Cellular responses to nanoscale substrate topography of TiO2 nanotube arrays: cell morphology and adhesion

doi:10.12336/biomatertransl.2022.03.006

Biomaterials Translational ›› 2022, Vol. 3 ›› Issue (3): 221-233.doi: 10.12336/biomatertransl.2022.03.006

• RESEARCH ARTICLE • Previous Articles

Cellular responses to nanoscale substrate topography of TiO₂ nanotube arrays: cell morphology and adhesion

Monchupa Kingsak¹, Panita Maturavongsadit¹, Hong Jiang², Qian Wang¹^,^*()

1 Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC, USA
2 Computer Science, Physics, and Engineering Department, Benedict College, Columbia, SC, USA

Received:2022-08-26 Revised:2022-09-07 Accepted:2022-09-17 Online:2022-09-28 Published:2022-09-28
Contact: Qian Wang E-mail:Wang263@mailbox.sc.edu
About author:Qian Wang,Wang263@mailbox.sc.edu.

Abstract

Abstract:

Nanotopographical features can be beneficial in augmenting cell functions and increasing osteogenic potential. However, the relationships between surface topographies and biological responses are difficult to establish due to the difficulty in controlling the surface topographical features at a low–nanometre scale. Herein, we report the fabrication of well–defined controllable titanium dioxide (TiO₂) nanotube arrays with a wide range of pore sizes, 30–175 nm in diameter, and use of the electrochemical anodization method to assess the effect of surface nanotopographies on cell morphology and adhesion. The results show that TiO₂ nanotube arrays with pore sizes of 30 and 80 nm allowed for cell spreading of bone marrow–derived mesenchymal stem cells with increased cell area coverage. Additionally, cell adhesion was significantly enhanced by controlled nanotopographies of TiO₂ nanotube arrays with 80 nm pore size. Our results demonstrate that surface modification at the nano–scale level with size tunability under controlled chemical/physical properties and culture conditions can greatly impact cell responses. These findings point to a new direction of material design for bone–tissue engineering in orthopaedic applications.

Key words: cell adhesion, cellular responses, morphology, nanotopography, TiO₂ nanotube arrays

Cite this article

Figures/Tables 10

Figure 1. The synthesis of TNA by electrochemical anodization. (A) The experimental setup for the anodization of Ti. Ti was used as the anode and Pt was used as the cathode. The distance between Ti and Pt was controlled at 1.5 cm. (B) Schematic illustration showing the reactions occurring during anodization of the Ti sheet. Three chemical reactions (water decomposition, metal oxidation, and oxide dissolution) occur simultaneously to create nanotube–like structures during the anodization process. Figure 1B has been redrawn based on an original figure by Regonini et al.44 Pt: platinum; Ti: titanium; TiO2: titanium dioxide; TNA: TiO2 nanotube array.

Figure 2. The effect of differences in water content in the electrolyte on tube geometry. Scanning electron microscopy images of anodized TiO2 nanotubes prepared by anodizing Ti at 10 V for 3 hours in DEG electrolytes containing 0.5 wt% NH4F with different concentrations of 1%, 5%, 10% and 20% (v/v) H2O. Increasing the water content from 1% to 20% markedly reduced the time required to dissolve the native oxide during TNA formation. The insets show enlarged images of the anodized substrate prepared with different water contents in the electrolyte. All images and insets share the same scale bars at 200 nm and 50 nm, respectively. DEG: diethylene glycol; NH4F: ammonium fluoride; Ti: titanium; TiO2: titanium dioxide; TNA: TiO2 nanotube array.

Figure 3. The effect of anodization times on nanotube development and structure. Scanning electron microscopy images show the evolution of nanotube formation during different anodizing times at 20 V for 5, 10, 15, 20, or 30 minutes in DEG–based electrolyte containing 0.5 wt% NH4F and 20% (v/v) H2O. Nanopits were created at 5 minutes, nanoporous layer was generated over the surface at 10–20 minutes, and nanotubes were developed at 30 minutes of anodizing times. The insets show enlarged images of the anodized substrate at different anodizing times. All images and insets share the same scale bars at 400 nm and 100 nm, respectively. DEG: diethylene glycol; NH4F: ammonium fluoride.

Figure 4. Characterization of sample nanostructure and surface wetting. (A, B) Top–view (A) and side–view (B) scanning electron microscopy images of TNAs prepared by anodizing Ti in DEG–based electrolyte containing 0.5 wt% NH4F and 20% (v/v) H2O at 10, 20, 30, and 35 V for 3 hours. The tube diameter and length were increased respectively by raising the applied voltage of anodization. Scale bars: 200 nm in A and 2 μm in B. (C) Surface wettability of TNAs at different diameters of pore size (30, 80, 120, and 175 nm). Data are expressed as mean ± SD (n = 6). DEG: diethylene glycol; NH4F: ammonium fluoride; Ti; titanium; TNA: titanium dioxide nanotube array.

Table 1. The chemical components, diameters and lengths of the anodised TNAs using 0.5% NH4F with 20% (v/v) H2O in DEG–based electrolyte under different voltages

	Ti	TNA30 (10 V)	TNA80 (20 V)	TNA120 (30 V)	TNA175 (35 V)
Element (weight%)
O	–	23.75	23.09	22.48	22.74
F	–	6.57	6.47	6.28	6.31
Ti	100	69.68	70.44	71.24	70.96
Diameter (nm)	–	31.1±5.0	78.3±16.8	120.9±27.5	174.2±31.4
Length (µm)	–	0.6±0.1	1.0±0.1	1.7±0.1	2.3±0.4

Figure 5. Summary of the surface roughness of TNAs with different diameters (30, 80, 120, 175 nm). The roughness was characterized by atomic force microscopy. The data are expressed as mean ± SD (n = 4). No significant change was observed in any comparisons based on one–way analysis of variance followed by Tukey’s multiple comparisons test. Ra: mean roughness; RMS: root mean square roughness; TNA: titanium dioxide nanotube array.

Figure 6. Morphology of BMSCs cultured on Ti or TNA substrates in two–dimensional cell culture without foetal bovine serum. TNA30 and TNA80 enhanced cell spreading and percentage cell area coverage of BMSCs. (A) SEM images show the morphology of BMSCs cultured on Ti or TNA substrates prepared by anodizing Ti in a DEG–based electrolyte at 10, 20, 30, or 35 V for 3 hours. The cells cultured on TNA30 and TNA80 exhibited good spreading all over the surface, while cells on TNA120 and TNA175 adopted a rounded shape with poor spreading. All scale bars share a length of 100 µm. (B) Percentage area coverage of BMSCs on Ti or TNA substrates, calculated from SEM images using ImageJ. Data are expressed as mean ± SD (n = 3). *P < 0.05 (Student’s t–test or one–way analysis of variance followed by Tukey’s multiple comparisons test). BMSC: bone marrow–derived mesenchymal stem cell; DEG: diethylene glycol; SEM: scanning electron microscopy; Ti; titanium; TNA: titanium dioxide nanotube array.

Figure 7. Morphology of NIH3T3 cells cultured on Ti or TNA substrates in two–dimensional cell culture with and without FBS. FBS significantly affected cell morphology and proliferation. (A, B) Confocal laser scanning microscopy images of NIH3T3 cells cultured on Ti or TNA substrates after 1 day of incubation in serum–free medium (A), and in 10% serum–containing medium (B). The morphology of NIH3T3 cells cultured in serum–containing medium showed good spreading, while the cells cultured in serum–free medium showed poor spreading and fewer cells. Cells were fixed and stained with Hoechst 33342 (blue fluorescence), Calcein AM (green fluorescence), and Rhodamine phalloidin (red fluorescence). The scale bars indicate a length of 50 µm for all images and 25 µm for the enlarged images. FBS: foetal bovine serum; Ti; titanium; TNA: titanium dioxide nanotube array.

Figure 8. Assessment of the adhesion of NIH3T3 and BHK–21 cells by centrifugation assay. TNA80 promoted enhanced cell adhesion in the early stage of cell attachment. (A, B) Fluorescent images show NIH3T3 cells (A) and BHK–21 cells (B) attached on Ti, TNAs, and tissue culture plate (polystyrene) and incubated in serum–free medium for 1 and 2 hours, respectively, before and after spinning. After post spin, NIH3T3 and BHK–21 cells cultured on TNA80 detached from the substrate fewer than those on TNA120 and TNA175 after 1 and 2 hours of incubation time, respectively. The cells were stained with Calcein AM. The scale bar indicates a length of 100 µm. (C, D) The percentage of remaining NIH3T3 cells (C) and BHK–21 cells (D) after centrifugation following different incubation times. Data are expressed as mean ± SD (n = 3). *P < 0.05 (Student’s t–test and one–way analysis of variance followed by Tukey’s multiple comparisons test). ns: not significant; Ti; titanium; TNA: titanium dioxide nanotube array.

Table 2. Various techniques used to generate nanotopographical features and surface roughness on implant materials

Physical techniques	Chemical techniques	Coating techniques	Others
Grit blasting	Anodization	Sol–Gel coating	Lithography
Nanoparticle compaction	Acid treatment	Self–assembly of monolayers	Ultraviolet photofunctionalization
	Alkali treatment	Discrete crystalline deposition
	Hydrogen peroxide treatment	Plasma spray
	Chemical vapor deposition	Ion implantation
		Sputtering
		Pulsed laser deposition
		Electron beam evaporation

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Cellular responses to nanoscale substrate topography of TiO₂ nanotube arrays: cell morphology and adhesion

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