The influence of microbial colonization on inflammatory versus pro-healing trajectories in combat extremity wounds.

A combination of improved body armor, medical transportation, and treatment has led to the increased survival of warfighters from combat extremity injuries predominantly caused by blasts in modern conflicts. Despite advances, a high rate of complications such as wound infections, wound failure, amputations, and a decreased quality of life exist. To study the molecular underpinnings of wound failure, wound tissue biopsies from combat extremity injuries had RNA extracted and sequenced. Wounds were classified by colonization (colonized vs. non-colonized) and outcome (healed vs. failed) status. Differences in gene expression were investigated between timepoints at a gene level, and longitudinally by multi-gene networks, inferred proportions of immune cells, and expression of healing-related functions. Differences between wound outcomes in colonized wounds were more apparent than in non-colonized wounds. Colonized/healed wounds appeared able to mount an adaptive immune response to infection and progress beyond the inflammatory stage of healing, while colonized/failed wounds did not. Although, both colonized and non-colonized failed wounds showed increasing inferred immune and inflammatory programs, non-colonized/failed wounds progressed beyond the inflammatory stage, suggesting different mechanisms of failure dependent on colonization status. Overall, these data reveal gene expression profile differences in healing wounds that may be utilized to improve clinical treatment paradigms.

Cutaneous burn injury represents a major risk factor for the development of traumatic ectopic bone formation following blast-related extremity injury.

Blast-related traumatic heterotopic ossification (tHO) impacts clinical outcomes in combat-injured patients, leading to delayed wound healing, inflammatory complications, and reduced quality of life. Blast injured patients often have significant burns. This study investigated whether a partial thickness thermal burn injury exacerbates blast-related tHO in a clinically relevant polytrauma animal model. Adult male Sprague Dawley rats were subjected to an established model involving a whole-body blast overpressure exposure (BOP), complex extremity trauma followed by hind limb amputation (CET) followed by the addition of a 10 % total body surface area (TBSA) second degree thermal burn (BU). Micro-CT scans on post-operative day 56 showed a significant increase in HO volume in the CET + BU as compared to the CET alone injury group (p < .0001; 22.83 ± 3.41 mm3 vs 4.84 ± 5.77 mm3). Additionally, CET + BU concomitant with BOP significantly increased HO (p < .0001; 34.95 ± 7.71 mm3) as compared to CET + BU alone, confirming BOP has a further synergistic effect. No HO was detectable in rats in the absence of CET. Serum analysis revealed similar significant elevated (p < .0001) levels of pro-inflammatory markers (Cxcl1 and Il6) at 6 h post-injury (hpi) in the CET + BU and BOP + CET + BU injury groups as compared to naïve baseline values. Real-time qPCR demonstrated similar levels of chondrogenic and osteogenic gene expression in muscle tissue at the site of injury at 168 hpi in both the CET + BU and BOP+CET + BU injury groups. These results support the hypothesis that a 10 % TBSA thermal burn markedly enhances tHO following acute musculoskeletal extremity injury in the presence and absence of blast overpressure. Furthermore, the influence of BOP on tHO cannot be accounted for either in regards to systemic inflammation induced from remote injury or inflammatory-osteo-chondrogenic expression changes local to the musculoskeletal trauma, suggesting that another mechanism beyond BOP and BU synergistic effects are at play. Therefore, these findings warrant future investigations to explore other mechanisms by which blast and burn influence tHO, and testing prophylactic measures to mitigate the local and systemic inflammatory effects of these injuries on development of HO.

Inhibition of focal adhesion kinase 2 results in a macrophage polarization shift to M2 which attenuates local and systemic inflammation and reduces heterotopic ossification after polysystem extremity trauma.

Introduction: Heterotopic ossification (HO) is a complex pathology often observed in combat injured casualties who have sustained severe, high energy polytraumatic extremity injuries. Once HO has developed, prophylactic therapies are limited outside of surgical excision. Tourniquet-induced ischemia injury (IR) exacerbates trauma-mediated musculoskeletal tissue injury, inflammation, osteogenic progenitor cell development and HO formation. Others have shown that focal adhesion kinase-2 (FAK2) plays a key role in regulating early inflammatory signaling events. Therefore, we hypothesized that targeting FAK2 prophylactically would mitigate extremity trauma induced IR inflammation and HO formation. Methods: We tested whether the continuous infusion of a FAK2 inhibitor (Defactinib, PF-573228; 6.94 µg/kg/min for 14 days) can mitigate ectopic bone formation (HO) using an established blast-related extremity injury model involving femoral fracture, quadriceps crush injury, three hours of tourniquet-induced limb ischemia, and hindlimb amputation through the fracture site. Tissue inflammation, infiltrating cells, osteogenic progenitor cell content were assessed at POD-7. Micro-computed tomography imaging was used to quantify mature HO at POD-56. Results: In comparison to vehicle control-treated rats, FAK2 administration resulted in no marked wound healing complications or weight loss. FAK2 treatment decreased HO by 43%. At POD-7, marked reductions in tissue proinflammatory gene expression and assayable osteogenic progenitor cells were measured, albeit no significant changes in expression patterns of angiogenic, chondrogenic and osteogenic genes. At the same timepoint, injured tissue from FAK-treated rats had fewer infiltrating cells. Additionally, gene expression analyses of tissue infiltrating cells resulted in a more measurable shift from an M1 inflammatory to an M2 anti-inflammatory macrophage phenotype in the FAK2 inhibitor-treated group. Discussion: Our findings suggest that FAK2 inhibition may be a novel strategy to dampen trauma-induced inflammation and attenuate HO in patients at high risk as a consequence of severe musculoskeletal polytrauma.

Systemic inflammation induced from remote extremity trauma is a critical driver of secondary brain injury.

Blast exposure, commonly experienced by military personnel, can cause devastating life-threatening polysystem trauma. Despite considerable research efforts, the impact of the systemic inflammatory response after major trauma on secondary brain injury-inflammation is largely unknown. The aim of this study was to identify markers underlying the susceptibility and early onset of neuroinflammation in three rat trauma models: (1) blast overpressure exposure (BOP), (2) complex extremity trauma (CET) involving femur fracture, crush injury, tourniquet-induced ischemia, and transfemoral amputation through the fracture site, and (3) BOP+CET. Six hours post-injury, intact brains were harvested and dissected to obtain biopsies from the prefrontal cortex, striatum, neocortex, hippocampus, amygdala, thalamus, hypothalamus, and cerebellum. Custom low-density microarray datasets were used to identify, interpret and visualize genes significant (p < 0.05 for differential expression [DEGs]; 86 neuroinflammation-associated) using a custom python-based computer program, principal component analysis, heatmaps and volcano plots. Gene set and pathway enrichment analyses of the DEGs was performed using R and STRING for protein-protein interaction (PPI) to identify and explore key genes and signaling networks. Transcript profiles were similar across all regions in naïve brains with similar expression levels involving neurotransmission and transcription functions and undetectable to low-levels of inflammation-related mediators. Trauma-induced neuroinflammation across all anatomical brain regions correlated with injury severity (BOP+CET > CET > BOP). The most pronounced differences in neuroinflammatory-neurodegenerative gene regulation were between blast-associated trauma (BOP, BOP+CET) and CET. Following BOP, there were few DEGs detected amongst all 8 brain regions, most were related to cytokines/chemokines and chemokine receptors, where PPI analysis revealed Il1b as a potential central hub gene. In contrast, CET led to a more excessive and diverse pro-neuroinflammatory reaction in which Il6 was identified as the central hub gene. Analysis of the of the BOP+CET dataset, revealed a more global heightened response (Cxcr2, Il1b, and Il6) as well as the expression of additional functional regulatory networks/hub genes (Ccl2, Ccl3, and Ccl4) which are known to play a critical role in the rapid recruitment and activation of immune cells via chemokine/cytokine signaling. These findings provide a foundation for discerning pathophysiological consequences of acute extremity injury and systemic inflammation following various forms of trauma in the brain.

Systematic Identification of the Optimal Housekeeping Genes for Accurate Transcriptomic and Proteomic Profiling of Tissues following Complex Traumatic Injury.

Trauma triggers critical molecular and cellular signaling cascades that drive biological outcomes and recovery. Variations in the gene expression of common endogenous reference housekeeping genes (HKGs) used in data normalization differ between tissue types and pathological states. Systematically, we investigated the gene stability of nine HKGs (Actb, B2m, Gapdh, Hprt1, Pgk1, Rplp0, Rplp2, Tbp, and Tfrc) from tissues prone to remote organ dysfunction (lung, liver, kidney, and muscle) following extremity trauma. Computational algorithms (geNorm, Normfinder, ΔCt, BestKeeper, RefFinder) were applied to estimate the expression stability of each HKG or combinations of them, within and between tissues, under both steady-state and systemic inflammatory conditions. Rplp2 was ranked as the most suitable in the healthy and injured lung, kidney, and skeletal muscle, whereas Rplp2 and either Hprt1 or Pgk1 were the most suitable in the healthy and injured liver, respectively. However, the geometric mean of the three most stable genes was deemed the most stable internal reference control. Actb and Tbp were the least stable in normal tissues, whereas Gapdh and Tbp were the least stable across all tissues post-trauma. Ct values correlated poorly with the translation from mRNA to protein. Our results provide a valuable resource for the accurate normalization of gene expression in trauma-related experiments.

Tourniquet-induced ischemia creates increased risk of organ dysfunction and mortality following delayed limb amputation.

Tourniquets are critical for the control of traumatic extremity hemorrhage. In this study, we sought to determine, in a rodent blast-related extremity amputation model, the impact of prolonged tourniquet application and delayed limb amputation on survival, systemic inflammation, and remote end organ injury. Adult male Sprague Dawley rats were subjected to blast overpressure (120±7 kPa) and orthopedic extremity injury consisting femur fracture, one-minute soft tissue crush injury (20 psi), ± 180 min of tourniquet-induced hindlimb ischemia followed by delayed (60 min of reperfusion) hindlimb amputation (dHLA). All animals in the non-tourniquet group survived whereas 7/21 (33%) of the animals in the tourniquet group died within the first 72 h with no deaths observed between 72 and 168 h post-injury. Tourniquet induced ischemia-reperfusion injury (tIRI) likewise resulted in a more robust systemic inflammation (cytokines and chemokines) and concomitant remote pulmonary, renal, and hepatic dysfunction (BUN, CR, ALT. AST, IRI/inflammation-mediated genes). These results indicate prolonged tourniquet application and dHLA increases risk of complications from tIRI, leading to greater risk of local and systemic complications including organ dysfunction or death. We thus need enhanced strategies to mitigate the systemic effects of tIRI, particularly in the military prolonged field care (PFC) setting. Furthermore, future work is needed to extend the window within which tourniquet deflation to assess limb viability remains feasible, as well as new, limb-specific or systemic point of care tests to better assess the risks of tourniquet deflation with limb preservation in order to optimize patient care and save both limb and life.

Tourniquet use following blast-associated complex lower limb injury and traumatic amputation promotes end organ dysfunction and amplified heterotopic ossification formation.

BACKGROUND: Traumatic heterotopic ossification (tHO) is characterized by ectopic bone formation in extra-skeletal sites leading to impaired wound healing, entrapment of neurovascular structures, pain, and reduced range of motion. HO has become a signature pathology affecting wounded military personnel who have sustained blast-associated traumatic amputations during the recent conflicts in Iraq and Afghanistan and can compound recovery by causing difficulty with prosthesis limb wearing. Tourniquet use to control catastrophic limb hemorrhage prior to surgery has become almost ubiquitous during this time, with the recognition the prolonged use may risk an ischemia reperfusion injury and associated complications. While many factors influence the formation of tHO, the extended use of tourniquets to limit catastrophic hemorrhage during prolonged field care has not been explored. METHODS: Utilizing an established pre-clinical model of blast-associated complex lower limb injury and traumatic amputation, we evaluated the effects of tourniquet use on tHO formation. Adult male rats were subjected to blast overpressure exposure, femur fracture, and soft tissue crush injury. Pneumatic tourniquet (250-300 mmHg) applied proximal to the injured limb for 150-min was compared to a control group without tourniquet, before a trans-femoral amputation was performed. Outcome measures were volume to tHO formation at 12 weeks and changes in proteomic and genomic markers of early tHO formation between groups. RESULTS: At 12 weeks, volumetric analysis with microCT imaging revealed a 70% increase in total bone formation (p = 0.007) near the site of injury compared to rats with no tourniquet time in the setting of blast-injuries. Rats subjected to tourniquet usage had increased expression of danger-associated molecular patterns (DAMPs) and end organ damage as early as 6 h and as late as 7 days post injury. The expressions of pro-inflammatory cytokines and chemokines and osteochondrogenic genes using quantitative RT-PCR similarly revealed increased expression as early as 6 h post injury, and these genes along with hypoxia associated genes remained elevated for 7 days compared to no tourniquet use. CONCLUSION: These findings suggest that tourniquet induced ischemia leads to significant increases in key transcription factors associated with early endochondral bone formation, systemic inflammatory and hypoxia, resulting in increased HO formation.

Controlling axonal regeneration with acellular nerve allograft limits neuroma formation in peripheral nerve transection: An experimental study in a swine model.

BACKGROUND: Symptomatic neuromata are a common indication for revision surgery following amputation. Previously described treatments, including traction neurectomy, nerve transposition, targeted muscle re-innervation, and nerve capping, have provided inconsistent results or are technically challenging. Prior research using acellular nerve allografts (ANA) has shown controlled termination of axonal regrowth in long grafts. The purpose of this study was to determine the ability of a long ANA to prevent neuroma formation following transection of a peripheral nerve in a swine model. MATERIALS AND METHODS: Twenty-two adult female Yucatan miniature swine (Sus scrofa; 4-6 months, 15-25 kg) were assigned to control (ulnar nerve transection only, n = 10), treatment (ulnar transection and coaptation of 50 mm ANA, n = 10), or donor (n = 2) groups. Nerves harvested from donor group animals were treated to create the ANA. After 20 weeks, the transected nerves including any neuroma or graft were harvested. Both qualitative (nerve architecture, axonal sprouting) and quantitative histologic analyses (myelinated axon number, cross sectional area of nerve tissue) were performed. RESULTS: Qualitative histologic analysis of control specimens revealed robust axon growth into dense scar tissue. In contrast, the treatment group revealed dwindling axons in the terminal tissue, consistent with attenuated neuroma formation. Quantitative analysis revealed a significantly decreased number of myelinated axons in the treatment group (1232 ± 540) compared to the control group (44,380 ± 7204) (p < .0001). Cross sectional area of nerve tissue was significantly smaller in treatment group (2.83 ± 1.53 mm2 ) compared to the control group (9.14 ± 1.19 mm2 ) (p = .0012). CONCLUSIONS: Aberrant axonal growth is controlled to termination with coaptation of a 50 mm ANA in a swine model of nerve injury. These early results suggest further investigation of this technique to prevent and/or treat neuroma formation.

Cellular microRNAs correlate with clinical parameters in multiple injury patients.

INTRODUCTION: The pathophysiology of the inflammatory response after major trauma is complex, and the magnitude correlates with severity of tissue injury and outcomes. Study of infection-mediated immune pathways has demonstrated that cellular microRNAs may modulate the inflammatory response. The authors hypothesize that the expression of microRNAs would correlate to complicated recoveries in polytrauma patients (PtPs). METHODS: Polytrauma patients enrolled in the prospective observational Tissue and Data Acquisition Protocol with Injury Severity Score of >15 were selected for this study. Polytrauma patients were divided into complicated recoveries and uncomplicated recovery groups. Polytrauma patients' blood samples were obtained at the time of admission (T0). Established biomarkers of systemic inflammation, including cytokines and chemokines, were measured using multiplexed Luminex-based methods, and novel microRNAs were measured in plasma samples using multiplex RNA hybridization. RESULTS: Polytrauma patients (n = 180) had high Injury Severity Score (26 [20-34]) and complicated recovery rate of 33%. MicroRNAs were lower in PtPs at T0 compared with healthy controls, and bivariate analysis demonstrated that variations of microRNAs correlated with age, race, comorbidities, venous thromboembolism, pulmonary complications, complicated recovery, and mortality. Positive correlations were noted between microRNAs and interleukin 10, vascular endothelial growth factor, Acute Physiology and Chronic Health Evaluation, and Sequential Organ Failure Assessment scores. Multivariable Lasso regression analysis of predictors of complicated recovery based on microRNAs, cytokines, and chemokines revealed that miR-21-3p and monocyte chemoattractant protein-1 were predictive of complicated recovery with an area under the curve of 0.78. CONCLUSION: Systemic microRNAs were associated with poor outcomes in PtPs, and results are consistent with previously described trends in critically ill patients. These early biomarkers of inflammation might provide predictive utility in early complicated recovery diagnosis and prognosis. Because of their potential to regulate immune responses, microRNAs may provide therapeutic targets for immunomodulation. LEVEL OF EVIDENCE: Diagnostic Tests/Criteria; Level II.

Key early proinflammatory signaling molecules encapsulated within circulating exosomes following traumatic injury.

BACKGROUND: Assessment of immune status in critically ill patients is often based on serial tracking of systemic cytokine levels and clinical laboratory values. Exosomes are extracellular vesicles that can be secreted and internalized by cells to transport important cellular cargo in the regulation of numerous physiological and pathological processes. Here, we characterize the early compartmentalization profile of key proinflammatory mediators in serum exosomes in the steady state and following trauma. Adult male Sprague-Dawley rats (91 including naïve) were divided into one of four traumatic injury model groups incorporating whole-body blast, fracture, soft-tissue crush injury, tourniquet-induced ischemia, and limb amputation. Serum was collected at 1, 3, 6, and 24 h, and 3- and 7-day post-injury. Electrochemiluminescence-based immunoassays for 9 key proinflammatory mediators in whole serum, isolated serum exosomes, and exosome depleted serum were analyzed and compared between naïve and injured rats. Serum clinical chemistry analysis was performed to determine pathological changes. RESULTS: In naïve animals, substantial amounts of IL-1ß, IL-10, and TNF-α were encapsulated, IL-6 was completely encapsulated, and CXCL1 freely circulating. One hour after blast injury alone, levels of exosome encapsulated IFN-γ, IL-10, IL-6, IL-13, IL-4, and TNF-α increased, whereas freely circulating and membrane-associated levels remained undetectable or low. Rats with the most severe polytraumatic injuries with end organ complications had the earliest rise and most pronounced concentration of IL-1ß, IL-10, TNF-α, and IL-6 across all serum compartments. Moreover, CXCL1 levels increased in relation to injury severity, but remained almost entirely freely circulating at all timepoints. CONCLUSION: These findings highlight that conventional ELISA-based assessments, which detect only free circulating and exosome membrane-bound mediators, underestimate the full immunoinflammatory response to trauma. Inclusion of exosome encapsulated mediators may be a better, more accurate and clinically useful early strategy to identify, diagnose, and monitor patients at highest risk for post-traumatic inflammation-associated complications.

Longitudinal Analysis of Circulating Markers of Bone Turnover Across Multiple Decades in Osteoporotic Women.

PURPOSE: The objective of this study was to analyze changes in serum markers of bone turnover across multiple decades in osteoporotic women compared with nonosteoporotic controls, to determine their utility as potential predictors for osteoporosis. Early prediction of those at risk for osteoporosis can enable early intervention before the irreversible loss of critical bone mass. METHODS: Serum samples were obtained from 20 women given the diagnosis of osteoporosis after age 46 years and 20 age-matched women with normal bone mineral density from 4 time points in their life (ages 25-31, 32-38, 39-45, and 46-60 years). Serum levels of bone turnover markers (propeptide of type I collagen, parathyroid hormone, bone-specific alkaline phosphatase, osteocalcin, C-terminal telopeptide of type I collagen, sclerostin, osteoprotegerin, osteopontin, and 25-OH vitamin D) were measured using commercially available arrays and kits. We used logistic regression to assess these individual serum markers as potential predictors of osteoporosis, and mixed-effects modeling to assess the change in bone turnover markers between osteoporotic and control groups over time, then performed fivefold cross-validation to assess the classification ability of the models. RESULTS: Markers of bone turnover, bone-specific alkaline phosphatase, C-terminal telopeptide of type I collagen, sclerostin, and osteocalcin were all independent predictors at multiple time points; osteopontin was an independent predictor in the 39- to 45-year age group. Receiver operating characteristic analyses demonstrated moderately strong classification ability at all time points. Sclerostin levels among groups diverged over time and were higher in the control group than the osteoporotic group, with significant differences observed at time points 3 and 4. CONCLUSIONS: Serum markers of bone turnover may be used to estimate the likelihood of osteoporosis development in individuals over time. Although prospective validation is necessary before recommending widespread clinical use, this information may be used to identify patients at risk for developing low bone mineral density long before traditional screening would ostensibly take place. TYPE OF STUDY/LEVEL OF EVIDENCE: Diagnostic II.

Viral Micro-RNAs Are Detected in the Early Systemic Response to Injury and Are Associated With Outcomes in Polytrauma Patients.

OBJECTIVES: To evaluate early activation of latent viruses in polytrauma patients and consider prognostic value of viral micro-RNAs in these patients. DESIGN: This was a subset analysis from a prospectively collected multicenter trauma database. Blood samples were obtained upon admission to the trauma bay (T0), and trauma metrics and recovery data were collected. SETTING: Two civilian Level 1 Trauma Centers and one Military Treatment Facility. PATIENTS: Adult polytrauma patients with Injury Severity Scores greater than or equal to 16 and available T0 plasma samples were included in this study. Patients with ICU admission greater than 14 days, mechanical ventilation greater than 7 days, or mortality within 28 days were considered to have a complicated recovery. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Polytrauma patients (n = 180) were identified, and complicated recovery was noted in 33%. Plasma samples from T0 underwent reverse transcriptase-quantitative polymerase chain reaction analysis for Kaposi's sarcoma-associated herpesvirus micro-RNAs (miR-K12_10b and miRK-12-12) and Epstein-Barr virus-associated micro-RNA (miR-BHRF-1), as well as Luminex multiplex array analysis for established mediators of inflammation. Ninety-eight percent of polytrauma patients were found to have detectable Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus micro-RNAs at T0, whereas healthy controls demonstrated 0% and 100% detection rate for Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus, respectively. Univariate analysis revealed associations between viral micro-RNAs and polytrauma patients' age, race, and postinjury complications. Multivariate least absolute shrinkage and selection operator analysis of clinical variables and systemic biomarkers at T0 revealed that interleukin-10 was the strongest predictor of all viral micro-RNAs. Multivariate least absolute shrinkage and selection operator analysis of systemic biomarkers as predictors of complicated recovery at T0 demonstrated that miR-BHRF-1, miR-K12-12, monocyte chemoattractant protein-1, and hepatocyte growth factor were independent predictors of complicated recovery with a model complicated recovery prediction area under the curve of 0.81. CONCLUSIONS: Viral micro-RNAs were detected within hours of injury and correlated with poor outcomes in polytrauma patients. Our findings suggest that transcription of viral micro-RNAs occurs early in the response to trauma and may be associated with the biological processes involved in polytrauma-induced complicated recovery.

Culture and characterization of various porcine integumentary-connective tissue-derived mesenchymal stromal cells to facilitate tissue adhesion to percutaneous metal implants.

BACKGROUND: Transdermal osseointegrated prosthesis have relatively high infection rates leading to implant revision or failure. A principle cause for this complication is the absence of a durable impervious biomechanical seal at the interface of the hard structure (implant) and adjacent soft tissues. This study explores the possibility of recapitulating an analogous cellular musculoskeletal-connective tissue interface, which is present at naturally occurring integumentary tissues where a hard structure exits the skin, such as the nail bed, hoof, and tooth. METHODS: Porcine mesenchymal stromal cells (pMSCs) were derived from nine different porcine integumentary and connective tissues: hoof-associated superficial flexor tendon, molar-associated periodontal ligament, Achilles tendon, adipose tissue and skin dermis from the hind limb and abdominal regions, bone marrow and muscle. For all nine pMSCs, the phenotype, multi-lineage differentiation potential and their adhesiveness to clinical grade titanium was characterized. Transcriptomic analysis of 11 common genes encoding cytoskeletal proteins VIM (Vimentin), cell-cell and cell-matrix adhesion genes (Vinculin, Integrin ß1, Integrin ß2, CD9, CD151), and for ECM genes (Collagen-1a1, Collagen-4a1, Fibronectin, Laminin-α5, Contactin-3) in early passaged cells was performed using qRT-PCR. RESULTS: All tissue-derived pMSCs were characterized as mesenchymal origin by adherence to plastic, expression of cell surface markers including CD29, CD44, CD90, and CD105, and lack of hematopoietic (CD11b) and endothelial (CD31) markers. All pMSCs differentiated into osteoblasts, adipocytes and chondrocytes, albeit at varying degrees, under specific culture conditions. Among the eleven adhesion genes evaluated, the cytoskeletal intermediate filament vimentin was found highly expressed in pMSC isolated from all tissues, followed by genes for the extracellular matrix proteins Fibronectin and Collagen-1a1. Expression of Vimentin was the highest in Achilles tendon, while Fibronectin and Col1agen-1a1 were highest in molar and hoof-associated superficial flexor tendon bone marrow, respectively. Achilles tendon ranked the highest in both multilineage differentiation and adhesion assessments to titanium metal. CONCLUSIONS: These findings support further preclinical research of these tissue specific-derived MSCs in vivo in a transdermal osseointegration implant model.

Long Acellular Nerve Allografts Cap Transected Nerve to Arrest Axon Regeneration and Alter Upstream Gene Expression in a Rat Neuroma Model.

BACKGROUND: Treatments to manage painful neuroma are needed. An operative strategy that isolates and controls chaotic axonal growth could prevent neuroma. Using long acellular nerve allograft to "cap" damaged nerve could control axonal regeneration and, in turn, regulate upstream gene expression patterns. METHODS: Rat sciatic nerve was transected, and the distal nerve end was reversed and ligated to generate a model end-neuroma. Three groups were used to assess their effects immediately following this nerve injury: no treatment (control), traction neurectomy, or 5-cm acellular nerve allograft cap attached to the proximal nerve. Regeneration of axons from the injured nerve was assessed over 5 months and paired with concurrent measurements of gene expression from upstream affected dorsal root ganglia. RESULTS: Both control and traction neurectomy groups demonstrated uncontrolled axon regeneration revealed using Thy1-GFP rat axon imaging and histomorphometric measures of regenerated axons within the most terminal region of regenerated tissue. The acellular nerve allograft group arrested axons within the acellular nerve allograft, where no axons reached the most terminal region even after 5 months. At 5 months, gene expression associated with regeneration and pain sensitization, including Bdnf, cfos, and Gal, was decreased within dorsal root ganglia obtained from the acellular nerve allograft group compared to control or traction neurectomy group dorsal root ganglia. CONCLUSIONS: Long acellular nerve allografts to cap a severed nerve arrested axon regeneration within the acellular nerve allograft. This growth arrest corresponded with changes in regenerative and pain-related genes upstream. Acellular nerve allografts may be useful for surgical intervention of neuroma.

Alarming Cargo: The Role of Exosomes in Trauma-Induced Inflammation.

Severe polytraumatic injury initiates a robust immune response. Broad immune dysfunction in patients with such injuries has been well-documented; however, early biomarkers of immune dysfunction post-injury, which are critical for comprehensive intervention and can predict the clinical course of patients, have not been reported. Current circulating markers such as IL-6 and IL-10 are broad, non-specific, and lag behind the clinical course of patients. General blockade of the inflammatory response is detrimental to patients, as a certain degree of regulated inflammation is critical and necessary following trauma. Exosomes, small membrane-bound extracellular vesicles, found in a variety of biofluids, carry within them a complex functional cargo, comprised of coding and non-coding RNAs, proteins, and metabolites. Composition of circulating exosomal cargo is modulated by changes in the intra- and extracellular microenvironment, thereby serving as a homeostasis sensor. With its extensively documented involvement in immune regulation in multiple pathologies, study of exosomal cargo in polytrauma patients can provide critical insights on trauma-specific, temporal immune dysregulation, with tremendous potential to serve as unique biomarkers and therapeutic targets for timely and precise intervention.

High Frequency Spectral Ultrasound Imaging Detects Early Heterotopic Ossification in Rodents.

Heterotopic ossification (HO) is a devastating condition in which ectopic bone forms inappropriately in soft tissues following traumatic injuries and orthopedic surgeries as a result of aberrant mesenchymal progenitor cell (MPC) differentiation. HO leads to chronic pain, decreased range of motion, and an overall decrease in quality of life. While several treatments have shown promise in animal models, all must be given during early stages of formation. Methods for early determination of whether and where endochondral ossification/soft tissue mineralization (HO anlagen) develop are lacking. At-risk patients are not identified sufficiently early in the process of MPC differentiation and soft tissue endochondral ossification for potential treatments to be effective. Hence, a critical need exists to develop technologies capable of detecting HO anlagen soon after trauma, when treatments are most effective. In this study, we investigate high frequency spectral ultrasound imaging (SUSI) as a noninvasive strategy to identify HO anlagen at early time points after injury. We show that by determining quantitative parameters based on tissue organization and structure, SUSI identifies HO anlagen as early as 1-week postinjury in a mouse model of burn/tenotomy and 3 days postinjury in a rat model of blast/amputation. We analyze single cell RNA sequencing profiles of the MPCs responsible for HO formation and show that the early tissue changes detected by SUSI match chondrogenic and osteogenic gene expression in this population. SUSI identifies sites of soft tissue endochondral ossification at early stages of HO formation so that effective intervention can be targeted when and where it is needed following trauma-induced injury. Furthermore, we characterize the chondrogenic to osteogenic transition that occurs in the MPCs during HO formation and correlate gene expression to SUSI detection of the HO anlagen.

An Empirical Antigen Selection Method Identifies Neoantigens That Either Elicit Broad Antitumor T-cell Responses or Drive Tumor Growth.

Neoantigens are critical targets of antitumor T-cell responses. The ATLAS bioassay was developed to identify neoantigens empirically by expressing each unique patient-specific tumor mutation individually in Escherichia coli, pulsing autologous dendritic cells in an ordered array, and testing the patient's T cells for recognition in an overnight assay. Profiling of T cells from patients with lung cancer revealed both stimulatory and inhibitory responses to individual neoantigens. In the murine B16F10 melanoma model, therapeutic immunization with ATLAS-identified stimulatory neoantigens protected animals, whereas immunization with peptides associated with inhibitory ATLAS responses resulted in accelerated tumor growth and abolished efficacy of an otherwise protective vaccine. A planned interim analysis of a clinical study testing a poly-ICLC adjuvanted personalized vaccine containing ATLAS-identified stimulatory neoantigens showed that it is well tolerated. In an adjuvant setting, immunized patients generated both CD4+ and CD8+ T-cell responses, with immune responses to 99% of the vaccinated peptide antigens. SIGNIFICANCE: Predicting neoantigens in silico has progressed, but empirical testing shows that T-cell responses are more nuanced than straightforward MHC antigen recognition. The ATLAS bioassay screens tumor mutations to uncover preexisting, patient-relevant neoantigen T-cell responses and reveals a new class of putatively deleterious responses that could affect cancer immunotherapy design.This article is highlighted in the In This Issue feature, p. 521.

BMP Ligand Trap ALK3-Fc Attenuates Osteogenesis and Heterotopic Ossification in Blast-Related Lower Extremity Trauma.

Traumatic heterotopic ossification (tHO) commonly develops in wounded service members who sustain high-energy and blast-related traumatic amputations. Currently, no safe and effective preventive measures have been identified for this patient population. Bone morphogenetic protein (BMP) signaling blockade has previously been shown to reduce ectopic bone formation in genetic models of HO. In this study, we demonstrate the efficacy of small-molecule inhibition with LDN193189 (ALK2/ALK3 inhibition), LDN212854 (ALK2-biased inhibition), and BMP ligand trap ALK3-Fc at inhibiting early and late osteogenic differentiation of tissue-resident mesenchymal progenitor cells (MPCs) harvested from mice subjected to burn/tenotomy, a well-characterized trauma-induced model of HO. Using an established rat tHO model of blast-related extremity trauma and methicillin-resistant Staphylococcus aureus infection, a significant decrease in ectopic bone volume was observed by micro-computed tomography imaging following treatment with LDN193189, LDN212854, and ALK3-Fc. The efficacy of LDN193189 and LDN212854 in this model was associated with weight loss (17%-19%) within the first two postoperative weeks, and in the case of LDN193189, delayed wound healing and metastatic infection was observed, while ALK3-Fc was well tolerated. At day 14 following injury, RNA-Seq and quantitative reverse transcriptase-polymerase chain reaction analysis revealed that ALK3-Fc enhanced the expression of skeletal muscle structural genes and myogenic transcriptional factors while inhibiting the expression of inflammatory genes. Tissue-resident MPCs harvested from rats treated with ALK3-Fc exhibited reduced osteogenic differentiation, proliferation, and self-renewal capacity and diminished expression of genes associated with endochondral ossification and SMAD-dependent signaling pathways. Together, these results confirm the contribution of BMP signaling in osteogenic differentiation and ectopic bone formation and that a selective ligand-trap approach such as ALK3-Fc may be an effective and tolerable prophylactic strategy for tHO.

The role of neutrophil extracellular traps and TLR signaling in skeletal muscle ischemia reperfusion injury.

Ischemia reperfusion (IR) injury results in devastating skeletal muscle fibrosis. Here, we recapitulate this injury with a mouse model of hindlimb IR injury which leads to skeletal muscle fibrosis. Injury resulted in extensive immune infiltration with robust neutrophil extracellular trap (NET) formation in the skeletal muscle, however, direct targeting of NETs via the peptidylarginine deiminase 4 (PAD4) mechanism was insufficient to reduce muscle fibrosis. Circulating levels of IL-10 and TNFα were significantly elevated post injury, indicating toll-like receptor (TLR) signaling may be involved in muscle injury. Administration of hydroxychloroquine (HCQ), a small molecule inhibitor of TLR7/8/9, following injury reduced NET formation, IL-10, and TNFα levels and ultimately mitigated muscle fibrosis and improved myofiber regeneration following IR injury. HCQ treatment decreased fibroadipogenic progenitor cell proliferation and partially inhibited ERK1/2 phosphorylation in the injured tissue, suggesting it may act through a combination of TLR7/8/9 and ERK signaling mechanisms. We demonstrate that treatment with FDA-approved HCQ leads to decreased muscle fibrosis and increased myofiber regeneration following IR injury, suggesting short-term HCQ treatment may be a viable treatment to prevent muscle fibrosis in ischemia reperfusion and traumatic extremity injury.

Proteomic characterization of a trauma-based rat model of heterotopic ossification identifies interactive signaling networks as potential therapeutic targets.

Heterotopic ossification (HO) is the formation of ectopic bone in soft tissues observed in patients following blast injuries, orthopedic or head trauma, burns, or in the context of inborn mutations of genes involved in osteogenesis. There is no universally accepted therapy for HO. This study has used global unbiased mass spectrometry proteomic approaches, validated by western immunoblots, to interrogate skeletal muscle tissues obtained from a highly reproducible rat model of trauma induced HO. During early the phase of HO development, statistically significant modulation of proteins within the following pathways was identified: coagulation, cyclic AMP, extracellular matrix, immunity/inflammation, NADH metabolism, TGFß. These metabolic proteins and pathways have the potential to serve as diagnostic, prognostic, and therapeutic targets for this devastating orthopedic condition that has considerable impact on the patient's quality of life. Furthermore, the findings confirm and extend previous in vitro stromal/stem cell and clinical studies from the field. SIGNIFICANCE: This study confirms and extends the field's understanding of the protein pathways that are modulated in a rat model of trauma induced heterotopic ossification. The identification of specific proteins such as the AP1 transcription factor as well as protein families such as the complement/coagulation pathway and serine protease inhibitors as biomarkers have potential clinical translational value. These outcomes have relevance to the physiological and pathological mineralization processes contributing to the recovery of orthopedic trauma patients.