Vitamin E Phosphate Coating Stimulates Bone Deposition in Implant-related Infections in a Rat Model

Abstract

Background: Implant-related infections are associated with impaired bone healing and osseointegration. In vitro antiadhesive and antibacterial properties and in vivo antiinflammatory effects protecting against bone loss of various formulations of vitamin E have been demonstrated in animal models. However, to the best of our knowledge, no in vivo studies have demonstrated the synergistic activity of vitamin E in preventing bacterial adhesion to orthopaedic implants, thus supporting the bone-implant integration.

Questions/purposes: The purpose of this study was to test whether a vitamin E phosphate coating on titanium implants may be able to reduce (1) the bacterial colonization of prosthetic implants and (2) bone resorption and osteomyelitis in a rat model of Staphylococcus aureus-induced implant-related infection.

Methods: Twelve rats were bilaterally injected in the femurs with S aureus UAMS-1-Xen40 and implanted with uncoated or vitamin E phosphate-coated titanium Kirschner wires without local or systemic antibiotic prophylaxis. Eight rats represented the uninfected control group. A few hours after surgery, two control and three infected animals died as a result of unexpected complications. With the remaining rats, we assessed the presence of bacterial contamination with qualitative bioluminescence imaging and Gram-positive staining and with quantitative bacterial count. Bone changes in terms of resorption and osteomyelitis were quantitatively analyzed through micro-CT (bone mineral density) and semiquantitatively through histologic scoring systems.

Results: Six weeks after implantation, we found only a mild decrease in bacterial count in coated versus uncoated implants (Ti versus controls: mean difference [MD], -3.705; 95% confidence interval [CI], -4.416 to -2.994; p < 0.001; TiVE versus controls: MD, -3.063; 95% CI, -3.672 to -2.454; p < 0.001), whereas micro-CT analysis showed a higher bone mineral density at the knee and femoral metaphysis in the vitamin E-treated group compared with uncoated implants (knee joint: MD, -11.88; 95% CI, -16.100 to -7.664; p < 0.001 and femoral metaphysis: MD, -19.87; 95% CI, -28.82 to -10.93; p < 0.001). We found decreased osteonecrosis (difference between medians, 1.5; 95% CI, 1-2; p < 0.002) in the infected group receiving the vitamin E-coated nails compared with the uncoated nails.

Conclusions: These preliminary findings indicate that vitamin E phosphate implant coatings can exert a protective effect on bone deposition in a highly contaminated animal model of implant-related infection.

Clinical relevance: The use of vitamin E coatings may open new perspectives for developing coatings that can limit septic loosening of infected implants with bacterial contamination. However, a deeper insight into the mechanism of action and the local release of vitamin E as a coating for orthopaedic implants is required to be used in clinics in the near future. Although this study cannot support the antimicrobial properties of vitamin E, promising results were obtained for bone-implant osseointegration. These preliminary results will require further in vivo investigations to optimize the host response in the presence of antibiotic prophylaxis.

Fig. 1

The fluoroscopic examination that assesses the correct position of Kirschner wires in the femurs is shown.

Fig. 2 A-C

Clinical data report the raw data of body weight (grams) over time as the mean ± SD (A) as well as the percentage of relative body weight increase normalized to the baseline at Day 0 as the mean ± SD (B). The blood neutrophil count on Day 42 is reported in the histogram as the median with 95% CI (C). Data are referred to the Control group (Control, n = 6) and to the infected group (Xen40, n = 9). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Fig. 3

The figure depicts a representative panel of bioluminescence imaging analysis in the infected Xen40 group. Rats receiving the femoral canal injection of 3 x 10⁴ CFU S aureus Xen40 showed the signal of the bioluminescent bacteria with differences between the injected limbs on Days 0 and 3. On Day 42, no bioluminescent signal was detected. Data at Days 3 and 42 are reported as normalized to the bioluminescence signal on Day 0. The intensity of the bioluminescence signal is measured as normalized photon counts (NC).

Fig. 4

The figure reports the micro-CT-based quantitative analysis of the entire knee and metaphysis of femurs. BMD is reported as the percentage of relative decrease in the Xen40 group with respect to the control group on Day 42 and data are expressed as mean ± SD. Data are referred to the Xen40 group (Ti, n = 9; TiVE, n = 9); ^**p < 0.01, ^***p < 0.001.

Fig. 5

The representative panel shows qualitative micro-CT images of Day 42 samples in the uninfected control (Control) and infected groups (Xen40) implanted with Ti and TiVE. The sagittal and coronal planes include the entire knee; the axial plane reports information of the femoral metaphysis. The control group presents complete integrity of the bone structure both in the knee and femoral metaphysis. Severe signs of osteomyelitis are represented by a wide or mild bone rarefaction in the Xen40 group receiving Ti or TIVE treatment, respectively.

Fig. 6

The representative panel shows Day 42 three-dimensional isosurface reconstructions. Images are presented for the uninfected control (Control) and infected group (Xen40) implanted with Ti and TiVE. The sagittal and coronal planes include the entire knee, whereas the axial plane includes the femoral metaphysis. The control group presents a smooth bone surface both in the knee and femoral metaphysis. The Xen40 group treated with Ti shows an appreciable rough bone texture as a sign of osteomyelitis. The Xen40 group treated with TiVE shows a denser bone surface compared with Ti as a sign of bone deposition.

Fig. 7

The representative panel shows the histologic analysis (hematoxylin and eosin) of the control (Control) and the infected groups (Xen40) implanted with Ti and TiVE Kirschner wires. The control group shows an intact appearance of bone and articular cartilage of the knee as well as well-preserved cortical bone in terms of structure and thickness of the femoral metaphysis. The Xen40 group treated with Ti shows irregular contours of the joint with the presence of abscesses; abscesses are also present at the level of the femoral metaphysis characterized by enlargement of the medullary space together with a periosteal reaction. Despite the irregular contour of the joint, the Xen40 group treated with TiVE does not show any presence of abscesses; a similar periosteal reaction of Ti is depicted at the femoral metaphysis of TiVE. F = femur; T = tibia; P = patella; Ab = abscesses; Pe = periosteum. In the small boxes are depicted: (a) articular joint, original magnification, x 2, scale bar 1000 µm; (b) metaphyseal cortex, original magnification, x 4, scale bar 500 µm.

Fig. 8 A-H

This panel reports the following histopathologic details: (A) endomedullary abscess with necrotic bone sequestrum (Sq); (B) endomedullary abscess (Ab) with necrotic bone sequestrum (Sq) and osteoclasts (black arrow); (C) soft tissue abscess, infected tissue (blue arrow) and plasma cells (small box); (D) granulation tissue (GT) and several osteoblasts (green arrow) within the bone marrow (BM) and bone trabeculae (Bo); (E) hypervascularization within the bone tissue (Bo); (F) hypervascularization of the soft tissues (ST); (G) bone neogenesis with formation of woven bone (WB) alongside lamellar bone (LB) and osteoblasts (green arrow); and (H) Gram-positive staining of S aureus Xen40. Panel (A) original magnification, x 2, scale bar 1000 µm; Panels (B-G) original magnification, x 10, scale bar 200 µm; Panel (H) original magnification, x 100, scales bar 20 µm.

Fig. 9 A-D

Petty’s scale is reported as the total score comparing the Control group (Control) with the infected group (Xen40) differently treated with Ti or TiVE on Day 42 (A). The HOES scale measurements of the femoral metaphysis and joint are reported separated for the three main features analyzed such as osteonecrosis (B), inflammatory pattern (C), and bone neogenesis (D) on Day 42. Data are reported as median with 95% CI. ^***p < 0.001 with respect to the Control (Control) group.