Human preadipocyte proliferation and differentiation

Obesity represents a major health, social and economic burden to many developing and Westernized communities, with the prevalence increasing at a rate exceeding almost all other medical conditions. Despite major recent advances in our understanding of adipose tissue metabolism and dynamics, we still have limited insight into the regulation of adipose tissue mass in humans. Any significant increase in adipose tissue mass requires proliferation and differentiation of precursor cells (preadipocytes) present in the stromo-vascular compartment of adipose tissue. These processes are very complex and an increasing number of growth factors and hormones have been shown to modulate the expression of genes involved in preadipocyte proliferation and differentiation. A number of transcription factors, including the C/EBP family and PP ARy, have been identified as integral to adipose tissue development and preadipocyte differentiation. Together PP ARy and C/EBPa regulate important events in the activation and maintenance of the terminally differentiated phenotype. The ability of PP ARy to increase transcription through its DNA recognition site is dependent on the binding of ligands. This suggests that an endogenous PP ARy ligand may be an important regulator of adipogenesis. Adipose tissue functions as both the major site of energy storage in the body and as an endocrine organ synthesizing and secreting a number of important molecules involved in regulation of energy balance. For optimum functioning therefore, adipose tissue requires extensive vascularization and previous studies have shown that growth of adipose tissue is preceded by development of a microvascular network. This suggests that paracrine interactions between constituent cells in adipose tissue may be involved in both new capillary formation and fat cell growth. To address this hypothesis the work in this project was aimed at (a) further development of a method for inducing preadipocyte differentiation in subcultured human cells; (b) establishing a method for simultaneous isolation and separate culture of both preadipocytes and microvascular endothelial cells from the same adipose tissue biopsies; (c) to determine, using conditioned medium and co-culture techniques, if endothelial cell-derived factors influence the proliferation and/or differentiation of human preadipocytes; and (d) commence characterization of factors that may be responsible for any observed paracrine effects on aspects of human adipogenesis. Major findings of these studies were as follows: (A) Inclusion of either linoleic acid (a long-chain fatty acid reported to be a naturally occurring ligand for PP ARy) or Rosiglitazone (a member of the thiazolidinedione class of insulin-sensitizing drugs and a synthetic PPARy ligand) in differentiation medium had markedly different effects on preadipocyte differentiation. These studies showed that human preadipocytes have the potential to accumulate triacylglycerol irrespective of their stage of biochemical differentiation, and that thiazolidinediones and fatty acids may exert their adipogenic and lipogenic effects via different biochemical pathways. It was concluded that Rosiglitazone is a more potent inducer of human preadipocyte differentiation than linoleic acid. (B) A method for isolation and culture of both endothelial cells and preadipocytes from the same adipose tissue biopsy was developed. Adipose-derived microvascular endothelial cells were found to produce factor/s, which enhance both proliferation and differentiation of human preadipocytes. (C) The adipogenic effects of microvascular endothelial cells can be mimicked by exposure of preadipocytes to members of the Fibroblast Growth Factor family, specifically ~-ECGF and FGF-1. (D) Co-culture of human preadipocytes with endothelial cells or exposure of preadipocytes to either ~-ECGF or FGF-1 were found to 'prime' human preadipocytes, during their proliferative phase of growth, for thiazolidinedione-induced differentiation. (E) FGF -1 was not found to be acting as a ligand for PP ARy in this system. Findings from this project represent a significant step forward in our understanding of factors involved in growth of human adipose tissue and may lead to the development of therapeutic strategies aimed at modifying the process. Such strategies would have potential clinical utility in the treatment of obesity and obesity related disorders such as Type II Diabetes.

Modulation of dermal microvascular endithelial cell responses to growth factors and haemostatic factors in the presence of vitronectin

In order to effect permanent closure in burns patients suffering from full thickness wounds, replacing their skin via split thickness autografting, is essential. Dermal substitutes in conjunction with widely meshed split thickness autografts (+/- cultured keratinocytes) reduce scarring at the donor and recipient sites of burns patients by reducing demand for autologous skin (both surface area and thickness), without compromising dermal delivery at the wound face. Tissue engineered products such as Integra consist of a dermal template which is rapidly remodelled to form a neodermis, at which time the temporary silicone outer layer is removed and replaced with autologous split thickness skin. Whilst provision of a thick tissue engineered dermis at full thickness burn sites reduces scarring, it is hampered by delays in vascularisation which results in clinical failure. The ultimate success of any skin graft product is dependent upon a number of basic factors including adherence, haemostasis and in the case of viable tissue grafts, success is ultimately dependent upon restoration of a normal blood supply, and hence this study. Ultimately, the goal of this research is to improve the therapeutic properties of tissue replacements, through impregnation with growth factors aimed at stimulating migration and proliferation of microvascular endothelial cells into the donor tissue post grafting. For the purpose of my masters, the aim was to evaluate the responsiveness of a dermal microvascular endothelial cell line to growth factors and haemostatic factors, in the presence of the glycoprotein vitronectin. Vitronectin formed the backbone for my hypothesis and research due to its association with both epithelial and, more specifically, endothelial migration and proliferation. Early work using a platform technology referred to as VitroGro (Tissue Therapies Ltd), which is comprised of vitronectin bound BP5/IGF-1, aided keratinocyte proliferation. I hypothesised that this result would translate to another epithelium - endothelium. VitroGro had no effect on endothelial proliferation or migration. Vitronectin increases the presence of Fibroblast Growth Factor (FGF) and Vascular Endothelial Growth Factor (VEGF) receptors, enhancing cell responsiveness to their respective ligands. So, although Human Microvascular Endothelial Cell line 1 (HMEC-1) VEGF receptor expression is generally low, it was hypothesised that exposure to vitronectin would up-regulate this receptor. HMEC-1 migration, but not proliferation, was enhanced by vitronectin bound VEGF, as well as vitronectin bound Epidermal Growth Factor (EGF), both of which could be used to stimulate microvascular endothelial cell migration for the purpose of transplantation. In addition to vitronectin's synergy with various growth factors, it has also been shown to play a role in haemostasis. Vitronectin binds thrombin-antithrombin III (TAT) to form a trimeric complex that takes on many of the attributes of vitronectin, such as heparin affinity, which results in its adherence to endothelium via heparan sulfate proteoglycans (HSP), followed by unaltered transcytosis through the endothelium, and ultimately its removal from the circulation. This has been documented as a mechanism designed to remove thrombin from the circulation. Equally, it could be argued that it is a mechanism for delivering vitronectin to the matrix. My results show that matrix-bound vitronectin dramatically alters the effect that conformationally altered antithrombin three (cATIII) has on proliferation of microvascular endothelial cells. cATIII stimulates HMEC-1 proliferation in the presence of matrix-bound vitronectin, as opposed to inhibiting proliferation in its absence. Binding vitronectin to tissues and organs prior to transplant, in the presence of cATIII, will have a profound effect on microvascular infiltration of the graft, by preventing occlusion of existing vessels whilst stimulating migration and proliferation of endothelium within the tissue.

Assimilating cell sheets and hybrid scaffolds for dermal tissue engineering

Cell sheets can be used to produce neo-tissue with mature extracellular matrix. However, extensive contraction of cell sheets remains a problem. We devised a technique to overcome this problem and applied it to tissue engineer a dermal construct. Human dermal fibroblasts were cultured with poly(lactic-co-glycolic acid)-collagen meshes and collagen-hyaluronic acid foams. Resulting cell sheets were folded over the scaffolds to form dermal constructs. Human keratinocytes were cultured on these dermal constructs to assess their ability to support bilayered skin regeneration. Dermal constructs produced with collagen-hyaluronic acid foams showed minimal contraction, while those with poly(lactic-co-glycolic acid)-collagen meshes curled up. Cell proliferation and metabolic activity profiles were characterized with PicoGreen and AlamarBlue assays, respectively. Fluorescent labeling showed high cell viability and F-actin expression within the constructs. Collagen deposition was detected by immunocytochemistry and electron microscopy. Transforming Growth Factor-alpha and beta1, Keratinocyte Growth Factor and Vascular Endothelial Growth Factor were produced at various stages of culture, measured by RT-PCR and ELISA. These results indicated that assimilating cell sheets with mechanically stable scaffolds could produce viable dermal-like constructs that do not contract. Repeated enzymatic treatment cycles for cell expansion is unnecessary, while the issue of poor cell seeding efficiency in scaffolds is eliminated.

Bearing damage analysis by calculation of capacitive coupling between inner and outer races of a ball bearing

Bearing damage in modern inverter-fed AC drive systems is more common than in motors working with 50 or 60 Hz power supply. Fast switching transients and common mode voltage generated by a PWM inverter cause unwanted shaft voltage and resultant bearing currents. Parasitic capacitive coupling creates a path to discharge current in rotors and bearings. In order to analyze bearing current discharges and their effect on bearing damage under different conditions, calculation of the capacitive coupling between the outer and inner races is needed. During motor operation, the distances between the balls and races may change the capacitance values. Due to changing of the thickness and spatial distribution of the lubricating grease, this capacitance does not have a constant value and is known to change with speed and load. Thus, the resultant electric field between the races and balls varies with motor speed. The lubricating grease in the ball bearing cannot withstand high voltages and a short circuit through the lubricated grease can occur. At low speeds, because of gravity, balls and shaft voltage may shift down and the system (ball positions and shaft) will be asymmetric. In this study, two different asymmetric cases (asymmetric ball position, asymmetric shaft position) are analyzed and the results are compared with the symmetric case. The objective of this paper is to calculate the capacitive coupling and electric fields between the outer and inner races and the balls at different motor speeds in symmetrical and asymmetrical shaft and balls positions. The analysis is carried out using finite element simulations to determine the conditions which will increase the probability of high rates of bearing failure due to current discharges through the balls and races.

Colonization and osteogenic differentiation of different stem cell sources on electrospun nanofiber meshes

Numerous challenges remain in the successful clinical translation of cell-based therapies for musculoskeletal tissue repair, including the identification of an appropriate cell source and a viable cell delivery system. The aim of this study was to investigate the attachment, colonization, and osteogenic differentiation of two stem cell types, human mesenchymal stem cells (hMSCs) and human amniotic fluid stem (hAFS) cells, on electrospun nanofiber meshes. We demonstrate that nanofiber meshes are able to support these cell functions robustly, with both cell types demonstrating strong osteogenic potential. Differences in the kinetics of osteogenic differentiation were observed between hMSCs and hAFS cells, with the hAFS cells displaying a delayed alkaline phosphatase peak, but elevated mineral deposition, compared to hMSCs. We also compared the cell behavior on nanofiber meshes to that on tissue culture plastic, and observed that there is delayed initial attachment and proliferation on meshes, but enhanced mineralization at a later time point. Finally, cell-seeded nanofiber meshes were found to be effective in colonizing three-dimensional scaffolds in an in vitro system. This study provides support for the use of the nanofiber mesh as a model surface for cell culture in vitro, and a cell delivery vehicle for the repair of bone defects in vivo.

Differentially Expressed Protein Profile of Human Dental Pulp Cells in the Early Process of Odontoblast-like Differentiation In Vitro

Dental pulp cells (DPCs) are capable of differentiating into odontoblasts that secrete reparative dentin after pulp injury. The molecular mechanisms governing reparative dentinogenesis are yet to be fully understood. Here we investigated the differential protein profile of human DPCs undergoing odontogenic induction for 7 days. Using two-dimensional differential gel electrophoresis coupled with matrix-assisted laser adsorption ionization time of flight mass spectrometry, 2 3 protein spots related to the early odontogenic differentiation were identified. These proteins included cytoskeleton proteins, nuclear proteins, cell membrane-bound molecules, proteins involved in matrix synthesis, and metabolic enzymes. The expression of four identified proteins, which were heteronuclear ribonuclear proteins C, annexin VI, collagen type VI, and matrilin-2, was confirmed by Western blot and real-time realtime polymerase chain reaction analyses. This study generated a proteome reference map during odontoblast- like differentiation of human DPCs, which will be valuable to better understand the underlying molecular mechanisms in odontoblast-like differentiation.

Dwell-time and run-time control for DC mass rapid transit railways

Dwell times at stations and inter-station run times are the two major operational parameters to maintain train schedule in railway service. Current practices on dwell-time and run-time control are that they are only optimal with respect to certain nominal traffic conditions, but not necessarily the current service demand. The advantages of dwell-time and run-time control on trains are therefore not fully considered. The application of a dynamic programming approach, with the aid of an event-based model, to devise an optimal set of dwell times and run times for trains under given operational constraints over a regional level is presented. Since train operation is interactive and of multi-attributes, dwell-time and run-time coordination among trains is a multi-dimensional problem. The computational demand on devising trains' instructions, a prime concern in real-time applications, is excessively high. To properly reduce the computational demand in the provision of appropriate dwell times and run times for trains, a DC railway line is divided into a number of regions and each region is controlled by a dwell- time and run-time controller. The performance and feasibility of the controller in formulating the dwell-time and run-time solutions for real-time applications are demonstrated through simulations.