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Objectives To describe clinical characteristics, management and outcome of individuals with coronavirus disease 2019 (COVID-19); and to evaluate risk factors for all-cause in-hospital mortality. Methods This retrospective study from a University tertiary care hospital in northern Italy, included hospitalized adult patients with a diagnosis of COVID-19 between 25 February 2020 and 25 March 2020. Results Overall, 317 individuals were enrolled. Their median age was 71 years and 67.2% were male (213/317). The most common underlying diseases were hypertension (149/317; 47.0%), cardiovascular disease (63/317; 19.9%) and diabetes (49/317; 15.5%). Common symptoms at the time of COVID-19 diagnosis included fever (285/317; 89.9%), shortness of breath (167/317; 52.7%) and dry cough (156/317; 49.2%). An ‘atypical' presentation including at least one among mental confusion, diarrhoea or nausea and vomiting was observed in 53/317 patients (16.7%). Hypokalaemia occurred in 25.8% (78/302) and 18.5% (56/303) had acute kidney injury. During hospitalization, 111/317 patients (35.0%) received non-invasive respiratory support, 65/317 (20.5%) were admitted to the intensive care unit (ICU) and 60/317 (18.5%) required invasive mechanical ventilation. All-cause in-hospital mortality, assessed in 275 patients, was 43.6% (120/275). On multivariable analysis, age (per-year increase OR 1.07; 95% CI 1.04–1.10; p Conclusions COVID-19 mainly affected elderly patients with predisposing conditions and caused severe illness, frequently requiring non-invasive respiratory support or ICU admission. Despite supportive care, COVID-19 remains associated with a substantial risk of all-cause in-hospital mortality.

The majority of patients hospitalized for heart failure (HF) are admitted to internal medicine (IM) rather than to cardiology (CA) units, but to date few studies have analyzed the characteristics of these two populations. In this snapshot survey, we compared consecutive patients admitted for HF in six IM units vs. one non-intensive CA unit. During the 6-month survey period, 467 patients were enrolled (127 in CA, 27.2% vs. 340 in IM, 72.8%). IM patients were almost 10 years older (CA 75 ± 10, IM 82 ± 8 years; p

Arterial embolization, aimed at the mechanical occlusion of tumor-feeding vessels, represents a satisfactory palliative therapy for bone metastases. In this study, we evaluated if the circulating levels of three factors related to the metastatic process change in response to embolization. Seven patients who underwent embolization of a single skeletal metastasis from carcinomas were analyzed prospectively. Circulating levels of Vascular Endothelial Growth Factor A (VEGF-A), Fibroblast Growth Factor 2 (FGF-2), and Tartrate-Resistant Acid Phosphatase-5b Isoform (TRACP5b) were evaluated before and after embolization at 1, 3, and 6 months. According to morphological and clinical evaluations, all the embolizations were successful. VEGF-A and TRACP5b did not show significant changes after the treatment. On the contrary, FGF-2 signifi- cantly decreased 1 month after the treatment. FGF-2 appears as a promising candidate for monitoring the efficacy of emboli- zation in patients with osteolytic metastases.

The most significant results in experimental and clinical orthopaedic research in Italy within the last three years have been primarily in major congenital diseases, bone tumors, regenerative medicine, joint replacements, spine, tendons and ligaments. The data presented in the following discussion is comparable with leading international results, highlighting Italian orthopaedic research excellemce as well as its shortcomings.

Blood vessels have a fundamental role both in skeletal homeostasis and in bone repair. Angiogenesis is also important for a successful bone engineering. Therefore, scaffolds should be tested for their ability to favour endothelial cell adhesion, proliferation and functions. The type of endothelial cell to use for in vitro assays should be carefully considered, because the properties of these cells may depend on their source. Morphological and functional relationships between endothelial cells and osteoblasts are evaluated with co-cultures, but this model should still be standardized, particularly for distinguishing the two cell types. Platelet-rich plasma and recombinant growth factors may be useful for stimulating angiogenesis.

Platelet-rich plasma is used to accelerate bone repair for the release of osteogenic growth factors from activated platelets. To date, the effects on osteoclasts have been only scarcely investigated, even though these cells are crucial for bone remodeling. The aim of this research was the evaluation of the effects of thrombin-activated platelets (PRP) on osteoclastogenesis from human blood precursors. We evaluated both the ability to influence osteoclast differentiation induced by the receptor activator of nuclear factor-kappaB ligand (RANKL), and the ability to induce osteoclast differentiation without RANKL. In both assays, the incubation with PRP supernatant at 10% did not significantly affect the formation of tartrate-resistant acid phosphatase (TRACP)-positive multinucleated cells that were able to form the F-actin ring. However, when PRP at 25 and 50% was added to the medium without RANKL, the generation of TRACP-positive multinucleated cells was inhibited. PRP, even at 10%, reduced the osteoclast-mediated bone collagen degradation, suggesting inhibition of osteoclast activation. Similarly, after incubation with PRP supernatant, calcitonin receptor mRNA was lower than the untreated samples. In conclusion, PRP at 10% interfered with the complete differentiation process of human osteoclast precursors. At higher concentration it impaired osteoclast formation also at an early stage of differentiation.

A total of 29 consecutive knee joint arthroplasties in 24 patients who underwent previous high tibial osteotomy (HTO) for medial unicompartment osteoarthritis of the knee and followed up for a mean of 97 months were compared with a control group of 28 patients with 29 primary total knee arthroplasty (TKA) without previous HTO. Results for the osteotomy group were satisfactory in 96.5% of cases. In one patient loosening of the implant occurred after 37 months, which required prosthesis revision. Three patients underwent a further operation of secondary patella resurfacing for patella pain. The group without osteotomy reported a similar percentage of satisfactory results.

Alternative bearing surfaces for total hip arthroplasty, such as metal-on-metal and ceramic-on-ceramic, offer the potential to reduce mechanical wear and osteolysis. In the short and medium term, the second generation of metal-on-metal bearings demonstrated high systemic metal ion levels, whereas ceramic-on-ceramic bearings showed the lowest ones. We aimed to verify whether the long-term ion release in metal-on-metal subjects was still relevant at a median 10-year follow-up, and whether a fretting process at the modular junctions occurred in ceramic-on-ceramic patients and induced an ion dissemination. Serum levels were measured in 32 patients with alumina-on-alumina implants (group A), in 16 subjects with metal-on-metal implants (group B), and in 47 healthy subjects (group C). Group B results were compared with medium-term findings. Cobalt and chromium levels were significantly higher in metal-on-metal implants than in ceramic-on-ceramic ones and controls. Nevertheless, ion levels showed a tendency to decrease in comparison with medium-term content. In ceramic-on-ceramic implants, ion values were not significantly different from controls. Both in groups A and B, aluminum and titanium release were not significantly different from controls. In conclusion, negligible serum metal ion content was revealed in ceramic-on-ceramic patients. On the contrary, due to the higher ion release, metal-on-metal coupling must be prudently considered, especially in young patients, in order to obtain definitive conclusions.

Materials used for total knee arthroplasty (TKA), may elicit an immune response whose role in the outcome of the arthroplasty is still unclear. The aim of this study was to evaluate the frequency of sensitization in patients who had undergone TKA, and the clinical impact of this event on the outcome of the implant. Ninety-four subjects were recruited, including 20 patients who had not yet undergone arthroplasty, 27 individuals who had a well-functioning TKA, and 47 patients with loosening of TKA components. Sensitization was detected by using patch testing including haptens representative of cobalt-based alloys (CoCrMo), titanium-based alloys (TiAlV), and bone cements. The frequency of positive skin reactions to metals increased significantly after TKA, either stable or loosened (No Implant 20%; Stable TKA 48.1%, p = 0.05; Loosened TKA 59.6%, p = 0.001, respectively). We found a higher frequency of positive patch testing to vanadium in patients who had a Stable TKA with at least one TiAlV component (39.1%, p = 0.01). The medical history for metal allergy seems to be a risk factor, because the TKA failure was fourfold more likely in patients who had symptoms of metal hypersensitivity before TKA. The prognostic value was supported by survival analysis, because in these individuals the outcome of the implant was negatively influenced (the logrank test Chi square 5.1, p = 0.02). This study confirms that in patients with a TKA the frequency of positive patch testing is higher than in the normal population, although no predictive value is attributable to the sensitization because patch testing was not able to discriminate between stable and loose implants. On the contrary, the presence of symptoms of metal allergy before implantation should be taken into account as a potential risk factor for TKA failure.

BACKGROUND: Previous studies have suggested that the balance between receptor activator of nuclear factor-kappaB ligand (RANKL) and its decoy-receptor osteoprotegerin (OPG) in local tissue seems to play a crucial role in the loosening of the total hip replacement. The aim of this study was to evaluate whether the circulating levels of OPG and RANKL, as well as their ratio, could be different in patients with aseptic loosening compared with patients with stable implants. METHODS: One hundred and twenty-eight subjects were recruited. They included thirty-nine patients with osteoarthritis who had not yet undergone total hip arthroplasty, thirty-three patients who had undergone total hip arthroplasty and had clinically and radiographically stable implants, thirty-six patients with aseptic loosening of total hip arthroplasty components, and twenty healthy volunteers. Serum levels of OPG and RANKL were measured with use of an immunoenzymatic method, and in each individual the OPG-to-RANKL ratio was calculated. RESULTS: In every group, a significant correlation was detected between OPG concentration and age (r = 0.58, p < 0.0001), especially in individuals older than fifty years, while gender and underlying disease were not found to influence serum levels of the tested parameters. In comparison with the levels in healthy donors and patients with a stable total hip replacement, the serum levels of OPG were increased in the patients who had not yet had an arthroplasty, those with aseptic loosening of a total hip replacement, and those with a cemented total hip replacement. Moreover, the OPG serum level provided good diagnostic accuracy in detecting the implant failure. A correlation was found between the sum of the osteolytic areas seen radiographically around the femoral stem and the RANKL level (r = 0.38, p = 0.02) and the OPG-to-RANKL ratio (r = -0.29, p = 0.04). CONCLUSIONS: An increase in OPG levels may reflect a protective mechanism of the skeleton to compensate for the osteolytic activity that occurs in severe osteoarthritis and in aseptic loosening. Prospective studies are needed to determine whether serum OPG levels could be used as markers for monitoring the stability of the implant, as well as for predicting aseptic loosening. LEVEL OF EVIDENCE: Diagnostic study, Level III. See Instructions to Authors for a complete description of levels of evidence.

Current methods for the replacement of skeletal tissue involve the use of autografts, allografts and, recently, synthetic substitutes, which provide a proper amount of material to repair large bone defects. Engineered bone seems a promising approach, but a number of variables have to be set prior to any clinical application. In this study, four different poly caprolactone-based polymers (PCL) were prepared and tested in vitro using osteoblast-like Saos-2 cells. Differences among three-dimensional polymers include porosity, addition of hydroxyapatite (HA) particles, and treatment with simulated body fluid. Biochemical parameters to assess cell/material interactions include viability, growth, alkaline phosphatase release, and mineralization of osteoblastic cells seeded onto three-dimensional samples, while their morphology was observed using light microscopy and SEM. Preliminary results show that the polymers, though degrading in the medium, have a positive interaction with cells, as they support cell growth and functions. In the short-term culture (3-7 days) of Saos-2 on polymers, little differences were found among PCL samples, with the presence of HA moderately improving the number of cells onto the surfaces. In the long term (3-4 weeks), it was found that the HA-added polymers obtained the best colonization by cells, and more mineral formation was observed after coating with SBF. It can be concluded that PCL is a promising material for three-dimensional scaffold for bone formation, and the presence of bone-like components improves osteoblast activity.

We studied the plasma levels of coagulation inhibitors, of fibrinolysis and PDGF-AB, in patients with aseptic loosening of the hip replacement. 23 patients having loose hip prostheses were compared to patients having 15 stable hip prostheses, and 26 undergoing primary hip replacement. The levels of the coagulation inhibitors antithrombin III and protein C were determined by chromogenic assays. Fibrinolysis was evaluated by the changes in fibrin degradation products (D-dimer), determined by enzyme immunoassay, and in the plasminogen activator inhibitor-1 (PAl-1), by enzymatic assay. PDGF-AB was determined by enzyme immunoassay. In patients with failed prostheses, we found fibrinolysis activation, as shown by a statistically significant increase in D-dimer and a significant decrease of PAI-1. No significant differences were obseved in antithrombin III, protein C, and PDGF-AB. PAI-1 and D-dimer assays in failed prostheses may be useful for the pathogenetic evaluation, because the continuous inflammatory stimulus associated with fibrin deposition may also affect the systemic levels.

A copolymer of L-lactic acid and e-caprolactone (PLLACL) was synthesized with the aim of preparing a bioartificial, small-diameter and partially resorbable vascular graft. The material was submitted to surface functionalizations (i.e. chemical modification by means of hydrolytic 'etching' and plasma discharge) to promote endothelial cell (EC) adhesion and growth avoiding platelet adhesion or coagulation factor absorption. Furthermore, the behaviour of human microvascular endothelial cells (HMVEC) seeded on the untreated and treated copolymer is described, as well as the platelet adhesion and the modifications of coagulation factors determined by the copolymer itself. PLLACL in its native state provided little support for EC adhesion. Improved EC adherence was obtained when functional groups were provided on the polymer surface by surface chemical hydrolysis. HMVEC seeded and cultured on the polymer surface did not show any ultrastructural alteration, thus demonstrating the absence of the polymer cytotoxic...

The expression of thrombomodulin after contact with CMW 1 bone cement extracts was studied in human umbilical vein endothelial cells. Cement extracts after 1 h and 7-day curing induced no significant variations in thrombomodulin antigen levels and in mRNA expression. Significant increase of thrombomodulin was observed when endothelial cells were treated with all-trans retinoic acid (ATRA). ATRA induced the increase of thrombomodulin also in cells incubated with cement extracts. These results suggest that CMW 1 bone cement does not impair the expression of thrombomodulin in endothelial cells.

The aim of the study was to evaluate some aspects of the immunocompatibility of 10 acrylic bone cements. Mononuclear cells harvested from healthy individuals were cultured with cement extracts which were tested to assess their effect on the viability of lymphocytes, unstimulated and phytohaemoagglutinin (PHA)-stimulated, activating resting lymphocytes, and changing the reactivity of PHA-stimulated lymphocytes. After 24 h the extracts did not increase the percentage of dead cells in unstimulated or PHA-stimulated lymphocytes. The early apoptotic events of culture were evaluated after 4 and 24 h in PHA-stimulated lymphocytes: at 4 h three cements, namely Zimmer-dough type, Palacos s R and CMW-1, increased significantly the percentage of apoptotic cells, while at 24 h no differences were found. Cement extracts did not activate the resting lymphocytes, whereas the response of the PHA-stimulated cells was significantly modified. All cements decreased the expression of the interleukin 2 receptor (CD25) and the lymphocyte proliferation, whereas only two materials (Zimmer-dough type, CMW 1) affected the expression of early activation antigen (CD69). These findings show that the products released from bone cement are not able, by themselves, to elicit a specific immune response; on the contrary they hamper the function of lymphocytes activated by an exogenous stimulus. r 2001 Elsevier Science Ltd. All rights reserved.

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.

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.

The aim of this research was to evaluate the effect of polyethylene terephthalate (Woven Dacron) on the expression of endothelial integrins. Human umbilical vein endothelial cells were cultured on the material for 24 h. The integrins VLA-2 (α2β1-CD49b/CD29), receptor for laminin and collagen, VLA-5 (α5β1-CD49e/CD29), receptor for fibronectin, VLA-6 (α6β1-CD49f/CD29), receptor for laminin, and αVβ3-CD51/CD61 (receptor for vitronectin) were evaluated by flow cytometry. After contact with polyethylene terephthalate, a slight but significant decrease in the percentage of both CD29 and CD49e positive cells was observed, which suggests a lower number of cells expressing the fibronectin receptor α5β1. Moreover, a significant increase in the mean channel for CD49b and for the vitronectin receptor CD51/CD61 was observed. The reduction in the fibronectin receptor could account for the poor endothelialization observed in vivo on polyethylene terephthalate. The increased expression of the vitronectin receptor, favoring the migration of smooth muscle cells, could give some information about the pathogenesis of intimal hyperplasia, which is a complication of vascular grafts. © 2001 Kluwer Academic Publishers

In order to evaluate if carbon coated polyethylene terephthalate (C-PET) could favor inflammatory reactions, the expression of interleukin-6 (IL-6) by cultured human umbilical vein endothelial cells was tested in vitro. The cultures were put in contact with C-PET for 1, 24, 48 and 72 h. The same cells cultured on tissue culture-treated polystyrene without biomaterials were tested as the negative control; the same cells incubated with LPS were the positive control. The level of IL-6 in the conditioned medium was tested by enzyme immunoassay; the mRNA expression was evaluated by RT-PCR with specific primers. The cultures incubated with C-PET produced non significantly different amount of IL-6 compared to the negative control and did not induce the expression of IL-6 specific mRNA. LPS induced a significantly higher release of IL-6 in the medium and the expression of mRNA after 24, 48 and 72 h. We conclude that C-PET does not stimulate the synthesis of IL-6 and therefore does not favor inflammatory reaction through the release of this cytokine. © 2001 Kluwer Academic Publishers

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.

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.

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.

The authors evaluated the ability of bone cement to modify the pro"le of pro-in#ammatory 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-1b, TNFa, 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-1b was undetectable: the addition of cement extract increased both TNFa and GM-CSF release and decreased IL-6, sometimes signi"cantly. The most recurrent observation in PHA-stimulated PBMCs exposed to bone cement extract was the increase in both IL-1b and IL-6 release, while both the mean concentration and the index of release of TNFa 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-in#ammatory cytokines which are known to be involved in the bone resorption associated with aseptic loosening of hip prostheses. These "ndings allowed us to identify materials endowed with the highest in#ammatory power. ( 2000 Elsevier Science Ltd. All rights reserved.

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.

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.

In testing for the cytotoxicity of medical devices, crucial parameters are the type of cells, the duration of the exposure, the physical form of the device and the method of evaluation. Using cell cultures the changes of cell functions induced by artificial materials may be evaluated. In screening tests the effect of biomaterials on functions common to all cell types is investigated through both continuous lines and primary cultures. In supplementary tests the effect of biomaterials on specific functions is studied, employing cells of the same type of those which will be in contact with the device in vivo. Osteoblasts, either from primary culture or an established line, are used to study the interaction with materials for bone implants. We evaluated whether dental cements affect the replication cycle of osteoblast-like cells MG63. The S phase was inhibited by the cements with a phenolic group, even though to a different extent. Mononuclear cell cultures are used to investigate the immune response, which is important in the host reaction to implant. We investigated whether the chromium ions released in a biological fluid may induce PBMC to produce cytokines involved in bone resorption. Chromium extract did not impair TNFα release significantly, but inhibited IL-6 release significantly in stimulated PBMC. Endothelial cell cultures are useful to the evaluation of materials for vascular grafts. We studied the influence of some types of Dacron on endothelial functions. Collagen-coated Dacron decreased significantly the production of 6-keto-prostaglandin F1α. Knitted Dacron caused a significant increase of tissue factor, a significant reduction in PECAM-1 and a significant increase in ELAM-1.

The release of basic fibroblast growth factor (bFGF) and platelet derived growth factor AB (PDGF-AB) by in vitro cultured human umbilical endothelial cells in contact with carbon and collagen coated polyethylene terephthalate (PET + PC) was assessed by enzyme immunoassay. The same cells cultured on polystyrene without biomaterials were tested as negative control. PET + PC induced a significant increase in the release of bFGF after 72 h and a significative reduction in the release of PDGF-AB after 48 and 72 h, compared to the negative control.

The production of 6-keto prostaglandin F1alpha (6-keto-PGF1alpha), stable metabolyte of prostacyclin, by cultured human endothelial cells in contact with carbon- and collagen-coated polyethylene terephthalate (PC), was assessed by enzyme immunoassay. As control material, tissue culture-treated polystyrene was used. The cultures were put in contact with the materials for 48 h and then were stimulated with 0.1 mM arachidonic acid for 3 h. The stimulation induced a highly significant increase of 6-keto-PGF1alpha in the cultures in contact with the control material. PC induced only insignificant variations in stimulated cultures compared to unstimulated ones. In conclusion, PC determined a decrease in the endothelial cell response to stimulation with arachidonic acid.

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. © 1998 John Wiley & Sons, Inc. J Biomed Mater Res, 41, 455–460, 1998.

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.

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.

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 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.

In this research adhesive proteins are studied in order to evaluate the interference of woven Dacron in the endothelialization process and in the ability of endothelial cells to bind circulating leucocytes. Endothelial cells from human umbilical vein (HUVEC) were put in contact with woven Dacron for 24 h. PECAM-1, ELAM-1, ICAM-1 and VCAM-1 expression was then evaluated by flow cytometry, using indirect immunofluorescence reaction with monoclonal antibodies. The study of adhesive proteins was completed with the quantitative determination of surface antigens expressed as the antibody binding capacity (ABC). Antigenic density was calculated by the DAKO QFIT calibration system for indirect immunofluorescence. After contact with woven Dacron no significant change was observed in the percentage of positive cells or in the fluorescence intensity of the adhesins. No significant variation was also noted by calculating the surface antigen density by means of calibration fluorospheres. It can be concluded that the material examined does not significantly affect leucocyte adhesion to the endothelium.

The authors examined the modifications of some markers of platelet activation after contact with biomaterials. Glycoprotein GMP-140 (CD62) was evaluated by flow cytometry; β-thromboglobulin (β-TG) and thromboxane B2 (TXB2) were determined by radioimmunoassay. Polyethylene terephthalate (PET) induced a remarkable platelet adhesion and a significant increase in β-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 β-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. © 1997 John Wiley & Sons, Inc. J Biomed Mater Res, 36, 289–294, 1997.

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.

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.

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.

The functional response of peripheral blood lymphocytes isolated from 22 patients with silicone gel-filled breast implants was assessed after in vitro re-exposure to silicone. Using cell culture test methods to quantify proliferation and viability and/or activation of lymphocyte microcultures, i.e., the uptake of tritiated thymidine (3H-TdR uptake test) and the reduction of formazan salts (MTT assay), interesting data were obtained. Peripheral blood lymphocytes purified from patients wearing silicone gel-filled breast implants react in vitro to silicone showing a statistically significant increase of both proliferation and viability, while healthy subjects do not respond on in vitro exposure to silicone. Differences resulted even more statistically significant when patients were divided into two groups depending on the type of surgery they underwent: patients with breast augmentation for aesthetic reasons seem to have an increased responsiveness in vitro to silicone compared to patients who experienced a reconstructive surgery of the breast. Although they are still preliminary, being referred to a limited population, these results suggest that the lymphocytes of patients with silicone gel-filled breast implants could be sensitized in vivo toward silicone; the re-exposure of these cells to silicone leads to a higher functional response which could be looked for by using quantitative in vitro test methods. © 1995 John Wiley & Sons, Inc.

This research aims at evaluating the expression of some adhesive proteins on endothelial cell surface after contact with polyethylene terephthalate coated with pyrolytic carbon (PET + PC). Twenty-two different cultures of human umbilical vein endothelial cells (HUVECs) were put in contact with PET + PC. Both HUVECs grown without the biomaterial and HUVECs incubated with endotoxin were used as control. After 24 h, platelet endothelial cell adhesion molecule-1 (PECAM-1), endothelial leucocyte adhesion molecule-1 (ELAM-1), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were evaluated on the cells by monoclonal antibodies and flow cytometry. In agreement with the literature, after 24 h the culture incubated with endotoxin determined a significant increase in the percentage of positive cells for ELAM-1, a significant increase in fluorescence intensity for ICAM-1, and a significant increase in the percentage of positive cells and fluorescence intensity for VCAM-1. After 24 h of culture with PET + PC, no significant variations in the antigens examined were observed. This demonstrates that such material does not activate in vitro the proteins involved in the adhesion between leucocytes and endothelium or in the adhesion between endothelial cells themselves.

The in vitro biocompatibility of an experimental surface-treated polyurethane was compared with an untreated polyurethane already used for intravascular catheters. The experimental surface was coated with a fluorinated film using a glow discharge treatment. Neither of the catheters was cytotoxic for L929 murine fibroblasts, caused platelet adhesion or release reaction, or changed the mean platelet volume. The surface-treated polyurethane, however, caused a higher adhesion of Staphylococcus aureus than did the untreated one. Therefore, using in vitro testing, it has been ascertained that the examined material, though not being cytotoxic and not modifying platelet behaviour, could favour bacterial adherence.

An in vitro quantitative study of the adhesion of a Staphylococcus aureus strain to two types of disposable contact lenses has been carried out. The first type was an ionic/high-water-content (I-HWC) lens (42% Etafilcon A, 58% water) and the second was a non-ionic/low-water-content (Nl-LWC) lens (61.4% poly(2-hydroxyethyl methacrylate), 38.6% water). Adhesion to the two lens types was evaluated both in basic conditions and after treatment with lysozyme. The results showed that I-HWC lenses are more prone to Staphylococcus aureus adhesion than NI-LWC lenses, both untreated (+15.4%) and treated with lysozyme (+20.5%). Lysozyme increased bacterial adhesion by 30.5% on the lenses with lower water content, and by 36.3% on those with higher water content.

We report a systematic review and meta-analysis of the peer-reviewed literature focusing on metal sensitivity testing in patients undergoing total joint replacement (TJR). Our purpose was to assess the risk of developing metal hypersensitivity post-operatively and its relationship with outcome and to investigate the advantages of performing hypersensitivity testing. We undertook a comprehensive search of the citations quoted in PubMed and EMBASE: 22 articles (comprising 3634 patients) met the inclusion criteria. The frequency of positive tests increased after TJR, especially in patients with implant failure or a metal-on-metal coupling. The probability of developing a metal allergy was higher post-operatively (odds ratio (OR) 1.52 (95% confidence interval (CI) 1.06 to 2.31)), and the risk was further increased when failed implants were compared with stable TJRs (OR 2.76 (95% CI 1.14 to 6.70)). Hypersensitivity testing was not able to discriminate between stable and failed TJRs, as its predictive value was not statistically proven. However, it is generally thought that hypersensitivity testing should be performed in patients with a history of metal allergy and in failed TJRs, especially with metal-on-metal implants and when the cause of the loosening is doubtful.

Nanoparticles (NPs) formed from polymers conjugated with bisphosphonates (BPs) allow the bone targeting of loaded drugs, such as doxorubicin, for the treatment of skeletal tumours. The additional antiosteoclastic effect of the conjugated BP could contribute to the inhibition of tumour-associated bone degradation. With this aim, we have produced NPs made of poly(d,l-lactide-co-glycolide) (PLGA) conjugated with alendronate (ALE). To show if ALE retained the antiosteoclastic properties after the conjugation with PLGA and the production of NPs, we treated human osteoclasts, derived from circulating precursors, with PLGA-ALE NPs and compared the effects on actin ring generation, apoptosis and type-I collagen degradation with those of free ALE and with NPs made of pure PLGA. PLGA-ALE NPs disrupted actin ring, induced apoptosis and inhibited collagen degradation. Unexpectedly, also NPs made of pure PLGA showed similar effects. Therefore, we cannot exclude that in addition to the observed antiosteoclastic activity dependent on ALE in PLGA-ALE NPs, there was also an effect due to pure PLGA. Still, as PLGA-ALE NPs are intended for the loading with drugs for the treatment of osteolytic bone metastases, the additional antiosteoclastic effect of PLGA-ALE NPs, and even of PLGA, may contribute to the inhibition of the disease-associated bone degradation.

The aim of this study is the in vitro evaluation of the functional modifications of human endothelial cells in the presence of Dacron® impregnated with resorbable proteins. For this purpose, human endothelial cells isolated from the umbilical vein have been put in contact for 48 h with knitted Dacron® impregnated with collagen or gelatin and with nonimpregnated knitted Dacron® and double velour Dacron®. As control, endothelial cells cultured in the absence of material were used. After the contact time, cell counts were performed. In addition, the concentrations of two proteins synthesized by endothelium, tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1), were evaluated on the supernatants. In the cultures in contact with Dacron® impregnated with collagen or gelatin and in those in contact with knitted Dacron®, we have observed a smaller cell growth than that observed in cultures without materials. The synthesis of t-PA showed some significant variations between the control cultures and those in contact with the materials. PAI-1 production was significantly reduced in the cultures in contact with gelatin impregnated Dacron® and with knitted Dacron®. Double velour Dacron® caused no significant variation in any of the examined parameters. © 1994 John Wiley & Sons, Inc.

Currently, the use of silicone-filled devices, mainly in plastic surgery for breast reconstruction or augmentation, is being debated by the scientific community in connection with the risk to the patient. In this study the response of whole blood or isolated peripheral blood lymphocytes from patients with silicone-gel-filled breast implants was assessed in vitro, in order to verify the hypothesis of silicone material acting in vivo as a sensitizing agent. Both quantitative and qualitative changes of lymphocyte subpopulations of patients carrying silicone devices were assessed and compared with healthy subjects. Upon 24–72 h in vitro re-exposure of patients' lymphocytes to silicone extract, lymphocyte surface antigen expression was monitored by flow cytometry, and the functional response of lymphocytes was measured by radioactive tracer uptake and biochemical changes.

The use of a tissue adhesive for surgical procedures has prompted a large number of clinical and experimental studies. Alkyl-2-cyanoacrylate esters constitute a family of adhesives with good mechanical properties but their biological compatibility has to be assessed. In this study the cytotoxicity of three commercially available cyanoacrylates and one of unknown composition has been determined. The first part of the study deals with direct contact testing procedures using L 929 cells challenged with drops of adhesives: cell morphology, cell growth and bacterial growth inhibition were assayed. Testing methods included cell viability assay using vital dyes, cell growth measurement using crystal violet staining uptake and bacterial growth assay using S. aureus growth inhibition. All the cyanoacrylate adhesives tested were found to be cytotoxic and to inhibit cell proliferation: differences between the cyanoacrylates were found.

BACKGROUND AND PURPOSE: Thrombophilia represents a risk factor both for idiopathic and secondary osteonecrosis (ON). We evaluated whether clotting changes in idiopathic ON were different from corticosteroid-associated ON. As platelet-rich plasma has been proposed as an adjuvant in surgery, we also assessed whether platelet and serum growth factors were similar to those in healthy subjects. METHODS: 18 patients with idiopathic ON and 18 with corticosteroid-associated ON were compared with 44 controls for acquired and inherited thrombophilia. Platelet factor 4 (PF4), transforming growth factor-β1, platelet-derived growth factor-BB (PDGF-BB), and vascular endothelial growth factor were assayed in the supernatants of thrombin-activated platelets, in platelet lysates, and in serum from 14 ON patients and 10 controls. RESULTS: Idiopathic ON patients had higher plasminogen levels (median 118%) than controls (101%) (p = 0.02). Those with corticosteroid-associated ON had significantly higher D-dimer (333 ng/mL) and lower protein C levels (129%) than controls (164 ng/mL, p = 0.004; 160%, p = 0.02). The frequency of inherited thrombophilia was not different from the controls. No statistically significant differences were found between idiopathic and corticosteroid-associated ON. 20 of the 36 ON patients were smokers. (The controls were selected from smokers because nicotine favors hypercoagulability). ON patients had significantly higher serum PF4 levels (7,383 IU/mL) and PDGF-BB levels (3.1 ng/mL) than controls (4,697 IU/mL, p = 0.005; 2.2 ng/mL, p = 0.02). INTERPRETATION: Acquired hypercoagulability was common in both ON types, but the specific changes varied. The release of GF from platelets was not affected, providing a biological basis for platelet-rich plasma being used as an adjuvant in surgical treatment.

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.