Phenotypic Study of Human Gingival Fibroblasts Labeled With Superparamagnetic Anionic Nanoparticles

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Abstract
Background: A specific labeler of the human gingival fibroblast (HGF) does not exist. Anionic maghemite nanoparticles allow labeling of a wide cell variety and their recognition in cellular, organotypical, and animal models. Methods: We studied internalization effects of nanoparticles on an HGF phenotype in vitro, evaluating transcription and secretion of connective tissue remodeling molecules, i.e., matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), and cytokines controlling their activation/inhibition, i.e., transforming growth factor-β (TGF-β1), tumor necrosis factor-α (TNF-α), and interleukins 1β and 4 (IL-1β and IL-4). After proliferation kinetics, cellular uptake was studied by Perls coloration and magnetophoresis on labeled culture. Dot blotting, Western blotting, and zymography were used to detect MMP-1, -2, and -3 and TIMP-1 and -2 secretions in culture supernatants, and reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect the mRNA expression of these molecules. Enzyme-linked immunosorbent assay (ELISA) tests were used to determine TGF-β1, TNF-α, IL-1β, and IL-4 levels. Results: Our data indicated high (15.3 ± 5.8 pg/cell) but heterogeneous distribution of nanoparticles in HGF. Twenty-four hours after labeling, MMP-1, -2, and -3 and TIMP-2 secretion increased (P P P <0.01), but not TGF-β1 or TNF-α. Conclusions: After labeling with these maghemite nanoparticles, HGF increased secretion of IL-1β at D1, probably inducing the increase of MMP-1, -2, and -3 and TIMP-2. The increase of IL-4 secretion began with the decreased synthesis of MMPs and TIMPs at D3. Despite this transitory inflammatory reaction at 3 days following internalization, maghemite nanoparticles did not affect HGF phenotype, thereby authorizing their use as labelers.

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