Fracture healing in a polytrauma rat model is influenced by mtDNA:cGAS complex mediated pro-inflammation.

PURPOSE: The mitochondrial DNA (mtDNA) activated cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon genes (cGAS-STING) signaling pathway is a key player in mediating immune responses in autoimmune disorders and cancer. However, its role in severe trauma associated fracture healing is unknown. This study investigated if the cGAS-STING signaling pathway contributes to delayed bone healing in polytrauma (PT) fractures. METHODS: For preliminary analyses, therapeutic dosage of RU.521 (cGAS inhibitor) (n = 2) was determined in C57BL/6 J mice by mass spectrometry, and IFNß expression levels in serum and bronchioalveolar fluid (BALF) at 6 and 24 h (h) in RU.521/vehicle + mtDNA injected mice (n = 3/treatment and time point) was measured by ELISA. In the main study, plasma mtDNA was quantified by qPCR in a clinically relevant delayed fracture healing PT rat model with burn injury, blunt trauma, and a femoral fracture at 3 h post-trauma (hpt). Next, PT rats received either RU.521 (12 mg/kg in povidone; n = 8) or vehicle (povidone only; n = 5) immediately after injury and were followed up for 5 weeks post-trauma to assess bone regeneration by radiography and histology. RESULTS: IFNß levels were significantly decreased only at 24 h in BALF of RU.521 treated mice. At 3hpt mtDNA was significantly elevated in PT rats compared to rats without injury. When treated with RU.521, PT rats showed improvement in bone healing compared to vehicle control PT rats. CONCLUSIONS: These data reveal that the cGAS-STING signaling pathway influences trauma-induced delayed bone healing. However, further evaluation of this pathway at the cellular and molecular levels to augment PT associated detrimental effects is needed.

Preclinical evaluation of a commercially available biofilm disrupting wound lavage for musculoskeletal trauma.

BACKGROUND: Treatment of open fractures remains a significant challenge in trauma care as these fractures are accompanied by extensive soft tissue damage, exposing the wound site to contaminants and increasing infection risk. Formation of biofilm, a capsule-like environment that acts as a barrier to treatment, is a primary mode by which infecting pathogens persist at the wound site. Therefore, a pressing need exists to identify irrigation methods that can disrupt biofilm and expose pathogens to treatment. This study aims to evaluate the antibiofilm wound lavage, Bactisure™, in comparison with saline for care of severe musculoskeletal wounds and elucidate potential effects on antibiotic treatment success. METHODS: UAMS-1 Staphylococcus aureus biofilms were formed in vitro and treated with Bactisure™ wound lavage or sterile normal saline, alone, or in combination with sub-biofilm inhibitory levels of vancomycin. Characterization methods included quantification of biofilm biomass, quantification of viable biofilm bacteria, and biofilm matrix imaging. For in vivo assessment, a delayed treatment model of contaminated open fracture was used wherein a critical-sized defect was created in a rat femur and wound site inoculated with UAMS-1. Following a 6 h delay, wounds were debrided, irrigated with lavage of interest, and antibiotic treatments administered. Bacterial enumeration was performed on bone and hardware samples after two weeks. RESULTS: An immediate reduction in biofilm biomass was observed in vitro following antibiofilm lavage treatment, with a subsequent 2- to 3- log reduction in viable bacteria achieved after 24 h. Furthermore, biofilms treated with antibiofilm lavage in combination with vancomycin exhibited a minor, but statistically significant, decrease in viable bacteria compared to irrigation alone. In vivo, a minor, not statistically significant, decrease in median bioburden was observed for the antibiofilm lavage compared to saline when used in combination with antibiotics. However, the percentage of bone and hardware samples with detectable bacteria was reduced from 50 to 38%. CONCLUSIONS: These results suggest that the antibiofilm wound lavage, Bactisure™, may hold promise in mitigating infection in contaminated musculoskeletal wounds and warrants further investigation. Here, we proposed multiple mechanisms in vitro by which this antibiofilm lavage may help mitigate infection, and demonstrate this treatment slightly outperforms saline in controlling bioburden in vivo.

Neutralization of HMGB1 improves fracture healing and γδ T lymphocyte counts at the fracture site in a polytrauma rat model.

PURPOSE: Delayed fracture healing is a common consequence of polytrauma (PT) occurring in patients with multiple injuries. We believe that when early release of high mobility group box 1 (HMGB1) molecules from necrotic tissues exceed their normal levels in blood, they dysregulate immune responses associated with normal healing. This study investigates the detrimental effect of such dysregulate immune responses by targeting HMGB1 in a PT rat model with debilitating injuries. We hypothesized that neutralization of extracellular HMGB1 immediately post-trauma would ameliorate local immune dysregulation and improve fracture healing in a PT rat model. METHODS: PT rats received a single dose of either anti-rat HMGB1 polyclonal antibody (PT-Ab HMGB1) or IgY isotype (PT-IgY), were left untreated (PT-C), or had a single injury/osteotomy only (OST). Fracture healing was evaluated by micro-computed tomography (µCT) and histology at 5 weeks; and macrophages and T cell counts within the fracture site were determined with flow cytometry  at 1 week. RESULTS: Notably, bone regeneration within the fracture site in PT-Ab HMGB1 rats was improved with comparable connective tissue organization than PT-C rats. Further, only γδTCR+ T cells, but not macrophages and CD4+ and CD8+ T cells, were diminished at the fracture site in PT-C and PT-IgY rats. Interestingly, the PT-Ab HMGB1 rats had increased γδTCR+ T cells compared to PT-C and PT-IgY, suggesting their potential role in regulating fracture healing. CONCLUSIONS: Therefore, the initial burst of systemic HMGB1 following trauma may have a role in regulating bone regeneration via the modulation of a subclass of T cells within the fracture site, suggesting it's importance as a therapeutic target in PT to combat immune dysregulation and delayed fracture healing.

Burn and thoracic trauma alters fracture healing, systemic inflammation, and leukocyte kinetics in a rat model of polytrauma.

BACKGROUND: Singular traumatic insults, such as bone fracture, typically initiate an appropriate immune response necessary to restore the host to pre-insult homeostasis with limited damage to self. However, multiple concurrent insults, such as a combination of fracture, blunt force trauma, and burns (polytrauma), are clinically perceived to result in abnormal immune response leading to inadequate healing and resolution. To investigate this phenomenon, we created a model rat model of polytrauma. METHODS: To investigate relationship between polytrauma and delayed healing, we created a novel model of polytrauma in a rat which encompassed a 3-mm osteotomy, blunt chest trauma, and full-thickness scald burn. Healing outcomes were determined at 5 weeks where the degree of bone formation at the osteotomy site of polytrauma animals was compared to osteotomy only animals (OST). RESULTS: We observed significant differences in the bone volume fraction between polytrauma and OST animals indicating that polytrauma negatively effects wound healing. Polytrauma animals also displayed a significant decrease in their ability to return to pre-injury weight compared to osteotomy animals. Polytrauma animals also exhibited significantly altered gene expression in osteogenic pathways as well as the innate and adaptive immune response. Perturbed inflammation was observed in the polytrauma group compared to the osteotomy group as evidenced by significantly altered white blood cell (WBC) profiles and significantly elevated plasma high-mobility group box 1 protein (HMGB1) at 6 and 24 h post-trauma. Conversely, polytrauma animals exhibited significantly lower concentrations of plasma TNF-alpha (TNF-α) and interleukin 6 (IL-6) at 72 h post-injury compared to OST. CONCLUSIONS: Following polytrauma with burn injury, the local and systemic immune response is divergent from the immune response following a less severe singular injury (osteotomy). This altered immune response that follows was associated with a reduced capacity for wound healing.

Topical rifampin powder for orthopaedic trauma part II: Topical rifampin allows for spontaneous bone healing in sterile and contaminated wounds.

Infectious complications can reduce fracture healing rate. Broad spectrum antibiotics are commonly administered to prevent and treat musculoskeletal infections. Local antibiotics are applied to the wound site to increase therapeutic concentrations without increasing systemic toxicity, however, may hinder local tissue recovery. Rifampin has been shown to eradicate mature Staphylococcal biofilms and its use proven for treating musculoskeletal infections. In this study, a spontaneously healing defect model in a rat was used to investigate the impact rifampin powder has on endogenous bone healing in both a sterile and contaminated wound. No significant differences were identified in bone volume fraction via microcomputed tomography, radiological scoring, or histology between an empty defect and animals that received vancomycin or rifampin powder in a sterile wound. When applied to a contaminated musculoskeletal wound, the rifampin powder had significantly greater bone formation compared to the control, as measured by microcomputed tomography, plain radiology, and histology. In addition, the animals treated with rifampin powder had reduced bacteria, reduced white blood cell count and reduced number of clinical indications of infection. Interestingly, while the vancomycin group still displayed signs of infection via quantitative microbiology, plain radiology, and histology, there was significant bone formation within the defect and reduction of systemic signs of infection. We demonstrated that the use of rifampin powder allows bone to heal in both a sterile and contaminated model of musculoskeletal infection. To our knowledge, this is the first time the direct impact of local antibiotics on bone healing has been investigated. Published 2018. This article is a U.S. Government work and is in the public domain in the USA. J Orthop Res 36:3142-3150, 2018.