Phenotypic study of human gingival fibroblasts labeled with superparamagnetic anionic nanoparticles.
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-beta (TGF-beta1), tumor necrosis factor-alpha (TNF-alpha), and interleukins 1beta and 4 (IL-1beta 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-beta1, TNF-alpha, IL-1beta, 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<0.001) with RT-PCR confirmation at 12 hours, whereas TIMP-1 did not. IL-1beta increased at day 1 (D1) (P<0.001) and IL-4 at D3 (P<0.01), but not TGF-beta1 or TNF-alpha.


CONCLUSIONS
After labeling with these maghemite nanoparticles, HGF increased secretion of IL-1beta 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.
Naveau
Adrien
A
Laboratory of Arterial Development Research, Paris Descartes Medicine University, National Institute of Health and Medical Research Combined Team 0016, Paris, France. adrien.naveau@laposte.net
Smirnov
Pierre
P
Ménager
Christine
C
Gazeau
Florence
F
Clément
Olivier
O
Lafont
Antoine
A
Gogly
Bruno
B
D000328:Adult; D002455:Cell Division; D049109:Cell Proliferation; D002478:Cells, Cultured; D005260:Female; D052203:Ferrosoferric Oxide; D005347:Fibroblasts / Q000166:cytology*; D005881:Gingiva / Q000166:cytology*; D006801:Humans; D007202:Indicators and Reagents; D007375:Interleukin-1 / Q000235:genetics / Q000378:metabolism; D015847:Interleukin-4 / Q000235:genetics / Q000378:metabolism; D008297:Male; D020781:Matrix Metalloproteinase 1 / Q000235:genetics / Q000378:metabolism; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism; D019278:Matrix Metalloproteinase 3 / Q000235:genetics / Q000378:metabolism; D049329:Nanostructures; D010641:Phenotype; D011540:Prussian Blue Reaction; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics; D016212:Transforming Growth Factor beta / Q000235:genetics; D053773:Transforming Growth Factor beta1; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
D007202:Indicators and Reagents; D007375:Interleukin-1; C508960:TGFB1 protein, human; D019715:Tissue Inhibitor of Metalloproteinase-1; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D014409:Tumor Necrosis Factor-alpha; D015847:Interleukin-4; D019278:Matrix Metalloproteinase 3; D020778:Matrix Metalloproteinase 2; D020781:Matrix Metalloproteinase 1; D052203:Ferrosoferric Oxide