Design and Development of a Clot Burst Pressure Device to Investigate Resuscitation Strategies.

INTRODUCTION: A reduction in clot strength is a hallmark feature of trauma-induced coagulopathy. A better understanding of clot integrity can optimize resuscitation strategies. We designed a device to gauge clot strength by pressurizing fluids over a formed clot and measuring the pressure needed to dislodge the clot. We hypothesized that this device could distinguish between clots formed in hypocoagulable and hypercoagulable states by observing differences in the clot burst pressure. METHODS: Whole blood from healthy volunteers was collected into sodium citrate tubes and was treated with heparin or fibrinogen to generate clots in a hypocoagulable or hypercoagulable state, respectively. Small bore holes were drilled into polystyrene plates, and recalcified blood was pipetted into the holes. Plates were incubated at 37°C for 30 min to form clots. A pressure cap with an inlet for fluid from a syringe pump and an outlet leading to a measurement column was secured in the wells with a watertight seal. RESULTS: Clot burst pressure was normalized to individual baseline values to account for inherent differences in clot strength. The 1.0 g/L and 2.0 g/L fibrinogen groups were 1.65 ± 0.07 (P = 0.0078) and 2.26 ± 0.16 (P = 0.0078) times as strong as baseline, respectively. The 0.10, 0.15, or 0.20 USP units/mL groups were 0.388 ± 0.07 (P = 0.125), 0.31 ± 0.07 (P = 0.125), 0.21 ± 0.07 (P = 0.125) times as strong as baseline, respectively. Data were analyzed using Wilcoxon matched pairs signed rank testing. CONCLUSIONS: This device tests clot strength using burst pressure, an easily interpreted clinical parameter not measured in existing devices. Future work can test blood from trauma patients to better understand trauma pathophysiology.

Validation and characterization of a novel blood-brain barrier platform for investigating traumatic brain injury.

The Blood-Brain Barrier (BBB) is a highly-selective physiologic barrier responsible for maintaining cerebral homeostasis. Innovative in vitro models of the BBB are needed to provide useful insights into BBB function with CNS disorders like traumatic brain injury (TBI). TBI is a multidimensional and highly complex pathophysiological condition that requires intrinsic models to elucidate its mechanisms. Current models either lack fluidic shear stress, or neglect hemodynamic parameters important in recapitulating the human in vivo BBB phenotype. To address these limitations in the field, we developed a fluid dynamic novel platform which closely mimics these parameters. To validate our platform, Matrigel-coated Transwells were seeded with brain microvascular endothelial cells, both with and without co-cultured primary human astrocytes and bone-marrow mesenchymal stem cells. In this article we characterized BBB functional properties such as TEER and paracellular permeability. Our platform demonstrated physiologic relevant decreases in TEER in response to an ischemic environment, while directly measuring barrier fluid fluctuation. These recordings were followed with recovery, implying stability of the model. We also demonstrate that our dynamic platform is responsive to inflammatory and metabolic cues with resultant permeability coefficients. These results indicate that this novel dynamic platform will be a valuable tool for evaluating the recapitulating BBB function in vitro, screening potential novel therapeutics, and establishing a relevant paradigm to evaluate the pathophysiology of TBI.

PET imaging of microglia using PBR28suv determines therapeutic efficacy of autologous bone marrow mononuclear cells therapy in traumatic brain injury.

Traumatic brain injury (TBI) results in activated microglia. Activated microglia can be measured in vivo by using positron emission topography (PET) ligand peripheral benzodiazepine receptor standardized uptake values (PBR28suv). Cell based therapies have utilized autologous bone marrow mononuclear cells (BMMNCs) to attenuate activated microglia after TBI. This study aims to utilize in vivo PBR28suv to assess the efficacy of BMMNCs therapy after TBI. Seventy-two hours after CCI injury, BMMNCs were harvested from the tibia and injected via tail-vein at 74 h after injury at a concentration of 2 million cells per kilogram of body weight. There were three groups of rats: Sham, CCI-alone and CCI-BMMNCs (AUTO). One hundred twenty days after injury, rodents were imaged with PBR28 and their cognitive behavior assessed utilizing the Morris Water Maze. Subsequent ex vivo analysis included brain volume and immunohistochemistry. BMMNCs therapy attenuated PBR28suv in comparison to CCI alone and it improved spatial learning as measured by the Morris Water Maze. Ex vivo analysis demonstrated preservation of brain volume, a decrease in amoeboid-shaped microglia in the dentate gyrus and an increase in the ratio of ramified to amoeboid microglia in the thalamus. PBR28suv is a viable option to measure efficacy of BMMNCs therapy after TBI.

Enhancing Mesenchymal Stromal Cell Potency: Inflammatory Licensing via Mechanotransduction.

Mesenchymal stromal cells (MSC) undergo functional maturation upon their migration from bone marrow and introduction to a site of injury. This inflammatory licensing leads to heightened immune regulation via cell-to-cell interaction and the secretion of immunomodulatory molecules, such as anti-inflammatory mediators and antioxidants. Pro-inflammatory cytokines are a recognized catalyst of inflammatory licensing; however, biomechanical forces, such as fluid shear stress, are a second, distinct class of stimuli that incite functional maturation. Here we show mechanotransduction, achieved by exposing MSC to various grades of wall shear stress (WSS) within a scalable conditioning platform, enhances the immunomodulatory potential of MSC independent of classical pro-inflammatory cytokines. A dose-dependent effect of WSS on potency is evidenced by production of prostaglandin E2 (PGE2) and indoleamine 2,3 dioxygenase 1 (IDO1), as well as suppression of tumor necrosis factor-α (TNF- α) and interferon-γ (IFN-γ) production by activated immune cells. Consistent, reproducible licensing is demonstrated in adipose tissue and bone marrow human derived MSC without significant impact on cell viability, cellular yield, or identity. Transcriptome analysis of WSS-conditioned BM-MSC elucidates the broader phenotypic implications on the differential expression of immunomodulatory factors. These results suggest mechanotransduction as a viable, scalable pre-conditioning alternative to pro-inflammatory cytokines. Enhancing the immunomodulatory capacity of MSC via biomechanical conditioning represents a novel cell therapy manufacturing approach.

3D printing in aortic endovascular therapies.

Endovascular treatment of aortic disease, including aneurysm or dissection, is expanding at a rapid pace. Often, the specific patient anatomy in these cases is complex. Additive manufacturing, also known as three-dimensional (3D) printing, is especially useful in the treatment of aortic disease, due to its ability to manufacture physical models of complex patient anatomy. Compared to other surgical procedures, endovascular aortic repair can readily exploit the advantages of 3D printing with regard to operative planning and preoperative training. To date, there have been numerous uses of 3D printing in the treatment of aortic pathology as an adjunct in presurgical planning and as a basis for training modules for fellows and residents. In this review, we summarize the current uses of 3D printing in the endovascular management of aortic disease. We also review the process of producing these models, the limitations of their applications, and future directions of 3D printing in this field.

Bone marrow stromal cell therapy improves survival after radiation injury but does not restore endogenous hematopoiesis.

The only available option to treat radiation-induced hematopoietic syndrome is allogeneic hematopoietic cell transplantation, a therapy unavailable to many patients undergoing treatment for malignancy, which would also be infeasible in a radiological disaster. Stromal cells serve as critical components of the hematopoietic stem cell niche and are thought to protect hematopoietic cells under stress. Prior studies that have transplanted mesenchymal stromal cells (MSCs) without co-administration of a hematopoietic graft have shown underwhelming rescue of endogenous hematopoiesis and have delivered the cells within 24 h of radiation exposure. Herein, we examine the efficacy of a human bone marrow-derived MSC therapy delivered at 3 h or 30 h in ameliorating radiation-induced hematopoietic syndrome and show that pancytopenia persists despite MSC therapy. Animals exposed to radiation had poorer survival and experienced loss of leukocytes, platelets, and red blood cells. Importantly, mice that received a therapeutic dose of MSCs were significantly less likely to die but experienced equivalent collapse of the hematopoietic system. The cause of the improved survival was unclear, as complete blood counts, splenic and marrow cellularity, numbers and function of hematopoietic stem and progenitor cells, and frequency of niche cells were not significantly improved by MSC therapy. Moreover, human MSCs were not detected in the bone marrow. MSC therapy reduced crypt dropout in the small intestine and promoted elevated expression of growth factors with established roles in gut development and regeneration, including PDGF-A, IGFBP-3, IGFBP-2, and IGF-1. We conclude that MSC therapy improves survival not through overt hematopoietic rescue but by positive impact on other radiosensitive tissues, such as the intestinal mucosa. Collectively, these data reveal that MSCs could be an effective countermeasure in cancer patients and victims of nuclear accidents but that MSCs alone do not significantly accelerate or contribute to recovery of the blood system.

Injury intensifies T cell mediated graft-versus-host disease in a humanized model of traumatic brain injury.

The immune system plays critical roles in promoting tissue repair during recovery from neurotrauma but is also responsible for unchecked inflammation that causes neuronal cell death, systemic stress, and lethal immunodepression. Understanding the immune response to neurotrauma is an urgent priority, yet current models of traumatic brain injury (TBI) inadequately recapitulate the human immune response. Here, we report the first description of a humanized model of TBI and show that TBI places significant stress on the bone marrow. Hematopoietic cells of the marrow are regionally decimated, with evidence pointing to exacerbation of underlying graft-versus-host disease (GVHD) linked to presence of human T cells in the marrow. Despite complexities of the humanized mouse, marrow aplasia caused by TBI could be alleviated by cell therapy with human bone marrow mesenchymal stromal cells (MSCs). We conclude that MSCs could be used to ameliorate syndromes triggered by hypercytokinemia in settings of secondary inflammatory stimulus that upset marrow homeostasis such as TBI. More broadly, this study highlights the importance of understanding how underlying immune disorders including immunodepression, autoimmunity, and GVHD might be intensified by injury.

A Useful Device to Measure Kinetics of Platelet Contraction.

Platelet contraction provides a minimally invasive source for physiologic information. In this article, we describe a device that directly measures the kinetics of platelet contraction. Whole blood is injected between acrylic plates and an adherent clot forms. The bottom plate is fixed, and the top plate is attached to a wire cantilever. Platelet contraction drives deflection of the wire cantilever which is captured by a camera. Force generated by the clot with time is derived using beam equations. Force derivations were verified using a microelectromechanical (MEMS) force sensor. Kinetics of clot contraction were defined, including maximum contraction force (FMAX), lift-off time (TLIFTOFF), and contraction rate (CR). Metrics were compared with optical aggregometry and thromboelastography. FMAX correlates with optical aggregometry maximal amplitude with a Spearman's rho of 0.7904 and p = 0.0195 and thromboelastography maximal amplitude with a Spearman's rho of 0.8857 and p = 0.0188. Lift-off time correlates with optical aggregometry lag time with a Spearman's rho of 0.9048 and p = 0.002. This preliminary study demonstrates the repeatability of a useful platelet contraction device and its correlation with thromboelastography and optical aggregometry, the gold standard platelet function test.