Microelectromechanical System Measurement of Platelet Contraction: Direct Interrogation of Myosin Light Chain Phosphorylation.

Myosin Light Chain (MLC) regulates platelet contraction through its phosphorylation by Myosin Light Chain Kinase (MLCK) or dephosphorylation by Myosin Light Chain Phosphatase (MLCP). The correlation between platelet contraction force and levels of MLC phosphorylation is unknown. We investigate the relationship between platelet contraction force and MLC phosphorylation using a novel microelectromechanical (MEMS) based clot contraction sensor (CCS). The MLCK and MLCP pair were interrogated by inhibitors and activators of platelet function. The CCS was fabricated from silicon using photolithography techniques and force was validated over a range of deflection for different chip spring constants. The force of platelet contraction measured by the clot contraction sensor (CCS) was compared to the degree of MLC phosphorylation by Western Blotting (WB) and ELISA. Stimulators of MLC phosphorylation produced higher contraction force, higher phosphorylated MLC signal in ELISA and higher intensity bands in WB. Inhibitors of MLC phosphorylation produced the opposite. Contraction force is linearly related to levels of phosphorylated MLC. Direct measurements of clot contractile force are possible using a MEMS sensor platform and correlate linearly with the degree of MLC phosphorylation during coagulation. Measured force represents the mechanical output of the actin/myosin motor in platelets regulated by myosin light chain phosphorylation.

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 Novel Platelet Function Assay for Trauma.

BACKGROUND: Platelet function tests such as thrombelastography platelet mapping and impedance aggregometry have demonstrated universal platelet dysfunction in trauma patients. In this study, we introduce the measurement of platelet contraction force as a test of platelet function. We hypothesize that force will correlate with established coagulation tests such as thrombelastography, demonstrate significant differences between healthy subjects and trauma patients, and identify critically ill trauma patients. METHODS: Blood samples were prospectively collected from level 1 trauma patients at initial presentation, assayed for force of and time to contraction and compared with thrombelastography. Blood from healthy subjects was assayed to establish a reference range. Results from trauma patients were compared with healthy controls and trauma patients that died. RESULTS: The study includes one hundred trauma patients with mean age 45 y, 74% were male, and median injury severity score of 14 ± 12. Patients that survived (n = 90) demonstrated significantly elevated platelet contraction force compared with healthy controls (n = 12) (6390 ± 2340 versus 4790 ± 470 µN, P = 0.043) and trauma patients that died (n = 10) (6390 ± 2340 versus 2860 ± 1830 µN, P = 0.0001). Elapsed time to start of platelet contraction was faster in trauma patients that survived compared with healthy controls (660 ± 467 versus 1130 ± 140 s, P = 0.0022) and those that died (660 ± 470 versus 1460 ± 1340 s, P < 0.0001). CONCLUSIONS: In contrast with all existing platelet function tests reported in the literature, which report platelet dysfunction in trauma patients, contractile force demonstrates hyperfunction in surviving trauma patients and dysfunction in nonsurvivors. Platelet contraction reflects platelet metabolic reserve and thus may be a potential biomarker for survival after trauma. Contractile force warrants further investigation to predict mortality in severely injured trauma patients.

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.

Biomechanical Forces Promote Immune Regulatory Function of Bone Marrow Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSCs) are believed to mobilize from the bone marrow in response to inflammation and injury, yet the effects of egress into the vasculature on MSC function are largely unknown. Here we show that wall shear stress (WSS) typical of fluid frictional forces present on the vascular lumen stimulates antioxidant and anti-inflammatory mediators, as well as chemokines capable of immune cell recruitment. WSS specifically promotes signaling through NFκB-COX2-prostaglandin E2 (PGE2 ) to suppress tumor necrosis factor-α (TNF-α) production by activated immune cells. Ex vivo conditioning of MSCs by WSS improved therapeutic efficacy in a rat model of traumatic brain injury, as evidenced by decreased apoptotic and M1-type activated microglia in the hippocampus. These results demonstrate that force provides critical cues to MSCs residing at the vascular interface which influence immunomodulatory and paracrine activity, and suggest the potential therapeutic use of force for MSC functional enhancement. Stem Cells 2017;35:1259-1272.

3D Printed Surgical Instruments: The Design and Fabrication Process.

BACKGROUND: 3D printing is an additive manufacturing process allowing the creation of solid objects directly from a digital file. We believe recent advances in additive manufacturing may be applicable to surgical instrument design. This study investigates the feasibility, design and fabrication process of usable 3D printed surgical instruments. METHODS: The computer-aided design package SolidWorks (Dassault Systemes SolidWorks Corp., Waltham MA) was used to design a surgical set including hemostats, needle driver, scalpel handle, retractors and forceps. These designs were then printed on a selective laser sintering (SLS) Sinterstation HiQ (3D Systems, Rock Hill SC) using DuraForm EX plastic. The final printed products were evaluated by practicing general surgeons for ergonomic functionality and performance, this included simulated surgery and inguinal hernia repairs on human cadavers. Improvements were identified and addressed by adjusting design and build metrics. RESULTS: Repeated manufacturing processes and redesigns led to the creation of multiple functional and fully reproducible surgical sets utilizing the user feedback of surgeons. Iterative cycles including design, production and testing took an average of 3 days. Each surgical set was built using the SLS Sinterstation HiQ with an average build time of 6 h per set. CONCLUSIONS: Functional 3D printed surgical instruments are feasible. Advantages compared to traditional manufacturing methods include no increase in cost for increased complexity, accelerated design to production times and surgeon specific modifications.

Intravenous multipotent adult progenitor cell therapy attenuates activated microglial/macrophage response and improves spatial learning after traumatic brain injury.

We previously demonstrated that the intravenous delivery of multipotent adult progenitor cells (MAPCs) after traumatic brain injury (TBI) in rodents provides neuroprotection by preserving the blood-brain barrier and systemically attenuating inflammation in the acute time frame following cell treatment; however, the long-term behavioral and anti-inflammatory effects of MAPC administration after TBI have yet to be explored. We hypothesized that the intravenous injection of MAPCs after TBI attenuates the inflammatory response (as measured by microglial morphology) and improves performance at motor tasks and spatial learning (Morris water maze [MWM]). MAPCs were administered intravenously 2 and 24 hours after a cortical contusion injury (CCI). We tested four groups at 120 days after TBI: sham (uninjured), injured but not treated (CCI), and injured and treated with one of two concentrations of MAPCs, either 2 million cells per kilogram (CCI-2) or 10 million cells per kilogram (CCI-10). CCI-10 rats showed significant improvement in left hind limb deficit on the balance beam. On the fifth day of MWM trials, CCI-10 animals showed a significant decrease in both latency to platform and distance traveled compared with CCI. Probe trials revealed a significant decrease in proximity measure in CCI-10 compared with CCI, suggesting improved memory retrieval. Neuroinflammation was quantified by enumerating activated microglia in the ipsilateral hippocampus. We observed a significant decrease in the number of activated microglia in the dentate gyrus in CCI-10 compared with CCI. Our results demonstrate that intravenous MAPC treatment after TBI in a rodent model offers long-term improvements in spatial learning as well as attenuation of neuroinflammation.

Flexible thin-film PVDF-TrFE based pressure sensor for smart catheter applications.

We demonstrate the design of thin flexible pressure sensors based on piezoelectric PVDF-TrFE (polyvinyledenedifluoride-tetrafluoroethylene) co-polymer film, which can be integrated onto a catheter, where the compact inner lumen space limit the dimensions of the pressure sensors. Previously, we demonstrated that the thin-film sensors of one micrometer thickness were shown to have better performance compared to the thicker film with no additional electrical poling or mechanical stretching due to higher crystallinity. The pressure sensors can be mass producible using standard lithography process, with excellent control of film uniformity and thickness down to one micrometer. The fabricated pressure sensors were easily mountable on external surface of commercial catheters. Elaborate experiments were performed to demonstrate the applicability of PVDF sensors towards catheter based biomedical application. The resonant frequency of the PVDF sensor was found to be 6.34 MHz. The PVDF sensors can operate over a broad pressure range of 0-300 mmHg. The average sensitivity of the PVDF sensor was found to be four times higher (99 µV/mmHg) than commercial pressure sensor while the PVDF sensor (0.26 s) had fivefold shorter response time than commercial pressure sensor (1.30 s), making the PVDF sensors highly suitable for real-time pressure measurements using catheters.

Bone marrow-derived stromal cell therapy for traumatic brain injury is neuroprotective via stimulation of non-neurologic organ systems.

Bone marrow-derived mesenchymal stromal cells (MSCs) used as "MSC therapy" after traumatic brain injury act as remote "bioreactors" via stimulation of lung macrophages and augmention of T regulatory cell production by the spleen, leading to systemic increases in circulating anti-inflammatory cytokines and alteration of the locoregional milieu of the central nervous system. The altered intracerebral microenvironment leads to modulation of the resident microglia population, thereby stimulating an increase in the ratio of M2 (anti-inflammatory) macrophage to M1 (proinflammatory) macrophage, and it is this effect that accounts for the observed neuroprotection.

Effects of nonocclusive mesenteric hypertension on intestinal function: implications for gastroschisis-related intestinal dysfunction.

INTRODUCTION: Infants with gastroschisis (GS) have significant morbidity from dysmotility, feeding intolerance, and are at increased risk of developing intestinal failure. Although the molecular mechanisms regulating GS-related intestinal dysfunction (GRID) are largely unknown, we hypothesized that mechanical constriction (nonocclusive mesenteric hypertension (NMH)) from the abdominal wall defect acts as a stimulus for GRID. The purpose of this study was to determine the effect of NMH on intestinal function and inflammation. METHODS: Neonatal rats had placement of a silastic disk to the base of the mesentery (NMH) or no disk placement (Sham). At 24 and 72 h, mesenteric venous pressures (MVPs), intestinal transit, electric impedance, permeability, length, and tissue water content were measured. RESULTS: After placement of the silastic disk, there was a significant increase in MVP at both time points. There was also decreased intestinal transit. As compared to Sham animals, NMH animals had significant changes in bowel impedance without an increase in tissue water, suggesting significant intestinal remodeling. NMH rats had significantly increased smooth-muscle thickness and loss of intestinal length as compared with Sham rats. DISCUSSION: NMH may be an initiating factor for GRID. Measurement of MVP and/or bowel impedance may be a way to assess severity and monitor progression and/or resolution of GRID.

A novel mechanism for neutrophil priming in trauma: potential role of peritoneal fluid.

BACKGROUND: We sought to determine the effect of peritoneal fluid from a novel animal model of abdominal compartment syndrome (ACS) on the proinflammatory status of polymorphonuclear leukocytes (PMNs) and monocytes. We hypothesize that peritoneal fluid is a potential priming and/or activating agent for PMNs/monocytes. METHODS: ACS was induced in female Yorkshire swine, and peritoneal fluid was collected at the time of decompressive laparotomy. Naïve PMNs/monocytes were primed and/or activated with peritoneal fluid, phosphatidylcholine (PAF) plus peritoneal fluid, peritoneal fluid plus n-formyl-met-leu-phe (fMLP), and peritoneal fluid plus phorbol 12-myristate 13-acetate (PMA). Activation was determined by surface marker expression of integrins (CD11b an CD18) and selectins (CD62L). Additionally, proinflammatory cytokines in peritoneal fluid were analyzed. RESULTS: Peritoneal fluid did not activate PMNs but increased CD11b expression on monocytes. When used as a primer for fMLP- or PMA-induced activation, peritoneal fluid significantly increased CD11b and CD18 expression on PMNs and monocytes. Peritoneal fluid collected at 6 and 12 h post decompressive laparotomy had similar effects. Interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) levels were increased in peritoneal fluid. CONCLUSION: Peritoneal fluid represents a primer for PMNs/monocytes and seems to act through receptor-dependent and receptor-independent pathways. Strategies to reduce the amount of peritoneal fluid may decrease the locoregional and systemic inflammatory response by reducing priming and activation of neutrophils/monocytes.

A novel physiologic model for the study of abdominal compartment syndrome (ACS).

BACKGROUND: : Current abdominal compartment syndrome (ACS) models rely on intraperitoneal instillation of fluid, air, and other space-occupying substances. Although this allows for the study of the effects of increased abdominal pressure, it poorly mimics its pathogenesis. We have developed the first reported large animal model of ACS incorporating hemorrhagic shock/resuscitation. METHODS: : Hemorrhagic shock was induced and maintained (1 hour) in 12 Yorkshire swine by bleeding to a mean arterial pressure (MAP) of 50 mm Hg. The collected blood plus two additional volumes of crystalloid was then reinfused. Mesenteric venous hypertension was induced by tightening a previously placed portal vein snare in a nonocclusive manner to mimic the effects of abdominal packing. Crystalloids were infused to maintain MAP. Hemodynamic measurements, abdominal pressure, peak inspiratory pressures, urine output, and blood chemistries were measured sequentially. Animals were studied for 36 hours after decompression. RESULTS: : ACS (intra-abdominal pressure of > or =20 mm Hg with new organ dysfunction) developed in all animals. There were significant increases in peak inspiratory pressure, central venous pressure, and pulmonary artery pressure and decreases in MAP upon development of ACS. Urine output was significantly decreased before decompression. Mean blood lactate decreased and base excess increased significantly after decompression. CONCLUSIONS: : We have created the first reported physiologic animal ACS model incorporating hemorrhagic shock/resuscitation and the effects of damage control surgery.

Hydrostatic intestinal edema induced signaling pathways: potential role of mechanical forces.

BACKGROUND: Hydrostatic intestinal edema initiates a signal transduction cascade that results in smooth muscle contractile dysfunction. Given the rapid and concurrent alterations in the mechanical properties of edematous intestine observed with the development of edema, we hypothesize that mechanical forces may serve as a stimulus for the activation of certain signaling cascades. We sought to examine whether isolated similar magnitude mechanical forces induced the same signal transduction cascades associated with edema. METHODS: The distal intestine from adult male Sprague Dawley rats was stretched longitudinally for 2 h to 123% its original length, which correlates with the interstitial stress found with edema. We compared wet-to-dry ratios, myeloperoxidase activity, nuclear signal transduction and activator of transcription (STAT)-3 and nuclear factor (NF)-kappa B DNA binding, STAT-3 phosphorylation, myosin light chain phosphorylation, baseline and maximally stimulated intestinal contractile strength, and inducible nitric oxide synthase (iNOS) and sodium hydrogen exchanger 1-3 messenger RNA (mRNA) in stretched and adjacent control segments of intestine. RESULTS: Mechanical stretch did not induce intestinal edema or an increase in myeloperoxidase activity. Nuclear STAT-3 DNA binding, STAT-3 phosphorylation, and nuclear NF-kappa B DNA binding were significantly increased in stretched seromuscular samples. Increased expression of sodium hydrogen exchanger 1 was found but not an increase in iNOS expression. Myosin light chain phosphorylation was significantly decreased in stretched intestine as was baseline and maximally stimulated intestinal contractile strength. CONCLUSION: Intestinal stretch, in the absence of edema/inflammatory/ischemic changes, leads to the activation of signaling pathways known to be altered in intestinal edema. Edema may initiate a mechanotransductive cascade that is responsible for the subsequent activation of various signaling cascades known to induce contractile dysfunction.

Effect of needle diameter and flow rate on rat and human mesenchymal stromal cell characterization and viability.

INTRODUCTION: Current mesenchymal stromal cell (MSC) delivery methods require infusion/implantation through needles and/or catheters. Little investigation into the effect of delivery via catheter injection has been completed. We hypothesize that injection of rat and human MSCs through various clinically relevant-sized catheters and flow rates will not affect cell viability, characterization, or function. METHODS: Both rat and human MSCs were injected through 20-, 25-, and 30-gauge needles, as well through an SL-10 microcatheter at rates of 60, 120, 240, and 500 mL/h. MSC viability and apoptotic fraction was measured. MSCs were characterized 24 h after injection with flow cytometric immunophenotyping, and multilineage differentiation was completed. RESULTS: Catheter diameter or flow rate did not affect rat MSC viability. No clinically significant decrease in human MSC viability was observed immediately after injection; however, a delayed decrease in viability was observed at 24 h. No difference in the surface markers CD11b, CD45, CD29, CD49e, CD73, CD90, CD105, and Stro-1 or the capacity for multilineage differentiation (adipogenesis, osteogenesis, and chondrogenesis) was observed for either rat or human MSCs. CONCLUSION: The injection of human and rat MSCs through various clinically relevant catheters and flow rates did not have a clinically significant effect on viability immediately after injection, indicating compliance with recently published Food and Drug Administration guidelines (viability >70%). Further, no changes in cell characterization or function were observed via measurement of cell surface markers and the capacity for multilineage differentiation, respectively. These results ensure the biocompatibility of MSCs with commonly used delivery methods.

Regional differences in cerebral edema after traumatic brain injury identified by impedance analysis.

OBJECTIVE: Cerebral edema is a common and potentially devastating sequel of traumatic brain injury. We developed and validated a system capable of tissue impedance analysis, which was found to correlate with cerebral edema. METHODS: Constant sinusoidal current (50 microA), at frequencies from 500 to 5000 Hz, was applied across a bipolar electrode unit superficially placed in a rat brain after traumatic brain injury. Rats were randomized to three groups: severe controlled cortical injury (CCI), mild CCI, or sham injury. At 60 h post-CCI, cerebral voltage and phase angle were measured at each frequency at the site of injury, at the penumbral region, at the ipsilateral frontal region, and in the contralateral hemisphere. Impedance measurements were also obtained in vivo. The electrical properties of varied injuries and specified locations were compared using a repeated measures analysis of variance (RMANOVA), were correlated with regional tissue water percentage using regression analyses, and were combined to generate polar coordinates. RESULTS: The measured voltage was significantly different at the site of injury (P<0.0001), in the penumbra (P=0.002), and in the contralateral hemisphere (P=0.005) when severe, mild, and sham CCI rats were compared. Severely injured rats had statistically different voltage measurements when the various sites were compared (P=0.002). The ex vivo measurements correlated with in vivo measurements. Further, the impedance measurements correlated with measured tissue water percentage at the site of injury (R2=0.69; P<0.0001). The creation of a polar coordinate graph, incorporating voltage and phase angle measurements, enabled the identification of impedance areas unique to normal, mild edema, and severe edema measurements in the rat brain. CONCLUSIONS: Electrical measurements and tissue water percentages quantified regional and severity differences in rat brain edema after CCI. Impedance was inversely proportional to the tissue water percentage. Thus, impedance measurement can be used to quantify severity of cerebral edema in real time at specific sites.

Advances in progenitor cell therapy using scaffolding constructs for central nervous system injury.

Traumatic brain injury (TBI) is a major cause of morbidity and mortality in the United States. Current clinical therapy is focused on optimization of the acute/subacute intracerebral milieu, minimizing continued cell death, and subsequent intense rehabilitation to ameliorate the prolonged physical, cognitive, and psychosocial deficits that result from TBI. Adult progenitor (stem) cell therapies have shown promise in pre-clinical studies and remain a focus of intense scientific investigation. One of the fundamental challenges to successful translation of the large body of pre-clinical work is the delivery of progenitor cells to the target location/organ. Classically used vehicles such as intravenous and intra arterial infusion have shown low engraftment rates and risk of distal emboli. Novel delivery methods such as nanofiber scaffold implantation could provide the structural and nutritive support required for progenitor cell proliferation, engraftment, and differentiation. The focus of this review is to explore the current state of the art as it relates to current and novel progenitor cell delivery methods.

Bioimpedance analysis: a guide to simple design and implementation.

BACKGROUND: Bioimpedance analysis has found utility in many fields of medical research, yet instrumentation can be expensive and/or complicated to build. Advancements in electronic component design and equipment allow for simple bioimpedance analysis using equipment now commonly found in an engineering lab, combined with a few components exclusive to impedance analysis. MATERIALS AND METHODS: A modified Howland bridge circuit was designed on a small circuit board with connections for power and bioimpedance probes. A programmable function generator and an oscilloscope were connected to a laptop computer and were tasked to drive and receive data from the circuit. The software then parsed the received data and inserted it into a spreadsheet for subsequent data analysis. The circuit was validated by testing its current output over a range of frequencies and comparing measured values of impedance across a test circuit to expected values. RESULTS: The system was validated over frequencies between 1 and 100 kHz. Maximum fluctuation in current was on the order of micro-Amperes. Similarly, the measured value of impedance in a test circuit followed the pattern of actual impedance over the range of frequencies measured. CONCLUSIONS: Contemporary generation electronic measurement equipment provides adequate levels of connectivity and programmability to rapidly measure and record data for bioimpedance research. These components allow for the rapid development of a simple but accurate bioimpedance measurement system that can be assembled by individuals with limited knowledge of electronics or programming.

Portable arteriovenous rewarming for hypothermia: cardiovascular considerations.

In trauma patients, continuous arteriovenous (AV) rewarming can effectively reverse hypothermia even if associated with hypovolemia. In battlefield conditions, however, portable fluid warmers driven by battery power show limited capacities. We studied the efficacy and safety of a portable fluid warmer that utilizes controlled hydrocarbon combustion (nonflame) for heat generation during continuous AV rewarming in a large animal model of hypothermia and hemorrhagic shock. Six dogs (26.1 +/- 0.8 kg) were cooled to a core temperature of 30 degrees C (hypo 1). After rewarming to 37 degrees C, dogs were bled by 20% of their estimated blood volume and cooled again to 30 degrees C (hypo 2) followed by rewarming. We recorded temperature (blood, esophageal, rectal, and bladder), left ventricular performance, hemodynamic parameters including superior mesenteric artery (SMA) flow and blood flow through the fluid warmer. Especially, we measured the effect of the AV-shunt on cardiac output and regional blood flow (superior mesenteric artery). Rewarming after hypothermia took 45 +/- 6 minutes (hypothermia 1) and 55 +/- 6 minutes (hypothermia 2), respectively. The AV-shunt flow was correlated to the cardiac output and affected neither cardiac output nor regional blood flow at any time point during the experiment. Arteriovenous rewarming, using the tested portable fluid warmer, effectively reversed hypothermia without compromising hemodynamics or regional blood flow.