Shear effect on material induced blood cell activation

Restenosis that occurs after stent implantation is a great threat to the quality of life of patients with coronary artery diseases. Studies on thrombosis have revealed that platelet is a primary component of blood clots, and leukocytes and other plasma proteins also contribute to thrombus formation. However, the mechanism of material-induced activation, especially with metallic materials, is currently not well understood and the effect of blood flow on material-induced blood cell activation has not been well characterized. In vitro static and flow experiments were performed to assess the effect of flow on blood cell activation. Blood was taken from healthy donors. Two common stent metals, ST316L and TiAl6V4, were used as test materials. In static experiments, blood was incubated with metal disks at 37 ℃ for two hours. In flow experiments, blood was circulated in flow chambers preloaded with or without metal wires at shear rates of 100 s-1, 500 s-1, and 1500 s-1. Platelet and leukocyte activation, leukocyte-platelet aggregation, and tissue factor expression on monocytes were measured by using a three-color FACSCalibur flow cytometer. Fibrin deposition and blood cell adhesion on metal surfaces were evaluated by scanning electron microscopy (SEM). The results indicate that platelet and leukocyte activation under static condition was low. TiAl6V4 metal disk induced relative lower platelet and leukocyte activation, albeit no significant difference was found between control groups and the two metals tested. Shear stress significantly enhanced platelet activation as measured by an increase in platelet microparticle formation and platelet receptor CD61 expression. CD11b up-regulation, leukocyte-platelet aggregates, and tissue factor (TF) expression increased at 100 s-1. However as shear rate increased, lower leukocyte activation was observed. TiAl6V4-induced leukocyte activation was generally lower than that of ST316L. Significant fibrin deposition and blood cell adhesion were observed on metal surfaces under static condition and with shear rate of 500 s-1. Adhesion significantly decreased with increasing shear rate to 1500 s-1. The flow cytometry analysis results and SEM images demonstrated that materials with different surface properties can induce different blood cell activation and adhesion after incubation with blood. While shear may have an effect on blood cell activation, the effect of biomaterials on cell activation under physiological shear conditions remains unclear. Further studies are required to determine if an increase in biomaterial surface area will provide some answers into the mechanisms of material-induced blood activation in the presence of flow

Thrombotic fingerprints for the enhanced prediction of thrombosis

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2007.Includes bibliographical references (leaves 111-114).Controlled thrombosis initiates and regulates tissue repair and remodeling in the vessel wall. Processes from heart attack to stroke, and deep vein thrombosis to pulmonary embolism, are all derived from unrestricted clotting. The frustrating aspects of these diseases lie not only in the difficulty of their timely diagnosis, but in the selection of appropriate therapy, titration of intervention, and identification of patients at risk. Assays like the INR, PTT, and ACT can follow the course of specific pathways that govern activation of specific coagulation factors or platelet function, but they do not provide the insight into mechanism, risk and potential therapeutic benefit. We posit that elucidation of the complex dynamics of clot generation requires an integrated assessment that takes into account all of the factors driving thrombosis simultaneously. Virchow's Triad, has for years been identified as the three critical parameters of clot formation. We propose to examine an individual's clot propensity and response to therapy on minute blood samples, in virtual real time, across a range of flow rates, blood state, and wall conditions with the use of our newly developed in vitro high-throughput testing device.(cont.) In this manner we will generate a unique thrombotic fingerprint that defines an individual's risk of clotting at a specific point in time over a range of stresses. This fingerprint can aid in tailoring therapeutic clinical treatments, determining the duration and dose of therapy, and assist in clinical trial management and establishment of clinical norms.by Javier Jacobo Echenique.S.M

Ex vivo revascularization in liver bioengineering. A critical first step towards effective transplantation of bioengineered livers.

Hasta la fecha, el trasplante de hígado es la única opción disponible para pacientes con enfermedad hepática terminal. El problema es la gran falta de órganos para trasplante. Por lo tanto, nuevas terapias emergentes, como la medicina regenerativa y la bioingeniería de órganos, esperan resolver este problema de escasez. Sin embargo, hasta la fecha nadie ha podido generar hígados de bioingeniería que puedan ser trasplantados con éxito, debido a la falta de permeabilidad vascular lo que conduce a trombosis en el animal receptor. Por lo tanto, el objetivo principal de esta tesis era el de generar una vasculatura ensamblada in vitro que fuera estable y funcional. Paralelamente, también hemos trabajado en la creación de un modelo animal de regeneración hepática, en el que las piezas de bioingeniería revascularizadas pueden ser trasplantados. Además, en colaboración con el grupo de investigación del Dr. Bart Spee y del Dr. Hans Clevers de la Universidad de Utrecht, probamos a sembrar hepatocitos derivados de células somáticas y células madre hepáticas adultas Lgr5 + en ECM descelularizada hepática, mejorando su destino y función hepática cuando se siembran en estos scaffolds. Esto puede permitir la posibilidad de obtener una fuente de células hepáticas que se puede expandir para obtener los grandes números requeridos para la bioingeniería de órganos, aumentando la complejidad estructural y anatómica de nuestros hígados de bioingeniería. En resumen, estos resultados pueden proporcionar las herramientas necesarias (una vasculatura funcional estable, una nueva fuente de hepatocitos y un modelo animal para trasplante) para generar hígados de bioingeniería adecuados para su futura traslación a la clínica.To date, liver transplantation is the only available option for patients with terminal liver disease. The problem is the huge lack of organs for transplantation. Therefore, new emerging therapies such as regenerative medicine and organ bioengineering present hope to solve this problem of organ shortage. However, to date no one has been able to generate bioengineered livers that can successfully be transplanted, due to lack of vascular patency in these bioengineered organs, leading to thrombosis in the receptor animal. Thus, the main objective of this thesis was to generate an in vitro assembled vasculature that was stable and functional. In parallel, we have also worked on the creation of an animal model of liver regeneration, in which the revascularized bioengineering grafts can be transplanted. In addition, in collaboration with the research group of Dr. Bart Spee and Dr. Hans Clevers from the University of Utrecht, we tested seeding hepatocytes derived from somatic cells and Lgr5+ adult liver stem cells in liver decellularized ECM, enhancing their hepatic fate and function when seeded in these scaffolds. This may allow the possibility of obtaining a source of hepatic cells which can be expanded into the large numbers required for organ bioengineering, increasing the structural and anatomical complexity of our bioengineering livers. In summary, these results can provide the necessary tools (stable functional vasculature, a novel source of hepatocytes and an animal model for transplantation) to generate bioengineered livers suitable for future translation into the clinic.<br /

Role of Brain Angiotensin II in Cardiovascular Regulation and Erythropoiesis

Hypertension prevalence is still growing worldwide as well as the number of drug-therapy non-responsive patients. Therefore, improving the current and developing new therapies to control blood pressure (BP) is warranted. Growing literature points to the sympathetic nerve system as the major system controlling long-term BP homeostasis, and human hypertension is often accompanied by increased sympathetic nerve activity (SNA). The renin angiotensin system (RAS) is involved in cardiovascular control and beyond due to the broad actions of the powerful hormone angiotensin II (Ang II) produced in the circulation or within organs. The project aimed to further characterize the modulatory effects of the RAS on SNA and its impact on cardiovascular control and erythropoiesis, particularly focusing on the role of brain-borne Ang II. These questions were experimentally addressed with RAS gain and loss of function mouse models in the FVB/N background strain, and complementary experiments were performed with rats and mixed background mice. A transgenic line with brain-specific angiotensinogen (Agt) expression was validated (Agt-Tg) and used to demonstrate in vivo local brain Ang II formation. FVB/N mice cardiovascular regulation revealed predicted and surprising phenotypes. As expected FVB/N mice lacking the major Ang II receptor (AT1a-KO) presented reduced BP, and Agt-Tg displayed increased BP accompanied by increased SNA. Surprisingly, adult FVB/N mice lacking Agt (Agt-KO) were normotensive contrasting previous findings in other strains. Paradoxically, the genetic brain-rescue of Agt in Agt-KO (Agt-KO-Tg) strongly reduced BP. Using integrative physiology, we identified that the normotension in Agt-KO is driven by increased vascular tone due to exaggerated sympathetic outflow and impaired nitric oxide production which is reversed by brain Ang II in Agt-KO-Tg contrasting Agt-Tg. Agt-KO-Tg also presented better renal function compared to Agt-KO despite similar renal morphological damage. In addition, brain Ang II strongly activated renal SNA in Agt-KO-Tg, and renal denervation had minimal effect on Agt-KO and Agt-KO-Tg baseline BP. A genome-wide-association-study was employed to uncover the genetic alteration causing FVB/N Agt-KO to fail in reducing BP. For this, a mixed background (FVB/N x C57BL/6N) F2 Agt-KO line was generated. Strikingly, the phenotyping data of the F2 Agt-KO strongly indicates a single recessive allele responsible for the BP phenotype in FVB/N Agt-KO once the BP in the F2 presented a clear bimodal distribution with most mice being hypotensive. However, the association analyses demonstrated that several loci likely contribute to the phenotype in FVB/N Agt-KO. Finally, we identified one central and one peripheral mechanism of erythropoiesis regulation modulated by the RAS. In the brain, Ang II favors erythropoiesis via SNA. Agt-Tg with elevated brain Ang II displayed increased erythropoiesis that was reverted by peripheral sympathectomy, and rats with depleted brain Agt presented decreased baseline erythropoiesis. In the periphery, the experiments revealed that the RAS influences the iron homeostasis because Agt-KO mice presented microcytic anemia accompanied by reduced levels of circulating and tissue-stored iron. Altogether, the data set generated within the frame of my doctoral thesis supports local formation and function of brain Ang II. Furthermore, we provided evidence that brain Ang II is involved in differential SNA regulation to different organs impacting BP homeostasis. Finally, two unprecedented mechanisms of erythropoiesis control modulated by the RAS via SNA and iron homeostasis were revealed.Die Prävalenz von Bluthochdruck nimmt weltweit weiter zu, ebenso wie die Zahl der Patienten, die nicht auf eine medikamentöse Therapie ansprechen. Daher ist die Verbesserung der derzeitigen und die Entwicklung neuer Therapien zur Kontrolle des Blutdrucks gerechtfertigt. Zunehmend weist Literatur darauf hin, dass das sympathische Nervensystem das wichtigste System für die Steuerung langfristiger Blutdruckhomöostase ist, außerdem wird Bluthochdruck oft von einer erhöhten sympathischen Nervenaktivität (SNA) begleitet. Das Renin-Angiotensin-System (RAS) ist an der Kontrolle des Herz-Kreislauf-Systems beteiligt. Das potente Hormon Angiotensin-II (Ang-II) des RAS wird im Blutkreislauf oder in den Organen gebildet und besitzt vielfältige physiologische Effekte. Das Projekt zielte darauf ab, die modulatorischen Wirkungen des RAS auf die SNA und entsprechenden Folgen auf die kardiovaskuläre Kontrolle und die Erythropoese weiter zu charakterisieren, wobei der Schwerpunkt auf der Rolle des im Gehirn gebildeten Ang II lag. Dieses Ausgangsinteresse wurde experimentell mit RAS Gain und Loss-of-Function-Mausmodellen im FVB/N Hintergrundstamm untersucht. Ergänzend wurden Versuche mit Ratten und Mäusen mit gemischtem genetischen Hintergrund durchgeführt. Eine transgene Linie mit gehirnspezifischer Angiotensinogen-Expression (Agt) wurde validiert (Agt-Tg) und verwendet, um die lokale Ang-II-Bildung im Gehirn in vivo zu demonstrieren. Die Ergebnisse zur kardiovaskulären Regulation von FVB/N-Mäusen ergaben die vermuteten und überraschende Phänotypen. Wie erwartet hatten FVB/N-Mäuse, denen der wichtigste Ang-II-Rezeptor (AT1a-KO) fehlte, einen verminderten Blutdruck. Außerdem zeigte Agt-Tg einen erhöhten Blutdruck im Zusammenhang mit erhöhter SNA. Im Gegensatz zu früheren Befunden bei anderen Stämmen, waren erwachsene FVB/N-Mäuse ohne Agt (Agt-KO), überraschenderweise normotensiv. Paradoxerweise senkte die spezifische Wiederherstellung der Agt Genexpression im Gehirn von Agt-KO (Agt-KO-Tg) den Blutdruck dramatisch. Mithilfe von integrativer Physiologie konnten wir feststellen, dass die Normotonie bei Agt-KO durch einen erhöhten Gefäßtonus aufgrund eines übermäßigen Sympathikotonus und einer beeinträchtigten Stickoxidproduktion verursacht wird. Diese werden bei Agt-KO-Tg im Gegensatz zu Agt-Tg durch Ang II im Gehirn umgekehrt. Agt-KO-Tg wies im Vergleich zu Agt-KO trotz ähnlicher morphologischer Nierenschäden eine bessere Nierenfunktion auf. Des Weiteren aktivierte Ang II im Gehirn die renale SNA in Agt-KO-Tg stark, wohingegen eine renale Denervierung nur minimale Auswirkungen auf den Basisblutdruck von Agt-KO und Agt-KO-Tg hatte. Eine genomweite Assoziationsstudie wurde implementiert um die genetische Veränderung, die zu der Normotonie bei FVB/N Agt-KO führt, aufzudecken. Für diesen Zweck wurde eine F2 Agt-KO-Linie mit gemischtem Hintergrund (FVB/N x C57BL/6N) erzeugt. Bemerkenswerterweise wiesen die Phänotypisierungsdaten durch eine klare bimodale Verteilung des Basisblutdrucks in der F2 Agt-KO stark auf ein einzelnes rezessives Allel hin, das für den Blutdruck-Phänotyp in FVB/N Agt-KO verantwortlich ist, wobei die meisten Mäuse hypoton waren. Die Assoziationsanalysen zeigten jedoch, dass wahrscheinlich mehrere Loci zum Phänotyp bei FVB/N Agt-KO beitragen. Im letzten Teil der Arbeit identifizierten wir zwei Regulationsmechanismen der Erythropoese- (zentraler und peripherer), die durch das RAS moduliert werden. Im Gehirn begünstigt Ang II die Erythropoese über SNA, da Agt-Tg mit gesteigertem Ang-II im Gehirn eine erhöhte Erythropoese zeigte, die durch periphere Sympathektomie rückgängig gemacht wurde. Außerdem zeigten Ratten mit verminderter Agt-Expression im Gehirn eine verringerte Erythropoese. In der Peripherie zeigten die Experimente, dass das RAS die Eisenhomöostase beeinflusst, da Agt-KO-Mäuse eine mikrozytäre Anämie aufwiesen, die mit reduzierten Konzentrationen von zirkulierendem und im Gewebe gespeichertem Eisen einherging. Zusammenfassend zeigen die im Rahmen meiner Doktorarbeit erhobenen Daten die lokale Bildung und Funktion von Ang II im Gehirn. Darüber hinaus konnten wir nachweisen, dass Ang II im Gehirn an der differenzierten SNA-Regulierung in verschiedenen Organen beteiligt ist, was sich auf die Homöostase des Blutdrucks auswirkt. Schließlich wurden zwei neue Mechanismen der Erythropoese Kontrolle aufgedeckt, die durch das RAS über SNA und Eisenhomöostase moduliert werden

Assessment of goal-directed closed-loop management in intensive care medicine

Given an aging population, shortage of nursing staff and a continuously increasing workload, automation in the medical sector is an important aspect of future intensive care. Although automation and machine learning are current research topics, progress is still very limited in comparison to other application areas. Probably one of the most serious problems is data shortage in a heterogeneous landscape of medical devices with limited interfaces and various protocols. In addition, the recording of data or, even more so, the evaluation of automation is limited by a complex legal framework. Given these complications and the sensitive legal nature of medical records, only very limited data is accessible for further analysis and development of automated systems. For this reason, within the context of this thesis various solutions for data acquisition and automation were developed and evaluated concomitant to two clinical studies utilizing a large animal model in a realistic intensive care setting at the University Hospital Tübingen. Foremost, to overcome the problems of data availability and interconnection of medical devices, a software framework for data collection and remote control using a client-server architecture was developed and significant amounts of research data could be collected in a central database. Furthermore, a closed-loop controller based on fuzzy logic was developed and used for management of end-tital CO2, glucose, and other parameters to stabilize the animal subjects during therapy and reduce caregivers’ workload. In addition to the fuzzy controller, closed-loop management for temperature and anticoagulation could be established by developing hardware interfaces for a forced-air warming unit and a point-of-care analysis device, respectively. Besides further reduction of caregivers’ workload, such systems can provide additional patient safety and allow management in settings where human supervision may not be present at all times. One general and encountered problem for closed-loop control in a medical setting is limited availability of measurements, especially if manual blood withdrawals are required. As an initial step to address this problem, measured parameters from other devices as potential surrogates were evaluated in a comparison between different regression approaches. The required training data, a matched set of blood gas and monitoring parameters, was obtained by utilizing a developed algorithm for automated detection of withdrawal events. Yet, besides any specific implementations and analysis, many general aspects regarding the physical implementation of such a system and interaction with caregivers could be evaluated in the experimental setting and might guide further development of clinical automation.Angesichts der alternden Bevölkerung, des Mangels an Pflegekräften und der ständig steigenden Arbeitsbelastung ist Automatisierung ein wichtiger Aspekt zukünftiger Intensivmedizin. Obwohl Automatisierung und maschinelles Lernen aktuelle Forschungsthemen sind, ist der Fortschritt im Vergleich zu anderen Anwendungsbereichen jedoch noch sehr begrenzt. Eines der größten Probleme ist wohl die Datenknappheit in einer heterogenen Medizinproduktelandschaft mit begrenzten Schnittstellen und zahlreichen unterschiedlichen Protokollen. Darüber hinaus sind die Datenerfassung und erst recht die Erprobung einer Automatisierung durch ein komplexes rechtliches Rahmenwerk eingeschränkt. Aufgrund dieser Komplikationen und der sensiblen Rechtslage für Patientendaten sind diese nur sehr begrenzt für weitere Analysen und die Entwicklung automatisierter Systeme zugänglich. Im Rahmen dieser Dissertation wurden daher verschiedene Lösungen zur Datenerfassung und Automatisierung begleitend zu zwei klinischen Studien des Universitätsklinikums Tübingen am Großtiermodell in einer realitätsnahen Intensivstation entwickelt und evaluiert. Um die Probleme der Datenverfügbarkeit und Vernetzung medizinischer Geräte zu lösen, wurde vorrangig ein Software-Framework für die Datenerfassung und Steuerung mittels einer Client-Server-Architektur entwickelt und umfangreiche Forschungsdaten in einer zentralen Datenbank gesammelt. Darüber hinaus wurde ein auf Fuzzy-Logik basierender Regler entwickelt, welcher zur Stabilisierung des endtitalen CO2, Glukose und anderen Parametern verwendet wurde und damit die Arbeitsbelastung der Pflegekräfte reduzieren konnte. Zusätzlich zum Fuzzy-Regler konnten durch die Entwicklung von Hardware-Schnittstellen für Geräte zum Temperaturmanagement mittels luftbasierter Wärmedecken und zur Messung der Blutgerinnung geschlossene Regelkreise aufgebaut werden. Neben einer weiteren Arbeitserleichterung für die Pflegekräfte können solche Systeme zusätzliche Sicherheit für den Patienten bieten und die Anwendung in nicht ständig überwachten Bereichen ermöglichen. Ein allgemeines und auch beobachtetes Problem für Regelkreise im medizinischen Bereich ist die begrenzte Verfügbarkeit von Messwerten, insbesondere bei manuellen Blutentnahmen. Als erster Schritt zur Lösung dieses Problems wurden Messparameter anderer Geräte als potentielle Ersatzparameter mit verschiedenen Regressionsansätzen analysiert und verglichen. Die dazu erforderlichen Trainingsdaten, Paare von Blutgas- und weiteren Vitaldaten, wurden mit Hilfe eines entwickelten Algorithmus zur automatisierten Erkennung von Blutentnahmen erzeugt. Abgesehen von diesen konkreten Anwendungen und Analysen konnten in der experimentellen Evaluation auch viele generelle Aspekte der realen Implementierung eines solchen Systems und die Interaktion mit Ärzten und Pflegekräften untersucht werden und damit der Entwicklung weiterer klinischen Automatisierung dienen

Effect of Procedural Pain on Purines, MDA and Allantoin in Premature Infants

Identifying and measuring the biochemical effects of procedural pain in infants is a continuing challenge for clinicians. To address this issue we evaluated the relationship between procedural pain, ATP utilization (hypoxanthine and uric acid), and oxidative stress (MDA and allantoin) in premature infants. Through these studies we found that 1) a single heel-lance significantly increases plasma uric acid in rabbit kits, 2) tape removal increases MDA and prevents a decrease in uric acid in the plasma of premature infants, and 3) a single dose of oral sucrose before a heel-lance is sufficient to significantly increase plasma markers of ATP utilization and oxidative stress. These studies indicate that, in premature infants, ATP utilization and oxidative stress are altered by procedural pain and that oral sucrose, a common analgesia, may increase ATP breakdown. Next, we examined clinical factors that need to be considered when using the urinary concentrations of hypoxanthine, xanthine, uric acid, and allantoin to evaluate the biochemical effects of procedural pain. Specifically, we determined the effects of gestational age, weight for gestational age, and neonatal morbidity on the urinary concentration of purines and allantoin. We found infants born at earlier gestational ages had significantly higher urinary purine and allantoin concentrations compared to infants born at later gestations, after 31 weeks. Weight for gestational age also altered the urinary concentration of purines with small for gestational age infants having significantly lower urinary hypoxanthine compared to appropriate and large for gestational age infants. In addition, respiratory morbidity significantly increased urinary purines, but not allantoin, within the late preterm population. These data suggest that gestational age, weight for gestational age, and neonatal morbidity alter the urinary concentration of purine metabolites. Allantoin was also found to be significantly altered by gestational age. Based upon these data, we recommend conducting age-matched as well as weight for age-matched studies when using urinary purines and allantoin to evaluate the relationship between procedural pain and hypoxia on ATP utilization and oxidative stress. Lastly, it is important to stratify subjects based on health status or morbidity, due to the significant effects of respiratory diseases on ATP use and oxidative stress

Clot structure and plasma microparticles in atrial fibrillation

Chronic oral anticoagulation (OAC) is an important decision related with stroke thromboprophylaxis in non-valvular atrial fibrillation (NVAF). Almost a decade after the approval of Non vitamin K oral anticoagulants (NOACs) for prevention of thromboembolic events in NVAF, there is now confidence regarding their efficacy and safety as real word evidence complements the findings from the phase III pivotal trials. NOACs favourable safety profile against warfarin have changed the threshold of starting OAC, even with lower risk for systemic thromboembolism. Apixaban, one of the four licenced NOACs for stoke prevention in NVAF, even at the higher recommended dose (5mg BID), has significantly reduced the haemorrhagic complications but there is still a considerable risk of intracranial bleeding. This MD research thesis studies the influence of antithrombotics (aspirin, warfarin and apixaban) on the fibrin polymerisation and fibrinolysis pathway. To highlight antithrombotic activity variances, this analysis is based on dynamic assays and biomarker quantification related with clot structure features. Additionally, explores possible relationship between microparticle levels and physical status in NVAF patients. My findings suggest that NVAF is associated with impaired haemostasis and each antithrombotic class is related to different clot structure characteristics. Apixaban has distinctive anticoagulation dynamics and induces a reduction of coagulation biomarkers. My results also support that microparticles levels may be a useful marker of physical status as suggested by the relation between objective (cardiopulmonary exercise test) and subjective (quality of life questionnaire – EQ5D5L) evidence of fitness level