Mario Cannas

In vitro evaluation of the inflammatory potential of the silk fibroin

Silk fibroin membranes recently have been suggested as matrices for biomedical applications, such as guided tissue regeneration and burn wound dressings. The aim of this study was to evaluate the inflammatory potential of fibroin films and to compare the fibroin films with two model materials with completely different physico-chemical properties: poly(styrene) and poly(2-hydroxyethyl methacrylate). Fibroin bound lower levels of fibrinogen than did the two synthetic polymers while the same amounts of adsorbed human plasma complement fragment C3 and IgG were detected. Studies of the binding strength of C3 to fibroin, evaluated by a novel experimental procedure, indicated the occurrence of strong hydrophobic interactions at the interface. The activation of the mononuclear cells by fibroin, measured as interleukin 1beta production, was lower than the reference materials. Adhesion experiments showed the ability of the macrophages to adhere to fibroin by filopodia without a complete spreading of the cells. The results achieved in this study demonstrate that the interactions of fibroin with the humoral components of the inflammatory system were comparable with those of the two model surfaces while the degree of activation and adhesion of the immunocompetent cells appeared more limited.

In vitro mechanical compression induces apoptosis and regulates cytokines release in hypertrophic scars.

Hypertrophic scars resulting from severe burns are usually treated by continuous elastic compression. Although pressure therapy reaches success rates of 60–85% its mechanisms of action are still poorly understood. In this study, apoptosis induction and release of interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α) were evaluated in normal (n = 3) and hypertrophic (=7) scars from burns after in vitro mechanical compression. In the absence of compression (basal condition) apoptotic cells, scored using terminal deoxyribonucleotidyl transferase assay, were present after 24 hours in the derma of both normal scar (23 ± 0.4% of total cell) and hypertrophic scar (11.3 ± 1.4%). Mechanical compression (constant pressure of 35 mmHg for 24 hours) increased apoptotic cell percentage both in normal scar (29.5 ± 0.4%) and hypertrophic scar (29 ± 1.7%). IL‐1β released in the medium was undetectable in normal scar under basal conditions while in hypertrophic scar the IL‐1β concentration was 3.48 ± 0.2 ng/g. Compression in hypertrophic scar‐induced secretion of IL‐1β twofold higher compared to basal condition. (7.72 ± 0.2 ng/g). TNF‐α basal concentration measured in normal scar medium was 8.52 ± 4.01 ng/g and compression did not altered TNF‐α release (12.86 ± 7.84 ng/g). TNF‐α basal release was significantly higher in hypertrophic scar (14.74 ± 1.42 ng/g) compared to normal scar samples and TNF‐α secretion was diminished (3.52 ± 0.97 ng/g) after compression. In conclusion, in our in vitro model, mechanical compression resembling the clinical use of elastocompression was able to strongly increase apoptosis in the hypertrophic scar derma as observed during granulation tissue regression in normal wound healing. Moreover, the observed modulation of IL‐1β and TNF‐α release by mechanical loading could play a key role in hypertrophy regression induced by elastocompression. (WOUND REP REG 2003;11:331–336)

17‐B estradiol elicits an autocrine leiomyoma cell proliferation: Evidence for a stimulation of protein kinase‐dependent pathway

The mechanism by which estradiol (E2) acts on cell proliferation is still unclear. In this paper, we report the results of a series of experiments in an attempt to elucidate the effector pathway(s) involved in coupling the E2 receptors binding to cellular growth response in leiomyoma cells (LSMC). Under conditions of E2‐dependent growth, E2 treatment of LSMC triggers rapid and transient activation of the MAP‐kinase pathway. Interestingly, we demonstrate that the early downstream signal transduction events determined by E2‐stimulation in quiescent LSMC, including the rapid protein tyrosine phosphorylation of a subset of intracellular proteins, such GAP, PI‐3‐K, and PLCγ, and the concomitant activation of ancillary protein kinases, are related to E2‐induced PDGF secretion. Moreover, we identify the PDGF, alone or in association with other growth factors, as the main growth factor involved in the proliferation response of LSMC to E2 stimulation. The addition of neutralizing antibodies anti‐PDGF was able to inhibit the mitogenic activity present in LSMC conditioned media samples. On the other hand, E2 did not affect the constitutive expression as well as the ligand affinity of PDGF receptors on LSMC plasmamembrane. Cell treatment with the antiestrogen ICI 182780 correlate both with a perturbation of E2‐induced transductional circuit and with the disappearance of the mitogenic factor, PDGF, in LSMC conditioned media; the latter therefore, represents the main autocrine mediator of cell growth modulation, upregulated by E2 and down‐regulated by antiestrogenic compound. Our experiments suggest that growth factor secretion is an initial and integral part of the signaling events mediated by the estradiol receptors, not related, at least in part, to E2 transcriptional modulation.

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.

Effect of the urine conditioning film on ureteral stent encrustation and characterization of its protein composition

The goal of this study was to characterize the protein composition of the conditioning film deposited onto the surface of ureteral stents during in vivo implantation and to relate its presence to the precipitation of calcium crystals. The protein pattern of the conditioning film of implanted nonencrusted and encrusted urological stents was assessed by SDS-PAGE and Western blot of the desorbed species. The results obtained highlighted different electrophoresis profiles between nonencrusted and encrusted stents. Western blot showed the ubiquitous presence of albumin, while Tamm-Horsfall Protein and alpha1-microglobulin adsorption was limited to nonencrusted devices. By an in vitro dynamic model in which artificial urine was flowed through the lumen of control and retrieved nonencrusted stents, we demonstrated that the organic layer remarkably enhanced crystal precipitation and aggregation events on the surface.

Early stage reactivity and in vitro behavior of silica-based bioactive glasses and glass-ceramics

The surface reactivity of different sets of glasses and glass-ceramics belonging to the SiO2–P2O5–CaO–MgO–K2O–Na2O system have been investigated. The attention was focused on the role of their composition on the bioactivity kinetics, in terms of pH modifications, silica-gel formation and its evolution toward hydroxycarbonatoapatite, after different times of soaking in simulated body fluid. Glasses and glass ceramics have been characterized by thermal analysis, SEM-EDS observations and phase analysis (XRD). XPS measurements have been carried out on the most representative set of sample in order to evaluate the evolution of the surface species during the growth of silica-gel and hydroxycarbonatoapatite. The response of murine fibroblast 3T3 to the material before and after a conditioning pre-treatment (immersion in SBF) has been investigated on the same set of samples in order to point out the role of the bioactivity mechanism on cell viability. The main differences among the various glasses have been related to the modifier oxides ratio and to the MgO content, which seems to have an influence on the glass stability, both in terms of thermal properties and surface reactivity. The surface characterization and in vitro tests revealed few variations in the reactivity of the different glasses and glass-ceramics in their pristine form. On the contrary, the different surface properties before and after the pre-treatment in SBF seem to play a role on the biocompatibility of both glass and glass-ceramics, due to the different ion release and hydrophilicity of the surfaces, affecting both cell viability and protein adsorption.

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®.

The pattern of c-Fos immunoreactivity in the hindbrain of the rat following stomach distension

It has been previously shown that the walls of the stomach contain vagal and splanchnic afferents, connected to low and high threshold (LT and HT) gastric receptors, that convey physiological and noxious information to areas of the hindbrain involved mainly in the control of gastrointestinal function. Because distension of the stomach also reflexly increases the sympathetic drive to the cardiovascular system, the present study was planned to examine the pattern of activation of all nuclei encountered throughout the hindbrain in response to gastric distension. In anaesthetized rats, the stimulus was controlled by employing different transmural pressures and frequencies of distension, and c-Fos immunohistochemistry was used to characterize neuronal activation. Low intensity stimulation induced c-Fos expression in the cranial part of nucleus of solitary tract (NTS), the nucleus ambiguus (NA), the lateral reticular area (LRt) and the ventrolateral medulla (RVL/CVL). At low frequency of stimulation c-Fos positive nuclei (p.n.) were found in NTS only. At high frequency of stimulation an increase in c-Fos immunoreactivity was found. High intensity stimulation induced c-Fos expression in area postrema (AP), the lateral vestibular nucleus (LVe) and the caudal part of the NTS. At low frequency, only the number of c-Fos p.n. was increased. Increasing the frequency of stimulation induced c-Fos expression in further nuclei such as the parabrachial nucleus (PBN), the inferior olive subnuclei (IOn), the oral part of spinal trigeminal nucleus (Sp5O) and locus coeruleus (LC). At higher frequencies c-Fos immunoreactivity decreased in NTS and LRt, disappeared in VLM and increased in NA. Thus stomach distension activated several neuronal excitatory and inhibitory circuits that are involved in the control of gastrointestinal function as well as in cardiovascular, respiratory and pain regulation. The differences in c-Fos immunoreactivity induced by changing the distension patterns suggested interactions between groups of vagal and splanchnic afferents.

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.