A novel rodent model of chronic spinal implant-associated infection

Abstract

Background context: Bacterial infection of spinal instrumentation is a significant challenge in spinal fusion surgery. Although the intraoperative local application of powdered vancomycin is common practice for mitigating infection, the antimicrobial effects of this route of administration are short-lived. Therefore, novel antibiotic-loaded bone grafts as well as a reliable animal model to permit the testing of such therapies are needed to improve the efficacy of infection reduction practices in spinal fusion surgery.

Purpose: This study aims to establish a clinically relevant rat model of spinal implant-associated infection to permit the evaluation of antimicrobial bone graft materials used in spinal fusion.

Study design: Rodent study of chronic spinal implant-associated infection.

Methods: Instrumentation anchored in and spanning the vertebral bodies of L4 and L5 was inoculated with bioluminescent methicillin-resistant Staphylococcus aureus bacteria (MRSA). Infection was monitored using an in vivo imaging system (IVIS) for 8 weeks. Spines were harvested and evaluated histologically, and colony-forming units (CFUs) were quantified in harvested implants and spinal tissue.

Results: Postsurgical analysis of bacterial infection in vivo demonstrated stratification between MRSA and phosphate-buffered saline (PBS) control groups during the first 4 weeks of the 8-week infection period, indicating the successful establishment of acute infection. Over the 8-week chronic infection period, groups inoculated with 1 × 10⁵ MRSA CFU and 1 × 10⁶ MRSA CFU demonstrated significantly higher bioluminescence than groups inoculated with PBS control (p = 0.009 and p = 0.041 respectively). Histological examination at 8 weeks postimplantation revealed the presence of abscesses localized to implant placement in all MRSA inoculation groups, with the most pervasive abscess formation in samples inoculated with 1 × 10⁵ MRSA CFU and 1 × 10⁶ MRSA CFU. Quantification of CFU plated from harvested spinal tissue at 8 weeks post-implantation revealed the 1 × 10⁵ MRSA CFU inoculation group as the only group with a significantly greater average CFU count compared to PBS control (p = 0.017). Further, CFU quantification from harvested spinal tissue was greater than CFU quantification from harvested implants across all inoculation groups.

Conclusion: Our model demonstrated that the inoculation dosage of 1 × 10⁵ MRSA CFU exhibited the most robust chronic infection within instrumented vertebral bodies. This dosage had the greatest difference in bioluminescence signal from control (p < 0.01), the lowest mortality (0% compared to 50% for samples inoculated with 1 × 10⁶ MRSA CFU), and a significantly higher amount of CFUs from harvested spine samples than CFUs from control harvested spine samples. Further, histological analysis confirmed the reliability of this novel rodent model of implanted-associated infection to establish infection and biofilm formation of MRSA for all inoculation groups.

Clinical significance: This model is intended to simulate the infection of instrumentation used in spinal fusion surgeries concerning implant locality and material. This model may evaluate potential antimicrobial and osteogenic biomaterials and investigate the relationship between implant-associated infection and failed fusion.

Keywords: Implant-associated infection; Osteomyelitis; Rodent model; Spinal arthrodesis; Spinal instrumentation; Spine infection.

Fig. 1.

Titanium implant anchored across L4 and L5 vertebral bodies in rodent model. The implant served to mimic pedicle screw placement in clinical spinal fusion surgeries and provided an abiotic surface for bacterial growth and biofilm development after inoculation with S. aureus.

Fig. 2.

In vivo Bioluminescent flux by experimental group. Panel A quantifies bioluminescent flux by IVIS scan over an 8-week infection period for all inoculation groups. Total flux is graphed on a logarithmic scale for ease of comparison between groups. Error bars are obtained from standard deviations of average luminescence scans per group (n = 4 for the PBS Control inoculation group, n = 6 for the 1 × 10⁴ inoculation group, n = 8 for the 1 × 10⁵ inoculation group, n = 4 for the 1 × 10⁶ inoculation group). Contaminated PBS controls from the first surgery day and the week 2 outlier from the third surgery group were omitted from this graph for ease of comparison. Rats inoculated with PBS exhibited less luminescence than infected groups over weeks 2−4 and 6−8 of the infection period. Panel B depicts the average luminescence value for each rat and the mean + SEM of each experimental group. One-way ANOVA pairwise comparison analysis revealed the 1 × 10⁵ CFU inoculation group had significantly higher average luminescence per scan than the PBS control group, as denoted by a double asterisk (p = 0.009). The 1 × 10⁶ inoculation also showed significantly higher average luminescence per scan than the PBS control group after one-way ANOVA pairwise comparison denoted by a single asterisk (p = 0.041). The 1 × 10⁴ inoculation group did not show significantly higher average luminescence per scan than the PBS control group (p = 0.673).

Fig. 3.

Histological and gross examination of lumbar spines of rats with S. aureus inoculated implants 8 weeks postoperatively. Histological analysis was performed using Masson’s Trichrome Stain on harvested spines representative of bacterial growth in each experimental group. A) PBS Control: The L4 (left) and L5 (right) vertebrae depict cell infiltration (white arrows), likely due to the presence of a foreign body (spinal implant) and trauma of implantation. However, the absence of abscesses in this sample is indicative of a lesser establishment of infection compared to samples inoculated with MRSA, which is consistent with this sample’s noninfectious inoculation dose. B) MRSA 1 × 10⁴ CFUs: Sample demonstrates presence of established infection through the formation of abscesses (white arrows). C) MRSA 1 × 10⁵ CFUs: Sample demonstrates larger and more numerous abscesses (white arrows). D) MRSA 1 × 10⁶ CFUs: Abscesses indicative of severe vertebral infection are apparent throughout the spine sample (white arrows).

Fig. 4.

Gross specimen depicting osteomyelitis in 1 × 10⁵ CFU implanted rat. A) Ventral aspect of the spine with transverse processes numbered according to corresponding vertebrae. Arrows indicate abscesses protruding from vertebral bodies into the transverse processes at L4 and L5. B) Dorsal aspect of the spine of a rat inoculated with 1 × 10⁶ MRSA CFU. Circle indicates the area where infection and implant migrated from the intervertebral space to pierce the epidermis.

Fig. 5.

Implant and spinal tissue CFU quantification 8 weeks postoperatively. Error bars are derived from standard deviations of inoculation groups (n = 2). Panel A depicts the average number of CFU plated and grown from the implants retrieved from each inoculation group. After one-way ANOVA analysis, the quantity of CFU grown from implants retrieved from the 1 × 10⁴ MRSA CFU group was significantly higher than the number of CFU plated from implants retrieved from the PBS control group (p = 0.014). However, the quantity of CFU grown from implants retrieved from the 1 × 10⁵ and 1 × 10⁶ MRSA CFU groups did not differ significantly from the number of CFU grown from implants retrieved from the PBS control group (p = 0.173 and p = 0.171 respectively). Panel B depicts the average quantity of CFU plated and grown from the spinal tissue retrieved from each inoculation group. The number of CFU grown from spinal tissue of the 1 × 10⁵ MRSA CFU group was significantly higher than the quantity of CFU grown from spinal tissue retrieved from the PBS control group (p = 0.017). The number of CFU grown from spinal tissue retrieved from the 1 × 10⁴ and 1 × 10⁶ MRSA CFU groups did not differ significantly from the number of CFU grown from spinal tissue retrieved from the PBS control group (p = 0.121 and p = 0.087 respectively).