Paola Locci

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.

Effects of hydroxyapatite and biostite® on osteogenic induction of hMSC

When isolated from the iliac crest human mesenchymal stem cells (hMSC) differentiate into osteoblast-like cells with appropriate stimulation in culture. This in vitro study tested the hypothesis that Biostite® and hydroxyapatite (HA) affect proliferation and differentiation of hMSC into osteoblastic cells. Cell proliferation was determined by measuring 3H-thymidine incorporation into DNA and typical markers of osteoblastic phenotype were determined by RT-PCR assay. No differences emerged in cell proliferation cultures with Biostite® or hydroxyapatite (HA), but gene expression analysis revealed higher expression of collagen, alkaline phosphatase (ALP), osteopontin and bone sialoprotein (BSP) in the presence of Biostite®. TGFβ2 production, as assessed by an Elisa kit, and Runx2 expression by RT-PCR, were greater in Biostite cultures, suggesting Biostite® provides a better environment for hMSC differentiation into osteoblasts and is, potentially, a more promising bone-filling material than HA.

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.

Phenotype expression of human bone cells cultured on implant substrates

Bone cells derived from the human jaw were cultured on titanium, titanium coated with hydroxyapatite (THA) or with plasma spray (TPS) to study the behaviour of the cells anchored to implant substrates. Bone cells were cultured in MEM with the addition of [3H]‐thymidine to evaluate cellular proliferation, and [3H]‐glucosamine to evaluate GAG synthesis and accumulation in the extra‐cellular matrix (ECM). Moreover, to study the degradation of GAG bone cells were cultured in the presence of NH4Cl, an amine known to inhibit lysosomal activity. Our results show that TPS is the substrate that favours both cellular proliferation and the accumulation of GAG in the ECM.

Epithelial-mesenchymal interactions and lung branching morphogenesis. Role of polyamines and transforming growth factor ß1

Lung branching morphogenesis is a result of epithelial-mesenchymal interactions, which are in turn dependent on extracellular matrix composition and cytokine regulation. Polyamines have recently been demonstrated as able to modify chick embryo skin differentiation. In this work we have examined the effects of putrescine and spermidine during chick embryo lung morphogenesis in organotypic cultures by morphological, histochemical and biochemical examination. To verify the role of polyamines, we used specific inhibitors, such as bis-cyclohexylammonium sulphate and alfa-difluoromethylornithine, and transforming growth factor beta1, an ornithine decarboxylase and polyamine stimulator. Our data show that lung morphogenesis is significantly altered following the induced mesenchymal glycosaminoglycan changes. The increase of mesenchymal glycosaminoglycans is correlated with a stimulation of lung development in the presence of polyamines, and with its inhibition when transforming growth factor beta1 is added to the culture medium. The morphometric data show a uniform increase of both the mesenchyme and epithelial branching with spermidine and putrescine stimulus, whereas the mesenchymal substance alone is significantly increased in apical-median lung sections with transforming growth factor beta1 and transforming growth factor beta1 + spermidine lung cultures. Transforming growth factor beta1 and transforming growth factor beta1 + spermidine confirm the blocking of epithelial branching formations and fibroblast activation, and show that polyamines are unable to prevent the blocking of epithelial cells due to the inhibitory effect of transforming growth factor beta1.

Effects of transforming growth factor-β1 and tumour necrosis factor-α on cultured fibroblasts from skin fibroma as modulated by toremifene

To determine how toremifene, an anti‐oestrogen triphenylethylene derivate, reduces tumour mass, we investigated its modulation of TGF‐β1 and TNF‐α in fibroma fibroblasts. Normal and fibroma fibroblasts, isolated from patients affected by Gardners syndrome without or with fibroma manifestation, were cultured in vitro. Secretion of GAG, collagen and TGF‐β1 was increased in fibroma fibroblasts compared to healthy cells. The increase in TGF‐β1 secretion into the medium was associated with a parallel increase in TGF‐β1 gene expression and receptor number. Receptor cross‐linking studies using radiolabelled TGF‐β1 revealed more receptors, particularly types I and II, in fibroma fibroblasts than in normal cells. Normal and fibroma fibroblasts did not synthesise TNF‐α, but they had TNF‐α membrane receptors, as shown by TNF‐α assay. TNF‐α secreted by human monocytes, which may be present in the peritumoral area, increased cell proliferation and GAG accumulation and was, in turn, enhanced by TGF‐β1 treatment. Both growth factors increased angiogenesis, as shown by the CAM assay. Toremifene reduced TGF‐β1 secretion by fibroma fibroblasts and TNF‐α secretion by monocytes, thus downregulating cell proliferation, ECM macromolecule accumulation and angiogenic progression. We hypothesise that increased TGF‐β1 gene expression and TGF‐β1 secretion in fibroma fibroblasts as well as the subsequent rise in TNF‐α production by monocytes may facilitate fibroma growth and that toremifene inhibits autocrine and paracrine growth factor production.