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

May 2024
Journal of Biomedical Materials Research Part A

May 2024

DOI:10.1002/jbm.a.37751

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CC BY 4.0

Authors:

Luna Sánchez - López

Spanish National Research Council

Rosa M Lozano

Centro de Investigaciones Biológicas

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.

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RESEARCH ARTICLE

Macrophage proteomic analysis of covalent immobilization of

hyaluronic acid and graphene oxide on CoCr alloy in a

tribocorrosive environment

L. Sánchez-López

1,2,3

| B. Chico

| Maria Cristina García-Alonso

Rosa M. Lozano

Centro de Investigaciones Biológicas-Margarita Salas (CIB Margarita Salas), Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain

Centro Nacional de Investigaciones Metalúrgicas (CENIM), Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain

PhD Program in Advanced Materials and Nanotechnology, Doctoral School, Universidad Autónoma de Madrid, Madrid, Spain

Correspondence

Rosa M. Lozano, Centro de Investigaciones

Biológicas-Margarita Salas (CIB Margarita

Salas), Consejo Superior de Investigaciones

Científicas (CSIC), C/ Ramiro de Maeztu,

28040 Madrid, Spain.

Email: rlozano@cib.csic.es

Maria Cristina García-Alonso, Centro Nacional

de Investigaciones Metalúrgicas (CENIM),

Consejo Superior de Investigaciones

Científicas (CSIC), Avenida Gregorio del Amo,

8, 28040 Madrid, Spain.

Email: crisga@cenim.csic.es

Funding information

Ministerio de Ciencia, Innovación y

Universidades (MICIU/FEDER), Grant/Award

Numbers: PRE2019-090122,

RTI2018-101506-B-C31, RTI2018-101506-B-

C33

Abstract

In this work, a sequential covalent immobilization of graphene oxide (GO) and hya-

luronic 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 sur-

faces. 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 biocompatibil-

ity in vitro. On the other hand, disruption of the HA/GO nanocoating by tribocorro-

sion 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 macro-

phage proteome, revealing that disruption of the covalent nanocoating impacts the

macrophage proteome. Although macrophage inflammation induced by tribocorro-

sion of HA/GO/CoCr surfaces is observed, it is ameliorated by the covalently grafting

of HA, which provides immunomodulation by eliciting downregulations in characteris-

tic pro-inflammatory signaling involved in inflammation and aseptic loosening of CoCr

joint arthroplasties. Covalent HA/GO nanocoating on CoCr provides potential

Received: 14 February 2024 Revised: 6 May 2024 Accepted: 8 May 2024

DOI: 10.1002/jbm.a.37751

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,

provided the original work is properly cited.

J Biomed Mater Res. 2024;1–19. wileyonlinelibrary.com/journal/jbma 1

applications for in vivo joint prostheses led a reduced metal-induced inflammation

and degradation by wear-corrosion.

KEYWORDS

CoCr, covalent immobilization, graphene oxide, hyaluronic acid, macrophage proteome,

tribocorrosion

1|INTRODUCTION

Cobalt-chrome (CoCr) implants used as joint prostheses suffer from

wear-corrosion degradation processes at some degree upon implanta-

tion, resulting in the release of metal ions and particulate debris into

the surrounding environments. Metal tribocorrosion intensifies the

foreign body response (FBR) causing CoCr-mediated inflammation,

osteolysis, and aseptic loosening. These adverse effects may ulti-

mately lead to the replacement of CoCr implants through revision sur-

geries due to implant failure.

1–3

Both innate and adaptive immune

responses play a role in the overall immune response. Nevertheless,

the severe inflammatory reaction localized around the implant is

mainly characterized by an innate response marked by a prominent

infiltration of macrophages.

4–6

The interaction between macrophages

and the biomaterial significantly influences the success and long-term

functionality of the implant, ultimately impacting the risk of implant

rejection. Hence, the development of immunomodulatory biomaterials

can be crucial for regulating macrophage response, a key factor in

ensuring the extended viability and effectiveness of implanted bio-

medical devices.

7–9

Regarding potential approaches for manufacturing immunomodu-

latory biomaterial, surface chemistry modifications can be employed

to establish a beneficial cell-biomaterial interface that effectively

modulates immune responses, all while preserving the required bulk

properties of metallic implants.

Given that metal debris is the primary trigger for adverse inflam-

matory reactions, it is crucial to explore surface chemistry modifica-

tions with the goal of creating a surface that not only reduces the

release of metal debris but also minimizes the biological FBR to the

greatest extent possible. In an effort to improve the tribocorrosion

performance and minimize the release of metallic debris, researchers

have proposed the use of graphene-based chemical modifications.

This approach seeks to replicate the presence of carbon-enriched tri-

bological layers observed in worn areas of in vivo retrieved prosthe-

ses, as their existence is associated with wear reduction. The concept

is to implement a comprehensive graphene-based surface modifica-

tion that acts as a barrier against the release of metal debris while pre-

serving the necessary bulk mechanical properties of the metal implant

and ensuring a favorable biological response.

On the other hand, the integration of biomolecules such as pro-

teins, peptides, glycosaminoglycans, or other organic compounds can

further address the modulation of the biological response through bio-

mimetic signaling. This approach involves the incorporation of biomol-

ecules on the biomaterial surface, allowing for the precise interaction

of cellular membrane receptors with these biomaterial motifs to influ-

ence and fine-tune desired immune responses.

The authors studied the electrochemical reduction of graphene

oxide (GO) on CoCr surfaces by cyclic voltammetry and posterior bio-

functionalization with HA 3 g/L. Reduction of GO on the CoCr sur-

faces and the posterior physical adsorption of hyaluronic acid

(HA) provided a decreased inflammatory macrophage response,

however, the reduced GO increased the wear-corrosion of the CoCr

surfaces.

The type of immobilization mechanism applied, based on

physical or chemical adsorption methods, can greatly affect the final

implant outcome. Molecules that are physically immobilized can

exhibit spontaneous desorption, typically leading to short-term thera-

peutic effects at the implant surface. Besides, desorbed biomolecules

may disseminate to other body sites, potentially eliciting undesired

biological effects in ectopic locations.

Conversely, biomolecules that

are covalently immobilized by chemical adsorption methods can effec-

tively prevent desorption and diffusion from the intended site of

action, thereby minimizing the potential adverse effects of these bio-

molecules. Importantly, biomolecules bound irreversibly can be

retained at the biomaterial site for extended periods, allowing them to

elicit the desired cellular responses at the cell–biomaterial interface

over prolonged periods.

Furthermore, the stability of the target bio-

molecule is enhanced through multi-point covalent immobilization,

resulting in improved resilience to variations in temperatures and

resistance to organic solvents.

16–20

This immobilization also serves as

a barrier against proteases and proteolytic degradation, improving the

biomolecule resistance to biodegradation when it is attached to

organic or inorganic supports.

A wide variety of biomolecules has been covalently attached to

metal surfaces for this purpose.

21–27

This strategy holds special signif-

icance in the field of biomedical applications, especially concerning

HA, one of the main components of the synovial fluids involved in

lubrication joints.

HA is prone to rapid degradation under physiological conditions,

due to enzymatic and chemical degradation mechanisms by hyaluroni-

dases, oxidation, and hydrolysis.

28–30

The challenges involved in pre-

serving the durability and long-term effectiveness of HA emphasize

the critical importance of employing biomolecule immobilization on

the biomaterial surface as a strategic solution. Chemical modification

strategies, such as chemically anchored or cross-linked HA, aim to

improve the stability and resistance to degradation of HA.

Minimal

physicochemical alterations are introduced through one-point and

multi-point covalent immobilizations

while they can also improve

the degradation resistance of HA and other extracellular matrix (ECM)

2S´

ANCHEZ-LÓPEZ ET AL.

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components.

32–35

Nevertheless, it is important to be careful not to

alter the chemical structure and cross-linking density of HA, since

excessive changes can potentially induce cytotoxicity and trigger

immunogenic reactions.

36–38

In this work, the sequential covalent immobilization of HA and

GO nanosheets on the biomedical grade CoCr alloy is characterized in

terms of electrochemical response, tribocorrosion behavior, and the

analysis of the macrophage response to modified CoCr surfaces, both

with and without wear. Potential applications of covalently HA/GO

biofunctionalized CoCr surfaces for metal joint prostheses will be

analyzed in terms of the biological response of the proteome of mac-

rophages associated with metal-induced inflammation by wear-

corrosion processes.

2|MATERIALS AND METHODS

2.1 |Materials

Biomedical grade CoCr alloy was supplied by International Edge with

the nominal chemical composition (wt %): 27.25% Cr, 5.36% Mo,

0.69% Mn, 0.68% Si, 0.044% C, 0.02% W, 0.15% N, 0.002% Al,

0.001% S, 0.002% P, 0.002% B, 0.001% Ti, and balance Co. CoCr sur-

faces were polished with SiC abrasive papers from 600 to 2000 grain

size and then mirror-polished with 3 and 1 μm diamond paste (hereaf-

ter samples named CoCr) and rinsed with ultrapure water (Milli-Q

Direct 8 water purification system, Merck, Germany).

3-aminopropyl-triethoxysilane (APTES) agent, used as an interme-

diate compound for the covalent immobilization of GO on CoCr, was

purchased from Merck, Sigma–Aldrich. GO nanosheets were supplied

by GRAnPH. Biofunctionalization of GO nanocoating was provided by

using high molecular weight HA (HMW-HA, 1500–1800 kDa, refer-

ence 53,747, Sigma–Aldrich Chemie GmbH, Schnelldorf, Germany).

Adipic dihydrazide 98% (ADH) agent, used as intermediate compound

for the biofunctionalization of GO with HA, was purchased from

Thermo Fisher Scientific, Acros Organics.

2.2 |Sequential covalent immobilization of HA and

GO on CoCr surface

Covalent immobilization of GO on CoCr was obtained as previously

described.

Briefly, CoCr surfaces were hydroxylated by alkaliniza-

tion in NaOH 5 M for 2 h followed by self-assembly of silane mono-

layers using the amino-silane intermediate coupling agent APTES,

which was pre-mixed at 2% vol in isopropanol-water (200: 1 v/v) and

stirred for 1 h. After this time, hydroxylated CoCr surfaces were

immersed in the APTES solution at RT for 1 min and kept 24 h at the

curing temperature of 45C. Silanized CoCr surfaces were immersed

in GO suspension at 4 g/L during 24 h at 60C to trigger covalent

immobilization reaction of GO sheets on silanized CoCr (hereafter

samples named GO/CoCr).

Carboxylic groups of covalently immobilized GO in the

GO/CoCr samples were activated by immersing the GO/CoCr

surfaces in ultrapure water (50 mL) with N-(3-dimethylaminopro-

pyl)-N-ethylcarbodiimide hydrochloride (EDC, 250 mg) and N-

hydroxysuccinimide (NHS, 750 mg) during 24 h in mild orbital

agitation. After that, the GO/CoCr surfaces with activated carboxyl

groups were immersed in ultrapure water (100 mL) with dissolved

adipic dihydrazide (ADH, 1.04 g) and let to react for another 24 h in

mild orbital agitation (samples named ADH/GO/CoCr).

Further covalent grafting of high molecular weight HA was per-

formed on GO nanosheets on GO/CoCr samples through ADH as an

intermediate covalent GO-HA linker using EDC/NHS carbodiimide

chemistry.

41,42

Pre-activated HA-(EDC/NHS) solution was obtained by dissolving

HA (100 mg), EDC (253 mg), and NHS (754 mg) in 100-mL phosphate

buffer saline (PBS) and stirred for 24 h to activate the carboxylic acids

of HA. After that, ADH/GO/CoCr surfaces were immersed in pre-

activated HA-(EDC/NHS) solution and let to react for 24 h in mild

orbital motion (hereafter samples named HA/GO/CoCr). Surfaces

were rinsed with ultrapure water between each immobilization step

to remove excess reagents.

2.3 |Characterization of covalent

immobilizations on CoCr

GO/CoCr and HA/GO/CoCr surfaces were characterized by using a

field-emission Hitachi S-4800 equipment by scanning electron micros-

copy (SEM) and Raman spectroscopy. Secondary electron images at

SEM were taken at 15 kV. Raman spectra were collected with a

Raman spectrometer B&W TEK (BTC675N) using a 532 nm wave-

length laser. Exposure time was set at 100 ms per scan. Spectral data

were processed using BWSpec4 software with dark-frame subtrac-

tion. Spectra were collected from 50 to 4000 cm

1

with integration

times of 100 and 300 s.

The barrier effect of GO and HA/GO on the CoCr surfaces was

assessed through electrochemical impedance spectroscopy (EIS). EIS

data were recorded up to 23 days of immersion in HA aqueous elec-

trolyte at physiological concentration of 3 g/L simulating the biologi-

cal synovial fluid environment.

A three-electrode electrochemical

cell consisting of the GO/CoCr or HA/GO/CoCr surfaces as working

electrodes, a Pt wire as counter electrode and Ag/AgCl (3 M KCl) as

reference electrode were used. An Autolab32 potentiostat/

galvanostat was used to generate a sinusoidal wave of 10 mV ampli-

tude applied at the corrosion potential Ecorr in the 100 kHz–1 mHz

frequency range logarithmically spaced by 5 points/decade. Fitting of

the experimental impedance data into equivalent electric circuits was

performed by using Z-view software based on a nonlinear least-

squares program. The criteria used for selecting the optimal equiva-

lent circuits were the lowest chi-square value and lowest relative

errors (in %). A good reproducibility was obtained for EIS tests per-

formed in duplicate.

S´

ANCHEZ-LÓPEZ ET AL.3

2.4 |Wear-corrosion tests

To evaluate the tribocorrosion performance in the pair MoM used for

joint replacement, CoCr-CoCr tribosystems were assembled in a trib-

ometer (Microtest MT/30/NI) with a pin-on-disk (ball-on-plate) con-

figuration. CoCr balls of 8 mm diameter were used as counterpart

(pin) in contact with HA/GO/CoCr disks of 38 mm diameter. The use

of CoCr balls as counterpart allows a more suitable configuration in

the tribometer promoting a uniform contact between the surface

materials involved in tribocorrosion tests.

The applied load was 5 N and a rotation speed of 120 rpm. To

simulate physiologically significant tribological conditions, a total slid-

ing distance of 30,000 m was set in the tribometer to consider the

motion experienced by a hip prosthesis, with an average of one million

cycles per year.

Wear-corrosion tests were carried out in recirculating HA aque-

ous electrolyte at physiological concentration 3 g/L used as lubricant

in a recirculation pump system in the tribometer configuration. HA

constitutes one of the main components of synovial fluid, playing a

crucial role in joint lubrication.

This rationale supports the choice of

HA as an electrolyte for tribocorrosion tests, as it represents more

closely the physiological environment where the materials will be

implanted. Tribocorrosion tests were performed in triplicate.

The worn tracks were characterized with a 3D Profilm3D equip-

ment (Filmetrics—KLV Company).

2.5 |Modified CoCr surfaces-macrophage

cell cultures

Mouse macrophage J774A.1 cell line was provided by DSMZ Human

and Animal Cell Bank, Braunschweig, Germany. Three different groups

were analyzed in cell-biomaterial assays. A control group where macro-

phages were directly seeded on the surface of cell-culture treated poly-

styrene petri dishes (BD, 1 Becton Drive, NJ, USA) without additional

biomaterial (Polystyrene control). A second group, where macrophages

were seeded on intact HA/GO/CoCr disks, that were previously incu-

bated 4 days in the HA solution as used in tribocorrosion tests; and a

third group where macrophages were seeded on worn HA/GO/CoCr

disks, with the worn disk surface in contact with cells.

All types of CoCr disks were UV-sterilized prior to cell cultures.

The macrophage culture was seeding at a cell density of 12,500

cells/cm

for 72 h and 10,000 cells/cm

for 96 h cell-biomaterial

cultures in complete cell culture medium (DMEM 41966, 10% heat-

inactivated FBS, 100 units/mL penicillin, and 100 μg/mL streptomy-

cin, Gibco ThermoFisher Scientific, USA) and incubated in a cell cul-

ture chamber at 37C and 5% CO

2.6 |Macrophage cell extracts for proteomics

analysis

Macrophage cell extracts were taken for proteomic analysis after

72 and 96 h of interaction with the three groups described. Firstly,

macrophages cultures were washed with ice-cold PBS and then incu-

bated with a cell lysis buffer supplemented with protease inhibitors to

prevent protein degradation (hereafter complete cell lysis buffer).

Complete cell lysis buffer contained 5 mL RIPA lysis buffer (name of a

commercial cell lysis buffer from Santa Cruz Biotechnology, Santa

Cruz, CA, USA), 1:100 protease inhibitor cocktail provided in the RIPA

lysis buffer system reagent.

Cells extracts were scraped and transferred to a 15 mL conical

tube, where lysates in complete cell lysis buffer were incubated on

ice for 15 min. Then, cellular lysates were sonicated three times dur-

ing 2 s-pulses with 1 min of rest in ice between sonication pulses.

Lysates were then incubated on ice for additionally 15 min to

increase protein extraction yields and centrifuged at 12,100 gfor

5 min at 4C, collecting the supernatants into Eppendorf microtubes

and storing in aliquots at 20C, avoiding multiple freeze/thaw

cycles.

2.7 |LC–MS/MS

Preparation of samples for liquid chromatography tandem mass

spectrometry (LC–MS/MS) analysis was performed as previously.

Briefly, the total protein concentration in purified cell lysates

supernatants was determined using Thermo Scientific™Pierce™

660 nm protein assay reactive (Pierce Biotechnology, USA) and

quantified by absorbance spectroscopy at 660 nm in an iMark

microplate absorbance reader (Bio-Rad Laboratories, Inc., USA).

Then, 60 μg of protein from macrophage cell extracts was precipi-

tated in cold acetone and resuspended in loading buffer for sodium

dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE).

Identified proteome bands from SDS/PAGE gels were purified and

digested in trypsin (Pierce, Thermo Fisher Scientific, USA). The

digested peptides were purified and separated in a nLC 1000 nano-

system (Thermo Scientific, USA) using Acclaim PepMap 100 and

rapidseparationliquidchromatographyPepMapC18chromato-

graphic columns (Thermo Scientific, USA). Eluted peptides were

incorporated into a Q Exactive mass spectrometer (Thermo Scien-

tific, USA) at the previous described setting conditions

and label-

free quantification (LFQ) of three technical replicates. MS scans

were analyzed with the Proteome Discoverer software (Thermo

Fisher Scientific, USA) using standardized workflows. Protein IDs

from mass spectra *.raw files were searched using MaxQuant pro-

gram and the SwissProt Mus musculus database, generating the raw

proteome matrix data.

2.8 |Proteome data analyses

The proteome raw data matrix was analyzed using Perseus-MaxQuant

software. Histograms of total MS counts scatter plots displaying pro-

tein abundance were inspected previously to data analysis for each

sample. Processing of raw data matrix was performed by pre-filtering

of contaminants and redundant values followed by Log

transforma-

tion of the proteome matrix and multiple sample analysis of variance

4S´

ANCHEZ-LÓPEZ ET AL.

(ANOVA, FDR 0.05). Normalization of the matrix was obtained by

computation of Z-scores. Hierarchical clustering was then performed

using the Euclidian distance and average linkage method, to obtain

the hierarchical dendrogram and heatMap of the clustered matrix.

Principal component analysis (PCA) was computed applying 14 princi-

pal components and delta of 1 10

5

with visualization of only the

first five principal components (PC).

Differentially expressed proteins (DEPs) were searched in t-test

pairwise group comparisons with a p-value <.05 and a fold change of

2, where significantly upregulated (FC > 2) and downregulated

(FC < 2) DEPs were displayed on the right and left upper panels of

volcano plots, respectively. The presence/absence of protein expres-

sion between paired groups was also included at the extremes of vol-

cano Xaxes. Gene-ontology enrichment analysis of upregulated and

downregulated DEPs was performed with ShinyGO 0.77 software

(South Dakota State University, FDR 0.05).

2.9 |Inflammatory response of J774A.1

macrophages

The cytokine secretion levels of murine tumor necrosis factor α(TNF-

α) and interleukin-10 (IL-10) were measured to analyze the inflamma-

tory behavior of materials under study. Macrophages were cultured

on both intact modified surfaces and those after wear-corrosion tests.

The modified surfaces included CoCr surfaces covalently immobilized

with HA and GO (HA/GO/CoCr). Polystyrene was included as a con-

trol. After 72 h of cell-biomaterial culture, cell medium supernatants

were collected and centrifuged (1024 g, 5 min) and the cytokine

secretion levels were quantified with commercial ELISA kits (Diaclone,

Besançon, France). The TNF-α/IL-10 ratios were the average of three

independent assays in triplicate.

2.10 |Statistical analysis

For proteome statistical analysis, two biological replicates were taken

at 72 h of exposure while three replicates were taken at 96 h of bio-

material exposure (n=5) in each of the three groups under study. In

each proteome replicate, the mean LFQ intensity value was obtained

from three technical measurements by LC–MS/MS. One-way analysis

of variance (ANOVA) was applied for proteome data analysis of the

three different groups in Z-scoring and hierarchical clustering in Per-

seus software. T-test was applied for analysis of DEPs between paired

groups.

For statistical analysis of the inflammatory response, mean TNF-

α/IL-10 ratios were computed from biological replicates from three

independent assays in triplicates, taken after 72 h of biomaterial

exposure. Inflammatory ratios were statistically analyzed with

GraphPad Prism software via one-way ANOVA. If a significant

ANOVA p-value was obtained (p-value <.05), post-hoc multiple

Tukey test comparisons were performed to assess significance

between paired groups.

3|RESULTS

3.1 |Synthesis and Raman characterization

of HA/GO/CoCr surfaces

The covalent grafting of HA is performed on GO/silane nanocoating

through ADH that works as an intermediate covalent GO-HA linker

through EDC/NHS carbodiimide chemistry. When GO nanosheets are

covalently immobilized on silanized CoCr surfaces,

the GO carboxyl

groups are activated by EDC/NHS reagents and the intermediate

ADH linker is assembled via amidation reaction between reactive

ADH amine groups and the activated carboxyl groups of GO.

41,42

premixed HA solution containing EDC/NHS is covalently grafted to

the immobilized ADH layer via amidation reaction between activated

HA carboxyl groups

and the other free reactive amine group of

ADH to obtain the covalent immobilization forming the HA/GO nano-

coating on CoCr surfaces. A scheme of the complete nanocoating

assembly is depicted in Figure 1.

After completing the immobilization procedure of HA on GO, the

final HA/GO nanocoating on CoCr surfaces was characterized by

SEM and Raman spectroscopy. Figure 2A shows the secondary elec-

tron image of the HA/GO/CoCr surface where the dimensions of the

GO nanosheets of about 200-nm thickness and 1-μm length are

observed. Figure 2B–Dshow the Raman spectra of CoCr surfaces

with HA/GO and GO nanocoatings. GO and HA references have been

added for comparative purposes. GO reference was obtained by

drop-casting of a 4 mg/mL GO aqueous dispersion, allowing it to dry

to obtain a thick layer of GO on the CoCr surface. HA reference was

directly taken from powder HA.

CoCr surfaces with the GO and HA/GO nanocoatings (Figure 2B)

show the characteristic bands of GO compounds. The D band located

at 1339 cm

1

is associated with the disruption of the pristine aro-

matic ring lattice structure's symmetry. The G band sited at

1605 cm

1

is attributed to the in-plane bond-stretching motion of

pairs of sp2 carbon atoms. The 2D band and the D+G combination

band appear around 2700 cm and 2930 cm

1

, respectively. Upon

covalent immobilization of HA, the HA/GO/CoCr surfaces (Figure 2B)

also show the characteristic peaks of HA in Raman spectra

at higher

wavenumber, around 3250–3750 cm

1

(see HA reference in

Figure 2C). However, the two bands of high intensity at 2904 and

2933 cm

1

corresponding to the C H stretching band and N H

stretching in HA (Figure 2C) are not discernible, which might be

caused by the overlap with the D+G combination band of immobi-

lized GO on the surface, which locates around the same wavenumber.

It can be observed that the heights of the D and G bands are

comparable for both conditions: GO/CoCr and GO reference

(Figure 2D). However, upon functionalizing with HA (HA/GO/CoCr

spectrum), the heights of the D and G bands are lightly higher than

those in GO reference and GO/CoCr surfaces (Figure 2D). The D band

is usually associated with the concentration of defects whereas the G

band is related to the size of sp2 domains. Hence, the ID/IG intensity

ratio can be a good indicator of the degree of disorder for graphene

networks.

S´

ANCHEZ-LÓPEZ ET AL.5

FIGURE 1 Scheme of the sequential covalent immobilization on CoCr surfaces. CoCr, cobalt-chrome; EDC, N-(3-dimethylaminopropyl)-N-

ethylcarbodiimide; GO, graphene oxide; HA, hyaluronic acid; NHS, N-hydroxysuccinimide.

2500 3000 3500 4000

Intensity, a.u.

Raman shift, cm–1

HA reference

HA/GO/CoCr

(A) (C)

(B) (D)

1250 1500 1750

GO reference

GO/CoCr

HA/GO/CoCr

Intensity, a.u.

Raman shift, cm

–1

1000 1500 2000 2500 3000 3500

2933

HA reference

GO reference

GO/CoCr

HA/GO/CoCr

Intensity, a.u.

Raman shift, cm

–1

D+G HA peaks

2904

FIGURE 2 (A) Secondary electron image of the HA/GO/CoCr surface; (B) Raman spectra of the covalent immobilizations of GO/CoCr and

GO/HA/CoCr surfaces, respectively. Reference spectra are added for powder HA and drop-casted GO; (C) magnification at high wave number of

Raman spectra of HA reference and HA/GO/CoCr surface; (D) magnification of D and G bands in the Raman spectra for GO reference, GO/CoCr,

and HA/GO/CoCr surfaces. CoCr, cobalt-chrome; GO, graphene oxide; HA, hyaluronic acid.

6S´

ANCHEZ-LÓPEZ ET AL.

The estimation of D and G band intensity ratio, ID/IG ratios, for

GO/CoCr, HA/GO/CoCr, and GO reference are shown in Table 1.

The higher intensity ratio, the higher structural defects. On the

opposite way, lower intensity ratios suggest lower disorder of the

crystalline and symmetry graphene structure. The ID/IG ratio of GO

reference was 0.980. When GO is covalently immobilized on silanized

CoCr surfaces, the comparable I

ratio (0.998 in GO/CoCr) indi-

cates that the covalent attachment of GO does not induce a substan-

tial degree of disorder within the GO layer, and the size of sp2

domains remains largely unchanged. On the other hand, following the

immobilization of HA, a moderate decrease in the I

ratio is

observed (0.952 in HA/GO/CoCr), indicative of a lower disorder and

increase in the size of sp2 domains (Figure 2). It is tempting to suggest

that this observation can denote that structural changes in the GO

nanolayer are induced by the EDC/NHS treatment and posterior

immobilization of ADH and HA.

3.2 |Electrochemical characterization of HA/GO

nanocoating on CoCr

The barrier effect of the HA/GO nanocoating on CoCr surfaces was

analyzed by EIS. Experimental impedance data of CoCr surfaces func-

tionalized with the covalent HA/GO nanocoating are represented in

Nyquist diagrams and Bode plots for impedance modulus and phase

angle in Figure 3A–C, respectively, when these surfaces were

immersed in HA aqueous electrolyte at 3 g/L. The electrochemical

behavior of the GO/CoCr surfaces at the day 7 of immersion in HA

aqueous electrolyte is also added for comparative purposes.

Figure 3shows that the impedance results for HA/GO/CoCr sur-

faces reveal a high stability with minor differences during immersion

in the HA electrolyte up to 10 immersion days. Nyquist diagrams

show arcs of wide amplitude (Figure 3A) and stabilized impedance

modulus (Figure 3B) and phase angle (Figure 3C) in Bode diagrams

over immersion time. On the other hand, the Nyquist diagrams for the

HA/GO/CoCr surfaces show lower capacitive arcs than those

obtained for the GO/CoCr surfaces. This result indicates that the pos-

terior immobilization of HA on GO nanocoating changed the electro-

chemical properties of the GO/CoCr surfaces.

The experimental impedance data were fitted to electric equiva-

lent circuits (EEC) which assign physical meaning to the parameters of

the circuit. An EEC with two time-constants in series provided a

proper fitting of the metal surface with a thin nanocoating.

The pas-

sive electrical components shown in Figure 3A forming the EEC were:

the electrolyte resistance (R

), the coating resistance (R

COAT

) due to

HA/GO or GO nanocoating in parallel with a constant phase element

(CPE

) and the oxide layer resistance (R

) in parallel with its constant

phase element (CPE

). CPE

and CPE

simulate the nonideal capaci-

tive behavior associated with distributed time constants of the modi-

fied surfaces. The fitting values for each EEC element for HA/GO/

CoCr surfaces along with the immersion times up to 23 days are

shown in Table 2. Fitting values for GO/CoCr surfaces at 0 days and

after 7 days of immersion are also added for comparative reasons.

Following the immobilization of HA on GO, a decrease in the

magnitude of the R

COAT

is seen at the first 24 h of immersion, in com-

parison to the R

COAT

values of the GO/CoCr surfaces (Table 2). This

decline in resistivity may be attributed to the enhanced conductivity

induced by HA immobilization in the nanocoating. Nevertheless, with

prolonged immersion, the R

COAT

value in HA/GO/CoCr surfaces

TABLE 1 I

ratios of GO immobilized (GO/CoCr) and

immobilized with HA and GO (HA/GO/CoCr) in the nanocoatings on

CoCr surface and reference GO.

(a.u) I

ratio

GO reference 23,853 24,338 0.980

GO/CoCr 22,109 22,158 0.998

HA/GO/CoCr 41,034 43,122 0.952

Abbreviations: CoCr, cobalt-chrome; GO, graphene oxide; HA,

hyaluronic acid.

FIGURE 3 Experimental Nyquist diagrams (A) and Bode plots (B, C) of HA/GO/CoCr surfaces throughout immersion time in HA electrolyte.

Impedance data for GO/CoCr surfaces after 7 days of immersion is included for comparative reasons. Fitted lines were not included to enhance

the clarity of the figure. Replicate number n=2. CoCr, cobalt-chrome; GO, graphene oxide; HA, hyaluronic acid.

S´

ANCHEZ-LÓPEZ ET AL.7

increases up to 7 days, reaching values comparable to those in

GO/CoCr surfaces (Table 2). Notably, values rise significantly, up to

six times higher values at 23 days, compared with the initial days of

immersion. This behavior suggests the possibility of degradation

occurring in the HA chemically bonded to the GO. However, it is

noteworthy that the electrochemical response of the oxide layer (R

CPE

, and associated n values) remains nearly constant through the

immersion period indicating electrochemical stability of the oxide

layer. This stability may be attributed to the hindering effect of large

HA molecules anchored in the GO, limiting electrolyte entry. Notably,

the electrolyte used for electrochemical testing is a HA solution. The

adsorption of HA molecules onto the HA/GO nanocoating must not

be discarded, potentially increasing resistance in the HA/GO nano-

coating on the CoCr surfaces. The diffusion process of molecules

through the cross-linked HA/GO nanocoating is reflected in the

nvalues associated with the CPE

values in Table 2. The high CPE

values and an exponent nvalue between 0.5 and 1 denote that diffu-

sion plays a significant role in controlling the electrochemical process

of the system. Overall, the electrochemical response of the protective

nanocoating immersed in HA electrolyte demonstrated remarkable

stability over the chosen immersion period in this work.

The R

and CPE

values are associated with the oxide layer of the

CoCr in both HA/GO/CoCr and GO/CoCr samples. The HA/GO/CoCr

samples exhibit lower R

values compared with the GO/CoCr samples,

possibly due to hindered oxygen diffusion through the HA/GO nano-

coating into the CoCr oxide layer. However, R

and CPE

values remain

nearly constant for each sample, irrespective of the immersion time

(Table 2). This consistency highlights the electrochemical stability of the

modified CoCr surface. This sustained electrochemical stability on the

CoCr metal surface is also attributed to the strong anchoring of GO

with the silane layer and HA with GO/Si nanocoating. The formation of

a protective nanocoating based on GO anchoring imparts barrier prop-

erties, owing to the impermeable sp2 graphene network securely

adhered to the metal surface through GO/Si interfacial bonding,

39,43

further enhanced by HA immobilization.

3.3 |Characterization of worn surfaces

In Figure 4appears the profilometry of the worn tracks after wear-

corrosion tests of CoCr and HA/GO/CoCr disks against CoCr ball at

5 N load, 120 rpm for 30,000 m in 3 g/L HA medium.

The track length is practically the same on both surfaces, CoCr and

HA/GO/CoCr, as can be seen in the annexed table in Figure 4. However,

the depth of the track analyzed on CoCr surfaces is significantly higher

(24 microns) compared with the HA/GO/CoCr surface (about

16 microns). This difference in track depth results in a lower volume of

metal particles and ions released into the environment (annexed table in

Figure 4), indicating that the presence of the nanocoating exerts a signifi-

cant lubricating effect in the tribocorrosion process of the CoCr surface.

3.4 |Macrophage proteome characterization

Macrophage proteome characterization was carried out after 72 and

96 h interaction with polystyrene control surfaces, intact HA/GO/

CoCr and worn HA/GO/CoCr surfaces. Unsupervised classification

analyses were performed by hierarchical clustering and PCA to clas-

sify the macrophage proteomes expressed in response to the different

biomaterial surfaces.

3.4.1 | Hierarchical clustering

In an initial analysis aimed at offering a comprehensive overview of

trends, Figure 5shows the hierarchical clustering of J744A.1 macro-

phage proteome data when cultured on the polystyrene control, intact

HA/GO/CoCr, and worn HA/GO/CoCr surfaces. Exposure of macro-

phages to the different surfaces was maintained for 72 h (R1 and R2

replicates) and for 96 h (R3, R4, and R5 replicates).

The heatmap displays the replicates of each group within columns

(designates as R1–R5 for each group) with their corresponding

TABLE 2 Fitting values of experimental impedance data from day 0 to day 23 of immersion of HA/GO/CoCr and GO/CoCr at 0 and 7 days

of immersion using the equivalent electric circuit shown in Figure 3a.

Time, d

± abs.

error, Ω

COAT

± abs.

error, Ω

CPE

± abs.

error, μSsn

± abs.

error

± abs.

error, MΩ

CPE

± abs.

error, μSsn

± abs.

error Chi

GO/

CoCr

0 3328 ± 7.78 3532 ± 281.44 73.3 ± 4.49 0.68 ± 0.02 66.5 ± 0.35 16.0 ± 0.05 0.94 ± 0.001 4.02 ± 10

5

7 3449 ± 8.63 4549 ± 346.76 61.6 ± 3.42 0.67 ± 0.01 81.9 ± 0.52 14.96 ± 0.05 0.94 ± 0.001 4.49 ± 10

5

HA/

GO/

CoCr

0 2170 ± 7.02 1307 ± 545.14 253.7 ± 51.3 0.73 ± 0.05 12.0 ± 0.22 15.8 ± 0.09 0.91 ± 0.002 8.91 ± 10

5

1 2646 ± 8.87 1803 ± 479.84 154.7 ± 24.0 0.74 ± 0.04 14.0 ± 0.31 16.3 ± 0.09 0.92 ± 0.002 1.00 ± 10

4

2 2527 ± 8.95 3055 ± 918.35 139.5 ± 18.0 0.81 ± 0.04 12.1 ± 0.27 16.1 ± 0.13 0.92 ± 0.003 1.36 ± 10

4

3 2162 ± 7.05 2938 ± 890.6 161.0 ± 19.5 0.77 ± 0.03 13.2 ± 0.29 16.2 ± 0.12 0.92 ± 0.002 1.06 ± 10

4

4 2078 ± 6.67 3547 ± 1117.4 163.1 ± 19.0 0.76 ± 0.03 13.4 ± 0.29 16.1 ± 0.13 0.92 ± 0.002 1.02 ± 10

4

7 2145 ± 7.71 4041 ± 1573.4 173.2 ± 22.5 0.72 ± 0.03 12.6 ± 0.28 15.9 ± 0.16 0.92 ± 0.003 1.19 ± 10

4

23 2240 ± 6.39 25,088 ± 5945.4 82.9 ± 7.6 0.74 ± 0.01 16.8 ± 0.51 16.6 ± 0.29 0.93 ± 0.004 8.37 ± 10

5

Note:R

—electrolyte resistance; R

COAT

—coating resistance, and CPE

—constant phase element, associated with the HA/GO or GO nanocoating on CoCr

surfaces; R

—oxide layer resistance and CPE

—constant phase element related to the metal surface; absolute errors for each element and Chi-square (χ

)is

also added.

Abbreviations: CoCr, cobalt-chrome; GO, graphene oxide; HA, hyaluronic acid.

8S´

ANCHEZ-LÓPEZ ET AL.

macrophage proteome expression profile within rows (comprising

2375 proteins IDs) clustered in the associated dendrogram shown in

Figure 5A. Two main ancestor branches clearly separate macrophage

proteome profiles. Within the first ancestor branch, proteome repli-

cates in response to worn HA/GO/CoCr surfaces are clustered as a

separate population of macrophage proteome (first left dendrogram

branch). In the second hierarchical branch, proteome replicates in

response to both polystyrene control and intact HA/GO/CoCr are

clustered (first right dendrogram branch). This finding highlights the

similarity of macrophage proteomes expressed in polystyrene con-

trol and intact HA/GO/CoCr surfaces. The heatmap also categorizes

proteome expression into two primary clusters (as shown in

Figure 5B), reflecting opposing upregulations and downregulations

observed in the main dendrogram branches. These clusters are

defined as Cluster 1, encompassing proteins with increased expres-

sion following macrophage exposure to the worn HA/GO/CoCr, and

Cluster 2, comprising proteins with increased expression following

macrophage exposure to the intact nanocoating on CoCr or to poly-

styrene. Expression changes as distances from the cluster center are

plottedinFigure5B.

3.4.2 | Principal component analysis

To assess the relative effect of the different surface chemistries on

the overall expression of the macrophage proteome, a multiparametric

space PCA was conducted, as depicted in Figure 6. PCA determines

the direction with the largest total variance between parameters

(or samples) in the multidimensional space determined by PC axes.

The PC 1 determines the new x-axis in the PCA space describing the

direction of maximum percentage of variance between samples

(23.3% of percentage of variance), followed by the rest of PC contrib-

uting with lower percentages of variances, contributing up to the total

percentage (100%) of variance between all replicates in the multidi-

mensional PCA space. Plots of the first five PC and their PCA scores

(%) are shown in Figure 6, revealing that proteome replicates from the

polystyrene control and intact HA/GO/CoCr surfaces were clustered

around the PC1 origin, whereas proteome replicates from worn

HA/GO/CoCr surfaces were located at further PCA distances along

the PC1 axis and spread away from the previous clustered population.

This result reinforces that the covalent nanocoating on CoCr pro-

motes the same protein patterns as polystyrene control while the

wear-corrosion degradation of the nanocoating induces a modification

in the macrophage protein expression.

3.4.3 | Protein abundance

Figure 7shows scatter pairwise density plots that compare protein

abundances between replicate groups. It is important to note that when

comparing macrophage protein expression for each protein ID (each ID

represented by a black dot), those in the control group (x-axis value)

and other surface groups (y-axis value) often exhibit similar protein

abundances. This is evident when black circles cluster closely around

the red line. However, there are fewer instances where certain protein

IDs show increased or decreased abundances between the paired

groups, which is indicated by black circles positioned above or below

the red lines, respectively. In cases where the protein abundances are

exactly the same in two different macrophage groups, they would align

perfectly along the red line (when black dots overlap the red line).

These plots reveal a close similarity in protein abundances

between the polystyrene control and the intact HA/GO/CoCr

FIGURE 4 Profilometry of the worn tracks of CoCr (A) and HA/GO/CoCr (B) surfaces, against CoCr ball at 5 N load, 120 rpm for 30,000 m in

3 g/L HA medium. CoCr, cobalt-chrome; GO, graphene oxide; HA, hyaluronic acid.

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ANCHEZ-LÓPEZ ET AL.9

FIGURE 6 Principal component analysis (PCA) showing distribution of sample groups under study along PCA axis: PC1 versus PC2, PC3, PC4,

and PC5. Replicate number n=5.

FIGURE 5 Hierarchical clustering of high-throughput proteome data from the J744A.1 macrophage when cultured on polystyrene control

surface, intact HA/GO/CoCr, and worn HA/GO/CoCr surfaces during 72 h (R1 and R2 samples) or 96 h (R3, R4, and R5 samples).

(A) Dendrogram showing clustered groups with heatmap color representation of corresponding macrophage proteome expressions (2375 total

protein IDs detected); (B) Cluster profile plot with distances to the cluster center of proteins classified with reduced/increased expression (1201

protein IDs) or increased/reduced expression (1174 protein ID) in cluster 1 or cluster 2, respectively. Replicate number n=5. CoCr, cobalt-

chrome; GO, graphene oxide; HA, hyaluronic acid.

10 S´

ANCHEZ-LÓPEZ ET AL.

(Figure 7A), as indicated by the narrow linear slopes and high correla-

tion coefficients (R2 closer to 1, represented by the line y=x). On the

other hand, in worn HA/GO/CoCr surfaces, protein abundances

exhibit more significant differences in comparison to both the polysty-

rene control and intact HA/GO/CoCr surfaces. This is evident from a

greater number of data points which deviate from the y=xreference

line, resulting in smaller correlation coefficient (R2 values in

Figure 7B,C).

3.4.4 | Differentially expressed proteins and Gene-

ontology enrichment analysis

Figure 8shows the proteome profiling of J744A.1 macrophage in

response to each group surfaces from pairwise volcano plots upon

72 h (A) and 96 h (B) of exposure.

DEPs are shown in the context of pairwise surface group compari-

sons through the percentage of total upregulated (UP) and downregu-

lated(DOWN).TheanalysisofDEPswasalsoperformedwithGene-

ontology enrichment analysis. The intact HA/GO nanocoating on the

CoCr surface induced only marginal percentages of upregulated and

downregulated DEPs with respect to the polystyrene control. Specifically,

at 72 h, it resulted in 1.3% upregulated and 0.6% downregulated DEPs,

while at 96 h, it led to 0.04% upregulated and 0.3% downregulated DEPs

(Figure 8a.1, a.2). However, for worn HA/GO/CoCr surfaces, there is a

notable increase in the percentage of upregulated DEPs and, more signifi-

cantly, in the percentages of downregulated DEPs in comparison to the

polystyrene control (asdepictedinFigure8b.1, b.2). This trend is also

observed when comparing worn HA/GO/CoCr with the intact HA/GO/

CoCr surfaces (as shown in Figure 8c.1, c.2) at both 72 and 96 h.

When DEPs of intact HA/GO/CoCr and polystyrene control at

both exposure times are summed, the percentages were 0.3%

FIGURE 7 Scatter plots showing the comparison of proteome abundances in macrophages cultured on the three surface groups, (A) intact

HA/GO/CoCr vs control (B) worn HA/GO/CoCr vs control, and (C) worn HA/GO/CoCr vs intact HA/GO/CoCr. Normalized protein abundance of

sample replicates in x and y axes (Log2-normalization of LFQ values). To quantify the similarity/dispersion degree of the proteome abundances,

correlation coefficients (R2) are shown. Replicate number n=5. CoCr, cobalt-chrome; GO, graphene oxide; HA, hyaluronic acid; LFQ, label-free

quantification.

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ANCHEZ-LÓPEZ ET AL.11

upregulated and 0.7% downregulated (as illustrated in Figure 9A), with

respect to the entire macrophage proteome.

The same pattern of DEPs for worn HA/GO/CoCr versus polysty-

rene (Figure 9B) and versus intact HA/GO/CoCr (Figure 9C) is evident

when both exposure times are summed. The Gene-ontology enrich-

ment analysis of upregulated and downregulated DEPs can be

accessed in the Figures S1–S3.

3.5 |Macrophage inflammatory balance

The macrophage inflammatory ratio was determined by measuring

secreted pro-inflammatory and anti-inflammatory cytokines TNF-α

and IL-10, respectively, in cell culture extracts, after 72 h. To

assess the implication of the covalent immobilization of the

HA/GOonCoCrsurfacesonthemacrophageinflammatory

FIGURE 8 Macrophage proteome profiling upon 72 h (a.1, b.1, c.1) and 96 h (a.2, b.2, c.3) of exposure showing percentage of total

upregulated (UP) and downregulated (DOWN) DEPs from pairwise volcano comparisons between intact HA/GO/CoCr vs polystyrene control

(a.1, a.2); worn HA/GO/CoCr vs polystyrene control (b.1, b.2), and worn HA/GO/CoCr vs intact HA/GO/CoCr (c.1, c.2). Some labeled proteins

were identified in the samples analyzed. Replicate number n=2 for 72 h of cell-biomaterial exposure; replicate number n=3 for 96 h of cell-

biomaterial exposure. CoCr, cobalt-chrome; DEP, differentially expressed protein; GO, graphene oxide; HA, hyaluronic acid.

12 S´

ANCHEZ-LÓPEZ ET AL.

response, a comparative analysis of the TNF-α/IL-10 ratio was also

evaluated with different surface chemistry and using different

types of surface immobilization (covalent vs. physical). In

Figure 10, a comparative of modified CoCr surfaces with covalent

immobilization of HA and GO (HA/GO/CoCr) on intact modified

surfaces (Figure 10A) and after applying wear-corrosion

(Figure 10B), and physical immobilization of ErGO (ErGO/CoCr)

and HA (HA/ErGO/CoCr) is shown. Polystyrene is added as con-

trol. No significant differences were obtained in TNF-α/IL-10

ratios of macrophages cultures on intact HA/GO/CoCr versus

polystyrene control after 72 h (Figure 10A).

As it is shown in Figure 10A, the inflammatory response

decreases significantly upon the covalent immobilization of HA/GO

on CoCr in comparison with the physical immobilization of electro-

chemically reduced GO (ErGO) and further functionalized under

immersion with HA (ErGO/HA). Covalent immobilization of HA and

GO on CoCr surfaces provides the largest effect by recovering a TNF-

α/IL-10 ratio comparable with the basal ratios cultured on commercial

polystyrene surface, obtaining nonsignificant differences between

HA/GO/CoCr versus polystyrene control surfaces (Figure 10A). This

agrees with proteome analyses by PCA and hierarchical clustering,

which showed similar macrophage proteomes when they are cultured

FIGURE 9 Macrophage proteome profiling considering the data summed at 72 h and 96 h between (A) intact HA/GO/CoCr versus control

(B) worn HA/GO/CoCr versus control, and (C) worn HA/GO/CoCr versus intact HA/GO/CoCr. Some labeled proteins were identified in the

samples analyzed. Replicate number n=5. CoCr, cobalt-chrome; GO, graphene oxide; HA, hyaluronic acid.

S´

ANCHEZ-LÓPEZ ET AL.13

on treated polystyrene and on HA/ GO/CoCr surfaces (see sections

3.4.1,3.4.2).

It is known that wear-corrosion phenomena on CoCr surfaces fur-

ther exacerbates macrophage pro-inflammatory ratios and increases

TNF-αsecretion.

12,49,50

Likewise, a slight but no significant increase in

TNF-α/IL-10 ratio is observed upon wear-corrosion of HA/GO/CoCr

with respect to the polystyrene control (Figure 10B). The covalent

immobilization in HA/GO/CoCr surfaces is able to reduce wear-

corrosion-induced inflammation at the largest extent with respect to

other physical adsorption of ErGO on CoCr surfaces, such as ErGO/

CoCr, HA/ErGO/CoCr (Figure 10B).

4|DISCUSSION

The beneficial impact of HA in healthcare is evident, given its wide-

spread use in various biomedical applications. Among its remarkable

attributes, alongside its abundance in the human body, is its capacity

to reduce friction. These properties render it a vital component in

synovial fluid, playing a significant role in joint lubrication. Conversely,

graphene compounds, when employed as solid lubricants on metallic

substrates, exhibit lubrication properties that could effectively reduce

wear in joint prostheses. The challenge in this research has been to

characterize the CoCr surface immobilized with HA and GO assess

the macrophage response.

The use of ADH as an intermediate reagent to promote the cova-

lent immobilization of HA on the GO induces modifications in the GO

network. The functionalization of GO by ADH is reported to occur

through the amidation reaction between activated EDC/NHS carboxyl

groups of GO and the primary amine groups of the ADH agent.

41,42

This process results in the removal of an oxygen atom from GO and

the nitrogen functionalization of GO immobilized on CoCr. In this

step, the simultaneous reduction and functionalization of GO caused

by the removal of oxygen-containing groups is also well-known to

occur for other reagents.

51–57

The functionalization of GO with ADH

did not introduce defects in the carbon network, as the ID/IG inten-

sity ratio for HA/GO/CoCr surfaces (0.952) is lower than that of

GO/CoCr surfaces (0.998), suggesting that ADH treatment may indi-

cate the mitigation of sp3 defects.

This decrease in the ID/IG ratio

suggests potential defect repair by ADH, particularly at the edges of

the GO lattice where functionalization takes place. Similar linear

amino-containing agent also reported not only avoid introducing

defects but also repair defects and enlargement of the sp2 domains

by a decrease in ID/IG ratios upon functionalization of GO with the

ethylene diamine (EDA).

The reduction of oxygenated groups of GO is known to be

directly proportional to an increase in the electric conductivity of GO

and a decrease in its hydrophilic character. The heightened conductiv-

ity in HA/GO nanocoating on CoCr surfaces can also be associated

with the functionalization with ADH, as functionalization with

diamines partially restore the aromatic structure in GO and the amine

groups donate electron density to the aromatic rings of GO.

55,60,61

This restoration likely constitutes another contributing factor to the

improved conductivity which correlates with the low R

COAT

values

estimated in the HA/GO/CoCr samples within the first 24 h of immer-

sion in HA electrolyte (Table 2). According to Ref. 62, besides the

reduction of terminal carboxylic groups of GO upon GO/ADH functio-

nalization, activation by EDC/NHS carbodiimide chemistry can also

contribute to a decrease in the R

COAT

of this nanocoating.

The GO-based encapsulation of CoCr by the HA/GO nanocoating

(see section 3.2) works hindering metal ion release.

This is corre-

lated with a reduction in the toxicities observed in graphene- and GO-

encapsulated metal implants, attributed to reduced metal ion

release.

63–66

The electrochemical parameter of R

in the HA/GO/CoCr exhibits

remarkable stability in the aqueous electrolyte solution. This indicates

that the infiltration of the electrolyte into the oxide layer remains

unchanged. Furthermore, it implies that the covalent linkage of HA to

ADH can improve its stability against degradation processes. This is

especially valuable considering that HA degradation primarily proceeds

through nonenzymatic processes in synovial fluid, an environment

where hyaluronidase activity is barely detectable.

In this medium,

joints HA degradation is predominantly driven by oxidation and hydro-

lysis mechanisms under the physiological pH of the synovial fluid.

28,67–

The covalent binding of HA to ADH represents a promising approach

to enhance resistance to degradation. Studies show that covalently

functionalizing HA with ADH leads to considerably slower degradation

kinetics compared with untreated HA.

71,72

This suggests that covalent

immobilization of HA and GO could provide added benefits in terms of

safeguarding against hydrolytic degradation after implantation.

FIGURE 10 Comparative TNF-α/IL-10 macrophage ratio on

modified CoCr surfaces with physical immobilization of ErGO (ErGO/

CoCr) and physical immobilization of HA and ErGO (HA/ErGO/CoCr)

or covalent immobilization of HA and GO (HA/GO/CoCr) on intact

modified surfaces (A) and after applying wear-corrosion (B) after 72 h.

Polystyrene is used as control. p-value <.05 (*), p-value <.01 (**), p-

value <.005 (***), p-value <.001 (****), ns—nonsignificant. The TNF-

α/IL-10 ratios were the average of three independent assays in

triplicates. Replicate number n=3. CoCr, cobalt-chrome; GO,

graphene oxide; HA, hyaluronic acid; TNF-α, tumor necrosis factor α.

14 S´

ANCHEZ-LÓPEZ ET AL.

However, given that significantly altered HA chemical structures

can potentially induce cytotoxicity, the study of the macrophage

response to the modified HA structure immobilized in the nanocoat-

ing is needed. The characterization of the macrophage proteome and

the analysis of the pro-inflammatory and anti-inflammatory molecule

secretion ratio is a key point in the proposal of this immobilization as

a suitable stable and protective bionanocoating on CoCr surfaces.

Unsupervised analyses through hierarchical clustering and PCA

analysis offer an unbiased classification of massive proteomic data.

The outcomes of hierarchical clustering and PCA revealed highly com-

parable macrophage proteomes on both cell-culture treated polysty-

rene and CoCr surfaces covalently immobilized with the HA/GO

nanocoating. Hierarchical clustering analysis classified the proteomes

of macrophages cultured on polystyrene or intact HA/GO/CoCr in

the same dendrogram branches, as they present similar gene expres-

sions in cluster 2 (Figure 5). In a similar way, PCA placed proteome

replicates from both polystyrene control and intact HA/GO/CoCr in a

mixed population around the PC1 origin (Figure 6). These results

strongly indicate a significant homology in proteome expressions.

Likewise, macrophages cultured on polystyrene control and intact

HA/GO/CoCr surfaces exhibit highly comparable protein abundances,

with R2 correlation coefficients near 1 (Figure 7A), indicating very low

percentages of DEPs and, consequently, very similar proteomes

(Figure 8a.1, a.2; Figure 9A). Gene-ontology enrichment analysis of

DEPs revealed upregulations in adhesion-related proteins (Parvb,

Itgb4bp, Ctnnd1) after 96 h. This suggests the formation of stable

adhesions between macrophages and the intact HA/GO/CoCr sur-

faces through these adhesion proteins (see Figure S2a). The increased

abundance of integrin beta 4, (Itgb4bp or Eif6) can facilitate the direct

binding of cells to material surfaces via integrins.

The increased

presence of beta parvin (Parvb), a focal adhesion protein connecting

ECM-binding integrins to the actin cytoskeleton, suggests the forma-

tion of focal adhesions. These structures facilitate cell spreading and

the establishment of stable adhesions on material substrates by link-

ing integrins to the actin cytoskeleton.

74–76

Moreover, these focal

adhesions play a crucial role in in conveying physicochemical clues

from the substrate to the associated cytoskeletal network through

mechano-transduction at the focal adhesions.

Similarly, the

increased abundance of catenin (Ctnnd1) can also regulate integrin

cell-matrix adhesions.

Altogether, this indicates an enhanced cell

adhesion on the intact HA/GO/CoCr substrate. The covalent grafting

of HA on the nanocoating likely assumes a significant role in adhesion

on the functionalized metal surface, as HA enhances integrin-

mediated mechano-transduction and promotes focal adhesion forma-

tion.

Additionally, the deposition of anionic HMW-HA increases the

hydrophilicity at CoCr surface.

It is well-established that enhancing

the hydrophilicity of hydrophobic surfaces, such as metals, improves

cell adhesion on biomaterials.

The heightened response in the mac-

rophage proteome can be attributed to the increased surface hydro-

philicity provided by the HA/GO nanomodifications on CoCr. These

modifications are reminiscent of patented surface treatments

designed to improve cell adhesion on polystyrene surfaces. Polysty-

rene surfaces are typically highly hydrophobic, exhibiting low cellular

attachment in their unmodified state. Various surface treatments

employed on commercial cell-culturing plastic plates include plasma

treatments to increase surface hydrophilicity,

82,83

silica film

deposition,

and the application of ECM proteins and proteoglycans,

among other methods. The latter, involving ECM proteins and proteo-

glycans, may help explain the comparable outcomes observed with the

HA/GO nanocoating on CoCr surfaces.

In summary, these findings underscore the efficacy of the cova-

lently immobilized HA/GO nanocoating on CoCr surfaces in creating a

cell-culture-treated metallic surface with excellent biocompatibility

with macrophages.

However, the degradation of the HA/GO nanocoating during

wear-corrosion processes exposes certain bare CoCr areas to the cell

culture. The coexistence of both the intact HA/GO nanocoating and

the bare CoCr areas induces significant differences in the macrophage

proteome. Hierarchical clustering, in this context, classified the worn

HA/GO/CoCr surfaces as inducing proteome replicates in a distinct

cluster. Additionally, PCA positioned these surfaces in a different mul-

tiparametric space compared with the polystyrene control and intact

HA/GO/CoCr surfaces. Scatter plots depicting protein abundance,

characterized by R

correlation coefficients less than 1 (Figure 7B,C),

indicated more pronounced differences in protein abundance within

the wear-corrosion group compared with the other two groups. Con-

sequently, higher percentages of significantly upregulated and down-

regulated DEPs were found in the wear-corrosion group as opposed

to the other two groups. This suggests a distinctively expressed prote-

ome, particularly in the percentages of downregulated DEPs (Figure 8

b.1, b.2, c.1, c.2; Figure 9B,C). Notable downregulations in pro-

inflammatory pathways were evident in the macrophage proteome

after 72 and 96 h of exposure to the worn HA/GO/CoCr surfaces.

Significant enrichments were observed in the downregulated proteins

interacting with the TNF-αnF-kB signaling pathway (Pfdn2, Cdc37,

Psmd6, Hsp90aa1, Hsp90ab1, Smarca4, Smarcc1, Txlna Arhgef2, Skp1,

Ppp6c, Rack1, or Gnb2l1) (Figures S1c,S2b,c, and S3b,c).

86,87

Downre-

gulation enrichments in the NLRP3 inflammasome (Casp1, Pycard,

Sugt1), the IL-1 signaling pathway (Capn1, Casp1, Sirpa) and IFN sig-

naling (Ptpn6, Abce1) and IFN production (G3bp1, Tomm70a, Irf5,

Hmgb1, Pycard, Sirpa) were also observed (Figures S2b,c and 3b,c).

The downregulations stimulated by immunosuppressive HMW-

HA can effectively inhibit the induction of pro-inflammatory

responses in macrophages exposed to implant wear debris, potentially

leading to a reduction in inflammation. This hypothesis was further

corroborated by the analysis of pro- and anti-inflammatory TNF-α/IL-

10 cytokine secretion ratios. The findings support the idea that immu-

nosuppressive HMW-HA from the covalent nanocoating mitigates

TNF-α/IL-10 ratios during biomaterial tribocorrosion. Macrophages

present in different tissues are polarized according to changes in their

environment, resulting in distinct subtypes such as M1 and M2 macro-

phages.

88,89

M1 macrophages exhibit pro-inflammatory capabilities,

secreting factors like IL-6, IL-12, and TNF-α. Conversely, M2 macro-

phages demonstrate anti-inflammatory functions, aiding in tissue

repair and expressing cytokines as IL-10.

90,91

TNF-αis recognized as a

marker for the pro-inflammatory M1 phenotype, while IL-10 serves as

S´

ANCHEZ-LÓPEZ ET AL.15

a marker for the anti-inflammatory M2 phenotype. It is known that

CoCr biomaterial and specially CoCr particulate debris and ions fur-

ther exacerbates macrophage pro-inflammatory ratios and increases

TNF-αsecretion.

12,49,50

Previous studies have shown that macro-

phages cultured on bare CoCr display a highly inflammatory response,

inducing more than a 15-fold in the TNF-α/IL-10 ratios with respect

to the basal ratios on control cultures.

Chemical modifications of

the bare CoCr surface, achieved through the physical immobilization

of ErGO sheets

and HMW-HA,

partially alleviate the inflammation

typically associated with the surface. Nevertheless, the TNF-α/IL-10

ratios on these modified surfaces remain significantly elevated com-

pared to the basal level in macrophages (Figure 10A), indicative of a

shift toward the pro-inflammatory M1 macrophage phenotype.

Similarly, a marginal, though not statistically significant, rise in the

TNF-α/IL-10 ratio is noted when macrophages are cultured on CoCr

with the HA/GO nanocoating. This observation suggests a potential

presence of inflammation, possibly triggered by the CoCr metal implant

beneath the coating and/or by the release of metal ions or particles into

the cell culture. Nevertheless, under wear conditions, the covalent

immobilization of the HA/GO nanocoating significantly minimizes the

inflammatory ratio compared with the physical adsorption of ErGO and

HA on CoCr surfaces (Figure 10B). Therefore, covalent modification of

CoCr with GO/HA is more likely to induce macrophage polarization

toward the M2 phenotype anti-inflammatory compared with CoCr sur-

faces modified by the physical adsorption of ErGO and ErGO/HA.

The covalent immobilization of HA and GO on CoCr surface

exhibits the most significant biocompatible effect, leading to TNF-

α/IL-10 ratios that closely resemble the basal ratios of macrophages

cultured on commercial polystyrene control surfaces (ns differences,

Figure 10), suggesting compatibility with an anti-inflammatory M2

phenotype that promote tissue repair. This finding is consistent with

proteome analyses by PCA and hierarchical clustering, demonstrating

similar macrophage proteomes when cultured on treated polystyrene

and intact HA/GO/CoCr (see sections 3.3.1, 3.3.2).

The decrease in pro-inflammatory TNF-α/IL-10 ratios on the

worn HA/GO/CoCr surfaces correlates with observed downregula-

tions in pro-inflammatory signaling pathways within the macrophage

proteome. Notably, reductions were identified in TNF-α, NLRP3, IFN-

γ, and IL-1 signaling pathways, signifying the immunosuppression

effects of HMW-HA covalently grafted onto the functionalized metal

surface in response to tribocorrosion-induced inflammatory signals.

This modulation plays a role in suppressing the macrophage inflamma-

tion response.

In summary, the modification of the surface chemistry CoCr

through covalent immobilization of HA and GO shows both electro-

chemical stability and biocompatibility evidenced by analyses of mac-

rophage proteome expression and the TNF-α/IL-10 secretion ratio.

5|CONCLUSIONS

•The covalent immobilization of HA and GO and does not incorpo-

rate additional structural disorder in the graphene network. Even

more, a slight enhancement in the size of sp2 domains is verified.

•The electrochemical response corroborates a barrier effect of

HA/GO nanocoating on the CoCr surfaces.

•The macrophages cultured on CoCr surfaces covalently modified

with the HA/GO nanocoating expressed a proteome highly equiva-

lent to those cultured on polystyrene control.

•Disruption of covalent immobilization HA/GO nanocoating on

CoCr surfaces by tribocorrosion processes induces a significantly

different macrophage proteome. Nevertheless, the covalent

anchoring of the nanocoating on the metallic material represents

an advantage over the physical adsorption of the coating.

•The immune response exerted by HA/GO covalently immobilized

on the metal surface was able to reduce the macrophage inflamma-

tory response, indicating that the covalent HA/GO nanocoating on

CoCr provides potential applications for metal implants and pros-

theses associated with metal-induced inflammation and degrada-

tion by wear-corrosion in vivo.

AUTHOR CONTRIBUTIONS

All authors contributed to the study conception and design. Material

preparation, data collection, and analysis were performed by all

authors. The first draft of the manuscript was written by L. Sánchez-

López and all authors commented on previous versions of the manu-

script. All authors read and approved the final manuscript.

ACKNOWLEDGMENTS

The authors wish to thank Francisco García Tabares (F.G.T.), Tamar San

HipólitoMarín(T.S.H.M.),andViviandelosRíos(V.d.l.R)oftheProteo-

mic and genomic facility at the Centro de Investigaciones Biológicas Mar-

garita Salas (CIB-MS, CSIC) for technical assistance in the preparation of

samples for proteomic analysis (F.G.T. and T.S.H.M) and technical sup-

port for the analysis of proteomic data (V.d.l.R). We acknowledge support

of the publication fee by the CSIC Open Access Publication Support Ini-

tiative through its Unit of Information Resources for Research (URICI).

FUNDING INFORMATION

This wok was supported by RTI2018-101506-B-C31 and

RTI2018-101506-B-C33 from the Ministerio de Ciencia, Innovación y

Universidades (MICIU/FEDER) in Spain. Author L. Sánchez-López has

received financial support from MICIU for the predoctoral contract

PRE2019-090122.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are openly available in

Digital CSIC at https://digital.csic.es/.

ORCID

L. Sánchez-López https://orcid.org/0000-0002-5065-9239

B. Chico https://orcid.org/0000-0001-8697-6298

Maria Cristina García-Alonso https://orcid.org/0000-0003-0275-

4626

Rosa M. Lozano https://orcid.org/0000-0003-2762-6938

16 S´

ANCHEZ-LÓPEZ ET AL.

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How to cite this article: Sánchez-López L, Chico B,

García-Alonso MC, Lozano RM. Macrophage proteomic

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graphene oxide on CoCr alloy in a tribocorrosive environment.

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