Single trauma injuries or isolated fractures are often manageable and generally heal without complications. In contrast, high-energy trauma results in multi/poly-trauma injury patterns presenting imbalanced pro- and anti- inflammatory responses often leading to immune dysfunction. These injuries often exhibit delayed healing, leading to fibrosis of injury sites and delayed healing of fractures depending on the intensity of the compounding traumas. Immune dysfunction is accompanied by a temporal shift in the innate and adaptive immune cells distribution, triggered by the overwhelming release of an arsenal of inflammatory mediators such as complements, cytokines and damage associated molecular patterns (DAMPs) from necrotic cells. Recent studies have implicated this dysregulated inflammation in the poor prognosis of polytraumatic injuries, however, interventions focusing on immunomodulating inflammatory cellular composition and activation, if administered incorrectly, can result in immune suppression and unintended outcomes. Immunomodulation therapy is promising but should be conducted with consideration for the spatial and temporal distribution of the immune cells during impaired healing. This review describes the current state of knowledge in the spatiotemporal distribution patterns of immune cells at various stages during musculoskeletal wound healing, with a focus on recent advances in the field of Osteoimmunology, a study of the interface between the immune and skeletal systems, in long bone fractures. The goals of this review are to (1) discuss wound and fracture healing processes of normal and delayed healing in skeletal muscles and long bones; (2) provide a balanced perspective on temporal distributions of immune cells and skeletal cells during healing; and (3) highlight recent therapeutic interventions used to improve fracture healing. This review is intended to promote an understanding of the importance of inflammation during normal and delayed wound and fracture healing. Knowledge gained will be instrumental in developing novel immunomodulatory approaches for impaired healing.

Delayed healing of traumatic wounds often stems from a dysregulated immune response initiated or exacerbated by existing comorbidities, multiple tissue injury or wound contamination. Over decades, approaches towards alleviating wound inflammation have been centered on interventions capable of a collective dampening of various inflammatory factors and/or cells. However, a progressive understanding of immune physiology has rendered deeper knowledge on the dynamic interplay of secreted factors and effector cells following an acute injury. There is a wide body of literature, both in vitro and in vivo, abstracted on the immunomodulatory approaches to control inflammation. Recently, targeted modulation of the immune response via biotechnological approaches and biomaterials has gained attention as a means to restore the pro-healing phenotype and promote tissue regeneration. In order to fully realize the potential of these approaches in traumatic wounds, a critical and nuanced understanding of the relationships between immune dysregulation and healing outcomes is needed. This review provides an insight on paradigm shift towards interventional approaches to control exacerbated immune response following a traumatic injury from an agonistic to a targeted path. We address such a need by (1) providing a targeted discussion of the wound healing processes to assist in the identification of novel therapeutic targets and (2) highlighting emerging technologies and interventions that utilize an immunoengineering-based approach. In addition, we have underscored the importance of immune engineering as an emerging tool to provide precision medicine as an option to modulate acute immune response following a traumatic injury. Finally, an overview is provided on how an intervention can follow through a successful clinical application and regulatory pathway following laboratory and animal model evaluation.

We previously reported an early surge in high mobility group box protein 1 (HMGB1) levels in a polytrauma (PT) rat model. This study investigates the association of HMGB1 levels in mediating PT associated dysregulated immune responses and its influence on the cellular levels of receptor for advanced glycation end products (RAGE) and toll-like receptor 4 (TLR4). Using the same PT rat model treated with anti-HMGB1 polyclonal antibody, we evaluated changes in circulating inflammatory cytokines, monocytes/macrophages and T cells dynamics and cell surface expression of RAGE and TLR4 at 1, 3, and 7 days post-trauma (dpt) in blood and spleen. Notably, PT rats demonstrating T helper (Th)1 and Th2 cells type early hyper-inflammatory responses also exhibited increased monocyte/macrophage counts and diminished T cell counts in blood and spleen. In blood, expression of RAGE and TLR4 receptors was elevated on CD68⁺ monocyte/macrophages and severely diminished on CD4⁺ and CD8⁺ T cells. Neutralization of HMGB1 significantly decreased CD68⁺ monocyte/macrophage counts and increased CD4⁺ and CD8⁺ T cells, but not γδ⁺TCR T cells in circulation. Most importantly, RAGE and TLR4 expressions were restored on CD4⁺ and CD8⁺ T cells in treated PT rats. Overall, findings suggest that in PT, the HMGB1 surge is responsible for the onset of T cell exhaustion and dysfunction, leading to diminished RAGE and TLR4 surface expression, thereby possibly hindering the proper functioning of T cells.

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.

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 IFNβ 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: IFNβ 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.

TLR ligands Resiquimod (TLR7/8) and Motolimod (TLR8) are used to activate NK cells and enhance immune defenses. This study reports the differential gene expressions of innate immune markers and Natural Killer (NK) cell lysins in rag1 mutant (rag MT) zebrafish to TLR8 and TLR7/8 activation in liver, kidney, and spleen tissues. Rag MT zebrafish were intracoelomically injected with Motolimod (VTX), Resiquimod (R848), or control saline. Gene expressions of interferon gamma (ifnγ), T-box transcription factor 21 (t-bet), novel immune type receptor 9 (nitr9), and NK lysins a, b, c, and d (nkla, nklb, nklc, nkld) were quantified at 6-, 12-, and 24-hours post-injection using quantitative PCR. We observed that the effects of TLR7/8 and TLR8 stimulation vary depending on the tissue type. R848 significantly upregulated ifnγ, t-bet, nitr9, and NK lysins across various tissues. In contrast, VTX had a more limited effect and primarily influenced nklc and nkld in the kidney and spleen, and nkld in the liver, suggesting tissue-specific responsiveness to TLR8. No significant changes in ifnγ or nkla expression were noted with VTX in any tissues, highlighting the specificity of TLR7 over TLR8 in these responses. Tissue-specific responses revealed dominant activation by TLR7/8 in the liver, particularly influencing ifnγ, t-bet, nitr9, nklb, and nklc. The kidney had high responsiveness to both TLR7/8 and TLR8. The spleen demonstrated broad gene activation by TLR7/8, but only nklc and nkld were significantly upregulated by TLR8. These findings demonstrate that TLR8 has selective effects, while TLR7/8 more broadly activates genes across the liver, kidney, and spleen in rag MT zebrafish. These NK cell gene expression findings suggest exposure to TLR7/8 and TLR8 ligands elicited differential effects across liver, kidney, and spleen tissues of rag MT zebrafish.