A surface-tethered spheroid model for functional evaluation of 3T3-L1 adipocytes.

In order to effectively treat obesity, it must be better understood at the cellular level with respect to metabolic state and environmental stress. However, current two-dimensional (2D) in vitro cell culture methods do not represent the in vivo adipose tissue appropriately due to the absence of complex architecture and cellular signaling. Conversely, 3D in vitro cultures have been reported to have optimal results mimicking the adipose tissue in vivo. The main aim of this study was to examine the efficacy of a novel conjugate of a genetically engineered polymer, elastin-like polypeptide (ELP) and a synthetic polymer, polyethyleneimine (PEI), toward creating a 3D preadipocyte culture system. We then used this 3D culture model to study the preadipocyte differentiation and adipocyte maintenance processes when subjected to various dosages of nutritionally relevant free fatty acids with respect to total DNA and protein content, cell viability, and intracellular triglyceride accumulation. Our results showed that 3T3-L1 preadipocytes cultured on the ELP-PEI surface formed 3D spheroids within 72 h, whereas the cells cultured on unmodified tissue culture polystyrene surfaces remained in monolayer configuration. Significant statistical differences were discovered between the 3D spheroid and 2D monolayer culture with respect to the DNA and protein content, fatty acid consumption, and triglyceride accumulation, indicating differences in cellular response. Results indicated that the 3D culture may be a more sensitive modeling technique for in vitro adipocyte culture and provides a platform for future evaluation of 3D in vitro adipocyte function.

Suspension culture of hepatocyte-derived reporter cells in presence of albumin to form stable three-dimensional spheroids.

Several studies in the past have formed 3-dimensional (3D) spheroids of primary hepatocytes in suspension culture. Unfortunately, primary hepatocytes in a suspension environment tend to lose their differentiated function over time, generally due to damage from fluid shear stress and eventual spheroid settling. We have therefore created a novel suspension culture system, by seeding H35 rat hepatoma cells, a hepatocyte-derived cell line, in a 24-well tissue culture polystyrene (TCPS) plate placed atop an orbital shaker to create 3D spheroids. To provide stability to the formed spheroids, we used a long-chain polymer, bovine serum albumin (BSA), dissolved in the cell culture medium and/or coated on TCPS surfaces placed in suspension configurations. Our results demonstrate that BSA coating of culture surfaces resulted in uniform and well-defined spheroids with little spheroid settling or "flattening" of cell colonies in either static or suspension configurations. In BSA-coated suspension systems, spheroid size scaled with the amount of BSA dissolved in culture medium. In static uncoated cultures, the normalized rat albumin production levels were enhanced by addition of BSA within culture medium. Thus, both addition of BSA to culture medium and application of BSA as a surface coating appear to be meaningful avenues for tailoring spheroid morphology and function. This 24-well plate suspension culture system may be a valuable tool for high throughput investigations of liver cell behavior in a stable, uniform, 3D spheroid state.

Nitinol Release of Nickel under Physiological Conditions: Effects of Surface Oxide, pH, Hydrogen Peroxide, and Sodium Hypochlorite.

Nitinol is a nickel-titanium alloy widely used in medical devices for its unique pseudoelastic and shape-memory properties. However, nitinol can release potentially hazardous amounts of nickel, depending on surface manufacturing yielding different oxide thicknesses and compositions. Furthermore, nitinol medical devices can be implanted throughout the body and exposed to extremes in pH and reactive oxygen species (ROS), but few tools exist for evaluating nickel release under such physiological conditions. Even in cardiovascular applications, where nitinol medical devices are relatively common and the blood environment is well understood, there is a lack of information on how local inflammatory conditions after implantation might affect nickel ion release. For this study, nickel release from nitinol wires of different finishes was measured in pH conditions and at ROS concentrations selected to encompass and exceed literature reports of extracellular pH and ROS. Results showed increased nickel release at levels of pH and ROS reported to be physiological, with decreasing pH and increasing concentrations of hydrogen peroxide and NaOCl/HOCl having the greatest effects. The results support the importance of considering the implantation site when designing studies to predict nickel release from nitinol and underscore the value of understanding the chemical milieu at the device-tissue interface.

Controlled Growth Factor Delivery and Cyclic Stretch Induces a Smooth Muscle Cell-like Phenotype in Adipose-Derived Stem Cells.

Adipose-derived stem cells (ASCs) are an abundant and easily accessible multipotent stem cell source with potential application in smooth muscle regeneration strategies. In 3D collagen hydrogels, we investigated whether sustained release of growth factors (GF) PDGF-AB and TGF-ß1 from GF-loaded microspheres could induce a smooth muscle cell (SMC) phenotype in ASCs, and if the addition of uniaxial cyclic stretch could enhance the differentiation level. This study demonstrated that the combination of cyclic stretch and GF release over time from loaded microspheres potentiated the differentiation of ASCs, as quantified by protein expression of early to late SMC differentiation markers (SMA, TGLN and smooth muscle MHC). The delivery of GFs via microspheres produced large ASCs with a spindle-shaped, elongated SMC-like morphology. Cyclic strain produced the largest, longest, and most spindle-shaped cells regardless of the presence or absence of growth factors or the growth factor delivery method. Protein expression and cell morphology data confirmed that the sustained release of GFs from GF-loaded microspheres can be used to promote the differentiation of ASCs into SMCs and that the addition of uniaxial cyclic stretch significantly enhances the differentiation level, as quantified by intermediate and late SMC markers and a SMC-like elongated cell morphology.

Spheroid Culture System Confers Differentiated Transcriptome Profile and Functional Advantage to 3T3-L1 Adipocytes.

This study highlights functional differences between 2-D monolayer and 3-D spheroid 3T3-L1 adipocyte culture models and explores the underlying genomic mechanisms responsible for the different phenotypes present. The spheroids showed higher triglyceride accumulation than the monolayer culture and further increase with larger spheroid size. Whole transcriptome analysis indicated significant differential expression of genes related to adipogenesis, including adipocytokine signaling, fatty acid metabolism, and PPAR-γ signaling. Spheroids also showed downregulation of matrix metalloproteinases (MMPs), integrin, actin-cytoskeleton associated genes, and Rho/GTPase3 expression relative to 2-D monolayer, indicating suppression of the Rho-ROCK pathway and thereby promoting adipogenic differentiation. When exposed to linoleic acid (500 µM) and TNF-α (125 ng/mL) to promote chronic adiposity, linoleic acid treatment resulted in increased intracellular triglycerides and subsequent TNF-α treatment resulted in significantly altered adipocytokine signaling, fatty acid metabolism, and PPAR signaling, in addition to upregulation of multiple MMPs in spheroids vs. monolayer. Overall, 3-D spheroids showed enhanced adipogenic phenotype as indicated by triglyceride synthesis and transcriptome changes while retaining sensitivity to a pro-inflammatory stimulus. The 3-D spheroid culture thus may provide a simple, convenient, and sensitive in vitro model to study adipocyte response to metabolic stresses relevant to clinical pathologies.

Harnessing macrophage-mediated degradation of gelatin microspheres for spatiotemporal control of BMP2 release.

Biomaterials-based approaches to harnessing the immune and inflammatory responses to potentiate wound healing hold important promise. Bone fracture healing is characterized by an acute inflammatory phase, followed by a transition to a regenerative and repair phase. In this study, we developed genipin-crosslinked gelatin microspheres designed to be preferentially degraded by inflammatory (M1) macrophages. Highly crosslinked (>90%) microspheres allowed efficient incorporation of bioactive bone morphogenetic protein 2 (BMP2), a potent stimulator of osteogenesis in progenitor cells, via electrostatic interactions. Release of BMP2 was directly correlated with degradation of the gelatin matrix. Exposure of microspheres to polarized murine macrophages showed that degradation was significantly higher in the presence of M1 macrophages, relative to alternatively activated (M2) macrophages and unpolarized controls. Microsphere degradation in the presence of non-inflammatory cells resulted in very low degradation rates. The expression of matrix metalloproteinases (MMPs) and tissue inhibitors of MMP (TIMPs) by macrophages were consistent with the observed phenotype-dependent degradation rates. Indirect co-culture of BMP2-loaded microspheres and macrophages with isolated adipose-derived mesenchymal stem cells (MSC) showed that M1 macrophages produced the strongest osteogenic response, comparable to direct supplementation of the culture medium with BMP2. Controlled release systems that are synchronized with the inflammatory response have the potential to provide better spatiotemporal control of growth factor delivery and therefore may improve the outcomes of recalcitrant wounds.

Growth factor sequestration and enzyme-mediated release from genipin-crosslinked gelatin microspheres.

Controlled release of growth factors allows the efficient, localized, and temporally-optimized delivery of bioactive molecules to potentiate natural physiological processes. This concept has been applied to treatments for pathological states, including chronic degeneration, wound healing, and tissue regeneration. Peptide microspheres are particularly suited for this application because of their low cost, ease of manufacture, and interaction with natural remodeling processes active during healing. The present study characterizes gelatin microspheres for the entrapment and delivery of growth factors, with a focus on tailored protein affinity, loading capacity, and degradation-mediated release. Genipin crosslinking in PBS and CHES buffers produced average microsphere sizes ranging from 15 to 30 microns with population distributions ranging from about 15 to 60 microns. Microsphere formulations were chosen based on properties important for controlled transient and spatial delivery, including size, consistency, and stability. The microsphere charge affinity was found to be dependent on gelatin type, with type A (GelA) carriers consistently having a lower negative charge than equivalent type B (GelB) carriers. A higher degree of crosslinking, representative of primary amine consumption, resulted in a greater negative net charge. Gelatin type was found to be the strongest determinant of degradation, with GelA carriers degrading at higher rates versus similarly crosslinked GelB carriers. Growth factor release was shown to depend upon microsphere degradation by proteolytic enzymes, while microspheres in inert buffers showed long-term retention of growth factors. These studies illuminate fabrication and processing parameters that can be used to control spatial and temporal release of growth factors from gelatin-based microspheres.

Adipogenic Differentiation of Human Adipose-Derived Stem Cells Grown as Spheroids.

Understanding the process of adipogenesis is critical if suitable therapeutics for obesity and related metabolic diseases are to be found. The current study presents proof of feasibility of creating a 3-D spheroid model using human adipose-derived stem cells (hASCs) and their subsequent adipogenic differentiation. hASC spheroids were formed atop an elastin-like polypeptide-polyethyleneimine (ELP-PEI) surface and differentiated using an adipogenic cocktail. Spheroids were matured in the presence of dietary fatty acids (linoleic or oleic acid) and evaluated based on functional markers including intracellular protein, CD36 expression, triglyceride accumulation, and PPAR-γ gene expression. Spheroid size was found to increase as the hASCs matured in the adipocyte maintenance medium, though the fatty acid treatment generally resulted in smaller spheroids compared to control. A stable protein content over the 10-day maturation period indicated contact-inhibited proliferation as well as minimal loss of spheroids during culture. Spheroids treated with fatty acids showed greater amounts of intracellular triglyceride content and greater expression of the key adipogenic gene, PPAR-γ. We also demonstrated that 3-D spheroids outperformed 2-D monolayer cultures in adipogenesis. We then compared the adipogenesis of hASC spheroids to that in 3T3-L1 spheroids and found that the triglyceride accumulation was less profound in hASC spheroids than that in 3T3-L1 adipocytes, correlated with smaller average spheroids, suggesting a relatively slower differentiation process. Taken together, we have shown the feasibility of adipogenic differentiation of patient-derived hASC spheroids, which with further development, may help elucidate key features in the adipogenesis process.

Three-dimensional spheroid cell model of in vitro adipocyte inflammation.

To improve treatment of obesity, a contributing factor to multiple systemic and metabolic diseases, a better understanding of metabolic state and environmental stress at the cellular level is essential. This work presents development of a three-dimensional (3D) in vitro model of adipose tissue displaying induced lipid accumulation as a function of fatty acid supplementation that, subsequently, investigates cellular responses to a pro-inflammatory stimulus, thereby recapitulating key stages of obesity progression. Three-dimensional spheroid organization of adipose cells was induced by culturing 3T3-L1 mouse preadipocytes on an elastin-like polypeptide-polyethyleneimine (ELP-PEI)-coated surface. Results indicate a more differentiated phenotype in 3D spheroid cultures relative to two-dimensional (2D) monolayer analogues based on triglyceride accumulation, CD36 and CD40 protein expression, and peroxisome proliferator-activated receptor-γ (PPAR-γ) and adiponectin mRNA expression. The 3T3-L1 adipocyte spheroid model was then used to test the effects of a pro-inflammatory microenvironment, namely maturation in the presence of elevated fatty acid levels followed by acute exposure to tumor necrosis factor alpha (TNF-α). Under these conditions, we demonstrate that metabolic function was reduced across all cultures exposed to TNF-α, especially so when pre-exposed to linoleic acid. Further, in response to TNF-α, enhanced lipolysis, monitored as increased extracellular glycerol and fatty acids levels, was observed in adipocytes cultured in the presence of exogenous fatty acids. Taken together, our 3D spheroid model showed enhanced adipogenic differentiation and presents a platform for elucidating the key phenotypic responses that occur in pro-inflammatory microenvironments that characterize obesogenic states.

Spheroid organization kinetics of H35 rat hepatoma model cell system on elastin-like polypeptide-polyethyleneimine copolymer substrates.

Though two-dimensional systems have yielded some success in deriving morphological and functional markers of hepatocyte culture, they largely fail to capture the three-dimensional organization, long-term viability, and functionality of the hepatic tissue. We have engineered a system for inducing self-assembly of model H35 rat hepatoma spheroids using a copolymer comprised of biocompatible elastin-like polypeptide (ELP) chemically conjugated to positively charged polyethyleneimine (PEI). We have achieved a conjugation ratio of 30 mol %, though our studies analyzing spheroid organization kinetics indicate conjugate ratios of 5 mol % and greater to be optimal for cell culture based on least variability in spheroid sizes and minimum incidence of overgrown aggregates. Furthermore, our ELP-PEI system indicated the potential for influencing ultimate spheroid dimensions, with spheroid size inversely related to polyelectrolyte conjugation. Overall, this study provides a good starting point to investigate functional correlations between spheroid size and functional markers and their future use as an in vitro diagnostic or tissue engineering tool.

Multiple views reveal the complexity of dementia diagnosis.

OBJECTIVE: To reveal views about dementia diagnosis derived from a larger study of information needs of carers of people with dementia in Tasmania, Australia. METHODS: Over 100 participants, including family carers, health professionals and dementia service personnel, met as discrete focus groups. Data pertinent to dementia diagnosis were segregated and subjected to across-group comparative analysis. RESULTS: The term dementia held connotations of stigma and futility, despite stated benefits of having a diagnosis. General practitioners were regarded as pivotal but having inadequate diagnostic and treatment options. While most health professionals advocated a longitudinal diagnostic process, this created considerable stress for family carers who sought a speedy process. Without a diagnosis, some dementia-specific services were undeliverable. CONCLUSION: Dementia diagnosis is steeped in deep-rooted difficulties and stressful implications, compounded by carers' differing needs and interests. Better understanding between care providers of their conflicting and consistent views could contribute to better dementia care.