No genotoxicity of a new nickel-free stainless steel.

Stainless steel is a metallic alloy largely employed in orthopedics, maxillofacial surgery and orthodontic therapy. However, the presence in its composition of a high quantity of nickel, an agent known to trigger toxic, allergic and cancerogenous responses in humans, is cause of some concern. In this study, we have investigated the in vitro mutagenicity and genotoxicity of a new nickel-free stainless steel, namely P558, in comparison to the conventional stainless steel AISI 316L. The cytogenetic effects were evaluated by studying the frequency of Sister Chromatid Exchanges (SCE) and chromosomal aberrations. Ames test was performed to detect the mutagenic activity. Both P558 and AISI 316L did not cause any significant increase in the average number of SCE and in chromosomal aberrations, either with or without metabolic activation. Furthermore, the Ames test showed that the extracts of both P558 and of AISI 316L are not mutagenic. Overall, these findings prove that P558 is devoid of genotoxicity and mutagenicity. The present results, together with other previous interesting observations that P558 promotes osseointegration, suggest that this new nickel-free stainless steel can represent a better alternative to other conventional steel alloys.

In vitro effects on MG63 osteoblast-like cells following contact with two roughness-differing fluorohydroxyapatite-coated titanium alloys.

The chances of integration between an implant and the surrounding bone tissue depend on the surface characteristics of the implant itself. Particularly, chemical composition and surface roughness of the material have emerged as crucial factors in affecting the behaviour of cells in contact with the material. Among various surfaces, calcium phosphate coatings seem to favour a rapid initial integration, but their dissolution by extracellular fluids raises some concern about the long-term stability at the bone-implant interface. Fluorinated apatites are known to be more stable than other ceramic coatings, but, at present, little is known on their effects on human cells. In this study, MG63 osteoblast-like cells were seeded onto two fluorohydroxyapatite (FHA)-coated titanium alloy (Ti6Al4V) materials differing in roughness, respectively, LR-FHA (Ra = 5.6 microm) and HR-FHA (Ra = 21.2 microm). Quantification of the cells in contact with the FHA-coated materials by conventional methods involved some technical difficulties, on which we report. Only the indirect esteem by the measure of total content of proteins and a procedure based on cell count, following a double enzymatic treatment to detach the cells, offered plain results, indicating no significant differences between cellular growth in contact with test materials and with plastic control. Differentiation and functionality of the cells were comparatively evaluated by analysis of alkaline phosphatase activity and osteocalcin production. As far as osteocalcin release is concerned, only slight variations were detected on FHA-coated materials in comparison with the control. Both types of coatings showed a significant increase in alkaline phosphatase activity with respect to the control, the roughest surface exhibiting a more prolonged effect on the time.

Staphylococci in orthopaedic surgical wounds.

From 50 infected surgical wounds of orthopaedic patients, 43 (86%) staphylococcal strains were isolated. 34 of all these staphylococci belonged to Staphylococcus aureus species (i.e. 68 % of all isolates from surgical wounds; 79% of staphylococcal isolates); 9 were coagulase-negative staphylococci (i.e. 21% of all isolates from surgical wounds; 18% of staphylococcal isolates). Among microorganisms isolated from the wounds we also found 2 (4%) of the Enterobacteriaceae family; 2 (4%) of the Pseudomonas genus; 3 (6%) of the Streptococcus genus. Thus, orthopaedic surgical wounds were infected by staphylococci (mainly S. aureus) more frequently than by other micro-organisms. All the staphylococcal strains were screened for methicillin resistance by agar disk diffusion testing and for the presence of mecA gene responsible for methicillin resistance by PCR. 32% of the S. aureus and 33% of the S. epidermidis strains resulted methicillin resistant and mecA-positive. The data confirm the diffusion of methicillin resistant S. aureus in surgical site infections and shows that the so-called "new pathogens", i.e. S. epidermidis and other coagulase-negative staphylococci, also exhibit a frequent and hazardous methicillin-resisting ability.

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.

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.

Quantitative evaluation by image analysis of platelet morphological modifications after contact with polyvinylacetate.

BACKGROUND: In order to investigate platelet activation after contact with artificial materials, which is an important aspect of biocompatibility especially for the blood-contacting devices, platelet morphological modifications and spread area were evaluated by light microscopy and image analysis after contact with glow discharge-treated polybutylene terephthalate coated with a polymer for platelet concentrate filtration. METHODS: A hydrophilic polymer made of partially hydrolyzed polyvinyl-acetate containing polyethylene oxide/poly-propylene oxide copolymer block as lateral chains (PVA) (Biofil S.r.l., Cavezzo, Modena, Italy) was evaluated. After contact with PVA, platelets were allowed to settle on a siliconized slide and then fixed and stained. The specimens were analyzed by image analysis. The percentages of spreading, round and dendritic shapes, as well as the presence of aggregates, were evaluated, and the mean area of the spread platelets was measured. RESULTS: PVA induced significant variations neither in the percentages of shape change distribution, nor of the mean spread area. However it determined a statistically significant reduction in platelets with the area from 60 to 70 mu 2. Such minimal variations agree with the results we obtained in the past, namely a non significant platelet adhesion induced by the same material. CONCLUSIONS: The method confirms the results of platelet adhesion and release reaction (the study of release reaction needs more refined but more expensive methods). However, the study of morphological modifications by image analysis is not suitable for testing materials that induce massive platelet adhesion, because the number of the residual platelets could be too low for the microscopic evaluation.

In vitro investigation of aluminum and fluoride release from compomers, conventional and resin-modified glass-ionomer cements: a standardized approach.

The amount of fluoride release from dental cements necessary for an anticariogenic effect is not established: moreover, the possible toxic effects due to high fluoride and aluminum release are not well known and the results are still controversial. The aim of our study was to evaluate fluoride (F) and aluminum (Al) release from dental cements using a 'standardized approach' according to the end-use of the materials, i.e. biocompatibility testing. Two polyacid-modified resin composites of recent application, commonly called compomers (Dyract and Dyract Cem), were compared with two conventional acid-based (Fuji I, Ketac-Cem) and two resin-modified (Vitremer, Vitrebond) glass-ionomer cements (GICs). All types of cement are used in dentistry and are commercially available. Extracts of the cements into minimum essential medium, after setting over a 1-h (group A) and 1-week (group B) period, were performed. The extraction conditions were rigorously standardized. Mean values +/- standard deviation of F- and Al-levels in such extracts were measured and were expressed as microg g(-1) (micrograms of ions per gram of cement). A great difference in the amount of ion release, both F and Al, was shown among the tested materials. The GICs, as well as Ketac-Cem, released more F and Al than the compomers. All of the materials released the greatest proportion of ions when the extraction was performed in the first hour after mixing (group A). Al- and F-values showed a highly significant positive correlation, independently from the curing time. We conclude that the biological assessment of dental cements can be performed only if a pre-evaluation of the leachables is obtained by applying a standardized protocol which allows a useful comparison between the different materials.

In vitro sister chromatid exchange induced by glass ionomer cements.

The genotoxicity of three glass ionomer cements used in dentistry, manufactured by American (Vitrebond), Japanese (Fuji I), and European (Ketac Cem) companies were examined. The cement components were mixed according to the manufacturers' instructions and allowed to set for two defined times: 1 h or 1 week, before extracting them, as established by ISO standard 10993 part 12. To highlight sister chromatid exchange during mitosis, the extracts then were tested with human peripheral lymphocytes in the presence or absence of metabolic activation with S9 mix. The test performed was a genotoxicity test as provided for in standard EN 30993 part 3. Vitrebond resulted in direct genotoxicity and was strongly cytotoxic both in the extracts performed at 1 h and those at 1 week if they were allowed to set without photoactivation. Fuji I was noncytotoxic and showed only uncertain indirect genotoxicity in the extracts at 1 h; genotoxicity was not present in the extracts at 1 week. Ketac Cem cement was not genotoxic nor was it cytotoxic either at 1 h or 1 week. The authors concluded that of the three cements tested the European cement Ketac Cem passed one of the tests suggested by the EEC standard for assessing genotoxicity.

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.

Detection of mutagenic potential of some glass-ionomer cements through Ames testing.

The mutagenic potential of three commercially available glass-ionomer cements used in dentistry was examined. The cement components were mixed according to the manufacturers indications and set for two defined times: 1 h or, alternatively, 1 wk. Cements B and C set spontaneously; in the case of cement A, the manufacturer suggests the use of a lamp to trigger also a photopolymerization. Photopolymerization, however, was not used. Ames tests were performed on the dimethyl sulphoxide extracts of cements by using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537, TA 1538 and TA 102. Cement A showed mutagenicity only against TA 1537 strain, either in the presence or absence of metabolic activation with microsomial fraction S9. The other two cements showed no mutagenic potential. We conclude that glass-ionomer cements are, on the whole, safe materials from the viewpoint of genotoxicity, and hypothesize that the mutagenicity observed in cement A could depend on its polymerization performed without light activation.

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.

The effects of metal corrosion debris on immune system cells.

Authors evaluated the correlation between immune system and metal ions release in blood of 17 subjects with Cr/Co/Ni joint prostheses. For the purpose Chromium (Cr), Cobalt (Co) and Nickel (Ni) serum levels were measured and, at the same time some immunological parameters (Leukocytes, Lymphocytes and Lymphocytes T, B and Natural Killer cells sub-populations) were evaluated. The results showed a significant decrease of Leukocytes, Lymphocytes and of T Lymphocytes sub-populations. At the same time it was demonstrated a significant increase of Chromium, Cobalt and Nickel levels in patients with joint prostheses as compared to control population (23 patients). In conclusion, ions release from metallic surface of the prostheses is correlated with a depression of immune system. This correlation could depend on a toxic action on immune system caused by the products released by the implant. It could also depend on a lymphocytes compartimentalization in periprosthetic tissues as a consequence of a cell-mediated hypersensitivity reaction towards implants corrosion products.

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.

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.

[In vitro tissue factor production by human endothelial cells put in contact with polyethylene terephthalate with different texture]. / Produzione di tissue factor da parte di cellule endoteliali umane poste a contatto in vitro con polietilene tereftalato a diversa tessitura.

The aim of this study is the evaluation of the in vitro tissue factor production by endothelial cells cultured in the presence of Woven or Knitted Dacron. The spectrophotometric evaluation of the protein content of the cultures and the both indirect assay and enzyme immune assay of tissue factor in cellular lysates were carried out after 72 hour contact between the cells and the materials under examination. The endothelial cell contact with Knitted Dacron did not determine variation in protein content, but it induced a significant increase in tissue factor production. The endothelial cell contact with Woven Dacron determined no significant variations neither in the protein content nor in the tissue factor concentration. It is concluded that Knitted Dacron, through the induction of tissue factor synthesis, can favour the extrinsic pathway of coagulation and therefore the production of thrombi. Woven Dacron, not inducing tissue factor formation, does not activate the extrinsic pathway of coagulation, as for the mechanism considered.

[Variations in the antibiotic sensitivity of bacterial strains after contact with the metals used for the construction of prosthetic implants]. / Variazioni della sensibilità agli antibiotici di alcuni ceppi batterici dopo contatto con metalli impiegati per la costruzione di impianti protesici.

The antibiotic sensitivity of 19 Gram-positive bacterial strains (11 Staphylococcus aureus and 8 Staphylococcus epidermidis) and 16 Gram-negative strains (8 Escherichia coli and 8 Proteus species) was evaluated after contact with stainless steel and with some metals compounding the alloys used for prosthetic devices. The hypothesis was that the resistance to antibiotic therapy of infections associated with prosthetic implants is also due to a modification in the sensitivity of microorganisms. The results, compared to those obtained from control tests, showed only slight variations in the antibiotic sensitivity of the strains put in contact with the metals. In Gram-positive strains, after contact with metals, the increase in sensitivity occurred more frequently than the reduction. In Gram-negative strains, the decrease in sensitivity was more frequent than the increase. Proteus strains showed sensitivity variations more frequently than Escherichia coli strains. Titanium and nickel induced the highest number of variations, both in Gram-positive and Gram-negative strains.

Effects of an acute protein load on urinary albumin excretion in kidney donors.

In six kidney donors with normal baseline urinary albumin excretion (UAE) we studied the behavior of the UAE after 150 g of a meat protein meal and after a carbohydrate meal of equal caloric (1,370 kcal), water (1 liter) and sodium content (51 mEq). The mean creatinine clearance (Ccr) increased significantly after a protein load at the first (p less than 0.01) and second hour (p less than 0.05), while it did not change after the carbohydrate meal. The mean UAE increased significantly after the protein meal at the first (p less than 0.05) and second hour (p less than 0.05), while after the carbohydrate meal the mean values were increased at the first (p less than 0.05), second (p less than 0.05) and third hour (p less than 0.05). Furthermore, the mean values of UAE after the protein meal were significantly higher (p less than 0.05) than those found at the same time after the carbohydrate meal. Diuresis and natriuresis increased significantly after both meals. These findings show that the increased UAE after the protein meal may be due to a further increase in Ccr in the hyperfiltering remaining kidney, while the smaller increase in the UAE observed after the carbohydrate meal may be due to water load and increased urine flow, which impairs albumin tubular reabsorption. The prognostic importance of microalbuminuria after either meal is therefore uncertain.

Keratinophilic fungi isolated from Antarctic soil.

In the present study, 10 soil samples were collected aseptically from an equal number of areas of the Antarctic in the zone occupied by the 1986-1987 Italian expedition for research on keratinophilic fungi. Of particular interest was the isolation of a pathogenic fungus, Microsporum gypseum, from two sites in the base camp occupied by men and by skuas. Trichophyton terrestre was isolated from a site in which people worked and through which penguins and skuas passed. The most widespread fungal species were members of the genus Chrysosporium. Some of these species were isolated but not identified and this part of the study was still be completed. Another significant finding was the absence of fungi in one sample, while in another the widespread and abundant growth in all the seeded dishes of a single species of Chrysosporium. Other species in genera of general diffusion in many environments were also isolated: Aspergillus spp., Malbranchea sp., Mycelia sterilia spp., Paecilomyces sp., Penicillium spp. and Scopulariopsis spp.