Tuning the surface immunomodulatory functions of polyetheretherketone for enhanced osseointegration

Journal Pre-proof Tuning the surface immunomodulatory functions of polyetheretherketone for enhanced osseointegration Ang Gao, Qing Liao, Lingxia Xie, Guomin Wang, Wei Zhang, Yuzheng Wu, Penghui Li, Min Guan, Haobo Pan, Liping Tong, Paul K. Chu, Huaiyu Wang PII:

S0142-9612(19)30741-0

DOI:

https://doi.org/10.1016/j.biomaterials.2019.119642

Reference:

JBMT 119642

To appear in:

Biomaterials

Received Date: 9 August 2019 Revised Date:

14 November 2019

Accepted Date: 19 November 2019

Please cite this article as: Gao A, Liao Q, Xie L, Wang G, Zhang W, Wu Y, Li P, Guan M, Pan H, Tong L, Chu PK, Wang H, Tuning the surface immunomodulatory functions of polyetheretherketone for enhanced osseointegration, Biomaterials (2019), doi: https://doi.org/10.1016/j.biomaterials.2019.119642. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

Tuning the Surface Immunomodulatory Functions of Polyetheretherketone for Enhanced Osseointegration

Ang Gaoa,b, Qing Liaoa, Lingxia Xiea, Guomin Wangb, Wei Zhangc, Yuzheng Wua, Penghui Lia,b, Min Guana, Haobo Pana, Liping Tonga,*, Paul K. Chub,*, Huaiyu Wanga,*

a

Institute of Biomedicine and Biotechnology, Shenzhen Institute of Advanced Technology,

Chinese Academy of Science, Shenzhen 518055, China b

Department of Physics, Department of Materials Science and Engineering, and Department

of Biomedical Engineering, City University of Hong Kong, Tat Chee Avenue, Hong Kong, China c

Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190,

China

* Corresponding authors E-mail addresses: [email protected] (Liping Tong), [email protected] (Paul K. Chu), [email protected] (Huaiyu Wang)

1

Abstract The adverse macrophage-mediated immune response elicited by the surface of polyetheretherketone (PEEK) is responsible for the formation of fibrous encapsulation and resulting inferior osseointegration of PEEK implants in the dental and orthopedic fields. Therefore, endowing the PEEK surface with immunomodulatory ability is an appealing strategy to enhance implant-bone integration.

Herein, a reliable and cost-effective method to

construct adherent films with tunable nanoporous structures on PEEK is described.

The

functionalized surface not only suppresses the acute inflammatory response of macrophages, but also provides a favorable milieu for osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs).

Whole genome expression analysis reveals that the

suppression effect arises from synergistic inhibition of focal adhesion, Toll-like receptor, and NOD-like receptor signaling pathways, as well as the attenuating loop through the JAK-STAT and TNF signaling pathways in macrophages.

Further in vivo studies confirm

that the functionalized surface induces less fibrous capsule formation and an improved bone regeneration.

The nanoporous films fabricated on PEEK harmonize the early

macrophage-mediated inflammatory response and subsequent hBMSCs-centered osteogenic functions consequently yielding superior osseointegration.

Keywords: immunomodulation, polyetheretherketone, osseointegration, surface modification, orthopedic implants

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1. Introduction

As promising alternative materials to conventional metals used in dental and orthopedic implants, polyetheretherketone (PEEK) possess superior characteristics including the excellent mechanical properties, good chemical stability and biocompatibility, as well as natural radiolucency [1,2].

Moreover, the elastic modulus of PEEK is less than those of

metals and comparable to that of cortical bone.

This attribute alleviates the stress shielding

effect plaguing metallic implants thus avoiding peri-implant bone resorption, loosening and even failure of implants [1,3].

However, PEEK implants still suffer from the inherent

bioinertness which severely impedes integration with bone tissues [4, 5].

Up to now,

considerable efforts have been made to functionalize the PEEK surface, for instance, sulfonation in concentrated sulfuric acid [6,7], deposition of silicate coatings by electron beam evaporation [8], and surface fluorination by plasma treatment [9].

Previous studies

have mainly focused on the osteoblastic lineage cells that enhance osteogenesis, but the host immune response crucial to the long-term survival and performance of implants has seldom been investigated and the related mechanisms are not well understood. After implantation of biomaterials in vivo, a cascade of inflammatory responses is initiated by immune cells [10].

Among the various immune cells, macrophages play a

central role in orchestrating the host immune response by releasing a repertoire of cytokines and growth factors to determine the fate of biomaterials [11,12].

Macrophages exhibit a

wide spectrum of polarization states which can be categorized into the pro-inflammatory M1 phenotype and anti-inflammatory, healing-related M2 phenotype [13].

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Generally,

macrophages persist in the pro-inflammatory M1 phenotype on the PEEK surface, leading to fusion of macrophages into multinucleated giant cells with increased release in fibrosis-enhancing cytokine.

These molecular signals thereafter contribute to the formation

of fibrous encapsulation and inferior osseointegration [14].

In contrast, transient and

appropriate inflammatory responses (M1 polarization) followed by the timely switch to the M2 phenotype, which releases cytokines and chemokines to resolve inflammation, recruit osteoprogenitor cells, and activate osteogenesis, will contribute to superior osseointegration of the implants [15].

In this respect, functionalization of the PEEK surface with the

immunomodulatory ability to provide a favorable osteo-immune environment is highly desirable. Several strategies have been proposed to modulate the inflammatory response elicited by implanted biomaterials [16].

For example, local delivery of interleukin-4 (IL-4)

cytokines from the surface has been applied to promote macrophage polarization towards the M2 phenotype [17].

The “marker of self” CD47 protein is covalently immobilized on the

surface to shield the implant from recognition by the host immune system [18].

However,

these strategies suffer from drawbacks such as the complicated preparation procedures, high cost of recombinant proteins, as well as short product shelf-life.

In fact, the physical

properties, particularly the surface topography of biomaterials, are increasingly recognized as a robust cue to regulate various cell functions.

Specifically, topographical cues have been

shown to modulate the immune response by selectively polarizing macrophages [19].

For

instance, the surface nanotopography mitigates the activation of macrophages, leading to decreased secretion of pro-inflammatory cytokines [20,21].

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On the other hand, surface

nanostructures with different features can be used to precisely guide the differentiation of bone mesenchymal stem cells into osteogenic lineage [22].

Many examples are available in

orthopedic and dental applications where titanium is widely used, as the nanotube arrays can be easily fabricated on titanium-based implants by electrochemical anodization [23,24]. With regard to PEEK, strategies such as sandblasting [25], porogen leaching [26], and plasma immersion ion implantation [27] have been proposed to enhance osseointegration via surface roughening.

However, PEEK is quite inert rendering surface functionalization difficult and

few studies have focused on the inflammatory responses. As an effective technique involving the electrostatic interactions between oppositely charged species [28], layer-by-layer (LBL) self-assembly has been adopted.

By dipping in

solutions of complementary polyelectrolytes successively, multilayered films with tailored chemical compositions and architectures can be constructed [29].

In fact, LBL films have

been fabricated on different types of biomaterials for drug delivery [30], gene therapy [31], anti-biofouling/antibacterial resistance [32, 33], as well as mediation of cellular functions [34]. In this study, this reliable and cost-effective technique is implemented to render PEEK with the immunomodulatory ability in additon to better osteogenesis and subsequent osseointegration. In this work, the LBL self-assembly technique is employed to fabricate adherent films with tunable nanoscale porosity on PEEK.

The in vitro acute inflammatory response of

macrophages and osteogenic performance of human bone marrow mesenchymal stem cells (hBMSCs) cultured on the modified surfaces are investigated systematically and in vivo experiments are carried out on rat femurs bone to verify the osseointegration ability.

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In

addition, whole genome expression analysis is performed to elucidate the underlying mechanisms why macrophages respond differently on the modified surface.

2. Materials and Methods

2.1 Materials

The poly(acrylic acid) solution (PAA; average molecular weight ~100,000, 35 wt.% in H2O), 2-mercaptoethanol, phorbol-12-myristate-13-acetate (PMA), lipopolysaccharides (from Escherichia coli), ascorbic acid 2 phosphate, β-glycerophosphate, and dexamethasone were purchased from Sigma-Aldrich.

Poly(allylamine hydrochloride) (PAH; average molecular

weight 120,000 ~ 200,000) was obtained from Alfa Aesar.

Water purified by the Milli-Q

water system (Millipore) was used in the experiments and the other reagents were obained from Sigma-Aldrich unless stated otherwise.

2.2 Surface preparation

Medical grade PEEK rods were supplied by GEHR Plastics Inc. (Mannheim, Germany) and cut into pieces with dimensions of Φ 15 mm × 2 mm.

The samples were ground with

sandpapers progressively up to 2000 grits, ultrasonically cleaned with acetone, ethanol, and deionized water, dried with nitrogen (N2) flow, and used as the PEEK control.

The pristine

PEEK underwent O2 plasma treatment at 150 W and 100 Pa for 2 minutes in a plasma cleaner (PT-10s, Potentlube, Shenzhen, China) and the sample was denoted O2.

The polyelectrolyte

multilayered films of PAH and PAA were deposited on the O2 samples by the LBL technique. 6

Both polyelectrolytes were prepared with a concentration of 0.01 M (based on the repeat unit of the polymer) in an aqueous solution.

The pH of PAH and PAA was adjusted with 0.1 M

sodium hydroxide (NaOH) or hydrochloric acid (HCl) to be 8.5 and 3.5, respectively.

The

LBL-assembled PAH/PAA films were fabricated by alternately dipping the O2 samples into aqueous PAH and PAA for 1 minute in each solution, followed by gentle agitation in deionized water each time.

The procedures were repeated until a total of 21 layers were

deposited with both the first and last layer being PAH.

Afterwards, the samples were rinsed

with the pH1.8 solution for 30 seconds followed by immersion in deionized water for 30 seconds or rinsed with a pH2.4 solution for 30 seconds.

The wet films were blown dry with

high-purity N2 and introduced into an oven at 180 °C for 2 hours.

The samples were denoted

as PH1.8 and PH2.4 and the processing procedures are schematically illustrated in Fig. 1.

Fig. 1.

Schematic illustration of the sample processing procedures.

treated with an O2 plasma for 2 minutes.

The pristine PEEK is

Afterwards, polyelectrolyte multilayers of PAH

and PAA are deposited by dipping the samples successively into the aqueous PAH and PAA for 1 minute each, followed by gentle agitation in deionized water each time.

7

The process

was repeated until a total of 21 layers are deposited and the first and last layers are PAH. Afterwards, different nanoporous topographies are constructed by either rinsing with the pH1.8 solution for 30 seconds followed by immersion in deionized water for 30 seconds or rinsing with the pH2.4 solution for 30 seconds. cross-linking at 180 °C for 2 hours.

The nanostructures are produced by thermal

The sample designations are underlined.

2.3 Sample characterization

The surface morphology was observed by field-emission scanning electron microscopy (FE-SEM; Supra 55, Zeiss, Germany) after the specimens were dried and sputter-coated with platinum.

The surface topography and roughness were determined by atomic force

microscopy (AFM; NanoScope V MultiMode 8 system, Bruker, Germany) performed on an area of 5 µm × 5 µm.

The surface chemical states were determined by X-ray photoelectron

spectroscopy (XPS; Escalab 250Xi, Thermo Fisher, USA).

The surface hydrophilicity was

measured with 5 µl of distilled water by the sessile drop method on the Rame'-Hart instrument (USA) under ambient conditions.

The surface zeta potential was measured on the Surpass

electrokinetic analyzer (Anton Parr, Austria).

A 0.001 M potassium chloride (KCl) solution

was used as the medium and the pH was adjusted in the range between 4.0 and 10.0 by HCl or NaOH.

In the electrical measurements, the electrolyte solution flowed on the sample surface

and the potential resulting from the motion of ions in the diffusion layer was determined according to the following Helmholtze-Smoluchowski equation: ζ =

   × ×  ×



8

where ζ is the zeta potential, dI/dP represents the slope of the streaming current versus pressure difference, η, ε, and ε0 denote the viscosity, dielectric constant, and vacuum permittivity of the electrolyte, respectively, and L and A are the length and cross-section of the streaming channel, respectively.

2.4 Nano-scratch test

The nano-scratch test was carried out to assess the adhesion strength of the multilayered films on different substrates.

The PEEK substrates were polished to a mirror finish before

film fabrication to alleviate the influence of surface roughness.

The test was performed on

the Keysight Nano Indenter G200 equipped with a cube corner indenter tip.

The indenter

was drawn across the sample surface using a ramp loading setup from 0 mN to 10 mN at a constant scratch velocity of 0.5 µm/s.

A total scratch length of 50 µm was generated and

three scratches were performed on each samples.

The fractures were observed by SEM after

sputter-coating with platinum.

2.5 Acute inflammation response of RAW264.7 2.5.1 Cell culture

The murine-derived macrophage cell line RAW264.7 was obtained from the American Type Culture Collection (ATCC) and used as the cell model to evaluate the acute inflammatory response to the samples.

The cells were maintained in the high-glucose

dulbecco's modified eagle medium (DMEM, Hyclone) supplemented with 10% fetal bovine serum (FBS; Corning) and 1% penicillin-streptomycin (Invitrogen) at 37 °C in humidified 5%

9

CO2 incubator.

Unless otherwise stated, the cells were seeded onto different samples with

an initial density of 1 × 105 cells/well on 24-well tissue culture plates (TCP) as holders.

The

TCP wells and TCP wells added with 10 ng/ml lipopolysaccharides in medium served as the negative and positive controls, respectively.

2.5.2 Cell morphology and proliferation After seeding 2 × 104 cells and incubating for 24 hours, the morphology of cells was examined by FE-SEM.

Prior to FE-SEM observation, the cells were fixed with 2.5%

glutaraldehyde, dehydrated through a graded ethanol series, critical-point dried, and sputter-coated with platinum.

Proliferation of RAW264.7 after culturing for 1 and 3 days

was determined by the CCK-8 (Donjindo, Japan) assay.

At each time point, the medium was

refreshed with the serum-free medium containing 10% CCK-8 and incubated at 37 °C for 4 hours.

Subsequently, the supernatant with a volume of 100 µl was collected and the

absorbance was determined on a microplate reader at a wavelength of 450 nm.

2.5.3 Polarization of macrophages

The expression of M1- and M2-related genes was analyzed by the real-time polymerase chain reaction (RT-PCR).

After RAW264.7 had been seeded on the different samples for 1,

3, and 5 days, the total RNA was extracted from the cultured cells by TRIzol Reagent (Invitrogen, USA).

The RNA was then reversely transcribed into complementary DNA

(cDNA) using a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher) following the manufacturer’s instruction.

The expressions of M1-related genes including inducible nitric

oxide synthase (iNOS), tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), interleukin 1 10

beta (IL-1β), and M2-related genes such as the mannose receptor (CD206), transforming growth factor beta 1 (TGF-β1), bone morphogenetic protein 2 (BMP-2), and vascular endothelial growth factor (VEGF) were analyzed by RT-PCR on the Bio-Rad CFX 96 Real-Time System using a mixture of SYBRGreen Realtime PCR Master Mix (Transgen Biotech, China), as well as the forward and reverse primers listed in Table S1.

The relative

gene expression levels were calculated using the 2-∆∆Ct method with the mean threshold cycle (Ct) values normalized to the house-keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

2.5.4 Determination of TNF-α secretion

The level of TNF-α released by RAW264.7 after culturing for 3 days was quantified by the enzyme-linked immunosorbent assay (ELISA) using a commercial kit (DuoSet, R&D systems, USA) according to the manufacturer’s instructions.

Briefly, a goat anti-mouse

TNF-α capture antibody was pre-coated on the bottom of a 96-well plate and the supernatant was pipetted into the wells and incubated at room temperature for 2 hours.

Each well was

sequentially incubated with the biotinylated goat anti-Mouse TNF-α detection antibody for 2 hours, horseradish-peroxidase conjugated streptavidin solution for 20 minutes, and substrate solution (1:1 mixture of H2O2 and tetramethylbenzidine) for 20 minutes.

After adding Stop

Solution (2 N of H2SO4), the absorbance was monitored on a microplate spectrophotometer at 450 nm.

The amount of released TNF-α was calculated according to the standard curve of

TNF-α and then normalized to the DNA content in the cell lysate as pg/µg DNA. The

DNA content

was

determined

11

by

adding

4’,6-Diamidino-2-phenylindole

dihydrochloride (DAPI), a specific and sensitive fluorescing DNA stain, to the cell lysate at a concentration of 0.5 µg/ml.

The fluorescent signals were detected by a spectrofluorometer

(MD Gemini EM, Molecular Devices, USA) with excitation at 365 nm and emission at 460 nm.

The concentration of DNA was calculated according to the standard curve established

by different concentrations of salmon DNA.

2.5.5 TRAP activity

To evaluate osteoclastic differentiation of RAW264.7 after culturing for 8 days, the intracellular tartrate-resistant acid phosphatase (TRAP) activity was determined quantitatively using a commercial TRAP Assay Kit (Beyotime, China) following the manufacturer‘s protocol.

The results were normalized to the total intracellular protein content determined

by the bicinchoninic acid (BCA) Protein Assay Kit (Pierce, Thermo Fisher, USA) and expressed as nmol/min/mg protein.

2.5.6 Determination of NO production and intracellular ROS content

The relative content of nitric oxide (NO) in the cell culture supernatant after incubating RAW264.7 on different samples for 3 days was quantitatively determined using the Total NO Assay Kit (Beyotime, China).

The intracellular reactive oxygen species (ROS) was

evaluated by the cell-permeant fluorescence probe dichlorofluorescin diacetate (DCFH-DA) provided by the ROS Assay Kit (Beyotime, China).

Briefly, the cells incubated for 3 days

were rinsed with PBS and incubated with 10 µM DCFH-DA for 30 minutes at 37 °C in darkness.

After diffusion into cells, DCFH-DA was deacetylated by cellular esterases to a

nonfluorescent compound which was later oxidized by intracellular ROS into a highly 12

fluorescent compound dichlorofluorescein.

The cells on the samples were detached, washed,

and resuspended in the PBS solution for flow cytometry analysis of over 10,000 cells on a BD Bioscience Canto II System.

The cells cultured on TCP were treated with Rosup (ROS

inducer provided by the ROS Assay Kit) as the positive control.

2.6 Whole genome expression analysis

The whole genome expression analysis was carried out after culturing the RAW264.7 cells on the PEEK and PH1.8 samples in triplicate for 3 days.

The total RNA was extracted

from the cells using Trizol reagent (Life Technologies, USA) followed by purification with the RNeasy Mini Kit (Qiagen, USA).

The double-stranded cDNA was synthesized from the

total RNA using primers containing a T7 promoter sequence and then converted into cRNA by in vitro transcription.

After purification, the cRNA was again reverse-transcribed into

sense-strand cDNA which was subsequently fragmented and hybridized to Mouse Clariom® S Array (Affymetrix, USA).

Afterwards, the gene chips were washed and stained in the

GeneChip Fluidics Station 450 (Affymetrix, USA).

All the arrays were scanned by the

GeneChip Scanner 3000 7G (Affymetrix, USA) equipped with the Affymetrix® GeneChip Command Console (Affymetrix, USA). protocols provided by Affymetrix.

All the procedures were performed according to the

The expression values were normalized using the Robust

Multiarray Average (RMA) algorithm and log2-transformed.

The differentially expressed

genes were defined by a fold change (FC) > 1.3 or < -1.3 and a false discovery rate-corrected p < 0.05 identified based on the student’s t-test for comparison of PH1.8 with the PEEK control.

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To elucidate the biological implications of the differentially expressed genes, gene ontology (GO) analysis was performed and the enriched terms were identified using the Fisher’s exact test.

Pathway analysis were also carried out to identify the significant

pathways of the differential genes according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.

The pathway-act network was constructed based on the KEGG

database to reveal the interaction among these significant enriched pathways.

The degree

centrality was defined as the number of links to upstream and downstream pathways, which were shown as in-degree and out-degree, respectively.

A higher degree indicates that the

pathway has a strong correlation with the other one implying a more important role in the signaling network.

The experimental design and data analysis in the whole genome

expression analysis are described in the flow chart in Fig. S1.

2.7 Effects of macrophage-conditioned medium on hBMSCs

The human monocytic THP-1 cells obtained from ATCC were cultured in the RPMI 1640

medium

(Hyclone)

supplemented

with

10%

FBS

2-mercaptoethanol, and 1% penicillin-streptomycin (Invitrogen).

(Corning),

0.05

mM

The cells were seeded on

differnt samples with an initial density of 3 × 105 cells/well using 24-well TCP as the holders and maintained under 5% CO2 at 37 °C.

The THP-1 monocytes were induced to

differentiate into macrophages after incubation for 48 h with 160 nM PMA in RPMI 1640 medium, followed by immersion in the PMA-free α-minimum essential medium (αMEM, Hyclone) for another day. surfaces and spread.

Upon differentiation, the suspended THP-1 cells attached to the

The TNF-α production levels in the culture supernatants were assessed

14

using a commercial ELISA Kit (Human TNF-Alpha valukine ELISA, R&D systems, USA) following the kit protocols. The monocyte-derived macrophages conditioned medium was prepared by maintaining the induced macrophages in αMEM (Hyclone) for 24 hours.

The conditioned medium was

collected, centrifuged, and frozen at -80 °C for further use.

Following incubation of

hBMSCs in the 24-well TCP for 4 days, the medium was replaced by the macrophage conditioned medium supplemented with 50 µg/ml ascorbic acid 2 phosphate, 10 mM β-glycerophosphate, and 10 nM dexamethasone for osteogenic induction.

The conditioned

medium was collected everyday and supplied to hBMSCs every 2 days for a total of 7 days. The intracellular alkaline phosphatase (ALP) activities in the cell lysis were quantitatively assayed on day 3 and day 7 using an ALP Assay Kit (Beyotime, China) according to the procedure recommended by the supplier.

The results were normalized to the total

intracellular protein content determined by the BCA Protein Assay Kit (Pierce, Thermo Fisher, USA) and expressed as nmol/min/mg protein.

2.8 Viability and osteogenic behavior of hBMSCs 2.8.1 Cell culture The hBMSCs obtained from ATCC were maintained in αMEM (Hyclone) supplemented with 10% FBS (Corning) and 1% penicillin-streptomycin (Invitrogen).

The

cells were incubated in a humidified atmosphere of 5% CO2 at 37 °C and the medium were refreshed every other day.

Unless stated otherwise, the hBMSCs were seeded on the

samples with a density of 2 × 104 cells/well using 24-well TCP as the holders.

15

For

osteogenic induction, the hBMSCs seeded onto different samples were allowed to proliferate for 3 days and the medium was further supplemented with 50 µg/ml ascorbic acid 2 phosphate, 10 mM β-glycerophosphate, and 10 nM dexamethasone.

2.8.2 Proliferation and osteogenic differentiation of hBMSCs

The cell morphology was observed by FE-SEM after culturing for 24 hours.

At least 12

randomly selected areas per sample were examined from three independent experiments and the representative images were shown.

All the images were analyzed by ImageJ software to

quantitatively compare the coverage of hBMSCs on the different samples.

Cell proliferation

was determined by the CCK-8 assay after culturing for 1, 3, and 5 days.

The ALP activities

were evaluated after osteogenic induction for 3, 7, and 14 days.

These assays were

performed per the protocols mentioned in the above sections.

The gene expressions of ALP,

type-1 collagen (COL), osteocalcin (OCN), and runt-related transcription factor 2 (Runx2) after osteogenic induction of hBMSCs for 7 and 14 days were analyzed by RT-PCR.

The

forward and reverse primers are listed in Table S2.

2.8.3 Collagen secretion

The sirius red staining assay was performed to evaluate collagen secretion from hBMSCs to the surfaces.

After osteogenic induction for 14 and 21 days, the cells were fixed with 4%

paraformaldehyde for 30 minutes and stained with Picro sirius red stain Kit (Solarbio, China), which contained 0.1% sirius red in a saturated picric acid solution, for 2 hours.

The

unbound stain was removed by rinsing with 0.1 M acetic acid thoroughly and observed by optical microscopy.

In the quantitative assay, the remaining stain on the samples was eluted 16

in a destaining solution (0.2 M NaOH/methanol 1:1) and the absorbance was measured at 570 nm on the microplate reader.

2.8.4 ECM mineralization

The degree of extracellular matrix (ECM) mineralization was evaluated by alizarin red S staining.

After osteogenic induction for 14 and 21 days, the cells on the surfaces were fixed

with 75% ethanol for 1 hour and stained with 1% alizarin red S solution (pH = 4.2, Solarbio, China) at room temperature for 30 minutes.

The unbound stain was removed by rinsing with

distilled water and observed by optical microscopy.

In the quantitative assay, the bound

stains were eluted with 500 µl/well of 10% cetylpyridinium chloride in 10 mM sodium phosphate and the OD values of the solution were measured at 570 nm.

2.9 In vivo evaluation 2.9.1 Surgical procedure

The animal procedures and experiments were approved by the Ethics Committee for Animal Research, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences.

The 12 weeks old female Sprague Dawley rats with a mean body weight of 350 g

were used in this study.

After general anesthesia, the distal aspects of the femurs were

carefully exposed via skin incision and muscle blunt dissection. mm was drilled longitudinally into the distal femurs.

A hole with a diameter of 2

PEEK, O2, and PH1.8 rods (Φ 2 mm ×

7 mm) were then randomly implanted in the bilateral femurs of the rats.

The contingent

gaps were padded with bone wax and the surgical sites were then carefully closed in layers.

17

8 weeks after surgical operation, the bilateral femurs containing cylindrical implants were harvested from the sacrificed rats and fixed in 4% polyformaldehyde.

2.9.2 Sequential Fluorescent Labeling

The new bone formation and mineralization process around the implant rods was evaluated by the polychrome sequential fluorescent labeling assay.

At 2, 4, and 6 weeks

post implantation, 3 different fluorochromes were intraperitoneally administered into the rats at a sequence of 30 mg/kg alizarin red S, 25 mg/kg tetracycline hydrochloride, and 20 mg/kg calcein, respectively.

2.9.3 Micro-CT evaluation

The newly formed bone around the implants was detected and imaged using Micro-CT (Quantum FX, PerkinElmer). program (SkyScan).

After scanning, the 3D images were reconstructed with CTvol

A hollow cylinder with the thickness of 120 µm from the implant

surface and length of middle 5 mm of the implant (1 mm each away from the implant ends) was defined as the volume of interest for analysis.

The bone volume/total volume (BV/TV),

trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp) were determined by 3D bone morphometric analysis.

2.9.4 Histological evaluation

After Micro-CT scanning, the fixed femur specimens were dehydrated in a graded series of ethanol, embedded in polymethylmetacrylate, and cut into longitudinal sections with a thickness of 100 µm using a Exakt system (310 CP, Exakt).

18

The sections were subsequently

ground and polished to a final thickness of about 50 µm for histological evaluation. Fluorescence observation was performed under a confocal laser scanning microscope (TCS SP8, Leica, Germany).

The excitation/emission wavelengths used to observe the chelating

fluorochromes were 543/620 nm, 405/575 nm and 488/520 nm for alizarin red S (red), tetracycline hydrochloride (yellow), and calcein (green), respectively.

Afterwards, the

sections were stained by Van Gieson’s picrofuchsin to visualize the bone-implant interfaces.

2.10 Statistical analysis

All the experiments were performed at least in triplicate and the results were presented as mean ± standard deviation.

One-way analysis of variance (ANOVA) followed by

Student-Newman-Keuls post hoc test was performed to determine the statistical significance. A difference at *p < 0.05 was considered to be significant and that at

**

p < 0.01 was

considered to be highly significant.

3. Results

3.1. Surface characterization

The PEEK specimens with different surfaces are prepared as shown in Fig. 1.

The

PEEK samples are first treated with an O2 plasma and then thin films with nanoporous structures are fabricated by the LBL technique followed by immersion in aqueous solutions and thermal cross-linking.

The samples are designated as O2 (O2 plasma treatment only),

PH1.8, and PH2.4 (1.8 and 2.4 are the solution pH values and please refer to the experimental 19

section for more details).

There is very little visual difference among the samples from the

top but the oblique views of PH1.8 and PH2.4 show light blue and yellow tinges, respectively (Fig. 2a).

According to the FE-SEM observation shown in Fig. 2b, scratches resulting from

sandpaper grinding are clear on the pristine PEEK and the O2 surface shows additional features arising from O2 plasma etching.

After deposition of LBL films, both PH1.8 and

PH2.4 exhibit homogeneous surfaces without scratches and the high-resolution images disclose nanoporous structures.

The pore size of PH1.8 is several tens of nanometers and

that of PH2.4 is in the range of 200 - 500 nm.

These topographical features are confirmed

by AFM (Fig. S2) and the surface roughness follows the order of O2 > PEEK > PH2.4 > PH1.8.

According to the cross-sectional SEM images in Fig. S3, the thickness of the PH1.8

film is about 120 nm and that of the PH2.4 film is about 200 nm. Fig. 2c shows the survey XPS spectra and the atomic percentages are presented in Fig. 2d.

Only the C 1s and O 1s peaks are detected from PEEK while the N 1s peak emerges

gradually from O2, PH1.8, and PH2.4.

O2 sample has the largest oxygen content and some

nitrogen is introduced because the active radicals generated during the O2 plasma treatment react with atmospheric oxygen and nitrogen after air exposure [35].

According to the fitted

high-resolution spectra (Fig. S4), the O 1s peak can be deconvoluted into three components at 531.2 eV, 532.2 eV, and 533.3 eV, corresponding to O=C, O-H, and O-C, respectively. particular, the larger O-H peak on O2 contributes to better wettability.

In

In the N 1s spectra,

the peak at 401 eV corresponding to amide bonds (N-C=O) is found from PH1.8 and PH2.4, confirming crosslinking between the carboxylate and amine groups after the 180 °C heat treatment.

Additionally, the spectra of PH1.8 and PH2.4 are quite similar and the similar

20

O/C and N/C ratios (Table S3) indicate that different post-treatment only affects the surface topography but not the surface chemistry. The surface hydrophilicity of the samples is evaluated by static contact angle measurement.

As shown in Fig. 2e, the water contact angle of pristine PEEK is 87.6° and

decreases to 32.0° after the O2 plasma treatment. A slight increase in the water contact angle is observed after deposition of the LBL films.

The changes in water wettability stem from

the variation of surface functional polar groups as shown by the XPS spectra and different topographies.

Since PH1.8 and PH2.4 show similar surface chemistry, the difference in

contact angles can be ascribed to the changes in the surface roughness. The zeta potentials are determined to investigate the electrical state on the samples, which significantly influence the biological responses. show descending zeta potentials with increasing pH.

As shown in Fig. 2f, the surfaces At a pH of 7.4 similar to that of the

physiological environment, the zeta potentials follow the order of PH1.8 > O2 > PH2.4 > PEEK.

21

Fig. 2.

Characterization of different samples: (a) Photographs taken from the top and an

oblique angle; (b) SEM images; (c) XPS survey spectra; (d) Atomic ratios; (e) Water contact angles; (f) Zeta potentials versus pH plots.

3.2 Film adhesion

The film adhesion strength is evaluated by nano-scratch tests by drawing a diamond indenter across the thin films under increasing normal loads. SEM images of the 50 µm scratch tracks.

Fig. 3 presents the panoramic

Without the pre-treatment of O2 plasma, the films

are readily damaged and delaminated once the indenter comes into contact with the surface and moves laterally (Figs. 3a and 3c).

A large area is scraped off from the film by the

moving stylus along the entire length of the scratch track.

In addition, cracks are visible

along the edges of the scratch track and they propagate a considerable distance to either side of the track.

At the end of the track, buckling and pile-up occur ahead of the indenter.

these observations indicate poor film adhesion.

All

As for PH1.8 and PH2.4 surfaces, plastic

deformation (plowing) is observed at a small load (Fig. 3b and 3d).

Delamination then

occurs after the indenter moves about 4.33 µm and 3.18 µm on PH1.8 and PH2.4 (marked by the red dotted lines), corresponding to normal loads of 0.87 mN and 0.63 mN, respectively. However, spallation after scratching is not conspicuous and generally confined within the scratch track and the edges of the tracks on PH1.8 and PH2.4 maintain the integrity without distortion.

No cracks are visible along the edges of the tracks on PH1.8, whereas minor

cracks without propagation are observed from PH2.4. 22

The plowed regions can also be found

from the track of PH1.8, indicating that the adhesion strength is even better than the cohesive strength of the film.

Fig. 3.

Panoramic SEM images of the nanoscratch test tracks after loading with the sliding

direction from left to right: (a) PH1.8 and (c) PH2.4 films fabricated on PEEK without the pretreatment of O2 plasma; (b) PH1.8 and (d) PH2.4 films fabricated on PEEK following the procedures shown in Fig. 1.

23

3.3 Acute inflammatory response of RAW264.7

The murine macrophage cell line RAW264.7 is used to evaluate the acute inflammatory response on the different samples and the 24-well TCP, and TCP with 10 ng/ml lipopolysaccharides supplemented in the culture medium (denoted as LPS) are employed as the negative and positive controls, respectively. h is examined by FE-SEM (Fig. 4a).

The cell morphology after incubation for 24

The cells on PEEK have a flattened appearance and the

cytoplasmic protrusions spread across the surface.

Spindle-shaped macrophages with

numerous protrusions are observed from O2 sample, whereas those on PH1.8 and PH2.4 display a normal spherical shape and low spreading with only a few pseudopodia.

For

comparison, the macrophages on the TCP surface have a round appearance with weak filopodial attachments, whereas under stimulation of lipopolysaccharides, they exhibit an irregular shape and extended pseudopodia. Proliferation of macrophages after culturing on for 1 and 3 days is assessed by the CCK-8 assay.

As shown in Fig. 4b, the PEEK group exhibits the highest rate of cell

proliferation at both time points.

The O2 surface slightly inhibits proliferation after the first

day but the difference disappears after 3 days.

Significantly lower cell proliferation is

observed from PH1.8 and PH2.4 than PEEK, while the difference is not significant in comparison with the negative TCP control. To evaluate the effects of different surfaces on macrophage polarization, the gene expression of function-associated markers of M1- and M2-type macrophages after culturing for 1, 3, and 5 days is determined by RT-PCR.

The results are presented as a heat map in

Fig. 4c with the original quantification data provided in Fig. S5. 24

The fold changes in gene

expressions are normalized to those of PEEK and the TCP and LPS groups are shown for reference.

The iNOS and pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β are

referred to as the M1-related genes, while the CD206, and anti-inflammatory growth factors including TGF-β1, BMP-2, and VEGF are considered as the M2-related genes.

All four

kinds of M1 gene expressions, except IL-6 at day 1, are notably down-regulated on PH1.8 and PH2.4 in the first 3 days.

The fold changes in the M1 gene expressions are comparable or

even lower than those of TCP.

When the culturing time is extended to 5 days, the PH1.8 and

PH2.4 groups still show significant down-regulation of the iNOS and TNF-α levels.

These

results indicate that the functionalized surfaces inhibit early pro-inflammatory M1 polarization of macrophages.

With regard to the M2-related genes, the expression levels of

the canonical M2 marker CD206 are up-regulated on the modified surfaces at all 3 time points. In particular, after culturing for 5 days, the gene expression of CD206 on PH1.8 is four times higher than that on PEEK.

The expression of other M2-related genes like TGF-β1, BMP-2,

and VEGF are also significantly up-regulated after culturing the macrophages on PH1.8 and PH2.4 for 5 days, implying that the functionalized surfaces not only facilitate the switch of macrophages to the M2 phenotype, but also induce secretion of anti-inflammatory growth factors as the culture time is increased.

The results demonstrate that polarization of

macrophages on samples is modulated in a time-dependent manne and the fabricated films, particularly those on PH1.8, possess the ability to inhibit early pro-inflammatory M1 polarization and promote the expression of M2-related gene with prolonged culture time. Fig. 4d shows the secretion of extracellular pro-inflammatory cytokines TNF-α determined by ELISA after culturing the macrophages for 3 days.

25

As expected, release of

TNF-α is greatly suppressed on PH1.8 and PH2.4 and there is no significant difference in TNF-α secretion between the PH1.8 group and TCP control.

The other cytokines such as

pro-inflammatory cytokines IL-6 and IL-1β as well as the anti-inflammatory cytokine IL-4 and interleukin 10 (IL-10) are further measured but their concentrations in the supernatant are below the detection limit (results not shown).

The NO production by macrophages after 3

days culture is also evaluated and the results show that the NO contents are lower on all the modified samples (Fig. S6).

The ROS results provided by flow cytometric analysis reveal

that the proportion of ROS positive cells decreases from 35.7% on PEEK to 24.4% on O2 sample, while PH1.8 and PH2.4 further reduce the level to 15.7% and 19.6%, respectively (Fig. S7).

The osteoclastogenic differentiation of RAW264.7 cells is detemined by the

TRAP assay which shows that osteoclast formation on PH1.8 and PH2.4 is suppressed (Fig. 4e).

Interestingly, the TCP group does not show significant inhibitory effects on the TRAP

activity in comparison with the PEEK group.

26

Fig. 4.

Acute inflammatory response of RAW264.7 on different samples: (a) SEM images

showing the cell morphology after culturing for 24 hours; (b) Cell proliferation determined by the CCK-8 assay after incubation for 1 and 3 days; (c) Heat map depicting the expression of the M1- and M2-related genes based on the RT-PCR results after incubation for 1, 3, and 5 days;

(d) TNF-α level in the supernatant after culturing for 3 days; (e) TRAP activity after

culturing for 8 days.

p < 0.05, **p < 0.01.

*

3.4 Whole genome expression analysis

To elucidate the mechanisms why macrophages respond differently on the different surfaces, the whole genome expression analysis is performed after the macrophages are cultured on the pristine PEEK and PH1.8 for 3 days. 27

Of the total 22,206 genes analyzed,

2,815 differentially expressed genes are identified between PEEK and PH1.8 (Fig. S8). Then GO analysis is performed to characterize the physiological significance of these changes. The primary down-regulated Biological Process category involves terms like “cell adhesion”, “regulation of cell proliferation”, “innate immune response”, “inflammatory response”, “immune system process”, and “positive regulation of ROS metabolic process” (Table S4), which are in full agreement with the observed cellular response in vitro.

Meanwhile, the

Cellular Component category, which indicates the locations in the macrophages where the regulated gene products perform the functions [36], are mainly associated with the plasma membranes (Table S5).

The regulated genes are also enriched into the pathways according

to the KEGG database (Table S6).

The pathway-act network (Fig. S9) is subsequently

constructed and the top 10 signaling pathways with high degree centrality are listed in Table S7.

The most significant interactions are related to the mitogen-activated protein kinase

(MAPK) and phosphoinositide 3-kinase/protein kinase B (PI3K-Akt) signaling pathways.

3.5 Osteogenic differentiation of hBMSCs induced by THP-1 macrophages

To facilitate homologous interactions, human monocytic THP-1 cells instead of RAW264.7 are adopted to study the effects of the macrophage conditioned medium on osteogenic differentiation of hBMSCs.

The THP-1 derived macrophages in the resting state

are obtained following the procedures illustrated in Fig. 5a.

The proinflammatory cytokine

TNF-α released into the culture medium by induced macrophages is examined using ELISA. Fig. 5c shows that secretion of TNF-α on all the modified samples is suppressed in comparison with the PEEK control.

The magnitude of the suppression effects is similar to

28

that of RAW264.7 as shown in Fig. 4d, indicating the consistency of macrophage response to the various samples between THP-1 derived macrophages and RAW264.7.

The conditioned

medium from the THP-1 derived macrophages is collected daily and supplied to hBMSCs every two days following the protocols described in Fig. 5b.

Subsequently, the ALP activity,

an early osteogenic differentiation marker of hBMSCs, is examined.

As shown in Fig. 5d,

the TCP group shows the highest ALP activity, followed by the PH1.8 and PH2.4 groups, with inferior ALP levels detected from the PEEK and O2 samples.

Hence, the macrophages

in contact with PH1.8 and PH2.4 create a more favorable microenvironment for osteogenic differentiation of hBMSCs than the PEEK and O2 groups.

29

Fig. 5.

Experimental design and analysis strategy: (a) Differentiation of THP-1 in the

macrophages; (b) Effects of the macrophage-conditioned medium on osteogenic differentiation of hBMSCs; (c) TNF-α secretion level of THP-1 derived macrophages in supernatant;

(d)

Normalized

ALP

activity

macrophage-conditioned medium for 3 and 7 days.

of

hBMSCs

cultured

in

the

p < 0.05, **p < 0.01.

*

3.6 Osteogenesis of hBMSCs

In vitro osteogenesis of hBMSCs cultured on the different samples is evaluated in terms of the cell morphology, proliferation, osteogenic gene expression, ALP activity, collagen secretion, as well as ECM mineralization.

As shown in Fig. 6a, slender cells with an

elongated shape are observed from the PEEK sample indicative of poor spreading.

The cells

cultured on O2 sample show better spreading but still attach poorly with few pseudopodia extensions.

In comparison, the cells on PH1.8 and PH2.4 are much more extended and have

a polygonal geometry.

The images with higher magnification disclose abundant filopodia

extending from the leading edges, indicating that they attach closely to the samples. According to the statistical analysis, the coverage of hBMSCs on PH1.8 and PH2.4 is significantly larger than that on PEEK and O2 (Fig. S10).

The time-dependent cell

proliferation is measured by the CCK-8 assay as shown in Fig. 6b.

There is no obvious

difference among the groups at day 1, but after 3 days, all the modified samples and TCP control show significantly better cell proliferation than PEEK. except that the O2 group falls behind.

This trend coninues to day 5

The hBMSCs cultured on the various samples after

30

osteogenic induction for 7 and 14 days are assessed for the expression of osteogenic genes including ALP, COL, OCN, and Runx2 as shown in Fig. S11.

Generally, the PH1.8 group

shows higher expressions of all the genes at both time points.

Subsequently, the ALP

activity of hBMSCs is evaluated after osteogenic induction for 3, 7, and 14 days.

Fig. 6c

shows that compared with PEEK, the O2 group slightly up-regulates the ALP activities, which are further enhanced on PH1.8 and PH2.4. day 14 is even higher than that of TCP.

The ALP activity of hBMSCs on PH1.8 at

Collagen secretion of the hBMSCs and the

corresponding staining images are displayed in Fig. 6d and Fig. 6f, respectively.

The

secreted collagen is denser on PH1.8 and PH2.4 than that on PEEK and O2 at both time points, and it is even comparable to the collagen secretion level of TCP.

Meanwhile, the

results of ECM mineralization based on alizarin red S staining shows a similar trend.

Fig. 6e

and Fig. 6g show more pronounced mineralized nodules on PH1.8 and PH2.4 than the PEEK and O2 groups and there is no statistical significance in ECM mineralization between the PH1.8 group and TCP control.

31

Fig. 6.

Osteogenic ability of hBMSCs cultured on different samples:

(a) SEM images (200

×, 500 ×, and 2000 × magnifications) showing the cell morphology after culturing for 24 hours with the cells at higher magnification shown with a pseudo green color; (b) Cell proliferation determined by the CCK-8 assay after incubation for 1, 3, and 5 days; (c) Normalized ALP activity after osteogenic induction for 3, 7, and 14 days; (d) Quantitative results and (f) Corresponding images of collagen secretion stained by sirius red after osteogenic induction for 14 and 21 days; (e) Quantitative results and (g) Images of extracellular matrix mineralization stained by alizarin red S after osteogenic induction for 14 and 21 days.

p < 0.05, **p < 0.01.

*

32

3.7 In vivo assays of osseointegration

Based on the superior performance observed in vitro, PH1.8 is selected as the representative sample for in vivo evaluation.

The reconstructed transverse, sagittal, coronal,

and 3D micro-CT images demonstrate that thin bone layers are formed around the implants within cancellous bone after implantation for 8 weeks.

The bone volume is visually larger

surrounding the PH1.8 implants (Fig. 7a) and quantitative analysis shows that both the bone volume ratio (Fig. 7b) and trabecular numbers (Fig. 7d) are highest for the PH1.8 group.

In

addition, although there is no significant difference in the trabecular thickness among the groups (Fig. 7c), a notable decrease in trabecular separation (Fig. 7e) which corresponds to the distance between adjacent trabecula is observed from the PH1.8 group.

The results

indicate that the quality and quantity of newly formed bone surrounding the PH1.8 implants are drastically better than those of the PEEK and O2 groups. The process of new bone formation surrounding the three kinds of implants is monitored by sequential fluorescent labeling.

As shown in Figs. 7f and 7g, the fluorescent

areas follow the order of PH1.8 (4.20 ± 0.46%) > O2 (2.33 ± 0.55%) ≈ PEEK (2.26 ± 0.36%), indicating that PH1.8 is more effective in facilicating bone deposition and remodeling than the other samples at each time point.

Fig. 7h shows the histological sections of different

experimental groups after being stained by van Gieson’s Picrofuchsin. layers can be observed from the peri-implant.

Newly formed bone

However, fibrous connective tissues are

observed from the bone-implant interface with PEEK (red arrows) and the bone layer in contact with O2 is discontinuous (yellow arrows).

In contrast, the PH1.8 group shows a

great degree of continuous bone apposition which bonds directly with the surface (white 33

arrows) and a few fibrous connective tissues (red arrow) are observed.

According to the

quantitative analysis shown in Fig. 7i, the PH1.8 group has a significantly higher percentage of bone-implant contact than the PEEK and O2 groups, thereby corroborating the superior ability in promoting osseointegration.

Fig. 7.

In vivo osseointegration of different implants: (a) Reconstructed transverse, sagittal,

coronal, and 3D Micro-CT images; (b-e) Quantitative analysis of Micro-CT data including (b) BV/TV, (c) Tb.Th, (d) Tb.N and (e) Tb.Sp; (f) Sequential fluorescent labeling of new bone formation [Red, yellow, and green represent labeling by alizarin red S (week 2), tetracycline hydrochloride (week 4), and calcein (week 6), respectively]; (g) Histogram of percentages of the areas of fluorochromes stained bone; (h) Histological observations of the tissue sections stained with Van Gieson’s picrofuchsin [Images at 100 × magnification are taken from yellow rectangular marked areas of images at 40 × magnification, white arrows mark the direct 34

contact between implants and new bones, and red and yellow arrows indicate the area covered with fibrous tissue and discontinuous bone layer, respectively]; (i) Histogram of percentages of the bone-implant contact ratios.

4. Discussion

PEEK offers a set of characteristics superior to metallic implants in dental and orthopedic fields.

However, its wide application is limited by its inert surface properties

which elicit detrimental host immune response resulting in the formation of fibrous encapsulation.

Therefore, surface modification of PEEK to endow it with the

immunomodulatory ability to provide a favorable environment for implant-bone integration is of great interest.

LBL self-assembly is selected here as it is a versatile technique for

fabrication of multilayered films even on substrates with a complex geometry [28].

In fact,

LBL self-assembly has been adopted in various biomedical applications [29] such as drug delivery [30], tissue engineering [37], and biosensors [38].

In this study, the LBL technique

is utilized to develop a reliable and cost-effective method to construct well-adhered films on PEEK with predesigned immunomodulatory ability. Prior to film deposition, the surface of pristine PEEK is treated by an O2 plasma to produce better surface wettability (Fig. 2e) and negative surface charges (Fig. 2f).

Then

LBL assembled multilayers are deposited by alternately dipping the pre-treated substrates into weak polyelectrolyte solutions of PAH and PAA and ionization is pH-dependent thus facilitating subsequent acid-induced formation of the nanoporous structures. 35

In order to

improve the processing efficiency, the dipping time for each layer is only 1 minute compared to 15 minutes or even longer per step reported in the literature [39,40].

The shorter dipping

time also suppresses interlayer diffusion to reduce the film thickness [41]. After deposition, the scratches on pristine PEEK are concealed giving rise to a smooth surface which is featureless under SEM observation (Fig. S12).

However, from the perspective of cellular

response, biomaterials with the nanoscale surface topography can better guide cell functions by resembling the natural ECM for cell residing and interaction [42]. film with porous structures is desirable.

In this regard, the LBL

A multilayered film is assembled by electrostatic

attractions between the cationic NH4+ of PAH chains and anionic COO- of PAA chains.

If

the deposited films are exposed to acid solutions, the interchain ionic bonds will be partially cleaved due to the protonation of carboxylate groups and charge repulsion among the free and positively charged amine groups.

Consequently, rearrangement of the polymer chains is

induced by the post-treatments of acid solutions allowing dramatic swelling of the multilayers forming pores in the LBL films [43].

In our study, the LBL films soaked in solutions with

pH ranging from 2.6 to 2.0 for 30 seconds show surface holes and pits, whereas the porous structure is homogenous after the treatment at a pH of 2.4 (Fig. S13). lower, the multilayers are completely dissolved (Fig. S13).

At a pH of 1.6 or

Although the LBL films exposed

to the pH 1.8 solution are quite flat, nanoscale pores emerge after immersion in water for 30 seconds and immediate drying (Fig. S14).

Formation of pores can be ascribed to the

re-protonation of carboxylate groups and reformation of electrostatic interactions in the films leading to recompression of the swollen films. Apatially inhomogeneous rejection of water during the compression process gives rise to the nanoporous structure after the films are dried

36

[44].

In conclusion, the emergence of nanopores can be attributed to the swelling and

deswelling of the LBL films due to protonation and deprotonation of the PAA acid groups [45].

The changes in the surface morphology after the post-treatment are also reflected by

the substantial changes in interference color in Fig. 2a. As aforementioned, the electrostatic interactions between polyelectrolytes in the LBL films are susceptible to the variation of ambient conditions such as the pH, ionic strength, and temperature.

For instance, the nanoporous structures in the LBL films vanish after

immersion in PBS for 12 hours (Fig. S15).

Therefore, in order to preserve the desirable

morphological features, the films treated at pH of 1.8 and 2.4 are thermally cross-linked at 180 °C for 2 hours to form amide bonds between the carboxylate and amine groups [46]. The amide bonds formed during the thermal treatment are confirmed by XPS (Fig. S4) and the porous structures are preserved (Fig. S16).

The long-term durability of the thermally

treated LBL films is corroborated by that little morphological change is observed after immersion in PBS for 7 days (Fig. S15).

Moreover, as a high-performance thermoplastic

polymer, PEEK has been reported to endure a temperature of up to 250 °C [1] and so the transient thermal treatment at 180 °C is not expected to degrade the mechanical properties of PEEK. Besides the stability, the adhesion strength of LBL films is a critical factor as the improvement of osseointegration will be meaningless if the film delaminates from the PEEK substrate.

Furthermore, film failure under physiological stress will release fragments and

debris to activate macrophages in the peri-prosthetic tissues and produce possible chronic inflammation and osteoclastogenesis [47, 48] and osteoresorption predominates over

37

peri-implant osteogenesis to increase the incidence of bone loss (osteolysis) and aseptic loosening [49].

In this respect, the O2 plasma pre-treatment yields much stronger film

adhesion and superior scratch and abrasion resistance (Fig. 3).

The plasma treatment not

only removes contaminants from the sample surface, but also leaves active sites at the substrate surface [50], either for further oxidation in the ambient atmoshpere (Fig. S3) or forming covalent bonds with the polyelectrolytes during LBL assembly [51]. adhesion strength of fabricated films is significantly improved.

As a result, the

In addition, the abrasion

resistance of PH1.8 is better than that of PH2.4 (Fig. 3), which can be ascribed to their difference in surface roughness (Fig. S2) [52].

All in all, the LBL films fabricated following

the procedures shown in Fig. 1, particularly the PH1.8 group, have superior adhesion strength as well as scratch and abrasion resistance. IMacrophages play a central role in orchestrating the innate immune response against implanted biomaterials [11].

Depending on the specific cytokine patterns and local

microenvironment, macrophages exhibit high levels of functional and phenotypic plasticity to regulate a complex cascade of events throughout the implant lifetime, including the host inflammatory response, wound healing process, as well as long-term integration and survival of

the

implants

[14].

Therefore,

designing

orthopedic

biomaterials

with

a

immunomodulatory surface is an innovative strategy to strengthen osseointegration of bone implants [20,53].

According to the macrophage morphology, proliferation, gene expression,

TNF-α secretion, NO production, and intracellular ROS content, PH1.8 and PH2.4 are effective in inhibiting the acute inflammatory response of macrophages.

As no exogenous

regulation factors are released, the different performance of macrophages can be ascribed

38

solely to the variation in the surface physicochemical properties which affect the amount, type, conformation, and orientation of proteins nonspecifically adsorbed on the surface [54].

The

anchored proteins are recognized by a repertoire of pattern recognition receptors expressed on the macrophage membrane followed by initiation of the innate immune response [55].

To

elucidate the underlying molecular mechanism, whole-genome expression and enriched signaling pathways are analyzed. Integrins are the primary adhesion receptors involved in macrophage adhesion and cytoskeletal remodeling [56] and they also mediate the inflammatory responses by activating signal transduction through focal adhesion and PI3K-Akt signaling cascades [57]. According to the whole-genome expression analysis, the lower expressions of integrin (ITG) subunits (α4, α5, αM, αX, αD, β2, and β7) and adhesion complexes (actinin, filamin, paxillin) on PH1.8 can explain why macrophages show a normal round shape on PH1.8 and cells with an elongated and spread morphology are observed from PEEK (Fig. 4a).

These

differences also translate into substantial downstream effects such as reduced proliferation of macrophages on PH1.8 (Fig. 4b).

In addition, the Toll-like receptor (TLR) signaling

pathway is downregulated on PH1.8 (Table S6).

It has been reported that macrophages

recognize synthetic biomaterials in a similar way as gram-negative bacteria or lipopolysaccharides via TLR [58-60].

PH1.8 is found to suppress the expression of

lymphocyte antigen 96 (MD-2), which is necessary for the response of TLR-4 to LPS, thus negatively regulating the TLR signaling pathway as well as downstream signaling cascades [61].

Besides integrins and TLR, the NOD-like receptor (NLR) signaling pathway is

involved in the recognition of PH1.8 surface (Table S6), although NLR is a cytoplasmic

39

receptor that senses intracellular cytosolic pathogens [62].

Downregulation of the NLR

signaling pathway is realized mainly by attenuating the activation of receptor-interacting protein 2 (RIP2). The attenuated focal adhesion, TLR, and NLR signaling pathways ultimately converge into downstream MAPK and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling cascades, resulting in reduced transcription of inflammation-related genes such as inducible nitric oxide synthase (NOS2), prostaglandin-endoperoxide synthase 2 (COX2), C-C motif chemokine ligand 3/4 (MIP-1α/β), and colony stimulating factor 1/2 (CSF1/2).

In particular, the gene expression of proinflammatory cytokines TNF-α is

suppressed on PH1.8 (Fig. 4c) and decreased secretion is confirmed by the ELISA results (Fig. 4d).

TNF-α together with other pro-inflammatory cytokines in turn triggers transient

activation of the TNF-α signaling pathway and JAK-STAT signaling pathway, which are also found to be downregulated on PH1.8 (Table S6 and S7) [63,64].

Therefore, it is reasonable

to hypothesize that the autocrine response of macrophage on PH1.8 falls into an attenuating feedback loop that gradually silences the acute inflammatory response.

This notion is

partially supported by the time-dependent changes in the expression of M1/M2 related genes (Fig. 4c).

The mechanism by which PH1.8 alleviates the acute inflammatory response is

illustrated in Fig. 8.

40

Fig. 8.

Schematic illustration of the key signaling transduction cascades on the PH1.8

sample in comparison with pristine PEEK.

The downregulated genes are highlighted in

green, and upregulated genes are highlighted in red and differentially expressed genes are screened by the whole genome expression analysis.

The signaling pathways are constructed

based on the KEGG database.

After resolving the acute inflammatory response at early stage, the LBL surfaces promote polarization of the macrophages into the M2 phenotype (Fig. 4c), which further possesses the ability to enhance osteogenic differentiation of hBMSCs by secreting a series of cytokines [65].

Therefore, the ALP activity of hBMSCs is enhanced after they are cultured

in the macrophage-conditioned medium collected from the LBL surfaces (Fig. 5d). Furthermore, macrophages fuse together and differentiate into osteoclasts as the culture time is increased.

As shown in Fig. 4e, the LBL surfaces exhibit inhibitory effects on

osteoclastogenesis thereby avoiding excessive bone resorption in early phase of bone 41

remolding [66].

All in all, the immune response of macrophages mediated by the LBL

surfaces synergistically contributes to upregulated osteogenesis and downregulated osteoclastogenesis which shows great potentialino facilitating peri-implant osseointegration. HBMSCs are another type of cells that play a critical role in osseointegration. Compared with the pristine PEEK and O2 samples, the LBL films provide more favorable milieus for hBMSCs adhesion, spreading (Fig. 6a), and proliferation (Fig. 6b).

The

hBMSCs cultured on the LBL films show a tendency of osteogenic differentiation as indicated by the enhanced ALP activity (Fig. 6c), improved collagen secretion and ECM mineralization (Fig. 6d-g), as well as numerous up-regulated osteogenic genes (ALP, COL, OCN, and RUNX2) (Fig. S7).

Besides the surface chemistry, these improvements can be

mainly ascribed to the porous structures constructed on the LBL films [67].

Osteoblasts

prefers rougher surfaces [68-70] and the topographical cues of orthopedic implants can be tailored to improve osseointegration [71,72].

Moreover, PH1.8 shows a superior osteogenic

ability than PH2.4 (Figs. 6c and 6e) and given the similar chemical composition, the difference in surface topography appears to be the main reason.

In fact, it has been reported

that surface nanoporous structures with a disordered arrangement, like that on PH1.8, deliver better performance than micrometer-scale features in promoting stem cell differentiation towards the osteogenic lineage [73,74].

Here, PEEK, O2, and PH1.8 are chosen for in vivo

evaluation of osseointegration and micro-CT, sequential fluorescent labeling, and histological analysis corroborate the in vitro findings revealing superior new bone formation without the presence of fibrous tissues for the PH1.8 group.

42

The comprehensive results provide

unambiguous evidence that LBL films with nanoporous structures provide a more promising milieu than pristine PEEK for better peri-implant osseointegration.

5. Conclusion

A reliable and cost-effective method to fabricate adherent films with tunable structures on PEEK is described.

The simplicity and reliability of this technique allows commercial

production on a large scale even for biomedical implants with a complex shape.

The in vitro

cellular response and in vivo osseointegration are investigated systematically.

The results

indicate that the LBL films, especially PH1.8 with a nanoporous structure, endow PEEK with immunomodulatory capability to inhibit the early acute inflammatory response of macrophages and induce macrophages to create a favorable microenvironment for osteogenesis.

This temporal response of macrophages is consistent with that in the natural

healing process of bone fracture.

In vivo results corroborate the improved osseointegration

on the functionalized PEEK sample.

Our findings provide not only new knowledge about

the regulation of the host immune response on biomaterials, but also insights into the design of high-performance implants for orthopedic applications.

Acknowledgements

The authors acknowledge financial support from the National Natural Science Foundation of China (No. 31922040), Shenzhen Science and Technology Research Funding 43

(No. JCYJ20180507182637685), Youth Innovation Promotion Association of Chinese Academy of Sciences (No. 2017416), Youth Talents of Guangdong Science and Technology Innovation (No. 2015TQ01C534), Leading Talents of Guangdong Province Program (No. 00201520), Shenzhen Peacock Program (No. KQTD2016030111500545), China Postdoctoral Science Foundation (No. 2018M631007), Science and Technology Service Network Initiative of Chinese Academy of Sciences (No. KFJ-STS-QYZX-035), Guangdong - Hong Kong Technology Cooperation Funding Scheme (TCFS) (No. GHP/085/18SZ), as well as Hong Kong Research Grants Council (RGC) (No. CityU 11205617).

Appendix A. Supplementary Data

Supplementary data related to this article can be found in the document named Supplementary Information.

Data availability

The data are available from the corresponding author on reasonable request.

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All authors have read and approved the manuscript and it has not been submitted to another journal for publication. We declare that there is no conflict of interest.

Paul K Chu