Biomedical subjects

D Granchi

Publications and source records attributed to D Granchi.

At least 55 records · Page 3Linked to original sources

PubMed1999

Cd 34+ cells and clonogenicity of peripheral blood stem cells during chemotherapy treatment in association with granulocyte colony stimulating factor in osteosarcoma.

The introduction of aggressive chemotherapy in the treatment of osteosarcoma has improved the long-term outcome for these patients. With the increasing aggressiveness of chemotherapy protocols, hematopoietic growth factors have emerged as useful adjuncts involving, in some cases, rescue by peripheral blood stem cell (PBSC) infusion to assist faster recovery and maintain relative dose intensity. To evaluate the number of PBSCs needed, we analyzed the number of CD34+ cells and hematopoietic progenitor cells in the peripheral blood of 16 patients with osteoblastic, condroblastic and fibroblastic osteosarcoma enrolled in an Istituto Ortopedico Rizzoli-Scandinavian Sarcoma Group (IOR-SSG) pilot study, consisting of two cycles of preoperative high dose chemotherapy. The blood samples were studied at different times. The CD34+ cells were analyzed by flow cytometry and the hematopoietic progenitor cells were analyzed by tissue culture clonogenic assay. In comparing the two courses of chemotherapy, we observed that modification of the mean values of WBC, CD34+ and CFU-GM were very similar. The second course of chemotherapy seemed to induce greater hematological toxicity. All three parameters showed good correlation. The results demonstrated that the best time to collect PBSC by means of leukapheresis is post G-CSF used as rescue after ifosfamide treatment. We verified the ability of G-CSF to mobilize PBSCs in patients with osteosarcoma through cytofluorimetric analysis of CD34+ cells and their clonogenic capability. Moreover, during this preoperative treatment, we identified the best time to collect a sufficient number of PBSCs, that is after 9-10 days of G-CSF treatment following the first cycle of ifosfamide.

Adolescent↗

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

X-ray diffraction of bone at the interface with hydroxyapatite-coated versus uncoated metal implants.

The microstructural characteristics of the newly formed bone tissue at the interface with hydroxyapatite-coated and uncoated stainless steel pins used in an external fracture fixation system have been evaluated. The bone far from the interface was used as a control. Pins were transversally inserted into the diaphyses of sheep tibiae and were loaded in for six weeks. Three sheep received coated pins and two received uncoated pins. Crystallographic habit and mineralization of the implant-facing bone were evaluated. Moreover, lattice parameters of bone apatite were measured and hydroxyapatite (HA) coating degradation was investigated, by means of conventional and microbeam X-ray diffraction (XRD). In coated pins, six weeks after the implantation the newly formed bone tissue at the interface did not reach complete maturation, but the presence of the implant did not alter the apatite lattice structure; the lattice parameters did not show statistically significant variations with respect to those observed in the control bone. In uncoated pins, bone tissue rarely appeared totally mineralized and lattice parameters were significantly different with respect to those observed in the bone far from the implant. HA particles were observed spreading in the bone-facing coated pins; the XRD pattern of bone apatite surrounding HA particles was unmodified. It was concluded that HA coatings improved the bone remodelling process during pin fixation in comparison to uncoated pins and did not alter the crystallographic habit of apatite.

Journal Article↗

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

CD62, thromboxane B2, and beta-thromboglobulin: a comparison between different markers of platelet activation after contact with biomaterials.

The authors examined the modifications of some markers of platelet activation after contact with biomaterials. Glycoprotein GMP-140 (CD62) was evaluated by flow cytometry; beta-thromboglobulin (beta-TG) and thromboxane B2 (TXB2) were determined by radioimmunoassay. Polyethylene terephthalate (PET) induced a remarkable platelet adhesion and a significant increase in beta-TG and TXB2, with no increase in CD62 on the nonadherent platelets. Pyrolytic carbon-coated PET (PC) did not induce platelet adhesion after 15 min of contact, but a significant increase in CD62 was detected. After 30 min a significant increase in platelet adhesion as well as the release of beta-TG and TXB2 were noted. The increase was lower than that observed for uncoated PET, and after 30 min of contact with PC the increase no longer was observed.

Biocompatible Materials↗

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↗

PubMed1996

Assessment of metal extract toxicity on human lymphocytes cultured in vitro.

In this study the toxic effects of chromium, nickel, and cobalt extracts on in vitro cultured lymphocytes were evaluated. Graphite furnace atomic absorption spectrometry was used to measure the ion concentration. After serial dilution of the extracts, the viability of lymphocytes at 24, 48, and 72 h was estimated by flow cytometry, including propidium iodide staining and light scatter property assessment, and by MTT reduction test. The results of the investigation allowed us to conclude that 1) standardization of the procedure for preparing extracts is fundamental to obtaining repeatability of results; 2) the toxicity of an extract cannot be evaluated with a single viability assay; a combination of functional and structural tests is required; 3) when methods based on enzymatic reactions are performed, e.g. MTT test, it is advisable to replace the extract containing metal ions with fresh medium in order to avoid any interference with viability testing; 4) the amount of Co and Ni in the extract is similar, but the Cr release is very poor; 5) the lower toxicity of Cr extract probably is due to the lower ion concentration; 6) the assessment of 50% cytotoxic concentration (TC50) allows quantification of materials toxicity and comparison of various metals; and 7) the determination of a noncytotoxic concentration, i.e., a concentration lower than TC10, is required for subsequent investigation of cell functions because such studies can be carried out only on viable cell population.

Cell Survival↗

PubMed1996

Cytokine production and adhesive protein expression by endothelial cells after contact with polyethylene terephthalate.

The authors have evaluated adhesive protein expression and cytokine production by human umbilical vein endothelial cells cultured in contact with polyethylene terephthalate (PET). ELAM-1, ICAM-1 and VCAM-1 expression was determined by flow cytometry; the concentration of interleukin-1 alpha (IL-1 alpha), interleukin-6 (IL-6), granulocyte colony stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF) in the supernatant was determined by enzyme immunoassay. The contact with PET determined a significant increase in ELAM-1 expression and insignificant increase in cytokine production, demonstrating that PET had a limited capability to stimulate endothelial cells in a pro-inflammatory sense.

Cell Adhesion Molecules↗

PubMed1996

In vitro assessment of phagocytosis of bovine collagen by human monocytes/macrophages using a spectrophotometric method.

The use of a wound dressing with covering and haemostatic properties significantly improves wound healing. In this study, a lyophilized bovine collagen sponge used for the treatment of wounds and ulcerae has been tested in a cell culture system. Phagocytosis of collagen fragments by human blood monocytes/macrophages has been investigated. For the assessment of collagen ingestion by mononuclear phagocytes, a picrosirius dye specific for collagen molecules has been used. By adapting this histochemical technique to microplate cell culture system, replicate monocyte cultures are assayed. Collagen content is determined by evaluating spectrophotometrically at 540 nm the absorbance of a sirius red/picric acid solution. Using this simple and sensitive method, the phagocytosis of bovine collagen by LPS-stimulated monocytes/macrophages has been ascertained.

Analysis of Variance↗

PubMed1996

Application of a combination of neutral red and amido black staining for rapid, reliable cytotoxicity testing of biomaterials.

Cell viability and growth for cytotoxicity evaluation of materials for prosthetic devices has been tested using various methods. The aim of this study was to extend the choice of reliable methods to quantify cytotoxicity of materials in vitro. By measuring both viability and growth of cells exposed to biomaterials in vitro, two different parameters are analysed and quantified upon reading of the absorbance of coloured solutions in a spectrophotometer. Neutral red uptake and amido black staining of cells have been used for cell viability and cell number measurement, respectively: they have been found to be well correlated with the number of surviving cells. These methods have been adjusted to a 96-well microplate cell culture system and re-evaluated as simple and reliable methods for the quantitative assessment of biomaterial effect on cells.

Amido Black↗