Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) are the two most popular surfactants among perfluorinated compounds (PFCs), with a wide range of uses. Growing evidence suggests that PFCs have the potential to interfere with estrogen homeostasis, posing a risk of endocrine-disrupting effects. This in vitro study aimed to investigate the estrogenic effect of these compounds on T47D hormone-dependent breast cancer cells. PFOS and PFOA (10(-12) to 10(-4) M) were not able to induce estrogen response element (ERE) activation in the ERE luciferase reporter assay. The ERE activation was induced when the cells were co-incubated with PFOS (10(-10) to 10(-7) M) or PFOA (10(-9) to 10(-7) M) and 1 nM of 17β-estradiol (E2). PFOS and PFOA did not modulate the expression of estrogen-responsive genes, including progesterone (PR) and trefoil factor (pS2), but these compounds enhanced the effect of E2-induced pS2 gene expression. Neither PFOS nor PFOA affected T47D cell viability at any of the tested concentrations. In contrast, co-exposure with PFOS or PFOA and E2 resulted in an increase of E2-induced cell viability, but no effect was found with 10 ng ml(-1) EGF co-exposure. Both compounds also intensified E2-dependent growth in the proliferation assay. ERK1/2 phosphorylation was increased by co-exposure with PFOS or PFOA and E2, but not with EGF. Collectively, this study shows that PFOS and PFOA did not possess estrogenic activity, but they enhanced the effects of E2 on estrogen-responsive gene expression, ERK1/2 activation and the growth of the hormone-deprived T47D cells. Copyright © 2015 John Wiley & Sons, Ltd.
The dedifferentiation of articular chondrocytes during in vitro expansion deteriorates the hyaline cartilage regeneration. Many approaches have been developed to enhance the redifferentiation of chondrocytes. In this study, a new and effective protocol to improve the redifferentiation of porcine chondrocytes in a pellet form was established. Pellets were initially treated in the modified culture media containing ternary mixtures, binary mixtures, or single reagents of sodium citrate (SCi), sodium chloride (SCh), and ethylenediaminetetraacetic acid (EDTA) at varied concentrations during the first 3 days of culture, followed by a normal culture medium until 21 days.Viability, proliferation, cartilaginous gene expression, extracellular matrix formation, and morphology of treated cell pellets were comparatively examined. Chondrocytes exposed to SCi, SCh, and EDTA individually or in combinations of two or three chemicals were non-cytotoxic when the concentration ranges of the chemicals were 1.83-2.75, 5.00-7.50, and 1.00-1.50 mM, respectively. Cells treated with the modified media containing EDTA alone and EDTA-containing mixtures enhanced glycosaminoglycan production as well as upregulated cartilaginous gene expression, despite their low proliferation rates. Overall, when all three reagents were in use, a pronounced synergistic effect on the activations of glycosaminoglycan accumulation and type II collagen production was explicitly observed at most, particularly when cells were cultured in the medium containing SCi, SCh, and EDTA at concentrations of 2.20, 6.00, and 1.20 mM, respectively. With a use of this protocol, the redifferentiation of articular chondrocytes for regeneration of hyaline cartilage for tissue engineering applications could be readily achieved.
Bone morphogenic protein-2 (BMP-2) knuckle epitope peptide has been recently discovered and known to activate chondrogenesis. However, the applications of this soluble peptide remain very limited due to rapid diffusion resulting in poor cellular uptake into target cells. We herein designed nanoparticles made from hyaluronic acid functionalized gold nanorods (GNRs) to conjugate with thiolated BMP-2 knuckle epitope peptide via a two-step reaction. Hyaluronic acid was modified to have thiol functional groups to replace the cetyl trimethylammonium bromide ligands on the surface of GNRs. The thiolated peptides were subsequently reacted with hyaluronic acid on the surface on GNRs via a maleimide-hydrazide crosslinker. The conjugation was confirmed by the change of surface charge of GNRs and the plasmon shift. A colorimetric peptide assay suggested more than 69% of the thiolated peptides were conjugated with the hyaluronic acid coated gold nanorods. Moreover, in vitro cell viability showed that BMP-2 conjugated hyaluronic acid functionalized gold nanorods (B2HGR) were cytocompatible and did not cause cytotoxicity to fibroblast cells. The B2HGRs also significantly promote cellular uptake of the BMP-2 peptides in both human mesenchymal stem cells and porcine chondrocytes due to multivalent ligand binding to the BMP receptors on the cell surface resulting in receptor-mediated endocytosis. The enhanced cellular uptake was clearly observed under a confocal microscope resulting in the significant activation of type II collagen gene expression and glucosaminoglycan secretion in those cells. Furthermore, our delivery system is a proof-of-concept of using scaffolds in combination with nanodelivery platform to enhance cartilaginous repair. The peptide loading capacity and the release is not limited by the scaffolds. Therefore, our delivery platform has potential applications for cartilage regeneration in a preclinical and clinical setting in the future.
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