Development of a hematogenous implant-related infection in a rat model

Abstract

Background: Implant-related osteomyelitis is a major complication that requires immediate treatment, often involving removal of the implant, prolonging patient recovery and inflating expenses. Current research involving interventions to diminish the prevalence of such measures include investigating prophylactic and therapeutic remedies. A proper and accurate animal model is needed to thoroughly investigate such treatments. The scope of this project was to develop an animal model in which a consistent and measurable infection can be formed on an orthopedic implant when bacteria is introduced via a hematogenous source.

Methods: Titanium Kirschner-wires were implanted into the intramedullary canals of both femurs. Staphylococcus aureus, ranging from10(4) to 10(9) colony forming units, was injected into a tail vessel. After a designated time (3, 7, 14, or 42 days) the femurs were harvested and bacterial numbers determined for both the femur and the implanted K-wire. In addition, histology and micro-computed tomography were used as subjective tools to further characterize the infection.

Results: Consistent infection, that is infection of ≥75% of the femurs, wasn't achieved until 10(7) CFU S. aureus was injected. At 10(7) CFU, the femurs contained 4.6x10(6) CFU/g bone tissue at day 3 and 4.8×10(8) CFU/g bone tissue by day 14. The wire showed comparable contamination with 4.8×10(4) CFU/mm(2) at day 3 and 3.7×10(5)/mm(2) by day 14. After 42 days, the bacteria number decreased but was still occupying at 1.9×10(5) CFU/g bone tissue. There were morphological changes to the bone as well. At day 42, there were signs of osteonecrosis and active bone formation when compared to control animals that received a K-wire but were inoculated with saline.

Conclusions: A model for hematogenous osteomyelitis, a common complication associated with implants, has been introduced. A reproducible, preclinical model is essential to evaluate future methods used to mitigate blood-borne bacteria hardware and bone infections.

Fig. 1

Surgical placement of a 1.25 mm diameter Ti alloy K-wire, 24 mm length

Fig. 2

Weight Change of animals that received a bilateral K-wire with a hematogenous injection of Staphylococcus aureus or saline as a control. Group 10⁷, 10⁸, and 10⁹ were different from the control at day 7 with p < 0.05, 0.01, and 0.001, respectively. Groups 10⁸ and 10⁹ were different from control at day 14 with p < 0.001

Fig. 3

a Bacteria colony forming units within the bone. CFU(log10)/g bone tissue and (b) Bacteria colony forming units on the K-wire. CFU(log10)/mm² surface area of K-wire. One-way ANOVA performed amongst each inoculum over each time point. Day 3 bone had statistical difference between 10⁴- 10⁶ and 10⁸ & 10⁹. 10⁷ was different than 10⁹ within the bone. Within the wire samples. 10⁸ and 10⁹ groups were significantly greater than the rest of the groups at day 3. There was no difference in the bone or wire at day 7 or 14

Fig. 4

Bacteria CFU in bone when hematogenously inoculated with 10⁷ CFU of S. aureus. CFU(log10)/g bone tissue

Fig. 5

longitudinal μCT sections of femurs at 3, 7, 14, and 42 days for the control (saline) and infected (10⁷ CFU S. aureus injected via tail vein) and longitudinal histological sections of bacterial colonization at 42 days post inoculation (stained with H&E) (a) progression of morphological changes within the bone between 3 and 42 days post inoculation as seen by μCT. Comparing the control and the infected limbs, the infection is evident by the periosteal reaction seen progressing (arrow) forming an involucrum (arrowhead). By 42 days, large amounts of boney remodeling are obvious in the infected bone compared to the control (*). b 42 days post inoculation with 10⁷ CFU S. aureus. The femoral condyle shows signs of bone remodeling

Fig. 6

Histological evidence of bacterial colonization. a arrows identify loci of colonization with the characteristic fibrous tissue surrounding the infection. b Necrotic bone tissue evident by the empty lacunae (arrow), localized bacteria (arrowhead), and agglomeration of neutrophils (*)

Fig. 7

Bacteria colony forming units in bone comparing the bone with an implanted K-wire and the bone with a sham surgery after post-surgical inoculation of 10⁷ CFU S. aureus CFU(log10)/g bone tissue. Comparing animals with infection (≥10³ CFU) to animals without infection, there is a statistical difference between the implanted limb and the sham limb at 14 and 42 days. Fisher’s exact p < 0.05