High-Throughput Screening of Rat Mesenchymal Stem Cell Behavior

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Bio-interactions and Biocompatibility

High-throughput Screening of Rat Mesenchymal Stem Cell Behavior on Gradient TiO Nanotubes 2

Ping Mu, Yanran Li, Yanmei Zhang, Yun Yang, Ren Hu, Xulin Zhao, Anhua Huang, Ruofan Zhang, Xiang Yang Liu, Qiaoling Huang, and Changjian Lin ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00488 • Publication Date (Web): 23 Jun 2018 Downloaded from http://pubs.acs.org on June 23, 2018

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ACS Biomaterials Science & Engineering

High-throughput Screening of Rat Mesenchymal Stem Cell Behavior on Gradient TiO2 Nanotubes Ping Mu† ‡ , Yanran Li† ‡, Yanmei Zhang§, Yun Yang§, Ren Hu§, Xulin Zhao†, Anhua Huang†, Ruofan Zhang†, Xiangyang Liu†∥, Qiaoling Huang† ‡* and Changjian Lin§ †

Research Institute for Biomimetics and Soft Matter, Fujian Provincial Key Laboratory for Soft

Functional Materials Research, Department of Physics, College of Physical Science and Technology, Xiamen University, Xiamen 361005, China ‡

Shenzhen Research Institute of Xiamen University, Shenzhen 518057, China

§

State Key Laboratory for Physical Chemistry of Solid Surfaces, and Department of Chemistry,

College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China ∥

Department of Physics, National University of Singapore, 2 Science Drive 3, Singapore,

117542, Singapore, Singapore Corresponding Author *E-mail: [email protected].

KEYWORDS: bipolar electrochemistry; gradient TiO2 nanotubes; high-throughput screening; cell behavior

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ABSTRACT: The dimension of TiO2 nanotubes (TNTs) ranges from several nanometers to hundreds of nanometers. This variety raises the difficulty of screening suitable nanotube dimension for biomedical applications. Herein, we report the use of a simple one-step bipolar anodization method for fabrication of TNT gradients with diameter range from 30 to 100 nm. The gradient TNTs were successfully applied for high-throughput screening of TNT size effect on cell responses, including cell adhesion, proliferation and differentiation. Results reveal that no significant difference in adherent cell number could be found within the range of 30-87 nm in both the presence and absence of serum proteins. On the contrary, large nanotubes (with outer diameter >87 nm) profoundly reduce cell adhesion in both the presence and absence of serum proteins, indicating TNT size could affect cell adhesion directly without the adsorbed proteins. The size effect on cell behavior becomes prominent with time that cell proliferation and differentiation decrease with increasing nanotube size. This size effect can be comprehended by protein adsorption and the formation of focal adhesion. Another two sample applications of gradient TNTs demonstrate gradient TNTs are promising for high-throughput screening.

INTRODUCTION

In the past decade, TiO2 nanotubes (TNTs) have attracted numerous interests owing to their high

surface

area,

cost

effective

synthesis,

hydrophilicity

property

and

excellent

biocompatibility. The TNTs are promising for miscellaneous applications, such as solar cells, sensors, biomedical field, storage device, photocatalysis

1-2

, etc. As for biomedical applications,

TNTs have been extensively applied in orthopedic and dental implants, drug delivery, antimicrobial agent and bio-sensing

1, 3-8

. For example, it has been demonstrated that TNTs are


2

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promising for orthopedic applications as they can not only promote cell adhesion and differentiation5-6, 9 but also enhance hydroxyapatite nucleation and formation 10-11. TNTs can be fabricated by electrochemical anodization, hydrothermal, templating, and electrospinning 2, 12. With the development of techniques, it is easy to fabricate TNTs with varied properties, such as different tube diameter, different tube lengths, different crystal structures, etc. On the other hand, however, it raises the challenge of optimizing TNTs properties for specific biomedical applications. And to date, it remains a mystery as to what kind of TNT is optimum for particular application even though numerous endeavors have been made. For instance, abundant researches showed that TNTs would be a promising candidate for bone implant applications. Schmuki’s group demonstrated that TNTs with diameter of 15 nm can preferentially enhance bone cells (rat mesenchymal stem cells, osteoblasts and osteoclasts) adhesion, proliferation and differentiation

13-16

. However, Jin’s group reported striking opposite

results that the highest osteogenic differentiation of human mesenchymal stem cells (hMSCs) was on larger TNTs with diameter of ~100 nm 17-18. Conflicting results continue to emerge in the literature 19-25 and the optimal dimension of TiO2 nanotubes for bone implants remains unknown. The controversies can come from different cell types, cell seeding density, different experimental protocols or different nature of the TNTs used

18

. Furthermore, most studies selectively

investigated several TNTs with specific dimensions. This is because investment of surface biocompatibility requires multiple samples for statistical analysis that it is difficult and tedious to handle a large amount of samples 6. Therefore, it is urgent to develop/apply effective methods for optimizing parameters of TiO2 nanotubes for biomedical applications. Micropatterning miniaturizes different samples into one pattern which allows high throughput screening of material properties. Schmuki’s group tried to resolve the above


3


mentioned conflict by defined micropattern with diameters of 15 and 100 nm

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19, 26-28

. Their

results confirmed and explained why cells favor small diameter nanotubes by extracellular matrix spreading behavior

26

. Chen et al. integrated four different nanotubes (60, 150, 250, and

350 nm) in one single substrate for investigating cell responses to coexisting nanotubes

19

. Our

recent study created a gradient TiO2 nanotube arrays with four different nanotubes for high throughput evaluation of biocompatibility and antibacterial property (to be published). Indeed, those micropatterns allow high-throughput simultaneous screening of multiple dimensions and reduce sample sizes and errors. But still, those studies investigated nanotubes with specific dimensions rather than a wide range of dimensions. In this work, we aim to construct TNT gradients with a wide range of dimensions and utilize it for high throughput screening of TNTs dimensions in cellular responses. Several studies have made the endeavor to construct gradient TiO2 nanotubes with a wide range of dimensions 29-35

. Li’s group fabricated gradient TNTs with outer diameter ranged from 100 nm to 190 nm by

asymmetric anodization

29-30

. Schmuki’s group manufactured gradient TNTs with a diameter

range from 45 nm to 180 nm via bipolar electrochemistry and utilized it for photocurrent screening

33

. In this study, TNT gradients with dimension of 30~100 nm were fabricated by

bipolar electrochemistry. In the meantime, we show that the gradient TiO2 nanotubes can be used for high-throughput screening of cell behaviors, platelet adhesion and octacalcium phosphate (OCP) deposition.

EXPERIMENTAL SECTION

Fabrication of gradient TNTs. Titanium foils (0.1 mm) of 99.6% purity were cut to 1.8 cm × 1 cm pieces. Titanium foils were ultrasonicated in acetone, deionized water and ethanol for


4

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20 minutes, respectively. Then the titanium foils were dried under air stream. TNTs gradients were fabricated by bipolar electrochemical anodization as described in Figure 1. The electrolyte solution was composed of 0.5 wt% hydrofluoric acid. Two Pt feeder electrodes (30 mm × 30 mm) were vertically positioned with a distance of 20 mm. The titanium foil was tapped with Kapton tape on the weighing boat at a distance of 1 mm from Pt electrodes. A constant potential of 35 V was applied by GW Instek power supply (PSW160-7.2) for 20 minutes under stirring at 4 ℃. After anodization, the titanium foil was thoroughly water-rinsed and air-dried. The top and cross-sectional morphologies of TNTs gradient were evaluated using a scanning electron microscope (FESEM OXFORD ZEISS SIGMA). Octacalcium phosphate deposition. Gradient TNTs were further electrodeposited from an aqueous electrolyte constituted of Ca(NO3)2 (0.042 mol/L) and NH4H2PO4(0.025 mol/L) at pH 4.2

36

. Pt was utilized as counter electrode. Electrodeposition was carried out at galvanostatic

mode with a current density of 0.5 mA/cm2 at 60 ℃. After five minutes reaction, samples were rinsed with DI water and air-dried. Protein binding. Fluorescein isothiocyanate (FITC) labeled bovine serum albumin proteins (BSA, Sigma) and serum proteins were used to study protein binding. BSA at a concentration of 10 mg/ml or 25% v/v serum proteins were mixed with 500 mM sodium carbonate. Then 5 mg/ml fluorescein isothiocyanate (FITC, Sigma) in DMSO was added slowly and kept stirring in the darkness for 2 h at room temperature. Then free FITC molecules were removed by centrifuging the protein mixture in Amicon Ultra-4 centrifugal filter tubes. Labeled proteins were resuspended in PBS and kept at 4 ℃ before use. 300 µl of FITC-labeled BSA (5 mg/ml) were incubated on TNT gradients at 37 ℃ for 30 min. The gradient samples were washed by deionized water and air dried before imaging under fluorescence microscopy.


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Cell culture. Rat mesenchymal stem cells (RMSCs) were segregated and expanded from tibias and femurs of Sprague-Dawley (SD) rats (~100 g). Cells were cultured in cell culture flasks (Corning, TCPS) and maintained at 37 ℃ in a humidified incubator containing 5% CO2. The growth medium was composed of alpha-modified minimum essential medium (a-MEM) supplemented with 10 vol% fetal bovine serum (FBS, Gibco) and 1 vol% antibiotics penicillin– streptomycin. Cells were harvested from flasks when cell density reached 80% to 100% confluence. Before cell seeding, all gradient samples were disinfected by UV-irradiation for 30 min. Cell adhesion and proliferation. Cells were seeded on the gradient TNTs with a density of 1.5×104 cells/cm2 as measured by hemocytometry. After cultured for 1 and 3 days, cells were incubated with Calcein-AM (Sigma) for fluorescence observation. Cell number was counted using ImageJ. To visualize F-actins, vinculins and nuclei, cells were fluorescently stained after 24 h. Briefly, cells on TNTs were washed by PBS and fixed in 4% paraformaldehyde in PBS at room temperature for 15 minutes. Then cells were permeabilized with 0.5% Triton X-100 (Sigma) solution for 15 minutes. Specimens were blocked with 5% bovine serum albumin (BSA, ExCell) in PBS to avoid nonspecific protein staining for 1 h. For vinculin staining, fixed cells were incubated with mouse monoclonal antivinculin primary antibody (1:100, Sigma) at 4 °C overnight and Anti-mouse IgG-FITC antibody produced in rabbit (1:200, Sigma) for 4 h at room temperature. To visualize filamentous actins (F-actins), samples were incubated with 1 µg/mL tetramethylrhodamine-conjugated phalloidin (Phalloidin-TRITC, Sigma) for 30 min followed by PBS wash. Nuclei were dyed blue with 2 µg/mL 4′,6-diamidino-2-phenylindole (DAPI, Sigma) for 8 min. The fluorescently stained cells were photographed by a laser scanning confocal microscope (Leica TCS SP).


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Characterization of osteogenic differentiation. Cells were cultured on the gradient samples at a density of 104 cells/cm2. After 1 day culture, the cell culture media was replaced by induction media consisting of a-MEM, 10 vol% FBS, 50 µM ascorbic acid-2-phosphate (Sigma), 100 nM dexamethasone (Sigma), and 10 mM β-glycerophosphate (Sigma). After 3 and 7 days culture, alkaline phosphatase (ALP) activity was investigated by ALP kit (Sigma) according to the instruction of the manufacturer. For collagen secretion investment, cells were cultured for two weeks. The cells were rinsed by PBS for three times and fixed in 4% paraformaldehyde in PBS for 1h. Then cells were incubated with 1% Sirius Red (Sigma) for 18 h and rinsed with distilled water thoroughly before observing under microscope. Extracellular matrix (ECM) mineralization was investigated by Alizarin Red staining. Cells on TNT gradients were fixed by 75% medical alcohol after two weeks growth. Then cells were stained with 40 mM Alizarin Red (Sigma) in carbazotic acid (saturated) at PH 4.2 for 10 min followed by thorough water wash and image acquiring. The expression of osteopontin (OPN) was also examined after two weeks culture. Cells were washed by PBS and immobilized with 4% paraformaldehyde solution for 15 min. Then cells were washed by 0.05% Tween-20 in PBS twice and permeabilized with 0.1% Triton X-100 diluted in PBS for 10 min before PBS wash. Samples were immersed in 1% BSA in PBS for 1 h to avoid non-specific binding before incubating with anti-osteopontin antibody (Santa Cruz) at 4 °C for 12 h. Then cells were triple washed by 0.05% Tween-20 in PBS and stained with antimouse IgG-FITC antibody for 4 h at room temperature. Thereafter, cells were thoroughly washed and incubated with 1 µg/mL rhodamine phalloidin for 30 min and DAPI for 8 min. Short-term Cell attachment. Cells were harvested and suspended in DMEM supplemented with or without serum proteins. A cell density of 1.5×104 cells/cm2 was cultured on the gradient


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TNTs for two hours. Then cells were fixed and stained for visualizing vinculin, actin and nucleus as described above. The numbers of adherent cells were quantified by counting calcein stained cells in ImageJ software. Platelet adhesion test. Human platelet rich plasma (PRP) was obtained from outdated (within 1 day of expiration) lots from Xiamen Maternity & Child Care Hospital. 3 ml PRP was added onto the TNT gradient placed in a 6-well plate and incubated in a CO2 incubator for 1 h. Then samples were gently washed with PBS three times before fixing in glutaraldehyde and serial dehydration. Samples were coated with platinum and observed using SEM after critical point drying. Statistical Analysis. At least three samples were investigated for statistical analysis. The data were illustrated as mean ± standard deviation. One-way analysis of variance (ANOVA) was conducted to evaluate the difference among groups followed by Turkey post hoc tests. P value of less than 0.05 indicated a significant difference.

RESULTS AND DISCUSSION

Mechanism of bipolar electrochemical anodization. Figure 1 is the illustration of the bipolar electrochemical approach for fabricating TNT gradient. Different from conventional electrochemical anodization, bipolar anodization utilizes two Pt foils as feeder electrodes and the wireless titanium lies in the electrolyte perpendicular to Pt foils. A polarization potential emerges along the titanium surface when applying an electric field between two Pt feeder electrodes. The potential is largest at the edge of the titanium foil toward the anodic region and reduces gradually toward the cathodic pole. Thus, oxidation takes place at the anodic region of the titanium foil, whereas reduction occurs at the cathodic site. As the anodization potential decreases constantly


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from the edge of the anodic pole towards the edge of the cathodic pole of the foil, this mechanism allows growing TNTs with dimension (nanotube diameter and length) gradient on the anodic pole. Characterizations of TNT gradient. As shown in Figure 1c, the TNT gradient took up more than 80% of the surface after bipolar anodization. Figure 2A showed surface morphologies of highly ordered, vertically aligned TNTs from different positions of one single sample. The nanotube outer diameter ranged from 30 nm to 100 nm (Figure 2B) and the inner diameter ranged from 23 nm to 100 nm (data not shown). The wall thickness increased from ~4.5 nm to 8.5 nm with the rising of nanotube size. The nanotube length ranged from 90 nm to 370 nm across the gradient. Apparently, large nanotubes were acquired at the sample edge nearest the anodic region. And both tube diameter and length decreased with the potential drop across the gradient. This is because the polarization potential gradient was generated along the Ti surface with the highest potential located at the edge toward cathodic pole. TiO2 nanotube diameter could be adjusted by applied potential. We attempted to obtain larger TiO2 nanotubes by changing applied potential. However, with the increase of voltage, nonorganized morphology was observed at the sample edge nearest the anodic region (Figure S1). This is probably due to the limitation of aqueous electrolytes

2, 33

.

The water contact angle of the gradient TNTs increased slightly from small nanotubes (30 nm: 11.9º) until 87 nm nanotubes (24.4º), and then reduced slightly on the largest nanotubes (23º, Supplementary Figure S2). Protein binding. Protein adsorption on biomaterial surfaces plays an important role in biological responses as which usually begins with instantaneous molecule adsorption, including water, ions and proteins. For example, adsorbed proteins act as intermediate layer which could


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further intervene cell adhesion and proliferation. Herein, gradient TNTs were used to screen protein adsorption. Bovine serum albumin and serum proteins were labeled by a fluorescent dye FITC and immunofluorescence was used for protein adsorption study. It is important to stress that the rinsing process after protein adsorption can remove loosely-adsorbed proteins. Thus, the intensity of fluorescence signal represents the amount of bound proteins instead of adsorbed proteins. As shown in Figure 3A, the green fluorescence was uniformly distributed for all diameters across the gradient. The fluorescence intensity of bound BSA decreased slightly with the rise of TNTs diameter until nanotube with diameter of 70 nm, and then remained low to the end of the largest nanotubes. This is probably owing to the larger total perimeters of smaller nanotubes (Figure S3). The wall thickness of nanotubes ranges from 4.5 to 8.5 nm which is of similar size to BSA protein (Dimensions: 140 × 40 × 40 Å)

37

. Thus, the total perimeter of the

nanotubes might dominate the amount of bound BSA at the top of nanotubes. Besides, the spacing of small nanotubes are more inappropriate for protein aggregation 17. On the contrary, serum proteins binding on the gradient was more complex as the fluorescence intensity was relatively high in the middle regions (Figure 3B). And the intensity was comparatively low on both smallest and largest diameter nanotubes. This intricacy may be due to the complexity of serum proteins (FBS). More than 1000 different proteins exist in the serum with concentration range over three orders of magnitude

38

. Usually competitive

adsorption occurs as small proteins adsorb on the surface earlier than larger proteins. Then larger proteins could replace small proteins, i.e. Vroman effect

39

. Thus, it is understandable that the

adsorption of serum proteins is different from BSA. Further investigation is necessary for better understanding of multi-protein adsorption on nanotubes.


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Cell adhesion and proliferation. To initially apply the TNTs gradients for high throughput screening in cell behavior study, rat mesenchymal stem cells (rMSCs) were cultured on the gradients for 1 day and 3 days as shown in Figure 4. The number and area of adherent cells on the gradient TNTs were statistically analyzed by Image J software as shown in Figure 4B and Figure 4C. A significant difference in cell density and morphology from different regions could be observed. On day 1, with the increase of nanotube diameter, only a small change in adherent cell number was observed with the range of 30-60 nm. Then adherent cell number decreased with increasing nanotube diameter (Figure 4B, day 1). It is conspicuous that the cells were well spread with more filopodia at the region of small diameter nanotubes. On the contrary, with the enlargement of the nanotube diameter, adherent cells were less spread with small amounts of short filopodia (Figure 4A). A slight increase in cell spreading area was observed when nanotube diameter increased from 30 nm (~39 µm2) to 40 nm (~44.4 µm2) and 60 nm (~43.8µm2). But it reduced sharply from ~43.8 µm2 to ~15.8 µm2 when tube diameter increased from 60 nm to 100 nm. (Figure 4C, day 1). After three days proliferation, cell density of viable cells increased ~2-fold for the smallest nanotubes (Figure 4B, from ~2×104 cells/cm2 for day 1 to ~4×104 cells/cm2 for day 3). The cell density decreased linearly with increasing tube size. No remarkable change in cell density was observed for 100 nm nanotubes (Figure 4B). Cell spreading area was reduced for most nanotubes especially for nanotube with diameter of 30 nm and 100 nm (Figure 4C, day 3). This is probably because the surface has become too crowded for high cell density on small nanotubes. And the cell density is too low for large diameter nanotubes that cells lack cell-cell communication 40. It is important to stress that cell density used in this paper is within the common density range of


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cell proliferation assay. As cell density plays an important role in cell proliferation, a different outcome would be found if cells were seeded at higher density as demonstrated by Oh et al 41. Cell differentiation. Cell differentiation has been examined by ALP staining, collagen secretion, ECM mineralization and osteopontin (OPN) expression. As shown in Figure 5A, ALP production could be seen across the gradient as early as 3 days after incubation. The ALP activities on nanotubes with smaller diameter were higher than those on nanotubes with large diameter. After 7 days of incubation, ALP activities were significantly enhanced across the sample (Figure 5A, compare 7 days to 3 days), with the highest on the nanotubes with diameter range of 30-40 nm. The ALP activities continued to decline on the gradient nanotubes from diameter of 40 to 100 nm. Collagen secretion was investigated by Sirius red staining as presented in Figure 5B. Abundant collagen was secreted by rMSCs on TNTs with small nanotubes (30-40 nm) after two weeks culture. And the secretion reduced as the diameter increased. Similarly, small nanotubes with outer diameter of 30-40 nm presented the highest ECM mineralization which reduced with increasing nanotube diameter at the range of 40 to 100 nm. Osteopontin (OPN) is a common biochemical marker for bone formation. TNTs with diameter scope of 30-40 nm demonstrated greater OPN staining comparing to larger nanotubes. By contrast, osteopontin immunoreactivity became weak and faint as the nanotube diameter increased from 40 to 100 nm. Together, ALP activities, collagen secretion, ECM mineralization and OPN expression indicate that small nanotubes (30-40 nm) present the best osteogenesis property which decreases with increasing nanotube diameter at the range of 40-100 nm. Focal adhesion. As mentioned in the introduction, cell behaviors on TNTs remain controversial in the literature. Our results show that the smallest diameter ~30 nm was the


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optimal dimension for the adhesion, proliferation and ECM, ALP, collagen production of rMSCs. In order to have a better understanding of this phenomenon, focal adhesion formation was investigated. Vinculin is a key cell membrane-cytoskeletal protein in focal adhesion which can connect integrin to actin filaments. Herein, cells were further stained for visualization of filamentous actin (F-actin), nucleus, and vinculin after 1 day culture (Figure 6). Cells presented a more spread morphology on the small diameter nanotubes, consistent with Figure 4. Abundant focal contacts formed around cell periphery on small nanotubes as shown by immunostaining for vinculin. Actin filaments were clearer and highly-ordered for small nanotubes, indicating higher cell tension

42

. However, cell spreading area of F-actin inclined to shrink with increasing

nanotube diameter. The expression of focal contact formation was also reduced with the increasing nanotube diameter, implying less interaction between cell receptors and ECM ligands 43

. It has been suggested that higher amount of focal adhesion and higher cell tension could

stimulate integrin-mediated signaling pathways and further controls cell behaviors such as cell proliferation, differentiation, and migration

14, 44

. Thus, it is not surprising that small nanotubes

could induce more focal adhesion and possess better ability to facilitate the proliferation and differentiation of rMSCs. Short-term cell attachment. It is understandable that cells have better proliferation and differentiation rate when more focal adhesions are formed. But why small nanotubes could induce higher focal adhesion and further stimulate cell proliferation and differentiation, Park et al. suggested that a spacing of 15 nm is optimal for integrin assembly and focal contact formation 14. And larger diameter nanotubes completely block integrin from clustering and focal contact formation

14

. Indeed, it will be easier for proteins to anchor and assemble into focal

adhesion when the spacing is appropriate. And the focal adhesion could facilitate cell spreading


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and the following proliferation and differentiation. But it remains unclear whether the nanotubular structure could affect cell adhesion directly rather than through indirect effect of adsorbed proteins or focal adhesion. In order to better understand this process, cells were cultured for two hours with or without serum proteins supplemented in the culture media. As shown in Figure 7A and Figure 7B, cells could attach on the TNTs in both conditions. No obvious focal adhesion was found for adherent cells across the gradient with or without serum supplementation. Cells were partially spread out when cell culture media was supplemented with serum proteins. The spreading area reduced with the increase of nanotube diameter. On the contrary, cell spreading was significantly restricted when cell culture media lacks of serum proteins. It suggests that serum proteins facilitate cell spreading. Adherent cell number was quantified by ImageJ as revealed in Figure 7C. Interestingly, only a slight difference in cell number with similar attachment trend was observed when comparing serum free condition to serum-containing condition. It suggests that serum proteins only have small contribution to adherent cell number within the first two hours. Surprisingly, there was no significant variation in cell number at the range between 30 nm and 87 nm (Figure 7A). But a dramatic drop in cell number was observed when tube diameter was larger than 87 nm (Figure 7). It implies that the direct and/or indirect nanotube size effect on adherent cell number within the first two hours is not significant until the range of 87-100 nm. But this size effect becomes remarkable with time during cell proliferation (Figure 4) and differentiation (Figure 5) process. Cell attachment starts from cell gravitation to within close to the surface (Figure 8). The forces received by cells are mainly van der Waal’s forces (>50 nm) and electrostatic force (10-20 nm) before specific protein mediation starts. For cell attachment from serum containing media,


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specific interactions between cell membrane integrin and adsorbed serum proteins (such as fibronectin, vitronectin) facilitate cell adhesion. The binding of integrin and extracellular proteins could assemble into focal adhesion which further stimulates cell spreading, proliferation and differentiation. It has been demonstrated that integrin can form clusters when the nanospacing is smaller than 70 nm while the clustering is inhibited on surfaces with spacing larger than 70 nm

45

. Thus, it is no surprising that cell adhesion is restricted on nanotube with

outer diameter larger than 87 nm (inner diameter > 80 nm). Moreover, serum protein adsorption was suppressed when the nanotube outer diameter is larger than 87 nm (Figure 7, serum protein condition). For cell adhesion from serum free media, even though without adsorbed serum proteins, cellular membrane proteins (such as integrin) could anchor through weak electrostatic force, hydrophobic interaction and van der Waal’s forces. As cellular proteins can interact directly with biomaterials without competition with other proteins, this interaction might be similar to single protein adsorption. Similar to BSA adsorption, the reduction of cell adhesion on 100 nm nanotubes might be due to insufficient interaction between cell membrane proteins and surface. Other applications of TNTs. As illustrated in the introduction section, TNTs have wide applications. Gradient TNTs have already been demonstrated to be useful for dye-sensitized solar cells

33, 46

， hybrid energy storage devices

44

. Herein, we give another two sample

biomedical applications of gradient TNTs, including platelet interaction and OCP deposition. High-throughput screening of platelet behaviors. TNTs have been reported to be promising for the application as blood contact biomaterial. However, it is still obscure how the TNTs affect blood compatibility

45, 47-50

. And we found it is extremely tedious and difficult to

systematically investigate the size effect of TNTs on blood compatibility as large amount of


15


samples were required for statistical analysis

6

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. Herein, we applied gradient TNTs for

preliminary high-throughput screening of TNT size on platelet behavior as the response of platelet rich plasma (PRP) reflects the combined results of contact activation of blood coagulation cascade and platelets. As shown in Figure 9A, abundant platelets adhered and activated on larger diameter nanotubes. Adherent platelet number increased with the increment of nanotube size. The platelets mainly displayed a spreading dendritic shape and no discernable shape changes could be observed across the gradient. It indicates that the blood compatibility of TNTs decreases with increasing diameter. However, further comprehensive investigation is warranted to fully reveal the relationship between blood compatibility and nanotube dimensions. High-throughput screening of OCP deposition. Further chemical and biochemical modification on TNTs have been widely explored to further improve osteointegration of titanium. Among all modification methods, deposition of octacalcium phosphate (OCP) has been extensively exploited as OCP acts as a precursor for biomineralization 36, 51-52. However, little is known about the effect of TNTs size on the formation of OCP. In view of this, we investigated the size effect of TNTs on OCP electrochemical deposition using gradient TNTs. As presented in Figure 9B, abundant acicular scattered flowers were deposited on the small size nanotubes with diameter of 30 nm. Interestingly, porous structures were formed on all other size nanotubes. No significant difference in pore size could be found for nanotubes with diameter range of 40-87 nm whereas smaller pore size was formed for nanotubes with diameter of 100 nm. Thus, the dimension of TNTs significantly affects OCP morphologies during electrodeposition. The OCP crystals were confirmed by Raman spectra and energy spectrum as shown in Figure S4.


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CONCLUSIONS

In conclusion, bipolar electrochemistry has been proved to be a convenient and efficient method for fabricating self-organized TNT gradients with a wide range of length and diameter on a single sample. Cell behaviors of rMSCs have been correlated to the TNT gradients. There was no significant difference in two hours short-term cell attachment for nanotubes with diameter range of 30-87 nm. However, 100 nm nanotubes notably reduced cell adhesion. And serum proteins only had small contribution to adherent cell number, but greatly enhanced cell spreading during the first two hours. The size effect of TNTs became remarkable with time as cell proliferation decreased with increasing nanotube size. Cell differentiation on TNTs also had a similar downward trend except small nanotubes with diameter range of 30-40 nm did not show significant osteogenesis difference. Cell responses to TNTs could be interpreted by protein adsorption and the formation of focal adhesion. Two sample applications (platelet adhesion and OCP deposition) were performed to further illustrate the applications of gradient TNTs. Thus, this study demonstrated that the application of bipolar electrochemistry could provide a simple high-throughput assay for exploring size effect of TNTs in biomedical applications.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: SEM images, surface wettability, calculation of total perimeters, analysis of energy spectrum, and Raman spectra


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AUTHOR INFORMATION Corresponding Author ORCID Qiaoling Huang: 0000-0001-9665-1542 Notes The authors declare no competing financial interest. ACKNOWLEDGMENT Authors gratefully acknowledge financial supports from the State Key Project of Research and Development (2016YFC1100300), National Natural Science Foundation of China (51571169, 21773199, 21621091), Natural Science Foundation of Guangdong Province, China (2016A030310370), The Fundamental Research Funds for the Central Universities (20720150218), 111 Project (B16029) and Doctoral Fund of the Ministry of Education (20130121110018). The authors would like to thank Shenshi Guo, Rui Yu, Hao Wang and Likun Yang for their technical supports.

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Table of Contents graphic High-throughput Screening of Rat Mesenchymal Stem Cell Behavior on Gradient TiO2 Nanotubes Ping Mu, Yanran Li, Yanmei Zhang, Yun Yang, Ren Hu, Xulin Zhao, Anhua Huang, Ruofan Zhang, Xiangyang Liu, Qiaoling Huang and Changjian Lin


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Figure 1. (a) Detailed schematic description of the bipolar anodization setup. (b) The potential gradient generated on the titanium. (c) Photograph of the gradient TiO2 nanotubes after bipolar anodization. 215x78mm (100 x 100 DPI)


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Figure 2. (A) Top (first row) and cross-sectional (second row) SEM images of gradient TNTs photographed from different positions starting from the edge of the titanium foil facing feeder anode: (a, g) 3 mm, (b, h) 6 mm, (c, i) 9 mm, (d, j) 12 mm, (e, k) 15 mm and (f, l) 18 mm. (B) The nanotube outer diameter, inner diameter and length varying along positions. (C) Wall thickness of gradient TNTs rising along positions. 326x224mm (100 x 100 DPI)



Figure 3. Immunofluorescence microscopy images of FITC stained BSA (A) and serum proteins (B) bound on gradient TNTs. 195x67mm (300 x 300 DPI)


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Figure 4. (A) Fluorescence micrographs of calcein labeled rMSC on gradient TNTs after 1 and 3 days incubation. Statistical results of cell density (B) and cell spreading area (C) along the gradients at day 1 and day 3. Asterisks indicated significant difference by ANOVA (*p < 0.05, **p < 0.01) compared to 30nm. 314x226mm (300 x 300 DPI)



Figure 5. (A) Representative optical photographs of ALP staining (purple) after 3days (a~f) and 7days (g~l) incubation on gradient TNTs. Collagen secretion (B) and extracellular matrix mineralization (C) by rMSCs on the gradient TNTs after 14 days of culture. (D) Immunofluorescence staining of osteopontin (green), actin (red) and DAPI (blue) for cells cultivated on gradient TNTs for 14 days. 364x338mm (300 x 300 DPI)


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Figure 6. Typical confocal fluorescence images of cells stained for cell nuclei (blue), vinculins (green), and Factins (red) after 1 day culture. 266x175mm (300 x 300 DPI)



Figure 7. Immunofluorescent staining of adherent cells for visualization of vinculins (green), nuclei (blue) and F-actin after 2 hours culture with (A) and without (B) the presence of serum proteins. (C) Quantitative analysis data of adherent cell density. Asterisks indicated significant difference by ANOVA (**p < 0.01) compared to 30nm. 240x299mm (300 x 300 DPI)


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Figure 8. Hypothetical mechanism of cell adhesion on nanotubes with small and large diameters. 857x972mm (70 x 70 DPI)



Figure 9: (A) SEM photographs of adherent platelets on gradient samples. (B) SEM images of OCP coatings on gradient TNTs. 403x119mm (300 x 300 DPI)


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High-Throughput Screening of Rat Mesenchymal Stem Cell Behavior

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