An Antibacterial Strategy of Mg-Cu Bone Grafting in Infection-Mediated Periodontics

Abstract

Periodontal diseases are mainly the results of infections and inflammation of the gum and bone that surround and support the teeth. In this study, the alveolar bone destruction in periodontitis is hypothesized to be treated with novel Mg-Cu alloy grafts due to their antimicrobial and osteopromotive properties. In order to study this new strategy using Mg-Cu alloy grafts as a periodontal bone substitute, the in vitro degradation and antibacterial performance were examined. The pH variation and Mg²⁺ and Cu²⁺ release of Mg-Cu alloy extracts were measured. Porphyromonas gingivalis (P. gingivalis) and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), two common bacteria associated with periodontal disease, were cultured in Mg-Cu alloy extracts, and bacterial survival rate was evaluated. The changes of bacterial biofilm and its structure were revealed by scanning electron microscopy (SEM) and transmission electronic microscopy (TEM), respectively. The results showed that the Mg-Cu alloy could significantly decrease the survival rates of both P. gingivalis and A. actinomycetemcomitans. Furthermore, the bacterial biofilms were completely destroyed in Mg-Cu alloy extracts, and the bacterial cell membranes were damaged, finally leading to bacterial apoptosis. These results indicate that the Mg-Cu alloy can effectively eliminate periodontal pathogens, and the use of Mg-Cu in periodontal bone grafts has a great potential to prevent infections after periodontal surgery.

Figure 1

(a) pH value of the Mg-Cu alloy and Mg extracts. The results are in line with the immersion time. (b) Mg²⁺ concentration in the Mg-Cu alloy and Mg extracts and (c) Cu²⁺ concentration in Mg-Cu alloy extracts. The concentration of Cu²⁺ was positively related to the Cu content in the Mg-Cu alloy. The Mg-0.4Cu alloy released much more Cu²⁺ than the other Mg-Cu alloys. # refers to a significant difference between Mg-Cu alloy groups and the Mg group (P < 0.05, n = 3).

Figure 2

The responses of P. gingivalis (Pg) to the Mg-Cu alloy and Mg and β-TCP extracts: (a) bacterial concentration; (b) live bacterial colonies; (c) optical images of the bacterial colonies in nutrition agar plates and the responses of A. actinomycetemcomitans (Aa) to the Mg-Cu alloy and Mg and β-TCP extracts; (d) bacterial concentration; (e) live bacterial colonies; (f) optical images of the bacterial colonies in nutrition agar plates. ∗ refers to a significant difference between Mg-Cu alloy groups (Mg-0.1Cu, Mg-0.2Cu, and Mg-0.3Cu) and the BHI broth and β-TCP groups. # refers to a significant difference between Mg-Cu alloy groups and the Mg group (P < 0.05, n = 3).

Figure 3

Live/dead imaging of Pg and Aa after culturing in Mg-0.1Cu extracts for 24 h under a confocal fluorescence microscope. The green color indicates live bacterial while the red color indicates dead bacterial. Majorities of both Pg and Aa were dead in the Mg-0.1Cu alloy extract after culturing for 24 h.

Figure 4

SEM images of bacterial biofilm after culturing in the extract for 24 h. (a) Single-species biofilm of Pg and Aa, (b) multispecies biofilm of Sg, Fn, and Pg and Sg, Fn, and Aa, scale bar = 5 μm. Single arrow shows the normal bacterial biofilm. Double arrows show the damaged bacterial biofilm. The bacteria in the Mg-0.1Cu alloy group lost connection with each other, and the biofilm collapsed completely due to death of bacteria.

Figure 5

TEM images of the cell membrane and structure of Pg (a) and Aa (b) after culturing in extracts and BHI broth for 24 h, scale bar = 500 μm. Single arrow shows the change of bacterial membrane in the Mg group. The cell membrane showed folds and stratification in the pure Mg group, where the membrane was separated from cytoplasm. Double arrows show the bacteria change in the Mg-Cu alloy group. The dead bacteria showed almost intact cell membranes with little cytoplasm remaining.