Determining Which Combinatorial Combat-Relevant Factors Contribute to Heterotopic Ossification Formation in an Ovine Model.

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.

Development and validation of a large animal ovine model for implant-associated spine infection using biofilm based inocula.

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.

Developing a combat-relevant translatable large animal model of heterotopic ossification.

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.

In vivo efficacy of a unique first-in-class antibiofilm antibiotic for biofilm-related wound infections caused by Acinetobacter baumannii.

Wounds complicated by biofilms challenge even the best clinical care and can delay a return to duty for service members. A major component of treatment in wounded warriors includes infected wound management. Yet, all antibiotic therapy options have been optimized against planktonic bacteria, leaving an important gap in biofilm-related wound care. We tested the efficacy of a unique compound (CZ-01179) specifically synthesized to eradicate biofilms. CZ-01179 was formulated as the active agent in a hydrogel, and tested in vitro and in vivo in a pig excision wound model for its ability to treat and prevent biofilm-related wound infection caused by Acinetobacter baumannii. Data indicated that compared to a clinical standard-silver sulfadiazine-CZ-01179 was much more effective at eradicating biofilms of A. baumannii in vitro and up to 6 days faster at eradicating biofilms in vivo. CZ-01179 belongs to a broader class of newly-synthesized antibiofilm agents (referred to as CZ compounds) with reduced risk of resistance development, specific efficacy against biofilms, and promising formulation potential for clinical applications. Given its broad spectrum and biofilm-specific nature, CZ-01179 gel may be a promising agent to increase the pipeline of products to treat and prevent biofilm-related wound infections.

System Setup to Deliver Air Impact Forces to a Sheep Limb: Preparation for Model Development of Blast-Related Heterotopic Ossification.

BACKGROUND: Heterotopic ossification (HO) is a significant complication for wounded warriors with traumatic limb loss. Although this pathologic condition negatively impacts the general population, ectopic bone has been observed with higher frequency for service members injured in Iraq and Afghanistan due to blast injuries. Several factors, including a traumatic insult, bioburden, tourniquet and wound vacuum usage, and bone fractures or fragments have been associated with increased HO for service members. A large combat-relevant animal model is needed to further understand ectopic bone etiology and develop new pragmatic solutions for reducing HO formation and recurrence. OBJECTIVE: This study outlines the optimization of a blast system that may be used to simulate combat-relevant trauma for HO and replicate percussion blast experienced in theater. METHODS: We tested the repeatability and reproducibility of an air impact device (AID) at various pressure settings and compared it with a model of blunt force trauma for HO induction. Furthermore, we assessed the ability of the higher-power air delivery system to injure host tissue, displace metal particulate, and disperse bone chips in cadaveric sheep limbs. RESULTS: Data demonstrated that the air delivery setup generated battlefield-relevant blast forces. When the AID was charged to 40, 80, and 100 psi, the outputs were 229 (SD 13) N, 778 (SD 50) N, and 1085 (SD 114) N, respectively, compared with the blunt force model which proposed only 168 (SD 11) N. For the 100-psi AID setup, the force equaled a 5.8-kg charge weight of trinitrotoluene at a standoff distance of approximately 2.62 m, which would replicate a dismounted improvised explosive device blast in theater. Dispersion data showed that the delivery system would have the ability to cause host tissue trauma and effectively disperse metal particulate and host bone chips in local musculature compared with the standard blunt force model (13 mm vs 2 mm). CONCLUSIONS: Our data showed that a high-pressure AID was repeatable or reproducible, had the ability to function as a simulated battlefield blast that can model military HO scenarios, and will allow for factors including blast trauma to translate toward a large animal model.

In vivo analysis of a first-in-class tri-alkyl norspermidine-biaryl antibiotic in an active release coating to reduce the risk of implant-related infection.

Prosthetic joint infection (PJI) is a well-known and persisting problem. Active release coatings have promise to provide early protection to an implant by eradicating small colony biofilm contaminants or planktonic bacteria that can form biofilm. Traditional antibiotics can be limited as active release agents in that they have limited effect against biofilms and develop resistance at sub-lethal concentrations. A unique first-in-class compound (CZ-01127) was assessed as the active release agent in a silicone (Si)-based coating to prevent PJI in a sheep model of joint space infection. Titanium (Ti) plugs contained a porous coated Ti (PCTi) region and polymer-coated region. Plugs were implanted into a femoral condyle of sheep to assess the effect of the Si polymer on cancellous bone ingrowth, the effect of CZ-01127 on bone ingrowth, and the ability of CZ-01127 to prevent PJI. Microbiological results showed that CZ-01127 was able to eradicate bacteria in the local region of the implanted plugs. Data further showed that Si did not adversely affect bone ingrowth. However, bacteria that reached the joint space (synovium) were not fully eradicated. Outcomes suggested that the CZ-01127 coating provided local protection to the implant system in a challenging model, the design of which could be beneficial for testing future antimicrobial therapies for PJI. STATEMENT OF SIGNIFICANCE: Periprosthetic joint infection (PJI) is now commonplace, and constitutes an underlying problem that patients and physicians face. Active release antibiotic coatings have potential to prevent these infections. Traditional antibiotics are limited in their ability to eradicate bacteria that reside in biofilms, and are more susceptible to resistance development. This study addressed these limitations by testing the efficacy of a unique antimicrobial compound in a coating that was tested in a challenging sheep model of PJI. The unique coating was able to eradicate bacteria and prevent infection in the environment adjacent to the implant. Bacteria that escaped into the joint space still caused infection, yet benchmark data can be used to optimize the coating and translate it toward clinical use.

In vivo efficacy of a silicone‒cationic steroid antimicrobial coating to prevent implant-related infection.

Active release antimicrobial coatings for medical devices have been developed to prevent and treat biofilm implant-related infections. To date, only a handful of coatings have been put into clinical use, with limited success. In this study, a novel antimicrobial compound was incorporated into a silicone (polydimethylsiloxane or PDMS) polymer to develop a novel active release coating that addressed several limitations of current device coatings. The efficacy of this coating was optimized using an in vitro flow cells system, then translated to an animal model of a simulated Type IIIB open fracture wherein well-established biofilms were used as initial inocula. Results indicated that the novel coating was able to prevent infection in 100% (9/9) of animals that were treated with biofilms and the novel coating (treatment group). In contrast, 100% (9/9) of animals that were inoculated with biofilms and not treated with the coating (positive control), did develop infection. Nine animals were used as negative controls, i.e., those that were not treated with biofilms, and showed a rate of infection of 11% (1/9). Eight animals were treated with the novel coating only to determine its effect on host tissue. Results indicated that the novel active release coating may have significant promise for future application to prevent biofilm implant-related infections in patients.