M. Bosetti

Prevention of Pin Track Infection in External Fixation with Silver Coated Pins: Clinical and Microbiological Results

Pin tract infection is a frequent complication of external fixation; according to literature its frequency ranges from 2-30%. The recent introduction of silver coating of polymeric materials was found to decrease bacterial adhesion; its clinical use with Foley catheters and central venous catheters led to significant results. To verify the ability of the same silver coating to decrease the bacterial colonization on external fixation screws, a prospective randomized study was carried out on 24 male patients; a total of 106 screws were implanted in the lower limb to fix femoral or tibial diaphyseal fractures: 50 were coated with silver and 56 were commercially available stainless steel screws. Although the coated screws resulted in a lower rate of positive cultures (30.0%) than the uncoated screws (42.9%), this difference was not statistically significant (p = 0.243). The clinical behavior of the coated screws did not differ from that of the uncoated ones. Furthermore, the implant of silver-coated screws resulted in a significant increase in the silver serum level. These results led us to consider it ethically unacceptable to continue this investigation.

In vitro characterisation of zirconia coated by bioactive glass

An in vitro evaluation of a biomedical device, which combines the mechanical properties of zirconia substrates with the bioactivity of two different glass layers (AP40 and RKKP), was performed. In this work, data on different kinds of analysis were reported both on as-sintered zirconia samples and on RKKP- and AP40-coated zirconia substrates. Structure, composition and morphology of the apatite layer growth on the coated samples after 30 days of soaking in an acellular simulated body fluid, serum protein adsorption, fibroblasts and human osteoblast-like cells adhesion, growth, morphology and biochemical aspects were studied. Results of soaking test in SBF, revealed the growth of an apatite layer on the surface of the glass-coated samples. Proteins adsorbed to the materials were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and results evidenced that the two glass-coated materials bound a higher amount of total protein than did the zirconia substrate. Fibroblasts and osteoblast-like cells cultured on RKKP- and AP40-coated zirconia showed a higher proliferation rate, leading to confluent cultures with higher cell density and a generally better expression of osteoblast alkaline phosphatase activity in comparison with zirconia substrate. In conclusion, our results indicate that the surface chemical characteristics of the two glass coatings AP40 and RKKP, with no great differences between them, substantially enhance zirconia integration with bone cells at least in vitro. This effect may be of significance in the stability of glass-coated zirconia orthopaedic and dental implants.

Properties of Zinc Releasing Surfaces for Clinical Applications

Two series of glasses of general formula (2-p) SiO2·1.1Na 2O·CaO·pP2O5·xZnO (p=0.10, 0.20; x=0.0, 0.16, 0.35, and 0.78) have been analyzed for physico-chemical surface features before and after contact with simulated body fluid, morphological characteristics, and osteoblast-like cells behavior when cultured on them. The resulted good cell adhesion and growth, along with nonsignificant changes of the focal contacts, allow the authors to indicate HZ5 and HP5Z5 glasses as the ones having optimal ratio of Zn/P to maintain acceptable cell behavior, comparable to the bioactive glass (Bioglass®) used as a control; results are also rationalized by means of three-dimensional models derived by molecular dynamic simulations, with decomposition and conversion rates optimized with respect to the parent Henchs Bioglass®.

In vitro evaluation of the inflammatory activity of ultra-high molecular weight polyethylene

To understand the inflammatory potential of oxidised ultra high molecular weight polyethylene (ox-UHMWPE) compared with the virgin one (UHMWPE), we analysed in vitro the predisposition of their interaction with plasma proteins and cells involved in the inflammatory response. The adsorption on the surface of the two materials of adhesion proteins (Fibronectin and Albumin), and pro-inflammatory proteins (IgG and IgA) have been studied. Moreover, we have evaluated the materials effect on complement activation and on macrophages and monocytes-neutrophils behaviour. The two UHMWPE chemical forms adsorbed all the proteins studied; the only difference was in complement activation. Enzyme immunoassay results evidenced higher levels of factor Bb and iC3b in plasma after the contact with the oxidised form. Physico-chemical properties of the oxidised UHMWPE affected the attachment of the cells as demonstrated by macrophages adhesion experiments. UHMWPE favoured only a limited peritoneal macrophages (PMs) spreading (round-shaped cells); the cell spreading and presence of microvilli on the cell membranes was evident in the case of the oxidised form, suggesting the activation of these cells on this chemical form. Ox-UHMWPE evidenced a statistically significative increase in chemiluminescence values respect to human unstimulated peripheral blood mononuclear cells, an index of increased cell release of reactive oxygen metabolites. In conclusion, UHMWPE oxidative degradation with its chemical modification induces monocytes-neutrophils chemiluminescence activation and PMs morphology changes correlated with macrophage activation, data consistent with the complement activation results obtained in this study; such modifications, along with changes in mechanical properties, are related to implant failure.

Dynamic fibroblast cultures: response to mechanical stretching.

Mechanical forces play an important role in the organization, growth and function of tissues. Dynamic extracellular environment affects cellular behavior modifying their orientation and their cytoskeleton. In this work, human fibroblasts have been subjected for three hours to increasing substrate deformations (1-25%) applied as cyclic uniaxial stretching at different frequencies (from 0.25Hz to 3Hz). Our objective was to identify whether and in which ranges the different deformations magnitude and rate were the factors responsible of the cell alignment and if actin cytoskeleton modification was involved in these responses. After three hours of cyclically stretched substrate, results evidenced that fibroblasts aligned perpendicularly to the stretch direction at 1% substrate deformation and reached statistically higher orientation at 2% substrate deformation with unmodified values at 5-20%, while 25% substrate deformation induced cellular death. It was also shown that a percentage of cells oriented perpendicularly to the deformation were not influenced by increased frequency of cyclical three hours deformations (0.25-3Hz). Cyclic substrate deformation was shown also to involve actin fibers which orient perpendicularly to the stress direction as well. Thus, we argue that a substrate deformation induces a dynamic change in cytoskeleton able to modify the entire morphology of the cells.

Fluoroapatite glass-ceramic coatings on alumina: structural, mechanical and biological characterisation

The aim of this work was to realise bioactive coatings on full density alpha-alumina substrates. An SiO2-CaO-based glass (SC) and an SiO2-Al2O3-P2O5-K2O-CaO-F--based glass-ceramic (SAF) were used for this purpose. Specifically, SAF is a fluoroapatite containing glass-ceramic and previous studies have shown that it is a highly bioactive biomaterial. Furthermore, these fluoroapatite crystals possess a needle-shaped morphology which mimics that of hydroxylapatite found in human hard tissues, particularly in teeth. SAF is a very viscous glass-ceramic and for this reason an intermediate, less viscous, SC layer was interposed in direct contact with alumina aiming to obtain a good coating adhesion. Moreover, this intermediate layer strongly lowers the Al3+ diffusion and thus minimises both compositional changes in the SAF outer layer and the risk of detrimental modifications of the nature of the crystalline phases. A complete characterisation of the coated samples was performed by means of X-ray diffraction, optical and scanning microscopy. Coating adhesion on alumina was tested by comparative shear tests while biocompatibility was investigated on alumina. bulk SAF and on the realised coatings. For this purpose, cytotoxicity, adhesion and proliferation of human osteoblast-like cells were cultured onto the three materials. Results showed that the interposition of the SC layer was successful in allowing a good softening and spreading of the SAF outer layer and in avoiding the crystallisation of undesired crystalline phases maintaining the good bioactive properties of the bulk one. In vitro results on the coatings showed osteoblast-like cell behaviour similar to bulk fluoroapatite glass-ceramic and better respect to alumina substrates, being a promising index of bone material integration and of its in vivo possible applications.

The genotoxicity of PEI-based nanoparticles is reduced by acetylation of polyethylenimine amines in human primary cells.

The ultrasmall size and unique properties of polymeric nanoparticles (NPs) have led to raising concerns about their potential cyto- and genotoxicity on biological systems. Polyethylenimine (PEI) is a highly positive charged polymer and is known to have varying degree of toxic effect to cells based on its chemical structure (i.e., amount of primary and secondary amine). Herein, drug delivery carriers such as PEI-PLGA nanoparticles (PEI-NPs) and acetylated PEI-PLGA nanoparticles (AcPEI-NPs) were utilized to examine the effect of acetylation on NPs biocompatibility and genotoxicity, using human primary cells as in vitro model. Cell uptake of NPs was characterized along with their effects on cellular viability. The results indicate that both NPs showed an equivalent behavior in terms of uptake and biocompatibility. In depth analysis of NP uptake on cell biology evidenced that these nanoparticles induced dose dependant genotoxic effects. This phenomenon was significantly reduced by PEI acetylation. Endocytosed PEI-NPs trigger an oxidative stress on cells by inducing the production of reactive oxygen species (ROS), which cause DNA damage without apparently affecting cell viability. Thus, the genotoxicity of nanoparticles, that could be used as non-viral drug carriers, should be evaluated based on the intracellular level of ROS generation and DNA damage even in absence of a significant cell death.

Chondrogenic induction of human mesenchymal stem cells using combined growth factors for cartilage tissue engineering.

The objective of this study was to evaluate whether growth factors (FGF‐2, FGF‐4 and FGF‐6) used alone or in combination with TGFβ2 are able to increase the proliferation and induce the differentiation of human bone marrow mesenchymal stem cells (hMSCs) to chondrocytes, with a view to using them in cartilage tissue engineering. Cells cultured in monolayer, used to test the activity of the growth factors on cell proliferation, showed that a combination of FGFs with TGFβ2 increases cell proliferation compared to cells cultured in control medium or in the presence of growth factors alone. The chondrogenic potential, evaluated in three‐dimensional (3D) cell aggregates, showed that FGF‐2 and FGF‐6, when used in combination with TGFβ2 increased the size and glycosaminoglycan content of the cell aggregates without increasing cell number. Extracellular matrix (ECM) also showed higher collagen type II immunoreactivity, which was particularly evident in an area similar to a germinative pole that was observed only in pellets cultured with FGF‐2 and FGF‐6 combined with TGFβ2, or in pellets cultured with FGF‐2 alone. Moreover, the RT‐PCR assay has highlighted an increased expression of collagen type II and Sox9, used as gene markers for chondrogenesis. We can conclude that combinations of FGF‐2 or FGF‐6 with TGFβ2 may provide a novel tool to induce the differentiation of adult human mesenchymal stem cells for applications in cartilage tissue engineering. Copyright

Effects and differentiation activity of IGF-I, IGF-II, insulin and preptin on human primary bone cells

The importance of the complex interrelated regulatory pathways involving IGF factors and pancreatic hormones can be observed in several metabolic diseases, where the deregulation of these factors has a wide impact on bone health. These findings have stimulated us to compare the effect of IGF-I, IGF-II, insulin and preptin on human bone cells. The effect on cell differentiation and cell activity of osteoblasts and osteoclasts has been analysed. We have observed a significant effect by IGF-I, a modest effect by IGF-II and preptin and no effect after insulin administration on human primary osteoblast-like cells. All studied factors have shown an induction on human primary osteoclast differentiation and bone resorption activity, with IGF-I being the most potent factor. We hypothesize that these findings may be on the basis of decreased bone mass density observed in several diseases.

The Role of Mechanical Stretching in the Activation and Localization of Adhesion Proteins and Related Intracellular Molecules

The molecular complexity of the processes which lead to cell adhesion includes membrane and cytoskeletal proteins, involved in the focal adhesion formation, as well as signaling molecules tightly associated with the main intracellular regulatory cascades (Akt/PKB and MAPK/Erk). Dynamic environments, which create substrate deformations at determined frequencies and timing, have significant influences on adhesion mechanisms and in general in cellular behavior. In this work, we investigated the role of mechanical stretching (10% substrate deformation, 1 Hz frequency applied up to 60 min) on adhesion proteins (vinculin and focal adhesion kinase—FAK), related RhoGTPases (Rac1 and RhoA), and intracellular pathways (Akt/PKB and MAPK/Erk) in terms of activation and membrane recruitment in relation with cytoskeletal changes observed (membrane ruffling and filopodia formation). These changes are due to intracellular molecular rearrangements, acting with sequential concerted dynamics, able to modify the cytoskeletal conformation. The observed cellular response adds some important issues for better understanding the cellular behavior in environment which mimic as close as possible the physiological conditions. J. Cell. Biochem. 112: 1403–1409, 2011.