[In vitro evaluation of bactericidal activity of an over-heated saturated dry atomized steam electro-medical device on inert contaminated surfaces]. / Valutazione in vitro dell'efficacia battericida di un'apparec- chiatura elettro-medicale con erogazione di vapore saturo secco surriscaldato ad alta temperatura applicata a superfici contaminate.

Surface disinfection together with cleaning practises and aseptic procedures are established measures for effective prevention of infectious diseases reducing infection risks. In this study, we evaluated the bactericidal effectiveness in vitro of the electro-medical device Sani System, an over-heated saturated dry-steam disinfection system, against predetermined bacterial load dried on inert surfaces. In particular we have tested different materials, representative of operating rooms furnishing and walls commonly used in healthcare setting, with Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii. As a result of treatment, the mean bacterial load on all the test surfaces was reduced by a factor of 102 in the contact plate experimental study and by a factor of 10 in the total bioburden experimental study. The Sani System Polti proved to be efficacious in killing 100% of the bacteria in all experimental conditions, therefore it could help to reduce the risk of spreading nosocomial infections in healthcare facilities.

Study of Staphylococcus aureus adhesion on a novel nanostructured surface by chemiluminometry.

In recent years the progress in the field of nanotechnologies has offered new possibilities to control the superficial features of implant materials down to a nanoscale level. Several studies have therefore tried to explore the effects of nanostructured biomaterial surfaces on the behavior of eukaryotic cells. However, nanotopography could exert an influence also on the behavior of prokaryotic cells, with relevant implications concerning the susceptibility of implant surfaces to infection. Aim of this study was to examine the behavior of Staphylococcus aureus on polyethylene terephthalate (PET) surfaces either cylindrically nanostructured (PET-N) or flat ion-etched (PET-F), and on tissue culture-grade polystyrene (PS). Microbial adherence was assessed by chemiluminometry under 4 different conditions: (a) bacteria suspended in MEM medium, (b) bacteria in MEM supplemented with 10% fetal bovine serum (FBS), (c) test surfaces preconditioned in FBS, and (d) post-exposure of colonised surfaces to serum-supplemented MEM. Under all circumstances, PET-F and PET-N specimens showed identical bacterial adhesion properties. In the absence of serum, all 3 test materials showed a very high adhesivity to microbial cells and both PET surfaces exhibited greater adhesion than PS. On the contrary, the presence of 10% serum in solution significantly affected cell behavior: the number of microbial cells on all surfaces was drastically reduced, and the adhesion properties of PET surfaces with respect to PS were reversed, with PET being less adhesive. Overall, the specific cylindrical nanostructures created on PET did not significantly influence microbial behavior. Ongoing studies are verifying whether other nanotopographies with different geometry could have more substantial effects.

Prevalence and antibiotic resistance of 15 minor staphylococcal species colonizing orthopedic implants.

Several species belonging to Staphylococcus genus (non Sau/ non Sep species) exhibit increasing abilities as opportunistic pathogens in colonisation of periprosthesis tissues. Here we report on antibiotic resistance of 193 strains, belonging to non Sau/ non Sep species, consecutively collected from orthopedic implant infections in a period of about 40 months. The 193 strains (representing 17% of all staphylococci isolated) were analysed for their antibiotic resistance to 16 different drugs. Five species turned out more prevalent, ranging from 1 to 5%: S. hominis (4.2%), S. haemolyticus (3.7%), S. capitis (2.7%), S. warneri (2.6%), and S. cohnii (1.6%). Among these, the prevalence of antibiotic resistance to penicillins was similar, ranging from 51% to 66%. Conversely, significant differences were observed for all the remaining antibiotics. For S. haemolyticus the resistances to oxacillin and imipenem, the four aminoglycosides and erythromycin were at least twice that of the other three species which were compared. S. warneri was on the contrary the species with the lowest occurrence of resistant strains. Ten species appeared only rarely at the infection sites: S. lugdunensis, S. caprae, S. equorum, S. intermedius, S. xylosus, S. simulans, S. saprophyticus, S. pasteuri, S. sciuri, and S. schleiferi. The behaviours of these species, often resistant to penicillins, were individually analysed. Differences in both the frequencies and the panels of antibiotic resistances observed among the non Sau/ non Sep species: i) suggest that horizontal spreading of resistance factors, if acting, was not sufficient per se to level their bio-diversities; ii) highlight and confirm the worrisome appearance within the Staphylococcus genus of emerging ""new pathogens"", not homogeneous for their virulence and antibiotic resistance prevalence, which deserve to be recognised and treated individually.

Etiology of implant orthopedic infections: a survey on 1027 clinical isolates.

In spite of the recent achievements derived from modern protocols of prophylaxis, orthopedic surgical infections still remain unacceptably frequent, especially in light of the often devastating outcomes of septic complications. The spectrum and the prevalence of the bacteria most frequently involved in orthopedic infections are here explored, with particular reference to those infections associated to implant biomaterials, which were grouped based on device typology. During a 30 months period (from September 2000 to April 2003), 1027 microbial strains were consecutively isolated from 699 patients undergoing revision surgery at the Rizzoli Orthopedic Institute. 775 (75.5%) of all these microorganisms were identified as belonging to the Staphylococcus genus, 82 (8%) to the Enterobacteriaceae family, 75 (7.3%) to the Pseudomonas genus, 54 (5.3%) to the Enterococcus genus and 20 (1.9%) to the Streptococcus genus. While confirming the importance of staphylococci as the most diffuse cause of infection, our data indicate an unexpectedly high prevalence of S. epidermidis on infected hip and knee arthroprostheses, respectively of 42% and 44%. The spectrum of bacteria infecting either internal or external fracture fixation devices appears to differ from that of hip and knee arthroprostheses and more closely resembles that of infections non-associated to medical devices, being characterized by a relatively higher prevalence of Staphylococcus aureus (over 40%) and Pseudomonas aeruginosa. Enterobacteriaceae and members of the Streptococcus and Corynebacterium genera are frequently associated with implants in which surgical incisions were made near the perineum, determining a completely altered spectrum.

Nitric oxide synthase in tissues around failed hip prostheses.

Nineteen patients who had undergone hip revision surgery for aseptic loosening of joint prostheses were studied. Tissue samples were harvested at the interface between bone and implant, either at the stem or at the cotyle level. Immunohistochemistry was performed on tissue sections to detect nitric oxide synthase (NOS), the enzyme which enables the synthesis of nitric oxide (NO), a molecule which can activate bone resorption. Quantitative analysis of the positive cells and correlation with the presence of particulate wear debris and radiological data were performed. The authors observed a trend towards a moderate increase in positive cells due to inducible NOS in tissues containing particulate wear debris, especially of a plastic material. This increase, however, did not achieve statistical significance. On the contrary, there was a statistical correlation between iNOS (inducible NOS) and the severity of osteolysis around the prosthetic implant. Pharmacological control of the biosynthesis of NO may be considered in the prevention or treatment of loosening.

Congo red agar plate method: improved accuracy and new extended application to Staphylococcus aureus.

In the last decade an increasing number of research studies have focused on the role of slime formation in Staphylococcus epidermidis and, more recently, also in S. aureus. In this context, much attention is being paid to evaluating the prevalence of slime production among bacteria strains isolated from clinical infections in an attempt to assess the role and the diagnostic value of this well recognised virulence marker. Such types of investigations require reliable techniques to identify slime producing strains. For years, even though based on a subjective chromatic evaluation, the Congo red agar plate (CRA) represented a reference phenotypic test for S. epidermidis. Only with the new introduction of PCR-based techniques, able to specifically identify the genes necessary for slime production, did the accurate genetic classification of slime producing bacteria become possible. In the present investigation, a comparison with new PCR methods confirmed the validity of the classic CRA test, implemented with minor refinements. Thanks to a few modifications it was also possible to adapt and extend the CRA test, making it also suitable to screen S. aureus strains.

Adhesion to a polymeric biomaterial affects the antibiotic resistance of Staphylococcus epidermidis.

The antibiotic-resistance both of adherent bacteria to polymethylmethacrylate (PMMA) and of bacteria which, although exposed to the material, had not undergone adhesion, was measured as bacterial growth inhibition area onto a plate antibiogram, according to Kirby-Bauer and using a dedicated image analyzer system. The adhesion onto PMMA induces a marked (about 30%) increase in resistance to beta-lactam antibiotics (cefamandole, cefazolin, imipenem and ampicillin) and a lower (about 15%) but significant increase to the macrolide erythromycin, to two aminoglycosides (amikacin, netilmicin) and to vancomycin, chloramphenicol and trimethoprim-sulfamethoxazole.

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.

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.

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.

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.

Hydroxyapatite-coated orthopaedic screws as infection resistant materials: in vitro study.

The authors evaluated in vitro the adherence of a Staphylococcus epidermidis strain to hydroxyapatite-coated stainless-steel screws-used in orthopaedic surgery for external fracture fixation-in comparison with the adherence to uncoated screws. Evaluations were also performed on analogous groups of screws immersed for 72 and 168 h in a solution at 37 degrees C, in order to simulate the interstitial fluid in a simplified way. Adherence values on coated prostheses resulted significantly lower compared with those observed on metal prostheses, both in basal conditions and after immersion in saline solution. Moreover, both on coated and on uncoated screws a significant reduction in bacterial adherence was noted related to the duration of the prosthesis permanence in saline solution.

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.

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.

Effects of orally administered bisphosphonates on bone loss in a disuse osteopenia model involving the rat.

OBJECTIVE: This study was carried out to evaluate the effectiveness of two newly synthesized bisphosphonates (BPs) [Alendronate (4-amino-1-hydroxybutylidene-1, 1-bisphosphonic acid (AHBuBP)) and Neridronate (6-amino-1-hydroxyhexylidene-1,1-bisphosphonic acid (AHHexBP)], administered orally, in reducing experimentally induced bone loss. METHODS: Unilateral sciatic nerve section was performed on the Sprague-Dawley rat to induce osteopenia in one of the hind limbs. Histomorphometric measurements of the tibial trabecular bone and femur ash content determinations were effected to assess the degree of osteopenia. For comparison Chlodronate (dichloromethylene-1-bisphosphonic acid (Cl2MBP) was employed as the reference drug. CONCLUSIONS: The results of this investigation show that both BPs were significantly active in reducing the osteopenic process in the involved limb and were more active than Chlodronate.

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.

Microhardness of bone at the interface with ceramic-coated metal implants.

We evaluated bone microhardness at the interface with hydroxyapatite-coated stainless-steel pins used in an external fracture fixation system. Pins were transversally inserted into the diaphyses of sheep tibiae and were loaded in for 6 weeks. Uncoated pins were implanted as controls. Microhardness analysis, based on the measure of the resistance of the bone to the penetration of a small diamond pyramid, yielded an accurate and reproducible measure of the mineralization degree and of the orientation of collagen fibers. Bone tissue close to the pin is less hard than bone tissue far from it. Moreover, the presence of hydroxyapatite coating on the pins did not significantly affect bone hardness; actually, the mean hardness at the interface with the pins was 56.9 Vickers degrees, whereas at the interface with the uncoated pins it was 62.2. It can be concluded that, 6 weeks postsurgery, the bone growing into the threadings of a loaded screwed implant reached maturity at a degree lower than that of the host bone in both uncoated and coated implants.

Microstructural investigation of bone-cement interface.

We evaluated the microstructural characteristics of newly formed bone tissue at the interface with cement. The bone-cement interfaces of the femoral components of nine hip prostheses retrieved after loosening were investigated by means of X-ray diffraction on microareas and microhardness. The bone far from the interface of two stable prostheses was used as a control. The newly formed bone adjacent to cement in the loosened prostheses showed a maturity degree lower than that of bone adjacent to cement in stable prostheses. The lattice parameters of bone apatite did not show significant variations as compared to the reference values. Bone trabeculae at the interface with loosened prostheses often showed an osteoid lining characterized by a strongly demineralized lamellar and haversian structure. Radioopaque cement particles are sometimes found in the trabecular bone tissue around the prosthesis.

X-ray diffraction of newly formed bone close to alumina- or hydroxyapatite-coated femoral stem.

The study was focused on the bone tissue response to two types of ceramic coating [alumina and hydroxyapatite (HA)] obtained with a plasma-spray technique. The HA coating was performed on titanium stems, while the alumina coating was on chromium-cobalt-molybdenum stems. The investigation was carried out by means of micro-area X-ray diffractometric analyses and microdiffractometric analyses at the bone-implant interface. Moreover, the coating before and after implantation was analysed by means of the conventional powder X-ray diffractometric technique. This study demonstrated that 1 yr after implantation in animals the newly formed bone adjacent to the HA-coated hip prosthesis stems was mineralized as much as the pre-existing bone, while 24-64 months after surgery in humans the newly formed bone close to the alumina-coated hip prosthesis stems showed a lamellar and Haversian structure strongly demineralized, probably due to the release of aluminum ions from the alumina covering. The authors conclude that the X-ray diffraction allows the evaluation of the structural modifications of the ceramic coating, the bone formation rate close to the coating and the chemical nature of the particles released from the coating.