Trauma patients with type O blood exhibit unique multi-omics signature with decreased lectin pathway of complement levels.

BACKGROUND: Patients with type O blood may have an increased risk of hemorrhagic complications due to lower baseline levels of von Willebrand Factor (vWF) and factor VIII, but the transition to a mortality difference in trauma is less clear. We hypothesized that type O trauma patients will have differential proteomic and metabolomic signatures in response to trauma beyond vWF and FVIII alone. METHODS: Patients meeting the highest level of trauma activation criteria were prospectively enrolled. Blood samples were collected upon arrival to the emergency department. Proteomic and metabolomic (multi-omics) analyses of these samples were performed using liquid chromatography-mass spectrometry. Demographic, clinical, and multi-omics data were compared between patients with type O blood versus all other patients. RESULTS: There were 288 patients with multi-omics data; 146 (51%) had type O blood. Demographics, injury patterns, and initial vital signs and laboratory measurements were not different between groups. Type O patients had increased lengths of stay (7 vs. 6 days, p = 0.041) and a trend towards decreased mortality secondary to traumatic brain injury compared to other causes (TBI, 44.4 % vs. 87.5%, p = 0.055). Type O patients had decreased levels of mannose-binding lectin (MBL) and MBL associated serine proteases 1 and 2 which are required for the initiation of the lectin pathway of complement activation. Type O patients also had metabolite differences signifying energy metabolism and mitochondrial dysfunction. CONCLUSION: Blood type O patients have a unique multi-omics signature, including decreased levels of proteins required to activate the lectin complement pathway. This may lead to overall decreased levels of complement activation and decreased systemic inflammation in the acute phase possibly leading to a survival advantage, especially in TBI. However, this may later impair healing. Future work will need to confirm these associations, and animal studies are needed to test therapeutic targets. LEVEL OF EVIDENCE: Retrospective Comparative Study, Level IV.

Platelet releasates mitigate the endotheliopathy of trauma.

BACKGROUND: Platelets are well known for their roles in hemostasis, but they also play a key role in thromboinflammatory pathways by regulating endothelial health, stimulating angiogenesis, and mediating host defense through both contact dependent and independent signaling. When activated, platelets degranulate releasing multiple active substances. We hypothesized that the soluble environment formed by trauma platelet releasates attenuates thromboinflammation via mitigation of trauma induced endothelial permeability and metabolomic reprogramming. METHODS: Blood was collected from injured and healthy patients to generate platelet releasates and plasma in parallel. Permeability of endothelial cells when exposed to trauma platelet releasates (TPR) and plasma (TP) was assessed via resistance measurement by Electric Cell-substrate Impedance Sensing (ECIS). Endothelial cells treated with TPR and TP were subjected to mass spectrometry-based metabolomics. RESULTS: TP increased endothelial permeability, whereas TPR decreased endothelial permeability when compared to untreated cells. When TP and TPR were mixed ex vivo, TPR mitigated TP-induced permeability, with significant increase in AUC compared to TP alone. Metabolomics of TPR and TP demonstrated disrupted redox reactions and anti-inflammatory mechanisms. CONCLUSION: TPRs provide endothelial barrier protection against TP-induced endothelial permeability. Our findings highlight a potential beneficial action of activated platelets on the endothelium in injured patients through disrupted redox reactions and increased antioxidants. Our findings support that soluble signaling from platelet degranulation may mitigate the endotheliopathy of trauma. The clinical implications of this are that activated platelets may prove a promising therapeutic target in the complex integration of thrombosis, endotheliopathy, and inflammation in trauma. LEVEL OF EVIDENCE: Prognostic/Epidemiological, Level III.

Smoking Primes the Metabolomic Response in Trauma.

INTRODUCTION: Smoking is a public health threat due to its well described link to increased oxidative stress-related diseases including peripheral vascular disease and coronary artery disease. Tobacco use has been linked to risk of inpatient trauma morbidity including acute respiratory distress syndrome, however its mechanistic effect on comprehensive metabolic heterogeneity has yet to be examined. METHODS: Plasma was obtained on arrival from injured patients at a Level 1 Trauma Center and analyzed with modern mass spectrometry-based metabolomics. Patients were stratified by non-smoker, passive smoker and active smoker by lower, inter-quartile and upper quartile ranges of cotinine intensity peaks. Patients were sub-stratified by High Injury/High Shock (Injury Severity Score ≥ 15, Base Excess<-6) and compared to healthy controls. P-value <0.05 following FDR correction of t-test was considered significant. RESULTS: 48 patients with High Injury/High Shock (7 (15%) non-smokers, 25 (52%) passive smokers and 16 (33%) active smokers) and 95 healthy patients who served as controls (30 (32%) non-smokers, 43 (45%) passive smokers and 22 (23%) active smokers) were included. Elevated metabolites in our controls who were active smokers include enrichment in chronic inflammatory and oxidative processes. Elevated metabolites in active smokers in high injury/high shock include enrichment in the malate-aspartate shuttle, tyrosine metabolism, carnitine synthesis, and oxidation of very long-chain fatty acids. CONCLUSIONS: Smoking promotes a state of oxidative stress leading to mitochondrial dysfunction which is additive to the inflammatory milieu of trauma. Smoking is associated with impaired mitochondrial substrate utilization of long-chain fatty acids, aspartate and tyrosine all of which accentuate oxidative stress following injury. This altered expression represents an ideal target for therapies to reduce oxidative damage toward the goal of personalized treatment of trauma patients. LEVEL OF EVIDENCE: Level III, Prognostic/Epidemiological.

Biological and Genetic Determinants of Glycolysis: Phosphofructokinase Isoforms Boost Energy Status of Stored Red Blood Cells and Transfusion Outcomes.

Mature red blood cells (RBCs) lack mitochondria, and thus exclusively rely on glycolysis to generate adenosine triphosphate (ATP) during aging in vivo and during storage in vitro in the blood bank. Here we identify an association between blood donor age, sex, ethnicity and end-of-storage levels of glycolytic metabolites in 13,029 volunteers from the Recipient Epidemiology and Donor Evaluation Study. Associations were also observed to ancestry-specific genetic polymorphisms in regions encoding phosphofructokinase 1, platelet (which we detected in mature RBCs), hexokinase 1, and ADP-ribosyl cyclase 1 and 2 (CD38/BST1). Gene-metabolite associations were validated in fresh and stored RBCs from 525 Diversity Outbred mice, and via multi-omics characterization of 1,929 samples from 643 human RBC units during storage. ATP levels, breakdown, and deamination into hypoxanthine were associated with hemolysis in vitro and in vivo, both in healthy autologous transfusion recipients and in 5,816 critically ill patients receiving heterologous transfusions. Highlights: Blood donor age and sex affect glycolysis in stored RBCs from 13,029 volunteers;Ancestry, genetic polymorphisms in PFKP, HK1, CD38/BST1 influence RBC glycolysis;RBC PFKP boosts glycolytic fluxes when ATP is low, such as in stored RBCs;ATP and hypoxanthine are biomarkers of hemolysis in vitro and in vivo.

Regulation of kynurenine metabolism by blood donor genetics and biology impacts red cell hemolysis in vitro and in vivo.

ABSTRACT: In the field of transfusion medicine, the clinical relevance of the metabolic markers of the red blood cell (RBC) storage lesion is incompletely understood. Here, we performed metabolomics of RBC units from 643 donors enrolled in the Recipient Epidemiology and Donor Evaluation Study, REDS RBC Omics. These units were tested on storage days 10, 23, and 42 for a total of 1929 samples and also characterized for end-of-storage hemolytic propensity after oxidative and osmotic insults. Our results indicate that the metabolic markers of the storage lesion poorly correlated with hemolytic propensity. In contrast, kynurenine was not affected by storage duration and was identified as the top predictor of osmotic fragility. RBC kynurenine levels were affected by donor age and body mass index and were reproducible within the same donor across multiple donations from 2 to 12 months apart. To delve into the genetic underpinnings of kynurenine levels in stored RBCs, we thus tested kynurenine levels in stored RBCs on day 42 from 13 091 donors from the REDS RBC Omics study, a population that was also genotyped for 879 000 single nucleotide polymorphisms. Through a metabolite quantitative trait loci analysis, we identified polymorphisms in SLC7A5, ATXN2, and a series of rate-limiting enzymes (eg, kynurenine monooxygenase, indoleamine 2,3-dioxygenase, and tryptophan dioxygenase) in the kynurenine pathway as critical factors affecting RBC kynurenine levels. By interrogating a donor-recipient linkage vein-to-vein database, we then report that SLC7A5 polymorphisms are also associated with changes in hemoglobin and bilirubin levels, suggestive of in vivo hemolysis in 4470 individuals who were critically ill and receiving single-unit transfusions.

Mitigation of trauma-induced endotheliopathy by activated protein C: A potential therapeutic for postinjury thromboinflammation.

BACKGROUND: Activated Protein C (aPC) plays dual roles after injury, driving both trauma-induced coagulopathy (TIC) by cleaving, and thus inactivating, factors Va and VIIIa and depressing fibrinolysis while also mediating an inflammomodulatory milieu via protease activated receptor-1 (PAR-1) cytoprotective signaling. Because of this dual role, it represents and ideal target for study and therapeutics after trauma. A known aPC variant, 3K3A-aPC, has been engineered to preserve cytoprotective activity while retaining minimal anticoagulant activity rendering it potentially ideal as a cytoprotective therapeutic after trauma. We hypothesized that 3K3A-aPC would mitigate the endotheliopathy of trauma by protecting against endothelial permeability. METHODS: We used electric cell-substrate impedance sensing to measure permeability changes in real time in primary endothelial cells. These were cultured, grown to confluence, and treated with a 2 µg/mL solution of 3K3A-aPC at 180 minutes, 120 minutes, 60 minutes, 30 minutes prior to stimulation with ex vivo plasma taken from severely injured trauma patients (Injury Severity Score > 15 and BD < -6) (trauma plasma [TP]). Cells treated with thrombin and untreated cells were included in this study as control groups. Permeability changes were recorded in real time via electric cell-substrate impedance sensing for 30 minutes after treatment with TP. We quantified permeability changes in the control and treatment groups as area under the curve (AUC). Rac1/RhoA activity was also compared between these groups. Statistical significance was determined by one-way ANOVA followed by a post hoc analysis using Tukey's multiple comparison's test. RESULTS: Treatment with aPC mitigated endothelial permeability induced by ex vivo trauma plasma at all pre-treatment time points. The AUC of the 30-minute 3K3A-aPC pretreatment group was higher than TP alone (mean diff. 22.12 95% CI [13.75, 30.49], p < 0.0001) (Figure). Moreover, the AUC of the 60-minute, 120-minute, and 180-minute pretreatment groups was also higher than TP alone (mean diff., 16.30; 95% confidence interval [CI], 7.93-24.67; 19.43; 95% CI, 11.06-27.80, and 18.65; 95% CI, 10.28-27.02;, all p < 0.0001, respectively). Rac1/RhoA activity was higher in the aPC pretreatment group when compared with all other groups ( p < 0.01). CONCLUSION: Pretreatment with 3K3A-aPC, which retains its cytoprotective function but has only ~5% of its anticoagulant function, abrogates the effects of trauma-induced endotheliopathy. This represents a potential therapeutic treatment for dysregulated thromboinflammation for injured patients by minimizing aPC's role in trauma-induced coagulopathy while concurrently amplifying its essential cytoprotective function. LEVEL OF EVIDENCE: Prognostic and Epidemiological; Level III.

ECM-Focused Proteomic Analysis of Ear Punch Regeneration in Acomys Cahirinus.

In mammals, significant injury is generally followed by the formation of a fibrotic scar which provides structural integrity but fails to functionally restore damaged tissue. Spiny mice of the genus Acomys represent the first example of full skin autotomy in mammals. Acomys cahirinus has evolved extremely weak skin as a strategy to avoid predation and is able to repeatedly regenerate healthy tissue without scar after severe skin injury or full-thickness ear punches. Extracellular matrix (ECM) composition is a critical regulator of wound repair and scar formation and previous studies have suggested that alterations in its expression may be responsible for the differences in regenerative capacity observed between Mus musculus and A. cahirinus , yet analysis of this critical tissue component has been limited in previous studies by its insolubility and resistance to extraction. Here, we utilize a 2-step ECM-optimized extraction to perform proteomic analysis of tissue composition during wound repair after full-thickness ear punches in A. cahirinus and M. musculus from weeks 1 to 4 post-injury. We observe changes in a wide range of ECM proteins which have been previously implicated in wound regeneration and scar formation, including collagens, coagulation and provisional matrix proteins, and matricryptic signaling peptides. We additionally report differences in crosslinking enzyme activity and ECM protein solubility between Mus and Acomys. Furthermore, we observed rapid and sustained increases in CD206, a marker of pro-regenerative M2 macrophages, in Acomys, whereas little or no increase in CD206 was detected in Mus. Together, these findings contribute to a comprehensive understanding of tissue cues which drive the regenerative capacity of Acomys and identify a number of potential targets for future pro-regenerative therapies.

Patterns of Electrocardiographic Abnormalities in Children with Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM), a common cardiomyopathy in children, is an important cause of morbidity and mortality. Early recognition and appropriate management are important. An electrocardiogram (ECG) is often used as a screening tool in children to detect heart disease. The ECG patterns in children with HCM are not well described.ECGs collected from an international cohort of children, and adolescents (≤ 21 years) with HCM were reviewed. 482 ECGs met inclusion criteria. Age ranged from 1 day to 21 years, median 13 years. Of the 482 ECGs, 57 (12%) were normal. The most common abnormalities noted were left ventricular hypertrophy (LVH) in 108/482 (22%) and biventricular hypertrophy (BVH) in 116/482 (24%) Of the patients with LVH/BVH (n = 224), 135 (60%) also had a strain pattern (LVH in 83, BVH in 52). Isolated strain pattern (in the absence of criteria for hypertrophy) was seen in 43/482 (9%). Isolated pathologic Q waves were seen in 71/482 (15%). Pediatric HCM, 88% have an abnormal ECG. The most common ECG abnormalities were LVH or BVH with or without strain. Strain pattern without hypertrophy and a pathologic Q wave were present in a significant proportion (24%) of patients. Thus, a significant number of children with HCM have ECG abnormalities that are not typical for "hypertrophy". The presence of the ECG abnormalities described above in a child should prompt further examination with an echocardiogram to rule out HCM.

METABOLOMIC AND PROTEOMIC CHANGES IN TRAUMA-INDUCED HYPOCALCEMIA.

ABSTRACT: Background: Trauma-induced hypocalcemia is common and associated with adverse outcomes, but the mechanisms remain unclear. Thus, we aimed to characterize the metabolomic and proteomic differences between normocalcemic and hypocalcemic trauma patients to illuminate biochemical pathways that may underlie a distinct pathology linked with this clinical phenomenon. Methods: Plasma was obtained on arrival from injured patients at a Level 1 Trauma Center. Samples obtained after transfusion were excluded. Multiple regression was used to adjust the omics data for injury severity and arrival base excess before metabolome- and proteome-wide comparisons between normocalcemic (ionized Ca 2+ > 1.0 mmol/L) and hypocalcemic (ionized Ca 2+ ≤ 1.0 mmol/L) patients using partial least squares-discriminant analysis. OmicsNet and Gene Ontology were used for network and pathway analyses, respectively. Results: Excluding isolated traumatic brain injury and penetrating injury, the main analysis included 36 patients (n = 14 hypocalcemic, n = 22 normocalcemic). Adjusted analyses demonstrated distinct metabolomic and proteomic signatures for normocalcemic and hypocalcemic patients. Hypocalcemic patients had evidence of mitochondrial dysfunction (tricarboxylic acid cycle disruption, dysfunctional fatty acid oxidation), inflammatory dysregulation (elevated damage-associated molecular patterns, activated endothelial cells), aberrant coagulation pathways, and proteolytic imbalance with increased tissue destruction. Conclusions: Independent of injury severity, hemorrhagic shock, and transfusion, trauma-induced hypocalcemia is associated with early metabolomic and proteomic changes that may reflect unique pathology in hypocalcemic trauma patients. This study paves the way for future experiments to investigate mechanisms, identify intervenable pathways, and refine our management of hypocalcemia in severely injured patients.

Trans-Omics analysis of post injury thrombo-inflammation identifies endotypes and trajectories in trauma patients.

Understanding and managing the complexity of trauma-induced thrombo-inflammation necessitates an innovative, data-driven approach. This study leveraged a trans-omics analysis of longitudinal samples from trauma patients to illuminate molecular endotypes and trajectories that underpin patient outcomes, transcending traditional demographic and physiological characterizations. We hypothesize that trans-omics profiling reveals underlying clinical differences in severely injured patients that may present with similar clinical characteristics but ultimately have very different responses to treatment and clinical outcomes. Here we used proteomics and metabolomics to profile 759 of longitudinal plasma samples from 118 patients at 11 time points and 97 control subjects. Results were used to define distinct patient states through data reduction techniques. The patient groups were stratified based on their shock severity and injury severity score, revealing a spectrum of responses to trauma and treatment that are fundamentally tied to their unique underlying biology. Ensemble models were then employed, demonstrating the predictive power of these molecular signatures with area under the receiver operating curves of 80 to 94% for key outcomes such as INR, ICU-free days, ventilator-free days, acute lung injury, massive transfusion, and death. The molecularly defined endotypes and trajectories provide an unprecedented lens to understand and potentially guide trauma patient management, opening a path towards precision medicine. This strategy presents a transformative framework that aligns with our understanding that trauma patients, despite similar clinical presentations, might harbor vastly different biological responses and outcomes.

Mesenchymal Stem Cells Delivered Locally to Ischemia-Reperfused Kidneys via Injectable Hyaluronic Acid Hydrogels Decrease Extracellular Matrix Remodeling 1 Month after Injury in Male Mice.

The translation of stem cell therapies has been hindered by low cell survival and retention rates. Injectable hydrogels enable the site-specific delivery of therapeutic cargo, including cells, to overcome these challenges. We hypothesized that delivery of mesenchymal stem cells (MSC) via shear-thinning and injectable hyaluronic acid (HA) hydrogels would mitigate renal damage following ischemia-reperfusion acute kidney injury. Acute kidney injury (AKI) was induced in mice by bilateral or unilateral ischemia-reperfusion kidney injury. Three days later, mice were treated with MSCs either suspended in media injected intravenously via the tail vein, or injected under the capsule of the left kidney, or MSCs suspended in HA injected under the capsule of the left kidney. Serial measurements of serum and urine biomarkers of renal function and injury, as well as transcutaneous glomerular filtration rate (tGFR) were performed. In vivo optical imaging showed that MSCs localized to both kidneys in a sustained manner after bilateral ischemia and remained within the ipsilateral treated kidney after unilateral ischemic AKI. One month after injury, MSC/HA treatment significantly reduced urinary NGAL compared to controls; it did not significantly reduce markers of fibrosis compared to untreated controls. An analysis of kidney proteomes revealed decreased extracellular matrix remodeling and high overlap with sham proteomes in MSC/HA-treated animals. Hydrogel-assisted MSC delivery shows promise as a therapeutic treatment following acute kidney injury.

Understanding the Effects of Trenbolone Acetate, Polyamine Precursors, and Polyamines on Proliferation, Protein Synthesis Rates, and the Abundance of Genes Involved in Myoblast Growth, Polyamine Biosynthesis, and Protein Synthesis in Murine Myoblasts.

Research suggests that androgens increase skeletal muscle growth by modulating polyamine biosynthesis. As such, the objective of this study was to investigate effects of anabolic hormones, polyamine precursors, and polyamines relative to proliferation, protein synthesis, and the abundance of mRNA involved in polyamine biosynthesis, proliferation, and protein synthesis in C2C12 and Sol8 cells. Cultures were treated with anabolic hormones (trenbolone acetate and/or estradiol), polyamine precursors (methionine or ornithine), or polyamines (putrescine, spermidine, or spermine). Messenger RNA was isolated 0.5 or 1, 12, or 24 h post-treatment. The cell type had no effect (p > 0.10) on proliferation, protein synthesis, or mRNA abundance at any time point. Each treatment increased (p < 0.01) proliferation, and anabolic hormones increased (p = 0.04) protein synthesis. Polyamines increased (p < 0.05) the abundance of mRNA involved in polyamine biosynthesis, proliferation, and protein synthesis. Treatment with polyamine precursors decreased (p < 0.05) the abundance of mRNA involved in proliferation and protein synthesis. Overall, C2C12 and Sol8 myoblasts do not differ (p > 0.10) in proliferation, protein synthesis, or mRNA abundance at the time points assessed. Furthermore, anabolic hormones, polyamines, and polyamine precursors increase proliferation and protein synthesis, and polyamines and their precursors alter the abundance of mRNA involved in growth.

A proteomic analysis of NETosis in trauma: Emergence of serpinB1 as a key player.

BACKGROUND: Release of neutrophil extracellular traps (NETosis) may mediate postinjury organ dysfunction, but mechanisms remain unclear. The intracellular serine protease inhibitor (serpin) B1 is vital to neutrophil function and has been shown to restrict NETosis in inflammatory settings. In this study, we used discovery proteomics to identify the proteomic signature of trauma-induced NETosis. We hypothesized that serpinB1 would be a major component of this NET protein profile and associated with adverse outcomes. METHODS: This was a post hoc analysis of data collected as part of the COMBAT randomized clinical trial. Blood was collected from injured patients at a single Level I Trauma Center. Proteomic analyses were performed through targeted liquid chromatography coupled with mass spectrometry. Abundances of serpinB1 and known NETosis markers were analyzed with patient and injury characteristics, clinical data, and outcomes. RESULTS: SerpinB1 levels on emergency department (ED) arrival were significantly correlated with proteomic markers of NETosis, including core histones, transketolase, and S100A8/A9 proteins. More severely injured patients had elevated serpinB1 and NETosis markers on ED arrival. Levels of serpinB1 and top NETosis markers were significantly elevated on ED arrival in nonsurvivors and patients with fewer ventilator- and ICU-free days. In proteome-wide receiver operating characteristic analysis, serpinB1 was consistently among the top proteins associated with adverse outcomes. Among NETosis markers, levels of serpinB1 early in the patient's course exhibited the greatest separation between patients with fewer and greater ventilator- and ICU-free days. Gene Ontology analysis of top predictors of adverse outcomes further supports NETosis as a potential mediator of postinjury organ dysfunction. CONCLUSION: We have identified a proteomic signature of trauma-induced NETosis, and NETosis is an early process following severe injury that may mediate organ dysfunction. In addition, serpinB1 is a major component of this NET protein profile that may serve as an early marker of excessive NETosis after injury.

Platelet and cryoprecipitate transfusions from female donors improve coagulopathy in vitro.

BACKGROUND: Females are relatively hypercoagulable compared with males, with increased platelet aggregation and improved clot dynamics. However, sex differences in coagulation have not yet been considered in transfusion guidelines. Therefore, our objective was to evaluate hemostatic differences in sex concordant and sex discordant cryoprecipitate and platelet transfusions. We hypothesized that transfusion of blood products from female donors results in improved coagulopathy compared with male blood products. METHODS: This was a cohort study evaluating sex dimorphisms in coagulation assays and clotting factors in healthy volunteer plasma and cryoprecipitate. Sex dimorphisms in transfusions were evaluated using an in vitro coagulopathy model. Female or male platelets or single-donor cryoprecipitate was added to "recipient" whole blood after dilution of recipient blood with citrated saline to provoke a coagulopathic profile. Citrated native thromboelastography was then performed. Liquid chromatography/mass spectroscopy was performed on single-donor cryoprecipitate to evaluate sex dimorphisms in the proteome of cryoprecipitate. RESULTS: Females have an increased proportion of functional fibrinogen. Transfusion of female-donor platelets and cryoprecipitate induces a larger decrease in R time and greater increase in angle than male-donor platelets or cryoprecipitate. Female-donor cryoprecipitate has increased factor V and factor XIII compared with male cryoprecipitate, and comprehensive proteomics revealed sex differences in several proteins with potential immunological significance. CONCLUSION: Platelets and cryoprecipitate from female donors improve coagulopathy more than male blood products in vitro. Increased factor V and factor XIII activity as well as increased fibrinogen activity in female donors appears to drive this disparity. Sex differences in the proteome of cryoprecipitate may influence how transfusions modulate the thromboinflammation of trauma. The differing hemostatic profiles of female and male blood products suggest the potential role of sex-specific transfusions guidelines in hemostatic resuscitation.

Matrix from urine stem cells boosts tissue-specific stem cell mediated functional cartilage reconstruction.

Articular cartilage has a limited capacity to self-heal once damaged. Tissue-specific stem cells are a solution for cartilage regeneration; however, ex vivo expansion resulting in cell senescence remains a challenge as a large quantity of high-quality tissue-specific stem cells are needed for cartilage regeneration. Our previous report demonstrated that decellularized extracellular matrix (dECM) deposited by human synovium-derived stem cells (SDSCs), adipose-derived stem cells (ADSCs), urine-derived stem cells (UDSCs), or dermal fibroblasts (DFs) provided an ex vivo solution to rejuvenate human SDSCs in proliferation and chondrogenic potential, particularly for dECM deposited by UDSCs. To make the cell-derived dECM (C-dECM) approach applicable clinically, in this study, we evaluated ex vivo rejuvenation of rabbit infrapatellar fat pad-derived stem cells (IPFSCs), an easily accessible alternative for SDSCs, by the abovementioned C-dECMs, in vivo application for functional cartilage repair in a rabbit osteochondral defect model, and potential cellular and molecular mechanisms underlying this rejuvenation. We found that C-dECM rejuvenation promoted rabbit IPFSCs' cartilage engineering and functional regeneration in both ex vivo and in vivo models, particularly for the dECM deposited by UDSCs, which was further confirmed by proteomics data. RNA-Seq analysis indicated that both mesenchymal-epithelial transition (MET) and inflammation-mediated macrophage activation and polarization are potentially involved in the C-dECM-mediated promotion of IPFSCs' chondrogenic capacity, which needs further investigation.

TRAUMA INDUCES INTRAVASCULAR HEMOLYSIS, EXACERBATED BY RED BLOOD CELL TRANSFUSION AND ASSOCIATED WITH DISRUPTED ARGININE-NITRIC OXIDE METABOLISM.

ABSTRACT: Background: Severe injury can provoke systemic processes that lead to organ dysfunction, and hemolysis of both native and transfused red blood cells (RBCs) may contribute. Hemolysis can release erythrocyte proteins, such as hemoglobin and arginase-1, the latter with the potential to disrupt arginine metabolism and limit physiologic NO production. We aimed to quantify hemolysis and arginine metabolism in trauma patients and measure association with injury severity, transfusions, and outcomes. Methods: Blood was collected from injured patients at a level I trauma center enrolled in the COMBAT (Control of Major Bleeding After Trauma) trial. Proteomics and metabolomics were performed on plasma fractions through liquid chromatography coupled with mass spectrometry. Abundances of erythrocyte proteins comprising a hemolytic profile as well as haptoglobin, l -arginine, ornithine, and l -citrulline (NO surrogate marker) were analyzed at different timepoints and correlated with transfusions and adverse outcomes. Results: More critically injured patients, nonsurvivors, and those with longer ventilator requirement had higher levels of hemolysis markers with reduced l -arginine and l -citrulline. In logistic regression, elevated hemolysis markers, reduced l -arginine, and reduced l -citrulline were significantly associated with these adverse outcomes. An increased number of blood transfusions were significantly associated with elevated hemolysis markers and reduced l -arginine and l -citrulline independently of New Injury Severity Score and arterial base excess. Conclusions: Severe injury induces intravascular hemolysis, which may mediate postinjury organ dysfunction. In addition to native RBCs, transfused RBCs can lyse and may exacerbate trauma-induced hemolysis. Arginase-1 released from RBCs may contribute to the depletion of l -arginine and the subsequent reduction in the NO necessary to maintain organ perfusion.

Blockade of Osteoclast-Mediated Bone Resorption With a RANKL-Inhibitor Enhances Bone Formation in a Rat Spinal Fusion Model.

STUDY DESIGN: Rat spine fusion model. OBJECTIVE: The present study aimed to determine whether administration of osteoprotegerin (OPG) in a rat model of spinal fusion increases bone volume, bone density, and decreases osteoclasts in the fusion mass. SUMMARY OF BACKGROUND DATA: OPG is a soluble RANK-ligand inhibitor that blocks osteoclast differentiation and activation. This makes it a potential agent to control the remodeling process and enhance bone mass during spinal fusion. MATERIALS AND METHODS: Forty-eight male Sprague-Dawley rats received a one-level spinal fusion of L4-L5 with bone allograft. Rats were then divided into four groups according to initiation of treatment: (1) saline on day 0 (saline), (2) OPG on day 0 (OPG D0), (3) OPG on day 10 (OPG D10), and (4) OPG on day 21 (OPG D21) postsurgery. After their initial injection, rats received weekly subcutaneous injections of OPG (10 mg/kg) and were euthanized six weeks postsurgery. MicroCT analysis of the fusion site and histological analysis of bone surface for quantification of osteoclast lining was performed. RESULTS: Increased bone volume in the fusion site and around the spinous process was seen in OPG D0 and OPG D10 when compared with saline. Mean trabecular thickness was greater in all groups receiving OPG compared with saline, with OPG D0 and OPG D10 having significantly greater mean trabecular thickness than OPG D21. All OPG groups had less bone surface lined with osteoclasts when compared with Saline, with OPG D0 and OPG D10 having fewer than OPG D21. CONCLUSIONS: This study indicates that OPG inhibited osteoclast bone resorption, which led to greater bone at the fusion site. Future studies investigating OPG on its own or in combination with an osteogenic factor to improve spinal fusion outcomes are warranted to further elucidate its potential therapeutic effect.

Analysis of Physeal Fractures from the United States National Trauma Data Bank.

BACKGROUND: Pediatric long-bone physeal fractures can lead to growth deformities. Previous studies have reported that physeal fractures make up 18-30% of total fractures. This study aimed to characterize physeal fractures with respect to sex, age, anatomic location, and Salter-Harris (SH) classification from a current multicenter national database. METHODS: A retrospective cohort study was performed using the 2016 United States National Trauma Data Bank (NTDB). Patients ≤ 18 years of age with a fracture of the humerus, radius, ulna, femur, tibia, or fibula were included. RESULTS: The NTDB captured 132,018 patients and 58,015 total fractures. Physeal fractures made up 5.7% (3291) of all long-bone fractures, with males accounting for 71.0% (2338). Lower extremity physeal injuries comprised 58.6% (1929) of all physeal fractures. The most common site of physeal injury was the tibia comprising 31.8% (1047), 73.9% (774) of which were distal tibia fractures. Physeal fractures were greatest at 11 years of age for females and 14 years of age for males. Most fractures were SH Type II fractures. DISCUSSION AND CONCLUSIONS: Our analysis indicates that 5.7% of pediatric long-bone fractures involved the physis, with the distal tibia being the most common. These findings suggest a lower incidence of physeal fractures than previous studies and warrant further investigation.

Zone 1 REBOA in a combat DCBI swine model does not worsen brain injury.

BACKGROUND: Zone 1 resuscitative endovascular balloon occlusion of the aorta has been recommended for refractory shock after a dismounted complex blast injury for the austere combat scenario. While resuscitative endovascular balloon occlusion of the aorta should enhance coronary perfusion, there is a potential risk of secondary brain injury due to loss of cerebral autoregulation. We developed a combat casualty relevant dismounted complex blast injury swine model to evaluate the effects of resuscitative endovascular balloon occlusion of the aorta zone I on intracranial pressure and cerebral edema. We hypothesized that zone 1 aortic occlusion with resuscitative endovascular balloon occlusion of the aorta would increase mean arterial pressure transmitted in excessive intracranial pressure, thereby worsening brain injury. METHODS: 50 kg male Yorkshire swine were subjected to a combination dismounted complex blast injury model consisting of blast traumatic brain injury (50 psi, ARA Mobile Shock Laboratory), tissue injury (bilateral femur fractures), and hemorrhagic shock (controlled bleeding to a base deficit goal of 10 mEq/L). During the shock phase, pigs were randomized to no aortic occlusion (n = 8) or to 30 minutes of zone 1 resuscitative endovascular balloon occlusion of the aorta (zone 1 aortic occlusion group, n = 6). After shock, pigs in both groups received a modified Tactical Combat Casualty Care-based resuscitation and were monitored for an additional 240 minutes until euthanasia/death for a total of 6 hours. Intracranial pressure was monitored throughout, and brains were harvested for water content. Linear mixed models for repeated measures were used to compare mean arterial pressure and intracranial pressure between zone 1 aortic occlusion and no aortic occlusion groups. RESULTS: After dismounted complex blast injury, the zone 1 group had a significantly higher mean arterial pressure during hemorrhagic shock compared to the control group (41.2 mm Hg vs 16.7 mm Hg, P = .002). During balloon occlusion, intracranial pressure was not significantly elevated in the zone 1 aortic occlusion group vs control, but intracranial pressure was significantly lower in the zone 1 group at the end of the observation period. In addition, the zone 1 aortic occlusion group did not have increased brain water content (zone 1 aortic occlusion: 3.95 ± 0.1g vs no aortic occlusion: 3.95 ± 0.3 g, P = .87). Troponin levels significantly increased in the no aortic occlusion group but did not in the zone 1 aortic occlusion group. CONCLUSION: Zone 1 aortic occlusion using resuscitative endovascular balloon occlusion of the aorta in a large animal dismounted complex blast injury model improved proximal mean arterial pressure while not significantly increasing intracranial pressure during balloon inflation. Observation up to 240 minutes postresuscitation did not show clinical signs of worsening brain injury or cardiac injury. These data suggest that in a dismounted complex blast injury swine model, resuscitative endovascular balloon occlusion of the aorta in zone 1 may provide neuro- and cardioprotection in the setting of blast traumatic brain injury. However, longer monitoring periods may be needed to confirm that the neuroprotection is lasting.

Fabrication of Size-Controlled and Emulsion-Free Chitosan-Genipin Microgels for Tissue Engineering Applications.

Chitosan microgels are of significant interest in tissue engineering due to their wide range of applications, low cost, and immunogenicity. However, chitosan microgels are commonly fabricated using emulsion methods that require organic solvent rinses, which are toxic and harmful to the environment. The present protocol presents a rapid, non-cytotoxic, non-emulsion-based method for fabricating chitosan-genipin microgels without the need for organic solvent rinses. The microgels described herein can be fabricated with precise size control. They exhibit sustained release of biomolecules, making them highly relevant for tissue engineering, biomaterials, and regenerative medicine. Chitosan is crosslinked with genipin to form a hydrogel network, then passed through a syringe filter to produce the microgels. The microgels can be filtered to create a range of sizes, and they show pH-dependent swelling and degrade over time enzymatically. These microgels have been employed in a rat growth plate injury model and were demonstrated to promote increased cartilage tissue repair and to show complete degradation at 28 days in vivo. Due to their low cost, high convenience, and ease of fabrication with cytocompatible materials, these chitosan microgels present an exciting and unique technology in tissue engineering.