Engineering nano-structures with controllable dimensional features on micro-topographical titanium surfaces to modulate the activation degree of M1 macrophages and their osteogenic potential

doi:10.1016/j.jmst.2021.03.078

Abstract

Abstract:

Modulating the activation state and degree of macrophages still remains as a challenge for the topographical design of Ti-based implants. In this work, micro/nano-structured coatings were prepared on Ti substrates by micro-arc oxidation (MAO) and subsequent hydrothermal (HT) treatment. By varying the HT conditions, plate-like nano-structures with an average length of 80, 440 or 780 nm were obtained on MAO-prepared micro-topographical surfaces. Depending on the dimensional features of nano-plates, the specimens were noted as Micro, Micro/Nano-180, Micro/Nano-440 and Micro/Nano-780, respectively. The in vitro results showed that the activation state and degree of macrophages could be effectively modulated by the micro/nano-structured surfaces with various dimensional features. Compared to the Micro surface, the Micro/Nano-180 surface activated both M1 and M2 phenotype in macrophages, while the Micro/Nano-440 and Micro/Nano-780 surfaces polarized macrophages to their M1 phenotype. The activation degree of M1 macrophages followed the trend: Micro < Micro/Nano-180 < Micro/Nano-440 < Micro/Nano-780. However, the osteogenic potential of the activated macrophages in response to various surfaces were in the order: Micro ≈ Micro/Nano-780 < Micro/Nano-180 < Micro/Nano-440. Together, the findings presented in this work indicate that engineering nano-structures with controllable dimensional features is a promising strategy to modulate macrophage activation state and degree. In addition, it is essential to determine the appropriate activation degree of M1 macrophages for enhanced osteogenesis.

Key words: Titanium, Micro/nano-structures, Macrophages activation, Inflammatory response, Osteogenesis

Luxin Liang, Qianli Huang, Hong Wu, Hao He, Guanghua Lei, Dapeng Zhao, Kun Zhou. Engineering nano-structures with controllable dimensional features on micro-topographical titanium surfaces to modulate the activation degree of M1 macrophages and their osteogenic potential[J]. J. Mater. Sci. Technol., 2022, 96: 167-178.

Figures/Tables 11

Fig. 1. SEM images showing the (a) surface and (b) cross-sectional morphology of (a1) MAO-treated Ti (a1) without and with HT for (a2, b1) 8, (a3, b2) 36 and (a4, b3) 72 h; (c) the contact angles (CAs) of various surfaces.

Fig. 2. (a) XRD patterns as well as high-resolution XPS spectra of (b) Ti 2p, (c) Ca 2p and (d) Si 2p in various groups.

Fig. 3. (a) TEM and its corresponding EDX mapping images of a single nano-plate in the Micro/Nano-440 group as well as (b) the quantiatative EDX results.

Fig. 4. Concentrations of (a) Ca2+ and (b) SiO32+ released from various coatings after immersion in PBS for 1, 3, 5 and 7 days.

Table 1 Dimensional features of nano-plates prepared by HT for various durations.

Sample name	Length (nm)	Width (nm)	Thickness (nm)
Micro/Nano-180	180±20	100±20	16±3
Micro/Nano-440	440±50	120±30	20±4
Micro/Nano-780	780±50	120±30	22±4

Fig. 5. Viability and proliferation of macrophages in response to various surfaces evaluated by (a) calcein-AM staining and (b) CCK-8 assay for 1, 3 and 5 days; (c) SEM images showing the morphology of RAW 264.7 cells cultured on various coatings for 8 and 24 h.

Fig. 6. Immunofluorescent staining images showing the expressions of Arg-1(green, M2 marker) and iNOS (red, M1 marker) as well as the presence of cell nuclei (blue) in macrophages grown on various surfaces. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

Fig. 7. Gene expressions of RAW 264.7 cells in response to various surfaces analyzed by RT-qPCR. *p < 0.05 and **p < 0.01 compared to Micro group; #p < 0.05 and ##p < 0.01 compared to Micro/Nano-180 group; ζp < 0.05 and ζζp < 0.01 compared to Micro/Nano-440 group.

Fig. 8. Productions of cytokines including TNF-α and IL-10 as well as osteogenic growth factors including BMP-6, BMP-2 and OSM by macrophages in response to various surfaces for 24 h. *p < 0.05 and **p < 0.01 compared to Micro group; #p < 0.05 and ##p < 0.01 compared to Micro/Nano-180 group; ζp < 0.05 and ζζp < 0.01 compared to Micro/Nano-440 group.

Fig. 9. ALP activity, COL synthesis and in vitro Min of SaOS-2 cultured by various CM for 4, 7 and 10 days, respectively. *p < 0.05 and **p < 0.01 compared to Micro group; #p < 0.05 and ##p < 0.01 compared to Micro/Nano-180 group; ζp < 0.05 and ζζp < 0.01 compared to Micro/Nano-440 group.

Fig. 10. Schematics showing nucleation and growth mechanisms of nano-structures on MAO-treated Ti surfaces.

References 49

[1]	W. Tang, D. Lin, Y. Yu, H. Niu, H. Guo, Y. Yuan, C. Liu, Acta Biomater. 32 (2016) 309-323. DOI URL PMID
[2]	Q. Huang, Z. Ouyang, Y. Tan, H. Wu, Y. Liu, Acta Biomater. 100 (2019) 415-426. DOI URL
[3]	L. Bai, Y. Liu, Z. Du, Z. Weng, W. Yao, X. Zhang, X. Huang, X. Yao, R. Crawford, R. Hang, D. Huang, B. Tang, Y. Xiao, Acta Biomater. 76 (2018) 344-358. DOI URL
[4]	L. Bai, Z. Du, J. Du, W. Yao, J. Zhang, Z. Weng, S. Liu, Y. Zhao, Y. Liu, X. Zhang, X. Huang, X. Yao, R. Crawford, R. Hang, D. Huang, B. Tang, Y. Xiao, Biomaterials 162 (2018) 154-169. DOI URL
[5]	D.J. Lin, L.J. Fuh, W.C. Chen, Mater. Sci. Eng. C Mater.Biol. Appl. 107 (2020) 110204.
[6]	Z.t. Chen, T. Klein, R. Z., R.C Murray, J. Chang, C. Wu, a.Y. Xiao, Mater. Today 19 (2016) 304-321. DOI URL
[7]	R.J. Miron, D.D. Bosshardt, Biomaterials 82 (2016) 1-19.
[8]	R. Sridharan, A.R. Cameron, D.J. Kelly, C.J. Kearney, F.J. O’Brien, Mater. Today 18 (6) (2015) 313-325. DOI URL
[9]	Z. Chen, A. Bachhuka, F. Wei, X. Wang, G. Liu, K. Vasilev, Y. Xiao, Nanoscale 9 (46) (2017) 18129-18152. DOI URL
[10]	F.O. Martinez, S. Gordon, F100 0Prime Rep. 6 (2014).
[11]	C. Yin, Q. Zhao, W. Li, Z. Zhao, J. Wang, T. Deng, P. Zhang, K. Shen, Z. Li, Y. Zhang, Acta Biomater. 102 (2020) 416-426. DOI URL
[12]	B. Li, H. Cao, Y. Zhao, M. Cheng, H. Qin, T. Cheng, Y. Hu, X. Zhang, X. Liu, Sci. Rep. 7 (2017) 42707. DOI URL
[13]	T. Hu, H. Xu, C. Wang, H. Qin, Z. An. Sci. Rep. 8 (1) (2018) 3406.
[14]	Z. Chen, A. Bachhuka, S. Han, F. Wei, S. Lu, R.M. Visalakshan, K. Vasilev, Y. Xiao, ACS Nano 11 (5) (2017) 4494-4506. DOI URL
[15]	L. Rifas, J. Cell. Biochem. 98 (4) (2006) 706-714. DOI URL
[16]	P. Guihard, Y. Danger, B. Brounais, E. David, R. Brion, J. Delecrin, C.D. Richards, S. Chevalier, F. Redini, D. Heymann, H. Gascan, F. Blanchard, Stem. Cells. 30 (4) (2012) 762-772. DOI URL PMID
[17]	K. Hess, A. Ushmorov, J. Fiedler, R.E. Brenner, T. Wirth, Bone 45 (2) (2009) 367-376. DOI URL
[18]	J. Ding, O. Ghali, P. Lencel, O. Broux, C. Chauveau, J.C. Devedjian, P. Hardouin, D. Magne, Life Sci. 84 (15-16) (2009) 499-504. DOI URL PMID
[19]	M. Feldmann, R.N. Maini, J. Immunol. 185 (2) (2010) 791-794. DOI URL PMID
[20]	W.L. Lu, N. Wang, P. Gao, C.Y. Li, H.S. Zhao, Z.T. Zhang, Cell Prolif. 48 (1) (2015) 95-104. DOI URL
[21]	L.M. Chamberlain, K.S. Brammer, G.W. Johnston, S. Chien, S. Jin, J. Biomater. Nanobiotechnol. 02 (03) (2011) 293-300. DOI URL
[22]	E. Lamers, X.F. Walboomers, M. Domanski, L. Prodanov, J. Melis, R. Luttge, L. Winnubst, J.M. Anderson, H.J.G.E. Gardeniers, J.A. Jansen, Biol. Med. 8 (3) (2012) 308-317.
[23]	X. Li, Q. Huang, X. Hu, D. Wu, N. Li, Y. Liu, Q. Li, H. Wu, Mater. Sci. Eng. C 110 (2020).
[24]	Q. Huang, X. Liu, T.A. Elkhooly, R. Zhang, X. Yang, Z. Shen, Q. Feng, Mater. Sci. Eng. C Mater.Biol. Appl. 60 (2016) 308-316.
[25]	X. Li, Q. Huang, L. Liu, W. Zhu, T.A. Elkhooly, Y. Liu, Q. Feng, Q. Li, S. Zhou, Y. Liu, H. Wu, Coll. Surf. B Biointerfaces (171) (2018) 276-284.
[26]	Q. Huang, X. Li, T. Liu, H. Wu, X. Liu, Q. Feng, Y. Liu, Appl. Surf. Sci. 447 (2018) 767-776. DOI URL
[27]	Q. Huang, X. Liu, T.A. Elkhooly, R. Zhang, Z. Shen, Q. Feng, Coll. Surf. B Bioint- erfaces 134 (2015) 169-177.
[28]	Q. Huang, X. Liu, R. Zhang, X. Yang, C. Lan, Q. Feng, Y. Liu, Appl. Surf. Sci. 465 (2019) 575-583. DOI URL
[29]	R. Zhou, Y. Han, J. Cao, M. Li, G. Jin, Y. Du, H. Luo, Y. Yang, L. Zhang, B. Su, ACS Appl. Mater. Interfaces 10 (36) (2018) 30191-30200. DOI URL
[30]	G. Li, H. Cao, W. Zhang, X. Ding, G. Yang, Y. Qiao, X. Liu, X. Jiang, ACS Appl. Mater. Interfaces 8 (6) (2016) 3840-3852. DOI URL
[31]	Q. Huang, T.A. Elkhooly, X. Liu, R. Zhang, X. Yang, Z. Shen, Q. Feng, Coll. Surf. B Biointerfaces 145 (2016) 37-45. DOI URL
[32]	H. Wu, Y. Yin, X. Hu, C. Peng, Y. Liu, Q. Li, W. Huang, Q. Huang, ACS Biomater. Sci. Eng. 5 (10) (2019) 5548-5557. DOI URL
[33]	F.Y. McWhorter, T. Wang, P. Nguyen, T. Chung, W.F. Liu, Proc. Natl. Acad. Sci. USA. 110 (43) (2013) 17253-17258. DOI URL
[34]	M. Mohiuddin, H. Pan, Y. Hung, G. Huang, Nanoscale Res. Lett. 7 (2012) 1-9. DOI URL
[35]	T.U. Luu, S.C. Gott, B.W. Woo, M.P. Rao, W.F. Liu, ACS Appl. Mater. Interfaces 7 (51) (2015) 28665-28672. DOI URL
[36]	G. Guo, Q. Xu, C. Zhu, J. Yu, Q. Wang, J. Tang, Z. Huan, H. Shen, J. Chang, X. Zhang, Appl. Mater. Today 22 (2021) 100888.
[37]	S. Cheng, D. Zhang, M. Li, X. Liu, Y. Zhang, S. Qian, F. Peng, Bioact. Mater. 6 (1) (2021) 91-105. DOI URL PMID
[38]	J.K. Park, Y.J. Kim, J. Yeom, J.H. Jeon, G.C. Yi, J.H. Je, S.K. Hahn, Adv. Mater. 22 (43) (2010) 4857-4861.
[39]	P. Palmqvist, E. Persson, H.H. Conaway, U.H. Lerner, J. Immunol. 169 (6) (2002) 3353-3362. URL PMID
[40]	Y. Huang, C. Wu, X. Zhang, J. Chang, K. Dai, Acta Biomater. 66 (2018) 81-92. DOI URL PMID
[41]	C. Troidl, G. Jung, K. Troidl, J. Hoffmann, H. Moellmann, H. Nef, W. Schaper, C.W. Hamm, T. Schmitz-Rixen, Curr. Vasc. Pharmacol. 11 (2013) 5-12. URL PMID
[42]	M. Shi, Z. Chen, S. Farnaghi, T. Friis, X. Mao, Y. Xiao, C. Wu, Acta Biomater. 30 (2016) 334-344. DOI URL
[43]	C.M. Kemmis, A. Vahdati, H.E. Weiss, D.R. Wagner, Biochem. Biophys. Res. Commun. 401 (1) (2010) 20-25. DOI URL
[44]	G.E. Glass, J.K. Chan, A. Freidin, M. Feldmann, N.J. Horwood, J. Nanchahal, Proc. Natl. Acad. Sci. 108 (4) (2011) 1585-1590. DOI URL
[45]	H.Y J.Hashimoto, N.S K.Takaoka, T.T K.Masuhara, K.O S.Miyamoto., Bone 10 (6) (1989) 453-457. URL PMID
[46]	H. Yoshikawa, J. Hashimoto, K. Masuhara, K. Takaoka, K. Ono, Bone 9 (1988) 391-396. URL PMID
[47]	A. Al-Rasheed, H. Scheerens, D.M. Rennick, H.M. Fletcher, D.N. Tatakis, J. Dent. Res. 82 (8) (2003) 632-635. URL PMID
[48]	A. Al-Rasheed, H. Scheerens, A.K. Srivastava, D.M. Rennick, D.N. Tatakis, J. Peri- odontal Res. 39 (3) (2004) 194-198.
[49]	W. Liu, L. Liang, B. Liu, D. Zhao, Y. Tian, Q. Huang, H. Wu, There-sponse of macrophages and their osteogenic potential modulated by mi- cro/nano-structured Ti surfaces, Colloids and Surfaces B: Biointerfaces 205 (2021) 111848 In press.