Modulating endothelial adhesion and migration impacts stem cell therapies efficacy.

BACKGROUND: Limited knowledge of stem cell therapies` mechanisms of action hampers their sustainable implementation into the clinic. Specifically, the interactions of transplanted stem cells with the host vasculature and its implications for their therapeutic efficacy are not elucidated. We tested whether adhesion receptors and chemokine receptors on stem cells can be functionally modulated, and consequently if such modulation may substantially affect therapeutically relevant stem cell interactions with the host endothelium. METHODS: We investigated the effects of cationic molecule polyethylenimine (PEI) treatment with or without nanoparticles on the functions of adhesion receptors and chemokine receptors of human bone marrow-derived Mesenchymal Stem Cells (MSC). Analyses included MSC functions in vitro, as well as homing and therapeutic efficacy in rodent models of central nervous system´s pathologies in vivo. FINDINGS: PEI treatment did not affect viability, immunomodulation or differentiation potential of MSC, but increased the CCR4 expression and functionally blocked their adhesion receptors, thus decreasing their adhesion capacity in vitro. Intravenously applied in a rat model of brain injury, the homing rate of PEI-MSC in the brain was highly increased with decreased numbers of adherent PEI-MSC in the lung vasculature. Moreover, in comparison to untreated MSC, PEI-MSC featured increased tumour directed migration in a mouse glioblastoma model, and superior therapeutic efficacy in a murine model of stroke. INTERPRETATION: Balanced stem cell adhesion and migration in different parts of the vasculature and tissues together with the local microenvironment impacts their therapeutic efficacy. FUNDING: Robert Bosch Stiftung, IZEPHA grant, EU grant 7 FP Health.

Saliva-coated titanium biosensor detects specific bacterial adhesion and bactericide caused mass loading upon cell death.

Bacteria adhering to implanted medical devices can cause invasive microbial infections, of e.g. skin, lung or blood. In dentistry, Streptococcus gordonii is an early oral colonizer initiating dental biofilm formation and also being involved in life-threatening infective endocarditis. To treat oral biofilms, antibacterial mouth rinses are commonly used. Such initial biomaterial-bacteria interactions and the influence of antibacterial treatments are poorly understood and investigated here in situ by quartz crystal microbalance with dissipation monitoring (QCM-D). A saliva-coated titanium (Ti) biosensor is applied to analyze possible specific signal patterns indicating microbial binding mechanisms and bactericide-caused changes in bacterial film rigidity or cell leakage caused by a clinically relevant antibacterial agent (ABA), i.e., a mouth rinse comprising chlorhexidine (CHX) and cetylpyridinium chloride (CPC). Apparent missing mass effects during the formation of microscopically proven dense and vital bacterial films indicate punctual, specific binding of S. gordonii to the saliva-coated biosensor, compared to unspecific adhesion to pure Ti. Coincidentally to ABA-induced killing of surface-adhered bacteria, an increase of adsorbed dissipative mass can be sensed, contrary to the prior mass-loss. This suggests the acoustic sensing of the leakage of cellular content caused by bacterial cell wall rupturing and membrane damage upon the bactericidal attack. The results have significant implications for testing bacterial adhesion mechanisms and cellular integrity during interaction with antibacterial agents.

QCM-D surpassing clinical standard for the dose administration of new oral anticoagulant in the patient of coagulation disorders.

The study focuses the dose administration of dabigatran to avoid the deaths due to hemorrhagic complications and thromboembolic stroke in clinics worldwide. To target the issue, a novel emerging acoustic technology, namely ''Quartz Crystal Microbalance with Dissipation'' (QCM-D) has been applied, while the acoustic assays namely ''activated Partial Thromboplastin Time'' (aPTT) and ''Prothrombinase complex-induced Clotting Test'' (PiCT) have been compared with the standard methods in parallel. Both techniques have been applied to 300 samples, including 220 plasma samples of patients suffering coagulation disorders and 80 plasma samples of non-patients. In comparison, the coagulation times of the acoustic aPTT and PiCT yielded an excellent correlation with the standard methods with in analytical standard deviation limits. Finally, the acoustic aPTT assay is the ''gold standard'' for a dose administration of the new oral anticoagulant, where the Δf/ΔΓ ratio of the acoustic assay demonstrates that dabigatran with FEIBA 50 combination could be a safe remedy to avoid the deaths in clinics.

Real-time measurement of Plasmodium falciparum-infected erythrocyte cytoadhesion with a quartz crystal microbalance.

BACKGROUND: An important virulence mechanism of the malaria parasite Plasmodium falciparum is cytoadhesion, the binding of infected erythrocytes to endothelial cells in the second half of asexual blood stage development. Conventional methods to investigate adhesion of infected erythrocytes are mostly performed under static conditions, many are based on manual or semi-automated read-outs and are, therefore, difficult to standardize. Quartz crystal microbalances (QCM) are sensitive to nanogram-scale changes in mass and biomechanical properties and are increasingly used in biomedical research. Here, the ability of QCM is explored to measure binding of P. falciparum-infected erythrocytes to two receptors: CD36 and chondroitin sulfate A (CSA) under flow conditions. METHODS: Binding of late stage P. falciparum parasites is measured in comparison to uninfected erythrocytes to CD36- and CSA-coated quartzes by QCM observing frequency shifts. CD36-expressing cell membrane fragments and CSA polysaccharide were coated via poly-L-lysine to the quartz. The method was validated by microscopic counting of attached parasites and of erythrocytes to the coated quartzes. RESULTS: Frequency shifts indicating binding of infected erythrocytes could be observed for both receptors CD36 and CSA. The frequency shifts seen for infected and uninfected erythrocytes were strongly correlated to the microscopically counted numbers of attached cells. CONCLUSIONS: In this proof-of-concept experiment it is shown that QCM is a promising tool to measure binding kinetics and specificity of ligand-receptor interactions using viable, parasite-infected erythrocytes. The method can improve the understanding of the virulence of P. falciparum and might be used to cross-validate other methods.

Utilisation of Quartz Crystal Microbalance Sensors with Dissipation (QCM-D) for a Clauss Fibrinogen Assay in Comparison with Common Coagulation Reference Methods.

The determination of fibrinogen levels is one of the most important coagulation measurements in medicine. It plays a crucial part in diagnostic and therapeutic decisions, often associated with time-critical conditions. The commonly used measurement is the Clauss fibrinogen assay (CFA) where plasma is activated by thrombin reagent and which is conducted by mechanical/turbidimetric devices. As quartz crystal microbalance sensors with dissipation (QCM-D) based devices have a small footprint, can be operated easily and allow measurements independently from sample transportation time, laboratory location, availability and opening hours, they offer a great opportunity to complement laboratory CFA measurements. Therefore, the objective of the work was to (1) transfer the CFA to the QCM-D method; (2) develop an easy, time- and cost-effective procedure and (3) compare the results with references. Different sensor coatings (donor's own plasma; gold surface) and different QCM-D parameters (frequency signal shift; its calculated turning point; dissipation signal shift) were sampled. The results demonstrate the suitability for a QCM-D-based CFA in physiological fibrinogen ranges. Results were obtained in less than 1 min and in very good agreement with a standardized reference (Merlin coagulometer). The results provide a good basis for further investigation and pave the way to a possible application of QCM-D in clinical and non-clinical routine in the medical field.

Detection of HIT antibody dependent platelet aggregation using novel surface imprinting approach.

We present a fast, robust and straightforward spin force assisted surface imprinting approach for activated platelets and demonstrate that Heparin induced thrombocytopenia (HIT) platelet aggregation can be measured by this approach. A critical and challenging step in functional assays for HIT is platelet separation from the healthy donor's platelet-rich plasma (PRP). Our approach using surface imprinted polymer (MIP) for measurements on a quartz crystal microbalance with dissipation (QCM-D) enables monitoring of platelet aggregation directly in PRP thus eliminating the challenge of platelet separation. This is the first report of platelet imprinting. We also provide proof of principle that QCM-D technology can be applied for functional measurements of HIT antibodies.

QCM-D providing new horizon in the domain of sensitivity range and information for haemostasis of human plasma.

Monitoring of the haemostasis status is significant for proper therapeutic directions and decisions in surgery and innate coagulation disorders. In this regard, to gain a general overview of the plasmatic coagulation, prothrombin time (PT) tests are frequently combined with tests for activated partial thromboplastin time (aPTT). For aPTT we report for the first time that a QCM-D (Quartz Crystal Microbalances with Dissipation) based technique offers a better alternative to the standard coagulometer method in the perspective of range and information. We used heparin as anticoagulant to generate different coagulation times for human plasma. QCM-D astonishingly proved to be more sensitive and reliable than the standard coagulometer for aPTT range of upper limits of coagulation times. The established platform can monitor the fibrinogen concentration ranging from 1-6g/L (yielding R(2)=0.98 in calibration curves) along with aPTT from frequency and dissipation shifts together in a single set of measurements. Additionally the sensor layers have been tested for reusability, demonstrating no loss in sensor characteristics up to ten times measurements.

NCO-sP(EO-stat-PO) coatings on gold sensors--a QCM study of hemocompatibility.

The reliability of implantable blood sensors is often hampered by unspecific adsorption of plasma proteins and blood cells. This not only leads to a loss of sensor signal over time, but can also result in undesired host vs. graft reactions. Within this study we evaluated the hemocompatibility of isocyanate conjugated star shaped polytheylene oxide-polypropylene oxide co-polymers NCO-sP(EO-stat-PO) when applied to gold surfaces as an auspicious coating material for gold sputtered blood contacting sensors. Quartz crystal microbalance (QCM) sensors were coated with ultrathin NCO-sP(EO-stat-PO) films and compared with uncoated gold sensors. Protein resistance was assessed by QCM measurements with fibrinogen solution and platelet poor plasma (PPP), followed by quantification of fibrinogen adsorption. Hemocompatibility was tested by incubation with human platelet rich plasma (PRP). Thrombin antithrombin-III complex (TAT), ß-thromboglobulin (ß-TG) and platelet factor 4 (PF4) were used as coagulation activation markers. Furthermore, scanning electron microscopy (SEM) was used to visualize platelet adhesion to the sensor surfaces. Compared to uncoated gold sensors, NCO-sP(EO-stat-PO) coated sensors revealed significant better resistance against protein adsorption, lower TAT generation and a lower amount of adherent platelets. Moreover, coating with ultrathin NCO-sP(EO-stat-PO) films creates a cell resistant hemocompatible surface on gold that increases the chance of prolonged sensor functionality and can easily be modified with specific receptor molecules.

Platelet aggregation monitoring with a newly developed quartz crystal microbalance system as an alternative to optical platelet aggregometry.

The objective of this study was to establish a new test system for the monitoring of platelet aggregation during extracorporeal circulation (ECC) procedures. Even though extensive progress has been made in improving the haemocompatibility of extracorporeal circulation devices, activation of blood coagulation, blood platelets and inflammatory responses are still undesired outcomes of cardiopulmonary bypass. This study deals with an approach towards a platelet aggregation measuring system using a newly developed quartz crystal microbalance (QCM) system. Since QCM is a rarely used technique in the field of blood analytics, the challenge was to transfer the well established methods of aggregometry to the new test system. In a QCM system, either bare gold or fibrinogen-coated sensors were incubated with ADP or arachidonic acid (AA) stimulated platelet rich plasma. For negative controls the GPIIb/IIIa inhibitory antibody abciximab (Reopro®) was used as an inhibitor of platelet aggregation. During incubation, the frequency shifts of the sensors were recorded. The results gained from the QCM system were compared to results gained by optical platelet aggregometry (born aggregometry). For additional visualization of platelet adhesion to the sensor surfaces, fluorescent microscopy and scanning electron microscopy were used. The QCM sensor was able to detect platelet aggregation in both uncoated and fibrinogen coated sensors. The measuring curves of aggregation measurements and controls were clearly distinguishable from each other in terms of frequency shifts and kinetics. For aggregation measurements and inhibited controls the therapeutic diagnosis of platelet function is identical between aggregometer and QCM data. In future, QCM based measuring devices may become an alternative to established point of care methods for rapid bedside testing of platelet aggregation.

Investigation of prothrombin time in human whole-blood samples with a quartz crystal biosensor.

Monitoring of blood coagulation and fibrinolysis is an important issue in treatment of patients with cardiovascular problems and in surgery when blood gets into contact with artificial surfaces. In this work a new method for measuring the coagulation time (prothrombin time, PT) of human whole-blood samples based on a quartz crystal microbalance (QCM) biosensor is presented. The 10 MHz sensors used in this work respond with a frequency shift to changes in viscosity during blood clot formation. For driving and for readout of the quartz, both a network analyzer and an oscillator circuit were utilized. The sensor surfaces were specifically coated with a thin polyethylene layer. We found that both frequency analysis methods are suitable to measure exact prothrombin times in a very good conformity with a mechanical coagulometer as a reference. The anticoagulant effect of heparin on the prothrombin time was exemplarily shown as well as the reverse effect of the heparin antagonist polybrene. The change of the viscoelastic properties during blood coagulation, reflected by the ratio of frequency and dissipation shifts, is discussed for different dilutions of the whole-blood samples. In conclusion, QCM is a distinguished biosensor technique to determine prothrombin time and to monitor heparin therapy in whole-blood samples. Due to the excellent potential of miniaturization and the availability of direct digital signals, the method is predestinated for incorporation and integration into other devices and is thus opening the field of application for inline coagulation diagnostic in extracorporeal blood circuits.