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-? (PPAR-?) 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.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140235/1/ten.tea.2014.0531.pd

Milieu for Endothelial Differentiation of Human Adipose-Derived Stem Cells

Human adipose-derived stem cells (hASCs) have been shown to differentiate down many lineages including endothelial lineage. We hypothesized that hASCs would more efficiently differentiate toward the endothelial lineage when formed as three-dimensional (3D) spheroids and with the addition of vascular endothelial growth factor (VEGF). Three conditions were tested: uncoated tissue culture polystyrene (TCPS) surfaces that induced a 2D monolayer formation; elastin-like polypeptide (ELP)-collagen composite hydrogel scaffolds that induced encapsulated 3D spheroid culture; and ELP-polyethyleneimine-coated TCPS surfaces that induced 3D spheroid formation in scaffold-free condition. Cells were exposed to endothelial differentiation medium containing no additional VEGF or 20 and 50 ng/mL of VEGF for 7 days and assayed for viability and endothelial differentiation markers. While endothelial differentiation media supported endothelial differentiation of hASCs, our 3D spheroid cultures augmented this differentiation and produced more von Willebrand factor than 2D cultures. Likewise, 3D cultures were able to uptake LDL, whereas the 2D cultures were not. Higher concentrations of VEGF further enhanced differentiation. Establishing angiogenesis is a key factor in regenerative medicine. Future studies aim to elucidate how to produce physiological changes such as neoangiogenesis and sprouting of vessels which may enhance the survival of regenerated tissues

Elastin-Collagen Based Hydrogels as Model Scaffolds to Induce Three-Dimensional Adipocyte Culture from Adipose Derived Stem Cells

This study aimed to probe the effect of formulation of scaffolds prepared using collagen and elastin-like polypeptide (ELP) and their resulting physico-chemical and mechanical properties on the adipogenic differentiation of human adipose derived stem cells (hASCs). Six different ELP-collagen scaffolds were prepared by varying the collagen concentration (2 and 6 mg/mL), ELP addition (6 mg/mL), or crosslinking of the scaffolds. FTIR spectroscopy indicated secondary bonding interactions between collagen and ELP, while scanning electron microscopy revealed a porous structure for all scaffolds. Increased collagen concentration, ELP addition, and presence of crosslinking decreased swelling ratio and increased elastic modulus and compressive strength of the scaffolds. The scaffold characteristics influenced cell morphology, wherein the hASCs seeded in the softer, non-crosslinked scaffolds displayed a spread morphology. We determined that stiffer and/or crosslinked elastin-collagen based scaffolds constricted the spreading of hASCs, leading to a spheroid morphology and yielded an enhanced adipogenic differentiation as indicated by Oil Red O staining. Overall, this study underscored the importance of spheroid morphology in adipogenic differentiation, which will allow researchers to create more physiologically-relevant three-dimensional, in vitro culture models

Effect of Processing Temperature on the Morphology and Drug-Release Characteristics of Elastin-Like Polypeptide–Collagen Composite Coatings

Elastin-like polypeptides (ELPs) exhibit an inverse phase transition temperature (<i>T</i>_t) in response to changes in their environment. We hypothesized that processing ELP–collagen composites at temperatures higher than the T_t of ELP (∼32 °C) will affect their microstructure and subsequently, achieve tunable release of model drugs. The composite coatings were prepared by formation of ELP–collagen hydrogels at 37 °C, incubation at 37, 45, or 55 °C, and finally air-drying at 37 °C. Scanning electron micrographs revealed that the fabrication process affected both the collagen and ELP microaggregate phases. A gradual time dependent bovine serum albumin (BSA) release that followed the power law and a burst antibiotic doxycycline release followed by a linear zero-order release were observed. Importantly, BSA and doxycycline releases were dependent on the ELP microaggregate size, which was governed by the processing temperatures. This study lays the foundation to achieve optimized composite microstructures by controlling processing conditions for drug delivery applications

Photocatalytic activity and antibacterial efficacy of UVA-treated titanium oxides

Studies have shown ultraviolet-A (UVA) irradiation of crystalline titanium oxides leads to the production of reactive oxygen species (ROS) via a photocatalytic process. The ROS exhibit antimicrobial properties that may be of benefit in preventing bacterial attachment to implant devices. Recent studies have suggested a potential benefit of mixed anatase and rutile oxides and dopants on the photocatalytic properties of titanium oxides. The goal of this work was to compare the photocatalytic activity of different anodized commercially pure titanium grade 4 (CPTi4) surfaces. CPTi4 specimens were anodized in three mixed-acid electrolytes to create crystalline oxide surfaces that were either primarily anatase, primarily rutile, or a combination of anatase and rutile. Additionally, the primarily anatase and combination oxides incorporated some phosphorous from the phosphoric acid component in the electrolyte. The photocatalytic activity of the anodized specimens was measured using both methylene blue (MB) degradation assay and comparing the attachment of S. aureus under irradiation with UVA light of differing intensities (1 mW/cm2, 8 mW/cm2, and 23 mW/cm2). Primarily rutile oxides exhibited significantly higher levels of MB degradation after exposure to 1 mW/cm2 UVA lights. Primarily rutile specimens also had the largest reduction in bacterial attachment followed by the mixed phase specimens and the primarily anatase specimens at 1 mW/cm2 UVA lights. Phosphorous-doped, mixed phase oxides exhibited an accelerated MB degradation response during exposure to 8 mW/cm2 and 23 mW/cm2 UVA lights. All anodized and unanodized CPTi4 groups revealed similar S. aureus attachment at the two higher UVA intensities. Although MB degradation assay and the bacteria attachment assay both confirmed photocatalytic activity of the oxides formed in this study, the results of the MB degradation assay did not accurately predict the oxides performance against S. aureus

Influence of the Tissue Collection Procedure on the Adipogenic Differentiation of Human Stem Cells: Ischemic versus Well-Vascularized Adipose Tissue

Clinical and basic science applications using adipose-derived stem cells (ADSCs) are gaining popularity. The current adipose tissue harvesting procedures introduce nonphysiological conditions, which may affect the overall performance of the isolated ADSCs. In this study, we elucidate the differences between ADSCs isolated from adipose tissues harvested within the first 5 min of the initial surgical incision (well-vascularized, nonpremedicated condition) versus those isolated from adipose tissues subjected to medications and deprived of blood supply during elective free flap procedures (ischemic condition). ADSCs isolated from well-vascularized and ischemic tissues positively immunostained for several standard stem cell markers. Interestingly, the percent change in the CD36 expression for ADSCs isolated from ischemic versus well-vascularized tissue was significantly lower in males than females (p p < 0.05). These results indicate that ADSCs isolated from ischemic tissue either fail to uptake fatty acids or fail to efficiently convert those fatty acids into triglycerides. Therefore, more robust ADSCs suitable to establish in vitro adipose tissue models can be obtained by harvesting well-vascularized and nonpremedicated adipose tissues