Biocompatible Mesoporous Nanotubular Structured Surface to Control

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Biocompatible mesoporous nanotubular structured surface to control cell behaviors and deliver bioactive molecules Kapil Dev Patel, Chinmaya Mahapatra, Guang-Zhen Jin, Rajendra Kumar Singh, and Hae-Won Kim ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b09114 • Publication Date (Web): 12 Nov 2015 Downloaded from http://pubs.acs.org on November 13, 2015

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ACS Applied Materials & Interfaces

Biocompatible Mesoporous Nanotubular Structured Surface to Control Cell Behaviors and Deliver Bioactive Molecules

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Kapil D. Patel , Chinmaya Mahapatra , Guang-Zhen Jin , Rajendra K. Singh , Hae-Won Kim

1,2,3,*

1

Institute of Tissue Regeneration Engineering (ITREN), Dankook University, Cheonan 330-714, South Korea

2

Department of Nanobiomedical Science & BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook

University, Cheonan 330-714, South Korea 3

Department of Biomaterials Science, School of Dentistry, Dankook University, Cheonan 330-714, South Korea

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*

Correspondence to Prof. H.-W. Kim

tel) +82 41 550 3081; fax) +82 41 550 3085; e-mail)[email protected]

For: ACS Applied Materials & Interfaces

ACS Paragon Plus Environment


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Abstract: Biocompatible nanostructured surfaces control the cell behaviors and tissue integration process of medical devices and implants. Here we develop a novel biocompatible nanostructured surface based on mesoporous silica nanotube (MSNT) by means of an electrodeposition. MSNTs, replicated from carbon nanotubes of 25 nm x 1200 nm size, were interfaced in combination with fugitive biopolymers (chitosan or collagen) onto a Ti metallic substrate. The MSNT-biopolymer deposits uniformly covered the substrate with weight gains controllable by the electrodeposition conditions. Random nanotubular networks were generated successfully, which, alongside the high mesoporosity, provided unique nanotopological properties for the cell responses and the loading / delivery of biomolecules. Of note, the adhesion and spreading behaviors of mesenchymal stem cells (MSCs) were significantly altered, revealing more rapid cell anchorage and extensive nano-filopodia development along the nanotubular networks. Furthermore, the nanotubular surface improved the loading capacity of biomolecules (dexamethasone and bovine serum albumin) up to 5-7 times. The release of the biomolecules was highly sustained, exhibiting a diffusion-controlled pattern over 15 days. The therapeutic efficacy of the delivered biomolecules was also confirmed in the osteogenic differentiation of MSCs. While in vivo performance and applicability studies are needed further, the current biocompatible nanostructured surface may be considered as a novel bio-interfacing platform to control cellular behaviors and biomolecular delivery.

Keywords: Nanostructured coating; Electrodeposition; Mesoporous nanotubes; Implants; Drug loading; Cell adhesion


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1.

Introduction

How to control the bio-interfaces that link the materials with biological components including proteins and cells has been a fundamental issue to improve the success of the medical devices and implants [1, 2]. Interface properties such as chemistry, topology and rigidity can alter the reactions of biomolecules and the responses of cells, which ultimately is determinant to tissue healing and integration [3-5]. Tissue cells that are engaged in repair and regenerative events, such as progenitor and stem cells, have recently been shown to favor the interfaces that are tailored with nano-topologies and biomimetic compositions. For example, mesenchymal stem cells (MSCs) recognize the nano-patterned or nano-tubular structured surface of metallic implants and adopt their behaviors towards more osteogenic lineage [6-9]. A fine control over the physical and chemical nature of the medical interfaces can thus stimulate favorable functions of cells.

In principle, the strategies to control interfacial properties are either i) removal or etching the material surface or ii) deposition or functionalization with exogenous materials or molecules. The latter can provide additional materials properties such as bioactivity to the underlying substrates while combining and incorporating functional groups or therapeutic molecules to accelerate cell responses and tissue healing process [10, 11]. When the medical devices have the capacity to elicit drugs and growth factors the healing and repair reactions can be substantially accelerated [12-14]. However, the proper loading of therapeutic molecules safely at high quantities, as well as the release of them in a sustainable and controllable manner, should be considered in the design [15, 16]. Therefore, the role of deposited interfaces is two-fold; i) in stimulating cells through the controlled physico-chemical properties, and ii) in therapeutically functioning with the delivery of drugs or bioactive molecules.

For this, here we propose a novel nanostructured interface that is biocompatible and holds a capacity to deliver biomolecules. The interfacing material is mainly composed of mesoporous silica nanotubes (MSNTs) which replicated from the carbon nanotubes (CNTs). The mesoporous silica composition has been shown to be biocompatible as the interfacing material of metallic implants, finding usefulness in the culture of a variety of cells



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[17, 18]. The nanotubular form of this mesoporous silica is considered to provide the surface with unique nanotopological cue, and the nanotubular network has previously been shown to accelerate cellular anchorage and to induce tubular guidance of filopodia [6]. As the template material of MSNTs, the CNTs are considered to offer a well-defined nanotubular structure, on which a thin silica layer is easily formable by a simple sol-gel reaction [19, 20] and the inner CNT part can be ultimately removed by a thermal treatment, possibly to generate CNT-replicated MSNTs. A small amount of biopolymers is also used as the fugitive (binder) of the nanotubular networks to enhance structural integrity. We apply an electrophoretic deposition method to implement the nanostructure interfacing onto a metallic substrate. The electrophoretic deposition has been proven to be effective in depositing charged nanomaterials including nanoparticles, nanorods and nanotubes, upon the electrically conductive surfaces like metals [21-23]. The method is simple yet can generate a thin uniform film of nanomaterials with various compositions, thus is considered to be properly applied to the current nanotubular MSNTs combined with charged biopolymers.

The performance of the electrophoretic-deposited nanotubular-structured interface is addressed in terms of initial cellular events, as well as the loading and delivery of bioactive molecules, with which a novel therapeutic nanointerfacing platform can be considered.

2.

Experimental Section

2.1.

Materials reagents

Chitosan (85% deacetylated, Mw = 200000 Da, Sigma-Aldrich, USA), multiwall carbon nanotubes (MWCNTs) (EMPower Co., Korea), H2SO4 (90%, Reagent Duksan, Korea), HNO3 (70% Sigma-Aldrich, USA), acetic acid (SigmaAldrich, USA), Ti plate (commercially pure grade, Senulbio Biotech, Korea), tetraethyl orthosilicate (98% SigmaAldrich, China), hexadecyltrimethylammonium bromide (≥98% Sigma-Aldrich, China), dexamethasone 21-


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phosphate disodium salt (Sigma-Aldrich, China), albumin from bovine serum (Sigma-Aldrich, USA), toluene (99.8 Sigma-Aldrich, USA), and 3-Aminopropyl) triethoxysilane (Sigma-Aldrich, China).

2.2.

Synthesis of mesoporous silica nanotubes and functionalization

The nanotubular form of mesoporous silica was prepared by using multi-walled carbon nanotubes (MWCNTs) as a template. For this, 4 g of pristine MWCNTs was functionalized with carboxylic group in the mixture of sulfuric acid and nitric acid at 1:1 in a two-neck round bottle flask, and kept at 80 °C for 4 days, under constant magnetic stirring for refluxing. The carboxylated MWCNTs were vacuum-filtered through a 0.2 μm mixed cellulose ester membrane, and thoroughly rinsed with deionized water, until the pH of the falling filtrate became neutral, which were then dried under vacuum at 50 °C for 24 h.

To enable mesoporous silica shell formation, a sol-gel polymerization of tetraethyl orthosilicate (TEOS) was implemented on the surface of MWCNTs, according to the protocol detailed elsewhere with slight modification [24]. A freshly prepared carboxylated MWCNTs (15 mg) was added to 180 ml of ethanol and dispersed completely by sonication. After a complete dispersion of MWCNTs, 20 mL water, 2.5 mL of a 25% ammonia solution, and 250 mg of hexadecyltrimethylammonium bromide (CTAB; used as a surfactant for the mesoporous structure) were added. Then, 200 µL TEOS in ethanol was added drop-wise to the solution while stirring at 1200 rpm for 5 h at room temperature. The solution was decanted and the silica-shelled MWCNTs were washed with ethanol (x3) and distilled water (x3) and finally with ethanol, and then dried at room temperature overnight. The process resulted in the formation of a uniform layer of silica on MWCNT. Furthermore, the silica-shelled MWCNTs were heat-treated -1

at 700 °C for 5 h at a ramping rate of 1 °C min to remove the MWCNT template, resulting in MSNTs. Selectively (the samples used for the deposition with collagen biomolecule), the surface of the MSNTs was functionalized with 3-aminopropyl triethoxysilane (APTES). For this, 50 µg of MSNTs were re-suspended in 50 ml of toluene with sonication and 1 ml APTES added and refluxed at 80 °C for 24 h. After the amination, the solution was centrifuged at 12000 rpm and dried at 80 °C.



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2.3.

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Interfacing onto a metallic surface by electrodeposition of MSNTs

To enable the interfacing of metallic surface with the MSNTs, electrophoretic deposition was carried out. Titanium discs were prepared by polishing with a 2000-grit SiC sand paper, followed by a ultrasonication in acetone/ethanol and drying. The deposition solution was made of the MSNTs dispersed at 0.15 mg/ml concentration and the biopolymer molecules (chitosan used as a fugitive material) within water-ethanol solvent with vigorous sonication. The concentrations of MSNTs and chitosan in the deposition solutions were varied to optimize the nanostructure formation, and the solution pH was adjusted to 3.5. The suspensions were kept stirring for 2-3 h and sonicated in ice-water prior to the deposition process. For the optimal conditions, the deposition parameters (time and voltage) were determined by a trial and error method.

2.4.

Sample characterizations

The nanostructure morphology of the MSNTs was observed by transmission electron microscopy (TEM; JEOL-7100). The surface charge property of the samples was measured using a Zetasizer Nano from Malvern Instrument. Zeta o

potential was measured at pH = 7.0 at 25 C. The mesoporosity of the samples, including specific surface area and pore volume, were determined by N2 gas adsorption/desorption, using the Brunauer–Emmett–Teller (BET) method. The pore size distribution was obtained by the Barret-Joner-Halenda (BJH) method. The surface nanomorphology was observed by field-emission scanning electron microscopy (FE-SEM; Tescan Mira II LMH) at an accelerating voltage of 25 kV, after sputtering with gold. The phase of the samples was determined by X-ray o

o

diffraction (XRD; Ragaku). The samples were scanned in the range of diffraction angle 2θ = 5-60 at a rate of 2 /min o

with a step width of 0.02 2θ using Cu Kα1 radiation at 40 kV and 40 mA current strength. Attenuated total reflection Fourier–transform infrared (ATR-FTIR; Varian 640-IR) spectroscopic analysis was performed to analyze -1

-1

the chemical structure, over a range of 400-2000 cm at a resolution of 4 cm . Atomic force microscopic (AFM; Agilent 5500 series) analysis was performed to visualize the nanotopological images.


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The in vitro bioactivity of the nanostructured surface was investigated by means of an apatite mineral formation in a simulated body fluid [15,25]. The simulated body fluid medium consists of NaCl 142.0 mM, NaHCO3 4.2 mM, KCl 5.0 mM, K2HPO4.3H2O 1.0 mM, MgCl2.6H2O 1.5 mM, CaCl2 2.5 mM, and Na2SO4 0.5 mM, which is buffered at pH 7.4 with HOCH2CNH2 and 1N HCl. Each sample was soaked in 10 ml of the simulated body fluid at 37 ° C, for different periods. The weight change of the samples during the soaking period was measured. The samples taken out from the solution was investigated by means of the surface morphological change using FE-SEM, and chemical structural change using ATR FT-IR.

2.5.

Cell culture and initial adhesion assays

For the in vitro cellular responses to the nanostructured interfaces mesenchymal stem cells derived from rat bone marrow were used [26,27]. All protocols involving animals were conducted according to the guidelines approved by the Animal Ethics Committee of Dankook University. Rats (5-week–old male Sprague Dawley) were sacrificed by decapitation, after which the bone marrow was aspirated from the tibiae and femurs in Hank’s balanced salt solution (Gibco, Franklin Lakes, NJ) containing 0.1% collagenase Type I and 0.2% dispase II, and the mononuclear 3

cells were then discarded by a centrifugation at 1500 rpm. Next, the cells were plated at a density of 2 x 10 cells -2

cm in two parallel culture dishes (one for proteomic analysis and the other for sub-cultures), and were cultured in a normal growth medium composed of α-minimal essential medium (α-MEM; WelGene) supplemented with 10% -1

-1

fetal bovine serum (Hyclone, Logan UT), 2 mM l-glutamine, 100 U ml penicillin and 100 ml ml streptomycin (all from Sigma-Aldrich) at 37 °C in a humidified atmosphere containing 5% CO2 for 7 days. Cells sub-cultured for 2–3 passages were used for the experiments. Prior to cell seeding, the samples for the cell culture were sterilized under UV light for 24 h, and then were washed twice with PBS and subsequently immersed in the culture medium.

The responses of the cells to the nanostructures were assessed by means of initial cell adhesion and spreading. The cells were seeded onto each sample (nanostructured or bare substrate, with a dimension of 10 mm x 10 mm x 1 4

mm) at a density of 1 × 10 cells per each well of 24-well plates. First, the cell distribution and morphology were



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monitored by means of staining cell nuclei and F-actins. After culturing for different periods (2, 6, 12 and 24 h), the cell images were obtained by fluorescence microscopy. For this, the cell-cultured samples were fixed with 4% paraformaldehyde, and then treated with 0.1% Triton X-100. The samples were blocked in 1% (w/v) BSA in PBS for 30 min to prevent non-specific protein binding, and then treated with 20 nM Alexa Fluor 546-conjugated Phalloidin (Invitrogen A22283) diluted in PBS to stain F-actins. The nuclei were also stained with 4’, 6-diamidino-2phenylindole (DAPI). The sample images were captured using a fluorescent microscopy (IX7151Olympus, Japan). Based on the images, the number of DAPI-positive cells was counted, and the cell spreading area was measured using the region of interest (ROI) manager tool in ImageJ 1.45s (NIH, USA). More than 220 cells for each sample were assessed for the quantification.

The distribution of a representative adhesive molecule, focal adhesion kinase (FAK) was also visualized. Cells positive for FAK were immunofluorescence-stained. Cells cultured for 4 h on each substrate were fixed in 4% paraformaldehyde and then stained with primary antibody included anti-FAK (phospho Y576; pFAK, abcam, USA) o

for 12 h at 4 C. The stained cells were then incubated with FITC-conjugated secondary antibody (Santacruz, USA). The cells were counterstained with DAPI to observe the nuclear morphology. Fluorescent images were observed under a LSM 700 laser-scanning confocal microscope. The electron images of cells were then examined by SEM. For this, the cell-cultured samples were washed with PBS, fixed in 4% paraformaldehyde, dehydrated with increasing concentrations of ethanol (50%, 75%, 90% and 100%), and then treated hexamethyldisilazane for 30 min.

2.6.

Biomolecular loading and release tests

The effects of the nanostructured surfaces on the loading and delivery of biomolecules were examined. As the model biomolecules, bovine serum albumin (BSA; Sigma-Aldrich) and dexamethasone 21-phosphate disodium salt (DEX; Sigma-Aldrich) were used. For the loading study, each biomolecule was prepared at different concentrations in PBS; 0.5~2.5 mg/ml for BSA and 10~100 mg/ml for DEX. The nanostructured or bare substrate sample was


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soaked in 1 ml of the solution, and then left at 37 °C for 6 h. After the soaking, samples were taken out, and the remaining solution was assayed for the measurement of biomolecules not loaded; BSA and DEX at 278 nm and 243 nm, respectively, using UV-visible spectrophotometer (Libra S22, Biochrom, UK). The amount of loaded molecules was then calculated by subtracting from the initial amount.

The release behaviors of BSA and DEX were then carried out. Each sample loaded with BSA or DEX (loading achieved at saturation point) was dispersed in 10 ml distilled water and then incubated at 37° C for different time periods. At predetermined time points, 1 ml was withdrawn from the release medium for the analysis using UVvisible spectrophotometer.

The biological effects of the released biomolecules were further analyzed. DEX-loaded samples were cultured with rMSCs, and the osteogenic differentiation was examined. For this, cells were plated in each well of 12-well plates while the disc sample, either DEX-loaded or not, was placed onto a Transwell membrane which was kept in each well of the cell-seeded 12-well plates. This enables the indirect interactions of rMSCs with the DEX molecules possibly released from the disc sample through the permeable Transwell membrane. As the osteogenic differentiation index, the enzymatic activity of alkaline phosphatase (ALP) was determined. After culture for 7, 14 and 21 days, cell lysates from each sample were obtained by three cycles of freezing-thawing and followed by an addition of cell lysis buffer (0.2% Triton X-100). The cell lysate samples (50 μl) were added to 50 μl of working reagent containing equal parts (1:1:1) of 1.5 M 2-amino-2 methyl-1-propanol, 20 mM p-nitrophenol phosphate, and 1 mM magnesium chloride, and the samples were then incubated at 37°C. The reaction was stopped with the addition of 100 μl of 1 N NaOH on ice, and the absorbance was determined at 405 nm using microplate-reader (iMark, BioRad, CA). The reading for the sample was determined from a standard ALP activity curve prepared using p-nitrophenol stock standard, and the data were normalized to the total protein content. Each test was performed on six replicate samples (n = 6).

2.7.

Statistical Analysis



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Data were presented as mean ± standard deviation (SD), and were analyzed using Student’s t-test. Difference was considered statistically significant at p < 0.05.

3.

Results and Discussion

3.1.

Mesoporous nanotubes and the formation of nanostructured surface

The characteristics of the MSNTs used for the nanostructure interfaces were examined. The TEM image of samples revealed a nanotubular morphology of the silica shelled CNTs (Fig. 1a), where the inner CNT and outer mesoporous silica layers were clearly separated. When the inner CNT was removed by a thermal treatment, the TEM image showed a hollowed tubular nanostructure. The dimension of nanotubes was measured to be 50 ± 2 nm (outer diameter) and 27 ± 2 nm (inner diameter). The XRD patterns showed only silica phase for both samples (Fig. 1b). The mesoporous properties of the MSNTs were analyzed by BET method. The N2 adsorption/desorption curve showed a hysteresis loop of type II isotherm, a characteristic of silica-based mesoporous nanomaterials [28], and also contained additional H1 type large-spaced loop at the relative pressure of 0.45~0.95. This large-spaced loop resulted from the existence of inner hollow space of nanotubes, and has also been observed elsewhere in the porous materials containing large pores or internal cavity [29-32]. The surface area and pore volume calculated 2

3

were 614 and 0.43 cm /g and m /g, respectively, as well as the mesopore size was characterized to be 3.77 nm, which demonstrating a high mesoporosity level of the MSNTs.

We used this nanomaterial platform of MSNTs for the surface tailoring of metals, through the electrophoretic deposition method. The electrophoretic deposition has been widely applied to tailor metallic implants and devices with various compositions to provide the surface improved biological properties [33, 34]. To enable this technique, the materials should be charged to move toward a substrate under an electrical field. Here we use MSNTs


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suspension with negatively charged, and moreover, introduce chitosan molecules. Chitosan, a type of natural polysaccharide that is highly-positive charged, has been deposited effectively on metallic surfaces via the electrophoretic coatings [35]. The series of interactions and deposition processes for chitosan molecules and MSNTs that are employed to form MSNT-based nanostructure coating system are schematically shown (Fig. 2a). The MSNTs mixed with biomolecules (either chitosan or collagen) are considered to form biomolecules-decorated MSNTs, which are then electrodeposited onto a metallic substrate; the biomolecules used at additive contents can play an adhesive role that networks the MSNTs. While the bare (hydroxylated) MSNTs were used for positivelycharged chitosan, the aminated-MSNTs were effective for negatively-charged collagen, which could enable chargecharge interactions between the biomolecules and MSNTs; in fact, the ζ-potential values of MSNTs were measured to be -23.8 ± 1.78 mV and +32.6 ± 1.24 mV, respectively. The nanostructure yield was recorded by means of a weight increase (Fig. 2b). The weight gain presented as a function of time and voltage showed an ongoing increase. The nanostructures of MSNT deposits produced with chitosan at various contents (MSNT:chitosan=10:1, 4:1, 4:3, and 2:3) were then examined by a high resolution SEM (Fig. 2c). The MSNTs-driven nanotubular morphology was preserved well when the chitosan content was smaller (MSNT:chitosan up to 4:3), which however, was not readily observed if the chitosan content was higher (MSNT:chitosan=2:3). When the chitosan molecules were small enough to decorate the MSNTs surface in the emulsion, the deposited structure could preserve the nanotubular topology of MSNTs, however, the excessive chitosan molecules (at higher content) remaining in the emulsion would be deposited to blunt the nanostructure. The AFM image also supports the generation of nano-topological surface enabled by the MSNT-chitosan (Fig. 2d). Likewise, collagen can also generate similar nanostructured morphologies at low contents (MSNT:collagen = 10:1 shown for a representative example). The ratio of MSNT:chitosan = 4:1 was used as the representative composition for further experiments. While here we used MSNTs that were replicated from CNTs with a size of 25 nm × 1200 nm for the deposition as this dimension was considered to form a typical nanotubular networked structure due to the large aspect ratio, the use of different size or morphology of MSNTs can also be possible to modify the surface with different nanotopologies that remains as further intriguing study.



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3.2.

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In vitro bioactivity and the early interactions with cells

The biological properties of the prepared MSNT-based nanostructured interface were then investigated. First, the in vitro acellular bioactivity assay, involving the apatite mineral induction in a simulated body fluid, was carried out. During incubation of the nanostructured sample for up to 7 days, the morphological changes were examined by SEM (Fig. 3a). Tiny mineral crystallites were formed, as early as at day 1, evenly on the nanotubular surface which thus could preserve the initial nanotopological pattern. At day 3, the crystal induction was more profound, thickening the nanotubular dimension yet largely exhibiting the nanotopological pattern. At day 7, a substantial level of mineral induction occurred, covering the whole surface with large mineral crystals. The phase of the minerals induced in simulated body fluid was characterized by XRD to be a poorly crystallized hydroxyapatite, which became stronger in peak intensity with increasing immersion time (Fig. 3b). Moreover, the ATR FT-IR spectra of samples revealed the phosphate chemical bands associated with apatite (Fig. 3c). The weight gain of the nanostructured surface, measured up to 30 days, reflects the substantial and continual apatite mineral induction with immersion time. The results suggest a high level of a cellular bioactivity of the nanostructured MSNTs, and this is considered to result from the combined effects of the high surface area (reaction sites) of MSNTs and the charged surface of chitosan, on which the calcium ions are initially attracted and then followed by the induction of phosphate ionic groups [6, 36,37].

We next addressed the interactions of the nanostructured surface with tissue cells. For this, rMSCs were cultured on the surface and the initial cellular events including cell anchorage and spreading behaviors were examined. The fluorescence images of cells, adhered to the surface over time sequence (2, 6, 12 and 24 h), were first gathered by a confocal microscopy (Fig. 4a). Cells adhered to the surfaces were well revealed (nucleus in blue and F-actin in green). Based on the images, the cell number anchored to the surfaces was counted at culture times (Fig. 4b). There was significantly higher level of cells adhered to MSNT surface than to bare substrate through the culture period. The difference between the two surfaces had already been achieved as short as 2 h, which then appeared to maintain up to 24 h. As another index, the cell spreading behavior was also examined. The cell spreading area (cytoskeleton/nucleus area) was quantified from the images (Fig. 4c). Interestingly, the cell spreading was initially


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(2 h) higher on the MSNT surface, which however, became reversed with increasing time. The cell spreading area for the MSNT surface appeared to almost saturate after 2 h, demonstrating the cells on this nanotubular surface spread very quickly to a certain level, without further extensions. However, on the bare surface, cells continue to spread up to 24 h, with much slower spreading rate than the MSNT surface, a typical behavior of cells on smooth biocompatible surface. We further examined the cell anchorage and spreading by means of immunofluorescence staining with p-FAK, an intracellular adhesive molecule that is critically involved in the focal adhesions of cells onto a matrix at very early stage [38-39]. Confocal images of cells show the distribution of p-FAK positive signals for both surfaces. Initially at 2 h, the signals in bare substrate were highly diffused at the border of cell membrane, however, those in MSNT-surface showed dense spot-like patterns, distributed even perpendicular to a radial direction. At 4 h, the signals in bare surface followed the cytoskeletal networks to some extent; however, those in nanostructure were developed into much more intensified spots. At 8 h, the cytoskeletal extensions were more pronounced in bare substrate; however, many of cells on MSNT-surface showed highly narrow-directional extensions, and this appeared to follow the MSNT’s nanotubular patterns. The confocal images clearly reflect the significant influence of the MSNT-nanostructure surface on the cell spreading and FAK distribution. Particularly at the very early time points, the tiny yet many spot-like patterns of cell anchorage, are considered to show the nanofilopodia development, which results from the nano-range distributed adhesion sites [40, 41]. The nano-filopodial processes of cells have been indicative of specified initial responses with the underlying nanotopological patterns, such as nanogrooves, nanoislands, and vertical form of nanotubes, and this event has often been shown to exert profound effects on cellular differentiation, like osteogenesis of stem cells [42-46]. Therefore, while here we demonstrated the initial responses of rMSCs to the underlying MSNT surface including cell anchorage and spreading, subsequent differentiation of the stem cells into a specific lineage can also be an intriguing study to follow in the future.

3.3.

Capacity to deliver biomolecules and the possible therapeutic role

We next sought to address the capacity of the MSNT-based nanostructure surface to load and deliver biomolecules. DEX and BSA were used as the exemplar molecules to study. First, the loading amounts onto the nanostructured



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samples were recorded with varying the biomolecule concentration. Onto the bare substrate, the DEX loading was very minimal (less than 1.5 µg per disc sample); however, onto the MSNT-surface, the DEX loading increased substantially, recording a maximum level of ~8 µg (5 times more than bare surface) (Fig. 5a). For the case of BSA, the loading amount onto bare surface was ~25 µg; however, the loading amount was as high as 72 µg onto MSNTsurface. The data clearly showed significant improvement of the biomolecular loading capacity by the nanostructured surface, and this was due to the substantially enhanced surface area of the nanotubular networks. The charge-charge interaction of biomolecules (negative-charged) with the chitosan (positive-charged) decorated over the MSNTs enabled the loading onto the tailored nanostructured surface. Compared to DEX, the BSA molecules were considered to interact more strongly with the underlying nanostructured surface, primarily the positively-charged chitosan molecules, which ultimately resulting in higher incorporation of BSA.

The release behavior of the loaded biomolecules from the nanostructured surface was then investigated for up to 15 days. The DEX release from the nanostructure was shown to profile a gradual increase with time, and the BSA release was observed to have a similar pattern to the DEX release (Fig. 5b). However, those molecules were released very quickly from the bare substrate (almost completely within a day). The data were also well fitted according to the Ritger-Peppas empirical equation, which allows kinetics parameters (n and K ) to be determined. Based on the fittings, both release patterns of DEX and BSA are indicative of similar release mechanism, primarily diffusion-controlled (with an exponent n value of 0.23 and 0.34 for DEX and BSA, respectively). The biomolecules liberated via an ionic exchange in a saline solution might be diffused out slowly through the nano-sized channels of nanotubular networks, resulting in such a long-term release profile over 15 days.

Based on this sustained release pattern of those releasing molecules, we performed an experiment to verify the biological effects. The rMSCs were allowed to indirectly interact with the DEX-loaded nanostructured surface through the design of a Transwell membrane-based permeable culture (as depicted in Fig. 6a). The cells were cultured under the osteogenic supplement medium (free of DEX) to examine the DEX molecules delivered from the nanostructure. The ALP activity, an enzymatic marker recognized for an early osteogenic process of MSCs, was


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determined during the culture for up to 21 days. Compared to the cells cultured with DEX-free substrate, those with DEX-loaded substrate showed significant improvement in ALP level, particularly at day 14 (the period considered when the rMSCs underwent osteogenic differentiation most significantly). The results clearly demonstrated the therapeutic role played by the delivered drug molecules from the nanostructured surface. While here we carried out two individual sets of experiments on the engineered nanotubular surface, i.e., nanotopological-cued initial cell adhesion and drug-delivered osteogenesis control, future study may be designed to investigate the synergistic role of the stimuli of the surface nanotopology and therapeutic drug.

Lastly, we carried out one additional experiment on the cell viability of the MSNTs (in nanotubular form used for coating) in direct comparison with the CNTs. This test was aimed to examine the possible effects of the debris or fragments of the MSNT-based nanostructure surface on the biological (in vitro) safety. Various concentrations of MSNTs or CNTs in culture medium (from 10 µg/ml to 150 µg/ml) were treated to the rMSCs and the cell viability at 24 and 48 h was assessed. Interestingly, excellent cell viability was noticed for the MSNT over the concentration range used, whilst significant cellular toxicity was concerned for the CNTs in a dose-dependent manner. This supportive data informs the beneficial use of the MSNTs compared to CNTs in the engineering of nanostructured surface of metallic implants and devices. Although this information cannot provide any solid confirmation on the in vivo safety, the MSNTs can be considered to be at least much safer nanomaterial platform for the nano-structuring of biomedical surfaces.

4.

Conclusions

Here we successfully developed biocompatible MSNT-based nanostructured interfaces on metallic substrates by an electrodeposition. The nanotubular networked topology could accelerate the very early anchorage of stem cells and then drive them to develop nanofilopodial and directional spreading, implicating subsequent alteration in lineage differentiation, which yet to be furthered. The nanostructured interfaces demonstrated the potential to load drug and protein molecules effectively and further to deliver them in a sustained manner, with significant drug-releasing effect on cells. These findings suggest the biocompatible nanostructures may be a promising



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interfacial platform that controls cell behaviors while delivering therapeutic molecules when engineered on medical devices and implants.

Acknowledgements The authors gratefully acknowledged the grants from the Global Research Lab Program (2015032163) and the Priority Research Centers Program (2009-0093829) funded by National Research Foundation, and the Korea Research Institute of Bioscience and Bioengineering (KRIBB) Research Initiative Program, Republic of Korea.


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References (1) Nel, A. E.; Lutz, M.; Velegol, D.; Xia, T.; Hoek, E. M. V.; Somasundaran, P.; Klaessig, F.; Castranova, V.; Thompson, M. Understanding Biophysicochemical Interactions at the Nano-Bio Interface. Nat. Mater. 2009, 8, 543-557. (2) Dalby, M. J.; Gadegaard, N.; Oreffo, R. O. C. Harnessing Nanotopography and Integrin-Matrix Interactions to Influence Stem Cell Fate. Nat. Mater. 2014, 13, 558-569. (3) Wennerberg, A.; Albrektsson. Effects of Titanium Surface Topography on Bone Integration: A Systematic Review. Clin. Oral Impl. Res. 2009, 20, 172–184. (4) Yim, E. K.; Darling, E. M.; Kulangara, K.; Guilak, F.; Leong, K. W. Nanotopography-Induced Changes in Focal Adhesions, Cytoskeletal Organization, and Mechanical Properties of Human Mesenchymal Stem Cells. Biomaterials 2010, 31, 1299−1306. (5) Chrzanowski, W.; Kondyurin, A.; Lee, J. H.; Lord, M. S.; Jang, J. H.; Kim H. W.; Bilek, M. M. M. Nano-bioChemical Braille for Cells: The Regulation of Stem Cell Responses using Bi-Functional Surfaces. Adv Funct Mater 2015, 25, 193-205. (6) Patel, K. D.; Kim, T. H.; Lee, E. J.; Han, C. M.; Lee, J. Y.; Singh R. K.; Kim, H. W. Nanostructured

Biointerfacing of Metals with Carbon Nanotube/Chitosan Hybrids by Electrodeposition for Cell Stimulation and Therapeutics Delivery. ACS Appl. Mater. Interfaces 2014, 6, 20214-20224. (7) McNamara, L.E.; Sjöström, T.; Seunarine, K.; Meek, R.M.D.; Su, B.; Dalby, M.J. Investigation of the Limits of Nanoscale Filopodial Interactions. J. Tissue Eng. 2014, 5, 2041731414536177. (8) Teo, B. K. K.; Wong, S. T.; Lim, C. K.; Kung, T. Y. S.; Yap, C. H.; Ramagopal, Y.; Romer, L. H.; Yim, E. K. F. Nanotopography Modulates Mechanotransduction of Stem Cells and Induces Differentiation Through Focal Adhesion Kinase. ACS Nano 2013, 7 (6), 4785-4798. (9) Li, L. H.; Kong, Y. M.; Kim, H. W.; Kim, Y. W.; Kim, H. E.; Heo, S. J.; Koak, J. Y. Improved Biological Performance of Ti Implants Due to Surface Modification by Micro-Arc Oxidation. Biomaterials 2004, 25, 2867-2875.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(10) Ren, X.; Yan, T.; Zhang, S.; Zhang, X.; Gao, P.; Wu, D.; Du, B.; Wei, Q. Ultrasensitive Dual Amplification Sandwich Immunosensor for Breast Cancer Susceptibility Gene Based on Sheet Materials. Analyst 2014, 139, 3061-3608. (11) Lv, X.; Pang, X.; Li, Y.; Yan, T.; Cao, W. Du, B.; Wei, Q. Electrochemiluminescent Immune-Modified Electrodes Based on Ag2Se@CdSe Nanoneedles Loaded with Polypyrrole Intercalated Grapheme for Detection of CA72-4. ACS Appl. Mater. Interfaces 2015, 7, 867-872. (12) Wildemann, B.; Bamdad, P.; Holmer, C.; Haas, N. P.; Raschke, M.; Schmidmaier. Local Delivery of Growth Factors From Coated Titanium Plates Increases Osteotomy Healing in Rats. Bone 2004, 34, 862-868. (13) Sirivisoot, S.; Pareta, R.; Webster, T. J. Electrically Controlled Drug Release From Nanostructured Polypyrrole Coated on Titanium. Nanotechnology 2011, 22, 085101. (14) Patel, K. D.; Singh, R. K.; Lee, E. J.; Han, C. M.; Won, J. E.; Knowles, J. C.; Kim, H. W. Tailoring Solubility and Drug Release from Electrophoretic Deposition Chitosan-Gelatin Film on Titanium. Surf. Coat. Technol. 2014, 242, 232-236. (15) Patel, K. D.; Fiqi, A. E.; Lee, H. Y.; Singh, R. K.; Kim, D. A.; Lee, H. H.; Kim, H. W. Chitosan-Nanobioactive Glass Electrophoretic Coatings With Bone Regenerative and Drug Delivering Potential. J. Mater. Chem. 2012, 22, 24945-24956. (16) Pishbin, F.; Mourino, V.; Flor, S.; Kreppel, S.; Salih, V.; Ryan, M. P.; Boccaccini, A. R. Electrophoretic deposition of gentamicin loaded Bioactive Glass/Chitosan Composite Coatings for Orthopaedic Implants. ACS Appl. Mater. Interfaces 2014, 6, 8796-8806. (17) Hu, Y.; Cai, K.; Luo, Z.; Jandt, K. D. Layer-by-layer Assembly of β-Estradiol Loaded Mesoporous Silica Nanoparticles on Titanium Substrates and Its Implication for Bone Homeostasis. Adv. Mater. 2010, 22, 4146-4150. (18) Inzunza, D.; Covarrubias, C.; Marttens, A. V.; Leighton, Y.; Carvajal, J. C.; Valenzuela, F.; Dosque, M. D.; Méndez N.; Martinez, C.; Pino, A. M.; Rodriguez, J. P.; Cáceres M.; Smith, P. Synthesis of Nanostructured Porous Silica Coatings on Titanium and Their Cell Adhesive and Osteogenic Differentiation Properties. J. Biomed. Mater. Res. Part A 2014, 102A, 37-48.


Page 18 of 30

Page 19 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(19) Kim, M.; Hong, J.; Lee, J.; Hong, C. K.; Shim, S. E. Fabrication of Silica Nanotubes using Silica Coated MultiWalled Carbon Nanotubes as the Template. J. Colloid Interface Sci. 2008, 322, 321-326. (20) Lee, S. A.; Hong, S. H.; Kim, D. O.; Han, T. H.; Iijima S.; Nam, J. D. A Mesoporous Composite Template Composed of Self-Assembled Silica Nanotube and Multi-Walled Carbon Nanotube. Microporous Mesoporous Mater. 2008, 111, 192-299. (21) Boccaccini, A. R.; Cho, J.; Roether, J. A.; Thomas B. J. C.; Minay E. J.; Shaffer, M. S. P. Electrophoretic Deposition of Carbon Nanotubes. Carbon 2006, 44, 3149-3160. (22) Pang, X.; Zhitomirsky I. Electrophoretic Deposition of Composite Hydroxyapatite-Chitosan Coatings. Mater. Charact. 2007, 58, 339-348. (23) Besra, L.; Liu, Meilin. A Review on Fundamentals and Applications of Electrophoretic Deposition (EPD). Prog. Mater. Sci. 2007, 52, 1-61. (24) Singh, R. K.; Kim, T. H.; Kim, J. J.; Lee, E. J.; Knowles J. C.; Kim, H. W. Mesoporous Silica Tubular Nanocarriers for the Delivery of Therapeutic Molecules. RSC Avd. 2013, 3, 8692-8704. (25) Singh, R. K.; Jin, G. Z.; Mahapatra, C.; Patel, K. D.; Chrzanowski, W.; Kim, H. w. Mesoporous Silica-Layered Biopolymer Hybrid Nanofibrous Scaffold: A Novel Nanobiomatrix Platform for Therapeutics Delivery and Bone Regeneration. ACS Appl. Mater. Interfaces 2015, 7, 8088−8098. (26) Hu, W.; Crouch A. S.; Miller, D.; Aryal, M.; Luebke, K. J. Inhibited Cell Spreading on Polystyrene Nanopillars Fabricated by Nanoimprinting and In Situ Elongation. Nanotechnology 2010, 21, 385301. (27) Lee, J. H.; Yu, H. S.; Lee, G. S.; Ji, A.; Hyun, J. K.; Kim, H. W. Collagen Gel Three-Dimensional Matrices Combined with Adhesive Proteins Stimulate Neuronal Differentiation of Mesenchymal Stem Cells. J. R. Soc. Interface 2011, 8, 998-1010. (28) Grosman, A.; Ortega, C. Capillary Condensation in Porous Materials. Hysteresis and Interaction Mechanism without Pore Blocking/Percolation Process. Langmuir 2008, 24, 3977-3986. (29) Fiqi, E. L.; Kim, T. H.; Kim, M.; Eltohamy, M.; Won, J. E.; Lee, E. J.; Kim, H. W. Capacity of Mesoporous Bioactive Glass Nanoparticles to Deliver Therapeutic Molecules. Nanoscale 2012, 4, 7475-7488.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(30) Kwon, S.; Singh, R. K.; Kim, T. H.; Patel, K. D.; Kim, J. J.; Chrzanowski, C.; Kim, H. W. Luminescent Mesoporous Nanoreservoirs for the Effective Loading and Intracellular Delivery of Therapeutic drug. Acta Biomaterialia 2014, 10, 1431-1442. (31) Singh, R. K.; Kim, T. H.; Mahapatra, C.; Patel, K. D.; Kim, H. W. Preparation of Self-Activated Fluorescence Mesoporous Silica Hollow Nanoellipsoids for Theranostics. Langmuir 2015, 31, 11344-52. (32) Corni, I.; Ryan, M. P.; Boccaccini, A. R. Electrophoretic Deposition: From Traditional Ceramics to Nanotechnology. J. Eur. Cer. Soc. 2008, 28, 1353−1367. (33) Thomas, B. J. C.; Bocaccini, A. R.; Shaffer, M. S. P. Multi-Walled Carbon Nanotube Coatings Using Electrophoretic Deposition (EPD). J. Am. Ceram. Soc. 2005, 88, 980-982. (34) Roy, M.; Bandyopadhyay, A.; Bose, Susmita. Laser Surface Modification of Electrophoretically Deposited Hydroxyapatite Coating on Titanium. J. Am. Ceram. Soc. 2008, 91, 3517-3521. (35) Hartgerink, J. D.; Beniash, E.; Stupp, S. I. Self-Assembly and Mineralization of Peptide-Amphiphile Nanofibers. Science 2001, 294, 1684−1688. (36) Wei, G.; Reichert, J.; Bossert, J.; Jandt, K. D. Novel Biopolymeric Template for the Nucleation and Growth of Hydroxyapatite Crystals Based on Self-Assembled Fibrinogen Fibrils. Biomacromolecules 2008, 9, 3258−3267. (37) Kim, S. S.; Wen, W.; Prowse, P.; Hamilton, D. W. Regulation of Matrix Remodelling Phenotype in Gingival Fibroblasts by Substratum Topography. J. Cell. Mol. Med. 2015, 6, 1183-1196. (38) Mendonça, G.; Mendonça, D. B. S.; Aragaô, F. J. L.; Cooper, L. F. Advancing Dental Implant Surface Technology – From Micron to Nanotopography. Biomaterials 2008, 9, 3822-3835. (39) Diu, T.; Faruqui, N.; Sjöström, T.; Lamarre, B.; Jenkinson, H. F.; Su, B.; Ryadnov, M. G. Cicada-Inspired CellInstructive Nanopatterned Arrays. Scientific Reports 2014, 4, 7122. (40) Choi, C. H.; Hagvall, S. H.; Wu, B. M.; Dunn, J. C. Y.; Beygui, R. E., Kim, C. J. Cell Interaction with ThreeDimensional Sharp-Tip Nanotopography. Biomaterials 2007, 28, 1672-1679. (41) Biggs, M. J. P.; Richards, R. G.; Gadegaard N., McMurray, R. J.; Affrossman, S.; Wilkinson, C. D. W.; Oreffo, R. O. C.; Dalby, M. J. Interactions with nanoscale topography: Adhesion Quantification and Signal Transduction in Cells of Osteogenic and Multipotent Lineage. J. Biomed. Mater. Res. A 2009, 91, 195-208.


Page 20 of 30

Page 21 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(42) Biggs, M. J. P.; Richards, R. G.; McFarlane, S.; Wilkinson, C. D. W.; Oreffo, R. O. C.; Dalby, M. J. Adhesion Formation of Primary Human Osteoblasts and the Functional Response of Mesenchymal Stem Cells to 330 nm Deep Microgrooves. J. R. Soc. Interface 2008, 5, 1231-1242. (43) Pan, H. A.; Hung, Y. C.; Su, C. W.; Tai, S. M.; Chen, C. H.; Ko, F. H.; Huang, G. S. A Nanodot Array Modulates Cell Adhesion and Induces an Apoptosis-Like Abnormality in NIH-3T3 Cells. Nanoscale Res. Lett. 2009, 4, 903-912. (44) Perez, R. A.; Seo, S. J.; Won, J.E.; Lee E. J.; Jang, J. H.; Knowles, J. C.; Kim, H. W. Therapeutically Relevant Aspects

in

Bone

Repair

and

Regeneration.

Mater.

Today

2015,

http://dx.doi.org/10.1016/j.mattod.2015.06.011 (45) Baek, S.; Singh R. K.; Khanal D.; Patel, K. D.; Lee, E. J.; Leong, K. W.; Chrzanowski, W.; Kim, H. W. Smart Multifunctional Drug Delivery Towards Anticancer Therapy Harmonized in Mesoporous Nanoparticles. Nanoscale 2015, 7, 14191-14216.



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Figures and Tables

Fig. 1. Characteristics of MSNTs used for the formation of nanostructured surfaces. (a) TEM images, (b) XRD patterns, and (c) N2 adsorption/desorption curve included with mesoporosity properties. MSNT sample formed after the CNT removal was compared with the sample before the removal. Arrows in (a) indicate the inner CNT template and the outer mesoporous silica.

Fig. 2. (a) Schematic showing the series of processes to form MSNT-based nanostructured deposition system. MSNTs mixed with biomolecules (either chitosan or collagen) form biomolecular-decorated MSNTs which are then electrodeposited onto a metallic substrate. (b) Coating weight gain presented as a function of time and voltage. (c) High resolution SEM of the MSNT nanostructures produced with the help of chitosan at various contents (MSNT:chitosan=10:1, 4:1, 4:3, and 2:3), revealing the increase in chitosan attenuated the nanostructure feature of MSNTs. (d) Representative AFM of the MSNT nanotopology (MSNT:chitosan=4:1). (e) Similar nanostructured surface generated with the help of collagen (MSNT:collagen = 10:1).

Fig. 3. In vitro bioactivity assay of the MSNT-based nanostructured surface, as examined by the apatite mineral induction in a simulated body fluid for up to 7 days. (a) SEM images, (b) XRD patterns and (c) ATR FT-IR spectra of samples. Nanotubular structure was preserved well up to 3 days, which however changed into the apatite morphology after 7 days. Arrows in (c) indicate phosphate bands related with apatite. (d) Weight gain due to apatite mineral induction on the nanostructure, measured up to 30 days, showing a gradual increase with time.

Fig. 4. Effects on the initial rMSCs responses of the MSNT-based nanostructured surface, including cell anchorage and spreading. (a) Fluorescence images of cells adhered to the surface over time sequence (2, 6, 12 and 24 h), by confocal microscopy (DAPI-nucleus in blue and F-actin in green), and based on the images, (b) cell adhesion number (per area) and (c) cell spreading index (cytoskeleton/nucleus area) quantified. Total cells counted were at least 200 for each condition. Scale bar in (b) = 200 µm. Cell adhesion was superior on MSNT at all time points (*p

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