The Interplay between Surface Nanotopography and Chemistry

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The Interplay between Surface Nanotopography and Chemistry Modulates Collagen I and III Deposition by Human Dermal Fibroblasts Akash Bachhuka, John Dominic Hayball, Louise E. Smith, and Krasimir Vasilev ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b15932 • Publication Date (Web): 03 Feb 2017 Downloaded from http://pubs.acs.org on February 6, 2017

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ACS Applied Materials & Interfaces

The Interplay between Surface Nanotopography and Chemistry Modulates Collagen I and III Deposition by Human Dermal Fibroblasts Akash Bachhuka1,2, John Dominic Hayball3, Louise E. Smith1, 4*, Krasimir Vasilev1, 4* 1Future Industries Institute, University of South Australia-SA 5095 2ARC Centre of Excellence for Nanoscale Biophotonics, Institute for Photonics and Advanced Sensing, School of Physical Sciences, The University of Adelaide, Adelaide, SA, 5005, Australia 3Experimental Therapeutics Laboratory, Sansom Institute and Hanson Institute, School of Pharmacy and Medical Science, University of South Australia, Adelaide, SA, 5000, Australia 4School of Engineering, University of South Australia–SA 5095 *Corresponding author email address: [email protected] *Corresponding author email address: [email protected]

KEYWORDS: biomaterials, plasma polymerization, surface nanotopography, collagen I and III, foreign body response.

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ABSTRACT The events within the foreign body response are similar to, but ultimately different from the wound healing cascade. Collagen production by fibroblasts is known to play a vital role in wound healing and device fibrous encapsulation. However, the influence of surface nanotopography on collagen deposition by these cells has not been reported so far. To address this gap, we have developed model substrata having surface nanotopography of controlled height of 16, 38 and 68 nm and tailored outermost surface chemistry of amines, carboxyl acid and pure hydrocarbon. Fibroblast adhesion was reduced on nanotopographically modified surfaces compared to the smooth control. Furthermore, amine and acid functionalized surfaces showed increased cell proliferation over hydrophobic hydrocarbon surfaces. Collagen III production increased from day 3 to day 8 and then decreased from day 8 to day 16 on all surfaces, whilst collagen I deposition increased throughout the duration of 16 days. Our data shows that the initial collagen I and III deposition can be modulated by selecting desired combinations of surface nanotopography and chemistry. This study provides useful knowledge that could help in tuning fibrous capsule formation and in turn govern the fate of implantable biomaterial devices.


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INTRODUCTION Surface nanotopography and chemistry are critical factors in integration of implanted devices into tissues and in the satisfactory resolution of the wound healing process.1-3 However, at present, the majority of studies focus on the influence of topography on the FBR and wound healing by modulating the innate immune response4-6 and only very few interrogate the influence of surface nanotopography on the deposition of collagen or other extracellular matrix (ECM) proteins.7 After implantation, the surface is immediately coated by a corona of serum proteins. Smaller molecular weight and more highly concentrated proteins adsorb first, but may be displaced by other proteins, typically of a higher molecular weight.8 It is to these adsorbed proteins that haemopoetic and non-haemopoetic cells interact and attach. Neutrophils and macrophages arrive to ‘clean the site’9-14 and the macrophages can fuse to form foreign body giant cells (FBGC’s). These FBGCs secrete reactive oxygen and nitrogen species (RONS) in an attempt to eliminate the foreign body. In parallel a wound healing response is initiated, the magnitude of which is directly proportional to the injury inflicted on the site during implantation. The initial phases of wound healing including haemostasis and acute inflammation are part of the FBR. When investigating collagen deposition in and around implants the phases of cellular recruitment and proliferation and tissue remodeling are of greater interest. During the proliferation phase of wound healing, activated fibroblasts and other stromal cells are recruited to the area and a provisional matrix is deposited. This granulation tissue consists primarily of collagen III and during tissue remodeling it is slowly resorbed by fibroblasts and replaced with the stronger and more organized collagen I. Foreign body giant cells are trapped


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within the collagen matrix that is deposited around the implant and they continue to secrete RONS as well as pro-inflammatory cytokines resulting in chronic inflammation which can result in implant failure.15 Today, it is well understood and recognized that controlling the FBR and FBGC formation is a key to improving the function of implantable medical devices and attempts to modulate FBGC formation focuses mainly on manipulating the immune cells recruited to the site using steroids and other pharmacological agents including growth factors.16-19 The aim of this work is to shed light on the interplay between surface nanotopography and surface chemistry and the deposition of collagens I and III. The hypothesis behind this study is that surface nanotopography and chemistry can be important regulators in collagen deposition. This hypothesis is substantiated by our earlier studies demonstrating that surface chemistry alone can modify the amount and type of collagen deposited by human dermal fibroblasts20 and that surface nanotopography can modulate fibroblast adhesion and spreading.21 Herein, model substrata with precisely controlled nanotopography in terms of size, height and lateral spacing were generated. Importantly, we were also able to accurately tune and tailor the outermost surface chemistry using plasma polymer layers of controlled thickness and chemical composition. The key benefit of utilizing plasma deposition is that a diverse range of pinholefree, conformal coatings can be produced in a single-step process on any type of substrate material as wells as a range of advanced nanoscale structures.22-26 Human dermal fibroblasts were grown on these model substrata for up to 16 days and the quantity of collagen I and collagen III deposited was analyzed and we found that fibroblasts initially adhered better on smooth surfaces compared to nanotopographically modified substrates with the same chemistry. Furthermore, we demonstrate that a combination of nanotopography and chemistry can be used to modulate initial collagen I and III deposition.


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MATERIALS AND METHODS Materials Allylamine (AA) (98%, Aldrich), acrylic acid (AC) (99%, Aldrich), octadiene (OD) (98%, Aldrich), hydrogen tetrachloroaurate (99.9985%, ProSciTech), trisodium citrate (99%, BHD Chemicals, Australia Pty. Ltd.), 2-mercaptosuccinic acid (97%, Aldrich), were used as received. Plasma polymerization A custom-built reactor with a 13.56 MHz plasma generator was employed for plasma polymerization.26 Oxygen plasma was employed for 2 minutes at 50W to clean all substrates. The vapors of allylamine, acrylic acid and octadiene were used to deposit polymer film of thicknesses 23 nm, 20 nm and 25 nm respectively. For polymer deposition, a pressure of 0.2 mbar and a deposition time of 2 minutes was employed. The power used for deposition was 40 W, 10 W and 20 W for all three monomers respectively. To achieve an overcoating of 5 nm’s the deposition time for all reactions was kept constant at 20 seconds. Synthesis of gold nanoparticles (AuNP’s) Gold NP’s were synthesized using hydrogen tetrachloroaurate (HAuCl4). Particles of 16, 38 and 68 nm diameter were synthesized by varying the amount of 1% trisodium citrate from 1ml to 0.3 ml respectively.27 Surface modification of these nanoparticles was performed using 2mercaptosuccinic acid.28 Atomic force microscopy An NT-MDT NTEGRA SPM atomic force microscope (AFM) was used in non-contact mode to provide nanotopographical images. Silicon nitride non-contact tips coated with Au on the reflective side (NT-MDT, NSG03) were used and had resonance frequencies between 47 and 150


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kHz. The amplitude of oscillation was 10 nm, and the scan rate for 5 µm x 5 µm images was 0.5 Hz. X-ray photoelectron spectroscopy XPS was used to determine the surface composition of the plasma polymers with the deposited AuNP’s. All spectra were recorded using a Spec SAGE XPS spectrometer equipped with a monochromatic Mg radiation source operated at 10 kV and 20 mA. Atomic compositions of the samples were identified from survey spectra recorded over a 0-1000eV with pass energy of 100 eV at a resolution of 0.5 eV. All binding energies (BE) were corrected relative to a neutral C1s carbon peak at 285.0 eV. Processing and curve fitting was performed using CasaXPS. Cell Culture and Attachment Human dermal fibroblasts (HDFs) were harvested and grown as described previously29 from split thickness skin samples obtained from specimens following routine breast reductions and abdominoplasties. All patients gave informed consent for skin to be used for research through a protocol approved by the Ethical Committee at the Queen Elisabeth Hospital and the University of South Australia Human Ethics Committee. Fibroblasts were grown in fibroblast culture medium (FCM) consisting of DMEM high glucose (Gibco, Life Technologies, Australia), 10% v/v fetal calf serum (FCS, Ausgenex, Australia), 100 IU/mL penicillin and 100µg/mL streptomycin (Gibco, Life Technologies, Australia) in an incubator at 37 °C, 5% CO2 in a humidified atmosphere. The medium was changed every 3–4 days until the cells were 80% confluent. Fibroblasts were used between passages 3 and 9.


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Cell Seeding Glass coverslips with different nanotopography and chemistries were placed into 24 well plates. 50 x 103 cells were carefully seeded in 500 µl FCM and left for 1 day. The media was changed and replaced with crowding media containing ascorbic acid (1 mM), ficoll 70 (37.5 mg/ml) and ficoll 400 (25 mg/ml) as these have been reported to enhance collagen deposition by fibroblasts.30-32 The media was filter sterilized and changed every 3 days till day 16. Collagen Staining Collagen I and III were stained after fibroblasts had been grown for 3, 8 and 16 days. Samples were washed thrice with PBS and then were fixed in ice cold methanol and left to dry. The dried plates were then washed thrice with PBS. The samples were blocked using 1% bovine serum albumin (BSA, Sigma Aldrich) in PBS)-polyethylene glycol sorbitan monolaurate (Tween 20 solution) (0.01% Tween-20 in PBS, Sigma Aldrich) for 1 hour. The plates were again washed thrice with PBS and a solution of primary antibody (mouse monoclonal anti-collagen I and III antibody (Sigma Aldrich) (1:1000) in blocking media was added for 90 minutes at room temperature. The plates were washed thrice with PBS and were incubated at room temperature using secondary antibody (Alexa flour 647 F (ab’) 2 fragment of goat anti – mouse IgG (H+L)) (Molecular probes, Life Technologies, Australia) (1:400) and counterstained with 4´, 6diamidino-2-phenylindole (DAPI, Molecular probes, Life Technologies, Australia, λex 350 nm, λem 470 nm) (staining cell nuclei) (1:400) for 1 hour in blocking media. The plates were washed thrice with PBS and then the coverslips were carefully mounted on glass slides using glycerol.


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Confocal Microscopy Confocal microscopy was used for qualitative and quantitative analysis of collagen I and III. Glass coverslips mounted on glass slides were carefully kept on the sample holder. A laser of 405nm wavelength was employed for visualization of the cell nuclei while a 640 nm wavelength laser was employed to visualize the collagen I and III. All samples were scanned with a 10x objective and all acquisition parameters (laser power, pinhole size and gain) were kept constant for all samples. These images were analyzed using NIS-Element AR software for calculating the number of cells and area covered by collagen per cell. Statistical Analysis All statistics were performed using graph pad prism 6 software. All data was expressed as mean ± standard error mean (SEM). Statistical significance was determined using a 1 way and a 2-way ANOVA followed by Tukey’s multi comparison test. All experiments were performed twice in triplicates. 2 confocal images per sample were captured for quantitative analysis.


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RESULTS Generation of controlled surface nanotopography

Figure 1. Analysis of AFM data a) 3D Images of nanoparticles immobilized on AApp modified surface. Analysis of the surface nanotopography in terms of: (b) Number of particles per µm2, (c) % Increase in surface area, (d) % Surface Coverage, (e) Root mean square roughness, (f) Interparticle distance (nm).


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In order to generate nanotopographically modified surfaces, glass coverslips were first coated with a 20 nm, thin, layer of plasma polymerized allylamine (AApp). These AApp coated surfaces were immersed in a solution of nearly monodispersed gold nanoparticles of three different sizes (16, 38 and 68 nm). The AApp surfaces are positively charged upon immersion in aqueous medium due to the availability of a significant population of primary amines.33-35 The nanoparticles are capped with carboxylic acid functional 2-mercaptosuccinic acid (MSA) and thus acquire a negative charge in water.28 These opposite charges facilitate electrostatic binding between the gold nanoparticles and the AApp coated surfaces. Therefore, the resultant surfaces have a mixed chemistry resulting from the nitrogen rich AApp and the carboxylic acid functionalities of the gold nanoparticles. This is a problem for biological, and other studies, since such surface composition would not allow discrimination between the effects of chemistry and nanotopography. In order to ensure a uniform outermost surface chemistry on the nanotopographically modified surfaces, an overcoating of a 5 nm thin layer of plasma polymer generated from vapor of either allylamine, acrylic acid or octadiene (AApp, ACpp and ODpp) was deposited. These types of plasma polymer coatings were chosen because they represent chemical compositions that are abundant in biological tissue i.e. NH2 (AApp), COOH (ACpp) and CH3 (ODpp). Furthermore these coatings present different surface charges in aqueous medium as AApp is positively charged, ACpp has a net negative charge and ODpp carries no charge.34 Respectively, the different chemistries result in different wetting characteristics, described by typical water contact angles of 85◦ for ODpp, 60◦ for AApp and 35◦ for ACpp.36 The plasma polymer films, of only 5 nm in thickness, are demonstrated to be continuous and pinhole free37 and allow us to preserve the nanotopography generated by the gold nanoparticles. AFM images of these


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nanotopographically modified surfaces before and after overcoating are shown in Figure 1a. The images demonstrate that the particles are well dispersed on the surface and do not form aggregates. Furthermore, the overcoating used to tailor the outermost surface chemistry does not affect particles distribution or surface morphology. Figure 1b shows the number of particles per µm2 for of different nanotopographically modified surfaces calculated from the AFM images using ‘Image J’ software (U. S. National Institutes of Health, USA). The number of surface bound particles was 162, 82 and 21 per µm2 for nanoparticles size of 16 nm, 38 nm and 68 nm, respectively. Figure 1c shows the percentage increase in surface area which was calculated based on the surface area covered by the total number of nanoparticles. It increased from 13% to 36% which corresponded with increase in the size of nanoparticles from 16 to 38nm. However, it decreased from 36% to 30% when the nanoparticle size increased from 38 to 68 nm. Figure 1d shows the percentage surface coverage which was calculated based on the area covered by the circumference of the nanoparticles. Surface coverage increased from 3% to 9% with an increase in the size of nanoparticles from 16 to 38 nm. However, it decreased from 9% to 7.5% when the nanoparticles increased in size from 38 to 68 nm. Figure 1e shows the root mean square roughness (RMS) for surfaces with different size of nanoparticles. This was calculated using NTMDT Ntegra software. The plot shows that as the size of the nanoparticles increases so does the roughness. Therefore, with an increase in the height of nanoparticles from 16 to 68 nm, RMS increases from 5 nm to 15 nm respectively. Figure 1f shows the interparticle distance between different nanoparticles on the same surface. The interparticle distance increased from 72 nm to 179 nm with an increase in the size of nanoparticles from 16 to 68 nm.


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Characterization of surface chemistry

Figure 2. XPS survey spectra showing the surface chemical composition of unmodified glass coverslip (GCS) (a), and AApp coated GCS modified with (b) 16 nm (c) 38 nm (d) 68 nm Au nanoparticles. XPS analysis of GCS after application of an additional ACpp overcoating on the outermost surface of bare GCS (e), and GCS modified with AuNPs of size 16 nm (f), 38 nm (g), and 68 nm (h). XPS was used to analyze the elemental composition of the unmodified and modified surfaces as shown in Figure 2. Compared to uncoated glass cover slip (Figure 2a), two additional peaks corresponding to gold (Au4f) and nitrogen (N1s) appear after modification with AApp and gold nanoparticles immobilization (Figure 2 b-d). The disappearance of the Si signal demonstrates that the AApp coating is continuous, pinhole free and of sufficient thickness to completely


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prevent Si2p photoelectron ejection from the glass surface. The appearance of the nitrogen peak is due to the deposition of the layer of allylamine, which aids in electrostatic immobilization of different sized gold nanoparticles. The gold peak is due to the presence of the immobilized gold nanoparticles. These modified surfaces have mixed chemistries generated due to the carboxylic acid capped nanoparticles and the underlying amine layer. In order to generate homogeneous surface chemistry with defined nanotopography these modified surfaces were overcoated with a 5 nm thin layer of plasma polymerized ACpp, ODpp or AApp. The survey spectra demonstrating the elemental composition of nanoparticles overcoated with ACpp are shown in Figure 2f-h while elemental composition of ODpp and AApp overcoated substrates are shown in Figure S1. Due to the 5 nm overcoating of ACpp (Figure 2f-h) both the nitrogen and gold atomic concentrations have decreased whilst the atomic concentration of the carbon and oxygen peaks have increased. However, it was still possible to detect gold, XPS has an accepted sampling depth of 10 nm, which is greater than that of the overcoat employed. To give a greater overview of the atomic percentage of different elements present, before and after modification of these surfaces, a detailed table showing surface atomic composition of all model substrata used in this study is shown in Table S1. In this table an increase in N/C ratio was observed after overcoating with AApp, which is due to the presence of more N1s in AApp. In the case of the ODpp and ACpp overcoat, the N/C ratio decreased whilst the O/C ratio increased.


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The addition of nanotopography increases collagen I deposition

Figure 3. a) Laser scanning confocal microscopy analysis a(i-xii) Representative images of human dermal fibroblasts and deposited collagen I on chemically modified (ACpp) and


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nanotopographically modified surfaces (16ACpp, 38ACpp and 68ACpp) at days 3, 8 and 16. (Blue: nucleus/DAPI; Pink/Red: collagen I). All images have dimensions of 1300 x1300 µm. Quantitative analysis of collagen I deposition obtained from laser scanning confocal microscope images. b), c) and d) show the mean intensity of collagen I deposited on chemically and nanotopographically modified surfaces at days 3, 8 and 16 respectively. e), f) and g) show a comparison of the mean intensity of collagen I between days 3, 8 and 16 on the modified surfaces. * = p

The Interplay between Surface Nanotopography and Chemistry

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