Clinical Hemorheology and Microcirculation - Volume 64, issue 3

Authors: Braune, S. | Basu, S. | Kratz, K. | Johansson, J. Bäckemo | Reinthaler, M. | Lendlein, A. | Jung, F.

Article Type: Research Article

Abstract: Polymer-based microparticles are applied as non-thrombogenic or thrombogenic materials in a wide variety of intra- or extra-corporeal medical devices. As demanded by the regulatory agencies, the hemocompatibility of these blood contacting biomaterials has to be evaluated in vitro to ensure that the particle systems appropriately fulfill the envisioned function without causing undesired events such as thrombosis or inflammation. Currently described in vitro assays for hemocompatibility testing of particles comprise tests with different single cell types (e.g. erythrocytes or leukocytes), varying concentrations/dilutions of the used blood cells or whole blood, which are not standardized. Here, we report about …an in vitro dynamic test system for studying the hemocompatibility of polymeric microparticles utilizing fresh human whole blood from apparently healthy subjects, collected and processed under standardized conditions. Spherical poly(ether imide) microparticles with an average diameter of 140±30 μm were utilized as model systems. Reported as candidate materials for the removal of uremic toxins, these microparticles are anticipated to facilitate optimal flow conditions in a dialyzer with minimal backflow and blood cell damage. Pristine (PEI) and potassium hydroxide (PEI-KOH) functionalized microparticles exhibited similarly nanoporous surfaces (PEI: ØExternal pore  = 90±60 nm; PEI-KOH ØExternal pore  = 150±130 nm) but varying water wettabilities (PEI: θ adv  = 112±10° PEI-KOH θ adv  = 60±2°). The nanoporosity of the microparticle surfaces allows the exchange of toxic solutes from blood towards the interconnective pores in the particle core, while an immigration of the substantially larger blood cells is inhibited. Sterilized PEI microparticles were incorporated –air-free –in a syringe-based test system and exposed to whole blood for 60 minutes under gentle agitation. Thereafter, thrombi formation on the particles surfaces were analyzed microscopically. In the collected whole blood the non-adherent/circulating single blood cells were quantified via a differentiated complete blood cell count and the activation of platelets (P-Selectin expression, secretion and release), platelet function (PFA100 closure time) as well as thrombin formation (thrombin-antithrombin-complex) was analyzed. Free hemoglobin (HGB) levels were quantified as a measure of hemolysis. Microscopic evaluation revealed thrombi formation and particle aggregates for all tested microparticles. Reduction of circulating blood cells differed significantly between the particle types. Particularly, platelet and monocyte counts decreased up to 50% compared to the control (syringe filled with whole blood but without microparticles). In accordance, platelet activation, thrombin levels and degrees of hemolysis were clearly elevated in the particle loaded test systems and allowed a differentiation between the particle types. Increased PFA100 closure times (as activating agent a combination of collagen/ADP was used) indicated a similarly reduced ability of platelets to adhere and form stable aggregates independent from the particle type tested. This observation is most probably a consequence of the strong thrombus formation in the test system, which is associated with a reduction of the circulating blood cells. The reported in vitro dynamic whole blood test system allowed the sensitive analysis of the hemocompatibility of polymer-based microparticles and was successfully validated for porous PEI microparticles with different water wettabilities. Beyond the qualitative and quantitative analysis of cell-material interactions, the test also allowed the functional evaluation of platelets in whole blood. Show more

Keywords: Biomaterial, polymer, microparticle, thrombogenicity, hemocompatibility, dynamic in-vitro test

DOI: 10.3233/CH-168114

Citation: Clinical Hemorheology and Microcirculation, vol. 64, no. 3, pp. 345-353, 2016

Authors: Li, Zhengdong | Wang, Weiwei | Kratz, Karl | Küchler, Judit | Xu, Xun | Zou, Jie | Deng, Zijun | Sun, Xianlei | Gossen, Manfred | Ma, Nan | Lendlein, Andreas

Article Type: Research Article

Abstract: Induced pluripotent stem cells (iPSCs) own the capacity to develop into all cell types of the adult body, presenting high potential in regenerative medicine. Regulating and controlling the differentiation of iPSCs using the surface topographic cues of biomaterials is a promising and safe approach to enhance their therapeutic efficacy. In this study, we tested the effects of surface roughness on differentiation of human iPSCs into neural progenitor cells and dopaminergic neuron cells using polystyrene with different roughness (R0: flat surface; R1: rough surface, Rq ∼ 6 μm; R2: rough surface, Rq ∼ 38 μm). Neural differentiation of human …iPSCs could be influenced by surface roughness. Up-regulated neuronal markers were found in cells on rough surface, as examined by real-time PCR and immunostaining. Particularly, the R1 surface significantly improved the neuronal marker expression, as compared to R0 and R2 surface. This study demonstrates the significance of surface roughness, depending on the roughness level, in promoting differentiation of human iPSCs towards the neuronal lineage. Our study suggests the potential applications of surface roughness in iPSCs based treatment of neural disorder diseases, and highlights the importance of design and development of biomaterials with effective surface structures to regulate stem cells. Show more

Keywords: Human iPSCs, roughness, neural differentiation, regenerative medicine

DOI: 10.3233/CH-168121

Citation: Clinical Hemorheology and Microcirculation, vol. 64, no. 3, pp. 355-366, 2016

Authors: Xu, Xun | Wang, Weiwei | Li, Zhengdong | Kratz, Karl | Ma, Nan | Lendlein, Andreas

Article Type: Research Article

Abstract: The permanent loss of cardiomyocytes may lead to the irreversible damage of myocardium in cardiovascular diseases. The induced pluripotent stem cells (iPSCs) with the capacity of differentiation into a variety of cell types including cardiomyocytes showed high potential for efficient heart regeneration. The iPSCs and iPSC-derived embryoid bodies (EBs) as well as the differentiated cardiomyocytes are highly sensitive to the biophysical cues of their microenvironment, and accordingly their behavior and function can be largely modulated by microstructure of the cell culture surface. In this study, we investigated the regulatory effect of microscale roughness on both cardiomyogenesis and secretion of EBs …using poly(ether imide) (PEI) cell culture inserts with different levels of bottom roughness (R0: flat surface; R1: rough surface, Rq ∼ 4 μm; R2: rough surface, Rq ∼ 23 μm). The proliferation rate and cardiomyogenesis of EBs increased with the increase of surface roughness. The EB secretome derived from R2 surface remarkably enhanced the in vitro new vessel formation of endothelial cells, as compared to those from R0 and R1. These findings highlight the potential to improve the iPSC/EB-based restoration of cardiovascular function via microstructured biomaterials. Show more

Keywords: Microroughness, induced pluripotent stem cells, embryoid body, cardiomyogenesis, secretome

DOI: 10.3233/CH-168107

Citation: Clinical Hemorheology and Microcirculation, vol. 64, no. 3, pp. 367-382, 2016

Authors: Krüger-Genge, Anne | Jung, Friedrich | Fuhrmann, Rosemarie | Franke, Ralf-Peter

Article Type: Research Article

Abstract: BACKGROUND: Endothelial cells (EC) in vivo are strongly influenced by changes of the milieu exterieur. Under pathological conditions EC can become activated e.g. in hypoxic areas or during sepsis. In general, the endothelialization of implant materials is evaluated in vitro under physiological conditions. Though, in patients who receive implant materials pathological conditions are often present. An open question is therefore, how ECs seeded on a body foreign substrate behave in a pathologic microenvironment. In this in vitro study a microenvironment was created mimicking the conditions present in septic patients. To simulate this situation in vitro , …serum of patients with septic shock was added to the culture medium of EC cultured on glass. The samples were sheared in a cone-plate rheometer (shear rate of 6 dyn/cm2 ) with subsequent analysis of the morphology, the microfilament organization and the shear resistance and compared to control cultures of EC without shock serum supplementation. Aim of the study was to investigate whether this in vitro model provides information about the functionality of an EC monolayer on a body foreign surface under pathological conditions. RESULTS: Septic conditions induced severe changes of the morphology of the adherent cells: there was a strong induction of stress fibers. In addition, lots of cells or cell groups were detached visible as denuded areas in the EC monolayer. After shear stress exposure only 28.7% of EC seeded in cell medium supplemented with serum of septic patients remained adherent (control cells: 96.8%). CONCLUSION: The study demonstrates that the microenvironment is of extreme importance for the behavior of EC and that in vivo pathologies can be simulated in vitro . This opens the possibility to evaluate new implant materials under physiological but more important also under certain pathological conditions - simulating the implant size and the disease of the host. Show more

Keywords: Endothelial cells, in vitro, sepsis, shear rate

DOI: 10.3233/CH-168111

Citation: Clinical Hemorheology and Microcirculation, vol. 64, no. 3, pp. 383-389, 2016

Authors: Kaessmeyer, S. | Sehl, J. | Khiao In, M. | Hiebl, B. | Merle, R. | Jung, F. | Franke, R.P. | Plendl, J.

Article Type: Research Article

Abstract: INTRODUCTION: Clinical complications like thrombosis or anaphylaxis have been described to go along with the intra-venous or intra-arterial injection of iodinated contrast media (CM). It has been suggested that the administration of CM affects rheological parameters and thereby causes reduced blood velocity in microvessels. In vitro studies revealed significant buckling of endothelial cells after exposure to CM reducing the lumen of vessels. The aim of this study was to test the influence of CM on three-dimensional microvascular tubules with open lumina within an organotypic soft-tissue co-culture assay in vitro . This model, which is based on the co-culture …of endothelial cells and fibroblasts, allows the analysis and quantitation of different parameters of microvascular endothelial capillary structures. MATERIAL AND METHODS: Human dermal fibroblasts and human dermal microvascular endothelial cells were co-cultured for 10 days. Fibroblasts were adapted to the endothelial cell medium before co-culture and allowed to proliferate as well as produce extracellular matrix. The co-cultures were exposed to three different CM, i.e., Iomeprol (Imeron 400MCT), Iodixanol (Visipaque 320) or Iohexol (Accupaque 350) for 1.5 minutes or 5.0 minutes, respectively. For this, a mixture of CM and cell culture medium in a ratio of 30% CM by volume was prepared. After fixation in methanol/acetone, the endothelial cells were immunolabeled with the endothelial marker anti-CD31 and the tubular structures were assessed morphometrically. RESULTS: In the organotypic soft-tissue co-cultures with fibroblasts, the endothelial cells developed three-dimensional capillary-like structures which expanded via sprouting branches. After incubation with the different CM, the numbers of endothelial tubes (p  = 0.001) and their lengths (p  = 0.003) were significantly lower after the 5 minutes incubation time, when compared to the 1.5 minutes incubation time. The tubular diameters were significantly reduced after 5 minutes (p  < 0.001), when compared to the 1.5 minutes incubation duration. Interestingly, Iomeprol and Iodixanol induced an elongation of the tubular branches during incubation duration of 1.5 minutes (p  = 0.015). However, after 5 minutes incubation, the tubular branches were drastically shorter in the presence of Iomeprol and Iodixanol than the tubular branches of the control (p  = 0.007). SUMMARY AND CONCLUSION: All CM exerted a negative effect on the parameters of in vitro blood vessel development. Show more

Keywords: Soft-tissue co-cultures, microvascular endothelial tubes, angiogenesis, Iomeprol, Iodixanol, Iohexol, contrast media

DOI: 10.3233/CH-168119

Citation: Clinical Hemorheology and Microcirculation, vol. 64, no. 3, pp. 391-402, 2016

Authors: Hanke, Alexander | Prantl, Lukas | Wenzel, Carina | Nerlich, Michael | Brockhoff, Gero | Loibl, Markus | Gehmert, Sebastian

Article Type: Research Article

Abstract: INTRODUCTION: The stem cell rich Stromal Vascular Fraction (SVF) can be harvested by processing lipo-aspirate or fat tissue with an enzymatic digestion followed by centrifugation. To date neither a standardised extraction method for SVF nor a generally admitted protocol for cell application in patients exists. A novel commercially available semi-automated device for the extraction of SVF promises sterility, consistent results and usability in the clinical routine. The aim of this work was to compare the quantity and quality of the SVF between the new system and an established manual laboratory method. MATERIAL AND METHODS: SVF was extracted …from lipo-aspirate both by a prototype of the semi-automated UNiStation™ (NeoGenesis, Seoul, Korea) and by hand preparation with common laboratory equipment. Cell composition of the SVF was characterized by multi-parametric flow-cytometry (FACSCanto-II, BD Biosciences). The total cell number (quantity) of the SVF was determined as well the percentage of cells expressing the stem cell marker CD34, the leucocyte marker CD45 and the marker CD271 for highly proliferative stem cells (quality). RESULTS: Lipo-aspirate obtained from six patients was processed with both the novel device (d) and the hand preparation (h) which always resulted in a macroscopically visible SVF. However, there was a tendency of a fewer cell yield per gram of used lipo-aspirate with the device (d: 1.1×105 ±1.1×105 vs. h: 2.0×105 ±1.7×105 ; p  = 0.06). Noteworthy, the percentage of CD34+ cells was significantly lower when using the device (d: 57.3% ±23.8% vs. h: 74.1% ±13.4%; p  = 0.02) and CD45+ leukocyte counts tend to be higher when compared to the hand preparation (d: 20.7% ±15.8% vs. h: 9.8% ±7.1%; p  = 0.07). The percentage of highly proliferative CD271+ cells was similar for both methods (d:12.9% ±9.6% vs. h: 13.4% ±11.6%; p  = 0.74) and no differences were found for double positive cells of CD34+ /CD45+ (d: 5.9% ±1.7% vs. h: 1.7% ±1.1%; p  = 0.13), CD34+ /CD271+ (d: 24.1% ±12.0% vs. h: 14.2% ±8.5%; p  = 0.07). DISCUSSION: The semi-automated closed system provides a considerable amount of sterile SVF with high reproducibility. Furthermore, the SVF extracted by both methods showed a similar cell composition which is in accordance with the data from literature. This semi-automated device offers an opportunity to take research and application of the SVF one step further to the clinic. Show more

Keywords: Stromal vascular fraction, lipo-aspirate, adipose tissue, mesenchymal stem cells

DOI: 10.3233/CH-168124

Citation: Clinical Hemorheology and Microcirculation, vol. 64, no. 3, pp. 403-412, 2016

Authors: Bramhoff, Anja | Giers, Günther | Blessing, Frithjof | Wenzel, Folker

Article Type: Research Article

Abstract: INTRODUCTION: Referring to current standards the quality of an apheresis procedure is estimated by the quantity of collected cells. Nowadays a new kind of quality measurement could be found in the detection of cell volumina. Recent diagnostics have shown that stem cells and platelets – when separated – are likely to appear in a higher volume inside the cell product. Therefore, in this study the question should be discussed wether platelets of higher volume are more likely to be separated than platelets showing a lesser volume. METHODS: Blood samples of three different apheresis procedures could be observed: …allogenic platelet donations (n  = 7) (Trima, Terumo), autologous (n  = 5) and allogenic stem cell donations (n  = 5) (Cobe Spectra, Terumo). To examine the blood samples the Sysmex hematology analyser (XT-2000) has been used. RESULTS: During stem cell apheresis, the volume of the separated platelets was 1.2 fold increased compared to the platelet volume in the peripheral blood before separation. Before apheresis the mean platelet volume in the peripheral blood was found to be 6,21 fl, after apheresis 6,09 fl and inside the platelet concentrate 7,42 fl. The platelet number in the peripheral blood was also significantly decreased (before separation 180.1/nl and after separation 133.5/nl). In the blood products the concentration of platelets was nearly 8 fold higher than in the peripheral blood before separation. CONCLUSION: Overall, the observed apheresis procedures are more likely to separate platelets showing a higher voulme than common in the peripheral blood. This might indicate that not only the amount of separated cells reflects the quality of the apheresis procedure but also that the volume of the separated cells can be used as a parameter for quality assessment. Show more

Keywords: Platelet volume, apheresis procedure, stem cells, thrombocytes, quality assessment

DOI: 10.3233/CH-168109

Citation: Clinical Hemorheology and Microcirculation, vol. 64, no. 3, pp. 413-424, 2016

Authors: Konschake, Wolfgang | Riebe, Helene | Pediaditi, P. | Haase, Hermann | Jünger, Michael | Lutze, Stine

Article Type: Research Article

Abstract: BACKGROUND: Below knee two-component compression stockings (AD) have revealed as effective for compression treatment of venous leg ulcers. Upto groin, thigh length stocking (AG) may enhance clinical effects, however wear comfort of these stocking may be affected. OBJECTIVE: venous haemodynamic in relation to the length of compression stockings. METHODS: A two-component AD stocking (37 mmHg) and two thigh length stockings (AG 37, with an interface pressure of 37 mmHg; AG 45, with an interface pressure of 45 mmHg) were tested by 16 patients with CVI. Leg volume changes and venous ejection fraction and venous filling …index were measured, whilst quality of life and wear comfort were surveyed by questionnaires. RESULTS: Volume of both the lower limb and the thigh was reduced by AG stockings, whereas AD stockings reduced only the volume of the lower limb and increased thigh volume. Venous hemodynamic, ejection fraction and filling index were improved by AG and AD stockings, AG, however, was superior to AD. Quality of life and comfort of the stockings was assessed as good for AG 37 mmHg, AG 45 mmHg and AD 37 mmHg. CONCLUSIONS: Thigh length two component stockings (AG) were shown to be superior to below knee stocking (AD) with regard to volume reduction and venous hemodynamic, yet wear comfort was not impaired. These results imply that healing of trophic skin changes e.g. ulcers will be faster when thigh length two component stocking will be worn. Show more

Keywords: Chronic venous insufficiency, compression stocking, two-component compression stocking, thigh lengthcompression stocking, below knee compression stocking, venous ejection fraction, venous filling index, leg volume

DOI: 10.3233/CH-168122

Citation: Clinical Hemorheology and Microcirculation, vol. 64, no. 3, pp. 425-434, 2016

Authors: Haimerl, M. | Poelsterl, S. | Beyer, L.P. | Wiesinger, I. | Nießen, C. | Stroszczynski, C. | Wiggermann, P. | Jung, E.-M.

Article Type: Research Article

Abstract: PURPOSE: To compare the diagnostic performance of MRI-based T1 relaxometry with dynamic contrast-enhanced ultrasound (CEUS)-based liver microcirculation for evaluation of liver function. MATERIALS AND METHODS: 22 patients underwent Gd-EOB-DTPA-enhanced MRI with T1 relaxometry and previous or consecutive CEUS examinations. A transverse 3D VIBE sequence with an inline T1 calculation was acquired and the reduction rate of T1 relaxation time (rrT1) was evaluated. For CEUS measurements (1–6 MHz), a bolus injection of 1.4 ml sulfur hexafluoride microbubbles were administered and both cine loops and single images from arterial phase up to late phase were stored. Quantification of time to …peak (TTP), rise time (RT), Wash- In Area Under the Curve (WiAUC), mean transit time (mTTI), the wash- in rate (WiR) and Wash-in perfusion index (WIPI)) was performed using a novel quantification software (VueBoxTM ). To compare quantification parameters, patients were classified in patients representing a healthy population (rrT1 > 50%, n  = 8) and those representing patients with liver disease (rrT1 < 50%, n  = 14). RESULTS: Comparing perfusion parameters TTP, mTTI, and WiR were higher in patients without liver disease compared to patients with impaired liver function (p  = 0.10–0.21). RT, WiAUC and WIPI were significantly lower in patients with impaired liver function (RT, 14.8±1.5 s; WiAUC, 17288±6179 a.u., WIPI, 1243±423) compared to patients without liver disease (RT, 21.2±2.6 s, p  = 0.032; WiAUC, 71534±25600, p  = 0.034; WIPI, 4286±1748, p  = 0.04). In a simple linear regression model, none of the perfusion parameters correlated significantly with rrT1 (p  = 0.08–0.63). CONCLUSION: Within the framework of this study, CEUS-based perfusion parameters were not able to assess severity of liver disease, however, WiAUC, RT and WIPI were significant perfusion parameters to make a rough assessment of liver function. Show more

Keywords: Chronic liver disease, CEUS, microcirculation, liver function

DOI: 10.3233/CH-168112

Citation: Clinical Hemorheology and Microcirculation, vol. 64, no. 3, pp. 435-446, 2016

Authors: Marcon, J. | Trottmann, M. | Rübenthaler, J. | D’Anastasi, M. | Stief, C.G. | Reiser, M.F. | Clevert, D.A.

Article Type: Research Article

Abstract: INTRODUCTION: Shear wave elastography (SWE) and its derivative Supersonic Shear Imaging (SSI) are newer techniques for the determination of tissue elasticity by measuring the velocity of generated shear waves (SWV), which correlates positively with tissue stiffness. The techniques are integrated into many modern ultrasound systems and have been examined in the evaluation of viscoelastic properties of different organ systems. Two-dimensional shear wave elastography (2D SWE) of the testes has been found to be a useful tool in recent studies which included the determination of standard values in healthy volunteers. Three-dimensional shear wave elastography (3D SWE) is the latest …development in elastography and is made possible by generation of a multiplanar three-dimensional map via volumetric acquisition with a special ultrasound transducer. This technique allows the assessment of tissue elasticity in a three-dimensional, fully accessible organ map. The aim of this preliminary study was to both evaluate the feasibility of 3D SWE and to compare 2D and 3D SWE standard values in the testes of healthy subjects. MATERIAL AND METHODS: We examined the testes of healthy male volunteers (n  = 32) with a mean age of 51.06±17.75 years (range 25–77 years) by B-mode ultrasound, 2D and 3D SWE techniques in September of 2016. Volunteers with a history of testicular pathologies were excluded. For all imaging procedures the SL15-4 linear transducer (bandwidth 4–15 MHz) as well as the SLV16-4 volumetric probe (bandwidth 4–16 MHz) of the Aixplorer® ultrasound device (SuperSonic Imagine, Aix-en-Provence, France) were used. Seven regions of interest (ROI, Q-Box® ) within the testes were evaluated for SWV using both procedures. SWV values were described in m/s. Results were statistically evaluated using univariateanalysis. RESULTS: Mean SWV values were 1.05 m/s for the 2D SWE and 1.12 m/s for the 3D SWE. Comparisons of local areas delivered no statistically significant differences (p  = 0.11 to p  = 0.66), except for the region in the central portion in the superior part of the coronal plane (p  = 0.03). Testicular volume was significanty higher by a mean of 1.72 ml when measured with 3D SWE (p  = 0.001). CONCLUSION: 3D SWE proved to be a feasible diagnostic tool in the assessment of testicular tissue, providing the examiner with a fully accessible three-dimensional map in a multiplanar or multislice view. With this technique a more precise testicular imaging – especially if combined with the display of tissue stiffness in SWE – is available and therefore could improve the diagnostic work-up of scrotal masses or the routine investigation of infertile men. Further studies for a better understanding in the context of various testicular pathologies will be required. Show more

Keywords: Elastography, testes, infertility

DOI: 10.3233/CH-168115

Citation: Clinical Hemorheology and Microcirculation, vol. 64, no. 3, pp. 447-456, 2016