Mussel-Inspired Polydopamine Coating: A General Strategy To

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Surfaces, Interfaces, and Applications

Mussel Inspired Polydopamine Coating: a General Strategy to Enhance Osteogenic Differentiation and Osseointegration for Diverse Implants Hui Wang, Chu-Cheng Lin, Xinran Zhang, Kaili Lin, Xudong Wang, and Steve G.F. Shen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b21558 • Publication Date (Web): 28 Jan 2019 Downloaded from http://pubs.acs.org on February 5, 2019

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ACS Applied Materials & Interfaces

Mussel Inspired Polydopamine Coating: a General Strategy to Enhance Osteogenic Differentiation and Osseointegration for Diverse Implants

Hui Wanga,b,‡, Chucheng Lin,c,‡ Xinran Zhangb, Kaili Lina,*, Xudong Wanga,*, Steve Guofang Shena,*

aDepartment

of Oral and Cranio-maxillofacial Science, Shanghai Ninth People’s

Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology & Shanghai Research Institute of Stomatology, Shanghai 200011, China. bSchool & Hospital of Stomatology, Tongji University, Shanghai Engineering Research

Center of Tooth Restoration and Regeneration, Shanghai 200072, China. cShanghai

Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050,

China.

‡These

authors contributed equally.

*Corresponding author: Tel: 86-21-23272699; Fax: 86-21-63136856. E-mail: [email protected]

(K.

Lin);

[email protected]

(X.

Wang);

[email protected] (S.G. Shen).

Keywords: Polydopamine, Coating, Osteogenic Differentiation, Osseointegration, Cell Signaling Pathway 1

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Abstract Surface modifications play an important role in endowing implant surface with excellent biocompatibility and bioactivity. Among the bioinspired surface modifications, the mussel inspired polydopamine (PDA) has aroused great interest of researchers. Herein, we fabricated PDA on diverse implant surfaces, including biopolymer, biometal and bioceramic. Then the effects of PDA-coating on cell responsive behaviors in vitro and bone formation capacity in vivo were evaluated in details. The results showed that PDA-coating was fabricated on diverse samples surface successfully, which could significantly improve the hydrophilicity of different material surfaces. Furthermore, the results indicated that PDA-coating exerted direct enhancing on the adhesion, proliferation and osteogenic differentiation of bone marrow derived mesenchymal stromal cells (BMSCs) through FAK and p38 signaling pathways. During the process, the focal adhesion protein expression and osteogenic-related genes expression level (e.g. ALP, BMP2, BSP and OPN) were considerably upregulated. Most importantly, the in vivo study confirmed that PDA-coating remarkably accelerated new bone formation and enhanced osseointegration performance. Our study uncovered the biological responses stimulated by PDA-coating to make a better understanding of cell/tissue-PDA interactions, and affirmed that PDA, a bio-inspired polymer, has great potential as a candidate and functional bioactive coating medium in bone regeneration and orthopedic application. 2


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1. Introduction It is well known that the life span of implants deeply relies on the stability and favorable initial osseointegration or new bone formation ability. Whereas, in clinic field, the initial osseointegration performance of existing implants is still unsatisfactory and the duration required for bone to heal is still long. The poor initial osseointegration may primarily be attributed to the bioinert surface of implant biomaterials.1 Hence, finding suitable methods to improve the performance of initial osseointegration and shorten the osseointegration duration has aroused great interest among researchers. In fact, the initially direct contact and following cell biological responses take place on the interface between the implant and host bone tissue. The previous researches have confirmed that the physical and chemical characteristics of implant surface, for example, hydrophilicity, surface roughness, surface morphology and chemical component take important roles in cell interactions with implants and exert profound influence on osseointegration.2,3 These findings propel plenty of endeavors to make implant surface modifications in order to endow implant surface with excellent biocompatibility and bioactivity, such as osteoinductivity and osteoconductivity. In other words, the suited modification on implant surface can steer cell behaviors and might even guide cellular events along predetermined pathways.4 In the past, a great number of surface topography modifications, such as acidetching,5 alkali-heat-treatment3 and electrochemical anodization,6,7 have been 3



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developed to improve the bioactivity of implants. However, those techniques are complicated and may change the intrinsic surface properties. Apart from the topography modification strategies, chemical modifications including chemical immobilization of extra cellular matrix (ECM) molecules or cell recognition peptide, layer-by-layer assembly or physical adsorption have been studied. Nevertheless, these methods also have shortcomings.8 For example, the chemical immobilization procedure is intricate and the physical adsorption is nonspecific and inefficient.9 Most importantly, they are not suitable for all surfaces on diverse implants. Recently, the bioinspired polydopamine-coating is considered as a good paradigm for surface modifications, which raises great interest of researchers. In nature world, mussels can tightly adhere to various surfaces no matter the natural inorganic materials or organic materials to resist the strong wave.10 Through careful research, scientists discovered that the dopamine (DOPA) is a crucial kind of adhesive proteins for mussel adhesion.11 Enlightened by the potent bioadhesive capacity of the marine mussel, as an analogue of DOPA, dopamine can be readily oxidized and self-polymerized under alkaline aqueous solution in dark. Then the powerful adhesive polydopamine (PDA) layer can generate on the substrates. Interestingly, Lee et al.12 firstly found that the PDA can functionalize surfaces irrespective of material types. It is suggested that PDA is not only a simple, versatile and powerful synthesis strategy to modify various surface, but also has many advantages. First of all, PDA can improve the biocompatibility and hydrophilicity of the implant surface.13-15 Thus, PDA can promote diverse cell adhesion 4


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and spreading, such as human endothelial cell, osteoblast MC3T3 and chondrocytes, even on super-hydrophobic surface.8, 16-18 Moreover, PDA can also activate osteogenic differentiation of human mesenchymal stem cells by improving the expression levels of osteogenic genes. Furthermore, PDA is able to enhance the calcium mineralization of MSCs, which can mimic the biochemical environment of extra cellular matrix of natural bone tissue to accelerate the osteogenic differentiation of MSCs.19-21 Besides, PDA could be applied as universal biomineralization route to induce the hydroxyapatite deposition on various surface.15, 22,23 The ability of inducing hydroxyapatite deposition is crucial in new bone formation. In addition, PDA is usually used as a medium to conjugate and immobilize bioactive compounds. PDA is full of functional groups, for instance, amine, catechol and imine. Therefore, those functional groups can act as initial points for covalent immobilization bioactive compounds and chelate various metal ions.24-27 Moreover, PDA is stable owing to the strong covalent interactions with substrates. It is considered that PDA-coating has good durability for long-term operation in aqueous environment. For example, Xi et al.28 had studied and reported that the PDA-coating was durable after soaking in deionized water at 60 oC for 36 days, and the hydrophilic property can be well maintained after long time washing. Till now, few studies focused on the direct influence of PDA on cell adhesion, proliferation and differentiation. In addition, the underlying mechanisms of interactions between PDA-coating and stem cells and the PDA-coating osteogenic activity in vivo are still unknown. Thus, in this study, firstly, in order to explore the universal effect of PDA in bone generation field, we fabricated PDA-coating on diverse implant material 5



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surfaces, including biopolymers, biometals and bioceramics. Then the cell response stimulated by PDA-coating in vitro and the osseointegration performance in vivo were evaluated in details. More significantly, the underlying biologic mechanisms of cell adhesion and osteogenic differentiation stimulated by PDA-coating were further investigated. 2. Materials and Methods 2.1 Fabrication of PDA coated Samples In this study, polyetheretherketone (PEEK), Ti6Al4V (Ti) and hydroxyapatite (HA) were respectively chosen as the three typical substances of biopolymer, biometal and bioceramic. PEEK disks (10 mm in length of side and 1 mm in thickness) and rod-like implants (2 mm in diameter and 3 mm in length) were purchased from Jilin Joinature Polymer Company, China. The commercial Ti6Al4V disks (10 mm in length of side and 2 mm in thickness) were provided by Xi’an Saite Metal Materials Development Company, China. As for the HA bioceramics, they were fabricated as described in previous study.29 Before the deposition of PDA-coating, all samples were thoroughly ultrasonic cleaned in ethanol and distilled water. The cleaned disks were put in dopamine solution (2 mg/mL in 10 mM Tris-buﬀer, pH 8.5). Subsequently, the polymerization reaction was conducted in dark for 24 h at room temperature according to the previous studies, which was an optimized method provided by Xi et al. 28, 30 The dopamine monomers were subjected to self-initiated polymerization. Then the polydopamine (PDA) layer was formed on the surfaces. After the process, the disks were washed with distilled water in order to eliminate the 6


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uncombined dopamine monomers and then dried at 37 °C before use. For the convenience, the PDA coated PEEK, Ti and HA disks were abbreviated as PEEK-PDA, Ti-PDA and HA-PDA, respectively in the following content. 2.2 Surface Characterizations of the Samples The surface morphology of the PDA coated/uncoated specimens was observed by scanning electron microscope (SEM, Hitachi SU8220, Japan). The surface wettability was tested by a surface-contact angle machine (Optical Contact Angle & interface tension meter, SL200KS, SOLON TECH, China). Raman spectroscope (RW2000, Renishaw, England) with 524 nm source wavelength was applied to obtain the Raman spectroscopy. The three-dimensional morphology and roughness of the samples were detected by Atomic force microscope (AFM) (NT-MDT, Russia) in tapping mode. The surface element composition of the different samples was examined with X-ray photoelectron spectroscopy (XPS) (ESCALAB 250, Thermo VG Scientific, UK) with monochromated Al Kα as an X-ray source. The spectra were collected at a pass energy of 50 eV and were calibrated using the hydrocarbon C 1s peak (285 eV). 2.3 Rat BMSCs Separation and Culture All the animal experiments were conducted under the authorization of the Institutional Animal Care and the ethical committee of Tongji University (Shanghai, China). BMSCs were insulated from the bone marrow of the tibia and femora of twoweek-old female Sprague Dawley (SD) rats as described previously.3 Subsequently, the cells were cultured in α-MEM medium added with 10% fetal bovine serum (FBS, Gibco, USA) and 1% (v/v) penicillin/streptomycin in a humidified atmosphere containing 5% 7



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CO2 at 37 °C. When 80-90% confluence happened, those cells were sub-cultured. The passages 2 to 4 cells were used in the following in vitro experiments.3 2.4 The Stimulation of PDA-coating on Cell Adhesion BMSCs were seeded respectively onto PDA coated/uncoated samples (2 × 104/well). After seeding for 24 h, specimens were rinsed by phosphate buffer saline (PBS), immobilized in 2.5% glutaraldehyde at 4 °C overnight and then dehydrated in a graded ethanol series. After being freeze-dried and platinum sputter-coated, the specimens were observed by a scanning electron microscope (SEM, Hitachi SU8220, Japan). 2.5 The Stimulation of PDA-coating on the Proliferation and Osteogenic differentiation of BMSCs Cell multiplication was measured with cell counting kit-8 (cck-8, Beyotime, China) after culturing for 1, 4 and 7 days. At each timepoint, samples were incubated in culture medium with 10 % cck-8 solution for 3 h at 37 °C in dark. The quantitative assay was performed using a microplate spectrophotometer (Biotek, USA) at an absorbance of 450 nm. BMSCs (2 × 104/well) were cultured on the various samples. At 4 and 7 days, BMSCs were immobilized with 4 % paraformaldehyde (PFA) and stained by making use of a BCIP/NBT ALP kit (Beyotime, China). For the purpose of detecting the ALP activity, cells were rinsed with PBS, lysed with 1% TritonX-100 (Beyotime, China) and then centrifuged at 12000 rpm at 4 °C for 10 min to obtain the supernatants. Based on the instructions of manufacturers, the ALP kit (JianCheng Bioengineering Institute, Nanjing, China) was used to measure ALP activity and the BCA protein assay kit 8


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(Beyotime, China) was applied to measure total protein concentration. Finally, the results were normalized to level of total protein. Moreover, the PEEK and PEEK-PDA samples were further selected as typical materials to study the impact of PDA-coating on osteogenic gene expression. The BMSCs were seeded on each sample (2 × 105 /well). Total RNA was extracted by making use of Trizol reagent (Invitrogen, USA) after culturing for 4 and 7 days. Following the instructions of manufacturers, cDNA was synthesized using a PrimeScriptTM RT reagent kit (Takara Bio, Japan) and stored at -20 °C. The mRNA expression level of target osteogenic genes: osteopontin (OPN), bone morphogenetic protein-2 (BMP-2), alkaline phosphate activity (ALP) and bone sialoprotein (BSP) were determined by using SYBR green PCR reaction mix (Roche, Basel, Switzerland) on the Light Cycler® 96 Real-Time PCR System (Roche, Switzerland). β-actin was used to act as the reference. The RT-qPCR primer sequences used in this research are listed in Table 1. All the RT-qPCR primer sequences are synthesized and provided by Sangon Biotech (Shanghai) Co., Ltd.

Table 1 Primer sequences used for RT-PCR Gene

Primer sequences (F=forward; R=reverse)

ALP

F: 5'-TATGTCTGGAACCGCACTGAAC-3' R: 5'-CACTAGCAAGAAGAAGCCTTTGG-3'

BMP-2

F: 5'-GAAGCCAGGTGTCTCCAAGAG-3' R: 5'-GTGGATGTCCTTTACCGTCGT-3'

9



BSP

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F: 5'-AGAAAGAGCAGCACGGTTGAGT-3' R: 5'-GACCCTCGTAGCCTTCATAGCC-3'

OPN

F: 5'-CCAAGCGTGGAAACACACAGCC-3' R: 5'-GGCTTTGGAACTCGCCTGACTG-3'

β-actin

F: 5'-GTAAAGACCTCTATGCCAACA-3' R: 5'-GGACTCATCGTACTCCTGCT-3'

2.6 The Stimulation of PDA-coating on the Expression of Focal Adhesion Protein The PEEK and PEEK-PDA groups were selected as typical materials to explore the impact of PDA-coating on focal adhesion protein expression. After culturing the BMSCs for 6 h, the samples were removed and rinsed by PBS three times, fixed by 4 % PFA for 30 min at room temperature. After that, they were permeabilized in 0.3% Triton X-100 and blocked in 1% bovine serum albumin (BSA) solution. Samples were incubated by Primary rabbit-anti-rat vinculin monoclonal antibody (1:200, Abcam, USA) overnight at 4 °C for evaluation the expression level of focal adhesion protein. After that, specimens were stained with secondary antibody AlexaFluor® 594conjugated goat anti-rabbit IgG at 37 °C for 1 h. The cytoskeleton and cellular nucleus were stained by FITC-phalloidin and DAPI. Images were captured on confocal laser scanning microscopy (CLSM, Zeiss, Germany). 2.7 The Stimulation of PDA-coating on Protein Adsorption Behavior The PEEK and PEEK-PDA samples were selected to estimate the effect of PDA coating on protein adsorption activity with bovine serum albumin (BSA) as model. The samples were put in 24-well plate and immersed in 1 mg/mL BSA solution for 24 h at 10


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37 °C. Then the samples were taken out and rinsed in PBS for three times, and the samples were subsequently moved into new well and incubated with 1% SDS solution (1mL/per well). The plate was shaken at 37 °C in order to extract protein absorbed by samples. The protein concentration was detected by using BCA protein assay kit (Beyotime, China) according to the instructions of manufacturer. 2.8 Molecular Mechanism Evaluation 2.8.1 Protein Separation and Western Blot Experiments Western blot analysis was conducted to study the molecular mechanism regulated by PDA-coating. The protein expression level of focal adhesion kinase (FAK), phosphofocal adhesion kinase (p-FAK) and the MAPK signaling pathways: extracellular signalrelated kinase (ERK), phospho-ERK (p-ERK), p38, phospho-p38 (p-p38), c-Junamino-terminal kinase (JNK) and phospho-JNK (p-JNK) were detected, and β-actin was used as reference. In order to extract the protein, RIPA buffer with 1% phenylmethanesulfonyl (PMSF, beyotime, Jiangsu, China) was added after 48 h. Then the cell lysates were collected and quantified by BCA protein assay kit. Proteins were isolated on SDS-PAGE gels and then were shifted onto PVDF membranes (Millipore, USA). After being blocked in 5% Nonfat-Dried Milk for 1 h, the membranes were incubated with each primary antibody (CST, USA) at 4 °C overnight. Then the membranes were washed thrice in TBS-tween buffer before incubated with HRPconjugated secondary antibody (CST, USA) for 1 h. The protein bands were visualized and the generated images were taken using Image Quant LAS 4000 (GE, USA). 2.8.2 FAK and p38 Inhibitor Treatment Study 11



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Further studies were conducted to explore whether the FAK and p38 signaling pathway were associated with the osteogenic effects of the PDA- coating. BMSCs were seeded on the PEEK-PDA samples in the culture medium added with FAK signaling pathway inhibitor PF573228 (1 μM, Sigma, USA) and p38 signaling pathway inhibitor SB203580 (10 μM, CST, USA) severally. The western blot experiment was conducted as stated above. After culturing for 4 and 7 days, the ALP staining and ALP activity experiment were evaluated. The total RNA was isolated and synthesized cDNA after culturing for 4 and 7 days. The osteogenic genes of BMP2, ALP, OPN and BSP were chosen to perform real-time PCR to estimate their expression level. 2.9 Osseointegration Evaluation in Vivo 2.9.1 Animal Experiments The PEEK and PEEK-PDA implants were selected as the representative implant materials to estimate the effect of PDA-coating on osseointegration in vivo. All the animal experiments procedures were conducted under the authorization of the Institutional Animal Care and the ethical committee of Tongji University (Shanghai, China). Ten male SD Rats weighting around 300 g were used in this study to detect the effect of PDA coating on osseointegration in vivo. The surgical implantation was performed as previously demonstrated.3 At 4 weeks, the lethal dose of pentobarbital sodium was used to euthanatize the rats and the rats’ femoral condyles were explanted and fixed in 4% PFA. 2.9.2 Histological Analysis The samples were rinsed in water to scour off residual paraformaldehyde then they 12


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were dehydrated through using a series of graded ethanol. After being embedded in a light-curing one-component resin (Technovit 7200VLC, kulzers, Friedrichsdorf, Germany), samples were cut (parallel to long axis of the implants) into 200 µm thick sections by using a diamond circular saw system (Exakt 300 CL, Exakt Apparatebau, Germany). Subsequently, the sections were ground and polished until the final thickness reached nearly 30 µm with a grinding system (Exakt 400 CS, Exakt Apparatbau, Germany). Then Van Gieson’s picrofuchsine was applied to stain these sections. Afterwards, microscopy (Olympus, Japan) was used to observe the stained sections for histological examination. The histomorphometric analysis was conducted by Image-Pro Plus 6.0 software. Three slices from each sample were randomly selected to evaluate the percentages of bone-implant contact (BIC). 2.10 Statistical Analysis The data were demonstrated as mean ± standard deviation (SD). SPSS 22.0 statistical software was used to do t-test for statistical analysis. Values of p < 0.05 were deemed to be statistically significant. 3. Results and Discussions 3.1 Characterization of PDA Coated/uncoated Samples In the area of bone tissue regeneration, the physico-chemical properties of the biomaterial may have profound effects on its bioactivity and biofunctions, and take an important part in modulating diverse intracellular signal cascades of cell behaviors, such as migration, multiplication, differentiation and apoptosis. Consequently, 13



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designing the surface characteristics, i.e. the bioactive interfaces, is the key link in cell– material interactions, which could further affect the osseointegration between the implants and the host bone tissues. Figure 1A showed the surface topography of PDA coated/uncoated samples. There was distinct difference between these two kinds samples. It is evident that the granular PDA aggregates were uniformly formed and distributed on the PDA coated samples. Dopamine (DA) oxidation to result in 5, 6-dihydroxyindole (DHI) formation is the mechanism of PDA layer formation on the samples, results in the formation of PDA precipitation. The DHI monomer is irreversibly exhausted from the aqueous solution by the addition of eumelanin suspensions.31 Owing to the spontaneous polymerization of dopamine, the strong covalent and non-covalent interactions with substrates were formed.19 Wettability plays a key role in determining cell biological behaviors. The Figure 1B presents that the samples with PDA coating possess obviously lower (*p < 0.05) water contact angles compared to the uncoated groups (PEEK: 91.00° ± 1.60° vs. PEEK-PDA: 78.73° ± 3.15°; Ti: 98.58° ± 3.08° vs. Ti-PDA: 69.49° ± 2.78°; HA: 64.82° ± 4.17° vs. HA-PDA: 42.85° ± 9.89°). The declined water contact angles indicated that the PDA-coating provided a facile approach to improve the hydrophilic property on diverse implant surfaces. The hydrophilic surface facilitates the absorption of extracellular matrix (ECM) proteins, which has an important effect on cell adhesion, proliferation and differentiation, etc.32 Raman spectroscopy images presented in Figure 1C further confirmed that PDA was successfully coated on the samples. It is not difficult to find that new broad peaks 14


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emerged at around 1350 cm-1 (catechol stretch) and 1580 cm-1 (catechol deformation) after the polydopamine coating besides the intrinsic substrate peaks.33 The PEEK- PDA, Ti-PDA and HA-PDA exhibited the same situation. For PEEK, there was a low intrinsic substrate peak at approximate 1600 cm-1, after the PDA coating, this peak was wrapped by the new and stronger adsorption peaks of PDA.

Figure 1. (A) SEM images, (B) Water contact angle, (C) Raman spectra analysis of PEEK, PEEK-PDA, Ti, Ti-PDA, HA and HA-PDA samples.

As material surface morphology and roughness play important roles in altering the cell-material interactions,34 the high-resolution AFM imaging was used to demonstrate the three-dimensional morphology and the roughness of the different samples. In Figure 15



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2A, the samples with PDA-coating showed obvious granular appearance compared to the uncoated samples, which was consistent with the SEM observation results. The distinct morphology change was due to the formation of PDA films and granules on the implant surfaces after soaking in dopamine solution.35 Furthermore, the results demonstrated that the roughness of the samples with PDA-coating was increased apparently in comparison with the uncoated specimens (PEEK: 14.21 ± 2.28 nm vs. PEEK-PDA: 106.31 ± 6.64 nm; Ti: 18.22 ± 1.76 nm vs. Ti-PDA: 127.47 ± 5.84 nm; HA: 110.64 ± 4.42 nm vs. HA-PDA: 233.33 ± 6.66 nm). The XPS results of different samples were shown in Figure 2B. The uncoated samples and PDA coated samples both revealed the presence of carbon (C1s, 285 eV) and Oxygen (O1s, 533 eV). A considerable difference could be observed from the PDA coated samples with high resolution nitrogen peak (N1s, 399.8 eV), which coincided with previous studies.19,28 Collectively, all results above confirmed that the PDA was coated successfully on diverse sample surfaces.

16


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Figure 2. (A) AFM images, (B) XPS spectra of PEEK, PEEK-PDA, Ti, Ti-PDA, HA and HA-PDA samples.

3.2 The Stimulation of PDA-coating on Cell Adhesion Adhesion is the first step for cell interaction with biomaterial surface, which could alter the subsequently cellular response cascades. The initial adhesion and spread of BMSCs play critical roles in affecting following cellular biofunctions and osseointegration in vivo. It was distinct that BMSCs seeded on the PDA coated samples showed better attachment and much more filopodia and lamellipodia, compared with the bare samples (Figure 3). The results indicated that PDA-coating apparently promoted cell initial adhesion and spreading with a universal phenomenon.

17



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Figure 3. The SEM images of BMSCs adhesion on different sample surfaces after 1 day of culture.

3.3 The Stimulation of PDA-coating on Cell Proliferation and Osteogenic Differentiation Cell proliferation is crucial in assessing the material biocompatibility. The cck-8 results showed that the BMSCs exhibited higher vitality on PEEK-PDA samples in comparison with uncoated PEEK samples after cultured for 4 and 7 days (*p < 0.05). Similar tendency also can be observed on Ti-PDA and HA-PDA samples (Figure 4A). Cell growth and differentiation of MSCs are maintained and regulated stringently in bone tissue. In the proliferation stage, MSCs can synthesize several collagen extracellular matrices, which can subsequently support the cellular maturation and osteogenic differentiation.36 Hence, favorable proliferation in the initial stage is beneficial to the osteogenic differentiation of MSCs. ALP activity is the early marker of osteoblast differentiation. Figure 4B presented that the PEEK-PDA sample promoted ALP activity than that on PEEK at days 4 and 7 18


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(*p < 0.05). The analogous results were detected on Ti-PDA and HA-PDA samples in contrast with the samples without PDA-coating. In addition, ALP staining was also used to testify the activity of PDA-coating in enhancing osteogenic differentiation. As expected, the PEEK-PDA sample showed deeper color and larger dyeing area. The expected consequents also appeared on Ti-PDA and HA-PDA compared with the uncoated samples (Figure 4C).

Figure 4. (A) Cell proliferation of BMSCs on PEEK, PEEK-PDA, Ti, Ti-PDA, HA and HA-PDA after culturing for 1, 4 and 7 days. (B) ALP quantitative analysis of activity of BMSCs seeded on different samples at days 4 and 7. (C) The ALP staining images of BMSCs seeded on the different specimens at day 7. * p < 0.05.

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Moreover, PEEK-PDA and PEEK samples were further chosen as representative materials to estimate the influence of PDA-coating on osteogenic gene expression. The osteogenic genes of ALP, BMP2, OPN and BSP were determined by RT-PCR at days 4 and 7 (Figure 5). At each timepoint, PEEK-PDA sample showed remarkably increased expression compared with PEEK group. These four gene expression profiles exhibited the same tendency. The results suggested that PDA-coating can distinctly improve the osteogenic differentiation of BMSCs. MSCs are the ideal seed cells for bone tissue. As mentioned before, MSCs undergo three stages, preosteoblast, early osteoblast and finally develop as mature osteoblast.37 During the process, osteoblasts synthesize, and secrete bone matrices, and then bone matrices mineralize to form and model bone tissue. BMP2 is an important osteoinductive cytokines, which can induce MSCs to differentiate into osteoblasts.38 ALP is the early marker of MSCs osteogenic differentiation. The noncollagenous protein OPN facilitates osteogenic cell adhesion on extracellular matrix proteins, and the OPN was expressed both at primary stage of bone formation and mineralization and bone remodeling sites.39 BSP as matricellular protein, which was significant in modulating osteoprogenitor cell adhesion and differentiation. BSP also specifically facilitates the hydroxyapatite nucleation at the bone tissue mineralization front.40 In present study, the RT-PCR results revealed that PDA-coating could apparently stimulate the bioactivity of diverse materials. In summary, the cck-8 assay, ALP activity, ALP staining and the RT-PCR results coincidently confirmed that PDA-coating has outstanding biocompatibility, and the 20


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PDA-coating could significantly improve osteogenic differentiation of BMSCs on diverse implants.

Figure 5. Real-time PCR analysis of osteogenic differentiation genes of BMSCs after culturing for 4 and 7 days on PEEK and PEEK-PDA samples.

3.4 The Stimulation of PDA-coating on Focal Adhesion Protein Expression Focal adhesions (FAs) are the sites where cells develop tight attachment to the beneath extracellular matrix. They can provide structural connections between extracellular matrix and cell actin cytoskeleton. Focal adhesions played vital role in modulating the signal transduction of cell adhesion, proliferation and differentiation.41 As one of the most abundant FA proteins, the immunofluorescence staining of vinculin protein expression was conducted in order to visualize focal adhesions. The results showed that BMSCs on PEEK-PDA samples presented better attachment and spreading, and the intensity of vinculin expression was stronger on PDA coated samples (Figure 21



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6). PDA-coating alters the focal adhesions, which will exert profound influence on the following cell interactions with biomaterials.

Figure 6. The immunofluorescence images of vinculin expression (red fluorescence) of BMSCs on PEEK and PEEK-PDA samples after being cultured for 6 h. The cell nuclei exhibited blue signal stained with DAPI. The cytoskeleton showed green fluorescence stained with FITC-phalloidin.

3.5 The Improvement of PDA-coating on Protein Adsorption

Figure 7. Protein adsorption capacity on PEEK and PEEK-PDA groups. *p < 0.05.

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It is well known that various proteins, such as cell membrane proteins, extracellular matrix proteins and cytoskeleton proteins have diverse functions and take a significant part in cell adhesion and colonization.42,43 The result presented in Figure 7 demonstrated that the protein absorption amount on PEEK-PDA specimens was higher than that on the uncoated PEEK group. The higher protein adsorption capacity on PEEK-PDA samples may attribute to the hydrophilicity and higher specific surface area of PEEKPDA surfaces.20, 44 3.6 The Impact of PDA-coating on FAK/p38 Signaling Pathways FAK is one type of cytosolic non-receptor tyrosine kinase, which belongs to focal adhesion complex family. Focal adhesion kinase (FAK) is an important signaling constituent that can be activated via a lot of stimuli and acts as a integrator or biosensor to modulate cell behaviors.45 By means of numerous molecular links, FAK can impact the structures of cell adhesion sites, cell cytoskeleton, cell spreading and cell mobility.46 In this research, we aimed to study the role of FAK in the PDA coating on the stimulation of cell differentiation. Hence, we detected the FAK and p-FAK protein expression through the western bolt assay (Figure 8A). The quantitative results shown in Figure 8B indicated that p-FAK expression was obviously higher on PEEK-PDA compared with that on PEEK (*p < 0.05) It is well known that FAK involves in multiple signaling pathways, plays a critical role in intracellular and extracellular signal transduction.47 FAK in a central position to participate and modulate various downstream of intracellular signaling pathways, for instance, Mitogen-activated protein kinases (MAPKs) signaling pathways. MAPKs 23



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have three subfamilies in mammalian cells: the c-Jun-N-terminal kinases (JNKs), the extracellular signal regulated kinases (Erks) and the p38 MAP kinases (p38s). MAPK signaling pathway take significant role in mediating cellular behaviors to numerous extracellular stimuli.48 It is also reported that MAPK signaling pathway takes a critical part in cell proliferation and differentiation. In order to investigate whether MAPK signaling pathway is involved in BMSCs osteogenic differentiation mediated by PDAcoating, we examined the protein expression of ERK, p38 and JNK pathways through the western bolt assay (Figure 8A). The quantitative results showed that significant upregulation of p-p38 expression on PEEK-PDA group compared with PEEK samples (*p < 0.05). But p-ERK and p-JNK expression exhibited no obvious difference on both groups (Figure 8B).

Figure 8. (A) Western blot experiments showed the expression of FAK, p-FAK and 24


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MAPK signaling pathways proteins of BMSCs cultured for 48 h on PEEK and PEEKPDA samples. (B) The quantitative analysis of expression of FAK, p-FAK and MAPK signaling pathways proteins. (*p < 0.05) To verify whether FAK and p38 signaling pathways are essential in BMSCs osteogenic differentiation mediated by PDA-coating, BMSCs were seeded in PEEKPDA samples and treated with specific inhibitors. In brief, FAK and p38 signaling pathways were inhibited by PF573228 and SB203580, respectively. PF573228 has been confirmed to be a powerful and selective inhibitor of FAK. PF573228 is found that exists mutual effect in the ATP-binding pocket of FAK, which can result into an inhibition of the catalytic activity of the enzyme.49 SB203580 can act as a selective inhibitor combined with ATP binding pocket of p38, which prevents the activation of MAPKAP kinase-2.50-52 The results in Figure 9 (A, B) presented that the protein expression level of p-FAK and p-p38 was significantly inhibited by PF573228 and SB203580, respectively (*p < 0.05). Furthermore, the ALP activity, ALP staining (Figure 9 (C, D)) and osteogenic gene expression (Figure 9 (E, F)) results indicated that the osteogenic differentiation of BMSCs on PDA modified samples was dramatically inhibited by the specific inhibitors (*p < 0.05). Based on the above results, it can be concluded that FAK and p38 signaling pathways played vital roles in osteogenic differentiation of BMSCs stimulated by PDA-coating.

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Figure 9. (A, B) The expression of FAK and p-FAK of BMSCs cultured on PEEK-PDA samples treated with or without specific inhibitors by western blot assay and the corresponding quantitative analysis. (C, D) The ALP staining images of BMSCs seeded on PEEK-PDA samples at day 7 treated with or without specific inhibitors and corresponding ALP activity quantitative analysis at days 4 and 7. (E, F) The RT-PCR analysis of osteogenic differentiation genes of BMSCs after culturing on PEEK-PDA samples treated with or without specific inhibitors for 4 and 7 days. (*p < 0.05).

3.7 In Vivo Studies It is suggested that sufficient new bone formation is necessary for implants survival. The animal experiments were further performed to investigate the effect of PDAcoating on osseointegration in vivo. After implanting for 4 weeks, much more new bone 26


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was deposited on PEEK-PDA implant surface compared to PEEK implant surface (Figure 10A). From the higher magnification images (Figure 10B), tighter bone adhesion phenomenon on PEEK-PDA implant could be confirmed. The Figure 10C indicated that bone implant contact (BIC) percentage was dramatically higher for PEEK-PDA group than that for bare PEEK group. In fact, BIC takes a significant part in the process of osseointegration, and the excellent osseointegration guarantees the survival of implant.

Figure 10. (A) Histological section images of the in vivo osseointegration of PEEK and PEEK-PDA at low magnification and (B) high magnification, (C) histomorphometry analysis of BIC percentage of PEEK and PEEK-PDA implants. (*p < 0.05)

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Figure 11. The schematic diagram of PDA-coating on osteogenic differentiation and osseointegration.

The present study revealed that the implant surfaces modified by PDA-coating could improve BMSCs osteogenic differentiation and enhance the implant osseointegration in vivo (Figure 11) by activating the FAK and p-38 signaling pathways. Therefore, the PDA-coating provide a universal strategy for surface modifications on diverse implant materials to improve osseointegration and osteogenesis performance.

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Conclusions In present research, the diverse implant surfaces, including biopolymer, bioceramic and biometal, were modified by PDA-coating. Subsequently, we detect the cell responses stimulated by PDA-coating in vitro and the effect of PDA-coating on osseointegration capacity in vivo. Implant surfaces with PDA-coating become more hydrophilic, which does benefits to functional protein adsorption and cellular responses, such as the enhancing of cell focal adhesion, proliferation and osteogenic differentiation in vitro. In addition, the FAK/P38 pathways involve the enhancement of PDA-coating on osteogenic differentiation of BMSCs. Most importantly, implant with PDA-coating showed excellent osseointegration performance in vivo. The BIC percentage of PEEKPDA group reached around 33.4%, which was approximately 32.6% higher than that of uncoated PEEK group. Our results put forward new evidences that PDA-coating exert direct influence on BMSCs osteogenic differentiation, uncover the biological mechanism of cell response stimulated by PDA-coating and make a better understanding of cell-PDA interactions. As a bio-inspired polymer, PDA can be used as a potently functional bioactive coating for diverse implants in bone regeneration and orthopedic applications. Acknowledgements The authors gratefully acknowledge the support of the National Key R&D Program of China (2017YFB1104100), National Natural Science Foundation of China (81672134, 81871490, 81271122), Science and Technology Commission of Shanghai Municipality 29



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Figure 1. (A) SEM images, (B) Water contact angle, (C) Raman spectra analysis of PEEK, PEEK-PDA, Ti, TiPDA, HA and HA-PDA samples. 55x55mm (300 x 300 DPI)


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Figure 2. (A) AFM images, (B) XPS spectra of PEEK, PEEK-PDA, Ti, Ti-PDA, HA and HA-PDA samples. 62x45mm (300 x 300 DPI)



Figure 3. The SEM images of BMSCs adhesion on different sample surfaces after 1 day of culture. 75x41mm (300 x 300 DPI)


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Figure 4. (A) Cell proliferation of BMSCs on PEEK, PEEK-PDA, Ti, Ti-PDA, HA and HA-PDA after culturing for 1, 4 and 7 days. (B) ALP quantitative analysis of activity of BMSCs seeded on different samples at days 4 and 7. (C) The ALP staining images of BMSCs seeded on the different specimens at day 7. * p < 0.05. 55x52mm (300 x 300 DPI)



Figure 5. Real-time PCR analysis of osteogenic differentiation genes of BMSCs after culturing for 4 and 7 days on PEEK and PEEK-PDA samples. 63x49mm (300 x 300 DPI)


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Figure 6. The immunofluorescence images of vinculin expression (red fluorescence) of BMSCs on PEEK and PEEK-PDA samples after being cultured for 6 h. The cell nuclei exhibited blue signal stained with DAPI. The cytoskeleton showed green fluorescence stained with FITC-phalloidin. 80x39mm (300 x 300 DPI)



Figure 7. Protein adsorption capacity on PEEK and PEEK-PDA groups. *p < 0.05. 99x88mm (300 x 300 DPI)


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Figure 8. (A) Western blot experiments showed the expression of FAK, p-FAK and MAPK signaling pathways proteins of BMSCs cultured for 48 h on PEEK and PEEK-PDA samples. (B) The quantitative analysis of expression of FAK, p-FAK and MAPK signaling pathways proteins. (*p < 0.05) 59x59mm (300 x 300 DPI)



Figure 9. (A, B) The expression of FAK and p-FAK of BMSCs cultured on PEEK-PDA samples treated with or without specific inhibitors by western blot assay and the corresponding quantitative analysis. (C, D) The ALP staining images of BMSCs seeded on PEEK-PDA samples at day 7 treated with or without specific inhibitors and corresponding ALP activity quantitative analysis at days 4 and 7. (E, F) The RT-PCR analysis of osteogenic differentiation genes of BMSCs after culturing on PEEK-PDA samples treated with or without specific inhibitors for 4 and 7 days. (*p < 0.05). 65x46mm (300 x 300 DPI)


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Figure 10. (A) Histological section images of the in vivo osseointegration of PEEK and PEEK-PDA at low magnification and (B) high magnification, (C) histomorphometry analysis of BIC percentage of PEEK and PEEK-PDA implants. (*p < 0.05) 71x41mm (300 x 300 DPI)



Figure 11. The schematic diagram of PDA-coating on osteogenic differentiation and osseointegration. 55x65mm (300 x 300 DPI)


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Table of Contents (TOC) 67x37mm (300 x 300 DPI)


Mussel-Inspired Polydopamine Coating: A General Strategy To

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