Biomedical subjects

G Ciapetti

Publications and source records attributed to G Ciapetti.

At least 55 records · Page 3Linked to original sources

PubMed2000

Modulation of pro- and anti-apoptotic genes in lymphocytes exposed to bone cements.

The ability of bone cements to modify the apoptotic program in activated immune cells and the mechanisms by which they act were evaluated. Mononuclear cells were collected from healthy individuals, cultured for 4 and 24 h with phytohemoagglutinina-P and cement extracts and then tested to assess: (a) cell viability; (b) early apoptotic events, by Annexin V/propidium iodide staining; and (c) the expression of pro- (p53, c-myc, ICE) and anti-apoptotic (bcl-2) genes. After 4 h three cements were able to increase significantly the percentage of apoptotic cells, while after 24 h no differences were found. The proportion of dead cells was not significantly changed at either culture time. The simultaneous expression of both pro-apoptotic (ICE, c-myc, p53) and antiapoptotic genes (bcl-2) was investigated only with regard to the materials which induced significant changes in apoptosis: two cements induced the p53 expression, while the third down-regulated bcl-2. As apoptosis regulates the balance of immune response, the authors recommend that the interaction between materials and immune cells should be assessed, so that the use of pro-apoptotic materials may be avoided in patients with immune defects.

Annexin A5↗

PubMed2000

Biocompatibility and performance in vitro of a hemostatic gelatin sponge.

The biocompatibility of a hemostatic gelatin sponge (Cutanplast Standard) was evaluated in vitro. Cytotoxicity was assessed by neutral red uptake and amido black staining tests; genotoxicity was assayed using the Ames test, Sister Chromatides Exchanges (SCE) and chromosomal aberrations. The ability of the hemostatic gelatin sponge to induce platelet adhesion and release reaction was also determined. The undiluted extract of the test material was found to be cytotoxic, but cell viability was not affected by 1:2 and 1:10 diluted extract. The same extract was found to be non-genotoxic using the three assays for genotoxicity. A significant decrease of platelet number, as well as a significant release of platelet factor 4 was found to be caused by the solid material. In conclusion, Cutanplast Standard is neither cytotoxic nor genotoxic, while inducing platelet adhesion and release reaction when challenged with blood.

Absorbable Implants↗

PubMed1999

Effects of metal ions on white blood cells of patients with failed total joint arthroplasties.

In this study twenty-two patients who had revision surgery for aseptic loosening of joint prostheses were examined. The concentration in serum of soluble products of corrosion from the implant, that is, chromium (Cr), cobalt (Co), and nickel (Ni) ions, and the number of white blood cells (leucocytes, myeloid cells, lymphocyte subpopulations) were measured. Twenty patients with no implants were used as controls. The patients who had revision surgery showed normal Ni concentration whereas by statistical analysis that same patient group was shown to have serum Cr and Co levels significantly higher than those of the control. By flow cytometry, a significant decrease of leucocytes, myeloid cells, lymphocytes, and CD16 populations as found in patients versus controls whereas CD3, CD4, CD8, and CD20 positive cells were decreased, but not significantly. In the arthroplasty patients the Cr concentrations were inversely correlated with some of the immunologic parameters while no significant correlation was found between Co levels and decreased lymphocyte subpopulations. Only in revision surgery patients with high Cr concentrations did we find a significant decrease of lymphocytes, namely of CD4 and CD16 positive cells; revision surgery patients with normal Cr concentrations did not show a significant decrease of lymphocyte subpopulations. These data suggest that the presence of metal ions, especially chromium, released from prosthesis components could be associated with changes of lymphocyte subpopulations in patients with loosening of joint prostheses.

Adult↗

PubMed1999

Cytokine release in mononuclear cells of patients with Co-Cr hip prosthesis.

The aim of this study was the evaluation of the release of bone-resorbing cytokines in peripheral blood mononuclear cells (PBMC) of patients with aseptic loosening of Co-Cr hip prostheses. TNF-alpha, IL-6, and GM-CSF were measured in both unstimulated and PHA-stimulated PBMC, and in PBMC cultured in the presence of chromium and cobalt extracts. Blood samples from healthy donors were used as controls. Serum samples of both patients and healthy donors were tested to determine the concentration of metal ions. The proportion of lymphocyte, monocyte and lympocyte subpopulation in cultured PBMC did not differ in patients and the control group. In unstimulated PBMC the release of TNF was significantly higher in patients than in the control group, while IL-6 was significantly decreased and no change was observed for GM-CSF. When the PBMC were challenged with chromium extract, all the 'index of cytokine release' resulted higher in patients than in the control group; cobalt extract increased both the TNF and GM-CSF index, but not the index of IL-6 release. Metal concentrations in serum from patients were significantly increased and correlated with the TNF release in PBMC stimulated with both metal extract. Our results suggest that a CoCr-implant releases a large amount of metal ions which could mediate the priming or the renewal of a cell-mediated hypersensitivity reaction. The prevalence of circulating lymphocytes responsible for the delayed hypersensitivity, namely Thl, would justify both the significant increase of TNF and the significant decrease of IL6 in unstimulated PBMC of patients, as well as the significant increase of the 'index of cytokine release' after the challenge with metal ions.

Adult↗

PubMed1999

The effects of nicotine and age on replication and viability of human gingival fibroblasts in vitro.

The aim of the present study was to examine: (1) the effects of nicotine on human gingival fibroblasts (HGF); (2) differences between smokers (> or = 10 cigarettes/day at least for 5 years) and non-smokers; (3) differences between patients of different age. HGF were obtained, through biopsies during periodontal surgical procedures, from 15 patients which were divided in 4 groups: 4 patients, smokers aged < or = 25 years; 4 patients, non-smokers aged < or = 25 years; 3 patients, smokers aged > or = 40 years; 4 patients, non-smokers aged > or = 40 years. Nicotine has been tested in 3 different concentrations: 6 microg/ml; 60 microg/ml; 600 microg/ml. To assess cells viability, the neutral red (NR) test was used; to evaluate cell proliferation, the Hoechst test was employed. After 48 h of nicotine exposure, it was found that 600 microg/ml nicotine was strongly cytotoxic to HGF of all groups, with a significant reduction of both proliferation and viability of cells versus control. Comparison between groups of the same age: when comparing untreated HGF (i.e., control values) of smokers < or = 25 years versus non-smokers < or = 25 years, cell proliferation, but not viability, was found to be increased in smokers. Both viability and proliferation of control cells of smokers > or = 40 years were increased versus non-smokers > or = 40 years. HGF of non-smokers < or = 25 years, when exposed to nicotine 600 microg/ml, have less viability and proliferation than HGF of smokers of the same age. Comparison between groups of different age: In the smoker group, untreated HGF (i.e., control values) had similar viability and proliferation, irrespective of age, but nicotine 600 microg/ml kills more HGF in smokers < or = 25 years than in smokers > or = 40 years. In non-smokers, untreated HGF < or = 25 years replicate less, but are not less viable than HGF > or = 40 years. When challenged with nicotine 600 microg/ml, HGF < or = 25 years were less viable than HGF > or = 40 years. From this study, it appears that the smoking history and the patient age could be relevant for final evaluation of the results, since HGF from smokers are less sensitive to nicotine than HGF from non-smokers, and cells from older donors are more resistant to nicotine than cells from younger donors.

Adult↗

PubMed1999

[Cytokines of bone turnover in postmenopause and old age].

BACKGROUND: The aim of the study was the assessment of the influence of cytokines on bone ageing by measuring their level in serum and their secretion in vitro by monocytes from women of different age. METHODS: The levels of cytokines in 34 postmenopausal subjects and 14 old subjects were compared to those measured in 13 cycling subjects who were considered as control group. Subjects suffering from diseases inducing secondary osteoporosis, subjects taking medications that affect bone metabolism and alcohol- or tobacco-consumers were excluded from the study. The levels in serum of (i) the bone stimulating peptide insulin-like growth factor-I (IGF-I), (ii) the inhibitor of bone resorption interferon-gamma (IFN gamma), (iii) the stimulators of bone resorption interleukin-1 beta (IL-1 beta) tumor necrosis factor alpha (TNF alpha) and interleukin 6 (IL-6) were evaluated by immunoassay. IL-1 beta, TNF alpha and IL-6 secreted by monocytes (MO) cultured in vitro from peripheral blood of the same subjects were measured, too. Bacterial endotoxin (LPS) was used as stimulator for cytokine secretion by monocytes. RESULTS: Unlike IFN gamma, which was unaltered, circulating IGF-I level was found significantly diminished in postmenopausal and old subjects compared to control group. Among stimulators of bone resorption, IL-6 was greatly increased in postmenopausal and old subjects, while TNF alpha was reduced in postmenopausal group. In the supernatants of unstimulated monocytes the level of IL-1 beta was consistently decreased in old subjects; TNF alpha was found to be decreased in postmenopausal and old subjects. The stimulation index (SI), calculated as the ratio between the level of cytokines secreted by LPS-stimulated MO and the level of cytokines secreted by unstimulated MO, was found to be significantly increased for IL-1 beta and TNF alpha in postmenopausal subjects vs control group. In the old subjects the SI for IL-6 was enhanced. CONCLUSIONS: The data collected suggest that the measurement of cytokines in serum and supernatants from monocytes may give a picture of the mechanisms regulating bone aging.

Adult↗

PubMed1998

Cytotoxicity testing of materials with limited in vivo exposure is affected by the duration of cell-material contact.

Silicones for dental impression largely are used to record the geometry of hard and soft dental tissues. They are considered to be medical devices, and the assessment of cytotoxicity is a necessary step in the evaluation of their biocompatibility. Extracts of six addition-type and six condensation-type silicones have been tested with L929 cells according to the ISO 10993-Part 5 standard. The cytotoxicity was evaluated by three different methods: neutral red uptake, propidium iodide (PI) staining, and amido black staining. According to the selected specific assay, contact between cells and material extracts was maintained for 24 h in the first series of experiments; then, considering that in vivo application of these materials is restricted to a few minutes, additional experiments were performed after 1 h of cell/extract contact. Analysis of the results showed that the addition-type silicones are nontoxic even when tested after prolonged exposure of the cells to the materials while the condensation-type silicones were cytotoxic at 24 h of incubation. Nevertheless, harm to the patient actually could be negligible, considering its very short time of exposure in vivo. This is supported by our finding that most are not toxic after 1 h. We suggest that the experimental conditions of cytotoxicity testing have to be relevant to the in vivo situation; accordingly, the time of exposure should be designed carefully.

Amido Black↗

PubMed1998

Fluorescent microplate assay for respiratory burst of PMNs challenged in vitro with orthopedic metals.

This report describes a simple, rapid, automated microassay for measuring in vitro changes of oxidative burst of phagocytes following challenge with metals for orthopedic devices. The production of reactive oxygen species (ROS) by polymorphonuclear leukocytes (PMNs) was measured using 2',7'-dichlorofluorescin-diacetate (DCFH-DA) as fluorescent probe. DCFH-DA enters the cells and is oxidized by ROS to fluorescent DCF. The DCF generated was directly proportional to ROS produced intracellularly: The fluorescence intensity was read and converted to an index of ROS production by cells. In our experimental system, granulocytes (PMNs) were isolated from normal human blood and seeded in microplates. To verify if metals could influence ROS production, chromium, cobalt, nickel, molybdenum, titanium, aluminum, and vanadium prepared as aqueous extracts in phosphate-buffered saline were tested onto PMNs using phorbolmyristate acetate (PMA) as positive control. Molybdenum, aluminum, and vanadium increased ROS generation by PMNs, while signals not different from unstimulated PMNs were recorded for chromium, cobalt, nickel, and titanium. The DCFH-DA microplate-based assay provides an in vitro tool for the detection of oxygen-reactive species generated by PMNs as a response to metals.

Fluoresceins↗

PubMed1998

Flow-cytometric analysis of leukocyte activation induced by polyethylene-terephthalate with and without pyrolytic carbon coating.

Leukocyte activation is one test for the evaluation of blood-materials interaction. The expression of adhesion molecules analyzed by flow cytometry provides a simple method to evaluate leukocyte activation by biomaterials: any change in these molecules can be predictive of the inflammatory activity of the materials. In this study the contact between leukocytes and uncoated polyethylene terephthalate or pyrolytic carbon-coated polyethylene terephthalate (PET and PET-PC, respectively) was inspected by analyzing whether the expression of some adhesion molecules involved in leukocyte activation, namely LFA-1 (CD11a/ CD18), Mac-1/CR3 (CD11b/CD18), and LECAM-1 (CD62L) can be modified. By flow cytometry expression of the adhesion molecules can be studied separately on lymphocytes and myeloid cells. The materials tested reduced the total numbers of both leukocytes and neutrophils, although not significantly. Neither PET nor PET-PC changed the expression of the adhesion molecules in lymphocytes: this suggests that no specific immune response is stimulated. On the contrary, statistically significant changes were observed for monocytes and granulocytes: the percentage of cells expressing Mac-1 and the density of such antigens on cell membranes increased while the percentage of LECAM-1 positive cells decreased. Similar changes were observed when the cells underwent the inflammatory stimulus provided by an in vitro challenge with bacterial endotoxin. Our results demonstrated that polyethylene terephthalate activates leukocytes by modifying the expression in neutrophils of the molecules involved in the early phase of the inflammatory response. Even after coating PET with pyrolytic carbon, the ability of this material to activate circulating leukocytes was maintained.

Biocompatible Materials↗

PubMed1998

False positive results in cytotoxicity testing due to unexpectedly volatile compounds.

We investigated the cytotoxicity of different dental materials according to the study protocol adopted by our lab for the screening of new materials. Experimental parameters used in such testing are addressed mainly in documents EN 30993 "Biological evaluation of medical devices, Part 5: Tests for cytotoxicity: in vitro methods" and "Biological evaluation of medical devices, Part 12: Sample preparation and reference materials." Cells were cultured in microplates and challenged with aqueous extracts of the materials. The assay methods were neutral red- and propidium iodide-uptake assays, both indicative of cell viability and able to provide quantitative data. The observation of contrasting results for one material using the above-mentioned methods raised some concern about the assay system used. With further experimentation, it appeared that a sustained release of volatile substances still present in one extract exerted a toxic effect in neighboring cultures. It is concluded that in the microenvironment of a microplate the distribution of samples cannot be disregarded, as it may be responsible for toxicity cross-contamination. Moreover, the use of more than one single method has to be recommended in cytotoxicity testing, in order to avoid false positive results due to experimental artifacts.

Animals↗

PubMed1998

The interface of bone microstructure and an innovative coating: an X-ray diffraction study.

The in vivo compatibility and degradation aspects of an innovative coating to be sprayed onto titanium implants were investigated. The surface of fluorinated apatite (fHA), consisting of fluorhydroxyapatite plasma sprayed in a vacuum atmosphere, was treated with carbonate to improve its biological compatibility. fHA coating was compared with titanium implants coated (a) with hydroxyapatite (HA) by the traditional plasma spraying, and (b) with titanium oxide (TiOx). Screw-shaped implants were inserted in the cortical bone of sheep tibiae. X-ray diffraction (XRD) analysis of bone tissue and coatings was carried out at 2, 4, 12 and 36 weeks after surgery. The crystallographic habit of the implant-facing bone, as well as the structural stability of the coating, were evaluated. For each time period and type of ceramic bone apatite lattice at the interface, no significantly different reference apatite lattice and no foreign peak were recorded. Two weeks after implantation, the bone at the interface was strongly unmineralized in all samples; after 4 weeks, poorly mineralized bone microareas decreased. At 12 weeks, the newly formed bone tissue at the interface with both the new coating and HA coating was shown to be fully mineralized; this crystallographic habit was retained at 36 weeks, when particle release from the tested material was lower compared to the controls. The XRD pattern of bone apatite surrounding the coating particles was unmodified. The innovative coating did not alter the mineralization process at the interface. It improved implant osteointegration, mainly due to a limited release of particles. Consequently, clinical performance of external fixation treatment could be improved by modifying the chemical composition of the implant surface.

Animals↗

PubMed1998

Cell death induced by metal ions: necrosis or apoptosis?

We have evaluated if the cytotoxic effects of metals released from implants are due to necrosis or apoptosis. Peripheral blood mononuclear cells were exposed to different concentrations of chromium, nickel and cobalt extracts and the characteristics of both apoptosis and necrosis were evaluated by flow-cytometry at different culture endpoints. In order to define the prevalence of apoptosis or necrosis, the ratio cell death/apoptosis was calculated. A ratio of </=1 means the prevalence of apoptotic events; a ratio >1 indicates the acute toxicity of the tested substance (necrosis). The extracts of chromium, cobalt and nickel had a cytotoxic effect on the mononuclear cells; high concentrations of cobalt and nickel produced cell necrosis, whereas by lowering the extract concentration apoptotic phenomena were observed. High chromium concentrations can induce cell death by apoptosis. Our data suggest that when large amounts of nickel and cobalt are released from implanted metal devices, necrosis is produced and consequently a strong inflammatory tissue reaction is likely to occur. The release of either chromium or limited amounts of nickel and cobalt induces toxicity characterized by apoptotic phenomena, which allows an adaptation of the tissue to the implant.

Journal Article↗

PubMed1998

Bone-resorbing cytokines in serum of patients with aseptic loosening of hip prostheses.

Our aim was to determine if the serum levels of bone-resorbing cytokines (IL-1beta, TNF-alpha, IL-6, GM-CSF) are altered in patients with aseptic loosening of a total hip prosthesis, and if such levels are influenced by the type of implant. We determined cytokine levels in sera from 35 patients before revision for failed total hip arthroplasty and compared them with those in 25 healthy donors. We also assessed the soluble receptor of interleukin-2 (sIL-2r) in serum as an indication of a specific immune reaction against the implant. Our findings showed that the sIL-2r and TNF-alpha serum level did not change. The IL-6 level was not significantly altered, but was higher in patients with TiAIV prostheses than in those with a CrCoMo implant and in patients with cemented prostheses. The IL-1beta level was found to be higher in those with a TiAIV cemented prosthesis than in the control group (p=0.0001) and other groups of patients (p=0.003 v uncemented TiAIV, p=0.01 v cemented CrCoMo, p=0.001 v uncemented CrCoMo). The GM-CSF level significantly increased in patients compared with healthy subjects (p=0.008), and it was higher in those with cemented than with uncemented implants (p=0.01). Only patients with cementless CrCoMo prostheses had levels of GM-CSF similar to those of the control group. The highest GM-CSF concentrations were observed in patients treated with non-steroidal anti-inflammatory drugs (NSAIDs) in the last months before revision (p=0.04). In addition, when massive osteolysis was observed, the level of GM-CSF tended to decrease to that of the control group.

Acetabulum↗

PubMed1997

High-performance liquid chromatography assay of N,N-dimethyl-p-toluidine released from bone cements: evidence for toxicity.

Five commercially available bone cements were analysed by high-performance liquid chromatography for detecting the residual content of an accelerator, the amine N,N-dimethyl-p-toluidine (DMPT), after curing. It was found that the concentration of DMPT in aqueous extracts decreases with time, being almost absent 7 days after curing. Differences were noticed among the cements; residual DMPT is higher in cements prepared with higher content of the amine. It is verified that DMPT's toxic effect on cell cultures is dose-related; a delay in the cell replication cycle is induced in vitro. Damage is reversible, thus justifying the low bone cement toxicity that is clinically ascertained.

Bone Cements↗

PubMed1997

Expression of adhesion molecules on endothelial cells after contact with knitted Dacron.

The aim of this study is to evaluate the expression of some adhesion molecules on the surface of endothelial cells cultured in contact with knitted Dacron. These molecules, as mediators of cell adhesion, could play a role in the modulation of adhesion on the biomaterials, therefore conditioning the response of tissues to implant. Twenty different cultures of human umbilical vein endothelial cells (HUVECs) were cultured in contact with knitted Dacron. Both HUVECs grown without the material and HUVECs incubated with endotoxin were used as control. After 24 h, the cell adhesion molecules PECAM-1, ELAM-1, ICAM-1 and VCAM-1 were evaluated on the cells by monoclonal antibodies and flow cytometry. After 24 h of contact with knitted Dacron, a significant decrease in the proportion of cells expressing PECAM-1 was observed, as well as a significant increase in the proportion of cells expressing ELAM-1. The contact with knitted Dacron did not induce significant variations of ICAM-1 and VACM-1. The incubation with endotoxin determined a significant increase in the proportion of ELAM-1-positive cells, a significant increase in ICAM-1 fluorescence intensity, and a significant increase both in fluorescence intensity and in the proportion of VCAM-1-positive cells. The results obtained with the endotoxin are in agreement with those reported in the literature. The ELAM-1 increase, observed after contact with knitted Dacron, could favour leucocyte adhesion, while the decrease in PECAM-1 expression could result from an inhibiting effect on the endothelial cell adhesion so as to hinder the mechanisms involved in the endothelialization of the material. The variations were interpreted as inhibiting endothelialization and favouring the leucocyte adhesion effect by knitted Dacron.

Analysis of Variance↗

PubMed1997

In vitro complement activation after contact with pyrolytic carbon-coated and uncoated polyethylene terephthalate.

This study was undertaken to evaluate whether the pyrolytic carbon coating of polyethylene terephthalate induces complement activation. Complement activation induced by pyrolytic carbon-coated polyethylene terephthalate (PET+PC) in comparison with uncoated polyethylene terephthalate (PET) was assessed on whole blood collected with heparin. The activation of the classic pathway was evaluated by C4d fragment enzyme immunoassay. The activation of the alternative pathway was evaluated with Bb fragment enzyme immunoassay. The results show that uncoated PET activates the alternative pathway, but not the classic one. PET+PC does not induce complement activation, not even through the alternative pathway. Pyrolytic carbon coating therefore contributes to improving blood compatibility.

Journal Article↗

PubMed1997

[In vitro production of endothelin-1 and prostacyclin by cultured endothelial cells in the presence of polymers].

The aim of this study was to evaluate endothelin-1 and prostacyclin production by human endothelial cells cultured in the presence of polyethylene terephthalate and collagen-coated PET. Cell counting and the assay of endothelin-1 and 6-keto-prostaglandin F1 alpha, stable metabolite of prostacyclin, were carried out after 48 hour contact of the cells with the examined materials. Endothelial cell contact with uncoated PET caused a significant reduction in cell number, a significant increase in the production of endothelin-1 and a not significant increase in 6-keto-prostaglandin F1 alpha. The endothelial cell contact with collagen-coated PET caused a highly significant decrease in cell number and a not significant decrease in endothelin-1 and 6-keto-prostaglandin F1 alpha. It was concluded that PET causes both a decrease in cell number and a remarkable increase in endothelin-1. On the contrary, collagen-coated PET determines a decrease in cell number and a slight reduction of endothelin-1 and 6-keto-prostaglandin F1 alpha.

Cells, Cultured↗

PubMed1996

Activation of the plasma coagulation system induced by some biomaterials.

The ability of some biomaterials to activate plasma coagulation system was examined in vitro. After contact of platelet-rich plasma with biomaterials, some markers of the thrombin formation, i.e., fragment 1 + 2 and fibrinopeptide A, and some inhibitors of the blood coagulation mechanism were tested. Fragment 1 + 2 and fibrinopeptide A were found to be increased by all of the materials, though to a different extent. In particular, fragment 1 + 2 and fibrinopeptide A were significantly increased upon contact with polybutylene terephthalate and with collagen coated polyethylene terephthalate, respectively. Also antithrombin III was shown to decrease following exposure to biomaterials, but statistical significance was found only for polyethylene terephthalate and polyvinylacetate. As a results of this wide range of variability in the parameters, it is advisable to explore the plasma coagulation system with a multiparametric approach in which thrombin formation and coagulation inhibitors are thoroughly investigated.

Antithrombin III↗