Macrophage proteomic analysis of covalent immobilization of hyaluronic acid and graphene oxide on CoCr alloy in a tribocorrosive environment.

In this work, a sequential covalent immobilization of graphene oxide (GO) and hyaluronic acid (HA) is performed to obtain a biocompatible wear-resistant nanocoating on the surface of the biomedical grade cobalt-chrome (CoCr) alloy. Nanocoated CoCr surfaces were characterized by Raman spectroscopy and electrochemical impedance spectroscopy (EIS) in 3 g/L HA electrolyte. Tribocorrosion tests of the nanocoated CoCr surfaces were carried out in a pin on flat tribometer. The biological response of covalently HA/GO biofunctionalized CoCr surfaces with and without wear-corrosion processes was studied through the analysis of the proteome of macrophages. Raman spectra revealed characteristic bands of GO and HA on the functionalized CoCr surfaces. The electrochemical response by EIS showed a stable and protective behavior over 23 days in the simulated biological environment. HA/GO covalently immobilized on CoCr alloy is able to protect the surface and reduce the wear volume released under tribocorrosion tests. Unsupervised classification analysis of the macrophage proteome via hierarchical clustering and principal component analysis (PCA) revealed that the covalent functionalization on CoCr enhances the macrophage biocompatibility in vitro. On the other hand, disruption of the HA/GO nanocoating by tribocorrosion processes induced a macrophage proteome which was differently clustered and was distantly located in the PCA space. In addition, tribocorrosion induced an increase in the percentage of upregulated and downregulated proteins in the macrophage proteome, revealing that disruption of the covalent nanocoating impacts the macrophage proteome. Although macrophage inflammation induced by tribocorrosion of HA/GO/CoCr surfaces is observed, it is ameliorated by the covalently grafting of HA, which provides immunomodulation by eliciting downregulations in characteristic pro-inflammatory signaling involved in inflammation and aseptic loosening of CoCr joint arthroplasties. Covalent HA/GO nanocoating on CoCr provides potential applications for in vivo joint prostheses led a reduced metal-induced inflammation and degradation by wear-corrosion.

Biological Responses in the Blood and Organs of Rats to Intraperitoneal Inoculation of Graphene and Graphene Oxide.

BACKGROUND: The discrepancy among the in vivo results found in the literature regarding graphene's side effects led us to conduct an in vivo study with graphene. METHODS: In vivo tests involving intraperitoneal inoculation of graphene and graphene oxide nanosheets in rats were carried out to assess potential changes in the blood and organs after 15 and 30 days. Graphene and graphene oxide nanosheets at a concentration of 4 mg per kilogram were suspended in an aqueous solution of 0.9% NaCl at a 1:1 proportion (graphene or graphene oxide), i.e., 1 mg/mL. RESULTS: Optical microscopy of liver, kidney, spleen, and lung tissues revealed no visible histological changes. However, particle traces were found in the peritoneal cavity. Thirty days after inoculation, blood samples were collected for hematological analysis. The blood analysis showed changes indicating a hepatic inflammatory process. Hematological changes after 30 days consisted of alterations to the red series, including microcytosis or higher mean hemoglobin concentrations. In addition, changes in prothrombin and thromboplastin caused longer coagulation times. CONCLUSION: This study contributes to further clarifying the possible toxicity of graphene and its potential biomedical applications.

Wettability, Corrosion Resistance, and Osteoblast Response to Reduced Graphene Oxide on CoCr Functionalized with Hyaluronic Acid.

The durability of metal-metal prostheses depends on achieving a higher degree of lubrication. The beneficial effect of hyaluronic acid (HA) on the friction and wear of both natural and artificial joints has been reported. For this purpose, graphene oxide layers have been electrochemically reduced on CoCr surfaces (CoCrErGO) and subsequently functionalized with HA (CoCrErGOHA). These layers have been evaluated from the point of view of wettability and corrosion resistance in a physiological medium containing HA. The wettability was analyzed by contact angle measurements in phosphate buffer saline-hyaluronic acid (PBS-HA) solution. The corrosion behavior of functionalized CoCr surfaces was studied with electrochemical measurements. Biocompatibility, cytotoxicity, and expression of proteins related to wound healing and repair were studied in osteoblast-like MC3T3-E1 cell cultures. All of the reported results suggest that HA-functionalized CoCr surfaces, through ErGO layers in HA-containing media, exhibit higher hydrophilicity and better corrosion resistance. Related to this increase in wettability was the increase in the expressions of vimentin and ICAM-1, which favored the growth and adhesion of osteoblasts. Therefore, it is a promising material for consideration in trauma applications, with improved properties in terms of wettability for promoting the adhesion and growth of osteoblasts, which is desirable in implanted materials used for bone repair.

Macrophage Biocompatibility of CoCr Wear Particles Produced under Polarization in Hyaluronic Acid Aqueous Solution.

Macrophages are the main cells involved in inflammatory processes and in the primary response to debris derived from wear of implanted CoCr alloys. The biocompatibility of wear particles from a high carbon CoCr alloy produced under polarization in hyaluronic acid (HA) aqueous solution was evaluated in J774A.1 mouse macrophages cultures. Polarization was applied to mimic the electrical interactions observed in living tissues. Wear tests were performed in a pin-on-disk tribometer integrating an electrochemical cell in phosphate buffer solution (PBS) and in PBS supplemented with 3 g/L HA, an average concentration that is generally found in synovial fluid, used as lubricant solution. Wear particles produced in 3 g/L HA solution showed a higher biocompatibility in J774A.1 macrophages in comparison to those elicited by particles obtained in PBS. A considerable enhancement in macrophages biocompatibility in the presence of 3 g/L of HA was further observed by the application of polarization at potentials having current densities typical of injured tissues suggesting that polarization produces an effect on the surface of the metallic material that leads to the production of wear particles that seem to be macrophage-biocompatible and less cytotoxic. The results showed the convenience of considering the influence of the electric interactions in the chemical composition of debris detached from metallic surfaces under wear corrosion to get a better understanding of the biological effects caused by the wear products.

Corrosion behavior of surface modifications on titanium dental implant. In situ bacteria monitoring by electrochemical techniques.

The effects of surface modifications and bacteria on the corrosion behavior of titanium have been studied. Five surface modifications were analyzed: two acid etchings (op V, op N), acid etching + anodic oxidation (op NT), sandblasting + acid etching (SLA), and machined surfaces (mach). The corrosion behavior of the surface modifications was evaluated by following the standard ANSI/AAMI/ISO 10993-15:2000. Cyclic potentiodynamic and potentiostatic anodic polarization tests and ion release by ICP-OES after immersion for 7 days in 0.9% NaCl were carried out. Microbiologically induced corrosion (MIC) of low and high roughness (mach, op V) was assessed in situ by electrochemical techniques. Streptococcus mutans bacteria were resuspended in PBS at a concentration of 3 × 108 bacteria mL-1 and maintained at 37°C. MIC was measured through the open circuit potential, Eoc , and electrochemical impedance spectroscopy from 2 to 28 days. Potentiodynamic curves showed the typical passive behavior for all the surface modifications. The titanium ion release after immersion was below 3 ppb. In situ bacteria monitoring showed the decrease in Eoc from -0.065 (SD 0.067) Vvs. Ag/AgCl in mach and -0.115 (SD 0.084) Vvs. Ag/AgCl in op V, to -0.333 (SD 0.147) Vvs. Ag/AgCl in mach and -0.263 (SD 0.005) Vvs. Ag/AgCl in op V, after 2 and 28 days, respectively. A reduction of the oxide film resistance, especially in op V (54 MΩ cm2 and 6 MΩ cm2 , after 2 and 28 days, respectively) could be seen. Streptococcus mutans negatively affected the corrosion resistance of titanium. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 997-1009, 2018.

In situ electrochemical study of the interaction of cells with thermally treated titanium.

Micromotion and fretting wear between bone and Ti-based alloys in stem and dental implants breaks the passive film and exposes the metal to the action of the complex surrounding medium, generating substantial amounts of debris and continuous Ti ion release. In this work, oxidation treatments at low temperatures (277 °C, 5 h) have been used to promote the formation of wear-corrosion resistant titanium oxide on the Ti surface. The objective of this paper has been the study of the influence of live cells on the protectiveness of the oxide formed at these low temperatures. The interaction of cells with the modified surface has been studied by scanning electron microscopy, electrochemical impedance spectroscopy, polarization curves, and x-ray photoelectron spectroscopy (XPS). The chemical composition of the thermally treated Ti surface is mainly TiO2 as anatase-rich titanium dioxide with a low concentration of hydroxyl groups and a low mean nanoroughness that could promote good cell adhesion. The electrochemical results indicate that the cells alter the overall resistance of the thermally treated Ti surfaces by decreasing the oxide resistance with time. At the same time, the anodic current increases, which is associated with cathodic control, and is probably due to the difficulty of access of oxygen to the Ti substrate. XPS reveals the presence of proteins on the surface of the treated specimens in contact with the cells and a decrease in the Ti signal associated with the extracellular matrix on the surface and the reduction of the oxide thickness.

Response of MC3T3-E1 osteoblasts, L929 fibroblasts, and J774 macrophages to fluoride surface-modified AZ31 magnesium alloy.

The present work evaluates the biocompatibility of a fluoride surface-modified AZ31 magnesium alloy (AZ31HF) with different cell lines that coexist in the implant environment to test its potential use as a biodegradable and absorbable biomaterial for bone repair. A clear stimulation of cell proliferation and an enhancement of the mitochondrial respiratory activity were observed when mouse osteoblasts (MC3T3-E1), fibroblasts (L929), and macrophages (J774) cell lines were cultured with the modified alloy. No significant change in apoptosis or viability rates was observed when osteoblasts and fibroblasts cultures were grown in the presence of this alloy. A proteomic analysis of the MC3T3-E1 cell extracts cultured in the presence of AZ31HF showed an overexpression of proteins related with the mineralization process, which is a necessary step for bone repair. An increase in the lactate dehydrogenase activity was observed in the MC3T3-E1 and J774 cell cultures that could be a response of the oxidative stress produced by the presence of the material. This stress could be related to the increase observed in the respiratory mitochondrial activity or respiratory burst measured in theses cultures that indicate damage in the cell membranes and subsequently some cell death. Results reported here, for and against AZ31HF, should be taken into account when considering the potential use of this modified alloy in bone repair applications.

Determination of metallic traces in kidneys, livers, lungs and spleens of rats with metallic implants after a long implantation time.

Metallic transfer from implants does not stop at surrounding tissues, and metallic elements may be transferred by proteins to become lodged in organs far from the implant. This work presents an in vivo study of metallic implant corrosion to measure metallic element accumulation in organs located far from the implant, such as kidneys, livers, lungs and spleens. The studied metallic implant materials were CoCr alloy, Ti, and the experimental alloy MA956 coated with alpha-alumina. The implants were inserted in the hind legs of Wistar rats. Analysis for Co, Cr, Ti and Al metallic traces was performed after a long exposure time of 12 months by Inductively Coupled Plasma (ICP) with Mass Spectrometry (MS). According to the results, the highest Cr and Ti concentrations were detected in spleens. Co is mainly found in kidneys, since this element is eliminated via urine. Cr and Ti traces increased significantly in rat organs after the long implantation time. The organs of rats implanted with the alpha-alumina coated experimental MA956 did not present any variation in Al content after 12 months, which means there was no degradation of the alumina layer surface.