Nanograins on Ti-25Nb-3Mo-2Sn-3Zr alloy facilitate fabricating biological surface through dual-ion implantation to concurrently modulate the osteogenic functions of mesenchymal stem cells and kill bacteria

doi:10.1016/j.jmst.2020.07.048

Abstract

Abstract:

Surface mechanical attrition treatment (SMAT) method is an effective way to generate nanograined (NG) surface on Ti-25Nb-3Mo-2Sn-3Zr (wt.%) (named as TLM), a kind of β-type titanium alloy, and the achieved nanocrystalline surface was proved to promote positive functions of osteoblastic cells. In this work, to further endow the NG TLM alloy with both good osteogenic and antibacterial properties, magnesium (Mg), silver (Ag) ion or both were introduced onto the NG TLM surface by ion implantation process, as a comparison, the Mg and Ag ions were also co-implanted onto coarsegrained (CG) TLM surface. The obtained results show that subsequent ion implantation does not remarkably induce the surface roughness and topography alteration of the SMAT-treated layers, and it also has little impact on the microstructure of the SMAT-derived β-Ti nanograins. In addition, the implanted Mg and Ag ions are observed to exist as MgO and metallic Ag nanoparticles (NPs) embedding tightly in the β-Ti matrix with grain size of about 15 and 7 nm, respectively. Initial cell adhesion and functions (including proliferation, osteo-differentiation and extracellular matrix mineralization) of rabbit bone marrow mesenchymal stem cells (rBMMSCs) and the bacterial colonization of Staphylococcus aureus (S. aureus) on the different surfaces were investigated. The in-vitro experimental results reveal that the Mg and Ag single-ion implanted NG surface either significantly promotes the rBMMSCs response or inhibits the growth of S. aureus, whereas the Mg/Ag co-implanted NG surface could concurrently enhance the rBMMSCs functions as well as inhibit the bacterial growth compared to the NG surface, and this efficacy is more pronounced as compared to the Mg/Ag co-implantation in the CG surface. The SMAT-achieved nanograins in the TLM surface layer are identified to not only play a leading role in determining the fate of rBMMSCs but also facilitate fabricating dual-functional surface with both good osteogenic and antibacterial activities through co-implantation of Mg and Ag ions. Our investigation provides a new strategy to develop high-performance Ti-based implants for clinical application.

Key words: β-Type titanium, Nanograined surface, Ion implantation, Stem cells, Antibacterial property

Run Huang, Lei Liu, Bo Li, Liang Qin, Lei Huang, Kelvin W.K. Yeung, Yong Han. Nanograins on Ti-25Nb-3Mo-2Sn-3Zr alloy facilitate fabricating biological surface through dual-ion implantation to concurrently modulate the osteogenic functions of mesenchymal stem cells and kill bacteria[J]. J. Mater. Sci. Technol., 2021, 73: 31-44.

Figures/Tables 12

Table 1 Sequences of the specific primer sets.

Target	Primer sequences
Runx2	Forward primer: 5’-TGGTGTTGACGCTGATGGAA-3’
	Reverse primer: 5’-ATACCGCTGGACCACTGTTG-3’
ALP	Forward primer: 5’-CTGAGCGTCCTGTTCTGAGG-3’
	Reverse primer: 5’-GTTCCTGGGTCCCCTTTCTG-3’
BSP	Forward primer: 5’-GTCAGAACTGCTGGGACTCG-3’
	Reverse primer: 5’-TGGCATTAGGTGTACTTGACAGT-3’
OPN	Forward primer: 5’-GTGTACCCCACTGAGGATGC-3’
	Reverse primer: 5’-CACGTGTGAGCTGAGGTCTT-3’
OCN	Forward primer: 5’-CTTCGTGTCCAAGAGGGAGC-3’
	Reverse primer: 5’-CAGGGGATCCGGGTAAGGA-3’
Col-I	Forward primer: 5’-TGCAGGGCTCCAATGATGTT-3’
	Reverse primer: 5’-AGGAAGGGCAAACGAGATGG-3’
GAPDH	Forward primer: 5’-ATCAAGTGGGGTGATGCTGG-3’
	Reverse primer: 5’-TACTTCTCGTGGTTCACGCC-3’

Fig. 1. TEM observations of the typical surface microstructure on the NG series samples. NG sample: (a) typical bright-field image, (b) corresponding SAED pattern and (c) dark-field image; Ag-NG sample: (d) typical bright-field image and corresponding SAED pattern (inset), (e) dark-field image taken on the reflection of Ag (200) diffraction ring in the inset of (d) and (f) HRTEM image of typical Ag grains marked in (d) with a white square; Mg-NG sample: (g) typical bright-field image and corresponding SAED pattern (inset), (h) dark-field image taken on the reflection of MgO (111) diffraction spots in the inset of (g) and (i) HRTEM image of typical MgO grains marked in (g) with a white square; MgAg-NG sample: (j) typical bright-field image and corresponding SAED pattern (inset), (k) and (l) are the dark-?eld images derived on the reflections of MgO (111) diffraction spot and Ag (200) diffraction ring in the inset of (j), respectively.

Fig. 2. Depth profiles of (a) Ag and (b) Mg acquired from the Ag-NG, Mg-NG, MgAg-NG and MgAg-CG samples.

Fig. 3. AFM images derived from various sample surfaces.

Table 2 Roughness (nm) measured by AFM analysis of NG, Ag-NG, Mg-NG, MgAg-NG, CG and MgAg-CG surfaces, data are presented as mean ± SD (n = 4).

Roughness	NG	Ag-NG	Mg-NG	MgAg-NG	CG	MgAg-CG
R_a	38.7 ± 2.8	36.9 ± 2.1	37.9 ± 3.2	36.1 ± 1.8	39.2 ± 3.1	37.4 ± 2.5
R_q	62.8 ± 5.4	59.8 ± 5.1	61.7 ± 5.9	59.2 ± 5.3	63.1 ± 4.2	60.9 ± 4.7
R_z	135.9 ± 11.8	133.0 ± 9.6	131.8 ± 12.3	132.6 ± 9.5	136.7 ± 10.8	133.8 ± 9.9

Fig. 4. Water droplets contact angles of various sample surfaces.

Fig. 5. Concentrations of (a) Ag released into PBS and (b) Mg released into cell culture medium after immersion different time of the Ag-NG, Mg-NG, MgAg-NG and MgAg-CG samples.

Fig. 6. Number of MSCs cultured on various sample surfaces at different time points. §p < 0.05 and §§p < 0.01 versus NG surface, ^^p < 0.01 versus Ag-NG surface, ⊥p < 0.05 and ⊥⊥p < 0.01 versus Mg-NG surface, σσp < 0.01 versus MgAg-NG surface, φp < 0.05 and φφp < 0.01 versus CG surface.

Fig. 7. Typical morphologies of MSCs seeded on various sample surfaces for 5 h.

Fig. 8. Osteogenesis-related gene expressions of MSCs after 3, 7 and 14 days of culture on various sample surfaces. §p < 0.05 and §§p < 0.01 versus NG surface, ^p < 0.05 and ^^p < 0.01 versus Ag-NG surface, ⊥p < 0.05 and ⊥⊥p < 0.01 versus Mg-NG surface, σσp < 0.01 versus MgAg-NG surface, φp < 0.05 and φφp < 0.01 versus CG surface.

Fig. 9. (a) ALP activity and protein (OPN, OCN and Col-I) contents levels, (b) collagen secretion, and (c) ECM mineralization of MSCs after 3, 7 and 14 days of culture on various sample surfaces. §p < 0.05 and §§p < 0.01 versus NG surface, ^p < 0.05 and ^^p < 0.01 versus Ag-NG surface, ⊥p < 0.05 and ⊥⊥p < 0.01 versus Mg-NG surface, σp < 0.05 and σσp < 0.01 versus MgAg-NG surface, φp < 0.05 and φφp < 0.01 versus CG surface.

Fig. 10. (a) SEM morphologies and (b) counted viable number of S. aureus cultured for 4 h on various sample surfaces. §§p < 0.01 versus NG surface, ^p < 0.05 and ^^p < 0.01 versus Mg-NG surface, ⊥p < 0.05 and ⊥⊥p < 0.01 versus MgAg-NG surface, σσp < 0.01 versus Ag-NG surface and φφp < 0.01 versus CG surface.

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