An antibacterial mechanism of titanium alloy based on micro-area potential difference induced reactive oxygen species

doi:10.1016/j.jmst.2021.12.031

Abstract

Abstract:

Antimicrobial material is highly desired because of the increasing demand in biomedical application to prevent from the formation of biofilm. A common strategy for enhancing the antibacterial property of a metal material is to incorporate toxic metal such as Cu and Ag. However, the reported Cu²⁺ or Ag⁺ released concentration from antibacterial alloys was much less than the reported minimum inhibitory ion concentrations (MIC), revealing the existence of an unknown alternative antimicrobial mechanism not relying on the toxicity of the metal ions. Herein, we proposed a new antibacterial mechanism that the antibacterial effectiveness of the different alloys is proportional to the micro-area potential differences (MAPDs) on the surface of the alloys. We designed three kinds of Ti-M (M=Zr, Ta and Au) alloys to eliminate the potential antibacterial contribution from Cu and Ag ion. We demonstrated that high MAPDs are associated with great production of reactive oxygen species (ROS), resulting in the killing effect to the biofilm known to be associated with implant infections (Staphlococcus aureus and Escherichia coli). These results provide new insights for the design of antibacterial alloys.

Key words: Antibacterial alloy, Micro-area potential difference, Electron transfer, Antibacterial mechanism

Shan Fu, Yuan Zhang, Yi Yang, Xiaomeng Liu, Xinxin Zhang, Lei Yang, Dake Xu, Fuhui Wang, Gaowu Qin, Erlin Zhang. An antibacterial mechanism of titanium alloy based on micro-area potential difference induced reactive oxygen species[J]. J. Mater. Sci. Technol., 2022, 119: 75-86.

Figures/Tables 9

Fig. 1. Summary of antibacterial rates and the Cu ion or Ag ion released concentrations of the reported antibacterial alloys: Ti-Cu alloy [[31], [32], [33], [34]], Ti-Ag alloy [26,29,30,35,36], Ti6Al4V-Cu alloy [37,38], Ti-Mo-Cu alloy [39], Ti-Mo-Ag alloy [40], Cobalt alloy [41,42], Ti-Ni-Ag alloy [24], Ti-Nb-Zr-Ag alloy and Nb-Ag alloy [43,44].

Fig. 2. Antibacterial activities of Ti-M samples against various bacteria: (a) 3D morphology of the fluorescently stained S. aureus, E. coli biofilms on the sample surfaces. (b) SEM images of S. aureus and E. coli on different titanium samples at different time. (c, d) Antibacterial effective against S. aureus and E. coli during cultivation. (e) Thickness of the biofilms that developed on the different material surfaces. (*denotes p < 0.05). (f) Antibacterial rate of pure metals and the extracts of Ti-Zr, Ti-Ta and Ti-Au samples against S. aureus.

Fig. 3. Surface physical properties of Ti-M samples: (a) XPS full spectra for four samples. (b-d) XPS high-resolution spectra of Zr 3d, Ta 4f and Au 4f, respectively. (e) XRD patterns of Ti-M samples. (f) Water contact angles of the samples (p>0.05). (g) Surface roughness of the samples (p>0.05).

Fig. 4. Surface characterization of Ti-M alloy. (a) BSE images of Ti-M alloy. (b) Elements mappings of Zr, Ta and Au in the Ti-M alloy. (c) Surface potential images of the Ti-M alloy. (d) SVET results of current density distribution on the surface of Ti-M alloy in the 3.5% NaCl solution. The scan area is 500 μm × 500 μm (X × Y). (e) Tafel curves of Ti-M alloy. (f) Corrosion potential of different samples. (g) Corrosion current density of different samples.

Table 1. EDS results and the existing form of element in different Ti-M samples

Sample	Position	Elements (at.%)		Phase constitute
Ti-Zr		Ti	Zr
	Point A	51.91	48.09	α-Ti(Zr) with about 48.09 at% solid solution Zr
	Area B	93.55	6.45	α-Ti(Zr) with about 6.45 at% solid solution Zr
	Area C	0.63	99.37	Zr(Ti) with about 0.63 at% solid solution Ti
Ti-Ta		Ti	Ta
	Point D	72.16	27.84	α-Ti(Ta) with about 27.84 at% solid solution Ta
	Area E	1.07	98.93	Ta(Ti) with about 1.07 at% solid solution Ti
	Area F	99.85	0.15	α-Ti(Ta) with about 0.15 at% solid solution Ta
Ti-Au		Ti	Au
	Area G	99.79	0.21	α-Ti(Au) with about 0.21 at% solid solution Au
	Point H	73.97	26.03	Eutectoid phase Ti₃Au

Fig. 4 (c) shows the surface potential distribution of Ti-M samples. There was no obvious undulation in the potential distribution on the surface of Ti-Zr sample and the calculated micro area potential difference (MAPD) was about 74 mV, indicating that the potential of Zr-rich phase was close to that of titanium matrix. In contrast, obvious undulation in the potential distribution and much high MAPD was detected in Ti-Ta alloy and Ti-Au alloy. The calculated MAPD is approximately 207 mV for Ti-Ta alloy and 272 mV for Ti-Au alloy, respectively. The difference in the MAPD reflects the inhomogeneous composition of the samples and is consistent with the difference between the midpoint of the Ti2+/Ti redox couple (-1.628 V) and the midpoint redox potentials of the alloying metals (Zr4+/Zr, -1.529 V; Ta3+/Ta, -0.6 V; Au3+/Au, 1.498 V). The electrode potential of intermetallic compounds was theoretically in order of: Zr(Ti)< Ta(Ti)<Ti3Au. This was consistent with the result of SKPFM measurement.

Fig. 5. Live/dead and ROS staining images of (a) S. aureus and (b) E. coli, respectively (Scale bar=100 μm). (c) Representative galvanic corrosion current density curves of Ti-Zr(Ti) couple, Ti-Ta(Ti) couple and Ti-Ti3Au couple with live and dead bacteria. (d) The calculated electron transfers between two electrodes in Ti-Zr(Ti) couple, Ti-Ta(Ti) couple and Ti-Ti3Au couple with live and dead bacteria. (e) Electron transfer from metal surface to live bacteria on Ti-Zr(Ti) couple, Ti-Ta(Ti) couple and Ti-Ti3Au couple. (f) pH and (g) ORP changes in the culture medium on different samples.

Cytocompatibility of the samples: (a) ROS expression of MC3T3-E1 osteoblasts cultured on different samples for 4 h and 24 h. (b) cytoskeleton staining of MC3T3-E1 osteoblasts on Ti-M samples and cp-Ti after culturing for 4 h and 24 h. (c) OD value and (d) RGR of MC3T3-E1 osteoblasts by the CCK-8 test after culturing for 1, 3, and 5 days on Ti-M samples. (*denotes p<0.05).

Fig. 7. Schematic of antibacterial mechanism via micro-area potential difference. (a) Properties of material surface. (b) Effect of MAPD on properties of samples and solution after immersing in liquid. (c) Effect of MAPD on bacteria.

References 59

[1]	Z. Jing, R. Ni, J. Wang, X. Lin, D. Fan, Q. Wei, T. Zhang, Y. Zheng, H. Cai, Z. Liu, Bioact. Mater. 6 (2021) 4542-4557.
[2]	P. Makvandi, C.-y. Wang, E.N. Zare, A. Borzacchiello, L.-n. Niu, F.R. Tay, Adv. Funct. Mater. 30 (2020) 1910021.
[3]	G. Mabilleau, S. Bourdon, M. Joly-Guillou, R. Filmon, M. Baslé, D. Chappard, Acta Biomater 2 (2006) 121-129. PMID
[4]	H.J. Han, S. Kim, D.H. Han, Int. J. Oral Max. Impl. 29 (2014) 303-310.
[5]	M.L. Knetsch, L.H. Koole, Polymers 3 (2011) 340-366. DOI URL
[6]	R. Liu, K. Memarzadeh, B. Chang, Y. Zhang, Z. Ma, R.P. Allaker, L. Ren, K. Yang, Sci. Rep. 6 (2016) 1-10. DOI URL
[7]	E. Zhang, F. Li, H. Wang, J. Liu, C. Wang, M. Li, K. Yang, Mater. Sci. Eng. C 33 (2013) 4280-4287. DOI URL
[8]	S. Rajalakshmi, A. Fathima, J.R. Rao, B.U. Nair, RSC Advances 4 (2014) 32004-32012. DOI URL
[9]	S. Shrivastava, T. Bera, A. Roy, G. Singh, P. Ramachandrarao, D. Dash, Nanotech- nology 18 (2007) 225103.
[10]	A. Kumar, P.K. Vemula, P.M. Ajayan, G. John, Nat. Mater. 7 (2008) 236. DOI URL
[11]	K. Huo, X. Zhang, H. Wang, L. Zhao, X. Liu, P.K. Chu, Biomaterials 34 (2013) 3467-3478. DOI URL
[12]	K. Li, C. Xia, Y. Qiao, X. Liu, J. Trace Elem. Med. Biol. 55 (2019) 127-135. DOI URL
[13]	H. Qin, H. Cao, Y. Zhao, C. Zhu, T. Cheng, Q. Wang, X. Peng, M. Cheng, J. Wang, G. Jin, Y. Jiang, X. Zhang, X. Liu, P.K. Chu, Biomaterials 35 (2014) 9114-9125. DOI URL
[14]	I. Sondi, B. Salopek-Sondi, J. Colloid Interface Sci. 275 (2004) 177-182. DOI URL
[15]	G. Wang, W. Jin, A.M. Qasim, A. Gao, X. Peng, W. Li, H. Feng, P.K. Chu, Bioma- terials 124 (2017) 25-34.
[16]	H. Cao, X. Liu, F. Meng, P.K. Chu, Biomaterials 32 (2011) 693-705. DOI URL
[17]	J. Ye, B. Li, M. Li, Y. Zheng, S. Wu, Y. Han, Bioact. Mater. 11 (2021) 181-191.
[18]	J. Diendorf, K. Loza, C. Sengstock, J. Mater. Chem. B 2 (2014) 1634-1643. DOI PMID
[19]	A. Pietroiusti, L. Campagnolo, B. Fadeel, Small 9 (2013) 1557-1572. DOI PMID
[20]	R. Foldbjerg, P. Olesen, M. Hougaard, D.A. Dang, H.J. Hoffmann, H. Autrup, Tox- icol. Lett. 190 (2009) 156-162.
[21]	K. Soto, K.M. Garza, L.E. Murr, Acta Biomater 3 (2007) 351-3582. DOI URL
[22]	A. Nair, G. Mun, P. Hande, S. Valiyaveettil, ACS Nano 3 (2009) 279-290. DOI URL
[23]	E. Zhang, X. Zhao, J. Hu, R. Wang, S. Fu, G. Qin, Bioact. Mater. 6 (2021) 2569-2612.
[24]	Y. Zheng, B. Zhang, B. Wang, Y. Wang, L. Li, Q. Yang, L. Cui, Acta Biomater 7 (2011) 2758-2767. DOI PMID
[25]	Y. Yuan, R. Luo, J. Ren, L. Zhang, Y. Jiang, Z. He, Mater. Lett. 306 (2022) 130875. DOI URL
[26]	M. Chen, E. Zhang, L. Zhang, Mater. Sci. Eng. C 62 (2016) 350-360. DOI URL
[27]	J. Hu, H. Li, X. Wang, L. Yang, M. Chen, R. Wang, G. Qin, D.-F. Chen, E. Zhang, Mater. Sci. Eng. C 115 (2020) 110921. DOI URL
[28]	C. Peng, S. Zhang, Z. Sun, L. Ren, K. Yang, Mater. Sci. Eng. C 93 (2018) 495-504. DOI URL
[29]	M. Chen, L. Yang, L. Zhang, Y. Han, Z. Lu, G. Qin, E. Zhang, Mater. Sci. Eng. C 75 (2017) 906-917. DOI URL
[30]	A. Shi, C. Zhu, S. Fu, R. Wang, G. Qin, D.-F. Chen, E. Zhang, Mater. Sci. Eng. C 109 (2020) 110548. DOI URL
[31]	S.M. Javadhesari, S. Alipour, M. Akbarpour, Colloids Surf. B 189 (2020) 110889. DOI URL
[32]	J. Liu, X. Zhang, H. Wang, F. Li, M. Li, K. Yang, E. Zhang, Biomed. Mater. 9 (2014) 025013. DOI URL
[33]	M. Bao, Y. Liu, X. Wang, L. Yang, S. Li, J. Ren, G. Qin, E. Zhang, Bioact. Mater. 3 (2018) 28-38.
[34]	X. Wang, H. Dong, J. Liu, G. Qin, D. Chen, E. Zhang, Mater. Sci. Eng. C 100 (2019) 38-47. DOI URL
[35]	Z. Lei, H. Zhang, E. Zhang, J. You, X. Ma, X. Bai, Mater. Sci. Eng. C 92 (2018) 121-131. DOI URL
[36]	M.K. Kang, S.K. Moon, J.S. Kwon, K.M. Kim, K.N. Kim, Mater. Res. Bull. 47 (2012) 2952-2955. DOI URL
[37]	Z. Ma, L. Ren, R. Liu, K. Yang, Y. Zhang, Z. Liao, W. Liu, M. Qi, R. Misra, J. Mater. Sci. Technol. 31 (2015) 723-732. DOI URL
[38]	S. Guo, Y. Lu, S. Wu, L. Liu, M. He, C. Zhao, Y. Gan, J. Lin, J. Luo, X. Xu, Mater. Sci. Eng., C 72 (2017) 631-640. DOI URL
[39]	W. Xu, C. Hou, Y. Mao, L. Yang, M. Tamaddon, J. Zhang, X. Qu, C. Liu, B. Su, X. Lu, Bioact. Mater. 5 (2020) 659-666.
[40]	Y. Zhang, K. Chu, S. He, B. Wang, W. Zhu, F. Ren, Mater. Sci. Eng. C 106 (2020) 110165. DOI URL
[41]	W. Li, X. Wang, C. Liu, G. Qin, E. Zhang, J. Mater. Sci.-Mater. Med. 30 (2019) 112. DOI URL
[42]	R. Wang, R. Wang, D. Chen, G. Qin, E. Zhang, J. Alloy. Compd. 824 (2020) 153924. DOI URL
[43]	D. Cai, X. Zhao, L. Yang, R. Wang, G. Qin, D.-F. Chen, E. Zhang, J. Mater. Sci. Technol. 81 (2021) 13-25. DOI URL
[44]	T. Wan, K. Chu, J. Fang, C. Zhong, Y. Zhang, X. Ge, Y. Ding, F. Ren, J. Mater. Sci. Technol. 80 (2021) 266-278. DOI URL
[45]	B. Fe rhat, L.E. Buelbuel, Appl. Phys. A: Mater. Sci. Process. 122 (2016) 25. DOI URL
[46]	C. Vasilescu, S.I. Drob, P. Osiceanu, J.M. Calderon, Metall. Mater. Trans. A 48 (2016) 1-11. DOI URL
[47]	E. Zhang, J. Ren, S. Li, L. Yang, G. Qin, Biomed. Mater. 11 (2016) 065001. DOI URL
[48]	J. Liu, F. Li, C. Liu, H. Wang, B. Ren, K. Yang, E. Zhang, Mater. Sci. Eng. C 35 (2014) 392-400. DOI URL
[49]	S. Fu, Y. Zhang, G. Qin, E. Zhang, Mater. Sci. Eng. C 128 (2021) 112266. DOI URL
[50]	Y. Lekbach, T. Liu, Y. Li, M. Moradi, W. Dou, D. Xu, J.A. Smith, D.R. Lovley, R.K. Poole, D.J. Kelly, in:Advances in Microbial Physiology, Academic Press, 2021, pp. 317-390.
[51]	A. Medve ďová, Ľ. Valík, InTech 4 (2012) 71-102.
[52]	C. Dong, W. Feng, W. Xu, L. Yu, H. Xiang, Y. Chen, J. Zhou, Adv. Sci. 7 (2020) 2001549.
[53]	H. Zhu, J. Yin, X. Wang, H. Wang, X. Yang, Adv. Funct. Mater. 23 (2013) 1305-1312. DOI URL
[54]	M.J. Flores, R.J. Brandi, A.E. Cassano, M.D. Labas, Chem. Eng. J. 198 (2012) 388-396.
[55]	M. Song, Y. Cheng, Y. Tian, C. Chu, C. Zhang, Z. Lu, X. Chen, X. Pang, G. Liu, Adv. Funct. Mater. 30 (2020) 2003587.
[56]	C. Nathan, A. Ding, Cell 140 (2010) 871-882. DOI PMID
[57]	M.A. Kohanski, D.J. Dwyer, B. Hayete, C.A. Lawrence, J.J. Collins, Cell 130 (2007) 797-810. PMID
[58]	H. Cao, Y. Qiao, X. Liu, T. Lu, T. Cui, F. Meng, P.K. Chu, Acta Biomater 9 (2013) 5100-5110. DOI URL
[59]	G. Wang, H. Feng, L. Hu, W. Jin, Q. Hao, A. Gao, X. Peng, W. Li, K.-Y. Wong, H. Wang, Nat. Commun. 9 (2018) 2055. DOI URL