Use of genetically modified muscle and fat grafts to repair defects in bone and cartilage

We report a novel technology for the rapid healing of large osseous and chondral defects, based upon the genetic modification of autologous skeletal muscle and fat grafts. These tissues were selected because they not only possess mesenchymal progenitor cells and scaffolding properties, but also can be biopsied, genetically modified and returned to the patient in a single operative session. First generation adenovirus vector carrying cDNA encoding human bone morphogenetic protein-2 (Ad.BMP-2) was used for gene transfer to biopsies of muscle and fat. To assess bone healing, the genetically modified (“gene activated”) tissues were implanted into 5mm-long critical size, mid-diaphyseal, stabilized defects in the femora of Fischer rats. Unlike control defects, those receiving gene-activated muscle underwent rapid healing, with evidence of radiologic bridging as early as 10 days after implantation and restoration of full mechanical strength by 8 weeks. Histologic analysis suggests that the grafts rapidly differentiated into cartilage, followed by efficient endochondral ossification. Fluorescence in situ hybridization detection of Y-chromosomes following the transfer of male donor muscle into female rats demonstrated that at least some of the osteoblasts of the healed bone were derived from donor muscle. Gene activated fat also healed critical sized defects, but less quickly than muscle and with more variability. Anti-adenovirus antibodies were not detected. Pilot studies in a rabbit osteochondral defect model demonstrated the promise of this technology for healing cartilage defects. Further development of these methods should provide ways to heal bone and cartilage more expeditiously, and at lower cost, than is presently possible.

The effect of exercise on the skeletal muscle phospholipidome of rats fed a high-fat diet

The aim of this study was to examine the effect of endurance training on skeletal muscle phospholipid molecular species from high-fat fed rats. Twelve female Sprague-Dawley rats were fed a high-fat diet (78.1% energy). The rats were randomly divided into two groups, a sedentary control group and a trained group (125 min of treadmill running at 8 m/min, 4 days/wk for 4 weeks). Forty-eight hours after their last training bout phospholipids were extracted from the red and white vastus lateralis and analyzed by electrospray-ionization mass spectrometry. Exercise training was associated with significant alterations in the relative abundance of a number of phospholipid molecular species. These changes were more prominent in red vastus lateralis than white vastus lateralis. The largest observed change was an increase of similar to 30% in the abundance of 1-palmitoyl-2-linoleoyl phosphatidylcholine ions in oxidative fibers. Reductions in the relative abundance of a number of phospholipids containing long-chain n-3 polyunsaturated fatty acids were also observed. These data suggest a possible reduction in phospholipid remodeling in the trained animals. This results in a decrease in the phospholipid n-3 to n-6 ratio that may in turn influence endurance capacity.

Long-term stability of adipose tissue generated from a vascularized pedicled fat flap inside a chamber

Background Numerous studies demonstrate the generation and short-term survival of adipose tissue; however, long-term persistence remains elusive. This study evaluates long-term survival and transferability of de novo adipose constructs based on a ligated vascular pedicle and tissue engineering chamber combination. Methods Defined adipose tissue flaps were implanted into rats in either intact or perforated domed chambers. In half of the groups, the chambers were removed after 10 weeks and the constructs transferred on their vascular pedicle to a new site, where they were observed for a further 10 weeks. In the remaining groups, the tissue construct was observed for 20 weeks inside the chamber. Tissue volume was assessed using magnetic resonance imaging and histologic measures, and constructs were assessed for stability and necrosis. Sections were assessed histologically and for proliferation using Ki-67. Results At 20 weeks, volume analysis revealed an increase in adipose volume from 0.04 ± 0.001 ml at the time of insertion into the chambers to 0.27 ± 0.004 ml in the closed and 0.44 ± 0.014 ml in the perforated chambers. There was an additional increase of approximately 10 to 15 percent in tissue volume in flaps that remained in chambers for 20 weeks, whereas the volume of the transferred tissue not in chambers remained unaltered. Histomorphometric assessment of the tissues documented no signs of hypertrophy, fat necrosis, or atypical changes of the newly generated tissue. Conclusion This study presents a promising new method of generating significant amounts of mature, vascularized, stable, and transferable adipose tissue for permanent autologous soft-tissue replacement.

Intrinsics and dynamics of fat grafts : an in vitro study

Background Despite a revived interest in fat grafting procedures, clinicians still fail to demonstrate clearly the in vivo behavior of fat grafts as a dynamic tissue substitute. However, the basic principles in cellular biology teach us that cells can survive and develop, provided that a structural matrix exists that directs their behavior. The purpose of this in vitro study was to analyze that behavior of crude fat grafts, cultured on a three-dimensional laminin-rich matrix. Methods Nonprocessed, human fat biopsy specimens (approximately 1 mm) were inoculated on Matrigel-coated wells to which culture medium was added. The control group consisted of fat biopsy specimens embedded in medium alone. The cellular proliferation pattern was followed over 6 weeks. Additional cultures of primary generated cellular spheroids were performed and eventually subjected to adipogenic differentiation media. Results A progressive outgrowth of fibroblast-like cells from the core fat biopsy specimen was observed in both groups. Within the Matrigel group, an interconnecting three-dimensional network of spindle-shaped cells was established. This new cell colony reproduced spheroids that functioned again as solitary sources of cellular proliferation. Addition of differentiation media resulted in lipid droplet deposition in the majority of generated cells, indicating the initial steps of adipogenic differentiation. Conclusions The authors noticed that crude, nonprocessed fat biopsy specimens do have considerable potential for future tissue engineering-based applications, provided that the basic principles of developmental, cellular biology are respected. Spontaneous in vitro expansion of the stromal cells present in fat grafts within autologous and injectable matrices could create "off-the-shelf" therapies for reconstructive procedures.

Spontaneous large volume adipose tissue generation from a vascularized pedicled fat flap inside a chamber space

A novel method of spontaneous generation of new adipose tissue from an existing fat flap is described. A defined volume of fat flap based on the superficial inferior epigastric vascular pedicle in the rat was elevated and inset into a hollow plastic chamber implanted subcutaneously in the groin of the rat. The chamber walls were either perforated or solid and the chambers either contained poly(D,L-lactic-co-glycolic acid) (PLGA) sponge matrix or not. The contents were analyzed after being in situ for 6 weeks. The total volume of the flap tissue in all groups except the control groups, where the flap was not inserted into the chambers, increased significantly, especially in the perforated chambers (0.08 ± 0.007 mL baseline compared to 1.2 ± 0.08 mL in the intact ones). Volume analysis of individual component tissues within the flaps revealed that the adipocyte volume increased and was at a maximum in the chambers without PLGA, where it expanded from 0.04 ± 0.003 mL at insertion to 0.5 ± 0.08 mL (1250% increase) in the perforated chambers and to 0.16 ± 0.03 mL (400% increase) in the intact chambers. Addition of PLGA scaffolds resulted in less fat growth. Histomorphometric analysis rather than simple hypertrophy documented an increased number of adipocytes. The new tissue was highly vascularized and no fat necrosis or atypical changes were observed.

Offspring of mothers fed a high fat diet display hepatic cell cycle inhibition and associated changes in gene expression and DNA methylation

The association between an adverse early life environment and increased susceptibility to later-life metabolic disorders such as obesity, type 2 diabetes and cardiovascular disease is described by the developmental origins of health and disease hypothesis. Employing a rat model of maternal high fat (MHF) nutrition, we recently reported that offspring born to MHF mothers are small at birth and develop a postnatal phenotype that closely resembles that of the human metabolic syndrome. Livers of offspring born to MHF mothers also display a fatty phenotype reflecting hepatic steatosis and characteristics of non-alcoholic fatty liver disease. In the present study we hypothesised that a MHF diet leads to altered regulation of liver development in offspring; a derangement that may be detectable during early postnatal life. Livers were collected at postnatal days 2 (P2) and 27 (P27) from male offspring of control and MHF mothers (n = 8 per group). Cell cycle dynamics, measured by flow cytometry, revealed significant G0/G1 arrest in the livers of P2 offspring born to MHF mothers, associated with an increased expression of the hepatic cell cycle inhibitor Cdkn1a. In P2 livers, Cdkn1a was hypomethylated at specific CpG dinucleotides and first exon in offspring of MHF mothers and was shown to correlate with a demonstrable increase in mRNA expression levels. These modifications at P2 preceded observable reductions in liver weight and liver:brain weight ratio at P27, but there were no persistent changes in cell cycle dynamics or DNA methylation in MHF offspring at this time. Since Cdkn1a up-regulation has been associated with hepatocyte growth in pathologic states, our data may be suggestive of early hepatic dysfunction in neonates born to high fat fed mothers. It is likely that these offspring are predisposed to long-term hepatic dysfunction.

Glucocorticoid receptor haploinsufficiency causes hypertension and attenuates hypothalamic-pituitary-adrenal axis and blood pressure adaptions to high-fat diet

Glucocorticoid hormones are critical to respond and adapt to stress. Genetic variations in the glucocorticoid receptor (GR) gene alter hypothalamic-pituitary-adrenal (HPA) axis activity and associate with hypertension and susceptibility to metabolic disease. Here we test the hypothesis that reduced GR density alters blood pressure and glucose and lipid homeostasis and limits adaption to obesogenic diet. Heterozygous GR βgeo/+ mice were generated from embryonic stem (ES) cells with a gene trap integration of a β-galactosidase-neomycin phosphotransferase (βgeo) cassette into the GR gene creating a transcriptionally inactive GR fusion protein. Although GRβgeo/+ mice have 50% less functional GR, they have normal lipid and glucose homeostasis due to compensatory HPA axis activation but are hypertensive due to activation of the renin-angiotensin- aldosterone system (RAAS). When challenged with a high-fat diet, weight gain, adiposity, and glucose intolerance were similarly increased in control and GRβgeo/+ mice, suggesting preserved control of intermediary metabolism and energy balance. However, whereas a high-fat diet caused HPA activation and increased blood pressure in control mice, these adaptions were attenuated or abolished in GRβgeo/+ mice. Thus, reduced GR density balanced by HPA activation leaves glucocorticoid functions unaffected but mineralocorticoid functions increased, causing hypertension. Importantly, reduced GR limits HPA and blood pressure adaptions to obesogenic diet.

Liking for high fat foods in patients with Obstructive Sleep Apnoea

Excess weight and obesity are factors that are strongly associated with risk for Obstructive Sleep Apnoea (OSA).Weight loss has been associated with improvements in clinical indicators of OSA severity; however, patients’ beliefs about diet change have not been investigated. This study utilized a validated behaviour change model to estimate the relationship between food liking, food intake and indices of OSA severity. Two-hundred and six OSA patients recruited from a Sleep Disorders Clinic completed standardized questionnaires of: a) fat and fibre food intake, food liking, and food knowledge and; b) attitudes and intentions towards fat reduction. OSA severity and body mass index (BMI) were objectively measured using standard clinical guidelines. The relationship between liking for high fat food and OSA severity was tested with hierarchical regression. Gender and BMI explained a significant 20% of the variance in OSA severity, Fibre Liking accounted for an additional 6% (a negative relationship), and Fat Liking accounted for a further 3.6% of variance. Although the majority of individuals (47%) were currently “active” in reducing fat intake, overall the patients’ dietary beliefs and behaviours did not correspond. The independent relationship between OSA severity and liking for high fat foods (and disliking of high fibre foods) may be consistent with a two-way interaction between sleep disruption and food choice. Whilst the majority of OSA patients were intentionally active in changing to a healthy diet, further emphasis on improving healthy eating practices and beliefs in this population is necessary.

Enhanced preference for high-fat foods following a simulated night shift

Objectives Shift workers are prone to obesity and associated co-morbidities such as diabetes and cardiovascular disease. Sleep restriction associated with shift work results in dramatic endocrine and metabolic effects that predispose shift workers to these adverse health consequences. While sleep restriction has been associated with increased caloric intake, food preference may also play a key role in weight gain associated with shift work. This study examined the impact of an overnight simulated night shift on food preference. Methods Sixteen participants [mean 20.1, standard deviation (SD) 1.4 years; 8 women] underwent a simulated night shift and control condition in a counterbalanced order. On the following morning, participants were provided an opportunity for breakfast that included high- and low-fat food options (mean 64.8% and 6.4% fat, respectively). Results Participants ate significantly more high-fat breakfast items after the simulated night shift than after the control condition [167.3, standard error of the mean (SEM 28.7) g versus 211.4 (SEM 35.6) g; P=0.012]. The preference for high-fat food was apparent among the majority of individuals following the simulated night shift (81%), but not for the control condition (31%). Shift work and control conditions did not differ, however, in the total amount of food or calories consumed. Conclusions A simulated night shift leads to preference for high-fat food during a subsequent breakfast opportunity. These results suggest that food choice may contribute to weight-related chronic health problems commonly seen among night shift workers.

Reproducibility of fatmax and fat oxidation rates during exercise in recreationally trained males

Aerobic exercise training performed at the intensity eliciting maximal fat oxidation (Fatmax) has been shown to improve the metabolic profile of obese patients. However, limited information is available on the reproducibility of Fatmax and related physiological measures. The aim of this study was to assess the intra-individual variability of: a) Fatmax measurements determined using three different data analysis approaches and b) fat and carbohydrate oxidation rates at rest and at each stage of an individualized graded test. Fifteen healthy males [body mass index 23.1±0.6 kg/m2, maximal oxygen consumption () 52.0±2.0 ml/kg/min] completed a maximal test and two identical submaximal incremental tests on ergocycle (30-min rest followed by 5-min stages with increments of 7.5% of the maximal power output). Fat and carbohydrate oxidation rates were determined using indirect calorimetry. Fatmax was determined with three approaches: the sine model (SIN), measured values (MV) and 3rd polynomial curve (P3). Intra-individual coefficients of variation (CVs) and limits of agreement were calculated. CV for Fatmax determined with SIN was 16.4% and tended to be lower than with P3 and MV (18.6% and 20.8%, respectively). Limits of agreement for Fatmax were −2±27% of with SIN, −4±32 with P3 and −4±28 with MV. CVs of oxygen uptake, carbon dioxide production and respiratory exchange rate were <10% at rest and <5% during exercise. Conversely, CVs of fat oxidation rates (20% at rest and 24–49% during exercise) and carbohydrate oxidation rates (33.5% at rest, 8.5–12.9% during exercise) were higher. The intra-individual variability of Fatmax and fat oxidation rates was high (CV>15%), regardless of the data analysis approach employed. Further research on the determinants of the variability of Fatmax and fat oxidation rates is required.

Comparing fat oxidation in an exercise test with moderate-intensity interval training

This study compared fat oxidation rate from a graded exercise test (GXT) with a moderate-intensity interval training session (MIIT) in obese men. Twelve sedentary obese males (age 29 ± 4.1 years; BMI 29.1 ± 2.4 kg·m-2; fat mass 31.7 ± 4.4 %body mass) completed two exercise sessions: GXT to determine maximal fat oxidation (MFO) and maximal aerobic power (VO2max), and an interval cycling session during which respiratory gases were measured. The 30-min MIIT involved 5-min repetitions of workloads 20% below and 20% above the MFO intensity. VO2max was 31.8 ± 5.5 ml·kg-1·min-1 and all participants achieved ≥ 3 of the designated VO2max test criteria. The MFO identified during the GXT was not significantly different compared with the average fat oxidation rate in the MIIT session. During the MIIT session, fat oxidation rate increased with time; the highest rate (0.18 ± 0.11 g·min- 1) in minute 25 was significantly higher than the rate at minute 5 and 15 (p ≤ 0.01 and 0.05 respectively). In this cohort with low aerobic fitness, fat oxidation during the MIIT session was comparable with the MFO determined during a GXT. Future research may consider if the varying workload in moderate-intensity interval training helps adherence to exercise without compromising fat oxidation.

Effect of an estrogen receptor-α intron 4 polymorphism on fat mass in 11-year-old children

Context: in the ESR1 gene encoding estrogen receptor (ER)-α may be associated with fat mass in adults. Objectives: The objective of the study was to establish whether ESR1 polymorphisms influence fat mass in childhood. Design: This was a cross-sectional analysis after genotyping of rs9340799, rs2234693, and rs7757956 ESR1 polymorphisms. Setting: The Avon Longitudinal Study of Parents and Children (ALSPAC) was a population-based prospective study. Participants: Participants included 3097 11-yr-old children with results for ESR1 genotyping, puberty measures, and dual-energy x-ray absorptiometry results. Outcomes: Relationships between ESR1 polymorphisms and indices of body composition were measured. Results: The rs7757956 polymorphism was associated with fat mass (P = 0.002). Total body fat mass (adjusted for height) was reduced by 6% in children with TA/AA genotypes, and risk of being overweight (≥85th centile of fat mass) was decreased by 20%. This genetic effect appeared to interact with puberty in girls (P = 0.05 for interaction): in those with the TT genotype, total body fat mass (adjusted for height) was 18% higher in Tanner stages 3-5 vs. stages 1-2; the equivalent difference was 7% in those with TA/AA genotypes. Furthermore, the risk of being overweight was 36% lower in girls with TA/AA genotypes in Tanner stages 3-5, but no reduction was seen in those in stages 1-2. Neither rs9340799 nor rs2234693 polymorphisms were associated with body composition measures. Conclusions: Fat mass in 11-yr-old children was related to the rs7757956 ESR1 polymorphism. This association was strongest in girls in more advanced puberty, in whom the risk of being overweight was reduced by 36% in those with the TA/AA genotype.

Parameter estimation and sensitivity analysis of fat deposition models in beef steers using acslXtreme.

The Davis Growth Model (a dynamic steer growth model encompassing 4 fat deposition models) is currently being used by the phenotypic prediction program of the Cooperative Research Centre (CRC) for Beef Genetic Technologies to predict P8 fat (mm) in beef cattle to assist beef producers meet market specifications. The concepts of cellular hyperplasia and hypertrophy are integral components of the Davis Growth Model. The net synthesis of total body fat (kg) is calculated from the net energy available after accounting tor energy needs for maintenance and protein synthesis. Total body fat (kg) is then partitioned into 4 fat depots (intermuscular, intramuscular, subcutaneous, and visceral). This paper reports on the parameter estimation and sensitivity analysis of the DNA (deoxyribonucleic acid) logistic growth equations and the fat deposition first-order differential equations in the Davis Growth Model using acslXtreme (Hunstville, AL, USA, Xcellon). The DNA and fat deposition parameter coefficients were found to be important determinants of model function; the DNA parameter coefficients with days on feed >100 days and the fat deposition parameter coefficients for all days on feed. The generalized NL2SOL optimization algorithm had the fastest processing time and the minimum number of objective function evaluations when estimating the 4 fat deposition parameter coefficients with 2 observed values (initial and final fat). The subcutaneous fat parameter coefficient did indicate a metabolic difference for frame sizes. The results look promising and the prototype Davis Growth Model has the potential to assist the beef industry meet market specifications.

New genetic loci link adipose and insulin biology to body fat distribution

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10−8). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms.

Meta-analysis identifies 13 new loci associated with waist-hip ratio and reveals sexual dimorphism in the genetic basis of fat distribution

Waist-hip ratio (WHR) is a measure of body fat distribution and a predictor of metabolic consequences independent of overall adiposity. WHR is heritable, but few genetic variants influencing this trait have been identified. We conducted a meta-analysis of 32 genome-wide association studies for WHR adjusted for body mass index (comprising up to 77,167 participants), following up 16 loci in an additional 29 studies (comprising up to 113,636 subjects). We identified 13 new loci in or near RSPO3, VEGFA, TBX15-WARS2, NFE2L3, GRB14, DNM3-PIGC, ITPR2-SSPN, LY86, HOXC13, ADAMTS9, ZNRF3-KREMEN1, NISCH-STAB1 and CPEB4 (P = 1.9 × 10−9 to P = 1.8 × 10−40) and the known signal at LYPLAL1. Seven of these loci exhibited marked sexual dimorphism, all with a stronger effect on WHR in women than men (P for sex difference = 1.9 × 10−3 to P = 1.2 × 10−13). These findings provide evidence for multiple loci that modulate body fat distribution independent of overall adiposity and reveal strong gene-by-sex interactions.