Lorella Marinucci

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.

Novel poly(L-lactide) PLLA/SWNTs nanocomposites for biomedical applications: material characterization and biocompatibility evaluation.

Poly(L-lactide) (PLLA)/single-walled carbon nanotubes (SWNTs) nanocomposite films were produced using the solvent casting method, and morphological, thermal and mechanical properties were investigated. Biocompatibility was evaluated by using human bone cells, performing adhesion and proliferation studies. The role of single-walled nanotube incorporation and functionalization on PLLA bio-polymers was investigated. Pristine (SWNTs) and carboxylated (SWNTs–COOH) carbon nanotubes were considered in order to control the interaction between PLLA and nanotubes. SWNTs and SWNTs–COOH showed a good dispersion in the polymer matrix and improved the PLLA crystallinity. Thermal, morphological and dynamic-mechanical analyses revealed that carboxylic groups on the tube sidewalls increased compatibility between PLLA and nanostructures. Mechanical properties demonstrated an enhancement related to introduction and functionalization of carbon nanotubes. Biological investigations showed osteoblasts cultured on PLLA/SWNTs–COOH nanocomposites has higher cell adhesion and proliferation than osteoblasts cultured on PLLA and PLLA/SWNTs nanocomposites. These studies suggest that combination of biodegradable polymers and SWNTs opens a new perspective in the self-assembly of nanomaterials and nanodevices for biomedical applications with tunable properties.

Effects of sub-toxic Cadmium concentrations on bone gene expression program: Results of an in vitro study

Since occupational and environmental exposure to the heavy metal Cadmium (Cd) affects human health this study investigated the effects of exposure to a single, or multiple, sub-toxic Cd concentrations on sub-confluent and confluent human osteoblast growth and expression of specific bone differentiation markers. RT-PCR quantified gene expression of type I collagen, metalloprotease (MMP13), runt-related transcription factor-2 (RUNX2), osterix, osteocalcin, osteonectin, alkaline phosphatase, integrins and bone sialoprotein (BSP). Expression of fibroblast growth factors 1 and 2 (FGF1, FGF2), transforming growth factor-beta(3) (TGFbeta(3)) and bone morphogenetic protein-2 (BMP2) were also evaluated to determine whether Cd-related effects were mediated by an imbalance in expression. Depending on osteoblast concentration and maturation stages, Cd inhibited or stimulated cell growth, decreased type I collagen, increased MMP13, FGF1 and BMP2 gene expression and stimulated the mineralization process only in continuously exposed cultures. These results suggest that in vivo, acute or chronic exposure to sub-toxic Cd concentrations may affect bone formation differently and support the hypothesis that Cd-induced bone disorders may involve downstream changes in growth factor expression. The results are of interest in forensic and occupational medicine in establishing preventive measures to reduce professional exposure risks.

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.

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.

Sub-toxic nicotine concentrations affect extracellular matrix and growth factor signaling gene expressions in human osteoblasts.

Exposure to nicotine and other compounds contained in cigarette smoking affects human health. This study examined the effects of exposure to a single or multiple sub‐toxic nicotine concentrations on human osteoblasts. Cell growth and expression of genes involved in bone differentiation, extracellular matrix (ECM) metabolism, and growth factor signaling pathways were investigated in nicotine‐treated cells compared to untreated cells. Depending on osteoblast concentration and maturation stages, nicotine differently regulated cell growth. Real‐time PCR showed regulated expressions of genes expressed by nicotine‐treated osteoblasts compared to untreated cells. Among ECM genes, type I collagen was down‐regulated and osteonectin was up‐regulated in nicotine‐treated osteoblasts; similarly, fibroblast growth factor‐1 (FGF1) and fibroblast growth factor‐2 (FGF2), two members of FGF signaling system, were discordantly modulated; genes involved in osteoblast maturation and differentiation such as alkaline phosphatase (ALP), runt‐related transcription factor‐2 (RUNX2), and bone sialoprotein (BSP) were over‐expressed after drug treatment. Our results show a positive association between nicotine exposure and osteoblast phenotype and illustrate for the first time a mechanism whereby acute or chronic exposure to sub‐toxic nicotine concentrations may affect bone formation through the impairment of growth factor signaling system and ECM metabolism. J. Cell. Physiol. 229: 2038–2048, 2014.