Biomimetic hyaluronic acid-lysozyme composite coating on AZ31 Mg

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Biomimetic hyaluronic acid-lysozyme composite coating on AZ31 Mg alloy with combined antibacterial and osteoinductive activities Sankalp Agarwal, Mathieu Riffault, David A. Hoey, B Duffy, James Curtin, and Swarna Jaiswal ACS Biomater. Sci. Eng., Just Accepted Manuscript • Publication Date (Web): 03 Nov 2017 Downloaded from http://pubs.acs.org on November 3, 2017

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ACS Biomaterials Science & Engineering

Biomimetic hyaluronic acid-lysozyme composite coating on AZ31 Mg alloy with combined antibacterial and osteoinductive activities Sankalp Agarwal1, 2, Mathieu Riffault3,4,5, David Hoey3,4,5, Brendan Duffy1, James Curtin2, Swarna Jaiswal1* 1

Centre for Research in Engineering and Surface Technology, FOCAS Institute, Dublin

Institute of Technology, Kevin Street, Dublin 8, Ireland. 2

School of Food Science and Environmental Health, Cathal Brugha Street, Dublin Institute

of Technology, Dublin 1, Ireland. 3

Trinity Centre for Bioengineering, Trinity Biomedical Sciences Institute, Trinity College

Dublin, Dublin 2, Ireland. 4

Department of Mechanical and Manufacturing Engineering, School of Engineering, Trinity

College Dublin, Dublin 2, Ireland. 5

Advanced Materials and Bioengineering Research Centre, Royal College of Surgeons in

Ireland and Trinity College Dublin, Dublin 2, Ireland.

*Corresponding author: Dr Swarna Jaiswal Email: [email protected]

1 ACS Paragon Plus Environment


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Abstract This study presents the covalent grafting of a hyaluronic acid-lysozyme (HA-LZ) composite onto corrosion resistant silane coated AZ31 Mg alloy via EDC-NHS coupling reactions. The HA-LZ composite coatings created a smooth and hydrophilic surface with the increased concentration of functional lysozyme complexed to the hyaluronic acid group. This was confirmed by the measurement of AFM, water contact angle, and quantification of hyaluronic acid and lysozyme. The colonisation of S.aureus on HA-LZ composite coated substrates was significantly reduced as compared to the hyaluronic acid, lysozyme coated and uncoated AZ31 controls. Such activity is due to the enhanced antibacterial activity of lysozyme component as observed from thw spread plate assay, propidium iodide staining and scanning electron microscopy. Furthermore, morphology of the osteoblast cells, alkaline phosphatase activity and DNA quantification studies demonstrated the improved biocompatibility and osteoinductive properties of HA-LZ coated substrates. This was verified by comparing with the lysozyme coated and uncoated AZ31 substrates in terms of cell adhesion, proliferation and differentiation of osteoblastic cells. Therefore, such multifunctional composite coatings with antibacterial and osteoinductive properties are promising can be potentially used for the surface modifications of orthopaedic implants. Keywords: Hyaluronic acid-lysozyme, corrosion resistant, silane, antibacterial and osteoinductive


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1

Introduction

Traditionally, temporary orthopaedic medical devices such as pins, screws, rods and plates which are generally employed to secure the bone fracture, have been manufactured using conventional biostable metals such as titanium, stainless steel and cobalt–chromium alloys 1. These metal implants are prone to failure due to the mismatch of mechanical properties with the natural bone, in addition to complications arising due to an inflammatory response, physical irritation or stress shielding 2. To avoid these drawbacks, biodegradable metals have been widely explored as alternative materials. Amongst the various biodegradable metals, magnesium (Mg) based alloys have unique characteristics because of their osteoinductive and excellent physical and mechanical properties 3. However, the major factors hampering the clinical use of these alloys is their rapid degradation in physiological conditions, thereby causing toxicity to surrounding tissues and susceptibility to bacterial colonisation, ultimately causing implant failure 4,5. Several approaches such as ion implantation, micro-arc oxidation and selective use of alloying elements have been employed to improve the corrosion resistance and biocompatibility of Mg alloys 2. However, these surface modifications are unable to provide long-term corrosion resistance 4. Although alloying elements may improve the antimicrobial properties of the alloys, it can also cause potential toxicity to the surrounding tissues 6,7. Over the past decade, functional coatings have been extensively investigated as an effective approach to improve the corrosion resistance and biocompatibility of Mg alloys. In addition, coatings of antimicrobial agents on various biomaterials such as conventional non-degradable metals, Poly (methyl methacrylate) (PMMA), silicone, polyethylene terephthalate (PET) etc., for applications (orthopaedic implants, contact lenses, catheters etc.) have proven effective against the bacterial colonisation on the surface 8.



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Amongst coating materials, alkylorganosilanes have been widely employed to improve the corrosion resistance of Mg alloy due to their chemical stability and good adhesive properties to the metal surface that generates the hydrophobic siloxane network 9. Previously, we demonstrated the corrosion resistant properties of an amine-functionalised multilayer MTESTEOS-APTES

(methyltriethoxysilane–tetraethoxysilane-3-aminopropyltriexthoxy

silane)

coating on the AZ31 Mg alloy 9. The methyl group of MTES-TEOS and siloxane hydrophobic network of MTES-TEOS-APTES coating prevents the penetration of corrosive electrolytes, thereby increasing the corrosion resistance of the Mg alloy. In addition to enhancing the corrosion resistance of Mg alloy, the biocompatibility of the Mg alloys plays an important role in the biological acceptability of the implant. In previous studies, organofunctional silanes (e.g. APTES) was used to functionalise surface with bioactive polymers or peptides to enhance the biocompatibility of metal implants

9,10

. Therefore, such

approaches can be employed to fabricate multifunctional coatings by immobilising polymers such as hyaluronic acid and lysozyme which impart osteoinductive as well as antimicrobial properties to surfaces for orthopaedic applications. It has been reported that the corrosion of Mg-based alloy causes an increase in the pH of the surrounding medium, thereby inhibiting the growth of bacteria 11. However, biological fluids are stabilised by buffer systems that maintain physiological processes. Therefore, highly alkaline environments that arise due to corrosion of Mg alloys are buffered under in vivo conditions, thereby limiting the antimicrobial activity of the implant 12. Previous studies have also demonstrated that the bare and corrosion-resistant Mg-based alloys are highly susceptible to microbial growth on the surface

12,5,13

. Furthermore, it is important that the

orthopaedic implant should expedite the healing of any defective bone. The orthopaedic implant efficiency in restoring damaged bone is determined by its osteointegration. This is


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characterised by the tight bond between the bone and implant surface, thereby minimising the risk of implant loosening over time 14. To improve the biological acceptance of orthopaedic implants in terms of osteoinductivity and inhibiting bacterial colonisation on the surface, bio-macromolecules such as hyaluronic acid and lysozyme can be employed on the corrosion-resistant Mg-based alloys. The combination of hyaluronic acid and lysozyme have been explored previously on ocular lenses were used

16

. Hyaluronic acid is a polyanionic multifunctional polymer present as a

component of extracellular matrix which plays an important role in promoting osteoblast cell motility and proliferation 16,17. Additionally, its anionic and hydrophilic nature also prevents bacterial cell adhesion 16,18. However, the application of HA onto an implant surface can not completely avoid infections. Lysozyme is a lytic enzyme present in egg white, human tears as well as in other secretions 16. Its antimicrobial properties are associated with the hydrolysis of the peptidoglycan layer in bacterial cell wall, thereby perturbing the membrane functions. Moreover, as a part of a natural defence system, lysozyme is environmentally friendly and acts with more specificity than conventional biocides, such as quaternary ammonium salts and antibiotics, thereby preventing the risk of developing multidrug resistant bacteria. Lysozyme has been utilised to modify the surfaces of biomedical devices to prevent bacterial growth

16,19

. Previously, adsorption of lysozyme on hyaluronic acid functionalised

mesoporous SBA-15 particles was studied as a therapeutic agent 20. Therefore, the synergistic immobilisation of hyaluronic acid and lysozyme on the corrosion resistant silane coated AZ31 Mg alloy surface is proposed first time in this work to confer simultaneous antimicrobial and osteoinductive properties for challenging biodegradable orthopaedic applications. In the present study, the biomimetic hyaluronic acid-lysozyme (HA-LZ) composite coating was developed on the AZ31 Mg alloy and the combined antibacterial and osteoinductive 5 ACS Paragon Plus Environment


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activities were evaluated. The covalent binding of HA-LZ composite with the amine terminated surface AZ31-MTES-TEOS-APTES (AZ31-MT-A) was achieved via EDC-NHS coupling reactions. The antimicrobial activity of HA-LZ immobilised on AZ31-MT-A coated Mg alloy was evaluated against S. aureus bacteria. Further, the improved biocompatibility of HA-LZ coated surface has been demonstrated with murine osteoblasts.

2 2.1

Experimental Materials

AZ31 alloy sheets were purchased from Shaanxi Taipu Rare Metal Materials Ltd, China. Methyltriethoxysilane

and tetraethoxysilane, 3-aminopropyl-triethoxy silane, Hyaluronic

acid (HA) from rooster comb (1-4 million Daltons), lysozyme from chicken egg white, phosphate buffer saline (PBS), Dulbecco's Modified Eagle's Medium/Nutrient F-12 Ham (DMEM/F-12), fetal bovine serum (FBS), Penicillin-streptomycin antibiotics, phosphatase substrate and bisBenzimide H 33342 trihydrochloride, NHS (N-Hydroxysuccinimide), EDC (1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide) and MES (2-(N-Morpholino) ethane sulfonic acid) buffer were purchased from Sigma Aldrich. Mueller-Hinton agar and nutrient broth were purchased from Lab M. 2.2

Surface modification with hyaluronic acid-lysozyme composite on AZ31 Mg alloy

The pre-treatment and corrosion resistant silane coating on AZ31 Mg alloy used to immobilise HA-LZ composite coating was adopted from our previous study 21. Briefly, 2 mm thick sheets of AZ31 Mg alloy were polished progressively by finer SiC paper from 400 to 1200 grit. The samples were cleaned ultrasonically in ethanol and passivated in 5 N NaOH for 2 h at 60 °C and then cleaned with deionised water. This alkali treated AZ31 Mg alloys are referred to as AZ31-OH. 6 ACS Paragon Plus Environment

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Furthermore, the MTES and TEOS sols were prepared in the acidified ethanol solution containing 0.04N nitric acid as a catalyst. A molar ratio of MTES: TEOS/2:1 sol gel was used with R = 2.36, where R = [H2O]/[MTES+TEOS]. The APTES sol was prepared by adding 400mM APTES in ethanol solution with R = 4.5 (R = [H2O]/[APTES]). The AZ31-OH passivated substrates were dip-coated sequentially in MTES-TEOS (AZ31-MT) and APTES sol-gels and cured at 120°C for 1h at each step to achieve the amine terminated AZ31-MT-A substrates. Furthermore, the hyaluronic acid-lysozyme composite was functionalised to amine terminated AZ31-MT-A substrates. In this process, lysozyme (LZ) (10% and 20 % w/w of HA), HA (1 mg.ml-1), NHS (0.1M) and EDC (0.4 M) were sequentially added into a MES buffer (0.01 M, pH 5.5) and stirred for 2h

16

. Similar reactions were also performed with

respective LZ (10% and 20 % w/v) and HA (1 mg.ml-1) concentrations which were considered as controls. The AZ31-MT-A coating was immersed in the above solution for 4 h. After incubation, substrates were rinsed with MES buffer to remove physically adsorbed HA and LZ. The resultant HA-LZ (10% and 20%), LZ (10% and 20%) and HA coated AZ31MT-A surfaces were denoted as AZ31-HA-LZ-10%, AZ31-HA-LZ-20%, AZ31-LZ-10%, AZ31-LZ-20% and AZ31-HA respectively. 2.3

Characterisation of AZ31 modified substrates

2.3.1 Surface morphology and wettability of the substrate The surface morphology of all modified (AZ31-HA-LZ-10%, AZ31-HA-LZ-20%, AZ31-LZ10%, AZ31-LZ-20% and AZ31-HA) and uncoated AZ31 substrates were characterised by atomic force microscopy in tapping mode (AFM, Asylum MFP-3D-BIO, USA). The wettability of samples was determined by static water contact angle measurements (FTÅ-200 system) using a sessile drop method (2µl, Milli-Q water).



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2.3.2 Quantification of immobilised hyaluronic acid and lysozyme on the AZ31 surface The effective concentration of HA immobilised on the AZ31-HA-LZ-10%, AZ31-HA-LZ20% and AZ31-HA surfaces was determined by a Morgan-Elson fluorometric enzyme assay 22

. The substrates were exposed to the hyaluronidase enzyme (reconstituted in phosphate

buffer, pH 5.5) at 37.5 °C for 1 hr. After incubation, the reaction mixture was heated in a boiling water bath for 5 mins to inactivate the enzyme. After cooling to room temperature, 25µl of tetraborate reagent (0.8M K2B4O7.4H2O) was added and incubated for 3 mins in a boiling water bath to start the Morgan-Elson fluorometric reaction. After cooling to room temperature, 0.75ml of p-dimethylaminobenzaldehyde reagent (10 % w/v dissolved in solution of glacial acetic acid and 10 N HCl (87.5/12.5 %v/v)) was added and incubated for 20 mins at 37.5 °C. The release of GlcNAc was then measured against a blank by fluorescence (Ex/Em: 545/604nm) using a Spectramax-M3 microplate reader (Molecular Devices). The immobilised HA concentration was determined using a standard curve of HA. The lysozyme activity of HA-LZ and LZ coatings was determined using 4methylumbelliferyl β-D-N,N’, N”-triacetylchitotrioside (4-MU-GlcNAc3) as a fluorimetric enzyme substrate

23,24

. The substrate was dissolved at a concentration of 100 µM in 50 mM

sodium acetate buffer of pH 5 and incubated with the HA-LZ coated samples at 37 °C for 3060 mins. The lysozyme activity was stopped by using a glycine-carbonate buffer at a pH of 10.5 and the fluorescence of 4-methyl umbelliferone was measured at Ex/Em =360/445 nm. The amount of 4-MU produced during the enzymatic reaction was determined using its standard curve. One unit of the lysozyme activity for 4-MU-GlcNAc3 hydrolysis was defined as the amount of enzyme that generates 1 µM of 4-MU per minute at 37 ˚C at pH 5.


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2.4

Cytocompatibility

2.4.1 DNA quantification The murine osteoblast cell line (MC3T3-E1) (ATCC® CRL-2593™, LGC standard) was used for in vitro cytotoxicity and osteogenesis experiments. Cells were cultured under 37°C, 5% CO2 and 95% relative humidity in standard growth medium consisting of DMEM/F-12 containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin (P/S). The DNA quantification of MC3T3-E1 osteoblast cells was determined as a measure of cell proliferation

25

. Cells were seeded at a density of 2.5 x104 cells.cm-2 on the coated and

uncoated AZ31 substrates and cultured in standard growth media (1cm2 = 1.25ml) for 3 days before replacing with a differentiation medium and further culturing the cells at 3, 7 and 14 days. The differentiation medium consisted of the standard growth media with the addition of 50 µM ascorbic acid, 100 nM dexamethasone and 10 mM β-glycerophosphate 25. After 3, 7 and 14 days of culture, the substrates were washed with PBS and the resultant cells were lysed by using cell lysis buffer. The fluorescent dye, Hoechst 33258 (Sigma Aldrich) was used to quantify the cellular DNA according to manufacturer’s instruction (DNA quantification kit, Sigma Aldrich). 2.4.2 Alkaline phosphatase (ALP) assay The differentiation of osteoblasts was studied by measuring intracellular ALP enzyme activity using a para-nitrophenyl substrate (p-NPP, Sigma) 25. After culturing osteoblast cells for 3, 7 and 14 days in the differentiation medium, cells were lysed and the lysate was incubated with the p-NPP substrate for 1 h at 37 °C. The enzymatic reaction was stopped by adding 1N NaOH and the ALP activity was measured by the absorbance of para-nitrophenol (p-NP) at the 405 nm wavelength. A standard curve of different p-NP concentrations was prepared in a 0.02 N NaOH solution. The measured para-nitrophenol was normalised against



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total protein concentration. The total protein content was determined using the bicinchoninic acid protein assay kit (Sigma Aldrich). 2.4.3 Actin Phalloidin and DAPI staining The morphology of actin cytoskeleton of osteoblast cells was investigated 24 h after seeding. Briefly, cells were washed with PBS, fixed with 10% formalin, and permeabilised with 0.1% Triton (Sigma) in PBS. Actin cytoskeleton was stained with Phalloidin (1/40 in PBS, AlexaFluor488-Phalloidin, Life Technologies) for 20 minutes, and nuclei were stained with DAPI (1/2000 in PBS, Sigma). All cells were imaged using an Olympus IX83 epifluorescence microscope fitted with a 10x objective. 2.4.4 Cell morphology imaging by SEM The morphology of osteoblast cells seeded at a density of 104 cells.cm-2 on the experimental substrates was examined after 1 and 3 days of culture

26

. Cell culture conditions were

maintained as described earlier. After incubation, substrates were washed with PBS, fixed with 10% formalin and dehydrated in alcohol gradients. Substrates were sputter coated with Au-Pd and observed by SEM (Hitachi SU-70 FESEM) operating at 5.0 kV. 2.5

Antibacterial activity

2.5.1 Spread plate assay The antibacterial activity of uncoated and coated AZ31 substrates was determined by using S. aureus, the most common pathogen associated with orthopaedic implant infections. The substrates were immersed in sterile plastic tubes with 20 ml (1 cm2 = 20 ml) of 1 x 106 cells.ml-1 of initial bacterial suspension in PBS and incubated at 37 °C with a constant shaking at 200 rpm. The substrates were removed from the tubes after 2, 4 and 12 h to evaluate the antibacterial activity. The substrates were gently washed thrice with PBS and sonicated in PBS for 5 mins to dislodge the cells from the surface. The 0.2 ml of bacterial


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suspension in PBS was plated onto the triplicate agar plate and incubated at 37 °C for 24 h. The colonies were counted and results were expressed as a mean of colony forming units (CFU)/ml/cm2 27. 2.5.2 Propidium iodide (PI) exclusion assay The coated and uncoated substrates (Size: 1 cm2) were incubated with S.aureus bacteria (106 cells/ml) in PBS for 2, 4 and 12 h. After the incubation, the substrates were treated with PI at a concentration of 60 µM for 30 minutes in the dark. The substrates were washed with PBS twice and placed in the black bottom 24 well plate to measure the fluorescence (Ex /Em: 535/617 nm) using Spectramax-M3 microplate reader. 2.5.3 Bacterial cell morphology by SEM All the coated and uncoated substrates were incubated for 4 h with bacteria and gently washed with the PBS. The bacterial cells were fixed in 10% formalin and dehydrated in alcohol gradients before being sputter coated with Au/Pd and imaged using SEM. 2.6

Statistical analysis

All of the experiments were conducted in triplicate. All data are expressed as mean ± S.D. The differences between the groups were analysed using one-way analysis of variance (ANOVA) followed by post hoc Tukey test. Statistically significance was considered at **p < 0.01 and *p < 0.05.

3 3.1

Results and discussion Characterisation of AZ31 modified substrates

Prior to application of HA and LZ coatings, the AZ31 surface was treated with the sequential steps of MTES-TEOS and APTES silanes to achieve amine functionalised coating. In previous research, it has been demonstrated that the amino group of such coatings can be



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functionalised with various biomolecules such as hyaluronic acid, bovine serum albumin, RGD peptides etc., through amide bonds by EDC-NHS chemistry

28,29,30

. The

functionalisation of HA-LZ coating onto the amine group of APTES coatings was confirmed by AFM, contact angle measurement as well as quantification of HA and LZ by enzymatic methods as shown in Fig 1 and Table 1. The surface roughness (Ra) of the AZ31 and AZ31MT-A are 74.5 ± 8.2 nm and 45.7 ± 0.82 nm respectively. The roughness reduced considerably after coating with HA-LZ composite and LZ coatings on AZ31-MT-A substrate ranging from 17.1 ± 1.84 nm to 25.7 ± 7.3 nm. However, the increase in surface roughness for the composite coatings as compared to the AZ31-HA substrate with Ra of 4.86 ± 0.07 nm could be attributed to the increased content of LZ. The fibrillar morphology of the HA and HA-LZ coated surfaces was observed and can be attributed to the tendency of drying induced aggregation of HA chains 31. Static water contact angle measurement was used to determine the hydrophilic changes of modified AZ31 substrates (Table 1). When compared to the AZ31 alloy, the AZ31-MT-A showed a lower contact angle of 46.4 ± 2.1°, indicating the presence of polar amine groups on the surface. The wettability of the HA-LZ and LZ modified surfaces decreased ranging from 48.85 ± 1.07° to 55.67 ± 0.92°, when compared to the AZ31-HA surface with a contact angle of 41.24 ± 2.66°. However, these coated substrates showed a considerable increase in wettability when compared to the AZ31 substrate (74.5 ± 8.2°). The wettability of the substrates is strongly related to the surface roughness and can play an important role in bacteria cell adhesion 32. Furthermore, the concentration of immobilised HA and lysozyme activity of the coatings were quantified ( Table 1). It was found that the concentration of HA immobilised on AZ31HA, AZ31- HA-LZ-10% and AZ31-HA-LZ-20% substrates were very similar and found to be ~ 48 µg/ml/cm2. The lysozyme activity of AZ31- HA-LZ-10% and AZ31-HA-LZ-20% 12 ACS Paragon Plus Environment

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substrates were higher and found to be 0.534 ± 0.042 U/ml/cm2 and 0.588 ± 0.021 U/ml/cm2 respectively when compared to the activity of AZ31-LZ-10% and AZ31-LZ-20% substrates at 0.465 ± 0.03 U/ml/cm2 and 0.513 ± 0.027 U/ml/cm2 respectively. Previous studies have shown that hyaluronic acid can interact electrostatically with lysozyme without affecting the activity of lysozyme in the HA-LZ composite

33,34

. Therefore, these characterisation studies

confirmed the surface modifications of AZ31 Mg alloy.with HA-LZ composite. 3.2

Antibacterial properties

Analysis of bacterial colonisation on HA, HA-LZ, LZ coated and uncoated AZ31 substrates were determined by using a spread plate assay as shown in Fig. 2. The results demonstrated that bacterial cells adhered to the AZ31 and AZ31-HA surfaces markedly increased by 144.6 % and 110.6% respectively over the 12 h period. However, the number of bacteria on the AZ31-HA surface is significantly less than the AZ31 alloy. On the other hand, the HA-LZ composite coatings showed a significant reduction in bacteria cell adhesion by over 82% correlating with increases in LZ content as compared to the uncoated AZ31 during the incubation period of 12h. The AZ31-HA-LZ-10% and AZ31-HA-LZ-20% substrates also showed a significant decrease in bacterial cell adhesion as compared to the AZ31- LZ-10% and AZ31-LZ- 20% controls. The overall decrease in the bacterial colonies of the HA-LZ composite coatings is an indication of the lysozyme concentration dependent anti-adhesive and/or anti-bacterial properties over uncoated AZ31 and other modified substrates. Furthermore, the bactericidal activity of the coated and uncoated AZ31 substrates was determined fluorometrically by using PI staining or exclusion of the bacteria adhered to the substrates as shown in Fig.3. The uncoated AZ31 substrate demonstrated antibacterial activity in the initial period of incubation but it decreases in the remaining period, whereas AZ31-HA-LZ-10% and AZ31-HA-LZ-20% showed a significant increase in antibacterial activity as compared to the bare AZ31, AZ31-LZ-10% and AZ31-LZ-20% substrates over 12 13 ACS Paragon Plus Environment


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h of incubation. The AZ31-HA substrate showed minimal antibacterial activity, as there are no bactericidal functional groups present in HA. Fig. 4 shows the morphology of the bacteria on the uncoated and coated substrates after 4 h of incubation in bacterial suspension. From Fig. 4(a)-(b), the bacteria adhered to the AZ31 and AZ31-HA surfaces maintained good shape indicating cytocompatibility of the bacteria. On the HA-LZ composite (Fig. 4c-d) and LZ coated (Fig.4e-f) substrates, only a few bacteria maintained their native shape and morphology (indicated by green arrows). In addition, cell debris (indicated by yellow arrows) and affected cells presenting distorted morphology (indicated by red arrows) are observed on HA-LZ composite and LZ coated substrates. Many studies have reported that antibacterial activity of Mg-based alloys being directly related to the increased pH of the medium

11,35

. However, recent studies showed that in

buffered environments, Mg-based alloys are susceptible to bacterial colonisation under in vitro and in vivo conditions 5,36,12. In this study, an increased bacteria cell adhesion or reduced antibacterial activity of uncoated AZ31 substrates during the incubation period in bacterial suspension in PBS was observed. This is likely due to the insufficient increase in the pH of the medium (8.16 ± 0.15), in addition to the greater roughness and contact angle of the surfaces (Fig. 2-4 and Table 1). In contrast, the enhanced antibacterial activity of AZ31-HALZ-10% and AZ31-HA-LZ-20% substrates over respective controls AZ31-LZ-10% and AZ31-LZ-20% was observed as demonstrated in Fig.2-4 could be attributed to the higher content of lysozyme immobilisation with hyaluronic acid (Table 1). The reduced bacterial cell adhesion on the hyaluronic acid coated AZ31-HA surface can be attributed to the bacterial anti-adhesive characteristics of HA polymer due to the anionic nature and lubricity of the coating as well as low surface roughness 18.


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The most common pathogen associated with the orthopaedic implants is S.aureus 18. Several studies reported the bactericidal activity of lysozyme against S.aureus

37,38

. The antibacterial

activity of lysozyme involves its disruptive interaction with the bacterial membrane. Lysozyme disrupts the cell wall by catalysing the hydrolysis of the β 1-4 linkages between the N-acetylmuramic acid and N-acetyl-D-glucosamine residues in chitodextrins, thereby increasing the permeability of bacterial cell wall and causing the cells to rupture. According to the contact killing mechanism of lysozyme, bacteria first adhere to the surface leading to the breakdown of the bacterial cell wall 16. Previous study showed the efficient antibacterial activity of the HA-LZ coated intraocular lenses against S.aureus for preventing the endophthalmitis 16. However, there is limited information available about the efficacy of HALZ as a composite coating to prevent the bacterial colonisation for orthopaedic applications and their effect on the osteoblast cell response. Having demonstrated the antimicrobial properties of HA-LZ against S aureus, the most common pathogen associated with orthopaedic implants, we next investigated the cytocompatibility of HA-LZ with osteoblast cultures. 3.3

Cytocompatibility studies

Fig. 5 shows the morphology of osteoblast cells on the uncoated and coated AZ31 substrates at day 1. The osteoblast cells that attached to the uncoated AZ31 substrate are sparse and circular in shape as shown in SEM image Fig. 5 I (a), demonstrating the poor adhesion to the substrate. The AZ31-HA (Fig. 5(b)), AZ31-HA-LZ-10% (Fig. 5(c)) and AZ31-HA-LZ-20% (Fig. 5(d)) surfaces demonstrate flattened, extended morphology and greater cell-to-cell interaction of adhered osteoblasts. In contrast, AZ31-LZ-10% (Fig. 5(e)) and AZ31-LZ-20% (Fig. 5(f)) coated substrates displayed an elongated morphology. Fig. 5 (II) series show the actin staining of osteoblast cells on different substrates after 24 h of incubation. The cellular morphology of osteoblast cells is circular and poorly defined on the 15 ACS Paragon Plus Environment


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uncoated AZ31 substrate (Fig 5(a`)), without strong actin filament staining, indicating the cells adhered poorly and did not develop a filamentous actin cytoskeleton on the AZ31 substrate. Similar osteoblast morphology on the uncoated AZ31 substrate can be observed from SEM image (Fig. 5(a)). On the AZ31-HA, AZ31-HA-LZ-10% and AZ31-HA-LZ-20% substrates, osteoblast cells appear cuboidal in morphology, showing strong actin filament organisation extending in different directions as shown in Fig. 5b`, 5c` and 5d` respectively, indicative of an osteoblastic phenotype. On the AZ31-LZ-10% (Fig. 5e`) and AZ31-LZ-20% (Fig. 5f `) substrates, cells displayed somewhat elongated actin filaments. Overall, these results indicate that HA-LZ composite coated substrates facilitate improved adhesion and morphological phenotype of osteoblast cells as compared to the uncoated substrates. Fig. 6 shows the DNA quantification of osteoblast cells cultured on the uncoated and coated AZ31 substrates which are considered to be a direct measure of cell proliferation over a period of 14 days. The DNA concentration is significantly lower than the other coated surface for the AZ31 substrate alone throughout the culture period. On the other hand, AZ31-HA-LZ10% and AZ31-HA-LZ-20% substrates showed a significant increase in DNA concentration as compared to the AZ31, AZ31-LZ-10% and AZ31-LZ-20% over a period of 14 days. This indicated the improved osteoblast adhesion and proliferation on the HA-LZ coated substrates. These results showed that even though the osteoblast adhered to AZ31-LZ coatings (Fig. 5), the proliferation of osteoblast cells was not observed. Moreover, the change in DNA concentration for the AZ31-HA and AZ31-HA-LZ coated substrate is insignificant, thereby indicating that the osteoblast cell proliferation was not affected by lysozyme in the HA-LZ composite coatings. Furthermore, the ALP activity of osteoblast cells cultured on the uncoated and coated AZ31 substrate for 14 days is presented in Fig. 7. The ALP activity of osteoblast cells cultured on HA coated substrates (i.e. AZ31-HA, AZ31-HA-LZ-10% and AZ31- HA-LZ-20%) 16 ACS Paragon Plus Environment

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demonstrated significantly higher activity as compared to AZ31, AZ31-LZ-10% and AZ31LZ-20% substrates. However, the ALP activity of AZ31-HA coated surface was significantly higher when compared to the HA-LZ coated equivalents particularly on day 7. The decrease in the ALP activity of the HA-LZ coated surface is likely due to the presence of lysozyme itself as the similar trend was also observed for AZ31-LZ-10% and AZ31-LZ-20% coated substrates. This may indicate that the level of ALP activity is affected by the presence of lysozyme. However, at a later stage of differentiation (day 14), the ALP activity of HA-LZ coated substrate is comparable to the HA coated equivalent and significantly higher when compared to AZ31 Mg alloy alone. Bone regeneration is a complex process which involves the early stages of cell adhesion and proliferation, and later stages of cell differentiation and function

39

. From this study, it can be observed that HA-LZ coated surface showed

comparable and enhanced cell adhesion and proliferation when compared to HA-coated and uncoated AZ31 equivalents respectively (Fig. 5 and 6). Osteoblast cells express ALP in the early stage of osteoblast differentiation and participate in the mineralisation of the extracellular matrix through the generation of phosphate ions from the hydrolysis of organic substrates 40. HA is one of the major components of the extracellular matrix (ECM) of many cells including osteoblasts which not only plays a structural role in the bone matrix but regulates cell adhesion, proliferation, migration, wound healing and expression of osteoinductive factors including ALP. It also possesses the receptors for CD 44 (cell surface glycoprotein) and RHAMM (receptor for HA-mediated motility) which promote the differentiation of osteoblast cells 41,39. Therefore, the decrease in the ALP activity on HA-LZ composite coated substrate in the early stage of differentiation is likely due to the interference in the above-mentioned interactions of osteoblast cells with the HA due to the lysozyme component. However, an in-depth study will require to elucidating the mechanisms regulating the differentiation of osteoblast cells on such composite coatings.



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4

Conclusion

In this study, the HA-LZ composite was functionalised onto a silane-based corrosion resistant coating on the AZ31 Mg alloy. AFM, contact angle measurement and quantification of hyaluronic acid and lysozyme immobilised on the surface confirmed the successful functionalisation of the coatings. Among all coated substrates, the AZ31-HA-LZ-20% substrate showed a significant higher bactericidal activity against S. aureus bacteria without greatly compromising the cytocompatibility of osteoblasts. Improved cell adhesion with flattened cuboidal morphology, proliferation and differentiation of osteoblast cells was observed on the AZ31-HA-LZ composite coated substrates relative to the uncoated AZ31 and AZ31-LZ substrates. At the later stages of osteoblast differentiation, the HA-LZ and HA functionalised AZ31 substrates showed a similar level of ALP activity. Collectively, these results indicate that such composite coatings are of great interest and may prevent bacterial colonisation whilst enhance the cytocompatibility of AZ31 Mg alloy for orthopaedic implant applications.

5

Acknowledgement

The authors acknowledge Dublin Institute of Technology, Dublin for the financial support through Fiosraigh Scholarship Programme 2014 (to S.A.). This work was also supported by a European Research Council Grant 336882 (to D.A.H.); Science Foundation Ireland European Research Council (ERC) Support Grant SFI 13/ERC/L2864 (to D.A.H.).


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Biomimetic hyaluronic acid-lysozyme composite coating on AZ31 Mg alloy with combined antibacterial and osteoinductive activities Sankalp Agarwal, Mathieu Riffault, David Hoey, Brendan Duffy, James Curtin, Swarna Jaiswal

For Table of Contents Use Only


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Biomimetic hyaluronic acid-lysozyme composite coating on AZ31 Mg alloy with combined antibacterial and osteoinductive activities Sankalp Agarwal, Mathieu Riffault, David Hoey, Brendan Duffy, James Curtin, Swarna Jaiswal

For Table of Contents Use Only



1 2 3 4 5 6 7 8 Table 1: Summary of characterization parameters of different substrates. 9 10 11 12 Parameters AZ31 AZ31-MT-A AZ31-HA AZ31-HA-LZ-10% 13 14 74.5 ± 8.2 45.7 ± 0.82 4.86 ± 0.07 17.1 ± 1.84 15 Roughness (Ra, nm) 16 17 46.4 ± 2.1 41.24 ± 2.66 48.85 ± 1.07 Contact angle (degrees) 74.57 ± 0.92 18 19 2 20 ----48.42 ± 0.05 48.22 ± 0.10 HA (µg/ml/cm ) 21 22 Lysozyme activity 23 ------0.534 ± 0.042 2 24 (U/ml/cm ) 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 ACS Paragon Plus Environment 47 48

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AZ31-HA-LZ-20% AZ31-LZ-10% AZ31-LZ-20% 21.14 ± 6.45

24.3 ± 3.15

25.7 ± 7.3

51.20 ± 3.36

53.37 ± 5.29

55.67 ± 0.92

47.53 ± 0.05

---

---

0.588 ± 0.021

0.465 ± 0.03

0.513 ± 0.027

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Figures Fig.1: Surface morphology of uncoated and coated substrates was determined using AFM for (a) AZ31, (b) AZ31-HA, (c) AZ31-HA-LZ-10%, (d) AZ31-HA-LZ-20%, (e) AZ31-LZ-10% and (f) AZ31-LZ-20% substrates.

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Fig. 2: Viability of adhered S. aureus on different substrates was measured using CFU assay 2 h, 4 h and 12 h after immersion in bacterial cultures. One way ANOVA with posthoc Tukey test with significance level of ** p < 0.01, * p < 0.05.


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Fig. 3: S. aureus viability was measured by fluorescence using the PI exclusion assay following incubation on different substrates. One way ANOVA with posthoc Tukey test with significance level of ** p < 0.01, * p < 0.05.



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Fig. 4: Representative SEM images of S.aureus on (a) AZ31, (b) AZ31-HA, (c) AZ31-HALZ-10%, (d) AZ31-HA-LZ-20%, (e) AZ31-LZ-10% and (f) AZ31-LZ-20% substrates after 4h of incubation (Red, yellow and green arrows are indicating affected cells, cell debris and unaffected cells respectively)


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Fig. 5: (I) Representative SEM and (II) fluorescent images of the actin cytoskeleton of osteoblasts cells cultured on AZ31 (a-a`), AZ31-HA (bb`), AZ31-HA-LZ-10% (c-c`), AZ31-HA-LZ-20% (d-d`), AZ31-LZ-10% (e-e`) and AZ31-LZ-20% (f-f`) substrates for day 1 respectively. Actin cytoskeleton was stained with Phalloidin (green) and nuclei were stained with DAPI (blue). (Scale bar of SEM images is 50 µm)



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Fig. 6: Total DNA quantification per cm2 was measured following culture of osteoblast cells on AZ31, (b) AZ31-HA, (c) AZ31-HA-LZ-10%, (d) AZ31-HA-LZ-20%, (e) AZ31-LZ-10% and (f) AZ31-LZ-20% substrates for 3, 7 and 14 days respectively, One way ANOVA with posthoc Tukey test with significance level of ** p < 0.01, * p < 0.05.


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Fig. 7: Intracellular ALP activity was used to measure the differentiation of osteoblast cells cultured on AZ31, (b) AZ31-HA, (c) AZ31-HA-LZ-10%, (d) AZ31-HA-LZ-20%, (e) AZ31LZ-10% and AZ31-LZ-20% substrates for 3, 7 and 14 days respectively, One way ANOVA with posthoc Tukey test with significance level of ** p < 0.01, * p < 0.05.



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Figures Fig.1: Surface morphology of uncoated and coated substrates was determined using AFM for (a) AZ31, (b) AZ31-HA, (c) AZ31-HA-LZ-10%, (d) AZ31-HA-LZ-20%, (e) AZ31-LZ-10% and (f) AZ31-LZ-20% substrates.


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Fig. 2: Viability of adhered S. aureus on different substrates was measured using CFU assay 2 h, 4 h and 12 h after immersion in bacterial cultures. One way ANOVA with posthoc Tukey test with significance level of ** p < 0.01, * p < 0.05.



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Fig. 3: S. aureus viability was measured by fluorescence using the PI exclusion assay following incubation on different substrates. One way ANOVA with posthoc Tukey test with significance level of ** p < 0.01, * p < 0.05.


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Fig. 4: Representative SEM images of S.aureus on (a) AZ31, (b) AZ31-HA, (c) AZ31-HALZ-10%, (d) AZ31-HA-LZ-20%, (e) AZ31-LZ-10% and (f) AZ31-LZ-20% substrates after 4h of incubation (Red, yellow and green arrows are indicating affected cells, cell debris and unaffected cells respectively)



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Fig. 5: (I) Representative SEM and (II) fluorescent images of the actin cytoskeleton of osteoblasts cells cultured on AZ31 (a-a`), AZ31-HA (bb`), AZ31-HA-LZ-10% (c-c`), AZ31-HA-LZ-20% (d-d`), AZ31-LZ-10% (e-e`) and AZ31-LZ-20% (f-f`) substrates for day 1 respectively. Actin cytoskeleton was stained with Phalloidin (green) and nuclei were stained with DAPI (blue). (Scale bar of SEM images is 50 µm)


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Fig. 6: Total DNA quantification per cm2 was measured following culture of osteoblast cells on AZ31, (b) AZ31-HA, (c) AZ31-HA-LZ-10%, (d) AZ31-HA-LZ-20%, (e) AZ31-LZ-10% and (f) AZ31-LZ-20% substrates for 3, 7 and 14 days respectively, One way ANOVA with posthoc Tukey test with significance level of ** p < 0.01, * p < 0.05.



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Fig. 7: Intracellular ALP activity was used to measure the differentiation of osteoblast cells cultured on AZ31, (b) AZ31-HA, (c) AZ31-HA-LZ-10%, (d) AZ31-HA-LZ-20%, (e) AZ31LZ-10% and AZ31-LZ-20% substrates for 3, 7 and 14 days respectively, One way ANOVA with posthoc Tukey test with significance level of ** p < 0.01, * p < 0.05.


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Biomimetic hyaluronic acid-lysozyme composite coating on AZ31 Mg

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