(M-506) Systemic Dysregulation of the Inflammatory Response following Composite Muscle and Bone Trauma

Introduction:: Composite extremity trauma involves severe damage to both the soft tissue and adjacent bone. These complex injuries are associated with a high morbidity rate and/or amputation, and burdens both the general and military populations. The regeneration of composite injuries is made more challenging due to the mounting of a multi-tissue altered wound healing environment that is exacerbated by dysregulation of the immune response. While there is evidence that the persistence of a local inflammatory milieu after injury impedes tissue regeneration, less is understood about the impacts of severe trauma on systemic inflammation. This study aims to quantify the systemic inflammatory and immune responses following severe composite injury. Findings from this study may enable the engineering of therapeutics that restore the pro-regenerative immune environment as well as support the discovery of new biomarkers that may predict healing outcomes.


Materials and Methods:: All animal procedures received approval from the Oregon Health and Science University’s Institutional Animal Care and Use Committee (IACUC). 10-12 week C57BL/6J wild-type mice (n = 22) underwent a composite bone-muscle injury consisting of a 2-mm tibial osteotomy fixed with a custom 3D-printed bone plate and subsequent 20-30% removal of the tibialis anterior (Figure 1A-D). Limb Morbidity Score (LMS)—a custom scoring index from 1 to 5 with lower integers indicating a healthy limb and higher necrosis—was recorded to quantify the healing progress post-injury. Plasma was isolated from blood samples obtained via retro-orbital blood collection before surgery (baseline) and 6-, 14-, 28-, and 56- days post-surgery (Figure 1E). 32 unique cytokines and chemokines related to the inflammatory and immune response were quantified using multiplex assays performed with MILLIPEX MAP magnetic bead panels and read on a Luminex MAGPIX platform. Data processing and statistical analysis were performed in R Studio and visualized in GraphPad Prism. Multiple comparisons between cytokine levels at baseline and each longitudinal timepoint were performed using non-parametric one-way ANOVA and corrected with Dunn’s test. Spearman correlations were calculated between LMS and cytokine levels due to the non-normality of the data with p-values adjusted using Bonferroni’s correction.


Results, Conclusions, and Discussions:: Of the 32 quantified proteins, 25 had median fluorescent intensities higher than the noise threshold and were used for analysis. Compared to each animal’s baseline, the following proteins were elevated 6-days post injury: Il-6, IP-10, Eotaxin, LIF, and LIX. Three proteins—G-CSF, Il-6, and Eotaxin—continued to be significantly elevated 14-days post injury with LIX being the only elevated marker 28-days post injury (Figure 2). By 56-days after injury, all measured cytokines had returned to baseline concentration levels. 11 of the analyzed proteins, including G-CSF, Tnf-a, Il-1b, and VEGF, demonstrated a positive correlation with LMS (p < 0.05, Table 1).



This study suggests that composite extremity trauma causes measurable dysregulation of the systemic immune response evidenced by significantly elevated levels of pro-inflammatory cytokines in the circulating plasma following injury. Increased inflammation peaked at 6-days post-injury and was completely resolved by 56- days post injury suggested by all cytokines returning to baseline levels. It should be noted that timepoints earlier than 6-days after injury were not evaluated in an effort to preserve limb recovery. Correlation between higher LMS (more necrosis) and circulating inflammatory cytokines further validates that the trauma caused by the composite injury to bone and soft tissue significantly impacts the systemic immune response. Given GCS-F and IFN-g are both highly expressed in poorly healing limbs (high LMS), these circulating pro-inflammatory cytokines may serve as predictors of poor limb healing at later timepoints (after 28-days post injury). Studies are currently underway to further correlate the inflammatory cytokine response with the muscle and bone functional outcomes.




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