Health hazards of particles in additive manufacturing: a cross-disciplinary study on reactivity, toxicity and occupational exposure to two nickel-based alloys.

The increasing use of additive manufacturing (AM) techniques (e.g., 3D-printing) offers many advantages but at the same time presents some challenges. One concern is the possible exposure and health risk related to metal containing particles of different sizes. Using the nickel-based alloys Hastelloy X (HX) and Inconel 939 (IN939) as a case, the aim of this cross-disciplinary study was to increase the understanding on possible health hazards and exposure. This was done by performing in-depth characterization of virgin, reused and condensate powders, testing in vitro toxicity (cytotoxicity, genotoxicity, oxidative stress), and measuring occupational airborne exposure. The results showed limited metal release from both HX and IN939, and slightly different surface composition of reused compared to virgin powders. No or small effects on the cultured lung cells were observed when tested up to 100 µg/mL. Particle background levels in the printing facilities were generally low, but high transient peaks were observed in relation to sieving. Furthermore, during post processing with grinding, high levels of nanoparticles (> 100,000 particles/cm3) were noted. Urine metal levels in AM operators did not exceed biomonitoring action limits. Future studies should focus on understanding the toxicity of the nanoparticles formed during printing and post-processing.

Toxicity assessment and health hazard classification of stainless steels.

Stainless steels are widely used iron-based alloys that contain chromium and, typically, other alloying elements. The chromium(III)-rich surface oxide of stainless steels efficiently limits the release (bioaccessibility) of their metal constituents in most physiological environments, influencing the toxicity of the alloy. Of the constituents and impurities of stainless steels, nickel and cobalt are of particular interest, primarily due to skin sensitization and repeated-dose inhalation toxicity of nickel, and (inhalation) carcinogenicity of cobalt. A review of the available toxicological data on stainless steels, and the toxicological, mechanistic, and bioaccessibility data on their constituent metals supports the low toxicity and non-carcinogenicity of stainless steels. The comparative metal release, rather than the bulk composition of stainless steels, needs to be considered when assessing their health hazard classification according to the UN Globally Harmonized System, and the corresponding EU CLP regulation. As an illustrative example, a 28-day inhalation toxicity study on stainless steel powder showed no signs of lung toxicity at exposure levels at which significant toxicity would have been expected on the basis of its bulk nickel content. This finding is associated with the low bioaccessibility of nickel from the alloy in the lungs.

Toxicity evaluation of particles formed during 3D-printing: Cytotoxic, genotoxic, and inflammatory response in lung and macrophage models.

Additive manufacturing (AM) or "3D-printing" is a ground-breaking technology that enables the production of complex 3D parts. Its rapid growth calls for immediate toxicological investigations of possible human exposures in order to estimate occupational health risks. Several laser-based powder bed fusion AM techniques are available of which many use metal powder in the micrometer range as feedstock. Large energy input from the laser on metal powders generates several by-products, like spatter and condensate particles. Due to often altered physicochemical properties and composition, spatter and condensate particles can result in different toxicological responses compared to the original powder particles. The toxicity of such particles has, however, not yet been investigated. The aim of the present study was to investigate the toxicity of condensate/spatter particles formed and collected upon selective laser melting (SLM) printing of metal alloy powders, including a nickel-chromium-based superalloy (IN939), a nickel-based alloy (Hastelloy X, HX), a high-strength maraging steel (18Ni300), a stainless steel (316L), and a titanium alloy (Ti6Al4V). Toxicological endpoints investigated included cytotoxicity, generation of reactive oxygen species (ROS), genotoxicity (comet and micronucleus formation), and inflammatory response (cytokine/chemokine profiling) following exposure of human bronchial epithelial cells (HBEC) or monocytes/macrophages (THP-1). The results showed no or minor cytotoxicity in the doses tested (10-100 µg/mL). Furthermore, no ROS generation or formation of micronucleus was observed in the HBEC cells. However, an increase in DNA strand breaks (detected by comet assay) was noted in cells exposed to HX, IN939, and Ti6Al4V, whereas no evident release of pro-inflammatory cytokine was observed from macrophages. Particle and surface characterization showed agglomeration in solution and different surface oxide compositions compared to the nominal bulk content. The extent of released nickel was small and related to the nickel content of the surface oxides, which was largely different from the bulk content. This may explain the limited toxicity found despite the high Ni bulk content of several powders. Taken together, this study suggests relatively low acute toxicity of condensates/spatter particles formed during SLM-printing using IN939, HX, 18Ni300, 316L, and Ti6Al4V as original metal powders.

Corrosion Resistance and Cytocompatibility of Magnesium-Calcium Alloys Modified with Zinc- or Gallium-Doped Calcium Phosphate Coatings.

In orthopedic surgery, metals are preferred to support or treat damaged bones due to their high mechanical strength. However, the necessity for a second surgery for implant removal after healing creates problems. Therefore, biodegradable metals, especially magnesium (Mg), gained importance, although their extreme susceptibility to galvanic corrosion limits their applications. The focus of this study was to control the corrosion of Mg and enhance its biocompatibility. For this purpose, surfaces of magnesium-calcium (MgCa1) alloys were modified with calcium phosphate (CaP) or CaP doped with zinc (Zn) or gallium (Ga) via microarc oxidation. The effects of surface modifications on physical, chemical, and mechanical properties and corrosion resistance of the alloys were studied using surface profilometry, goniometry, scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), nanoindentation, and electrochemical impedance spectroscopy (EIS). The coating thickness was about 5-8 µm, with grain sizes of 43.1 nm for CaP coating and 28.2 and 58.1 nm for Zn- and Ga-doped coatings, respectively. According to EIS measurements, the capacitive response (Yc) decreased from 11.29 to 8.72 and 0.15 Ω-1 cm-2 sn upon doping with Zn and Ga, respectively. The Ecorr value, which was -1933 mV for CaP-coated samples, was found significantly electropositive at -275 mV for Ga-doped ones. All samples were cytocompatible according to indirect tests. In vitro culture with Saos-2 cells led to changes in the surface compositions of the alloys. The numbers of cells attached to the Zn-doped (2.6 × 104 cells/cm2) and Ga-doped (6.3 × 104 cells/cm2) coatings were higher than that on the surface of the undoped coating (1.0 × 103 cells/cm2). Decreased corrosivity and enhanced cell affinity of the modified MgCa alloys (CaP coated and Zn and Ga doped, with Ga-doped ones having the greatest positive effect) make them novel and promising candidates as biodegradable metallic implant materials for the treatment of bone damages and other orthopedic applications.

TiO2-Induced In Situ Reaction in Graphene Oxide-Reinforced AZ61 Biocomposites to Enhance the Interfacial Bonding.

Graphene oxide (GO) can improve the degradation resistance of biomedical Mg alloy because of its excellent impermeability and outstanding chemical inertness. However, the weak interfacial bonding between GO and Mg matrix leads to easily detaching during degradation. In this study, in situ reaction induced by TiO2 took place in the AZ61-GO biocomposite to enhance the interfacial bonding between GO and Mg matrix. For the specific process, TiO2 was uniformly and tightly deposited onto the GO surface by hydrothermal reaction (TiO2/GO) first and then used for fabricating AZ61-TiO2/GO biocomposites by selective laser melting (SLM). Results showed that TiO2 was in situ reduced by magnesiothermic reaction during SLM process, and the reduzate Ti, on the one hand, reacted with Al in the AZ61 matrix to form TiAl2 and, on the other hand, reacted with GO to form TiC at the AZ61-GO interface. Owing to the enhanced interfacial bonding, the AZ61-TiO2/GO biocomposite showed 12.5% decrease in degradation rate and 10.1% increase in compressive strength as compared with the AZ61-GO biocomposite. Moreover, the AZ61-TiO2/GO biocomposite also showed good cytocompatibility because of the slowed degradation. These findings may provide guidance for the interfacial enhancement in GO/metal composites for biomedical applications.

A biodegradable Zn-1Cu-0.1Ti alloy with antibacterial properties for orthopedic applications.

Zinc (Zn) alloys are receiving increasing attention in the field of biodegradable implant materials due to their unique combination of suitable biodegradability and good biological functionalities. However, the currently existing industrial Zn alloys are not necessarily biocompatible, nor sufficiently mechanically strong and wear-resistant. In this study, a Zn-1Cu-0.1Ti alloy is developed with enhanced mechanical strength, corrosion wear property, biocompatibility, and antibacterial ability for biodegradable implant material applications. HR and HR + CR were performed on the as-cast alloy and its microstructure, mechanical properties, frictional and wear behaviors, corrosion resistance, in vitro cytocompatibility, and antibacterial ability were systematically assessed. The microstructures of the Zn-1Cu-0.1Ti alloy after different deformation conditions included a η-Zn phase, a ε-CuZn5 phase, and an intermetallic phase of TiZn16. The HR+CR sample of Zn-1Cu-0.1Ti exhibited a yield strength of 204.2 MPa, an ultimate tensile strength of 249.9 MPa, and an elongation of 75.2%; significantly higher than those of the HR alloy and the AC alloy. The degradation rate in Hanks' solution was 0.029 mm/y for the AC alloy, 0.032 mm/y for the HR+CR alloy, and 0.034 mm/y for the HR alloy. The HR Zn-1Cu-0.1Ti alloy showed the best wear resistance, followed by the AC alloy and the alloy after HR + CR. The extract of the AC Zn-1Cu-0.1Ti alloy showed over 80% cell viability with MC3T3-E1 pre-osteoblast and MG-63 osteosarcoma cells at a concentration of ≤ 25%. The as-cast Zn-1Cu-0.1Ti alloy showed good blood compatibility and antibacterial ability. STATEMENT OF SIGNIFICANCE: This work repots a Zn-1Cu-0.1Ti alloy with enhanced mechanical strength, corrosion wear property, biocompatibility, and antibacterial ability for biodegradable implant applications. Our findings showed that Zn-1Cu-0.1Ti after hot-rolling plus cold-rolling exhibited a yield strength of 204.2 MPa, an ultimate tensile strength of 249.9 MPa, an elongation of 75.2%, and a degradation rate of 0.032 mm/y in Hanks' Solution. The hot-rolled Zn-1Cu-0.1Ti showed the best wear resistance. The extract of the as-cast alloy at a concentration of ≤ 25% showed over 80% cell viability with MC3T3-E1 and MG-63 cells. The Zn-1Cu-0.1Ti alloy showed good hemocompatibility and antibacterial ability.

The bioeffects of degradable products derived from a biodegradable Mg-based alloy in macrophages via heterophagy.

Biodegradable magnesium alloys are promising candidates for use in biomedical applications. However, degradable particles (DPs) derived from Mg-based alloys have been observed in tissue in proximity to sites of implantation, which might result in unexpected effects. Although previous in vitro studies have found that macrophages can take up DPs, little is known about the potential phagocytic pathway and the mechanism that processes DPs in cells. Additionally, it is necessary to estimate the potential bioeffects of DPs on macrophages. Thus, in this study, DPs were generated from a Mg-2.1Nd-0.2Zn-0.5Zr alloy (JDBM) by an electrochemical method, and then macrophages were incubated with the DPs to reveal the potential impact. The results showed that the cell viability of macrophages decreased in a concentration-dependent manner in the presence of DPs due to effects of an apoptotic pathway. However, the DPs were phagocytosed into the cytoplasm of macrophages and further degraded in phagolysosomes, which comprised lysosomes and phagosomes, by heterophagy instead of autophagy. Furthermore, several pro-inflammatory cytokines in macrophages were upregulated by DPs through the induction of reactive oxygen species (ROS) production. To the best of our knowledge, this is the first study to show that DPs derived from a Mg-based alloy are consistently degraded in phagolysosomes after phagocytosis by macrophages via heterophagy, which results in an inflammatory response owing to ROS overproduction. Thus, our research has increased the knowledge of the metabolism of biodegradable Mg metal, which will contribute to an understanding of the health effects of biodegradable magnesium metal implants used for tissue repair. STATEMENT OF SIGNIFICANCE: Biomedical degradable Mg-based alloys have great promise in applied medicine. Although previous studies have found that macrophages can uptake degradable particles (DPs) in vitro and observed in the sites of implantation in vivoin vivo, few studies have been carried out on the potential bioeffects relationship between DPs and macrophages. In this study, we analyzed the bioeffects of DPs derived from a Mg-based alloy on the macrophages. We illustrated that the DPs were size-dependently engulfed by macrophages via heterophagy and further degraded in the phagolysosome rather than autophagosome. Furthermore, DPs were able to induce a slight inflammatory response in macrophages by inducing ROS production. Thus, our research enhances the knowledge of the interaction between DPs of Mg-based alloy and cells, and offers a new perspective regarding the use of biodegradable alloys.

Effect of Lithium and Aluminum on the Mechanical Properties, In Vivo and In Vitro Degradation, and Toxicity of Multiphase Ultrahigh Ductility Mg-Li-Al-Zn Quaternary Alloys for Vascular Stent Application.

Magnesium alloys are the most widely studied biodegradable metals for biodegradable vascular stent application. Two major issues with current magnesium alloy based stents are their low ductility and fast corrosion rates. Several studies have validated that introduction of Li into the magnesium alloys will significantly improve the ductility while alloying with Al will improve the corrosion resistance and strength. In the present study, we studied the effects of alloying different amounts of Li and Al on the Mg-Li-Al-Zn (LAZ) quaternary alloy system. Rods were made from four different LAZ alloys, namely, LAZ611, LAZ631, LAZ911, and LAZ931 following melting, casting, and then extrusion. Systematic assessment of mechanical properties, in vitro corrosion, cytotoxicity, and in vivo degradation including local and systemic toxicity conducted demonstrated the beneficial effects of Li and Al on the mechanical properties. Our results specifically suggest that alloying with Li significantly improved the ductility while Al enhanced the strength of the LAZ alloys. Four of the LAZ alloys exhibited different corrosion rates in Hank's balanced salt solution depending on the chemical composition. Indirect in vitro cytotoxicity tests also showed lower cytotoxicity for the alloys exhibiting higher corrosion resistance. In vivo corrosion rates in the mouse subcutaneous model showed different corrosion rates compared to the in vitro tests. Nevertheless, all of the four LAZ alloys displayed no local and systemic toxicity based on the histology analysis. This research study, therefore, demonstrated the benefits of using Li and Al as alloying elements in LAZ alloys and the potential use of LAZ alloys for vascular stent application.

Bioaccessibility of nickel and cobalt in synthetic gastric and lung fluids and its potential use in alloy classification.

This study investigated nickel and cobalt ion release from the metals and several alloys in synthetic gastric, as well as interstitial and lysosomal lung fluids. Results were used to calculate the relative bioaccessible concentrations (RBCs) of the metals. Nickel release from SS 316L powder in gastric fluid was >300-fold lower than from a simple mixture of powders of the same bulk composition. Gastric bioaccessibility data showed 50-fold higher metal releases per gram of sample from powder than massive forms. RBCs of nickel and cobalt in the alloy powders were lower, equal, or higher in all fluids tested than their bulk concentrations. This illustrates the fact that matrix effects can increase or decrease the metal ion release, depending on the metal ingredients, alloy type, and fluid, consistent with research by others. Acute inhalation toxicity studies with cobalt-containing alloy powders showed that the RBC of cobalt in interstitial lung fluid predicted acute toxicity better than bulk concentration. This example indicates that the RBC of a metal in an alloy may estimate the concentration of bioavailable metals better than the bulk concentration, and the approach may provide a means to refine the classification of alloys for several human health endpoints.

Mechanical, corrosion, and biocompatibility properties of Mg-Zr-Sr-Sc alloys for biodegradable implant applications.

Magnesium (Mg) and its alloys are considered promising biodegradable implant materials because of their strength and natural degradation in the human body. However, the high corrosion rate of pure Mg in the physiological environment leads to rapid degradation before adequate bone healing. This mismatch between bone healing and the degradation of Mg implants supports the development of new Mg alloys with the addition of other suitable alloying elements in order to achieve simultaneously high corrosion resistance and desirable mechanical properties. This study systematically investigates the microstructure, mechanical properties, corrosion behavior, and biocompatibility of Mg-based alloys with the addition of different concentrations of scandium (Sc), i.e., Mg-0.6Zr-0.5Sr-xSc (x = 0.5, 1, 2, 3 wt.%). Results indicated that high concentration of Sc in strontium (Sr)-containing Mg alloys can alter their microstructures by suppressing the intermetallic phases along the grain boundaries and improve the corrosion resistance by forming chemically stable Sc oxide layers on the surfaces of the Mg alloys. Cytotoxicity assessment revealed that the Sc containing Mg alloys did not significantly alter the viability of human osteoblast-like SaOS2 cells. This study highlights the advantages of using Sc as an alloying element to simultaneously tune Mg alloys with higher strength and slower degradation. STATEMENT OF SIGNIFICANCE: Rare earth elements such as scandium (Sc) with both a high solid-solubility and strong affinity towards oxygen can improve the mechanical and corrosion properties of magnesium (Mg) alloys. However, the feasibility of Sc-containing Mg alloys as biodegradable implant materials is scarcely reported. This study investigates the effects of different Sc concentrations on the mechanical, corrosion, and biocompatibility properties of Mg-Zr-Sr-Sc alloys. Our findings indicated that the addition of Sc significantly improves the mechanical and corrosion properties of Mg-Zr-Sr alloys. Moreover, in vitro cytotoxicity assessment of the Mg-Zr-Sr-Sc alloys did not show any adverse effects on the viability of osteoblast-like cells.

Effect of TiO2 and Fe doped TiO2 nanoparticles on mitochondrial membrane potential in HBL-100 cells.

Titanium dioxide (TiO2) nanoparticles (NPs) have made unbelievable progress in the field of nanotechnology and biomedical research. The proper toxicological assessment of TiO2 NPs and the reduction of its cytotoxicity need to be addressed. Fe doping in TiO2 has been investigated to reduce the toxic effects of TiO2 NPs. Fe doped TiO2 powder samples were synthesized by sol-gel methods. The prepared samples were characterized by x-ray diffractometer (XRD), transmission electron microscope (TEM), and Raman spectroscopy to study their structure, morphology, and molecular conformation. XRD results revealed the coexistence of anatase (A) and rutile (R) phases of TiO2. The A-R transformation was observed with an increase in Fe doping along with the formation of α-Fe2O3 phase. TEM showed changes in morphology from spherical nanoparticles to elongated rod-shaped nanostructures with increasing Fe content. Shape variation of TiO2 nanoparticles after incorporation of Fe is a key reason behind the toxicity reduction. The authors observed that the toxicity of TiO2 nanoparticles was rescued upon Fe incorporation. The effect of NPs on the mitochondrial membrane potential (MMP) was assessed using flow cytometry. The MMP (%) decreased in TiO2 treated cells and increased by 1% Fe doped TiO2 NPs treated cells. Confocal imaging revealed the presence of functional mitochondria upon the exposure of Fe doped TiO2 NPs. The goal of the present study was to decrease the toxic effects induced by TiO2 NPs on mitochondrial potential and its prevention by Fe doping.

Ferroxidase-like and antibacterial activity of PtCu alloy nanoparticles.

Many metal nanoparticles are reported to have intrinsic enzyme-like activities and offer great potential in chemical and biomedical applications. In this study, PtCu alloy nanoparticles (NPs), synthesized through hydrothermal treatment of Cu2+ and Pt2+ in an aqueous solution, were evaluated for ferroxidase-like and antibacterial activity. Electron spin resonance (ESR) spectroscopy and colorimetric methods were used to demonstrate that PtCu NPs exhibited strong ferroxidase-like activity in a weakly acidic environment and that this activity was not affected by the presence of most other ions, except silver. Based on the color reaction of salicylic acid in the presence of Fe3+, we tested the ferroxidase-like activity of PtCu NPs to specifically detect Fe2+ in a solution of an oral iron supplement and compared these results with data acquired from atomic absorption spectroscopy and the phenanthroline colorimetric method. The results showed that the newly developed PtCu NPs detection method was equivalent to or better than the other two methods used for Fe2+ detection. The antibacterial experiments showed that PtCu NPs have strong antibacterial activity against Staphylococcus aureus and Escherichia coli. Herein, we demonstrate that the peroxidase-like activity of PtCu NPs can catalyze H2O2 and generate hydroxyl radicals, which may elucidate the antibacterial activity of the PtCu NPs against S. aureus and E. coli. These results showed that PtCu NPs exhibited both ferroxidase- and peroxidase-like activity and that they may serve as convenient and efficient NPs for the detection of Fe2+ and for antibacterial applications.

Synchrotron analysis of human organ tissue exposed to implant material.

BACKGROUND: Orthopaedic implants made of cobalt-chromium alloy undergo wear and corrosion that can lead to deposition of cobalt and chromium in vital organs. Elevated cardiac tissue cobalt levels are associated with myocardial injury while chromium is a well-established genotoxin. Though metal composition of tissues surrounding hip implants has been established, few investigators attempted to characterize the metal deposits in systemic tissues of total joint arthroplasty patients. METHODS: We report the first use of micro-X-ray fluorescence coupled with micro-X-ray absorption spectroscopy to probe distribution and chemical form of cobalt, chromium and titanium in postmortem samples of splenic, hepatic and cardiac tissue of patients with metal-on-polyethylene hip implants (n = 5). RESULTS: Majority of the cobalt was in the 2+ oxidation state, while titanium was present exclusively as titanium dioxide, in either rutile or anatase crystal structure. Chromium was found in a range of forms including a highly oxidised, carcinogenic species (CrV/VI), which has never been identified in human tissue before. CONCLUSIONS: Carcinogenic forms of chromium might arise in vital organs of total joint arthroplasty patients. Further studies are warranted with patients with metal-on-metal implants, which tend to have an increased release of cobalt and chromium compared to metal-on-polyethylene hips.

BMP-7 ameliorates cobalt alloy particle-induced inflammation by suppressing Th17 responses.

Metal wear debris has been shown to activate an aseptic osteolytic process that causes failure in total joint arthroplasty (TJA). This osteolysis is characterized by a proinflammatory, self-propagating immune response involving primarily macrophages, dendritic cells, and activated osteoclasts, as well as T cells and B cells. The human bone morphogenic protein (BMP)-7, on the other hand, was shown to promote osteoblast survival, and reversed the downregulation of anabolic Smad proteins and Runx2 following cobalt injury. Therefore, we investigated the effect and mechanism of BMP-7 on the proinflammatory immune responses in osteoarthritis patients with previous TJA. Cobalt-treated monocytes/macrophages presented significantly elevated levels of interleukin 6 (IL-6) and tumor necrosis factor (TNF), both of which were suppressed by the addition of exogenous BMP-7. In patients with TJA, the serum BMP-7 level was inversely associated with the level of IL-6 and TNF secreted by monocytes/macrophages. Cobalt-treated monocytes/macrophages effectively supported Th17 inflammation, by an IL-6-dependent but not TNF-dependent mechanism. BMP-7, however, significantly suppressed cobalt-induced Th17 inflammation. In patients with TJA, the risk of osteolysis development was positively associated with the frequency of Th17 cells and negatively associated with the level of BMP-7. Together, these results demonstrated that BMP-7 could serve as a therapeutic agent in treating patients with metal wear debris-induced inflammation.

In vitro inflammatory effects of hard metal (WC-Co) nanoparticle exposure.

Identifying the toxicity of nanoparticles (NPs) is an important area of research as the number of nanomaterial-based consumer and industrial products continually rises. In addition, the potential inflammatory effects resulting from pulmonary NP exposure are emerging as an important aspect of nanotoxicity. In this study, the toxicity and inflammatory state resulting from tungsten carbide-cobalt (WC-Co) NP exposure in macrophages and a coculture (CC) of lung epithelial cells (BEAS-2B) and macrophages (THP-1) at a 3:1 ratio were examined. It was found that the toxicity of nano-WC-Co was cell dependent; significantly less toxicity was observed in THP-1 cells compared to BEAS-2B cells. It was demonstrated that nano-WC-Co caused reduced toxicity in the CC model compared to lung epithelial cell monoculture, which suggested that macrophages may play a protective role against nano-WC-Co-mediated toxicity in CCs. Nano-WC-Co exposure in macrophages resulted in increased levels of interleukin (IL)-1ß and IL-12 secretion and decreased levels of tumor necrosis factor alpha (TNFα). In addition, the polarizing effects of nano-WC-Co exposure toward the M1 (pro-inflammatory) and M2 (anti-inflammatory) macrophage phenotypes were investigated. The results of this study indicated that nano-WC-Co exposure stimulated the M1 phenotype, marked by high expression of CD40 M1 macrophage surface markers.

Hard metal lung disease: a case series / Doença pulmonar por metal duro: uma série de casos

ABSTRACT Objective: To describe diagnostic and treatment aspects of hard metal lung disease (HMLD) and to review the current literature on the topic. Methods: This was a retrospective study based on the medical records of patients treated at the Occupational Respiratory Diseases Clinic of the Instituto do Coração, in the city of São Paulo, Brazil, between 2010 and 2013. Results: Of 320 patients treated during the study period, 5 (1.56%) were diagnosed with HMLD. All of those 5 patients were male (mean age, 42.0 ± 13.6 years; mean duration of exposure to hard metals, 11.4 ± 8.0 years). Occupational histories were taken, after which the patients underwent clinical evaluation, chest HRCT, pulmonary function tests, bronchoscopy, BAL, and lung biopsy. Restrictive lung disease was found in all subjects. The most common chest HRCT finding was ground glass opacities (in 80%). In 4 patients, BALF revealed multinucleated giant cells. In 3 patients, lung biopsy revealed giant cell interstitial pneumonia. One patient was diagnosed with desquamative interstitial pneumonia associated with cellular bronchiolitis, and another was diagnosed with a hypersensitivity pneumonitis pattern. All patients were withdrawn from exposure and treated with corticosteroid. Clinical improvement occurred in 2 patients, whereas the disease progressed in 3. Conclusions: Although HMLD is a rare entity, it should always be included in the differential diagnosis of respiratory dysfunction in workers with a high occupational risk of exposure to hard metal particles. A relevant history (clinical and occupational) accompanied by chest HRCT and BAL findings suggestive of the disease might be sufficient for the diagnosis.

RESUMO Objetivo: Descrever aspectos relacionados ao diagnóstico e tratamento de pacientes com doença pulmonar por metal duro (DPMD) e realizar uma revisão da literatura. Métodos: Estudo retrospectivo dos prontuários médicos de pacientes atendidos no Serviço de Doenças Respiratórias Ocupacionais do Instituto do Coração, localizado na cidade de São Paulo, entre 2010 e 2013. Resultados: Entre 320 pacientes atendidos no período do estudo, 5 (1,56%) foram diagnosticados com DPMD. Todos os pacientes eram do sexo masculino, com média de idade de 42,0 ± 13,6 anos e média de tempo de exposição a metal duro de 11,4 ± 8,0 anos. Os pacientes foram submetidos a avaliação clinica, história ocupacional, TCAR de tórax, prova de função pulmonar, broncoscopia com LBA e biópsia pulmonar. Todos apresentaram distúrbio ventilatório restritivo. O achado de imagem à TCAR de tórax mais frequente foi de opacidades em vidro fosco (em 80%). Em 4 pacientes, o LBA revelou presença de células gigantes multinucleadas. Em 3, foi diagnosticada pneumonia intersticial por células gigantes na biópsia pulmonar. Houve o diagnóstico de pneumonia intersticial descamativa associada à bronquiolite celular em 1 paciente e de pneumonite de hipersensibilidade em 1. Todos foram afastados da exposição e tratados com corticoide. Houve melhora em 2 pacientes e progressão da doença em 3. Conclusões: Apesar de ser uma entidade rara, a DPMD deve ser sempre considerada em trabalhadores com risco ocupacional elevado de exposição a metais duros. A história clínica e ocupacional associada a achados em TCAR de tórax e LBA sugestivos da doença podem ser suficientes para o diagnóstico.

Hard metal lung disease: a case series.

OBJECTIVE:: To describe diagnostic and treatment aspects of hard metal lung disease (HMLD) and to review the current literature on the topic. METHODS:: This was a retrospective study based on the medical records of patients treated at the Occupational Respiratory Diseases Clinic of the Instituto do Coração, in the city of São Paulo, Brazil, between 2010 and 2013. RESULTS:: Of 320 patients treated during the study period, 5 (1.56%) were diagnosed with HMLD. All of those 5 patients were male (mean age, 42.0 ± 13.6 years; mean duration of exposure to hard metals, 11.4 ± 8.0 years). Occupational histories were taken, after which the patients underwent clinical evaluation, chest HRCT, pulmonary function tests, bronchoscopy, BAL, and lung biopsy. Restrictive lung disease was found in all subjects. The most common chest HRCT finding was ground glass opacities (in 80%). In 4 patients, BALF revealed multinucleated giant cells. In 3 patients, lung biopsy revealed giant cell interstitial pneumonia. One patient was diagnosed with desquamative interstitial pneumonia associated with cellular bronchiolitis, and another was diagnosed with a hypersensitivity pneumonitis pattern. All patients were withdrawn from exposure and treated with corticosteroid. Clinical improvement occurred in 2 patients, whereas the disease progressed in 3. CONCLUSIONS:: Although HMLD is a rare entity, it should always be included in the differential diagnosis of respiratory dysfunction in workers with a high occupational risk of exposure to hard metal particles. A relevant history (clinical and occupational) accompanied by chest HRCT and BAL findings suggestive of the disease might be sufficient for the diagnosis. OBJETIVO:: Descrever aspectos relacionados ao diagnóstico e tratamento de pacientes com doença pulmonar por metal duro (DPMD) e realizar uma revisão da literatura. MÉTODOS:: Estudo retrospectivo dos prontuários médicos de pacientes atendidos no Serviço de Doenças Respiratórias Ocupacionais do Instituto do Coração, localizado na cidade de São Paulo, entre 2010 e 2013. RESULTADOS:: Entre 320 pacientes atendidos no período do estudo, 5 (1,56%) foram diagnosticados com DPMD. Todos os pacientes eram do sexo masculino, com média de idade de 42,0 ± 13,6 anos e média de tempo de exposição a metal duro de 11,4 ± 8,0 anos. Os pacientes foram submetidos a avaliação clinica, história ocupacional, TCAR de tórax, prova de função pulmonar, broncoscopia com LBA e biópsia pulmonar. Todos apresentaram distúrbio ventilatório restritivo. O achado de imagem à TCAR de tórax mais frequente foi de opacidades em vidro fosco (em 80%). Em 4 pacientes, o LBA revelou presença de células gigantes multinucleadas. Em 3, foi diagnosticada pneumonia intersticial por células gigantes na biópsia pulmonar. Houve o diagnóstico de pneumonia intersticial descamativa associada à bronquiolite celular em 1 paciente e de pneumonite de hipersensibilidade em 1. Todos foram afastados da exposição e tratados com corticoide. Houve melhora em 2 pacientes e progressão da doença em 3. CONCLUSÕES:: Apesar de ser uma entidade rara, a DPMD deve ser sempre considerada em trabalhadores com risco ocupacional elevado de exposição a metais duros. A história clínica e ocupacional associada a achados em TCAR de tórax e LBA sugestivos da doença podem ser suficientes para o diagnóstico.

Structure, mechanical characteristics and in vitro degradation, cytotoxicity, genotoxicity and mutagenicity of novel biodegradable Zn-Mg alloys.

Zn-(0-1.6)Mg (in wt.%) alloys were prepared by hot extrusion at 300 °C. The structure, mechanical properties and in vitro biocompatibility of the alloys were investigated. The hot-extruded magnesium-based WE43 alloy was used as a control. Mechanical properties were evaluated by hardness, compressive and tensile testing. The cytotoxicity, genotoxicity (comet assay) and mutagenicity (Ames test) of the alloy extracts and ZnCl2 solutions were evaluated with the use of murine fibroblasts L929 and human osteosarcoma cell line U-2 OS. The microstructure of the Zn alloys consisted of recrystallized Zn grains of 12 µm in size and fine Mg2Zn11 particles arranged parallel to the hot extrusion direction. Mechanical tests revealed that the hardness and strength increased with increasing Mg concentration. The Zn-0.8 Mg alloys showed the best combination of tensile mechanical properties (tensile yield strength of 203 MPa, ultimate tensile strength of 301 MPa and elongation of 15%). At higher Mg concentrations the plasticity of Zn-Mg alloys was deteriorated. Cytotoxicity tests with alloy extracts and ZnCl2 solutions proved the maximum safe Zn(2+) concentrations of 120 µM and 80 µM for the U-2 OS and L929 cell lines, respectively. Ames test with extracts of alloys indicated that the extracts were not mutagenic. The comet assay demonstrated that 1-day extracts of alloys were not genotoxic for U-2 OS and L929 cell lines after 1-day incubation.

Molecular basis of carcinogenicity of tungsten alloy particles.

The tungsten alloy of 91% tungsten, 6% nickel and 3% cobalt (WNC 91-6-3) induces rhabdomyosarcoma when implanted into a rat thigh muscle. To investigate whether this effect is species-specific human HSkMc primary muscle cells were exposed to WNC 91-6-3 particles and responses were compared with those from a rat skeletal muscle cell line (L6-C11). Toxicity was assessed by the adenylate kinase assay and microscopy, DNA damage by the Comet assay. Caspase 3 enzyme activity was measured and oligonucleotide microarrays were used for transcriptional profiling. WNC 91-6-3 particles caused toxicity in cells adjacent to the particles and also increased DNA strand breaks. Inhibition of caspase 3 by WNC 91-6-3 occurred in rat but not in human cells. In both rat and human cells, the transcriptional response to WNC 91-6-3 showed repression of transcripts encoding muscle-specific proteins with induction of glycolysis, hypoxia, stress responses and transcripts associated with DNA damage and cell death. In human cells, genes encoding metallothioneins were also induced, together with genes related to angiogenesis, dysregulation of apoptosis and proliferation consistent with pre-neoplastic changes. An alloy containing iron, WNF 97-2-1, which is non-carcinogenic in vivo in rats, did not show these transcriptional changes in vitro in either species while the corresponding cobalt-containing alloy, WNC 97-2-1 elicited similar responses to WNC 91-6-3. Tungsten alloys containing both nickel and cobalt therefore have the potential to be carcinogenic in man and in vitro assays coupled with transcriptomics can be used to identify alloys, which may lead to tumour formation, by dysregulation of biochemical processes.