Mar 16, 2018 - Gregorio del Amo 8, 28040 Madrid, ...... https://www.ncbi.nlm.nih.gov/pmc/articles/PMC348455/pdf/pnas00665-0524.pdf. 13. ... Dorr, L.D.; Bloebaum, R.; Emmanual, J.; Meldrum, R. Histologic, biochemical, and ion ... Ponthiaux, P.; Wenger
Apr 13, 2015 - School of Mechanical and Systems Engineering, Newcastle ... Keywords: wear analysis/testing; joint simulators; biotribology; .... articulating surfaces were then subject to further cleaning using isopropanol and lint free cloths.
Jun 1, 2004 - Processes of the Combustion Engines Possibilities ... information is estimated to average 1 hour per response, including the time for reviewing .... Their appearance may be similar to abnormal particles (severe wear particles), ...
Feb 22, 2013 - wear, wear particles, and reduced inflammatory potential of vitamin E ultrahigh-molecular-weight polyethylene for use in total .... All materi- als were nonirradiated. Isolation and analysis of UHMWPE wear debris from six-station pin o
The particles became incorporated in synovial tissue and persisted in the synovial fluid for many months. Smaller particles were phagocytosed by intimal cells, macrophages, multinucleate giant cells and fibroblasts; larger particles remained extracel
Jan 28, 2014 - We highlight the latest modifications and research directions that promise to more holistically design ..... surfaces coated with tropoelastin have shown improved blood biocompatibility, significantly reduced ... development promise to
of wear debris were observed with balls of different surface roughnesses ... grinding process and did not have any preferred orientation of the surface pattern. 2 .... ticle density of between 1 and 2 percent is observed. .... concentrations from the
Kodoya Y, Takaoka K. Characteristics of Hylamer polyethylene particles isolated from peri-prosthetic tissues of failed cemented total hip arthroplasties. J Biomed ...
immunogenic structural proteins is facilitating vaccine testing and the production of inexpensive diagnostic tests. .... urea, 0.05 M Tris-HCl (pH 6.8), 10% glycerol, and 0.0025% phenol red. Radiolabeled proteins were .... with the minimal protecting
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Sep 15, 2017 - phatase (TRAP), and cathepsin K . UHMWPE wear ... GTTGAGAA for cathepsin K (Cstk), TCCCCTG- ... Antibodies against NF-ÎºB p65.
Apr 20, 2015 - 1 Department of Biology, Chemistry and Materials Science, Office of Science and ... However, the role of endothelial cells in the generation of MGCs has not ... Under normal physiological conditions, bone structure and growth ... were
Jul 23, 2014 - tion and loosening of cemented hip implants, when Zr is present in the polymethylmethacrylate matrix.13. Other implant-derived metal ions ...
deliver therapeutics to the target cells, such as in the case of cancer.4. However, in other ... poly(sodium 4-styrenesulfonate) and poly(allylamine hydrochloride).
Ebola and Marburg VLPs Produced in Insect Cells â¢ JID 2007:196 (Suppl 2) â¢ S421 .... High Five (Hi5) insect cells, the VLPs were recovered from the.
Dec 5, 2012 - Journal of. Functional. Biomaterials. ISSN 2079-4983 www.mdpi.com/journal/jfb/. Review. Biocompatibility of Bacterial Cellulose Based Biomaterials ..... Ul-Islam et al.  prepared BC-Chitosan composites that showed improved water hol
Sep 19, 2017 - The best photocatalytic efficiency was found for the sample with iron atoms localized at the sample surface. ... Also, Bendali et al. showed that neuronal cells can survive on peptide-free graphene layers . .... we used pristine gr
Jul 11, 2017 - non-metallic: PEEK and Al2O3) by a 5-axes high-precision milling machine (US 20, Deckel Maho ... 2017, 18, 1489. 10 of 16 the implant-to-bone adhesion test and the railway rail profile was used (Figure 5A). After machining, implants re
Oct 9, 2009 - extracted from some of the infected tissues such as bone marrow and eyeballs. .... causes the greater stretching of molecular chains favoring the formation of Î²-form structure. Figure 2. SEM images of P(3HB) nanofiber electrospun from
Aug 18, 2009 - Maria Antonietta Croce. 2. , Daniela Quaglino. 2. , Deanna Guerra. 2 and. Roberta Tiozzo. 2,. *. 1 Department of Biomedical Sciences, Section ...
Dec 19, 2014 - Advanced powder manufacturing routes such as metal injection moulding (MIM) have emerged as techniques .... The aim of this review is to summarize existing literature and report on the use of advanced ..... The samples they manufacture
Feb 1, 2011 - tumor growth, decreases the time to a second detectible tumor, and increases the overall tumor burden of our model mice. In conclusion, we.
method to detect metallic wear particles is discussed, along with the effects of each method on subsequent debris examination. Keywords: Wear particles; filter debris; filter debris analysis; magnetic separation; oxidation; particle counting. INTRODU
Macrophage Biocompatibility of CoCr Wear Particles Produced under Polarization in Hyaluronic Acid Aqueous Solution Blanca Teresa Perez-Maceda 1 , María Encarnación López-Fernández 1 , Iván Díaz 2 , Aaron Kavanaugh 3 , Fabrizio Billi 3 , María Lorenza Escudero 2 , María Cristina García-Alonso 2 and Rosa María Lozano 1, * 1
Cell-Biomaterial Recognition Lab., Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas (CIB-CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain; [email protected] (B.T.P.-M.); [email protected] (M.E.L.-F.) Department of Surface Engineering, Corrosion and Durability, Centro Nacional de Investigaciones Metalúrgicas (CENIM-CSIC), Avda. Gregorio del Amo 8, 28040 Madrid, Spain; [email protected] (I.D.); [email protected] (M.L.E.); [email protected] (M.C.G.-A.) Department of Orthopaedic Surgery, David Geffen School of Medicine, University of California Los Angeles, Orthopaedic Hospital Research Center, 615 Charles E. Young Dr. South, Room 450A, Los Angeles, CA 90095, USA; [email protected] (A.K.); [email protected] (F.B.) Correspondence: [email protected]; Tel.: +34-918-373-112 (ext. 4208); Fax: 34-915-360-432
Received: 15 March 2018; Accepted: 2 May 2018; Published: 8 May 2018
Abstract: 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. Keywords: polarization; CoCr alloy; wear particles; hyaluronic acid; macrophages biocompatibility
1. Introduction Macrophages are cells involved in inflammatory processes . All orthopedic biomaterials may induce a biologic host response to generated wear debris, which is strictly dependent on the nature of the debris. Metal wear particles and metal ions from prosthetic devices may induce a cascade of adverse cellular reactions that may include inflammatory complications, macrophage activation, bone resorption, and, although rarely, neoplasia [2,3]. In this context, macrophages play a decisive role in the hostile inflammatory reactions that can lead to implant loosening and failure. Materials 2018, 11, 756; doi:10.3390/ma11050756
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Implanted metal surfaces in biological environments are exposed to cells and to physiological milieu interacting between them, an interaction that affects both the cells and the metallic surface. Implanted metallic materials, such as CoCr alloys, undergo dissolution and formation of a passive film that is affected by factors such as pH, ions present in the physiological medium, temperature, and biopotentials. Biopotentials are natural electrical properties that control the normal growth and development of different types of cells and tissues [4,5]. When a tissue is injured, its potentials undergo alterations to the normal potential of intact tissue [6,7]. Both biopotentials and injury potentials are found in bone and these potentials induced between injured and intact tissues persist until the tissue heals. Potentials in injured tissue can span over hundreds of microns and are generated by electric fields or ions flowing through the injured tissue [8,9] with a range of 10–100 mV/cm . Assuming the resistivity of soft tissues to be 100 Ω cm [9,11], the resulting current density is in the 1–100 µA/cm2 range [8,12]. Fukada and Yasuda had already described in 1957 the piezoelectric nature of the bone tissue . Endogenous electrical properties of bone may play a role in the feedback mechanism of bone remodeling and development [14,15]. In vivo, these electrical signals work in collaboration to provide the correct environment for normal bone growth and development, but can be disrupted or altered by an injury after a trauma and during the healing process. Moreover, the resulting voltage gradients may induce modifications in the electrochemical potential of metallic implants and consequently may affect their surface properties. Díaz et al.  recently characterized the CoCr alloy oxide films in a phosphate buffer solution containing 3 g/L of hyaluronic acid, the approximate concentration found in the synovial fluid of healthy joints , and under potentials with current density similar to those reported for injured tissues (1–100 µA/cm2 ). Potentiostatic pulses applied during the growth of the CoCr oxide film produced a modification of the film that affected its chemical composition, thickness, and structure compared to the passive film formed in air . These modifications induced surface heterogeneities at the atomic scale, geometric irregularities, such as nano-roughness, and a variation of the oxide composition . Moreover, application of potentials of 0.7 V vs. Ag/AgCl induced changes in the oxide layer with the formation of 10–50 nm diameter nanopores, uniformly distributed along the surface and an increase in Cr (VI) and Mo (VI) concentration . Despite the presence of the passive film, metals are susceptible to corrosion, particularly in aqueous environments, which may affect the surrounding tissue. Corrosion events generate electrical currents due to electron transfer from ions in the solution to the metallic surface where reactions are occurring. Wear-corrosion phenomena and micromotion or fretting-corrosion mechanically removes material, including the passive film, causing continuous activation/repassivation cycles . These continuous and dynamic processes not only weaken the surface performance but also lead to an increase in the debris around the implant. Wear debris is considered one of the main factors responsible for aseptic loosening of orthopedic endoprostheses [19,20]. Implant failure due to aseptic loosening, or osteolysis, may result from the release of wear debris or electrochemical ions generated during corrosion events [20–22]. From the electrochemical point of view, on the metallic surfaces of implants, the breakdown of the passive film under the wear-corrosion process causes a drastic decrease in the open circuit potential of the metal towards negative potentials, i.e., from the passive to active state. This situation can suppose a polarization of about 500–700 mV with respect to the original open circuit potential. The change from the passive to active state can be induced mechanically under wear and electrochemically applying anodic polarization on the tribological system. Several researchers have studied the wear corrosion processes by application of anodic potentiodynamic polarization under wear processes [23,24]. The object of this paper was to evaluate the biocompatibility of particles produced during wear-corrosion assays of a CoCr alloy at potentiodynamic range to cover a wide polarization window on the samples. The hyaluronic acid, the lubricant component of the synovial liquid, was selected as the electrolyte for the generation of wear particles in conditions that represent more closely the prosthesis environment. Since macrophages are the main cells involved in the primary response to
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bodies, cytotoxicity and biocompatibility of the wear particles were evaluated using these cells, 3 of 16 measuring lactate dehydrogenase and mitochondrial activity, respectively.
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2. Results and Discussion foreign bodies, cytotoxicity and biocompatibility of the wear particles were evaluated using these cells, measuring lactate dehydrogenase and mitochondrial activity, respectively. 2.1. Wear-Corrosion Tests 2. Results Discussion Theand interaction of physiological fluids with the bearing surfaces of hip implants is of great importance in the research of artificial joint lubrication, although this study has been so far little 2.1. Wear-Corrosion Tests explored. TheThe interaction physiological withball theon bearing surfaces of hip implants is of great importance effect ofofsliding of the fluids alumina the HCCoCr alloys is clearly shown in the drastic in the research of artificial joint lubrication, although this study has been so far little explored. change in the open circuit potential. As an example, Figure 1 shows the change in the open circuit The effect of the sliding of thesurfaces aluminainball the HCCoCrwith alloys is clearly shown in drastic to potential when HCCoCr PBSon supplemented 3 g/L HA (PBS-HA) arethe subjected change the open open circuit circuit potential. As an example, Figure 1 shows in the open circuit wear.inThe potential without wear was around −0.25the V change versus Ag/AgCl, decreasing potential when the HCCoCr surfaces in PBS supplemented with 3 g/L HA (PBS-HA) are subjected sharply when the alumina ball (pin) started the circular movement under 5 N load at 120 rpm. At this to moment, wear. Thethe open circuit potential without wearuntil was around −0.25 V versus Ag/AgCl, open circuit potential decreased achieving values of about −0.55 V decreasing vs. Ag/AgCl, sharply when300 the mV, alumina (pin) started the until circular 5 N reduction load at 120inrpm. this i.e., about and ball remained constant themovement end of theunder test. The the At potential moment, the open circuit potential decreased until achieving values of about − 0.55 V vs. Ag/AgCl, value towards more negative values indicates that the HCCoCr surface becomes electrochemically i.e.,active. aboutThis 300 mV, and remained until the of the test. reduction inpromoting the potential variation is due to constant the breakdown ofend the passive filmThe under sliding, thevalue release towards more negative values indicates that the HCCoCr surface becomes electrochemically active. of metallic ions and particles. This variation is due to the breakdown of the passive film under sliding, promoting the release of metallic ions and particles.
Potential, V vs. Ag/AgCl
-0.30 starting wear
-0.35 -0.40 -0.45 -0.50
Time, s Figure 1. 1. Open circuit potential of of HCCoCr disks under wear. Measurement of of thethe open circuit Figure Open circuit potential HCCoCr disks under wear. Measurement open circuit potential before and during the wear corrosion test of HCCoCr in PBS containing 3 g/L HA (PBS-HA). potential before and during the wear corrosion test of HCCoCr in PBS containing 3 g/L HA (PBS-HA).
Figure 2 panels a and b show coefficient of friction (COF) HCCoCr/alumina pair PBS Figure 2 panels a and b show thethe coefficient of friction (COF) for for HCCoCr/alumina pair in in PBS and PBS supplemented with 3 g/L HA (PBS-HA) during anodic potentiodynamic polarization and and PBS supplemented with 3 g/L HA (PBS-HA) during anodic potentiodynamic polarization and the the anodic polarization drawn 10 mV/min of HCCoCr in PBS containing 3 g/L HA anodic polarization curvescurves drawn at 10 at mV/min of HCCoCr in PBS andand PBSPBS containing 3 g/L HA under wear conditions (between point 1 and 2 Figure in Figure respectively. anodic polarization under wear conditions (between point 1 and 2 in 2a),2a), respectively. TheThe anodic polarization curve of HCCoCr PBS-HA without wear been also added Figure comparative reasons. curve of HCCoCr in in PBS-HA without wear hashas been also added in in Figure 2b2b forfor comparative reasons. It can seen that under sliding at the corrosion potential (before point in Figure COF was It can be be seen that under sliding at the corrosion potential (before point 1 in1 Figure 2a),2a), thethe COF was significantly higher in PBS than in PBS-HA. This result agrees with hypothesis that hyaluronic significantly higher in PBS than in PBS-HA. This result agrees with thethe hypothesis that thethe hyaluronic acid a known lubricant role the acting joint, as acting as aabsorber shock absorber  facilitating and thus facilitating acid hashas a known lubricant role in thein joint, a shock  and thus smooth smooth joint movement by friction reducingbetween friction both between both surfaces. At the next stagepoint (from1 point joint movement by reducing surfaces. At the next stage (from to 2, 1 to 2, in Figure 2a), the difference between both COF (in PBS and PBS-HA) remained, but higher in Figure 2a), the difference between both COF (in PBS and PBS-HA) remained, but higher fluctuations were detected. The fluctuations could be related to the continuous formation of hard particulate matter
fluctuations fluctuationswere weredetected. detected.The Thefluctuations fluctuationscould couldbe berelated relatedtotothe thecontinuous continuousformation formationofofhard hard particulate matter that enhances friction between both counterparts and decreases the friction when particulate matter thatbetween enhancesboth friction between both decreases friction when that enhances friction counterparts and counterparts decreases theand friction whenthe ejected from the ejected the areas ititaccumulates 3). load on ejected from thetrack trackto tosurrounding surrounding areaswhere where(Figure accumulates (Figure 3).The The load applied onthe the track tofrom surrounding areas where it accumulates 3). The(Figure load applied on theapplied CoCr surfaces CoCr surfaces while sliding activates mechano-chemical reactions, causing not only the detachment CoCr surfaces while sliding activates mechano-chemical reactions, causing not only the detachment while sliding activates mechano-chemical reactions, causing not only the detachment of the passive of passive film  but ofofCOF. hyaluronic acid ofthe the passive film material butalso alsobulk bulkmaterial material resulting inan anincrease increase COF.The The hyaluronic acidinin film  but also bulk resulting in anresulting increase in of COF. The hyaluronic acid in PBS maintains PBS maintains the lubricant effect during most of the wear corrosion tests. PBSlubricant maintains the lubricant effect most of the wear the effect during most of during the wear corrosion tests. corrosion tests. During DuringAPC APC 1 1 PBS PBS
Figure 2. Friction coefficient of HCCoCr/alumina pair (a) and anodic polarization curves (APC) Figure Figure2.2.Friction Frictioncoefficient coefficientofofHCCoCr/alumina HCCoCr/aluminapair pair(a) (a)and andanodic anodicpolarization polarizationcurves curves(APC) (APC)ofof of HCCoCr disks (b) during wear corrosion tests in PBS and PBS containing 3 g/L HA (PBS-HA). HCCoCr disks (b) during wear corrosion tests in PBS and PBS containing 3 g/L HA (PBS-HA). HCCoCr disks (b) during wear corrosion tests in PBS and PBS containing 3 g/L HA (PBS-HA). Measurement of the friction coefficient before, during, and after application of anodic polarization Measurement Measurementofofthe thefriction frictioncoefficient coefficientbefore, before,during, during,and andafter afterapplication applicationofofanodic anodicpolarization polarization current (APC). Anodic polarization curve for HCCoCr alloy in PBS-HA without wear is added for current (APC). Anodic polarization curve for HCCoCr alloy in PBS-HA without wear is current (APC). Anodic polarization curve for HCCoCr alloy in PBS-HA without wear isadded addedfor for comparative analysis. comparative analysis. comparative analysis.
Figure Figure3.3. 3.Secondary Secondaryelectron electronimages imagesof ofwear weartracks trackson onHCCoCr HCCoCrdisks. disks.Images Imagesby bySEM SEMof ofHCCoCr HCCoCr Figure Secondary electron images of wear tracks on HCCoCr disks. Images by SEM of HCCoCr samples in PBS-HA under wear: (a) at the open circuit potential (PBS-HA) and (b) applying samples in PBS-HA under wear: (a) at the open circuit potential (PBS-HA) and (b) applying anodic samples in PBS-HA under wear: (a) at the open circuit potential (PBS-HA) and (b) applyinganodic anodic potentiodynamic polarization (PBS-HA+POL). potentiodynamic polarization polarization (PBS-HA+POL). (PBS-HA+POL). potentiodynamic
As Asaaconsequence consequenceofofmechanically mechanicallyassisted assistedcorrosion, corrosion,the thepassive passivefilm filmon onthe theHCCoCr HCCoCrsurface surface As a consequence of mechanically assisted corrosion, the passive film on the HCCoCr surface was wasrapidly rapidlybroken brokenininboth bothmedia, media,PBS PBSand andPBS-HA, PBS-HA,producing producingan anincrease increaseofofapproximately approximately33orders orders was rapidly broken in both media, PBS and PBS-HA, producing an increase of approximately 3 orders ofofmagnitude in current (Figure 2b) with respect to the anodic polarization curve without wear. magnitude in current (Figure 2b) with respect to the anodic polarization curve without wear. of magnitude in current (Figure 2b) with respect to the anodic polarization curve without wear. Corrosion Corrosionprogresses progresseson onthe thewear weartrack trackdrawn drawnby bythe thesliding slidingofofalumina aluminaball ballon onthe theHCCoCr HCCoCrdisks disks Corrosion progresses on the wear track drawn by the sliding of alumina ball on the HCCoCr disks (Figure (Figure3). 3).Having Havingininmind mindthe thewide widepassive passiveregion regionseen seenininthe theanodic anodicpolarization polarizationcurve curvedrawn drawn (Figure 3). Having in mind the wide passive region seen in the anodic polarization curve drawn without withoutwear wear(Figure (Figure2), 2),the thepotential potentialapplied appliedcould couldbe beemployed employedininforming formingrapidly rapidlythe thenew newoxide oxide without wear (Figure 2), the potential applied could be employed in forming rapidly the new oxide film. film.However, However,the thesliding slidingrate rateisisquick quickenough enoughtotoavoid avoidthe therepassivation repassivationand andformation formationofofnew new film. However, the sliding rate is quick enough to avoid the repassivation and formation of new protective chromium oxides. The constant value of the current density around 1 mA (three orders protective chromium oxides. The constant value of the current density around 1 mA (three ordersofof protective chromium oxides. The constant value of the current density around 1 mA (three orders of magnitude magnitudehigher higherthan thanwithout withoutwear) wear)indicates indicatesthat thatunder underthese theseexperimental experimentalconditions conditions(5(5NNload load magnitude higher than without wear) indicates that under these experimental conditions (5 N load and andsliding slidingrate rateofof120 120rpm), rpm),the thepassive passivefilm filmisisdestroyed destroyedand andremains remainsininan anactive activestate stateuntil untilthe theend end ofofthe test. the test.
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and sliding rate of 120 rpm), the passive film is destroyed and remains in an active state until the end Materials 2018, 11, x FOR PEER REVIEW 5 of 17 of the test. Figure 3 shows the secondary electron (SE) images of the tracks HCCoCr in PBS-HA after wearafter Figure 3 shows the secondary electron (SE) images of the of tracks of HCCoCr in PBS-HA corrosion tests, at the corrosion potential (PBS-HA) and under anodic potentiodynamic polarization wear corrosion tests, at the corrosion potential (PBS-HA) and under anodic potentiodynamic (PBS-HA+POL). In both cases (a)In and (b),cases debris accumulated in accumulated the immediate of the wear polarization (PBS-HA+POL). both (a)isand (b), debris is invicinity the immediate vicinity tracks, butwear the surface inside trackinside is especially when anodic potential of the tracks, but the the surface the trackaltered is especially altered potentiodynamic when anodic potentiodynamic is applied. 4 shows, as an example, semiquantitative analysis taken by EDStaken of thebythree potentialFigure is applied. Figure 4 shows, as the an example, the semiquantitative analysis EDS of areas of interest in the HCCoCr alloy immersed in PBS-HA after wear corrosion under polarization the three areas of interest in the HCCoCr alloy immersed in PBS-HA after wear corrosion under (PBS-HA-POL): away from the track 1), immediate (spectrum 2), and inside 2), theand polarization (PBS-HA-POL): away(spectrum from the track (spectrumvicinity 1), immediate vicinity (spectrum track (spectrum 3). The most important feature found is the high % C content accumulated in the inside the track (spectrum 3). The most important feature found is the high % C content accumulated vicinity of vicinity the track. means that debris mainly composed of composed C and O, the proportion in the ofItthe track. It the means thatis the debris is mainly of greatest C and O, the greatest probably coming from the hyaluronic acid. proportion probably coming from the hyaluronic acid.
Figure 4. HCCoCr surface after wear corrosion applying anodic potentiodynamic polarization. Figure 4. HCCoCr surface after wear corrosion applying anodic potentiodynamic polarization. Secondary electron image and EDS analyses away from the track, in the immediate vicinity, and Secondary electron image and EDS analyses away from the track, in the immediate vicinity, and inside insideinthe in thesurface HCCoCr surface in PBS-HA+POL. the track thetrack HCCoCr in PBS-HA+POL.
The morphology and chemical characterization of the wear particles detached during wear The morphology and chemical characterization the wear particles wear corrosion tests revealed some interesting results. of Figure 5 shows, as andetached example,during the secondary corrosion tests revealed some interesting results. Figure 5 shows, as an example, the secondary electron electron image of wear particles collected from the tribocorrosion test in PBS containing 3 g/L HA image wear particles collected from tribocorrosion test in PBS g/L HA andofthe semiquantitative analysis ofthe some particles, identified fromcontaining 1 to 6 and 3marked in and bluethe color. semiquantitative analysis of some particles, identified from 1 to 6 and marked in blue color. The statistical results of the effect of the corrosive medium and polarization applied in the wear The statistical results of the effect of the corrosive medium and polarization appliedappear in the wear corrosion tests on the chemical composition of the wear-detached particles collected in Table corrosion teststable, on the chemical composition the wear-detached particles collected appear2,inthe Table 1. 1. In this three condition numbersofassigned to 1, the PBS corrosive medium, PBS-HA In this table, three condition numbers assigned to 1, the PBS corrosive medium, 2, the PBS-HA corrosive corrosive medium without applying polarization, and 3, the PBS-HA corrosive medium applying medium without(PBS-HA-POL), applying polarization, and 3,considered. the PBS-HA Mean, corrosive mediumdeviation, applying polarization polarization have been standard minimum and (PBS-HA-POL), haveC25 been considered. Mean, standard deviation, minimum and maximum value, maximum value, and C75, and median are shown. C25 and C75, and median are shown.
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Figure 5. Secondary electron images of wear particles. Particles were collected from wear corrosion Figure 5. Secondary electron images of wear particles. and Particles were on collected corrosion tests performed in PBS containing 3 g/L HA (PBS-HA) deposited silicon from waferwear to analyze the tests performed in PBS containing 3 g/L HA (PBS-HA) and deposited on silicon wafer to analyze the chemical composition of detached particles. Blue colors represent the particle where EDS has been chemical composition of detached represent theptparticle EDS attached. has been performed and the particle numberparticles. shown in Blue pink colors is correlated to the shown where in the table performed and the particle number shown in pink is correlated to the pt shown in the table attached. Table 1. Statistical analysis by Kruskal–Wallis test for the chemical composition (wt %) of wear Table 1. Statistical by Kruskal–Wallis test the samples chemicalin composition (wt %) of wear1),particles particles detachedanalysis during wear corrosion tests offor CoCr PBS (condition number PBS+ 3 detached during wear corrosion tests of CoCr samples in PBS (condition number 1), PBS+number 3 g/L HA g/L HA (condition number 2) and under anodic potentiodynamic polarization (condition 3), (condition 2) and anodic potentiodynamic polarization (condition 3), where is where n is number the number of under samples, mean is the average value, SD is the standardnumber deviation, C25 is nthe the number of samples, mean is the average value, SD is the standard deviation, C25 is the value of value of the 25% of the data, C75 is the value of the 75% of the data, and p * is the significant difference the 25%confidence of the data,level. C75 is the value of the 75% of the data, and p * is the significant difference at 0.95 at 0.95 confidence level.
Condition Number n Mean SD Minimum Maximum C25 Median C75 p* Condition Mean 6.92 SD Minimum Maximum Median 25 1 Number 7n 33.28 21.61 43.13 C29.33 34.38 C7538.45p * 33.28 6.92 21.61 43.13 Cr 21 247 28.53 8.73 14.06 48.82 29.33 21.28 34.38 27.52 38.4533.66 0.034 2 24 28.53 8.73 14.06 48.82 21.28 27.52 33.66 0.034 Cr 39.09 11.02 11.02 22.34 57.01 33 77 39.09 22.34 57.01 33.65 33.65 37.60 37.60 47.6747.67 4.31 6.02 0.00 13.81 11 77 4.31 6.02 0.00 13.81 0.000.00 0.000.00 11.5411.54 2 24 7.73 16.94 0.00 54.07 0.00 0.00 5.99 0.001 Co Co 23 247 7.73 0.00 54.07 17.57 0.00 32.120.00 51.485.99 0.001 36.08 16.94 17.59 15.14 58.64 31 77 36.08 15.14 58.64 0.00 17.57 0.0032.12 0.0051.48 0.00 17.59 0.00 0.00 0.00 2 24 0.45 1.54 0.00 5.63 0.00 Mo 1 7 0.00 0.00 0.00 0.00 0.00 0.000.00 0.00 0.000.006 3 7 2.71 2.60 0.00 5.48 0.00 4.00 5.45 21 247 0.45 1.54 0.00 5.63 15.76 0.00 16.870.00 23.160.00 0.006 Mo 18.59 5.02 11.46 26.36 14.11 5.90 0.96 27.48 32 724 2.71 2.60 0.00 5.48 11.64 0.00 13.244.00 17.835.450.051 P 3 7 9.40 7.30 1.30 19.57 1.89 8.79 16.10 1 7 18.59 5.02 11.46 26.36 15.76 16.87 23.16 1 7 1.52 4.01 0.00 10.62 0.00 0.00 0.00 P Al 22 24 5.90 0.96 27.48 0.00 11.64 0.0013.24 0.0017.830.1230.051 24 14.11 0.18 0.68 0.00 3.17 0.76 1.06 0.00 2.57 33 77 9.40 7.30 1.30 19.57 0.001.89 0.008.79 1.8516.10 40.10 10.67 23.79 54.36 11 77 1.52 4.01 0.00 10.62 33.72 0.00 41.310.00 51.570.00 2 24 41.82 17.72 3.52 72.84 34.64 49.54 51.69 0.002 O Al 23 247 0.18 0.68 0.00 3.17 0.00 8.280.00 16.050.00 0.123 10.91 7.03 3.76 23.11 4.83 2.19 5.80 0.00 15.35 0.00 31 77 0.76 1.06 0.00 2.57 0.00 0.000.00 0.00 1.85 2 24 6.98 7.49 0.00 19.94 0.00 6.39 14.16 0.022 C 13 77 40.10 23.79 54.36 0.00 33.72 0.0041.31 0.0051.57 0.00 10.67 0.00 0.00 0.00 2 24 41.82 17.72 3.52 72.84 34.64 49.54 51.69 0.002 O * p-value in the Kruskal–Wallis test. 3 7 10.91 7.03 3.76 23.11 4.83 8.28 16.05 1 7 2.19 5.80 0.00 15.35 0.00 0.00 It can be seen that the particles are mainly composed of Co, Cr, Mo, P, C and 0.00 O, with some C of Al in some 2 isolated24 6.98 The 7.49 Kruskal–Wallis 0.00 19.94 0.00 that 6.39 0.022 traces particles. test indicated there 14.16 are significant 3 7 0.00 0.00 0.00 0.00 0.00 0.00 0.00 differences in the levels of Cr, Co, Mo, O and C, comparing the different conditions, i.e., depending on * p-value in the Kruskal–Wallis test. the composition of corrosive medium (PBS-condition 1 or PBS-HA-condition 2) and the application of polarization in wear corrosion tests (PBS-HA-POL, condition 3, and PBS-HA, condition 2). However, It can be seen that theinparticles mainly composed no significant differences P and Alare levels were obtained.of Co, Cr, Mo, P, C and O, with some traces of Al in some isolated particles. The Kruskal–Wallis test indicated that there are significant differences in the levels of Cr, Co, Mo, O and C, comparing the different conditions, i.e., depending
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The results of the post hoc Mann–Whitney test used to determine which pairs differed among them are shown in Table 2. Cr levels are significantly higher in condition number 3 than 2 (p = 0.021). Co and Mo levels are significantly higher in condition number 3 than 1 and 2 (p = 0.002 and p = 0.001, p = 0.025 and p = 0.002, respectively). O levels are significantly lower in condition number 3 than 1 and 2 (p = 0.002 in both cases). C levels are significantly lower in condition number 3 than 2 (p = 0.017). Table 2. Post hoc Mann–Whitney analysis to determine which pairs differed among them (condition numbers: 1-PBS, 2-PBS-HA, and 3-PBS-HA+POL). Comparison between Pairwise
Cr Co Mo P Al O C
p ** 1 vs. 2
p ** 1 vs. 3
p ** 2 vs. 3
0.119 0.556 0.438 0.508 0.098
0.277 0.002 0.025 0.002 0.317
0.021 0.001 0.008 0.002 0.017
** p-value in the Mann–Whitney test.
In summary, the statistical analysis confirmed that factors such as “composition of the corrosive medium” and “polarization applied” have an influence on the dependent variable chemical composition of the particles that is discussed immediately below. The main significant effect of the addition of hyaluronic acid in the PBS to the wear particles detached is observed in the increase of the C content in the chemical composition of the particles. In both media (PBS, condition 1, and PBS-HA, condition 2), particles are mainly composed of Cr and O, followed by P and some Co. This chemical composition can be directly linked to the detachment of the native passive film during the wear corrosion test. It has been proven by XPS (data not shown) that the immersion of the HCCoCr surfaces in PBS-HA causes a decrease in the Co species in the passive film and the enrichment in chromium oxide where phosphorus is included. It has been reported in literature that phosphate is adsorbed upon freshly exposed metal at the same time that ions are released into the solution until the passive layer is formed, whose composition varies significantly depending upon the environment . Lewis et al. established that the corrosion, especially when associated with mechanical wear, is controlled by phosphate anions that absorb or react with the Co and Cr dissolution products. This promotes the formation of a mixed composition of phosphates, hydroxides, and oxides originating from the bulk metal. This means that most of the particles collected after the wear corrosion tests in PBS and PBS-HA come from the native passive film (whose thickness is about 5–7 nm) and are mainly composed of chromium oxide and phosphate. With respect to applying polarization during the wear corrosion tests in PBS-HA (condition 3), this factor has an important effect on the chemical composition of the wear particles detached. In this condition, particles are mainly composed of Cr and Co, followed by O, P, and Mo. The main significant effect of the polarization is the significant enrichment in Co, Cr, and Mo in the chemical composition of the detached particles. In this case, the wear particles produced under anodic polarization increased the Co/Cr ratio (with a value of 0.9 in comparison with a value of 0.3 found in PBS-HA without polarization). As wear particles obtained without polarization, these particles also contained P, although in a low proportion (Table 1). It has been reported in the literature  that the potential applied on the HCCoCr induces a change in the chemical composition of the passive film. Díaz et al. established that the increase in polarization (from 0.5 to 0.7 V) induced the preferential dissolution of cobalt whereas chromium was concentrated in the surface oxide film . The passive film grown at a potential of 0.5 V vs. Ag/AgCl (into the passive region of the anodic polarization curve) consisted
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film grown at a potential of 0.5 V vs. Ag/AgCl (into the passive region of the anodic polarization curve) consisted predominantly of Cr2O3 and Cr(OH)3. However, the oxidation at a potential of 0.7 V vs. Ag/AgCl caused the of3Cr (VI) in the filmatbut Co was not increased. In the predominantly of Cr . However, thepassive oxidation a potential of 0.7 V vs. Ag/AgCl 2 Oappearance 3 and Cr(OH) case of wear corrosion under anodic potentiodynamic polarization, continuous sliding of wear the caused the appearance of Cr (VI) in the passive film but Co was notthe increased. In the case of alumina ball on the HCCoCr surface did not allow the the regeneration the oxide film. Instead, corrosion under anodic potentiodynamic polarization, continuousofsliding of the alumina ballan on active state stimulated bynot polarization was inducedof onthe theoxide surface bulk wasstimulated directly the HCCoCr surface did allow the regeneration film.where Instead, anmaterial active state exposed and detached to the electrolyte. Considering thismaterial situation,was thedirectly results reveal that thedetached anodic by polarization was induced on the surface where bulk exposed and polarization on CoCr surfaces under wear-corrosion processes and polarization induced the on release to the electrolyte. Considering this situation, the results revealaccelerated that the anodic CoCr ofsurfaces larger metallic particles with processes higher Coaccelerated content coming from the material. under wear-corrosion and induced thebase release of larger metallic particles with higher Co content coming from the base material. 2.2. Macrophage Cell Response 2.2. Macrophage Cell Response Macrophages are a primary immune cell type and the main cellular type involved in Macrophages are a primary cell type and main cellular type involved inflammatory inflammatory processes and inimmune host response , sothe their biologic host response to in wear particles processesfrom  and host response , so biologic host response to wear particles generated from generated theinimplanted materials is their of great interest. the Macrophage implanted materials is to of great response wear interest. particles derived from the tribocorrosion assays was evaluated Macrophage response to wear particles derived from the tribocorrosion assays was evaluated by by measuring the effect on cell toxicity and respiratory activity. measuring the effect on cell andwear respiratory activity. Cytotoxicity induced bytoxicity HCCoCr particles was analyzed by measuring LDH activity Cytotoxicity induced by HCCoCr wear particles was measuring LDH activity released from cells (Figure 6), whose levels increase uponanalyzed plasma by membrane damage, a signreleased of cell from . cells As (Figure 6), whose levels6,increase upon plasma membrane damage, a sign of cellinduced death . death is shown in Figure exposure of macrophages cultures to wear particles a As is shown in Figurethat 6, exposure of macrophages to wear used particles induced a degree of degree of cytotoxicity was mainly dependent oncultures the conditions during wear-corrosion cytotoxicity that was mainly dependent conditions used duringconcentration wear-corrosion assays and assays and particle concentration. As shownoninthe Figure 6 panel A, particles of 0.5 mg/mL particle concentration. As almost shown in Figure 6 panel A, particles concentration of 0.5 mg/mL obtained obtained in PBS produced 58% cytotoxicity, a percentage that was significantly reduced to in PBS12% produced almost 58% cytotoxicity, a percentage that was significantly reduced to of almost almost when wear particles were generated from tribocorrosion tests in the presence 3 g/L12% of when wear particles werean generated from tribocorrosion tests in the presence of 3 g/L ofacid hyaluronic hyaluronic acid (PBS-HA), effect that could indicate a protective role of the hyaluronic on the acid (PBS-HA), an effect that could indicate(Table a protective role of the hyaluronic acid on the metallic metallic surface under wear stress conditions 3). Concentrations of 1 mg/mL of wear particles surface wear stress conditions (Table 3). Concentrations of 1 mg/mL wear75%, particles from from the under PBS test produced an increase in the macrophage cytotoxicity to of almost a value the PBS in testcomparison produced an increase in the macrophage to almost 75%, a value elevated elevated with the cytotoxicity inducedcytotoxicity by the wear particles obtained in PBS in comparison thewhere cytotoxicity induced by the14% wear particles obtained PBS containing 3 g/L containing 3 g/L with of HA, cytotoxicity reached (data not shown). Noinadditional increase in of HA, where cytotoxicity reached 14% (data not shown). No additional increase in the cytotoxicity the cytotoxicity was observed at higher concentrations of wear particles (2 mg/mL) generated in PBS observed at higher concentrations wear particlesto(2the mg/mL) generated PBS as macrophages aswas macrophages cytotoxicity appearedof comparable one elicited byinexposure to lower cytotoxicity appeared comparable the one elicited by exposure toalower concentrations particles concentrations of particles (0.5 and 1tomg/mL), where approximately 64% cytotoxicity wasofdetected (0.5 and 1 mg/mL), where approximately a 64% cytotoxicity was detected (data not shown). (data not shown).
Table 3. Statistical analyses of cytotoxicity data. Mean differences of cytotoxicity effects between P2 (0.5 mg/mL) vs. P3 (0.5 mg/mL) and P3 (2 mg/mL) vs. P6 (2 mg/mL) were studied with Student’s t tests (α = 0.05), respectively. (a) Wear particles obtained in PBS (P2) and in PBS containing 3 g/L HA (P3). (b) Wear particles obtained in PBS containing 3 g/L HA (P3) and in PBS containing 3 g/L HA with polarization application (P6). (a) P2 (0.5 mg/mL) vs. P3 (0.5 mg/mL) Mean of P2 57.84
P Value 0.015
Mean of P3 12.24 (b)
P3 (2 mg/mL) vs. P6 (2 mg/mL) Mean of P3 (2 mg/mL) 46.18
Mean of P6 (2 mg/mL) 23.9
P Value 0.248
Particles produced in PBS containing 3 g/L of HA at concentrations of 2 mg/mL elicited an increase in the macrophages cytotoxicity that reached almost 46% (Figure 6, panel B, PBS-HA). Although such an increase was higher than the one produced by particles concentrations of 0.5 and 1 mg/mL, which were 12% and 14%, respectively, it was reduced to 24% when polarization conditions characteristic of damaged tissue were applied (Figure 6, panel B, PBS-HA+POL). Although the statistical analysis of the data from Figure 6 panel B (Table 3) gave no significant differences between results analyzed here, the application of anodic polarization to HA aqueous solution seems to have important observable differences on the mean value of the cytotoxicity. This feature could be relevant and, for this reason, verification by other biocompatibility assays is required. With this purpose, the wear particles collected from the tribocorrosion assays of CoCr alloy in PBS-HA without and applying anodic polarization were tested on macrophages cultures by measuring the mitochondrial activity. It is well known that the mitochondrial activity measurement is directly proportional to the number of metabolically active cells in culture  constituting a measure of cell viability and biocompatibility. As it is shown with Figure 7 by white bars, wear particles collected in the PBS-HA produced a gradual and significant reduction in the mitochondrial respiratory response of macrophages. This result seemed to be directly related to the concentration of particles to which macrophages were exposed (Table 4). Nevertheless, no reduction in the mitochondrial respiratory activity was observed in macrophages exposed to wear particles generated when polarization was applied during wear-corrosion tests. No significant effects in respiratory activity were observed in the range of particles concentrations tested (Figure 7, black dotted bars, and Table 5). The results suggest that the polarization conditions in the wear-corrosion assays in PBS containing HA at the approximate concentration found in synovial fluid seem to be beneficial to macrophage viability and biocompatibility. Table 4. Statistical analyses of mitochondrial respiratory activity. The effects of the particle (P), the concentration, and their interaction on the changes in respiratory activity were analyzed with a two-way analysis of variance. A p value of ≤0.05 was considered significant. Mean pairwise comparisons were computed with a Tukey’s test (α = 0.05). All analyses were performed with the R software version 3.4.2 (R Core Team, Vienna, Austria, 2017). ANOVA
P conc P × conc Residuals
5745.2 2191.39 2648.33 236.91
1 2 2 11
266.75 50.87 61.48 -
4.64 × 10−9 2.76 × 10−6 1.07 × 10−6 -
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Figure 7. Mitochondrial respiratory activity of macrophages cell cultures exposed for 72 h to different
Figure 7. Mitochondrial respiratory activity of macrophages cell cultures exposed for 72 h to different doses of HCCoCr wear particles. Wear particles were obtained in PBS containing 3 g/L HA without doses of HCCoCr wear particles. Wear particles were obtained in PBS containing 3 g/L HA without (white bars) and with polarization (dotted black bars). Cell cultures were exposed to the following (white bars) and with polarization black bars). Cell cultures were exposed to triplicate. the following wear particles concentrations: 0.5, 1(dotted and 2 mg/mL. Experiments were done as independent wearBars particles concentrations: 0.5, 1 and 2 mg/mL. Experiments were done as independent labeled with different letters show statistically significant differences and bars labeled withtriplicate. the Barssame labeled with different letters show statistically significant differences and bars labeled with the letter (c) show nonsignificant differences. same letter (c) show nonsignificant differences. Table 4. Statistical analyses of mitochondrial respiratory activity. The effects of the particle (P), the and their interaction on the simple changeseffects in respiratory activity were analyzed two- letter Tableconcentration, 5. As the interaction was significant, were compared. Means withwith the asame way analysis of variance. A p value of ≤0.05 was considered significant. Mean pairwise comparisons are not significantly different. were computed with a Tukey’s test (α = 0.05). All analyses were performed with the R software version 3.4.2 (R Core Team, Vienna, Austria, 2017).
P3 P3 P3 P6 P6 P6
2 1 P 0.5 1 conc 2 P × conc 0.5 Residuals
Lower.CL Upper.CL Group ANOVA 25.7 17.1 34.3 a Sum 60.9 Sq Df 52.3 F Value 69.4Pr(>F) b 5745.2 177 266.75 98 4.64 × 10−9 c 87.5 89.8 2191.39 281.3 50.87 98.4 2.76 × 10−6 c 94.3 2648.33 285.8 61.48 102.9 1.07 × 10−6 c 95 86.4 103.5 c 236.91 11 -
Table 5. As the interaction was significant, simple effects were compared. Means with the same letter
The dose-dependence effect on mitochondrial respiratory activity by particles detached in PBS-HA are not significantly different. could be explained by the chemical composition of wear particles collected from wear-corrosion P Concin Co Mean Upper.CL tests in this solution as a decrease was Lower.CL observed, as well as anGroup enrichment in chromium oxide, P3 2 25.7 17.1 34.3 aof PBS-HA-POL wear particles. a compound with high toxicity  in comparison with the composition P3 1 conditions 60.9 52.3 b solution at the approximate The results suggest that polarization applied to an69.4 HA aqueous P3 0.5 87.5 77 98 c concentration found in synovial fluid produce changes in material tribocorrosion behavior inducing P6 1 89.8 81.3 98.4 c wear particles that seem toP6 be beneficial to macrophage viability and biocompatibility. Data that could 2 94.3 85.8 102.9 c explain the higher biocompatibility of95 wear particles generated could be related P6 0.5 86.4 103.5 in PBS-HA+POL c to the fact that under these conditions, wear processes did not allow the regeneration of the oxide film. ThisThe event could determine the of an active stateactivity where by CoCr base detached material in was directly dose-dependence effect oncreation mitochondrial respiratory particles PBSHA could explained without by the chemical of wear particles collected exposed to thebe electrolyte enoughcomposition time to build up the new oxide filmfrom that wear-corrosion induced the release tests in particles this solution as higher a decrease Co wasprobably observed,coming as well as an enrichment in chromium oxide, of metallic with Co in content from the base material. a compound with high toxicity  in comparison with the composition of PBS-HA-POL wear particles. and The Methods results suggest that polarization conditions applied to an HA aqueous solution at the 3. Materials approximate concentration found in synovial fluid produce changes in material tribocorrosion behavior inducing wear particles that seem to be beneficial to macrophage viability and 3.1. Material biocompatibility. Data that could explain the higher biocompatibility of wear particles generated in
A high carbon CoCr alloy (hereafter HCCoCr) that complies with ASTM F75 standard was used as material. HCCoCr composition is shown in Table 6. “Double heat-treated” disks, i.e., solution treatment (ST) followed by hot isostatically pressing (HIP), of 38 mm in diameter and 4 mm
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thickness, were obtained from BIOMET Spain Orthopaedic (Valencia, Spain). The sample preparation consisted of grinding on SiC paper, followed by mechanical polishing with 3 µm diamond paste. Table 6. Chemical composition (wt %) of High Carbon CoCr alloy (HCCoCr).
0.004 0.001 0.01
3.2. Wear-Corrosion Tests under Electrochemical Control Wear-corrosion experiments were carried out on a pin-on-disk tribometer, and 6-mm diameter alumina ball pins were used as HCCoCr disk counterpart. The HCCoCr disks were 38 mm in diameter and 4 mm thick. Both disks and pins were previously washed with double distilled water and cleaned in an ultrasonic ethanol bath for 10 min. The alumina pins were placed in a pin plastic holder and fixed on the load cell. A low normal load of 5 N was applied on the counterpart. The working electrode motion was provided by a rotating motor at a rotation rate of 120 rpm that produced, at the end of the alumina ball, a circular wear track (5 mm in diameter) on the HCCoCr disk surface. The tribometer configuration consisted of an integrated electrochemical cell (3-electrode cell) including the HCCoCr disk as working electrode, a ring-shaped Pt wire counter electrode and a saturated Ag/AgCl reference electrode. All the potentials of the HCCoCr disks during the wear corrosion tests were measured versus the reference electrode. Wear-corrosion tests were performed in Phosphate Buffer Solution (PBS) containing the following composition: 0.2 g/L KCl, 0.2 g/L KH2 PO4 , 8 g/L NaCl, and 1.150 g/L Na2 HPO4 (anhydrous) and this PBS solution was supplemented with 3 g/L hyaluronic acid, the approximate concentration reported for the synovial fluid of healthy joints . The wear-corrosion behavior was studied simultaneously measuring the friction coefficient and electrochemical parameters. The wear-corrosion tests were performed as follows (Figure 8): (a) before wear (no sliding) by the measurement of the corrosion potential for 10 min and (b) under sliding (at 120 rpm and 5 N load) in two different ways: one where a simultaneous measurement of the corrosion potential and the coefficient of friction (COF) were performed for 40 min without applying polarization and the other, applying anodic potentiodynamic polarization and simultaneous measurement of current and coefficient of friction (COF) for 200 min. The anodic potentiodynamic polarization was applied from the corrosion potential to a polarization of 1 V at a scanning rate of 10 mV/min, and back curve was drawn until reaching the corrosion potential. The back curve was also measured to analyze the repassivation ability of the HCCoCr alloy. For comparative purposes, the anodic potentiodynamic polarization without wear in PBS and PBS-HA was also measured. All experiments werePEER carried out in triplicate. Materials 2018, 11, x FOR REVIEW 12 of 17
Figure of the the wear-corrosion wear-corrosion tests. tests. Figure 8. 8. Schema Schema of of the the experimental experimental procedure procedure of
Surface characterization of the worn surface after wear-corrosion tests with polarization and without polarization was performed by profilometry and using a JEOL-6500F microscope equipped with a field Emission Gun (FEG) coupled to an Energy Dispersive X-ray (EDS) spectrometer. Secondary electron (SE) images were taken at 7.5 keV and EDS analysis was performed at 20 keV.
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Surface characterization of the worn surface after wear-corrosion tests with polarization and without polarization was performed by profilometry and using a JEOL-6500F microscope equipped with a field Emission Gun (FEG) coupled to an Energy Dispersive X-ray (EDS) spectrometer. Secondary electron (SE) images were taken at 7.5 keV and EDS analysis was performed at 20 keV. 3.3. Isolation and Characterization of Particles Debris from tribocorrosion tests performed in PBS, PBS containing 3 g/L hyaluronic acid (both without applying polarization), and in PBS supplemented with hyaluronic acid under anodic polarization was collected for the subsequent characterization. Metallic particles were isolated, purified, and characterized following the protocol developed by Billi et al. for metal particles . This procedure allows exhaustive removal of organic and inorganic impurities from the metallic particles. To completely digest the hyaluronic acid, metal particles in PBS supplemented with 3 g/L HA were digested adapting the protocol developed by Kavanaugh et al. . Wear-corrosion media (PBS and the digested hyaluronic acid solutions) containing metallic particles were rotated at 28 rpm in an orbital agitator for 24 h at room temperature to disperse the metal particles evenly before their isolation. The particles were then purified via density gradient centrifugation varying from 4446× g to 284,000× g (Beckman Optima L80 XP; Beckman Instruments, Fullerton, CA,USA) through multiple layers of denaturants and metal-selective high-density layers as was described [31,32]. This led to well-dispersed particles deposited onto a 5 mm × 5 mm featureless display silicon wafer (Ted Pella, Inc., Redding, CA, USA) coated with a monolayer of marine mussel glue (Cell-TakTM; BD Biosciences, San Jose, CA, USA). The silicon wafer was then coated with 10 Å iridium [31,32]. The morphology of metallic particles was studied with a field emission scanning electron microscope (FE-SEM) (Supra VP-40; Zeiss, Peabody, MA, USA) at a voltage of 15 kV and chemically analyzed by means of Energy-dispersive spectroscopy (EDS) analysis (Thermo Ultradry feature sizing system; Thermo Electron Scientific Instruments, Madison, WI, USA). 3.4. Macrophages Cell Cultures Assays The biocompatibility of wear particles was tested in a mouse macrophage cell line (J774A.1) from DSMZ Human and Animal Cell Bank. Macrophages cell cultures were exposed to different concentrations of wear particles. Wear particles obtained from the wear-corrosion tests were centrifuged and the particle pellet was weighted, UV sterilized for 15 min, and resuspended in sterile bidistillated water and maintained in aliquots at −20 ◦ C until use. Wear particles, just before cell cultures assays, were thawed, resuspended by vigorously mixing with a vortex, and diluted at a concentration of 20 mg/mL in Dulbecco’s Modified Eagle Medium (DMEM 41966; Gibco, BRL, Invitrogen, Thermofisher scientific, Paisley, UK) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, BRL) and with a mixture of antibiotics (penicillin at 100 units/mL and streptomycin at 100 g/mL, Gibco, BRL), named as complete cell culture medium. A concentration of 20 mg/mL was used as stock solutions for the particles concentration tested in different cell assays. To assure a polydisperse distribution of the particles vigorous vortexing was applied in all experimental steps that required particles manipulation. To evaluate the effect of HCCoCr particles on cell cultures, macrophages were seeded on 96-well culture plates at 75,000 cells/mL cell density in complete cell culture medium. A final volume of 100 µL of cell suspension in complete cell culture medium was added to each well of the 96-well plates. After 24 h in culture, cell media were removed and replaced by 100 µL of fresh complete cell culture medium containing the following concentrations of HCCoCr particles: 0, 0.5, 1 and 2 mg/mL. Cell cultures were maintained for 72 h in a cell culture chamber at 37 ◦ C and 5% CO2 . Incubation time was selected based on the set-up of cell cultures assays for metallic particles studies carried out in the lab and is the most commonly used time point for cell viability studies . Mitochondrial activity
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(WST-1 assay) and plasma membrane damage (LDH assay) were used to evaluate the biocompatibility and cytotoxicity, respectively, as described below . 3.5. Mitochondrial Activity Measurement Reduction of the WST-1 reagent (4-[3-4-iodophenyl)-2-(4-nitro-phenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (Roche Diagnostics GmbH, Mannheim, Germany)) was used to evaluate the effect of different concentrations of the HCCoCr wear particles on mitochondrial activity of macrophages cultures. The mitochondrial activity measurement is directly proportional to the number of metabolically active cells in culture. After 72 h in culture, 10 µL of the cell proliferation kit reagent WST-1 was added to each well containing 100 µL of fresh complete cell culture medium, and the mixture was incubated inside the cell culture incubator for 30 min. After incubation, 100 µL of each reaction mixture were transferred to a 96-well cell plate, and the absorbance of the samples was measured as differential absorbance, 415 nm minus 655 nm, in an iMark microplate absorbance reader (Bio-Rad, Hercules, CA, USA), using the absorbance given by complete cell culture medium as a blank. All experiments were carried out as independent triplicate. 3.6. Measurement of Lactate Dehydrogenase Activity To measure and quantify the effect of HCCoCr wear particles on cell death and cell lysis, lactate dehydrogenase (LDH) activity was measured in the supernatants of cell cultures by an enzymatic assay using the Cytotoxicity Detection Kitplus (Roche Diagnostics GmbH, Mannheim, Germany). Supernatants were collected from cell culture after being exposed for 72 h to different HCCoCr particles concentrations and were centrifuged for 5 min at 1024× g. The enzymatic assays were performed according to the LDH kit protocol provided by Roche Diagnostics (Mannheim, Germany). Complete cell culture medium was used as a control for absorbance baseline. LDH activity was measured based on differential absorbance, 490 nm minus 655 nm, in an iMark microplate absorbance reader (Bio-Rad, Hercules, CA, USA). LDH catalyzes the conversion of lactate to pyruvate, reducing NAD+ to NADH/H+ , which is used by the catalyst to reduce a tetrazolium salt to a formazan salt, which is responsible for the change in absorbance at 490 nm. Quantification of LDH activity is used as an indicator of plasma membrane damage, as is a stable cytoplasmic enzyme present in all cells and rapidly release into the cell culture supernatant when the plasma membrane is damaged being a sign of cell death. The percentage of cytotoxicity is calculated taking as control a total cell lysate in the absence of any particles. The percentage cytotoxicity is calculated as described in the LDH kit protocol provided by Roche Diagnostics: Cytotoxicity (%) = [(exp. value − low control)/(high control − low control)] × 100; where experimental value (exp. value) corresponds to the absorbance of the treated sample in the study exposed to wear HCCoCr particles, low control is the absorbance from the untreated cell cultures with no particles that corresponds to spontaneous LDH released, and high control is the absorbance value obtained after total cell cultures lysis that corresponds to the maximum releasable LDH activity. The background absorbance corresponding to complete cell culture media was subtracted from the absorbance of all samples before cytotoxicity calculations. All experiments were carried out as independent triplicate. 3.7. Statistical Analysis of Data 3.7.1. Wear Particles Analysis Data The experimental design used to determine the effect of two factors as the corrosive medium and the application of polarization on the dependent variable, that is, the chemical composition of the particles, was a 22 factorial design. In order to explain significant interaction, simple effects of one factor on the dependent variable at each single level of the other factor were computed. After this, simple effect pairwise comparisons were performed to detect levels of the second factor in which simple effects of the first factor on the dependent variable were significantly different.
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Kruskal–Wallis  and Mann–Whitney  nonparametric tests were used to confirm the ANOVA results. A p-value < 0.05 was considered as significant. All the statistical analyses were performed with the Minitab® 17.1.0 software (Minitab Inc., State College, PA, USA) . 3.7.2. Biocompatibility Analysis Data Mean differences on cytotoxicity effects between wear particles obtained in PBS (0.5 mg/mL) versus particles in PBS containing 3 g/L of hyaluronic acid (0.5 mg/mL) and between wear particles obtained in PBS containing 3 g/L HA (PBS-HA; 2 mg/mL) without versus with polarization application (PBS-HA+POL; 2 mg/mL) were studied with Student’s t tests (α = 0.05), respectively. The effects of the particles, the concentration, and their interaction on the changes in mitochondrial respiratory activity of macrophages were analyzed with a two-way analysis of variance. A p value of ≤0.05 was considered significant. Mean pairwise comparisons were computed with a Tukey’s test (α = 0.05). Means with the same letter are not significantly different and means with different letters are significantly different. All analyses were performed with the R software version 3.4.2 (R Core Team, Vienna, Austria, 2017) . 4. Conclusions 1. The wear particles collected after wear corrosion in PBS and PBS-HA were mainly composed of chromium oxide coming from the detachment of the passive film and phosphate adsorbed on the particle surface and/or adsorbed on the broken passive film. 2. Composition of the corrosive medium and polarization, applied to mimic the electrical interactions observed in living tissues, has an influence on the chemical composition of the particles. The wear particles detached after wear corrosion with polarization in PBS-HA have a chemical composition with a higher significant content of Cr and Co than those particles collected without polarization. 3. Biocompatibility in vitro assays here reported, measured by LDH release and mitochondrial respiratory activity, seem to indicate that particles from wear corrosion in PBS supplemented with 3 g/L of hyaluronic acid, an approximate concentration that is found in the synovial fluid of healthy joints, under anodic polarization produce in macrophages lower damage to the plasma membrane and are more biocompatible, most likely associated with particles chemical composition. 4. As more variables of the prosthesis environment are considered in in vitro assays to study cell-biomaterial interactions, as are the electric interactions, in order to have a closer view of the different processes that are taking place in vivo at the cell-biomaterial interface, a better knowledge of the biological consequences will be obtained. 5. Understanding these consequences of the electrical signals on the growth and development of cells and tissues should be applicable for the design of appropriate solutions and adequate treatments for orthopedic-bearing patients. Author Contributions: Conceptualization, B.T.P.-M., M.L.E., M.C.G.-A., R.M.L.; Methodology, B.T.P.-M., M.E.L.F., I.D., A.K., F.B., M.L.E., M.C.G.-A., R.M.L.; Software, B.T.P.-M., M.L.E., M.C.G.-A., R.M.L., G.P., J.G.; Validation, B.T.P.-M., I.D., A.K., F.B., M.L.E., M.C.G.-A., R.M.L; Formal Analysis, B.T.P.-M., M.E.L.F., I.D., M.L.E., M.C.G.-A., R.M.L.; Investigation, B.T.P.-M., M.E.L.F., I.D., A.K., F.B., M.L.E., M.C.G.-A., R.M.L.; Resources, B.T.P.-M., M.L.E., M.C.G.-A., R.M.L.; Data Curation, B.T.P.-M., M.L.E., M.C.G.-A., R.M.L.; Writing-Original Draft Preparation, B.T.P.-M., M.L.E., M.C.G.-A., R.M.L.; Writing-Review & Editing, B.T.P.-M., M.L.E., M.C.G.-A., R.M.L.; Visualization, B.T.P.-M., M.L.E., M.C.G.-A., R.M.L.; Supervision, B.T.P.-M., M.L.E., M.C.G.-A., R.M.L.; Project Administration, B.T.P.-M., M.L.E., M.C.G.-A., R.M.L.; Funding Acquisition, B.T.P.-M., M.L.E., M.C.G.-A., R.M.L. Funding: Financial support received through the MAT2015-67750-C3-2-R, MAT2015-67750-C3-1-R, MAT2011 -29152-C02-01 and the MAT2011-29152-C02-02 projects from the Ministerio de Economía y Competitividad (MINECO/FEDER) from Spain. Acknowledgments: Authors wish to thank Guillermo Padilla PhD G.P. (Bioinformatics and Biostatics facility at Centro de Investigaciones Biológicas, CIB-CSIC) and J. Garrido J.G. (U. Autonoma Madrid, Dept. Psicol. Social & Metodol., Fac. Psicol.) for technical assistance in the statistical analysis of macrophages data and wear particles.
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Conflicts of Interest: There are no conflicts of interest to declare. The authors will receive no benefit of any kind either directly or indirectly.
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