Cinzia Lilli

Ion release from orthodontic appliances

OBJECTIVE The microbiological and enzymatic characteristics of the oral cavity would seem to provide a suitable environment for the corrosion of metals. We assayed the release of metal ions from one orthodontic appliance which included two 304 and 316 steel molar bands, ten 316 steel brackets, one nickel-titanium archwire and a brazing alloy to connect the elements of molar bands and brackets. METHODS The orthodontic appliance was dipped in both inorganic (pH 3.5-6.5) and organic acid solutions (w/v 1% each of tartaric, citric and ascorbic acid at pH 2.2 or 1.5% each of lactic and acetic acid at pH 2.5). The release of nickel (Ni), chromium (Cr), copper (Cu), silver (Ag) and palladium (Pd) was determined using an atomic absorption spectrophotometer Varian AA 10. RESULTS The release of Ni, Cr and Cu was markedly less at pH 6.5 than at pH 3.5 at all time points in acid solution. Daily release/single appliance after the first day decreased. Contrary to expectations, appliances immersed in organic acid solutions at pH 2.2 or 2.5 after 28 days generally released an amount of ions similar to that observed in inorganic acid solution at pH 3.5, with the exception of Cu. Release of silver and palladium, two metals present in the brazing alloy, proved to be very low (approximately 0.2 microgram after 28 days). CONCLUSIONS The daily release of Ni, Cu and Cr by an orthodontic appliance in acid pH, particularly favourable to corrosion, was well below that ingested with a normal daily diet. It is therefore concluded that the quantities of metal ions released in our experimental conditions should not be cause for concern in utilising the appliance.

Transforming growth factor ß1 acid interaction

Chick embryo skin fibroblasts release transforming growth factor β1 that is able to modulate glycosaminoglycan synthesis and secretion. When incubated with individual classes of glycosaminoglycans, the factors modulatory activity was altered. To determine whether direct interactions between transforming growth factor β1 and glycosaminoglycans occur, we have assessed the activity of the growth factor after pre-incubation with single classes of glycosaminoglycans by assaying its inhibitory effect upon the proliferative response of thymocytes stimulated with interleukin-1. Untreated transforming growth factor β1 suppressed the proliferative response of thymocytes to interleukin-1, as did transforming growth factor β1 pre-incubated with sulphated glycosaminoglycans. By contrast, transforming growth factor β1 lost its inhibitory capacity when preincubated with high molecular weight hyaluronic acid. Digestion of transforming growth factor β1-hyaluronic acid complex with hyaluronidase released active transforming growth factor β1. Trypsin degraded transforming growth factor β1 alone, but did not degrade the transforming growth factor β1-hyaluronic acid complex. These results suggest that hyaluronic acid interacts with transforming growth factor β1, thus protecting the factor from tryptic degradation and may be a means of concentrating growth factor activity.

Biocompatibility of alloys used in orthodontics evaluated by cell culture tests.

The cytotoxicity of the most common alloys used in orthodontic appliances was determined by cell culture testing. Human gingival fibroblasts were cultured on 304 and 316 stainless steel, on brazing alloy composed of palladium (Pd), copper (Cu), and silver (Ag), and on plastic substrate (control). Studies were carried out with SEM and radiolabeled precursor incorporation. Cells were cultured in MEM without serum but with the addition of (3)H-thymidine to evaluate cell proliferation and (3)H-glucosamine to evaluate glycosaminoglycan (GAG) synthesis and secretion in the culture medium. Moreover, gingival fibroblasts were cultured in the presence of some metal ions generally released by orthodontic appliances to evaluate the cytotoxic effects of single ions. Morphologic observations with SEM and radiolabeled incorporation studies showed that 304 and 316 stainless steel were more biocompatible than the brazing alloy. Among the metal ions tested, Ag and Pd, constituents of the brazing alloy, showed the highest cytotoxicity.

P253R fibroblast growth factor receptor‐2 mutation induces RUNX2 transcript variants and calvarial osteoblast differentiation

Unregulated fibroblast growth factor 2 (FGF2) signaling caused by mutations in the fibroblast growth factor receptor (FGFR2) leads to human craniosynostosis such as the Apert syndrome. In an in vitro control model of calvarial osteoblasts from Apert patients carrying the FGFR2 P253R mutation, we studied the changes in cellular phenotype and evaluated the effects of FGF2. Compared with wild‐type controls, osteocalcin mRNA was down‐regulated in Apert osteoblasts, Runt‐related transcription factor‐2 (RUNX2) mRNA was differentially spliced, and FGF2 secretion was greater. Total protein synthesis, fibronectin and type I collagen secretion were up‐regulated, while protease and glycosidase activities and matrix metalloproteinase‐13 (MMP‐13) transcription were decreased, suggesting an altered ECM turnover. Adding FGF2 increased protease and glycosidase activities and down‐regulated fibronectin and type I collagen secretion in Apert osteoblasts. High affinity FGF2 receptors were up‐regulated in Apert osteoblasts and analysis of signal transduction showed elevated levels of Grb2 tyrosine phosphorylation and the Grb2‐p85 beta association, which FGF2 stimulation strongly reduced. All together these findings suggest increased constitutive receptor activity in Apert mutant osteoblasts and an autocrine loop involving the FGF2 pathway in modulation of Apert osteoblast behavior.

Basic fibroblast growth factor autocrine loop controls human osteosarcoma phenotyping and differentiation.

BackgroundWe focused on the phenotype of non-mineralizing MG63 and mineralizing TE85 human osteosarcoma cells and investigated the role of bFGF in modulating their differentiative responses. Basic FGF expression and bFGF effects on osteocalcin, runt-related transcription factor-2 (RUNX2), matrix molecular production and bFGF receptors, were evaluated.Materials and MethodsOsteocalcin and RUNX2 gene expression were studied by RT-PCR analysis. We evaluated cell proliferation by DNA content and performed differentiation studies on glycosaminoglican (GAG), collagen and proteoglican (PG) synthesis by using radiolabelled precursors and Northern blotting. BFGF receptors were quantified by bFGF receptor binding assay.ResultsOsteocalcin is expressed in MG63 and TE65. RUNX2 RNA is differentially spliced in the two cell lines. BFGF elicits the effects of differentially splicing RUNX2. Proliferation, GAG synthesis, bFGF and proteoglycan mRNA expression, high and low affinity bFGF receptors, were more marked in MG63 and differently affected by bFGF. Procollagen expression and alkaline phosphatase activity were significantly reduced. BFGF increased TE85 cell proliferation and reduced TE85 procollagen and osteocalcin production.ConclusionsThe different splice variants in RUNX2 gene in the two cell lines might be related to their different phenotypes. The less differentiated stage of MG63 could also be related to bFGF over-production and more bFGF receptors. The consequent increase in bFGF-bFGF receptor binding could explain the bFGF differentiative effects on MG63. We suggest an autocrine role of bFGF endogenous release in controlling the different osteosarcoma phenotypes.

In vitro cytotoxic effects of orthodontic appliances

The objective of this study was to evaluate the effects of an orthodontic appliance and of its components (brackets, bands, and arch wires) on some cell functions. Fibroblasts were cultured either in the presence of one unwashed orthodontic appliance, or one orthodontic appliance immersed in MEM for 28 days before use (washed appliance), or in the presence of MEM in which the appliances had been immersed. At the end of in vitro maintenance, morphological studies were carried out with SEM and TEM. Cell proliferation and GAG synthesis and secretion by radio-labeled precursors were assessed. The data indicated that unwashed appliances were more cytotoxic than washed ones. Moreover, the arch wire was the most biocompatible component of the orthodontic appliance, and the bracket was the least biocompatible. A comparative study into the effects on cell proliferation of the most common metal ions released by the appliances was also carried out. At the concentration released by one orthodontic appliance immersed for 28 days, the highest reduction in DNA synthesis was observed in the presence of Cu(++).

Differential in vitro phenotype pattern, transforming growth factor-β1 activity and mRNA expression of transforming growth factor-β1 in Apert osteoblasts

Abstract The phenotype of Apert osteoblasts differs from that of normal osteoblasts in the accumulation of macromolecules in the extracellular matrix. Apert osteoblasts increase type I collagen, fibronectin and glycosaminoglycans secretion compared with normal osteoblasts. Because the extracellular matrix macromolecule accumulation is greatly modulated by transforming growth factor-β1, we examined the ability of normal and Apert osteoblasts to secrete transforming growth factor-β1 by CCL-64 assay and to produce transforming growth factor-β1 by analysis of the mRNA expression of transforming growth factor-β1. Northern blot analysis revealed an increased amount of transforming growth factor-β1 mRNA expression in Apert osteoblasts compared with normal ones. Moreover, the level of the active transforming growth factor-β1 isoform was higher in Apert than in normal media. In pathologic cells, the increase in transforming growth factor-β1 gene expression was associated with a parallel increase in the factor secreted into the medium. The level of transforming growth factor-β1 was decreased by the addition of basic fibroblast growth factor. Transforming growth factor-β1 is controlled temporally and spatially during skeletal tissue development and produces complex stimulatory and inhibitory changes in osteoblast functions. We hypothesise that in vitro differences between normal and Apert osteoblasts may be correlated to different transforming growth factor-β1 cascade patterns, probably due to an altered balance between transforming growth factor-β1 and basic fibroblast growth factor.

Synthesis and Secretion of Transforming Growth Factor-β1 by Human Desmoid Fibroblast Cell Line and Its Modulation by Toremifene

The present study provides evidence that the in vitro cultured fibroblast cell line from desmoid tumors differs from normal fibrobasts in its extracellular matrix (ECM) macromolecule composition and is modulated by treatment with toremifene, an antiestrogen that reduces tumor mass by an unknown mechanism. The results showed increased transforming growth factor-beta 1 (TGF-beta1) production, TGF-beta1 mRNA expression, and TGF-beta1 receptor number in desmoid fibroblasts compared with normal cells. As desmoid fibroblasts did not produce tumor necrosis factor-alpha (TNF-alpha) but were sensitive to it, which enhanced glycosaminoglycans (GAG) accumulation, we assessed the TGF-beta1 effects on TNF-alpha production by human monocytes. Our results showed TGF-beta1 significantly increased TNF-alpha secretion by monocytes. Toremifene mediated its effects in desmoid fibroblasts via an estrogen receptor-independent pathway. It inhibited GAG accumulation and the secretion of both latent and active forms of TGF-beta1 and had an inhibitory effect on TNF-alpha production by monocytes. Our results suggest that in reducing TGF-beta1 production by desmoid fibroblasts and TNF-alpha production by monocytes, toremifene may restore the balance between the two growth factors.

Human cleft lip and palate fibroblasts and normal nicotine-treated fibroblasts show altered in vitro expressions of genes related to molecular signaling pathways and extracellular matrix metabolism†

Nonsyndromic cleft lip with or without cleft palate (CLP) is a frequent craniofacial malformation caused by both genetic and environmental factors. Maternal smoking during pregnancy is a known risk factor, due to the teratogenic role of nicotine. To assess and compare the impact of CLP and nicotine, we studied the quantitative expression of genes involved in signaling pathways and extracellular matrix (ECM) metabolism in human normal nicotine‐treated (NicN) and CLP fibroblasts compared to normal control (CTRL) cells. Palatal fibroblast cultures from seven CLP children and seven age‐matched CTRL subjects were established and subconfluent cells incubated for 24 h without (CTRL and CLP fibroblasts) or with (NicN fibroblasts) 0.6 mM nicotine. Gene expressions were analyzed by real‐time quantitative PCR. For the first time, a regulated cholinergic signaling in our human fibroblasts in vitro was demonstrated. Members of TGF‐beta, retinoic acid (RA), and GABA‐ergic signaling systems were also differently regulated. Among the ECM genes, fibronectin, syndecan, integrin α2, and MMP13 genes were concordantly modulated, while integrin β5, and decorin genes were discordantly modulated. Interestingly, nicotine treatment regulated gene expressions of CD44 and CLPTM1, two candidate genes for CLP. Our findings show a positive association between nicotine treatment and CLP phenotype. Results suggest that nicotine deranges normal palate development, which might contribute to the development of a CLP malformative phenotype, through the impairment of some important signaling systems and ECM composition. J. Cell. Physiol. 222: 748–756, 2010.

Reversal of experimental Laron Syndrome by xenotransplantation of microencapsulated porcine Sertoli cells.

Recombinant human IGF-1 currently represents the only available treatment option for the Laron Syndrome, a rare human disorder caused by defects in the gene encoding growth hormone receptor, resulting in irreversibly retarded growth. Unfortunately, this treatment therapy, poorly impacts longitudinal growth (13% in females and 19% in males), while burdening the patients with severe side effects, including hypoglycemia, in association with the unfair chore of taking multiple daily injections that cause local intense pain. In this study, we have demonstrated that a single intraperitoneal graft of microencapsulated pig Sertoli cells, producing pig insulin-like growth factor-1, successfully promoted significant proportional growth in the Laron mouse, a unique animal model of the human Laron Syndrome. These findings indicate a novel, simply, safe and successful method for the cell therapy-based cure of the Laron Syndrome, potentially applicable to humans.