Clinical Hemorheology and Microcirculation - Volume 61, issue 2

Authors: Manz, Patrick | Cadeddu, Ron-Patrick | Wilk, Matthias | Fischer, Johannes C. | Fritz, Birgit | Haas, Rainer | Wenzel, Folker

Article Type: Research Article

Abstract: INTRODUCTION: Phthalates are a group of synthetic plasticizers that are ubiquitous environmental pollutants with toxic and endocrine disrupting characteristics. DEHP is the most commonly used plasticizer in the world and is still applied to stem cell transfusion bags used for storage of hematopoietic stem and progenitor cells (CD34+ HSPC), which are transferred during stem cell transplantation. Here we examined the effect of DEHP on vitality of CD34+ HSPC as well as stem cell specific properties like migration and differentiation capacity – both important for successful stem cell transplantations. MATERIAL AND METHODs: CD34+ HSPC were incubated …for 24 h and 72 h with DEHP concentrations ranging from 1 μg/ml to 250 μg/ml. DEHP was diluted in DMSO. Migration rate was analyzed along an SDF-1α gradient using Transwell migration inserts. Differentiation of CD34+ HSPC was investigated after two weeks in methylcellulose with colony stimulating factors. Apoptosis rate was measured via Annexin V and 7-AAD staining. RESULTS: 24 h of incubation with 10 μg/ml DEHP led to a significant (p  <  0.01) decrease in migration rate of CD34+ HSPC (70.70% ± 7.53% ) with a minimum migration rate of 48.33% ± 6.72% in relation to control after incubation with 100 μg/ml DEHP for 72 h. Incubation with the highest tested DEHP concentrations (50 and 100 μg/ml) significantly (p  <  0.05) altered colony formation rate and cell type distribution. Apoptosis rate of CD34+ HSPC significantly (p  <  0.05) increased after incubation with concentrations of 10 μg/ml DEHP for 24 h (1.46 ± 0.19) with a maximum apoptosis rate of 2.71 ± 0.66 after 24 h incubation with the highest DEHP concentration (250 μg/ml) in relation to control. CONCLUSIONS: As shown, DEHP takes impact on migration rate, apoptosis rate, and differentiation of CD34+ HSPC. As these are functions with an important role in stem cell transplantations, the usage of DEHP-free stem cell transfusion bags should be considered. Show more

Keywords: Phthalates, DEHP, hematopoietic stem cells, CD34+ , vitality, migration, differentiation, apoptosis, apheresis, stem cell transplantation

DOI: 10.3233/CH-151984

Citation: Clinical Hemorheology and Microcirculation, vol. 61, no. 2, pp. 111-118, 2015

Authors: Vijaya Bhaskar, Thanga Bhuvanesh | Ma, Nan | Lendlein, Andreas | Roch, Toralf

Article Type: Research Article

Abstract: Silicones are widely used as biomaterials for medical devices such as extracorporeal equipments. However, there is often conflicting evidence about their supposed cell- and histocompatibility. Macrophages could mediate silicone-induced adverse responses such as foreign body reaction and fibrous encapsulation. The polarization behaviour of macrophages could determine the clinical outcome after implantation of biomaterials. Induction of classically activated macrophages (CAM) may induce and support uncontrolled inflammatory responses and undesired material degradation. In contrast, polarization into alternatively activated macrophages (AAM) is assumed to support healing processes and implant integration. This study compared the interaction of non-polarized macrophages (M0), CAM, and AAM …with commercially available tissue culture polystyrene (TCP) and a medical grade silicone-based biomaterial, regarding the secretion of inflammatory mediators such as cytokines and chemokines. Firstly, by using the Limulus amoebocyte lysate (LAL) test the silicone films were shown to be free of soluble endotoxins, which is the prerequisite to investigate their interaction with primary immune cells. Primary human monocyte-derived macrophages (M0) were polarized into CAM and AAM by addition of suitable differentiation factors. These macrophage subsets were incubated on the materials for 24 hours and their viability and cytokine secretion was assessed. In comparison to TCP, cell adhesion was lower on silicone after 24 hours for all three macrophage subsets. However, compared to TCP, silicone induced higher levels of certain inflammatory and chemotactic cytokines in M0, CAM, and AAM macrophage subsets. Conclusively, it was shown that silicone has the ability to induce a pro-inflammatory state to different magnitudes dependent on the macrophage subsets. This priming of the macrophage phenotype by silicone could explain the incidence of severe foreign body complications observed in vivo . Show more

Keywords: Biomaterials, silicone, macrophage subsets, cytokines/chemokines

DOI: 10.3233/CH-151991

Citation: Clinical Hemorheology and Microcirculation, vol. 61, no. 2, pp. 119-133, 2015

Authors: Niessen, C. | Jung, E.M. | Beyer, L. | Pregler, B. | Dollinger, M. | Haimerl, M. | Scheer, F. | Stroszczynski, C. | Wiggermann, P.

Article Type: Research Article

Abstract: PURPOSE: To evaluate the use of contrast-enhanced ultrasound (CEUS) after irreversible electroporation (IRE) of prostatic cancer tissue to assess the ablation status by depicting microvascularisation in the ablation area. MATERIALS AND METHODS: Retrospective evaluation of CEUS of 13 patients (mean age: 61.4 ± 7.5 years) with histologically confirmed prostatic cancer who underwent percutaneous IRE. In the course of clinical routine, the tumor lesions were documented before, immediately after, and 1 day after the ablationusing color-coded transabdominal and transrectal CEUS. The obtained image data (DICOM loops and images) were subsequently evaluated by 2 experienced radiologists and assessed with regard …to micro vascularisation by means of a 5-point scale. RESULTS: CEUS images showed significantly reduced microcirculation of the lesions (mean 0.9 ± 0.6 cm (0.5–1.5 cm) after IRE. Microcirculation was reduced from 2.15 ± 0.56 prior to ablation to 0.65 ± 0.63 (p  <  0.001) immediately after the ablation and to 0.27 ± 0.44 one day after IRE (p  <  0.001). CONCLUSION: This study showed rapid and significant reduction of the microcirculation in the ablation area afterpercutaneous IRE of prostatic cancer tissue. Show more

Keywords: Irreversible electroporation, prostate cancer, percutaneous ablation, contrast-enhanced ultrasound

DOI: 10.3233/CH-151985

Citation: Clinical Hemorheology and Microcirculation, vol. 61, no. 2, pp. 135-141, 2015

Authors: Paprottka, P.M. | Zengel, P. | Cyran, C.C. | Paprottka, K.J. | Ingrisch, M. | Nikolaou, K. | Reiser, M.F. | Clevert, D.A.

Article Type: Research Article

Abstract: PURPOSE: To evaluate the diagnostic benefits of multimodality imaging using image fusion with magnetic-resonance-imaging (MRI) and contrast-enhanced-ultrasound (CEUS) in an experimental small-animal-squamous-cell-carcinoma-model for the assessment of tissue hemodynamics and morphology. MATERIAL AND METHODS: Human hypopharynx-carcinoma-cells were injected subcutaneously into the left flank of 15 female athymic nude rats. After 10 daysof subcutaneous tumor growth, CEUS and MRI measurements were performed using a high-end-ultrasound-system and 3-T-MRI. After successful point-to-point or plan registration, the registered MR-images were simultaneously shown with the respective ultrasound sectional plane. Data evaluation was performed using the digitally stored video sequence data sets by two experienced …radiologists using a subjective 5-point scale. RESULTS: CEUS and MRI are well-known techniques for the assessment of tissue hemodynamics (score: mean 3.8 ± 0.4 SD and score 3.8 ± 0.4 SD). Real-time image fusion of MRI and CEUS yielded a significant (p  <  0.001) improvement in score (score 4.8 ± 0.4 SD). Reliable detection of small necrotic areas was possible in all animals with necrotic tumors. No significant intraobserver and interobserver variability was detected (kappa coefficient = +1). CONCLUSION: Image fusion of MRI and CEUS gives a significant improvement for reliable differentiation between different tumor tissue areas and simplifies investigations by showing the morphology as well as surrounding macro-/microvascularization. Show more

Keywords: Image fusion, multimodality imaging, magnetic resonance imaging (MRI), contrast-enhanced ultrasound (CEUS), experimental animal model

DOI: 10.3233/CH-151986

Citation: Clinical Hemorheology and Microcirculation, vol. 61, no. 2, pp. 143-150, 2015

Authors: Krüger-Genge, A. | Fuhrmann, R. | Jung, F. | Franke, R.P.

Article Type: Research Article

Abstract: BACKGROUND: The evaluation of the interaction of human, venous endothelial cells (HUVEC) with body foreign materials on the cellular level cannot be performed in vivo , but is investigated in vitro under standard culture conditions. To maintain the vitality, proliferation and morphology of HUVEC seeded on body foreign substrates over days, the cell culture medium is usually exchanged every second day. It is well known, that alterations in the microenvironment of cells bear the risk of influencing cell morphology and function. In the current study the influence of cell culture medium exchange on HUVEC cytoskeletal microfilament structure and function …was investigated. MATERIAL AND METHODS: HUVEC in the third passage were seeded on extracellular matrix (ECM) - which was secreted from bovine corneal endothelial cells on glass- until functional confluence was reached. The experiment started 11 days after HUVEC seeding with an exchange of the cell culture medium followed by a staining of the actin microfilaments with phalloidin-rhodamin 1.5 and 5 minutes after medium exchange. The microfilaments were documented by use of an Olympus microscope (IMT-2) equipped with a UV lamp and online connected to a TV chain (Sony XC 50 ST/monochrome) implying an OPTIMAS – Image analysis system. Prostacyclin was analysed in the cell culture supernatant. RESULTS: 1.5 min after culture medium exchange in the functionally confluent cultures a slight disturbance of the actin microfilament structure with a broadening of the marginal filament band, a partial disconnection of cell-cell contacts and the appearance of intercellular fenestrations were observed. 5 minutes after medium exchange a redevelopment of the slightly disturbed microfilament structure with a condensation and narrowing of the marginal filament band was seen. 12 h later a further consolidation of the microfilament structure occurred. In addition, a perturbation of the cultured HUVEC occurred after cell culture medium exchange. The prostacyclin concentration in the supernatant increased significantly after 1.5 min to 466 ± 543 pg·mL–1 (p  <  0.001) and after 5 min to 408 ± 458 pg·mL–1 (p  <  0.001), while in control cells the prostacyclin concentration did not change remaining in the range of 50 ± 48.9 pg·mL–1 . CONCLUSION: This study revealed that the exchange of the cell culture medium led to a rapid disturbance of the HUVEC with stress fiber formation, disconnection of cell-cell contacts and an altered prostacyclin secretion, which had regressed nearly completely after 12 hours. Therefore, the evaluation of HUVEC on body foreign materials should be performed not earlier than 12 hours after cell culture medium exchange to avoid a misinterpretation of the endothelial cell morphological state. This procedure minimizes the risk of a misinterpretation of the endothelial cell morphology – caused by the culture medium exchange and not by the interaction between biomaterials and HUVEC. Show more

Keywords: HUVEC, morphology, microfilaments, adherence, culture medium

DOI: 10.3233/CH-151992

Citation: Clinical Hemorheology and Microcirculation, vol. 61, no. 2, pp. 151-156, 2015

Authors: Khiao In, M. | Wallmeyer, L. | Hedtrich, S. | Richardson, K.C. | Plendl, J. | Kaessmeyer, S.

Article Type: Research Article

Abstract: INTRODUCTION: Inducing vascularization in three-dimensional skin constructs continues to be difficult. In this study, two variations of human full-thickness skin constructs were examined. Type KCFB consists of keratinocytes (epidermal equivalent) and fibroblasts that were embedded in a collagen matrix (dermal equivalent). Type KCFB-EC consists of keratinocytes as well as fibroblasts and vascular endothelial cells. The epidermal equivalent of KCFB-EC constructs underwent cellular alterations in their differentiation possibly induced by the presence of endothelial cells. The objective of the study was to assess the effect of endothelial cells, i.e., endothelialization of the dermal equivalent on the differentiation of keratinocytes by comparing …the morphology and ultrastructure of the two types of skin constructs, as well as to excised normal human skin. Hypothesis: The differentiation of keratinocytes is influenced by the presence of endothelial cells. METHODS, PATIENTS, MATERIAL: KCFB constructs (keratinocytes, fibroblasts) and KCFB-EC skin constructs(kera-tinocytes, fibroblasts, endothelial cells) were prepared according to Küchler et al. [25]. After two weeks, the skin constructs were processed for analysis by light microscopy (LM) and electron microscopy (TEM), followed by quantitative, semi-quantitative as well as qualitative assessment. For comparison, analysis by LM and TEM of excised normal human skin was also performed. RESULTS: Both KCFB and KCFB-EC skin constructs and the human skin had all strata of stratified soft-cornified epidermis present. The comparison of the respective layers of the skin constructs brought the following characteristics to light: The KCFB-EC constructs had significantly more mitotic cells in the stratum spinosum, more cell layers in the stratum granulosum and more keratohyalin granules compared to KCFB skin constructs. Additionally, the epidermal architecture was unorganized in the endothelialized constructs and features of excessive epidermal differentiation appeared in KCFB-EC skin constructs. CONCLUSION: The endothelialization of the dermal equivalent caused changes in the differentiation of the epidermis of KCFB-EC skin constructs that may be interpreted as an unbalanced, i.e., uncontrolled or enhanced maturation process. Show more

Keywords: Full-thickness skin constructs, human skin, epidermis, keratinocyte, fibroblast, endothelial cell

DOI: 10.3233/CH-151988

Citation: Clinical Hemorheology and Microcirculation, vol. 61, no. 2, pp. 157-174, 2015

Authors: Riebe, Helene | Konschake, Wolfgang | Haase, Hermann | Jünger, Michael

Article Type: Research Article

Abstract: Standard of care in the therapy of chronic venous insufficiency (CVI) is the use of graduated elastic stockings (GECS). This paper is based on a prospective, mono-centric, open randomised, controlled and cross over study and discusses the hemodynamic effects of GECS and inverse graduated compression stockings (PECS) in 32 healthy volunteers and 32 patients with CVI and the consecutive impact on comfort. The application of stockings was performed sequentially, the allocation of the first stocking was randomized and double blind, wearing period for each stocking type about 7 days with one week of break between wearing periods. Measurements of the …interface pressure were carried out by Picopress® (Microlab Elettronica, Italy) and the venous drainage were registered by strain gauge plethysmography. Mean interface pressure: GECS (level B1): 27.3 mmHg; GECS (level C): 19,6 mmHg; PECS (level B1): 17,8 mmHg; PECS (level C): 24,7 mmHg. Significant increase of EF and decrease of VFI by wearing both types of compression stockings, comparatively GESC resulted in a significantly greater improvement of EF than the PECS. PECS were significantly easier to don and put on compared to the GECS, the PECS tended to slip towards the foot more often. Show more

Keywords: Chronic venous insufficiency, compression stockings, graduated elastic stockings, GECS, progressive pressure, PECS, interface pressure profile

DOI: 10.3233/CH-151989

Citation: Clinical Hemorheology and Microcirculation, vol. 61, no. 2, pp. 175-183, 2015

Authors: Helling, H. | Stephan, B. | Pindur, G.

Article Type: Research Article

Abstract: Activation of coagulation and inflammatory response including the complement system play a major role in the pathogenesis of critical illness. However, only limited data are available addressing the relationship of both pathways and its assessment of a predictive value for the clinical outcome in intense care medicine. Therefore, parameters of the coagulation and complement system were studied in patients with septicaemia and multiple trauma regarded as being exemplary for critical illness. 34 patients (mean age: 51.38 years (±16.57), 15 females, 19 males) were investigated at day 1 of admittance to the intensive care unit (ICU). Leukocytes, complement factors C3a and …C5a were significantly (p  <  0.0500) higher in sepsis than in trauma, whereas platelet count and plasma fibrinogen were significantly lower in multiple trauma. Activation markers of coagulation were elevated in both groups, however, thrombin-antithrombin-complex was significantly higher in multiple trauma. DIC scores of 5 were not exceeded in any of the two groups. Analysing the influences on mortality (11/34; 32.35% ), which was not different in both groups, non-survivors were significantly older, had significantly higher multiple organ failure (MOF) scores, lactate, abnormal prothrombin times and lower C1-inhibitor activities, even more pronounced in early deaths, than survivors. In septic non-survivors protein C was significantly lower than in trauma. We conclude from these data that activation of the complement system as part of the inflammatory response is a significant mechanism in septicaemia, whereas loss and consumption of blood components including parts of the coagulation and complement system is more characteristic for multiple trauma. Protein C in case of severe reduction might be of special concern for surviving in sepsis. Activation of haemostasis was occurring in both diseases, however, overt DIC was not confirmed in this study to be a leading mechanism in critically ill patients. MOF score, lactate, C1-inhibitor and prothrombin time have been the only statistically significant predictors for lethal outcome suggesting that organ function, microcirculation, haemostasis and inflammatory response are essential elements of the pathomechanism and clinical course of diseases among critically ill patients. Show more

Keywords: Septicemia, multiple trauma, coagulation, complement, critical illness

DOI: 10.3233/CH-151993

Citation: Clinical Hemorheology and Microcirculation, vol. 61, no. 2, pp. 185-193, 2015

Authors: Haimerl, M. | Jung, E.-M. | Beyer, L.P. | Pregler, B. | Dollinger, M. | Sieroń, D. | Niessen, C. | Stroszczynski, C. | Wiggermann, P.

Article Type: Research Article

Abstract: PURPOSE: To evaluate whether changes of dynamic contrast-enhanced ultrasound (CEUS)-based liver microcirculation during chronic liver disease are correlated with the extent of impaired liver function, expressed by the indocyanine green (ICG) clearance. MATERIALS AND METHODS: 19 patients underwent CEUS examinations with previous or consecutive ICG clearance test. The ICG plasma disappearance rate (ICG-PDR) was determined using the noninvasive pulse-densitometric LiMON system. Quantification of peak enhancement (PE), rise time (RT) and the wash-in rate (WiR) was performed in the liver parenchyma (ROIliver ) as well as in the portal vein (ROIportal vein ) using a novel quantification software (VueBoxTM ). …To compare quantification parameters, patients were classified in patients representing a healthy population (ICG-PDR >16, n  = 8) and those representing patients with liver disease (ICG-PDR ≤16, n  = 11). RESULTS: ROIportal vein showed significant differences comparing healthy patients and patients with liver disease for all perfusion parameters: PE and WiR were significantly higher in patients without liver disease (p  = 0.048; p  = 0.039). RT was significantly lower in healthy population (p  = 0.039). Comparing perfusion parameters for ROIliver , PE was significantly higher in patients without liver disease (p  = 0.039). There was no significant difference for RT (p  = 0.804) and WiR (p  = 0.058), respectively. CONCLUSION: Within the framework of this study CEUS derived estimation of microcirculation did not prove to be a reliable estimator of liver function. RT, PE and WiR measured in the portal vein were significant perfusion factors in predicting liver function. Show more

Keywords: Chronic liver disease, CEUS, ICG-test, microcirculation, liver function

DOI: 10.3233/CH-151990

Citation: Clinical Hemorheology and Microcirculation, vol. 61, no. 2, pp. 195-204, 2015

Authors: Hiebl, Bernhard | Nennig, Ernst | Schiestel, Stefanie | Kovacs, Adalbert | Jung, Friedrich | Fischer, Harald

Article Type: Research Article

Abstract: Biomaterials made of zinc have been widely described to be antioxidative, hypothrombogenic, antiinflammatory and antiproliferative. Additionally in vivo zinc is toxic only in high concentrations and can completely be metabolized in vivo . Due to these properties zinc based vascular stents might be able to reduce the rate of restenosis in comparison to bare metal stents and zinc stents might be also able to limit the foreign body reaction. In the presented study we tested the biocompatibility and degradability of a stent made of zinc and characterized by a closed-cell-design to achieve high opening force and to increase stent …stiffness. After 100 days of enzymatic and hydrolytic degradation in 15 ml blood serum (fetal calf serum) a significant loss of weight (1.72 wt% ) was measured. Zinc was compared to other metals in terms of degradation rates. After six weeks of incubation in physiologic sodium chloride solution zinc showed the slowest degradation time, 6 times less than stainless steel and 4 times less than magnesium. In the tests for cytotoxic effects the degraded zinc stent caused no changes in the LDH-release and cell membrane integrity (3T3 cells, mouse fibroblasts) respectively, in the cell activity/proliferation (MTS assay) and in the morphological characteristics of the cells and cell layers in comparison to the control material (polystyrene). Based on these results the tested zinc stent proved to be non-cytotoxic and to be characterized by degradation characteristics which might be advantageous in comparison to magnesium and stainless steel. Show more

Keywords: zinc, stent, biocompatibility, degradation rate

DOI: 10.3233/CH-151983

Citation: Clinical Hemorheology and Microcirculation, vol. 61, no. 2, pp. 205-211, 2015