 24 hr. The MIXED procedure of SAS was utilized to determine the effect of treatment on in vitro angiogenic cellular processes. The addition of MA alone augmented cellular angiogenic processes including elongation, sprouting, formation of nascent tubes, and vessel tube formation (P < 0.05). However, vessel tube formation in the FGF2 treatment group was lower than MA alone (P < 0.05), which was ameliorated, in part, with FGF2 antibody treatment. Mature adipocytes appear to augment angiogenic processes; however, FGF2 may diminish or impair the progression of vessel tube formation.Support or Funding InformationUSDA LEADERS 2 Award #2017‐38422‐27298

Characterization of Naturally Occurring Bioactive Factor Mixtures for Bone Regeneration.

In this study, the bone-regenerative potential of bioactive factors derived from adipose tissue, platelet-rich plasma (PRP) and conditioned medium from hypoxia-treated human telomerase immortalized bone-marrow-derived mesenchymal stem cells (hTERT-MSC) was investigated in vitro with the aim to develop cost-effective and efficient bone substitutes for optimized regeneration of bone defects. Adipose tissue was harvested from human donors undergoing reconstructive surgery, and adipose tissue extract (ATE) was prepared. Platelet lysates (PL) were produced by repeated freeze-thaw cycles of PRP, and hypoxia-conditioned medium (HCM) was obtained by culturing human telomerase immortalized bone-marrow-derived mesenchymal stromal cells for 5 days with 1% O2. Besides analysis by cytokine and angiogenesis arrays, ELISA was performed. Angiogenic potential was investigated in cocultures of bone-marrow-derived (BM)-MSC and human umbilical vein endothelial cells. Multiple angiogenic proteins and cytokines were detected in all growth factor mixtures. HCM and ATE contained high amounts of angiogenin and CCL2/MCP-1, whereas PL contained high amounts of IGFBP-1. Culturing cells with HCM and ATE significantly increased specific ALP activity of BM-MSC as well as tubule length and junctions of endothelial networks, indicating osteogenic and angiogenic stimulation. To achieve a synergism between chemoattractive potential and osteogenic and angiogenic differentiation capacity, a combination of different growth factors appears promising for potential clinical applications.

Il trattamento delle condropatie degenerative con tessuto adiposo autologo microframmentato

Osteoarthritis is a widespread pathology, with a prevalence of over 50% of the population, which leads to pain and functional limitation. At the moment, no surgical technique has been shown to be capable of real regeneration of the chondral layer. The new regenerative frontier seems to be represented by the self-ability of the Stromal Vascular Fraction (SVF) to preserve the cellular network, which is essential for cell regeneration. Several micro-fragmented adipose tissue extraction and processing systems have been refined in the past years and recent literature data show the biological effectiveness of this tissue, which plays a key role in restoring joint homeostasis, through the modulation of inflammation and pain. It will be necessary and important to understand whether this injective method can really be considered a “regenerative” option.

Effects of nanofibrillated cellulose hydrogels on adipose tissue extract and hepatocellular carcinoma cell spheroids in freeze-drying

The aim of this study was to evaluate the effects of two nanofibrillated cellulose (NFC) hydrogels on two human derivatives during freeze-drying. Native NFC hydrogel is a suitable platform to culture 3D cell spheroids and a hydrogel processed further, called anionic NFC (ANFC) hydrogel, is an excellent platform for controlled release of proteins. Moreover, it has been shown to be compatible with freeze-drying when correct lyoprotectants are implemented. Freeze-drying is a method, where substance is first frozen, and then vacuum dried trough sublimation of water in order to achieve dry matter without the loss of the original three-dimensional structures.The first chosen human derivative was adipose tissue extract (ATE) which is a cell-free growth factor-rich preparation capable of promoting growth of regenerative cells. The release of growth factors from the freeze-dried mixture of ATE and ANFC was compared to that of non-freeze-dried control mixtures. The release profiles remained at the same level after freeze-drying. The second derivative was hepatocellular carcinoma (HepG2) cell spheroids which were evaluated before and after freeze-drying. The 3D structure of the HepG2 cell spheroids was preserved and the spheroids retained 18% of their metabolic activity after rehydration. However, the freeze-dried and rehydrated HepG2 cell spheroids did not proliferate and the cell membrane was damaged by fusion and formation of crystals.

Oscillating and static dietary crude protein supply: II. Energy and protein requirements of young Nellore bulls.

:The objective of this study was to evaluate whole body chemical composition and energy and protein nutrient requirements for maintenance and gain of Nellore bulls. Fifty young bulls, with an average age of 7 ± 1 mo and initial body weight (BW) of 260.0 ± 8.1 kg, were used in this experiment. Four bulls were used as baseline reference animals and were slaughtered at the beginning of the experiment. Four bulls were fed at maintenance (12 g dry matter [DM]/kg of BW), whereas 42 bulls were divided into six groups (n = 7/group) and were randomly assigned to the following dietary treatments 105 (low [LO]), 125 (medium [MD]), or 145 (high [HI]) g crude protein (CP)/kg DM, LO to HI (LH), LO to MD (LM), or MD to HI (MH) oscillating CP at a 48-h interval for 140 d. At the end of the experiment, bulls were slaughtered and samples of the whole body were collected. All samples were lyophilized, ground, and composed as percentage of component of empty body weight (EBW) from each bull. A power model was used to estimate carcass, non-carcass components, and gastrointestinal content of the shrunk body weight (SBW), and CP and water present in the empty body, whereas an exponential model was used to estimate adipose tissue and ether extract (EE) present in the EBW. Nonlinear regression equations were developed to predict heat production from metabolizable energy (ME) intake and retained energy (RE). The net energy requirements for maintenance and ME for maintenance were 77 and 122.75 kcal/EBW0.75/d, respectively. The efficiency of ME utilization for maintenance was 62.7%. The equation obtained for net energy for gain (NEg) was: NEg (Mcal/EBW0.75/d) = 0.0535 × EBW0.75 × EBG0.7131, where EBG is the empty body gain, and the efficiency was 24.25%. Net protein requirement for growth (NPg) was: NPg (g/d) = 227.372 × EBG – 19.479 × RE. There was a linear increase for carcass, CP, and water present in the EBW as the animal grew. The EE deposition exponentially increased as EBW increased.

Effects of Phaseolus vulgaris Extract on Lipolytic Activity and Differentiation of 3T3-L1 Preadipocytes into Mature Adipocytes: A Strategy to Prevent Obesity.

Background Overweight and obesity are defined as abnormal or excessive fat accumulation that may be harmful for health. A global trend in this area is the search for natural compounds that have a proven beneficial effect and no clinical complications. Phaseolus vulgaris (bean) is a vegetable highly consumed worldwide. One of its effects, the most reported, is weight reduction in overweight individuals. Objective The objective of this study was to investigate the antiobesity activity of this legume in mature 3T3-L1 adipocytes and in rat white adipose tissue in an ex vivo model. Design Mature adipocytes 3T3-L1 and rat adipose tissue were treated with bean extracts. We quantified lipolysis in mature 3T3-L1 adipocytes and in rat white adipose tissue in an ex vivo model. Results In an ex vivo assay with adipose tissue, methanolic and aqueous green bean extracts increased glycerol release to the medium compared to control (p < 0.05 and p < 0.001 respectively). Treatment of 3T3-L1 adipocytes with green bean extracts (800 and 1000 µg/mL) increased glycerol release significantly (p < 0.0001). Extracts at concentrations between 500 and 1000 µg/mL reduced intracellular triglyceride accumulation by 34.4% and 47.1% compared to control (p < 0.0001). Discussion Our results propose that bioactive compounds of green beans exert a direct mechanism on adipocytes through lipolysis. Conclusion We have identified a novel capacity of bean extracts related to lipolytic activity both in vitro and ex vivo, resulting in a powerful lipolytic effect. Moreover, we also found that bean extracts has an antiadipogenic effect during the differentiation of 3T3-L1 preadipocytes. These results suggest that bean is a good candidate for the development of functional ingredients that can help reduce the high rates of death from cardiovascular diseases associated with obesity.

Autologous adipose-derived stromal vascular fraction and scarred vocal folds: first clinical case report

Patients with scarred vocal folds (congenitally or following phonosurgery) are currently difficult to treat and present a dysphonia, often disabling in daily life. Several therapies are available on the market but the results of these are often disappointing. The autologous adipose-derived stromal vascular fraction (ADSVF) is recognized as an easily accessible source of cells with angiogenic, anti-inflammatory, immunomodulatory, and regenerative properties. We present here the case of a 43-year-old woman who had a severe dysphonia associated with scarred vocal folds after a phonosurgery and was resistant to conventional medical and surgical treatments. She received a local administration of autologous ADSVF. The protocol involved, on the same day, adipose tissue extraction, ADSVF preparation, and then local injection (0.45ml of ADSVF in each vocal fold, for a total of 12.2 million ADSVF viable cells). No serious adverse events have been described. One year following the surgery, the laryngoscopic aspect and the majority of voice parameters had improved, in particular the Voice Handicap Index decreasing from 75 to 9. The perceptual analysis found the voice to be less hoarse and more stable, without breathiness. The case of this patient highlights the therapeutic potential of ADSVF for such applications (trial registration, ClinicalTrials.gov NCT02622464; registered 4 December 2015).

Optimizing adipose tissue extract isolation with stirred suspension culture

ABSTRACTObjectives: Adherent culture which is used to collect adipose tissue extract (ATE) previously brings the problem of inhomogeneity and non-repeatability. Here we aim to extract ATE with stirred suspension culture to speed up the extraction process, stabilize the yield, and improve consistent potency metrics of ATE. Materials and methods: ATE was collected with adherent culture (ATE-A) and stirred suspension culture (ATE-S) separately. Protein yield and composition were detected by SDS-PAGE, while cytokines in ATE were determined with ELISA. The adipogenic and angiogenic potential of ATE were compared by western blot and qPCR. In addition, haematoxylin and eosin staining and lactate dehydrogenase (LDH) activity assays were used to analyze the cell viability of adipose tissue cultured with different methods. Results: The yield of ATE-S was consistent while ATE-A varied notably. Characterization of the protein composition and exosome-like vesicles (ELVs) indicated no significant difference between ATE-S and ATE-A. The concentrations of cytokines (VEGF, bFGF, and IL-6) showed no significant difference, while IGF in ATE-S was higher than that in ATE-A. ATE-S showed upregulated adipogenic and angiogenic potential compared to ATE-A. Morever, stirred suspension culture decreased the LDH activity of ATE while increased the number of viable adipocytes and reduced adipose tissue necrosis. Conclusion: Compared with adherent culture, stirred suspension culture is a reliable, time- and labor-saving method to collect ATE, which might be used to improve the downstream applications of ATE.