Regulation of RAW 264.7 macrophages behavior on anodic TiO<sub>2</sub> nanotubular arrays

doi:10.1007/s11706-017-0402-z

Front. Mater. Sci.

2017, Vol. 11

Issue (4) : 318-327 https://doi.org/10.1007/s11706-017-0402-z

RESEARCH ARTICLE

Regulation of RAW 264.7 macrophages behavior on anodic TiO₂ nanotubular arrays

Shenglian YAO^1,², Xujia FENG¹, Wenhao LI¹, Lu-Ning WANG¹(

), Xiumei WANG²(

)

¹. School of Materials Science and Engineering, University of Science and Technology Beijing, Beijing 100083, China
². School of Materials Science and Engineering, Tsinghua University, Beijing 100084, China

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Abstract

Titanium (Ti) implants with TiO₂ nanotubular arrays on the surface could regulate cells adhesion, proliferation and differentiation to determine the bone integration. Additionally, the regulation of immune cells could improve osteogenesis or lead in appropriate immune reaction. Thus, we evaluate the behavior of RAW 264.7 macrophages on TiO₂ nanotubular arrays with a wide range diameter (from 20 to 120 nm) fabricated by an electrochemical anodization process. In this work, the proliferation, cell viability and cytokine/chemokine secretion were evaluated by CCK-8, live/dead staining and ELISA, respectively. SEM and confocal microscopy were used to observe the adhesion morphology. Results showed that the small size nanotube surface was benefit for the macrophages adhesion and proliferation, while larger size surface could reduce the inflammatory response. These findings contribute to the design of immune-regulating Ti implants surface that supports successful implantation.

Keywords RAW 264.7 macrophages nanotopography TiO₂ nanotubular arrays inflammation

Corresponding Author(s): Lu-Ning WANG,Xiumei WANG

Online First Date: 03 November 2017 Issue Date: 29 November 2017

Cite this article:

Shenglian YAO,Xujia FENG,Wenhao LI, et al. Regulation of RAW 264.7 macrophages behavior on anodic TiO₂ nanotubular arrays[J]. Front. Mater. Sci., 2017, 11(4): 318-327.

URL:

https://academic.hep.com.cn/foms/EN/10.1007/s11706-017-0402-z
https://academic.hep.com.cn/foms/EN/Y2017/V11/I4/318

Fig.1 SEM images of the various sample surfaces (the insert images are the bottom of the TiO₂ nanotubular arrays): (a)flat Ti with native TiO₂ oxide layer; (b) 20 nm, (c) 50 nm and (d) 120 nm TiO₂ nanotubular surface.

Fig.2 Typical SEM morphologies of macrophage cells cultured on (a1)(a2)flat Ti and (b1)(b2)(c1)(c2)(d1)(d2)TiO₂ nanotubular surface with various sizes (20, 50 and 120 nm). The red block area in (a1)–(d1) are magnified in (a2)–(d2).

Fig.3 Macrophage cells adhesion and proliferation on the various sample surfaces by CCK-8 assay. Error bars represent standard deviation for three specimens for each piece of data. * Statistical significance (p<0.05).

Fig.4 Fluorescence images showing live/dead assays at 2 d post-seeding with/without the LPS pro-inflammatory. With the LPS stimulation (+LPS), the live cell density on all the surface ((a2) Ti, (b2)20 nm, (c2)50 nm and (d2)120 nm) is lower than the standard culture condition (−LPS) on (a1) Ti and (b1)(c1)(d1) TiO₂ nanotubular surfaces (20, 50 and 120 nm). Green= live cells; Red= dead cells.

Fig.5 Fluorescence micrographs of RAW 264.7 cells stained with rhodamine phalloidin (F-actin) and DAPI (nucleus) after 4 d of culture. Comparing to the standard culture condition (−LPS) on various surfaces ((a1) Ti, (b1) 20 nm, (c1) 50 nm and (d1) 120 nm), the stimulated cells (+LPS) on various surfaces ((a2) Ti, (b2) 20 nm, (c2) 50 nm and (d2) 120 nm) showed larger spread area.

Fig.6 Morphology of macrophage cells cultured on (a1)(a2)flat Ti and (b1)(b2)(c1)(c2)(d1)(d2) TiO₂ nanotubular surface with various sizes (20, 50 and 120 nm) with the LPS pro-inflammatory.

Fig.7 Mean (±SD) levels of (a) MCP-1 and (b) TNF-α secreted into culture media after 4 d culture of RAW 264.7 macrophages without the LPS pro-inflammatory, as detected by ELISA. * Statistical significance (p<0.05).

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