Antibacterial Ti-Cu implants: A critical review on mechanisms of action

Abstract

Titanium (Ti) has been widely used for manufacturing of bone implants because of its mechanical properties, biological compatibility, and favorable corrosion resistance in biological environments. However, Ti implants are prone to infection (peri-implantitis) by bacteria which in extreme cases necessitate painful and costly revision surgeries. An emerging, viable solution for this problem is to use copper (Cu) as an antibacterial agent in the alloying system of Ti. The addition of copper provides excellent antibacterial activities, but the underpinning mechanisms are still obscure. This review sheds light on such mechanisms and reviews how incorporation of Cu can render Ti-Cu implants with antibacterial activity. The review first discusses the fundamentals of interactions between bacteria and implanted surfaces followed by an overview of the most common engineering strategies utilized to endow an implant with antibacterial activity. The underlying mechanisms for antibacterial activity of Ti-Cu implants are then discussed in detail. Special attention is paid to contact killing mechanisms because the misinterpretation of this mechanism is the root of discrepancies in the literature.

Keywords: Anti-infection; Antibacterial mechanisms; Antimicrobial; Biomaterials; Contact killing; Ion releasing; Ti–Cu implants.

Graphical abstract

Scheme 1

(a) General structure of a bacteria cell, and the cell wall structure in (b) Gram-negative bacteria, and (c) Gram-positive bacteria.

Scheme 2

Different stages of biofilm formation and development on a susceptible surface. Attachment: Initial colonizers adhere to the implant surface due to van der Waals forces and reproduce, followed by secretion of a gel-like substance (extracellular polysaccharides). Growth: The substance tenaciously bounds with the surface and protects bacteria against external threats, providing them with a safe environment to reproduce. Maturity: The population of bacteria in EPS increases until a mature biofilm is formed. Dispersal: The biofilm is dispersed by releasing either a small part of it or planktonic bacteria to colonize other sites of the implant [27].

Scheme 3

Various strategies to fabricate an antibacterial implant. Anti-adhesive implants are obtained through either changing the surface chemistry or morphology of the implants. Contact killing implants are fabricated by immobilizing antimicrobial peptides (AMP) on the surface or utilizing antimicrobial chemical compounds. Release-killing implants are acquired by equipping the surface with an antibacterial agent, which could be released from a matrix coated on the surface.

Fig. 1

An assessment to detect the cell membrane integrity of MRSA bacteria inoculated with copper surfaces. Bacteria were stained with Backlight™, Systo 9 (a), and propidium iodide (b). Cells with undamaged membranes fluoresce green (a), while bacteria with damaged membranes fluoresce red (b). This assessment shows that copper exposure does not disrupt the cell membrane integrity of MRSA. However, other analyses reported in this work confirmed that bacteria were killed as a result of damages to their DNA and respiratory system [150].

Fig. 2

Scanning electron microscope (SEM) images of the contact array system designed by Mathews et al. [160] This system was used to show the significant role of the contact-killing mechanism in the antibacterial ability of a copper surface. The surface of the copper coupon was coated with an inert polymer to prevent contact of the E. hirae bacteria with the metal surface. The SEM images of the honeycomb-like structure of inert polymer coated on the surface of the copper coupon at relatively low (a) and high (b and c) magnifications. The size of the holes was designed to be smaller than that of bacteria to prevent the contact of bacteria with the copper surface. The red arrows point to E. hirae bacteria.

Fig. 3

Typical S. aureus and E. coli colonization images of (a)–(b) negative samples, (c)–(d) control samples (cp-Ti), and (e)–(f) Ti–10Cu samples after incubation for 24 ​h in the plate counting test method. A large number of bacteria were detected on the negative and cp-Ti samples, indicating that cp-Ti does not show antibacterial activity. However, the copper-containing sample (Ti–10Cu) killed nearly all S. aureus and E. coli bacteria, indicating that Ti–10Cu implants exhibit a strong antibacterial activity [19].

Fig. 4

TEM images of S. mutans and P. gingivalis bacteria. (a) S. mutans bacteria exposed to Ti. (b) P. gingivalis bacteria exposed to Ti. (c) S. mutans bacteria exposed to Ti–Cu alloy. (d) P. gingivalis bacteria exposed to Ti–Cu alloy. White and black arrows show the peptidoglycan layer and the cell membrane, respectively. The red arrow determines the separation of the cytoplasmic membrane from the cell wall and expels the contents of the cell. The bacteria exposed to the Cu-containing surfaces exhibit shriveled or cracked morphologies [39].

Scheme 4

Schematic of contact killing mechanism of Ti–Cu implants containing the intermetallic phase of Ti₂Cu in their microstructure. (a) The planktonic bacteria adhere to the surface of the implant. (b) After contact with the implant surface, the cell wall of bacteria is disintegrated and the cellular content leaks out. (c) The potential difference between Ti₂Cu and Ti matrix results in the formation of micro-galvanic cell and the generated electron transport between the anode and the cathode of the cell is conducive to the interfering of normal cell function such as ATP synthesis and disturbing the potential membrane. Another suggested mechanism is the disruption of balance in the internal-external osmotic pressure of the cell due to the excessive accumulation of K⁺ outside the bacteria which is culminated in the disintegration of the cell.

Fig. 5

The effect of copper concentration present in the Ti-x wt.% Cu alloys on the S. aureus bacterial colonies in the plate counting test method. (a) Negative sample, (b) cp-Ti sample (c) Ti–2Cu sample, (d) Ti–5Cu sample, (e) Ti–10Cu sample, and (f) Ti–25Cu sample. While colonies excited in the case of Ti-2.5Cu and Ti–5Cu alloys, by increasing the copper content in the alloys, the bacterial colonies decreased, and alloys that contained more than 10 ​wt.% copper killed almost all bacteria [208].

Fig. 6

The relationship between ion releasing of an alloy and its antibacterial performance estimated in various publications [19,39,177,178,190,207,208,257]. These data well show that the ion-releasing characteristic is not the only factor determining the antibacterial performance. For example, the ion releasing amount of Ti–10Cu(I) alloy [180] is more than that of Ti–5Cu(S) [180], however, the antibacterial rate of the latter is much higher than the former.

Fig. 7

SEM images from different microstructures formed by various heat treatments and the corresponding antibacterial activities observed in the plate counting test. (a) Ti–5Cu(I) with a high ratio of solid solution and low content of Ti₂Cu phase, and (b) the corresponding bacterial colonization. (c) Ti–5Cu(T4) alloys with fully solid solution microstructure, and (d) the corresponding bacterial colonization. (e) Ti–5Cu (T6) alloy with a high ratio of intermetallic phase and a small amount of solid solution, and (f) the corresponding bacterial colonization. (g) Ti–5Cu(S) alloy with an extremely high ratio of intermetallic phase and an extremely low content of the solid solution, and (h) corresponding bacterial colonization. With the increase in the intermetallic phase, the antibacterial efficacy of alloys enhances [190].

Scheme 5

Schematic of two existing forms of copper in Ti–Cu alloys and their different roles on the invading bacteria. (a) An alloy with a fully solid solution microstructure that is unable to kill all bacteria coming in contact with the implant surface, resulting in biofilm formation. (b) An alloy containing large concentrations of Ti₂Cu and a small fraction of solid solution showing effective antimicrobial performance. In this case, the Ti₂Cu particles prevent bacterial adhesion to the surface and thus inhibit the formation of biofilm. The Cu²⁺ ions kill planktonic bacteria near the surface. The schematic is inspired by Ref. [190].

Fig. 8

Typical S. aureus colonies on agar plate incubated with bacteria obtained from a, b) the suspension with cp-Ti and the suspension with Ti–3Cu; and c, d) the surface of cp-Ti and the surface of Ti–3Cu, respectively [177]. There is no difference in the cell number between the Cu-free and Cu-containing suspensions sample, indicating the Ti–Cu is unable to kill bacteria when they are in planktonic mode. However, bacteria that come in contact with Cu-containing samples are killed at a high rate.