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. 2016 May 19:11:2223-34.
doi: 10.2147/IJN.S102752. eCollection 2016.

In vivo evaluation of the anti-infection potential of gentamicin-loaded nanotubes on titania implants

Ying Yang  1 , Hai-Yong Ao  1 , Sheng-Bing Yang  1 , Yu-Gang Wang  1 , Wen-Tao Lin  2 , Zhi-Feng Yu  1 , Ting-Ting Tang  1
Affiliations

In vivo evaluation of the anti-infection potential of gentamicin-loaded nanotubes on titania implants

Ying Yang et al. Int J Nanomedicine. .

Abstract

Titanium-based implants have been widely used in orthopedic surgery; however, failures still occur. Our in vitro study has demonstrated that gentamicin-loaded, 80 nm-diameter nanotubes possessed both antibacterial and osteogenic activities. Thus, the aim of this study was to further investigate the in vivo anti-infection effect of the titanium implants with gentamicin-loaded nanotubes. Thirty-six male Sprague Dawley rats were used to establish an implant-associated infection model. A volume of 50 μL Staphylococcus aureus suspension (1×10(5) CFU/mL) was injected into the medullary cavity of the left femur, and then the titanium rods without modification (Ti), titanium nanotubes without drug loading (NT), and gentamicin-loaded titanium nanotubes (NT-G) were inserted with phosphate-buffered saline-inoculated Ti rods as a blank control. X-ray images were obtained 1 day, 21 days, and 42 days after surgery; micro-computed tomography, microbiological, and histopathological analyses were used to evaluate the infections at the time of sacrifice. Radiographic signs of bone infection, including osteolysis, periosteal reaction, osteosclerosis, and damaged articular surfaces, were demonstrated in the infected Ti group and were slightly alleviated in the NT group but not observed in the NT-G group. Meanwhile, the radiographic and gross bone pathological scores of the NT-G group were significantly lower than those of the infected Ti group (P<0.01). Explant cultures revealed significantly less bacterial growth in the NT-G group than in the Ti and NT groups (P<0.01), and the NT group showed decreased live bacterial growth compared with the Ti group (P<0.01). Confocal laser scanning microscopy, scanning electron microscopy, and histopathological observations further confirmed decreased bacterial burden in the NT-G group compared with the Ti and NT groups. We concluded that the NT-G coatings can significantly prevent the development of implant-associated infections in a rat model; therefore, they may provide an effective drug-loading strategy to combat implant-associated infections in clinic.

Keywords: animal model; gentamicin; implant-associated infection; titanium nanotubes.

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Figures

Figure 1
Figure 1
Radiographic images and analysis at 1 day, 3 weeks, and 6 weeks. Notes: (A) Lateral X-rays of the left femur. The red arrows indicate osteolysis and slight periosteal reactions around the distal femurs, while the white arrows indicate articular surface damage, osteosclerosis, and deformities. (B) Three-dimensional micro-CT images of the left femur at the time of sacrifice. The micro-CT evaluations of the middle femurs were confined to the red square region. (C) Radiographic scores of the X-ray images. *Denotes a significant difference compared with groups I and II (P<0.01, n=5). **Denotes a significant difference compared with group I (P<0.05, n=5). (D) Bone volume/total volume and (E) cortical bone mineral density of the selected regions of the left femurs evaluated by micro-CT. *Denotes a significant difference compared with groups I and II (P<0.01, n=5). Groups I, II, III, and IV indicate Ti + S. aureus, NT + S. aureus, NT-G + S. aureus, and Ti + PBS, respectively. Ti, titanium without modification. Abbreviations: CT, computed tomography; d, day; NT, nanotubes; NT-G, gentamicin-loaded nanotubes; PBS, phosphate-buffered saline; S. aureus, Staphylococcus aureus; w, weeks.
Figure 2
Figure 2
Gross appearance and scores of the left femur longitudinal sections at the time of sacrifice. Notes: (A) Gross appearance. The white arrows mark intramedullary pus formation. (B) Gross bone pathology scores evaluation. The mean gross bone pathological scores of groups I through IV were 3.33±1.16, 2.67±0.58, 1.00±0.00, and 0.67±0.58, respectively. *Denotes a significant difference compared with group I (P<0.01, n=3). **Denotes a significant difference compared with group II (P<0.05, n=3). ***Denotes a significant difference compared with group II (P<0.01, n=3). Groups I, II, III, and IV indicate Ti + S. aureus, NT + S. aureus, NT-G + S. aureus, and Ti + PBS, respectively. Ti, titanium without modification. Abbreviations: NT, nanotubes; NT-G, gentamicin-loaded nanotubes; PBS, phosphate-buffered saline; S. aureus, Staphylococcus aureus.
Figure 3
Figure 3
Microbiological evaluation of the implants and bones. Notes: (A) Rollover cultures obtained from the explanted rods. (B) Confocal laser scanning microscopy observations of the explanted rods. The live bacteria were stained with SYTO 9 and fluorescence green, and the dead bacteria were stained with propidium iodide and fluorescence red. The magnification is ×100. (C) Number of detached adhered bacteria and amount of biofilm after the rods were rolled over TSA. (D) Quantity of CFU/g of pulverized femur. *Denotes a significant difference compared with groups III and IV (P<0.01, n=3). **Denotes a significant difference compared with group I (P<0.05, n=3). Groups I, II, III, and IV indicate Ti + S. aureus, NT + S. aureus, NT-G + S. aureus, and Ti + PBS, respectively. Ti, titanium without modification. Abbreviations: NT, nanotubes; NT-G, gentamicin-loaded nanotubes; PBS, phosphate-buffered saline; S. aureus, Staphylococcus aureus; TSA, tryptone soy agar; CFU, colony-forming unit.
Figure 4
Figure 4
Scanning electron microscopy (SEM) observations of the explanted implants. Notes: The red arrowheads indicate the intact nanotubular structure on the titanium rods. The magnifications are ×50, ×3,000, and ×10,000, and the scale bars are 1.0 mm, 10.0 μm, and 5.0 μm, respectively. Groups I, II, III, and IV indicate Ti + S. aureus, NT + S. aureus, NT-G + S. aureus, and Ti + PBS, respectively. Ti, titanium without modification. Abbreviations: NT, nanotubes; NT-G, gentamicin-loaded nanotubes; PBS, phosphate-buffered saline; S. aureus, Staphylococcus aureus.
Figure 5
Figure 5
Representative histological images of longitudinal sections from middle femur with H&E staining, Masson’s trichrome staining, and Giemsa staining at 4 weeks after implantation. Notes: The black arrows indicate intracortical abscesses or inflammatory cells; the red arrowheads indicate bone cortex destruction; and the red arrows indicate bacteria. Groups I, II, III, and IV indicate Ti + S. aureus, NT + S. aureus, NT-G + S. aureus, and Ti + PBS, respectively. Ti, titanium without modification. Abbreviations: H&E, hematoxylin and eosin; NT, nanotubes; NT-G, gentamicin-loaded nanotubes; PBS, phosphate-buffered saline; S. aureus, Staphylococcus aureus.
Figure 6
Figure 6
Representative histological images of transverse sections from the middle femur at 4 weeks after implantation. Notes: (A) Decalcified sections without implants with H&E staining, Masson’s trichrome staining, and Giemsa staining. (B) Van Gieson-stained undecalcified sections with implants. The black arrows indicate intracortical abscesses or inflammatory cells; the red arrowheads indicate bone cortex destruction; the red arrows indicate bacteria; and the black arrowheads indicate new bone formation around the implants. Groups I, II, III, and IV indicate Ti + S. aureus, NT + S. aureus, NT-G + S. aureus, and Ti + PBS, respectively. Ti, titanium without modification. Abbreviations: H&E, hematoxylin and eosin; I, implant; NT, nanotubes; NT-G, gentamicin-loaded nanotubes; PBS, phosphate-buffered saline; S. aureus, Staphylococcus aureus.
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