Biomedical subjects

G Ciapetti

Publications and source records attributed to G Ciapetti.

At least 19 recordsLinked to original sources

PubMed2002

In vitro testing of the potential for orthopedic bone cements to cause apoptosis of osteoblast-like cells.

The purpose of this study was to investigate in vitro the apoptosis- and/or necrosis-inducing potential of polymethylmethacrylate (PMMA)-based bone cements for prosthetic surgery. Four bone cements widely used in orthopedics were tested as extracts onto osteoblast-like MG-63 cells and for comparison, HL-60 cells, which are remarkably sensitive to apoptotic stimuli. Neutral red uptake (NRU) was used to measure cell viability while Hoechst 33258 staining was used to detect DNA content. Apoptosis was characterized using a BrdU-based ELISA assay for DNA fragmentation and examined by fluorescence microscopy using acridine orange and propidium iodide staining of nuclei. The generation of reactive oxygen species (ROS), which could mediate apoptosis, was verified using dichlorofluorescein-diacetate (DCFH-DA) oxidation to DCF. After 24 h of challenge of the cells with the four cement extracts, the viability of either MG-63 or HL-60 cells was found to be unaltered, as recorded by NRU. Apoptotic cell death was induced by three cements in HL-60, whereas MG-63 cells were significantly affected by the four cements tested: the finding of DNA fragments both in the cytoplasm and supernatants of MG-63 after 24 h demonstrated that these cells underwent late-apoptosis secondary necrosis. Fluorescent staining of the nuclei confirmed the results obtained with the ELISA test. Oxygen free radicals were elicited by two cements in HL-60 cells, while MG-63 did not generate ROS in response to cements. This study helps to gain more insight into the mechanism of cell death induced by PMMA-based cements and suggests apoptosis of osteoblasts as a part of the tissue reaction around cemented prostheses.

Apoptosis↗

PubMed2001

Evaluation of tissue-factor production by human endothelial cells incubated with three acrylic bone cements.

The effect of three methacrylate-based cements used for the fixation of joint prostheses on tissue-factor production by human umbilical vein endothelial cells was evaluated in vitro. The extracts in the culture medium of the cements were tested after 1-h and 7-day curing. The endothelial cells were incubated with the cement extracts for 4 h, and then the tissue factor was determined in cell lysates with both the recalcification time and enzyme immuno assay. The cements did not induce significant production of tissue factor and, therefore, did not activate the extrinsic pathway of coagulation within the limits of the mechanism considered.

Ascorbic Acid↗

PubMed2001

Cytotoxicity, blood compatibility and antimicrobial activity of two cyanoacrylate glues for surgical use.

The biocompatibility of two cyanoacrylate surgical glues (Glubran and Glubran 2), supplied by General Enterprise Marketing, Viareggio, Lucca, Italy, was tested through cytotoxicity and blood compatibility tests and the evaluation of antimicrobial activity. Cytotoxicity and blood compatibility tests were performed on the polymerized glues. Using the neutral red uptake test, the extracts from Glubran and Glubran 2 after polymerization were non-toxic to L929 cells only when diluted 1: 10 with culture medium. Glubran and Glubran 2 induced a significant decrease of activated partial thromboplastin time (APTT), which is favourable with regard to the desired haemostasis. The APTT shortening determines a haemostatic effect and therefore contribute to the tissue adhesion induced by the glues. Otherwise, no significant variation of prothrombin activity, fibrinogen, platelet number, total and differential leukocyte count was induced by the glues, which, in addition, did not show haemolytic effect. There was no difference between Glubran and Glubran 2 regarding haemocompatibility. The antimicrobial ability of the unpolymerized glues was tested onto Bacillus subtilis var. niger for 3 weeks: neither Glubran nor Glubran 2 were found effective in this respect. In conclusion, we can assume that cytotoxicity was severe with the undiluted glues, but was acceptable when glues were diluted. On the contrary, blood compatibility was acceptable for the intended use of the glues. No difference was found between Glubran and Glubran 2 after polymerization.

Animals↗

PubMed2001

Evaluation of the effect of seven acrylic bone cements on erythrocytes and plasmatic phase of coagulation.

The haemolytic activity and the effect on the plasmatic phase of coagulation of seven bone cements were evaluated (Sulfix-60 from Sulzer Orthopedic Inc., a bone cement at low viscosity from Zimmer, a bone cement dough-type from Zimmer, Palacos R from Merck, CMW1, CMW2 and CMW3 from DePuy International Ltd.). Haemolytic activity was tested by adding the cement extracts in phosphate buffered saline to a suspension of erythrocytes. After 4 h incubation at 37 degrees C, the haemoglobin concentration was determined on the supernatants by colorimetric method. The effect on the plasmatic phase of coagulation was tested by adding the cement extracts in saline to human plasma. After 30 min incubation at room temperature activated partial thromboplastin time (APTT) was determined. All extracts induced non-significant variations of haemoglobin concentration and APTT. It was concluded that the tested cement extracts do not induce haemolysis and do not activate the intrinsic pathway of coagulation, at least in the tests that were performed.

Acrylates↗

PubMed2001

Serum concentrations of zinc and selenium in elderly people: results in healthy nonagenarians/centenarians.

Trace elements such as zinc (Zn) and selenium (Se) play an important role in maintaining the metabolic homeostasis in elderly people and the risk of deficiency seems to increase in proportion to the age. Zn and Se concentrations, as indices of the micronutrient status in healthy subjects over 90 years, are scarcely analyzed and could represent a model for studying the physiology of successful aging. Our aim was to investigate Zn and Se concentrations in the healthy persons over the age of 90 years. One hundred and fifty two subjects volunteered for the study. They were divided into two groups: 90 non-institutionalized nonagenarians/centenarians (91-110 years) (group A) and 62 elderly subjects (60-90 years) used for comparison (group B). Serum concentrations of Zn and Se were determined, respectively, by flame atomic absorption spectrophotometry (FAAS) and electrothermal atomic absorption spectrophotometry (ETAAS). The effect of age and sex on ion concentrations was investigated. Mean values+/-standard deviation of Zn and Se concentrations in the group A were 11.97+/-2.00 and 0.87+/-0.28 micromol/l, respectively. A significant decrease of Se and Zn values was demonstrated in group A, when compared with group B, in both males and females. However, 84.4% of the 'healthy' nonagenarians/centerians had both Zn and Se concentrations equal to or greater than the lowest values of the elderly group and only 3.3% of cases showed both Zn and Se deficiencies. Consequently, a prospective and follow-up evaluation of Zn and Se could be proposed as a good index for a correct monitoring of the micronutrient deficiencies, that could represent an early sign of disease.

Aged↗

PubMed2001

No effect of methacrylate-based bone cement CMW 1 on the plasmatic phase of coagulation, red blood cells and endothelial cells in vitro.

The compatibility of a methacrylate-based bone cement (CMW 1, DePuy International Ltd, England) used for the fixation of joint prostheses was evaluated on plasma, an erythrocyte suspension and cultured human endothelial cells. The extract of the cement was tested, following 1 hour and 7 days of curing. After the contact in vitro of the extract with plasma, activated partial thromboplastin time, antithrombin III, thrombin-antithrombin complexes and fibrin degradation products were assayed. Hemolytic activity was tested by adding the cement extracts to a suspension of erythrocytes. After 4 hours of incubation at 37 degrees C, the hemoglobin concentration was determined on the supernatants by the colorimetric method. The effect of the cement on tissue factor and thrombomodulin production was evaluated on human umbilical vein endothelial cell cultures. Tissue factor was determined in cell lysates by enzyme immunoassay, following 4 hours' incubation of cultures with the cement extract. Thrombomodulin was assayed in cell lysates by enzyme immuno assay, after 24 hours' incubation with the cement extract. The response to all trans-retinoic acid (ATRA) was tested. The cement caused no significant modifications of the coagulation tests, had no hemolytic activity, did not determine tissue factor production and did not modify thrombomodulin, compared to the negative control. The response to stimulation with ATRA was similar to that of the negative control. We conclude that the cement extract does not affect the plasmatic phase of coagulation, has no effect on erythrocytes, does not induce the expression of procoagulant activity by endothelial cells and does not impair their antithrombotic property, within the limits of the tests performed.

Blood Coagulation↗

PubMed2001

Interleukin-6 expression by osteoblast-like MG63 cells challenged with four acrylic bone cements.

Periprosthetic osteolysis is a major clinical problem in total hip and total knee arthroplasty and polymethylmethacrylate (PMMA) is a possible etiologic factor. Recently, increasing importance was ascribed to interleukin-6 (IL-6) as an agent favouring bone resorption. The aim of the present study was to investigate the role of bone cements on IL-6 production by MG63. The effect of four acrylic bone cements (Sulfix-60, CMW 1, CMW 2, and CMW 3) on the protein release and mRNA expression of IL-6 in osteoblast-like cell line MG63 was examined using IL-1beta (0.2 microg ml(-1)) as the positive control. The extracts in minimum essential medium (MEM) of the cements were tested, following 1-h and 7-day curing. CMW 1 and CMW 2 significantly increased the IL-6 release into the culture media (p < 0.01). The cells incubated with Sulfix-60 and CMW 3 produced no significantly different levels of IL-6 than the basal production. A positive correlation was found between the concentration of IL-6 and the contents of benzoylperoxide (p = 0.0003) and barium sulphate (p < 0.0001). MG63 expressed IL-6 mRNA constitutively, as demonstrated by the positivity of the negative controls too. We conclude that CMW 1 and CMW 2 increase the production of IL-6 in MG63 cells. The response to Sulfix-60 and CMW 3 is not significantly greater than the negative control.

Bone Cements↗

PubMed2001

Effect of CMW 1 bone cement on transforming growth factor-beta 1 expression by endothelial cells.

The present study examined the effects of in vitro challenge with an acrylic bone cement CMW 1 on the expression of transforming growth factor-beta 1 (TGF-beta 1) in human umbilical vein endothelial cells (HUVEC). The extracts in cell culture medium of the cements were tested, after 1 h and 7-day curing. Some cultures were also stimulated with interleukin-1 beta (IL-1 beta) or all-trans retinoic acid (ATRA). The expression of mRNA was evaluated by RT-PCR with specific primers. The release of TGF-beta 1 into the conditioned medium was evaluated by enzyme immunoassay. TGF-beta 1 mRNA was constitutively expressed by endothelial cells in the culture medium after 24 h. The incubation with the extracts of CMW 1, cured both for 1 h and 7 days, induced changes neither in mRNA expression, nor in the release of TGF-beta 1 into the conditioned medium, compared to the unstimulated cells. Even stimulation with ATRA, alone or added to the extracts at both curing times, affected neither mRNA expression nor TGF-beta 1 release, compared to the cells incubated with the cement alone or with the unstimulated cultures. The mRNA expression and the release were not changed by the stimulation with IL-1beta alone or added to the extract cured for 1 h. A significant decrease compared to the unstimulated cells was observed after the addition of IL-1 beta to the extract cured for 7 days. It was concluded that CMW 1 extract did not significantly modify TGF-beta 1 expression after 1-h curing, or after 7-day curing. Incubation with CMW 1 added with ATRA did not produce any changes in TGF-beta 1 synthesis. Incubation with cement extract after 7-day curing added with IL-beta 1 produced a significant reduction in TGF-beta 1 release.

Bone Cements↗

PubMed2000

Cytokine expression in vitro by cultured human endothelial cells in contact with polyethylene terephthalate coated with pyrolytic carbon and collagen.

In order to evaluate whether or not polyethylene terephthalate coated with pyrolytic carbon and collagen (PET+PC) favors inflammatory or hyperplastic reactions, the expression of mRNAs specific for interleukin-6 (IL-6), platelet-derived growth factor-A (PDGF-A), PDGF-B, transforming growth factor-beta1 (TGF-beta1), and TGF-beta2 were tested in vitro by cultured human umbilical vein endothelial cells (HUVEC). The cultures were put in contact with PET+PC for 1, 24, 48, and 72 h. The same cells cultured on polystyrene without biomaterials were tested as negative controls; cultures incubated with LPS were the positive control. The expression of mRNAs was evaluated by RT-PCR with specific primers. PET+PC did not determine any differences in the expression of IL-6-specific mRNA at any of the incubation times compared to the negative control while LPS (the positive control) induced expression after 24, 48, and 72 h. PET+PC induced a more precocious expression of mRNA specific for PDGF-A than did the negative control; however, the expression no longer was present after 48 h while in the negative control the expression stopped after 72 h. PET+PC induced a less frequent expression of PDGF-B-specific mRNA than did the negative control and LPS, especially after 24 h. PET+PC induced a later expression of TGF-beta2-specific mRNA than did the negative control and a less frequent expression of mRNA specific for TGF-beta1 after 24 and 72 h.

Biocompatible Materials↗

PubMed2000

Sister chromatid exchanges and ion release in patients wearing fracture fixation devices.

The quantification of sister chromatid exchange (SCE) during mitosis is a useful index for evaluating genotoxic effects in subjects occupationally or incidentally exposed to potentially toxic substances. The authors investigated the hypothesis that ions released by corrosion from prosthetic components of fracture fixation devices are associated with change in SCE incidence. In the present study, ten patients with implants were examined, and fifteen subjects with no implants were used as controls. SCE and high frequency cell (HFC) numbers were evaluated in circulating lymphocytes. In addition, nickel (Ni) and chromium (Cr) ion values in the serum were measured because, after iron, these metals are major components of stainless steel. A significant increase in SCE numbers was observed in patients compared to the control population (4.9 +/- 1.3 vs. 3.5 +/- 1.4). Ni concentration was 1.71 +/- 1.49 ng/mL in patients and 0.72 +/- 0.52 ng/mL in control subjects; Cr concentration was, respectively, 1.01 +/- 0.77 ng/mL and 0.19 +/- 0. 27 ng/mL. The increase of serum Cr and Ni was statistically significant. No correlation was found between the increased Cr concentrations and SCE number while Cr ion levels were found to be significantly correlated to HFC. An inverse correlation between Ni level and SCE numbers was observed. Our findings suggest that Cr release by stainless steel implants could have a genotoxic effect; thus it would be useful to carefully monitor implanted subjects with regard to serum ion dosage, SCE analysis, and HFC evaluation. In any case, it would be appropriate to remove the implant when fracture fixation is reached.

Adolescent↗

PubMed2000

Cytotoxic effect of bone cements in HL-60 cells: distinction between apoptosis and necrosis.

Ten PMMA-based bone cements used in prosthetic surgery have been studied with respect to the induction of programmed cell death (i.e., apoptosis) in HL-60 cells, which are remarkably sensitive to various apoptotic stimuli. Annexin V binding and propidium iodide (PI) exclusion were the methods for detection of early apoptotic changes, while PI entry was considered as a marker of necrosis. Hoechst 33342 staining was used to detect DNA fragmentation and Alamar blue was applied to measure oxide-reduction activity of cells. The production of reactive oxygen species (ROS) related to cell damage was verified using dichlorofluorescein-diacetate (DCFH-DA) oxidation to DCF. Under our experimental conditions, the cements tested, for the most part, were not toxic to leukemic cells at 4 and 24 h. After 24 h, three cements were able to induce cell death, with two eliciting both apoptosis and necrosis, and one cement acting mainly via apoptosis. Both processes of cell death are likely to be mediated by the production of oxygen-free radicals. These findings provide potential leads for investigation into the molecular mechanisms of cell death, which are responsible for tissue damage by cements and intolerance of cemented prostheses.

Apoptosis↗

PubMed2000

Cytokines and osteolysis around total hip prostheses.

The aim of this work is to assess the correlation between the osteolysis around the prosthesis and the presence of cytokines favouring inflammation in the tissues at the interface between loosened prosthesis and bone. In this study, twenty-nine patients that underwent revision surgery were examined. Bioptic samples were collected at the interface between bone and implant both at the stem and socket level. Semiquantitative immunohistochemistry was performed to detect interleukin 1 alpha, interleukin 1 beta, interleukin 6 and tumour necrosis factor, cytokines that directly cause bone resorption and indirectly induce synthesis of other bone resorbing cytokines. Wear particles were identified and quantified by light microscopy. Radiographic evidence for osteolysis was scored by the Engh and Bobyn score. In tissues collected at the interface, the percentage of cells positive to IL1, IL6 and particularly to TNF increased in relation to the tissues collected at the interface with stable components. The cells occurring in the new capsule do not secrete cytokines in quantities that can be related to severity of wear. Cemented prostheses showed higher incidence of severe osteolysis, and higher levels of cytokines. It can be concluded that TNF, and to a lesser extent IL1 and IL6, are positively related to the severity of osteolysis around the prosthesis and therefore a pharmacological treatment can be hypothesized with anti-inflammatory or anti-cytokine drugs in order to limit or to avoid prosthesis loosening.

Adult↗

PubMed2000

Microstructural analysis of severe bone lesions in seven thalassemic patients treated with deferoxamine.

Osteochondrodystrophic lesions, mainly affecting long bone metaphyses, can be radiologically evident in homozygotic thalassemic patients treated with deferoxamine, and their incidence rate varies among authors. The clinical and radiological appearance of these lesions is described in the literature, but microstructural data are still lacking. The aim of our research was to evaluate the microstructure of five tibial biopsy specimens from thalassemic patients with bone lesions (5 cases out of 180 patients followed for the last 10 years, i.e., 2.8%) and two bone biopsy specimens from thalassemic patients with no radiological alteration of the long bones. As control, bone tissue taken from autoptic tibiae of two subjects with no skeletal pathology was used. Using microradiography and X-ray diffraction (XRD), we found a reduced and irregular mineralization of the bone (compared with controls) in thalassemic subjects. Bone tissue microhardness was also significantly reduced. Nevertheless, bone apatite lattice was unaltered and no 'foreign' crystallographic phase was recorded by XRD. In conclusion, all the patients shared a similar picture of abnormal bone, even with no radiological evidence of lesion.

Adolescent↗

PubMed2000

Apoptosis in peri-implant tissue.

The authors examined 54 biopsies taken from the tissue surrounding loosened hip joint prostheses. In situ apoptotic cell identification was performed by the detection of single- and double-stranded DNA breaks that occurred in the early stages of apoptosis. Both types of breaks can be revealed by labeling the free 3'-OH termini with modified nucleotides (fluoresceine-dUTP) in an enzymatic reaction catalyzed by terminal deoxynucleotidyl transferase (TdT). Results were correlated with the presence of wear debris in the tissue and with the use of bone cement for prosthesis fixation. Apoptotic cells were present in a higher percentage in tissue sections where metal particles were present (24% apoptotic cells) if compared to areas where no wear (6%), or plastic wear (2.8%) or ceramic wear (1.5%) was observed. Apoptosis is neither related to bone cement, nor to the time it takes for the implant to fail. Cell death by apoptosis may be important in implants which release metal ions by corrosion or wear and may have been underestimated up to now, as it is a 'clean' way of cell death, leading to limited damage in the surrounding tissues.

Acetabulum↗

PubMed2000

In vitro cytokine production by mononuclear cells exposed to bone cement extracts.

The authors evaluated the ability of bone cement to modify the profile of pro-inflammatory cytokines secreted by the immune cells. Peripheral blood mononuclear cells (PBMC) collected from healthy individuals were cultured with cement extracts and tested to assess the release of IL-1beta, TNFalpha, GM-CSF and IL-6 in both unstimulated and PHA-stimulated PBMC. The cytokine release of unstimulated PBMC was very poor, and in particular the IL-1beta was undetectable: the addition of cement extract increased both TNFalpha and GM-CSF release and decreased IL-6, sometimes significantly. The most recurrent observation in PHA-stimulated PBMCs exposed to bone cement extract was the increase in both IL-1beta and IL-6 release, while both the mean concentration and the index of release of TNFalpha and GM-CSF were changeable. In conclusion our results showed that leachable components of some bone cements can induce in vitro the release of pro-inflammatory cytokines which are known to be involved in the bone resorption associated with aseptic loosening of hip prostheses. These findings allowed us to identify materials endowed with the highest inflammatory power.

Biocompatible Materials↗

PubMed2000

Expression of the CD69 activation antigen on lymphocytes of patients with hip prosthesis.

The aim of the study was to evaluate the sensitization to metals in patients with Co-Cr hip prosthesis. Peripheral blood mononuclear cells (PBMC) were collected from 14 healthy donors and three groups of patients: 10 candidates for primary total joint replacements, 11 patients with well-fixed implant and 13 patients with aseptic loosening of the hip prosthesis. PBMCs were cultured with the metal ions employed for implant manufacturing and the expression of CD69 activation antigen on CD3/T lymphocytes was detected by flow cytometry. Chromium extract increased significantly the expression of CD3/CD69 phenotype in patients with loosening of hip prosthesis. The chromium-induced 'activation index' was higher in patients with loosening of hip prosthesis than in healthy donors and in pre-implant patients. The cobalt-stimulated PBMC of patients with either well-fixed or loosened prosthesis had an 'activation index' significantly higher than healthy donors. The activation index values were used to graduate the PBMC-response as 'normal' (> or = 0.9 and < 2), 'low' (< 0.9) and 'high' (> or = 2): an high-activation index was observed only in chromium-exposed PBMC of patients with prosthesis. Our data show that chromium released from orthopedic implants could be responsible for the lymphocyte sensitization and flow cytometry is an easy and reliable method for monitoring the hypersensitivity state in patients with metal prostheses. Activated lymphocytes in the peri-implant tissue are likely to elicit a localized immune response and contribute to maintain the inflammatory process evolving in the implant failure.

Aged↗

PubMed2000

In vitro testing of ten bone cements after different time intervals from polymerization.

Biological response of cells to implanted bone cement is a fundamental but often neglected issue in successful cemented implants. In this study, ten acrylic bone cements for orthopedics were assayed using two different in vitro testing methods on L929 cells. The cements were mixed as prescribed, cured for either 1 h or 7 days and then extracted in minimum essential medium (MEM) according to the ISO standard for the preparation of samples. For the evaluation of cytotoxicity, the neutral red uptake assay (NRU) and the incorporation of propidium iodide (PI) were used to detect the viability/death of cells. The two methods were shown to be well correlated (p < 0.0001) in the case of both the 1-h and the 7-day extracts. Two cements, i.e. CERIM LT and CMW2, were found to be toxic after 1-h curing through both the spectrophotometric NRU assay and the cytofluorometric assay with PI. After 7-day curing, these two cements, as well as the Zimmer-low viscosity cement, were toxic according to the NRU assay. The toxic effect of all the cements disappeared after dilution of extracts 1:2 with MEM, except in the case of CERIM LT. In the search for the component inducing the toxic effect, the possible contribution of the residual monomer was discarded on the basis of literature data and the influence of various other factors was analyzed, including the contrast medium (barium sulphate or zirconium dioxide) and the concentrations of N,N-dimethyl-paratoluidine and of benzoyl peroxide (< 1% or > or = 1%). Unlike zirconium dioxide, barium sulphate was found to damage the cells at the 1-h endpoint. Benzoyl peroxide at concentration > or = 1% was found to affect cells at the same endpoint, whereas dimethylparatoluidine had no effect regardless of the proportion.

Animals↗

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↗