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Postoperative implant-associated spine infection remains poorly understood. Currently there is no large animal model using biofilm as initial inocula to study this challenging clinical entity. The purpose of the present study was to develop a sheep model for implant-associated spine infection using clinically relevant biofilm inocula and to assess the in vivo utility of methylene blue (MB) for visualizing infected tissues and guiding debridement. This 28-day study used five adult female Rambouillet sheep, each with two non-contiguous surgical sites– in the lumbar and thoracic regions– comprising randomized positive and negative infection control sites. A standard mini-open approach to the spine was performed to place sterile pedicle screws and Staphylococcus aureus biofilm-covered (positive control), or sterile (negative control) spinal fusion rods. Surgical site bioburden was quantified at the terminal procedure. Negative and positive control sites were stained with MB and staining intensity quantified from photographs. Specimens were analyzed with x-ray, micro-CT and histologically. Inoculation rods contained ∼10.44 log10 colony forming units per rod (CFU/rod). Biofilm inocula persisted on positive-control rod explants with ∼6.16 log10 CFU/rod. There was ∼6.35 log10 CFU/g of tissue in the positive controls versus no identifiable bioburden in the negative controls. Positive controls displayed hallmarks of deep spine infection and osteomyelitis, with robust local tissue response, bone resorption, and demineralization. MB staining was more intense in infected, positive control sites. This work presents an animal-efficient sheep model displaying clinically relevant implant-associated deep spine infection.

Traumatic heterotopic ossification (HO) is frequently observed in Service Members following combat-related trauma. Estimates suggest that ~65% of wounded warriors who suffer limb loss or major extremity trauma will experience some type of HO formation. The development of HO delays rehabilitation and can prevent the use of a prosthetic. To date there are limited data to suggest a standard mechanism for preventing HO. This may be due to inadequate animal models not producing a similar bone structure as human HO. We recently showed that traumatic HO growth is possible in an ovine model. Within that study, we demonstrated that 65% of sheep developed a human-relevant hybrid traumatic HO bone structure after being exposed to a combination of seven combat-relevant factors. Although HO formed, we did not determine which traumatic factor contributed most. Therefore, in this study, we performed individual and various combinations of surgical/traumatic factors to determine their individual contribution to HO growth. Outcomes showed that the presence of mature biofilm stimulated a large region of bone growth, while bone trauma resulted in a localized bone response as indicated by jagged bone at the linea aspera. However, it was not until the combinatory factors were included that an HO structure similar to that of humans formed more readily in 60% of the sheep. In conclusion, data suggested that traumatic HO growth can develop following various traumatic factors, but a combination of known instigators yields higher frequency size and consistency of ectopic bone.

Heterotopic ossification (HO) refers to ectopic bone formation, typically in residual limbs following trauma and injury. A review of injuries from Operation Iraqi Freedom (OIF) and Operation Enduring Freedom (OEF) indicated that approximately 70% of war wounds involved the musculoskeletal system, largely in part from the use of improvised explosive devices (IED) and rocket-propelled grenades (RPG). HO is reported to occur in approximately 63%–65% of wounded warriors from OIF and OEF. Symptomatic HO may delay rehabilitation regimens since it often requires modifications to prosthetic limb componentry and socket size. There is limited evidence indicating a mechanism for preventing HO. This may be due to inadequate models, which do not produce HO bone structure that is morphologically similar to HO samples obtained from wounded warfighters injured in theatre. We hypothesized that using a high-power blast of air (shockwave) and simulated battlefield trauma (i.e. bone damage, tourniquet, bacteria, negative pressure wound therapy) in a large animal model, HO would form and have similar morphology to ectopic bone observed in clinical samples. Initial radiographic and micro-computed tomography (CT) data demonstrated ectopic bone growth in sheep 24 weeks post-procedure. Advanced histological and backscatter electron (BSE) analyses showed that 5 out of 8 (63%) sheep produced HO with similar morphology to clinical samples. We conclude that not all ectopic bone observed by radiograph or micro-CT in animal models is HO. Advanced histological and BSE analyses may improve confirmation of HO presence and morphology, which we demonstrated can be produced in a large animal model.