CoCrMo-Nanoparticles induced peri-implant osteolysis by promoting osteoblast ferroptosis via regulating Nrf2-ARE signalling pathway.

OBJECTIVES: Aseptic loosening (AL) is the most common reason of total hip arthroplasty (THA) failure and revision surgery. Osteolysis, caused by wear particles released from implant surfaces, has a vital role in AL. Although previous studies suggest that wear particles always lead to osteoblast programmed death in the process of AL, the specific mechanism remains incompletely understood and osteoblast ferroptosis maybe a new mechanism of AL. MATERIALS AND METHODS: CoCrMo nanoparticles (CoNPs) were prepared to investigate the influence of ferroptosis in osteoblasts and calvaria resorption animal models. Periprosthetic osteolytic bone tissue was collected from patients who underwent AL after THA to verify osteoblast ferroptosis. RESULTS: Our study demonstrated that CoNPs induced significant ferroptosis in osteoblasts and particles induced osteolysis (PIO) animal models. Blocking ferroptosis with specific inhibitor Ferrostatin-1 dramatically reduced particle-induced ferroptosis in vitro. Moreover, in osteoblasts, CoNPs significantly downregulated the expression of Nrf2 (nuclear factor erythroid 2-related factor 2), a core element in the antioxidant response. The overexpression of Nrf2 by siKeap1 or Nrf2 activator Oltipraz obviously upregulated antioxidant response elements (AREs) and suppressed ferroptosis in osteoblasts. Furthermore, in PIO animal models, the combined utilization of Ferrostatin-1 and Oltipraz dramatically ameliorated ferroptosis and the severity of osteolysis. CONCLUSIONS: These results indicate that CoNPs promote osteoblast ferroptosis by regulating the Nrf2-ARE signalling pathway, which suggests a new mechanism underlying PIO and represents a potential therapeutic approach for AL.

Favorable modulation of osteoblast cellular activity on Zr-modified Co-Cr-Mo alloy: The significant impact of zirconium on cell-substrate interactions.

Cobalt-chromium-molybdenum alloys exhibit good mechanical properties (yield strength: ~530 MPa, ultimate tensile strength: ~1114 MPa, elongation-to-failure: ~47.3%, and modulus: ~227 GPa) and corrosion resistance. In recent years, from the perspective of osseointegration, they are considered to be lower in rank in comparison to the widely used titanium alloys. We elucidate here the significant and favorable modulation of cellular activity of Zr-modified Co-Cr-Mo alloys. The average grain size of Co-Cr-Mo alloy samples with and without Zr was 104 ± 27 and ~53 ± 11 µm, respectively. The determining role of small addition of Zr (0.04 wt. %) to the Co-Cr-Mo alloys in favorable modulation of cellular activity was accomplished by combining cellular biology and materials science and engineering. Experiments on the influence of Zr addition to Co-Cr-Mo alloys clearly demonstrated that the cell adhesion, spread and cell-substrate interactions were enhanced in the presence of Zr. The spread/growth rate of cells was ~120% on the Co-Cr-Mo alloy and 190% per day on the Co-Cr-Mo-Zr alloy. While the % area covered by the cells increased from ~5.1 to ~33.6% on Co-Cr-Mo alloy and ~19.2 to ~47.8% on Co-Cr-Mo-Zr alloy after 2 and 24 hr of incubation. Similarly, the cell density increased from ~1354 to ~3424 cells/cm2 on Co-Cr-Mo alloy and ~3583 to ~7804 cells/cm2 on Co-Cr-Mo-Zr alloy after 2 and 24 hr of incubation. Additionally, stronger vinculin focal adhesion contact and signals associated with actin stress fibers together with extracellular matrix protein, fibronectin, were noted.

Negative influence of biofilm on CoCrMo corrosion.

Minimal studies exist investigating biofilm-induced corrosion of orthopaedic implants. This study investigates potential contributions of Pseudomonas aeruginosa and Staphylococcus aureus biofilms on corrosion resistance of CoCrMo under static and fretting conditions. Biofilms were cultured on CoCrMo coupons for either 4 weeks (static culture) or 6 days (fretting culture; pin-on-disk with a Ti6Al4V hemispherical tip pin). Morphology of biofilms and corrosion of coupon surfaces were analyzed via SEM. Open circuit potential and electrochemical impedance spectroscopy measurements were collected for corrosion performance evaluation. Results showed no visible corrosion on coupon surfaces in static culture, which suggests these biofilms alone do not induce severe corrosion under the conditions of this study. However, electrochemical data showed biofilm presence lowered coupon electrochemical impedance in static and fretting cultures, suggesting resistive and capacitive characteristics of the metal oxide-biofilm-media interface were altered. Under fretting, the P. aeruginosa group exhibited a distinct damage morphology and Co:Cr:Mo ratio within the wear scar when compared with S. aureus and the bacteria-free control. These differences suggest the presence of P. aeruginosa biofilms may negatively impact corrosion resistance at the fretting interface. Taken together these results demonstrate biofilms can contribute to implant corrosion by influencing the electrochemical impedance of implant metal surfaces.

CoCrMo alloys ions release behavior by TiNbN coating: an in vitro study.

The aim of the study was to show in vitro the greater inertness to the corrosion body fluid of TiNbN coating than the CoCrMo alloy substrate. The prosthetic component under study was a femoral component of total knee prosthesis in CoCrMo alloy coated in TiNbN with Physical Vapor Deposition technique immersed in static Hank's balanced salt solution (HBS) (pH = 6) for at least 34 months at a constant temperature of 37 °C. Another uncoated prosthetic component of CoCrMo alloy with the same type and size was left in static immersion in the same solution and for the same period of time. Scanning electron microscope (SEM) analysis was performed to investigate adhesion and proliferation at 24, 48, 72 h after seeding of 104 sub-confluents osteoblast-like cells (SaOS-2) cells on scaffold. The results of the study showed a reduction in the concentration of the metal ions released from the TiNbN-coated femoral component surface compared to the uncoated surface in the HBS solution. The overall reduction of the ions for the TiNbN-coated femoral component compared to the uncoated one was 80.1 ± 2%, 62.5% ± 8% and 48% ± 10% for Co, Cr, Mo, respectively (p < 0.01). SEM analysis confirmed the healthy state of the cells, the cellular adhesion and proliferation of SaOS-2 on the TiNbN-coated specimen. Although the results observed in vitro for the TiNbN coating are encouraging, clinical studies are certainly needed to be performed in order to understand how these positive findings can be translated in vivo and to determine the clinical benefit of TiNbN coating.

Tribocorrosion of a CoCrMo alloy sliding against articular cartilage and the impact of metal ion release on chondrocytes.

Partial knee replacement and hemiarthroplasty are some of the orthopedic procedures resulting in a metal on cartilage interface. As metal implant material, CoCrMo based alloys are commonly used. The aim of the present study is to assess the role of biotribocorrosion on the CoCrMo-cartilage interface with an emphasis on metal release during sliding contact. The biotribocorrosion experiments were performed under controlled electrochemical conditions using a floating cell with a three electrode set up coupled to a microtribometer. Throughout the experiment the coefficient of friction and the open circuit potential were monitored. Analyses of the electrolyte after the experiment show that metal release can occur during sliding contact of CoCrMo alloy against articular cartilage despite the extraordinary low coefficient of friction measured. Metal release is attributed to changes in passive layer caused at the onset of sliding. The released metal was found to be forming compounds with potential cytotoxicity. Since the presence of metal ions in the cartilage matrix can potentially lead to cell apoptosis, the metabolic activity of human osteoarthritic chondrocytes (2D-cultures) was investigated in the presence of phosphate buffered saline containing metal ions using XTT-assay. The experiments indicate that critical concentrations of Co ions lead to a significant decrease in chondrocyte metabolic activity. Therefore, biotribocorrosion is a mechanism that can occur in partial replacements and lead to chondrocyte apoptosis thus playing a role in the observed accelerated degradation of the remaining cartilage tissue after the mentioned orthopedic procedures. STATEMENT OF SIGNIFICANCE: Partial replacements provide an alternative to total joint replacements. This procedure is less invasive, allows a faster rehabilitation and provides a better function of the joint. However, the remaining native cartilage experiences accelerated degradation when in contact with metallic implant components. This work investigates the role of tribocorrosion at the metal-cartilage interface during sliding. Tribocorrosion is a degradation process that can alter significantly the wear rates experienced by metallic implants and lead to the release of metal ions and particles. The released metal can form compounds with potential cytotoxicity on cartilage tissue. The knowledge gained in this work will serve to understand the mechanisms behind the failure of partial replacements and develop future biomaterials with an enhanced lifetime.

Response of Saos-2 osteoblast-like cells to laser surface texturing, sandblasting and hydroxyapatite coating on CoCrMo alloy surfaces.

Cobalt chrome alloys are commonly used in orthopaedic implants where high stiffness and wear resistance are required. This study proposes Laser Surface Texturing (LST) as a cost-effective mean for producing bioinspired surface textures in order to improve the performance of CoCrMo orthopaedic implants. Cobalt-chrome alloy disks were modified using three different LST strategies: i) micro-scale texturing using a nanosecond laser source; (ii) micro-scale texturing with an ultrashort laser source and (iii) bioinspired sub-micron scale texturing with an ultrashort laser source. The modified disks were characterized and compared to blasted, hydroxyapatite coated and polished surface finishes. Saos-2 osteoblast-like cells were seeded on the different surfaces and their proliferation and morphology was assessed. The laser modification increases the surface energy of the CoCrMo alloy disks when compared to their untreated counterparts. The bioinspired sub-micron textured surfaces exhibited the highest cell metabolic activity on day 7 of the MTT assay.

TiO2-coated CoCrMo: improving the osteogenic differentiation and adhesion of mesenchymal stem cells in vitro.

The current gold standard material for orthopedic applications is titanium (Ti), however, other materials such as cobalt-chromium-molybdenum (CoCrMo) are often preferred due to their wear resistance and mechanical strength. This study investigates if the bioactivity of CoCrMo can be enhanced by coating the surface with titanium oxide (TiO2 ) by atmospheric pressure chemical vapor deposition (CVD), thereby replicating the surface oxide layer found on Ti. CoCrMo, TiO2-coated CoCrMo (CCMT) and Ti substrates were used for this study. Cellular f-actin distribution was shown to be noticeably different between cells on CCMT and CoCrMo after 24 h in osteogenic culture, with cells on CCMT exhibiting greater spread with developed protrusions. Osteogenic differentiation was shown to be enhanced on CCMT compared to CoCrMo, with increased calcium ion content per cell (p < 0.05), greater hydroxyapatite nodule formation (p < 0.05) and reduced type I collagen deposition per cell (p < 0.05). The expression of the focal adhesion protein vinculin was shown to be marginally greater on CCMT compared to CoCrMo, whereas AFM results indicated that CCMT required more force to remove a single cell from the substrate surface compared to CoCrMo (p < 0.0001). These data suggest that CVD TiO2 coatings may have the potential to increase the biocompatibility of CoCrMo implantable devices.

Study of cellular dynamics on polarized CoCrMo alloy using time-lapse live-cell imaging.

The physico-chemical processes and phenomena occurring at the interface of metallic biomedical implants and the body dictate their successful integration in vivo. Changes in the surface potential and the associated redox reactions at metallic implants can significantly influence several aspects of biomaterial/cell interactions such as cell adhesion and survival in vitro. Accordingly, there is a voltage viability range (voltages which do not compromise cellular viability of the cells cultured on the polarized metal) for metallic implants. We report on cellular dynamics (size, polarity, movement) and temporal changes in the number and total area of focal adhesion complexes in transiently transfected MC3T3-E1 pre-osteoblasts cultured on CoCrMo alloy surfaces polarized at the cathodic and anodic edges of its voltage viability range (-400 and +500 mV (Ag/AgCl), respectively). Nucleus dynamics (size, circularity, movement) and the release of reactive oxygen species (ROS) were also studied on the polarized metal at -1000, -400 and +500 mV (Ag/AgCl). Our results show that at -400 mV, where reduction reactions dominate, a gradual loss of adhesion occurs over 24 h while cells shrink in size during this time. At +500 mV, where oxidation reactions dominate (i.e. metal ions form, including Cr6+), cells become non-viable after 5h without showing any significant changes in adhesion behavior right before cell death. Nucleus size of cells at -1000 mV decreased sharply within 15 min after polarization, which rendered the cells completely non-viable. No significant amount of ROS release by cells was detected on the polarized CoCrMo at any of these voltages.

Fabrication of thin film TiO2 nanotube arrays on Co-28Cr-6Mo alloy by anodization.

Titanium oxide (TiO2) nanotube arrays were prepared by anodization of Ti/Au/Ti trilayer thin film DC sputtered onto forged and cast Co-28Cr-6Mo alloy substrate at 400 °C. Two different types of deposited film structures (Ti/Au/Ti trilayer and Ti monolayer), and two deposition temperatures (room temperature and 400 °C) were compared in this work. The concentrations of ammonium fluoride (NH4F) and H2O in glycerol electrolyte were varied to study their effect on the formation of TiO2 nanotube arrays on a forged and cast Co-28Cr-6Mo alloy. The results show that Ti/Au/Ti trilayer thin film and elevated temperature sputtered films are favorable for the formation of well-ordered nanotube arrays. The optimized electrolyte concentration for the growth of TiO2 nanotube arrays on forged and cast Co-28Cr-6Mo alloy was obtained. This work contains meaningful results for the application of a TiO2 nanotube coating to a CoCr alloy implant for potential next-generation orthopedic implant surface coatings with improved osseointegrative capabilities.

Cobalt-alloy implant debris induce HIF-1α hypoxia associated responses: a mechanism for metal-specific orthopedic implant failure.

The historical success of orthopedic implants has been recently tempered by unexpected pathologies and early failures of some types of Cobalt-Chromium-Molybdenum alloy containing artificial hip implants. Hypoxia-associated responses to Cobalt-alloy metal debris were suspected as mediating this untoward reactivity at least in part. Hypoxia Inducible Factor-1α is a major transcription factor involved in hypoxia, and is a potent coping mechanism for cells to rapidly respond to changing metabolic demands. We measured signature hypoxia associated responses (i.e. HIF-1α, VEGF and TNF-α) to Cobalt-alloy implant debris both in vitro (using a human THP-1 macrophage cell line and primary human monocytes/macrophages) and in vivo. HIF-1α in peri-implant tissues of failed metal-on-metal implants were compared to similar tissues from people with metal-on-polymer hip arthroplasties, immunohistochemically. Increasing concentrations of cobalt ions significantly up-regulated HIF-1α with a maximal response at 0.3 mM. Cobalt-alloy particles (1 um-diameter, 10 particles/cell) induced significantly elevated HIF-1α, VEGF, TNF-α and ROS expression in human primary macrophages whereas Titanium-alloy particles did not. Elevated expression of HIF-1α was found in peri-implant tissues and synovial fluid of people with failing Metal-on-Metal hips (n = 5) compared to failed Metal-on-Polymer articulating hip arthroplasties (n = 10). This evidence suggests that Cobalt-alloy, more than other metal implant debris (e.g. Titanium alloy), can elicit hypoxia-like responses that if unchecked can lead to unusual peri-implant pathologies, such as lymphocyte infiltration, necrosis and excessive fibrous tissue growths.

Electrochemical control of cell death by reduction-induced intrinsic apoptosis and oxidation-induced necrosis on CoCrMo alloy in vitro.

Electrochemical voltage shifts in metallic biomedical implants occur in-vivo due to a number of processes including mechanically assisted corrosion. These excursions may compromise the biocompatibility of metallic implants. Voltages can also be controlled to modulate cell function and fate. The in vitro effect of static voltages on the behavior of MC3T3-E1 pre-osteoblasts cultured on CoCrMo alloy (ASTM-1537) was studied to determine the range of cell viability and mode of cell death beyond the viable range. Cell viability and morphology, changes in actin cytoskeleton, adhesion complexes and nucleus, and mode of cell death (necrosis, or intrinsic or extrinsic apoptosis) were characterized at different voltages ranging from -1000 to +500 mV (Ag/AgCl). Moreover, electrochemical currents and metal ion concentrations at each voltage were measured and related to the observed responses. Results show that cathodic and anodic voltages outside the voltage viability range (-400 < V < +500) lead to primarily intrinsic apoptotic and necrotic cell death, respectively. Cell death is associated with cathodic current densities of 0.1 µA cm(-2) and anodic current densities of 10 µA cm(-2). Significant increase in metallic ions (Co, Cr, Ni, Mo) was seen at +500 mV, and -1000 mV (Cr only) compared to open circuit potential. The number and total projected area of adhesion complexes was also lower on the polarized alloy (p < 0.05). These results show that reduction reactions on CoCrMo alloys leads to apoptosis of cells on the surface and may be a relevant mode of cell death for metallic implants in-vivo.

Calcineurin/nuclear factor of activated T cells (NFAT) signaling in cobalt-chromium-molybdenum (CoCrMo) particles-induced tumor necrosis factor-α (TNFα) secretion in MLO-Y4 osteocytes.

Aseptic loosening is the devastating long term complication of total hip arthroplasty and orthopedic implant debris has been shown to trigger an intense inflammatory reaction leading to resorption of the bone matrix. Inflammatory cytokines, such as tumor necrosis factor-α (TNFα), have been implicated in this process and osteocytes may play a role in its production. We previously demonstrated that cobalt-chromium-molybdenum (CoCrMo) particles upregulate TNFα production by MLO-Y4 osteocytes in vitro, but the underlying mechanism has not been elucidated. Based on previous studies by others, we hypothesized that the calcineurin-nuclear factor of activated T cells (NFAT) pathway mediates CoCrMo particle-induced TNFα production in MLO-Y4 osteocytes. MLO-Y4 osteocytes exposed to CoCrMo particle treatment resulted in a rapid and significant increase in calcineurin activity. We also demonstrate that CoCrMo particle-induced upregulation of TNFα is reduced to control levels with calcineurin-NFAT inhibitors and this was also confirmed at mRNA level. Moreover, we demonstrate the localization of NFATs in MLO-Y4 osteocytes and that NFAT1 and 2 translocate to the nucleus upon CoCrMo particle treatment. Our results suggest that calcineurin-NFAT signaling is involved in TNFα production by MLO-Y4 osteocytes after CoCrMo particle treatment.

Effect of synovial fluid, phosphate-buffered saline solution, and water on the dissolution and corrosion properties of CoCrMo alloys as used in orthopedic implants.

The corrosion and dissolution of high- and low-carbon CoCrMo alloys, as used in orthopedic joint replacements, were studied by immersing samples in phosphate-buffered saline (PBS), water, and synovial fluid at 37 degrees C for up to 35 days. Bulk properties were analyzed with a fine ion beam microscope. Surface analyses by X-ray photoelectron spectroscopy and Auger electron spectroscopy showed surprisingly that synovial fluid produced a thin oxide/hydroxide layer. Release of ions into solution from the alloy also followed an unexpected pattern where synovial fluid, of all the samples, had the highest Cr concentration but the lowest Co concentration. The presence of carbide inclusions in the alloy did not affect the corrosion or the dissolution mechanisms, although the carbides were a significant feature on the metal surface. Only one mechanism was recognized as controlling the thickness of the oxide/hydroxide interface. The analysis of the dissolved metal showed two mechanisms at work: (1) a protein film caused ligand-induced dissolution, increasing the Cr concentration in synovial fluid, and was explained by the equilibrium constants; (2) corrosion at the interface increased the Co in PBS. The effect of prepassivating the samples (ASTM F-86-01) did not always have the desired effect of reducing dissolution. The release of Cr into PBS increased after prepassivation. The metal-synovial fluid interface did not contain calcium phosphate as a deposit, typically found where samples are exposed to calcium rich bodily fluids.

Biocompatibilidade do "Duracrom MS" e "Vitálio Cirúrgico" / Duracrom MS and surgical vitallium biocompatibility

O objetivo desta pesquisa foi comparar "Duracrom MS" (SIMONETTI-1981) e Vitálio Cirúrgico (liga de Cr-Co) como possíveis materais de prótese interna, após testados na microestrutura pelo Instituto de Pesquisa Tecnológicas (IPT) de Säo Paulo os corpos de prova foram implantados abaixo do periósteo de 28 ratos Wistar, sendo 14 com cada uma das ligas. Os animais foram sacrificados no tempos experimentais de: 1, 3, 7, 14, 21, 28 e 90 dias. A liga "Duracrom MS" apresentou após exame histológico uma biocompatibilidade adequada como material de implante quando comparada com o padräo "Vitálio Cirúrgico"

[Assays of cytotoxicity of the Nickel-Titanium shape memory alloy]. / Essais de cytotoxicité sur l'alliage à mémoire de forme Nickel-Titane.

The equiatomic Nickel-Titanium (NiTi) alloy has exceptional mechanical properties such as shape memory and superelasticity. It already has applications in orthodontics and is a promising orthopaedic biomaterial. Cytocompatibility studies must therefore be undertaken. The objective of this study is to determine the biological response that NiTi elicits compared to other orthopaedic metals currently used in orthopaedic surgery. Cytotoxicity tests constitute an efficient first step in a biocompatibility study and contribute to reduce animal use in laboratory. Direct contact and agar diffusion cytotoxicity assays were performed following ASTM standards #F813-83 and #F895-84 respectively. Confluent L-929 fibroblasts culture plates were incubated (directly or under an agar bed) in presence of NiTi, titanium (Ti), vitallium (Co-Cr-Mo) and 316L stainless steel discs. Following exposition to specimens, a vital dye was added to the plates. All cultures were evaluated for cytotoxic reactions, under light microscopy. Direct contact and agar diffusion assays indicated that all metals tested induced a mild biological reaction. Specimens were ranked according to an index of biological response, they are enumerated here in decreasing order of cytotoxicity: NiTi approximately Co-Cr-Mo >> pure grade 4 Ti approximately pure grade 1 Ti approximately Ti 6A1 4V approximately 316L stainless steel. Furthermore, plasma surface modification increased the cytocompatibility of NiTi.

Reaçäo tecidual a material de implante: estudo experimental / Tissue reaction to implant material: experimental study

Estuda-se a reaçäo tecidual a alguns materiais de implante (Aço-Inoxidável, Vitallium, Tântalo e Polietileno) através de modelo experimental em cäes. Foram realizados estudos macro e microscópico das amostras obtidas. Os resultados mostraram que o Vitalliun é o menos reativo entre os tecidos, vindo a seguir o Aço-Inoxidável e o Polietileno, sendo o Tântalo o material menos inerte entre os testados

Chromium-associated 32P-labeling of proteins.

The incubation of proteins with chromium (Cr3+ or Cr6+) in the presence of 32P ([gamma-32P]ATP or H3(32)PO4) at room temperature for 10-30 min resulted in the labeling of these proteins with 32P. The 32P-labeled proteins could be separated by SDS-polyacrylamide gel electrophoresis and identified by exposure to X-ray film. The characteristics of this procedure included: the optimal chromium concentration was 100 microM; the minimum requirement of each protein was 1 microgram; the optimal pH value was between 6 and 8; metal ions such as V5+, Mn2+ and Fe3+ strongly inhibited the effect of chromium, whereas Ca2+ and Mg2+ had little effect. It was concluded that chromium binds to the proteins and forms a complex with 32P to achieve the 32P-labeling of the proteins. This technique can be applied for the rapid preparation of 32P labels on protein markers for gel electrophoresis and for the identification of unknown protein species.