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BACKGROUND: Physical training in patients with heart failure can affect hemodynamic, cardiac and angiogenesis parameters. OBJECTIVE: The aim of the present study was to investigate the effects of traditional moderate-intensity rehabilitation training and interval training on some angiogenesis factors in coronary artery bypass graft (CABG) patients. METHODS: Thirty CABG patients (mean age±SD, 55±3 years) were randomly assigned to one of three groups: high-intensity interval training (HIIT) or moderate-intensity continuous training (MICT) or the control group. After the initial assessments, eligible patients in the experimental groups (HIIT and MICT) performed exercise training for 8 weeks, while the control group did not. Angiogenesis and angiostatic indices, including pro-adrenomedullin (pro-ADM), basic fibroblast growth factor (bFGF), and endostatin, were then measured. RESULTS: The results showed no significant difference between pro-ADM in the HIIT and MICT groups (P = 0.99), but a significant difference was found between MICT and the control group and between HIIT and the control group (P = 0.001). There is also no significant difference between the bFGF levels in the HIIT and MICT training groups (P = 1.00), but the changes in this factor between the training groups and the control group were significant (P = 0.001). There was a significant difference between the levels of endostatin in all three groups. CONCLUSIONS: Two methods of cardiac rehabilitation (HIIT and MICT) may be useful for the recovery of patients with coronary artery bypass grafting. This improvement manifested itself in changes in angiogenesis and angiostatic indices in this study. However, more extensive studies are needed to investigate the effects of these two types of rehabilitation programs on other indicators of angiogenesis and angiostatic.

Sulfated biomolecules are known to influence numerous biological processes in all living organisms. Particularly, they contribute to prevent and inhibit the hypercoagulation condition. The failure of polymeric implants and blood contacting devices is often related to hypercoagulation and microbial contamination. Here, bioactive sulfated biomacromolecules are mimicked by sulfation of poly(glycerol glycidyl ether) (polyGGE) films. Autoclaving, gamma-ray irradiation and ethylene oxide (EtO) gas sterilization techniques were applied to functionalized materials. The sulfate group density and hydrophilicity of sulfated polymers were decreased while chain mobility and thermal degradation were enhanced post autoclaving when compared to those after EtO sterilization. These results suggest that a quality control after sterilization is mandatory to ensure the amount and functionality of functionalized groups are retained.

BACKGROUND: Ultrasound follow-up of transjugular intrahepatic portosystemic shunt (TIPS) is challenging due to the bent course of the stent-graft. OBJECTIVE: Aim of this retrospective study was to assess to which extent the combination of HR flow with Glazing Flow improves hemodynamic assessment in the ultrasound follow-up of TIPS. METHODS: Comparative studies with CCDS and High Resolution (HR)-Flow with Glazing Flow were evaluated regarding image quality and artifacts on a 5-point scale (0 = cannot be assessed up to 5 = maximum image quality without artifacts). In all cases, an experienced examiner performed the examinations with a 1–6 MHz probe (Resona 7, Mindray). RESULTS: 61 ultrasound examinations in 48 patients were performed; the mean patient age was 54±14.2 years. The use of HR-Flow with Glazing Flow resulted in an improved flow display in 55/61 cases (90.2%). Both methods correlated well (r = 0.71), but HR flow with Glazing flow values were in general higher than CCDS values. The reading resulted in an average value of 2.52±0.54 for CCDS and 3.52±0.57 for HR flow with Glazing flow (p = 0.013). CONCLUSION: The combination of HR-Flow and Glazing Flow results in improved flow representation and reduction of artifacts in the ultrasound follow-up of TIPS.

AIM: To examine to what extent the high frame rate contrast-enhanced ultrasound (HiFR) diagnostic enables the conclusive diagnosis of liver changes with suspected malignancy. MATERIAL/METHODS: Ultrasound examinations were performed by an experienced examiner using a multifrequency probe (SC6-1) on a high-end ultrasound system (Resona 7, Mindray) to clarify liver changes that were unclear on the B-scan. A bolus of 1–2.4 ml of the Sulphur hexafluoride ultrasound microbubbles contrast agent SonoVue™ (Bracco SpA, Italy) was administered with DICOM storage of CEUS examinations from the early arterial phase (5–15 s) to the late phase (5–6 min). Based on the image files stored in the PACS, an independent reading was performed regarding image quality and finding-related diagnostic significance (0 not informative/non-diagnostic to 5 excellent image quality/confident diagnosis possible). References were clinical follow-up, if possible, comparison to promptly performed computed tomography or magnetic resonance imaging, in some cases also to histopathology. RESULTS: We examined 100 patients (42 women, 58 men, from 18 years to 90 years, mean 63±13 years) with different entities of focal and diffuse liver parenchymal changes, which could be detected in all cases with sufficient image quality with CEUS and with high image quality with HiFR-CEUS. Proportionally septate cysts were found in n = 19 cases, scars after hemihepatectomy with local reduced fat in n = 5 cases, scars after microwave ablation in n = 19 cases, hemangiomas in n = 9 cases, focal nodular hyperplasia in n = 8 cases, colorectal metastases in n = 15 cases, hepatocellular carcinoma (HCC) in n = 11 cases, Osler disease in n = 8 cases. The size of lesions ranged from 5 mm to 200 mm with a mean value of 33.1±27.8 mm. Conclusive diagnoses could be made by the experienced investigator in 97/100 cases with CEUS, confirmed by reference imaging, in parts by histopathology or follow-up. The image quality for HiFR CEUS was rated with a score of 3 to 5; 62 cases were assessed with an average of good (4 points), 27 cases with very good (5 points), and in 11 cases (3 points) still satisfactory despite aggravated acoustic conditions. The specificity of HIFR-CEUS was 97%, the sensitivity 97%, the positive predictive value 94%, the negative predictive value 99% and the accuracy 97%. CONCLUSION: HIFR-CEUS has demonstrated has demonstrated an improved image quality resulting in a high diagnostic accuracy. In the hands of an experienced investigator, HiFR-CEUS allows the assessment of focal and diffuse unclear liver parenchymal changes on B-scan and dynamic assessment of microcirculation in solid and vascular changes.

Establishing an endothelial cell (EC) monolayer on top of the blood contacting surface of grafts is considered to be a promising approach for creating a hemocompatible surface. Here we utilized the high affinity interactions between the EC plasma membrane expressed enzyme called endothelin converting enzyme-1 (ECE-1) and its corresponding substrate big Endothelin-1 (bigET-1) to engineer an EC-specific binding surface. Since enzymatic cleavage of substrates require physical interaction between the enzyme and its corresponding substrate, it was hypothesized that a surface with chemically immobilized synthetic bigET-1 will preferentially attract ECs over other types of cells found in vascular system such as vascular smooth muscle cells (VSMCs). First, the expression of ECE-1 was significantly higher in ECs, and ECs processed synthetic bigET-1 to produce ET-1 in a cell number-dependent manner. Such interaction between ECs and synthetic bigET-1 was also detectible in blood. Next, vinyl-terminated self-assembled monolayers (SAMs) were established, oxidized and activated on a glass substrate as a model to immobilize synthetic bigET-1 via amide bonds. The ECs cultured on the synthetic bigET-1-immobilized surface processed larger amount of synthetic bigET-1 to produce ET-1 compared to VSMCs (102.9±5.13 vs. 9.75±0.74 pg/ml). The number of ECs bound to the synthetic bigET-1-immobilized surface during 1 h of shearing (5dyne/cm2) was approximately 3-fold higher than that of VSMCs (46.25±12.61 vs. 15.25±3.69 cells/100×HPF). EC-specific binding of synthetic bigET-1-immobilized surface over a surface modified with collagen, a common substance for cell adhesion, was also observed. The present study demonstrated that using the substrate-enzyme affinity (SEA) of cell type-specific enzyme and its corresponding substrate can be an effective method to engineer a surface preferentially binds specific type of cells. This novel strategy might open a new route toward rapid endothelialization under dynamic conditions supporting the long-term patency of cardiovascular implants.

Biofouling on medical device surfaces, which is initiated by protein adsorption and adhesion of microbes especially the antibiotic-resistant bacteria, attracts global attention for centuries due to its enduring challenges in healthcare. Here, the antifouling effect of hydrophilic poly(glycerol glycidyl ether) (polyGGE) film is explored in comparison to hemocompatible and protein-resistant control polymers. The chemical and thermomechanical stability of polyGGE in hydrated conditions at body temperature was achieved via adjusting UV curing and KOH quenching time. The polyGGE surface is inert to the plasma protein adsorption and interfered the metabolism conditions, biofilm formation and growth of both Gram negative (Gram–) and antibiotic-resistant Gram positive (Gram+) bacteria. These results indicate the potential application of polyGGE for combating the risk of hospital-acquired infections and preventing drug-resistant superbug spreading.

BACKGROUND: The laser Doppler fluxmetry (LDF) is a non-invasive method to assess skin blood perfusion, measuring the flow of blood cells inside a tissue volume without harming the tissue. In the diagnosis of skin circulation disorders, the results of the LDF measurement are generally used in such a way that “normal” (or non-ill) or “pathological” values are achieved by comparison with a reference sample, for example of apparently healthy subjects. MATERIAL AND METHODS: In this study, the values of LDF for the diagnosis of microcirculatory disorders in patients with coronary artery disease (n = 20) or in patients with microcirculatory disorders, already diagnosed by capillary microscopy (n = 46), were examined. RESULTS: The mean values of LD amplitudes in the four frequency windows for patients with coronary artery disease were in the reference range. However, some of the patients showed reduced LD values: in eleven of the twenty patients, one or more mean LD amplitudes were below the reference range. Four of the eleven patients had pathologically decreased capillary erythrocyte velocities of v ery = 0.09–0.21 [mm/s], while the other seven patients had normal blood circulation at rest. For all patients with a proven cutaneous microcirculatory disorder, the mean LD amplitude in at least one of the frequency windows FF2 to FF4 was pathologically reduced. CONCLUSION: The Laser-Doppler fluxmetry method used in the study allows the reliable diagnosis of cutaneous microcirculatory disorders.

AIM: Aim of this study was to firstly describe reproducible, objective perfusion parameters of contrast-enhanced ultrasound (CEUS) kinetics of parathyroid gland adenoma (PA) using perfusion analysis software (VueBox (R), Bracco, Italy). Thereby the efficiency of quantitative CEUS for characterization of PA should be evaluated comparing US to postoperative histopathological findings after PA resection. MATERIAL AND METHODS: 42 patients with symptoms/lab work suggestive of pHPT presented a parathyroid gland lesion in B-mode US, which was consequently analyzed by dynamic CEUS. CEUS was performed by one experienced examiner after i.v.-injection of max. 2.4 ml sulphurhexaflouride microbubbles saving digital DICOM cine loops (up to 25 s) and images. PA were evaluated during arterial, venous and late phase (up to 3 min.) for perfusion characterization. A retrospective, blinded VueBox (R) perfusion analysis of arterial phase of 28/42 PA was performed by a second, independent examiner placing 3 ROIs manually in the PA (center, rim of PA, surrounding thyroid gland tissue) to objectify findings. US findings were correlated to postoperative histology after PA resection. RESULTS: Out of 42 patients with PA findings in CEUS, perfusion analysis could be performed in 28/42 cases only as some CEUS cine loops had too much moving. In three cases the second examiner could not detect PA retrospectively, in 25 cases PA were characterized correctly resulting in a sensitivity rate of 89.3 %. VueBox (R) perfusion analysis confirmed that PA present a persisting hypervascularization of the rim with higher TTP (mean 7.93 s centrally, 8.36 s rim-sided), mTT (mean 56.6 s centrally, 64.5 s rim-sided) and lower PE (mean 10542.93 rm(2) centrally, 8909.21 rm(2) rim-sided) peripherally followed by a central wash-out during later phases. RT was comparable in all defined regions. CONCLUSION: VueBox (R) analysis of parathyroid gland CEUS examinations seemed to be a valuable tool for quantification of a PA's perfusion and can help to detect and localize hyperfunctional parathyroid glands prior to surgery.

Background Remote ischemic preconditioning (RIPC) has been demonstrated to induce potent cardioprotection in individuals experiencing coronary ischemia. A protocol combining limb ischemia and electronic muscle stimulation of the ischemic skeletal muscle (RIPC+), performed in advance of coronary artery occlusion, was superior in terms of infarct size reduction when compared to RIPC alone. Methods This study was performed to evaluate the benefit of RIPC + in humans compared to a standard RIPC protocol and a control group. Patients with a single vessel coronary artery disease undergoing elective PCI were eligible to participate in this study. ST-segment elevations from an intracoronary ECG during 3 brief episodes of coronary artery balloon occlusions/dilatation were used as the primary endpoint. Results ST-elevations significantly declined from the first to the third angioplasty in the control but remained at the same level in the RIPC and RIPC+groups. The RIPC group was characterized by the lowest ST-segment shift during coronary ischemia, which was comparable to coronary balloon occlusion number 3 in the control group, indicating successful preconditioning by the conventional RIPC method. In contrast, ST segment elevations were significantly higher in the RIPC + group. Troponin levels taken 24 h after the study procedure were significantly lower in the RIPC when compared to the control and the RIPC + group. Conclusion Our results again confirm the feasibility of remote ischemic preconditioning in patients undergoing coronary angioplasty. According to our results ischemia combined with electronic skeletal muscle stimulation was not superior to conventional RIPC cycles (skeletal muscle ischemia alone).

Implantation of synthetic small-diameter vascular bypass grafts is often associated with an increased risk of failure, due to thrombotic events or late intimal hyperplasia. As one of the causes an insufficient hemocompatibility of the artificial surface is discussed. Endothelialization of synthetic grafts is reported to be a promising strategy for creating a self-renewing and regulative anti-thrombotic graft surface. However, the establishment of a shear resistant cell monolayer is still challenging. In our study, cyto- and immuno-compatible poly(ether imide) (PEI) films were explored as potential biomaterial for cardiovascular applications. Recently, we reported that the initial adherence of primary human umbilical vein endothelial cells (HUVEC) was delayed on PEI-films and about 9 days were needed to establish a confluent and almost shear resistant HUVEC monolayer. To accelerate the initial adherence of HUVEC, the PEI-film surface was functionalized with an aptamer-cRGD peptide based endothelialization supporting system. With this functionalization the initial adherence as well as the shear resistance of HUVEC on PEI-films was considerable improved compared to the unmodified polymer surface. The in vitro results confirm the general applicability of aptamers for an efficient functionalization of substrate surfaces.

Copolyetheresterurethane (PDC) is a biodegradable, shape-memory biomaterial, which has been shown to be of low toxicity and pro-angiogenic in vitro. In the present study we examined the in vivo compatibility of PDC as a compression molded film and as electrospun scaffolds and its well established constituent, the homopolymer poly(p-dioxanone) (PPDO), which were compared with the clinically used poly[(vinylidene fluoride)-co-hexafluoropropene] (PVDF) as reference material. The materials were implanted in the subcutaneous tissue of mice and the host responses were analyzed histologically 7 and 28 days after implantation. All materials induced a foreign body response (FRB) including the induction of foreign body giant cells and a peripheral fibrous capsule. PDC, PPDO and PVDF films showed no signs of degradation after 28 days. PDC films showed a significantly reduced associated macrophage layer and fibrous capsule on their surface. Few fragments of PDC and PPDO scaffolds were present at the implantation site, while PVDF scaffolds were still present in large amounts at day 28. Especially aligned electrospun PDC scaffold induced a significantly thinner fibrous and a slightly reduced inflammatory response after 28 days of implantation. In addition, only PDC aligned fibrous scaffold structures induced a significant increase in angiogenesis. In summary, PDC films outperformed PPDO and PVDF films in terms of compatibility, especially in capsule and macrophage layer thickness. Through microstructuring of PDC and PPDO into scaffolds an almost complete degradation was observed after 28 days, while their respective films remained almost unchanged. However, the capsule thickness of all scaffolds was comparable to the films after 28 days. Finally, the parallel arrangement of PDC fibers enabled a strong enhancement of angiogenesis within the scaffold. Hence, material chemistries influence overall compatibility in vivo, while angiogenesis could be influenced more strongly by microstructural parameters than chemical ones.

The endothelialization of synthetic surfaces applied as cardiovascular implant materials is an important issue to ensure the anti-thrombotic quality of a biomaterial. However, the rapid and constant development of a functionally-confluent endothelial cell monolayer is challenging. In order to investigate the compatibility of potential implant materials with endothelial cells several in vitro studies are performed. Here, glass and tissue culture plates (TCP) are often used as reference materials for in vitro pre-testing. However, a direct comparison of both substrates is lacking. Therefore, a comparison of study results is difficult, since results are often related to various reference materials. In this study, the endothelialization of glass and TCP was investigated in terms of adherence, morphology, integrity, viability and function using human umbilical vein endothelial cells (HUVEC). On both substrates an almost functionally confluent HUVEC monolayer was developed after nine days of cell seeding with clearly visible cell rims, decreased stress fiber formation and a pronounced marginal filament band. The viability of HUVEC was comparable for both substrates nine days after cell seeding with only a few dead cells. According to that, the cell membrane integrity as well as the metabolic activity showed no differences between TCP and glass. However, a significant difference was observed for the secretion of IL-6 and IL-8. The concentration of both cytokines, which are associated with migratory activity, was increased in the supernatant of HUVEC seeded on TCP. This result matches well with the slightly increased number of adherent HUVEC on TCP. In conclusion, these findings indicate that both reference materials are almost comparable and can be used equivalently as control materials in in vitro endothelialization studies.

Pericytes reside within the basement membrane of small vessels and are often in direct cellular contact with endothelial cells, fulfilling important functions during blood vessel formation and homeostasis. Recently, these pericytes have been also identified as mesenchymal stem cells. Mesenchymal stem cells, and especially their specialized subpopulation of pericytes, represent promising candidates for therapeutic angiogenesis applications, and have already been widely applied in pre-clinical and clinical trials. However, cell-based therapies of ischemic diseases (especially of myocardial infarction) have not resulted in significant long-term improvement. Interestingly, pericytes from a hematopoietic origin were observed in embryonic skin and a pericyte sub-population expressing leukocyte and monocyte markers was described during adult angiogenesis in vivo. Since mesenchymal stem cells do not express hematopoietic markers, the latter cell type might represent an alternative pericyte population relevant to angiogenesis. Therefore, we sourced blood-derived angiogenic cells (BDACs) from monocytes that closely resembled hematopoietic pericytes, which had only been observed in vivo thus far. BDACs displayed many pericytic features and exhibited enhanced revascularization and functional tissue regeneration in a pre-clinical model of critical limb ischemia. Comparison between BDACs and mesenchymal pericytes indicated that BDACs (while resembling hematopoietic pericytes) enhanced early stages of angiogenesis, such as endothelial cell sprouting. In contrast, mesenchymal pericytes were responsible for blood vessel maturation and homeostasis, while reducing endothelial sprouting.Since the formation of new blood vessels is crucial during therapeutic angiogenesis or during integration of implants into the host tissue, hematopoietic pericytes (and therefore BDACs) might offer an advantageous addition or even an alternative for cell-based therapies.

BACKGROUND Physical and chemical characteristics of implant materials determine the fate of long-term cardiovascular devices. However, there is still a lack of fundamental understanding of the molecular mechanisms occurring in the material-tissue interphase. In a previous study, soft covalently crosslinked poly(n-butyl acrylate) networks (cPnBA) were introduced as sterilizable, non-toxic and immuno-compatible biomaterials with mechanical properties adjustable to blood vessels. Here we study the influence of different surface treatments in particular oxygen plasma modification and fibrinogen deposition as well as a combinatorial approach on the adhesion and viability of fibroblasts. MATERIAL AND METHODS Two types of cPnBA networks with Young's moduli of 0.19±0.01 MPa (cPnBA04) and 1.02±0.01 MPa (cPnBA73) were synthesized and post-modified using oxygen plasma treatment (OPT) or fibrinogen coating (FIB) or a combination of both (OPT+FIB). The water contact angles of the differently post-treated cPnBAs were studied to monitor changes in the wettability of the polymer surfaces. Because of the key role of vascular fibroblasts in regeneration processes around implant materials, here we selected L929 fibroblasts as model cell type to explore morphology, viability, metabolic activity, cell membrane integrity as well as characteristics of the focal adhesions and cell cytoskeleton on the cPnBA surfaces. RESULTS Compared to non-treated cPnBAs the advancing water-contact angles were found to be reduced after all surface modifications (p

In cancer therapy, a number of drugs with different mechanisms of action are in clinical use, which act directly after administration without metabolism, while others only become active in the metabolites produced in the liver. Such drugs/metabolites – especially when administered parenterally – interact in high concentrations with the endothelium. Whether this induces adverse responses of the endothelial cells (EC) is barely studied for many medicaments. This pilot in vitro study revealed that the addition of cyclophosphamide (CPA) to the culture medium (5 or 10 mM, respectively) showed a clear influence on EC compared to non-treated EC: The number of adherent human vein endothelial cells (HUVEC) decreased by the addition of CPA in a concentration-dependent manner compared to the untreated control, whereby the vitality of adherent cells was not affected. In addition, concomitant with activation of the adherent HUVEC, increased migratory activity occurred. These results are in agreement with clinical events like thromboses in patients in compromised condition under therapy with CPA, as the detachment of EC might induce responses of circulating platelets leading to the adherence and aggregation with the risk of the formation of thrombi. Whether CPA acts directly or via toxic metabolites on EC will be examined in more detail in following studies.

Ultrasound contrast agents (USCA) allows the dynamic detection of blood flow of both the macro and microvasculature. An obvious prerequisite for USCAs is the unhindered passage of clinically relevant dose levels through the microcirculation especially of the lungue, where they have to pass capillaries with diameters of around 4 μm. While smaller microbubbles rapidly passed through the microcirculation along with the red blood cells, larger microbubbles, however, were observed to coalesce and interrupt the blood flow. Whether this might influence the tissue oxygen tension is unclear up to now.To examine this question a bolus of 2.4 ml SonoVue™ was injected into the suprarenal aorta at a flow rate of 10 ml/s (a dosage usually applied in the clinic). The pO2 in the outer medulla of the kidney was continuously measured using a flexible pO2 microcatheter. In addition, the SonoVue™ injection and its passage through the renal vasculature were documented by the CEUS technology to assess whether the microbubbles passed the kidney.The study revealed that SonoVue™ induced no changes of the mean oxygen partial pressure in the outer medulla which confirms that these microbubbles on their way through the medullar capillaries did not hinder the co-flow of blood through the renal microvessels in a big animal model with a renal morphology and function comparable to human kidneys. These results demonstrate that the CEUS diagnostic itself did not influence the system to be examined which is a most important prerequisite for any diagnostic method.

In drug eluting stents the cytostatic drugs Sirolimus or Tacrolimus are used to inhibit blood vessel restenosis by limiting the proliferation of smooth muscle cells. However, the cytostatic activity of both drugs was shown to be not cell specific and could also affect the stent endothelialisation, respectively. Currently, only limited in vitro data are available about the impact of Sirolimus and Tacrolimus on endothelial cell proliferation over a broad concentration range. To answer this question the following study was performed.Commercially obtained HUVEC were expanded with DMEM cell culture medium (GIBCO, Germany) supplemented with 5 vol% fetal calf serum on non-coated regular polystyrene-based 24-multiwell plates. For drug testings 2×104 cells/cm2 were seeded and grown for 24 h until 30-40% of the multiwell surfaces were covered and then exposed to Sirolimus (1.0×10-11 - 1.0×10-5 mol/l) or Tacrolimus (2.0×10-8 - 6.2×10-5 mol/l), both dissolved in DMSO. 12, 24 and 48 h after adding the drugs cell numbers per area were quantified by counting the cells in six wells with four fields of view per well, representing 0.6 mm2, using a confocal laser microscope.After 48 h of cell growth in the drug-free cell culture medium, the HUVEC number increased from 2.0×104 to 3.55×104 cells/cm2 (mean cell doubling time: 53.6 h, n = 6). At lower concentrations (≤2.0×10-6 mol/l) Tacrolimus reduced the number of adherent HUVEC significantly less than Sirolimus (p < 0.05). However, at higher concentrations (≥2.07×10-5 mol/l) the effect of Tacrolimus on the number of adherent endothelial cells was significantly greater than that of Sirolimus (p < 0.05). At the highest concentration applied (6.22×10-5 mol/l), Tacrolimus induced detachment of all HUVECs within 12 h after drug application. The number of adherent HUVEC decreased only slightly (about 9%) after Sirolimus application at the highest concentration (1.09×10-5 mol/l).These data show that in a non-flow model the cytostatic drug Tacrolimus reduced the number of adherent endothelial cells less than Sirolimus, as long as the drug concentration did not surpass 10-6 mol/l. At the limits of solubility, Sirolimus (1×10-5 mol/l) reduced the number of adherent endothelial cells less than Tacrolimus (6×10-5 mol/l), which induced detachment of endothelial cells.

Cell-based therapies often face the challenge of low cell retention and viability upon transplantation. Hence, biomaterials, which can immobilize transplanted cells, while at the same time support cell viability, are essential for successful clinical application. Noteworthy, biomaterials in the micrometer range such as microcapsules or microspheres have the advantage of a minimally invasive introduction into tissue.Hence, we established an approach to generate gelatin-based cell carriers in the form of microspherical hydrogels. Fibroblasts were microencapsulated in glycidylmethacrylate (GMA)-functionalized gelatin by photopolymerization. While the degree of GMA-functionalization was kept constant, the hydrogel cross-linking density was adjusted by varying the time of irradiation or the average gelatin-chain length.Stable microspheres were synthesized from 10 wt% GMA-gelatin solutions for all irradiation periods tested (0.5 -2 min). Evaluation of cell viability revealed that microgels with the same weight content of biopolymer but with decreased cross-linking densities and thus decreased storage and E modulus, resulted in best cell support. Noteworthy, encapsulated cells partially migrated out of the microspheres and attached to the spherical surface.10 wt% GMA-gelatin-based hydrogels with E moduli comparable to the native cellular niche proved to be a promising biomaterial suitable for the production of cell-laden microspheres and shall be evaluated further for biomedical application.

Angiogenesis is one of the key processes during development, wound healing and tumor formation. Prerequisite for its existence is the presence of endogenous electrical fields (EFs) generated by active ion transport across polarized epithelia and endothelia, and appearance of the transcellular potentials. During angiogenesis cellular factor as endothelial growth factor (VEGF), synthesis of adhesive proteins and membrane metalloproteinases (MMPs) govern the angiogenic response to different external stimuli as biomaterials interactions and/or exogenous EF. Gelatin-based hydrogels with elasticities comparable to human tissues have shown to influence cell behavior as well as cell attachment, protein synthesis, VEGF and MMP's production after the application of EF. Gelatin-based matrices with 3 (G10_LNCO3), 5 (G10_LNCO5), and 8 (G10_LNCO8) fold excess of isocyanate groups per mol of amine groups present in gelatin were used. Human umbilical endothelial cells (HUVEC) (Lonza Basel, Switzerland) and highly invasive breast cancer MDA-MB-231 cells (ATCC®HTB-26TM) were used. For an estimation of the amount of VEGF released from cells a commercially available VEGF ELISA (Thermo Fisher Scientific, Germany) kit was used. Fibronectin (FN) enzyme immunoassay (EIA) was used to analyze the secreted amount of FN by cells seeded on the materials. Secreted MMPs were analyzed by zymography. Gelatin-based hydrogels attracted HUVEC adhesion and diminished the adhesion of MDA-MB-231 cells. The applied direct current (DC) EF induced an almost 5-fold increase in VEGF production by HUVEC seeded on gelatin-based hydrogels, while in contrast, the applied EF decreased the production of VEGF by cancer cells. FN synthesis was elevated in HUVEC cells seeded on gelatin-based materials in comparison to FN synthesis by cancer cells. HUVEC seeded on gelatin hydrogels showed an expression mainly of MMP-2. The application of EF increased the production of MMP-2 in HUVEC seeded on gelatin materials. In contrast, for MDA-MB-231 the production of MMPs on gelatin materials was lower compared to control materials. With the application of EF the levels of MMP-9 decreased but MMP-2 expression raised significantly for gelatin materials. Overall, the results showed that studied gelatin materials suppressed attachment of cancerous cells, as well as suppressed their angiogenic potential revealed by decreased VEGF and MMP production. Thus, this study approved gelatin-based hydrogels with proper elasticity characteristics and different degradation behavior as useful matrices for use in vascular tissue regeneration or in restriction of tumor growth after tumor resection.

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.

BACKGROUND Magnetocardiography (MCG) is a non-invasive technique and to characterize the magnetic field, a pseudo-current conversion was used. The role of MCG in detecting left atrial (LA) dysfunction in patients with paroxysmal atrial fibrillation (PAF) is unknown. OBJECTIVE The aim of this study was to evaluate LA function using MCG in patients with PAF and healthy subjects, to identify possible indices to diagnose PAF. METHODS We enrolled a total of 70 subjects including 26 healthy volunteers (group 1) and 22 marathon runners (group 2) who did not exhibit any cardiac abnormalities, and 22 patients with PAF (group 3) which was documented by electrocardiography (ECG). Spatiotemporal activation graph (STAG) in base-apex and left-right direction was reconstructed. The maximum value of LA pseudo-current under rest and peak exercise were measured between the end of the P wave and beginning of the Q wave. RESULTS LA pseudo-current increase at peak exercise in PAF patients was significantly lower than in healthy volunteers and marathon runners (0.4±0.3 pT in group 3 vs. 0.8±0.3 pT in group 1 vs. 1.1±0.5 pT in group 2, p

Polymers are often contaminated with lipopolysaccharides also known as endotoxins. Even small amounts of endotoxins can have strong effects on endothelial cell function so that the endothelialisation of cardiovascular implants might be hampered. An open question is how endothelial cells seeded on a body foreign substrate respond to shear load after adding Lipid A (LPA), the domain, which is responsible for much of the toxicity of gram-negative bacteria, and whether morphological changes of endothelial cells occur. LPA supplementation to the culture medium in increasing concentrations (5, 25 and 50μg/ml) resulted in progressive reductions of the density of adherent HUVEC after shear load (p

Circulating blood cells are prone to varying flow conditions when contacting cardiovascular devices. For a profound understanding of the complex interplay between the blood components/cells and cardiovascular implant surfaces, testing under varying shear conditions is required. Here, we study the influence of arterial and venous shear conditions on the in vitro evaluation of the thrombogenicity of polymer-based implant materials. Medical grade poly(dimethyl siloxane) (PDMS), polyethylene terephthalate (PET) and polytetrafluoroethylene (PTFE) films were included as reference materials. The polymers were exposed to whole blood from healthy humans. Blood was agitated orbitally at low (venous shear stress: 2.8 dyne. cm(-2)) and high (arterial shear stress: 22.2 dyne .cm(-2)) agitation speeds in a well-plate based test system. Numbers of non-adherent platelets, platelet activation (P-Selectin positive platelets), platelet function (PFA100 closure times) and platelet adhesion (laser scanning microscopy (LSM)) were determined. Microscopic data and counting of the circulating cells revealed increasing numbers of material-surface adherent platelets with increasing agitation speed. Also, activation of the platelets was substantially increased when tested under the high shear conditions (P-Selectin levels, PFA-100 closure times). At low agitation speed, the platelet densities did not differ between the three materials. Tested at the high agitation speed, lowest platelet densities were observed on PDMS, intermediate levels on PET and highest on PTFE. While activation of the circulating platelets was affected by the implant surfaces in a similar manner, PFA closure times did not reflect this trend. Differences in the thrombogenicity of the studied polymers were more pronounced when tested at high agitation speed due to the induced shear stresses. Testing under varying shear stresses, thus, led to a different evaluation of the implant thrombogenicity, which emphasizes the need for testing under various flow conditions. Our data further confirmed earlier findings where the same reference implants were tested under static (and not dynamic) conditions and with fresh human platelet rich plasma instead of whole blood. This supports that the application of common reference materials may improve inter-study comparisons, even under varying test conditions.

BACKGROUND Exercise electrocardiography (ECG) is frequently used as a diagnostic measure in patients with suspected coronary artery disease (CAD). However, it has low sensitivity for the detection of CAD. Magnetocardiography (MCG) has been proposed as an alternative tool to accurately diagnose CAD. OBJECTIVE To date, a direct comparison of MCG to ECG has not been performed. This study sought to compare them for predicting the presence of significantly obstructive CAD. METHODS The patients with chest pain or other symptoms suggestive of CAD were enrolled in the analysis. All the patients underwent a clinical evaluation, exercise ECG, MCG test, and coronary angiography (CA). CAD was defined as stenosis ≥70% in at least one major coronary artery on quantitative analysis of CA. RESULTS We prospectively enrolled 202 consecutive patients who suggested CAD. The prevalence of CAD on CA was 39.1%. Sensitivity and accuracy for CAD diagnosis was higher for MCG compared with exercise ECG (sensitivities 68.4% and 40.5%, p

BACKGROUND: The formation of a functionally-confluent endothelial cell (EC) monolayer affords proliferation of EC, which only happens in case of appropriate migratory activity. AIM OF THE STUDY: The migratory pathway of human umbilical endothelial cells (HUVEC) was investigated on different polymeric substrates. MATERIAL AND METHODS: Surface characterization of the polymers was performed by contact angle measurements and atomic force microscopy under wet conditions. 30,000 HUVEC per well were seeded on polytetrafluoroethylene (PTFE) (theta(adv) = 119 degrees +/- 2 degrees), on low-attachment plate LAP (theta(adv) = 28 degrees +/- 2 degrees) and on polystyrene based tissue culture plates (TCP, theta(adv) = 22 degrees +/- 1 degrees). HUVEC tracks (trajectories) were recorded by time lapse microscopy and the euclidean distance (straight line between starting and end point), the total distance and the velocities of HUVEC not leaving the vision field were determined. RESULTS: On PTFE, 42 HUVEC were in the vision field directly after seeding. The mean length of single migration steps (SML) was 6.1 +/- 5.2 mu m, the mean velocity (MV) 0.40 +/- 0.3 mu m.min(-1) and the complete length of the trajectory (LT) was 710 +/- 440 mu m. On TCP 82 HUVEC were in the vision field subsequent to seeding. The LT was 840 +/- 550 mu m, the SML 6.1 +/- 5.2 mu m and the MV 0.44 +/- 0.3 mu m.min(-1). The trajectories on LAP differed significantly in respect to SML (2.4 +/- 3.9 mu m, p

In the past years the microcirculation has gained increasing attention not only by basic scientists, but also by clinicians and translational researchers. In the clinical scenario, it has been convincingly described that the microcirculation is a key predictor of outcome and of central pathophysiological relevance. A vast body of evidence demonstrates the central role of the smallest vessels in inflammation, hyperviscosity, cell-cell-interaction, endothelial function, tissue edema, hemodynamic and blood flow regulation and its important role in the interaction with soluble factors. A central feature of different diseases and a strong regulator of different changes is hypoxia, the lack of oxygen. Also the microcirculation is on one hand a central component responding with dynamic changes to hypoxia but also the central place where hypoxia mediates its unfavorable effects. These changes and associated interactions are the topic of this special thematic issue "Hypoxia" in Clinical Hemorheology and Microcirculation and it seems logical that important and relevant findings are presented.

BACKGROUND: Magnetocardiography (MCG) has been proposed as a non-invasive and functional technique with high accuracy for diagnosis of myocardial ischemia. OBJECTIVE: This study sought to develop a novel scoring system of MCG for predicting the presence of significant obstructive coronary artery disease (CAD). METHODS: In a training set of 108 subjects, predictors of ≥70% stenosis in at least one major coronary vessel were prospectively identified from MCG variables. The final model was then retrospectively validated in a separate set of 45 subjects. RESULTS: In the multivariable logistic regression, among those in the training set, elevated scores were predictive of ≥70% stenosis in all subjects (OR: 40.85; 95% CI: 6.28–265.90; p

Background Contrast-induced acute kidney injury (CI-AKI), a potentially life-threatening complication of iodinated contrast media in patients with impaired renal function, has attracted increasing attention in recent years. There is overwhelming evidence that the most important pre-disposing factor for a contrast-medium induced nephropathy is the pre-existence of a renal impairment. Methods The registry was performed as a part of a quality management project in the Dresden-Friedrichstadt heart catheter laboratory. In compliance with the Declaration of Helsinki/Somerset West, 9,026 patients were included between 2010 and 2015. 100 patients of these were participants in a chronic dialysis program. All patients were dialyzed on the day before angiography. In all patients a coronary angiography, in 28 patients a stent implantation and in 12 patients a surgical reconstruction had to be performed. Prior to the intervention and one, two and three days thereafter the serum creatinine was measured. Results Up to the third day after application of the iodinated contrast medium no significant changes of the serum creatinine (baseline value: 423.3±42.6μmol/l) occurred (ANOVA for repeated measures: p = 0.507). On average, a slight decrease of the serum creatinine was found.All patients remained in their routine dialysis-program. 18 out of 100 died during the next three months after the procedure. Conclusion The study revealed that the coronary angiography using Iodixanol as iodinated contrast medium did not result in an increase of serum creatinine, which was drastically elevated in these patients before application of the iodinated contrast medium.

Introduction Concomitant mitral-regurgitation (MR) is frequently observed in patients undergoing trans-catheter aortic valve implantation (TAVI). The predictive value of MR etiology remains to be elucidated. Methods 600 patients with coincidental MR (≥moderate) undergoing TAVI were categorized according to a modified Carpentier classification [Groups: no/mild MR, n = 477; left atrial (LA) functional MR, n = 18; MR due to left ventricular dilatation, n = 29; degenerative MR, n = 50; MR with restricted leaflet motion n = 26]. MR improvement and patient outcome was compared among the groups in a retrospective analysis. Results MR regression was most pronounced in patients with restricted leaflet motion after 6 months, although a significant improvement was observed in all subgroups. MR relief was predominantly observed within the first 30 days after TAVI. Only patients with restricted leaflet motion experienced further improvement thereafter.In the entire cohort a total of 15 strokes (2.5%) during the first 30 days after TAVI were observed, with the highest incidence in the LA functional cohort (3 events, 17%; p = 0.008). In multivariate analysis, organic etiology was associated with an increased 1-year mortality.In conclusion, despite significant MR regression in all MR groups, some individuals may require additional mitralvalve repair after TAVI. According to our data the timing of these procedures should be based on the underlying MR etiology. The Carpentier classification in patients with coincidental MR undergoing TAVI for severe AS may also have prognostic implications as we found an increased incidence of strokes in our LA functional cohort and a worse mortality rates in organic MR.

Background Multiple symmetric lipomatosis is a rare disease characterized by the excessive growth of uncapsulated masses of adipose tissue. Although the etiology has yet to be elucidated, a connection to brown adipose tissue has been proposed recently. The mTOR pathway which is found to be regulated in lipomatous tissue as well as associated with brown adipose tissue can be inhibited by a compound called rapamycin. Methods We isolated adipose tissue derived stem cells from both affected and unaffected tissue and treated these cells with different concentrations of rapamycin. Results The differences in both proliferation and differentiation between adipose tissue derived stem cells (ASCs) from lipomatous and normal tissue decreased after mTOR pathway inhibition. In some patients regulation of mTOR genes was opposed in the ASCs from the two different tissues. Conclusions Treatment with rapamycin might be a novel therapeutical approach for patients suffering from multiple symmetric lipomatosis.

Laser tissue soldering (LTS) based on indocyanine green (ICG)-mediated heat-denaturation of proteins might be a promising alternative technique for micro-suturing, but up to now the problem of too weak shear strength of the solder welds in comparison to sutures is not solved. Earlier reports gave promising results showing that solder supported by carrier materials can enhance the cohesive strength of the liquid solder. In these studies, the solder was applied to the carriers by dip coating. Higher reliability of the connection between the solder and the carrier material is expected when the solder is bound covalently to the carrier material. In the present study a poly(ether imide) (PEI) membrane served as carrier material and ICG-supplemented albumin as solder substrate. The latter was covalently coupled to the carrier membrane under physiological conditions to prevent structural protein changes. As laser source a diode continuous-wave laser emitting at 808 nm with intensities between 250 mW and 1500 mW was utilized. The albumin functionalized carrier membrane was placed onto the tunica media of explanted pig thoracic aortae forming an overlapping area of approximately 0.5×0.5 cm2. All tests were performed in a dry state to prevent laser light absorption by water. Infrared spectroscopy, spectro-photometrical determination of the secondary and primary amine groups after acid orange II staining, contact angle measurements, and atomic force microscopy proved the successful functionalization of the PEI membrane with albumin. A laser power of 450 mW LTS could generate a membrane-blood vessel connection which was characterized by a shear strength of 0.08±0.002 MPa, corresponding to 15% of the tensile strength of the native blood vessel. Theoretically, an overlapping zone of 4.1 mm around the entire circumference of the blood vessel could have provided shear strength of the PEI membrane-blood vessel compound identical to the tensile strength of the native blood vessel. These in-vitro results confirmed the beneficial effects of solder reinforcement by carrier membranes, and suggest LTS with covalently bound solders on PEI substrates for further studies in animal models.

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.

The Laser Doppler technique (Laser-Doppler-Fluxmetry, LDF), a noninvasive method to estimate skin blood flow (LDF), is frequently used in research and clinical routine [1]. Here, the measurements were carried out with a new Laser Doppler system, the DOP-system, which allows to measure frequency spectra in four different frequency windows according to the velocities in venules (low velocity), capillaries (low to medium velocities), and in arteries (with high and very high velocities). However, the diagnostic reliability or the effectiveness of the LDF has not yet been evaluated sufficiently, which is indispensable, where medical diagnostics and therapy controls are concerned. For a valid interpretation of LDF values of individual patients, the knowledge of the reference range and the variability of the measured parameters is required.In four successive studies the reference range (62 apparently healthy subjects), the circadian variability (8 subjects), the variability from day-to-day (6 subjects) and over one year with monthly measurements (6 subjects) were evaluated.With the knowledge of the reference range, microcirculatory disorders can now be diagnosed using the DOP method. Following a standard measurement procedure there was no dependence of the measured data on the day or season of measurement.

Mesenchymal stem cells (MSCs) are targeted as vehicles for cell mediated gene therapy. Here we report on a macromolecular carrier, which was designed aiming at successful targeted gene delivery into MSCs through the mediation of folate receptor and reduced cytotoxicity compared to established cationic polymer vector – polyethylenimine with a weight average molecular weight (Mw) of 25,000 Dalton (PEI25K). The carrier PHPA-PEI1800-FA was synthesized in a two-step procedure. PHPA-PEI1800 was prepared by grafting polyethylenimine with a Mw of 1800 Dalton (PEI1800) onto the α,β-poly(N-3-hydroxypropyl)-D,L-aspartamide (PHPA) backbone. PHPA-PEI1800-FA was obtained by chemically conjugating folic acid onto PHPA-PEI1800. The grafting degree of PEI1800 was 3.9±0.2% in relation to the CH groups of PHPA and the molar ratio of folic acid conjugated to PEI1800 (χFA) was 1.8±0.1 as calculated by NMR spectroscopy. The copolymers were biodegradable and exhibited lower cytotoxicity than PEI25K. Compared to PHPA-PEI1800, PHPA-PEI1800-FA led to a significantly higher transfection efficiency in human MSCs, which could be attributed to the mediation of folate receptor during the transfection process as confirmed by folic acid competition assay. Both marker gene (GFP) and therapeutic gene (VEGF) were delivered into human MSCs from multi-donors using PHPA-PEI1800-FA. The percentage of GFP+ MSCs showed an average value of 2.85±1.60% but a large variation for different samples. The VEGF expression level of the PHPA-PEI1800-FA transfected cells was significantly higher than that of either untransfected or naked DNA transfected cells. Conclusively, PHPA-PEI1800-FA is a suitable vector to deliver genes into human MSCs through the interaction with folate receptor.

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Radiographic contrast media (RCM) can initiate microcirculatory disorders. This study was performed to investigate effects of Ioxaglate on the cutaneous microcirculation. The investigation was carried out as prospective randomized double-blind comparison in parallel-group design on two groups of n = 10 patients each who had to undergo a diagnostic coronary angiography. The confirmatory parameter of the study was mean erythrocyte capillary velocity [vRBC in mm/sec]. VRBC in the ipsilateral nail-fold capillaries was recorded continuously for 3 min before and 6 min after injection of RCM or isotonic saline solution in the A. axillaris respectively, and was evaluated off-line. VRBC in nailfold capillaries was found to be decreased by Ioxaglate by 34% 150 seconds after injection, while isotonic NaCl solution immediately induced a slight increase of 14%.

Controlling mesenchymal stem cells (MSCs) behavior is necessary to fully exploit their therapeutic potential. Various approaches are employed to effectively influence the migration capacity of MSCs. Here, topographic microstructures with different microscale roughness were created on polystyrene (PS) culture vessel surfaces as a feasible physical preconditioning strategy to modulate MSC migration. By analyzing trajectories of cells migrating after reseeding, we demonstrated that the mobilization velocity of human adipose derived mesenchymal stem cells (hADSCs) could be promoted by and persisted after brief preconditioning with the appropriate microtopography. Moreover, the elevated activation levels of focal adhesion kinase (FAK) and mitogen-activated protein kinase (MAPK) in hADSCs were also observed during and after the preconditioning process. These findings underline the potential enhancement of in vivo therapeutic efficacy in regenerative medicine via transplantation of topographic microstructure preconditioned stem cells.

BACKGROUND Occlusions of artificial small-diameter cardiovascular grafts are frequent events after implantation, often caused by clot formations. A main factor is the insufficient hemocompatibility of the inner artificial graft surface, which could be improved by endothelialization. Therefore, one challenge in cardiovascular graft engineering is the establishment of a shear-resistant endothelial cell layer to prevent cell detachment by shear forces after implantation. MATERIALS AND METHODS Recently, very smooth (Rq = 2.37 ± 1.40 nm) poly(ether imide) (PEI) films were introduced as a biocompatible candidate material for cardiovascular devices. In this study the stability of primary human umbilical vein endothelial cell (HUVEC) monolayer was investigated after long-term seeding (nine days) on PEI-films and subsequent exposure to a venous shear stress of 3 dyn/cm2 for up to six hours using the cone-and-plate shearing technique. Cell density, growth pattern and morphology of HUVEC were determined prior and after shearing compared to glass as control substrate. HUVEC adhering to the substrate after shear stress were counted and analyzed by fluorescent staining. Supernatants were collected and secretion profile analysis of vasoactive and inflammatory mediators was performed. RESULTS The cell density on PEI-films compared to the controls was slightly higher after long-term seeding and exposure to shear stress (glass: 71,656 ± 8,830 cells/cm2 and 42,239 ± 5,607 cells/cm2; PEI-film: 64,056 ± 2,829 cells/cm2 and 45,422 ± 2,507 cells/cm2 before and after shear stress, respectively). Actin- and vinculin-staining revealed a scattered re-organization of the cytoskeleton as well as a formation of stress fibers and focal adhesion points. Secretion of prostacyclin and thromboxane A2 was increased after application of shear stress, but no significant differences were detectable between cells growing on PEI-films or glass. Amounts of secreted inflammatory cytokines IL-6 and IL-8 in the supernatant were significantly lower for HUVEC seeded on PEI-films compared to glass before as well as after stress. CONCLUSION The study demonstrated that HUVEC were able to resist exposure to venous shear stress when seeded on smooth PEI-films with typical morphology and adhesion behavior. However, HUVEC adherence on PEI was not yet sufficient to retain a complete cell monolayer after shear stress exposure. Occasionally, single cells or cell plaques were disrupted resulting in cell free areas in the confluent HUVEC layer. Apart from this our data suggest that PEI is a suitable substrate for HUVEC under static and dynamic conditions and therefore a promising candidate material for cardiovascular applications. The next objective is a surface functionalization of the PEI-films in a cell specific manner to reach a functionally confluent, shear resistant HUVEC monolayer.

Angiogenically stimulated alternative monocytes (aMO2) could be established as cellular release system accelerating the endothelialization of polymers rendering their surfaces hemocompatibility in a short-term study. However, for their clinical application it is essential that aMO2 do not switch back to the MO1 state sustaining their capability as cellular release system over an extended period of time. We explored whether aMO2 can maintain their differentiation state over 21 days in a mono- and in a co-culture with HUVEC. In comparison, the influence of recombinant VEGF-A165 on the endothelialization of biomaterials was assessed including endothelial cell (HUVEC) density, organisation of the endothelial cytoskeleton, cytokine secretion profile and release of prostacyclin, thromboxane A2 and matrix metalloproteinases. In mono-culture aMO2 secreted high amounts of VEGF and other growth factors/cytokines. Co-cultured with HUVEC, aMO2 accelerated the formation of a confluent HUVEC monolayer. Furthermore, no pro-inflammatory cytokines were found, neither in aMO2-mono, nor in co-cultures with HUVEC indicating that the majority of the aMO2 remained stable in their aMO2 state during the 21 days of cultivation. In contrast, the addition of recombinant VEGF-A165 instead of the co-culture with aMO2 resulted in the formation of stress fibres, dissociated marginal filament bands, and a detachment of HUVEC. In addition, the profile of bioactive agents of HUVEC (e.g. prostacyclin, thromboxane A2, matrix metalloproteinases, IFN-γ and TNF-α) was influenced by the VEGF-A165 treatment inducing the detachment of HUVEC. In conclusion, in co-culture with HUVEC aMO2 remained stable in their type 2 state over 21 days confirming the suitability of aMO2 as biological release system for the endothelialization of biomaterial surfaces with constant release of angiogenic factors but without secretion of pro-inflammatory cytokines over three weeks. Therefore, this endothelialization approach seems to be appropriate to improve the hemocompatibility of cardiovascular implant materials in vitro, and proved to be superior to the use of recombinant VEGF-A165.

For in vitro studies assessing the interaction of platelets with implant materials, common and standardized protocols for the preparation of platelet rich plasma (PRP) are lacking, which may lead to non-matching results due to the diversity of applied protocols. Particularly, the aging of platelets during prolonged preparation and storage times is discussed to lead to an underestimation of the material thrombogenicity. Here, we study the influence of whole blood- and PRP-storage times on changes in platelet morphology and function. Blood from apparently healthy subjects was collected according to a standardized protocol and examined immediately after blood collection, four hours and twenty four hours later. The capability of platelets to adhere and form stable aggregates (PFA100, closure time) was examined in sodium citrate anticoagulated whole blood (WB) using the agonists equine type I collagen and epinephrine bitartrate (collagen/epinephrine) as well as equine type I collagen and adenosine-5'-diphosphate (collagen/ADP). Circulating platelets were quantified at each time point. Morphology of platelets and platelet aggregates were visualized microscopically and measured using an electric field multi-channel counting system (CASY). The percentage of activated platelets was assessed by means of P-selectin (CD62P) expression of circulating platelets. Furthermore, platelet factor 4 (PF4) release was measured in platelet poor plasma (PPP) at each time point. Whole blood PFA100 closure times increased after stimulation with collagen/ADP and collagen/epinephrine. Twenty four hours after blood collection, both parameters were prolonged pathologically above the upper limit of the reference range. Numbers of circulating platelets, measured in PRP, decreased after four hours, but no longer after twenty four hours. Mean platelet volumes (MPV) and platelet large cell ratios (P-LCR, 12 fL - 40 fL) decreased over time. Immediately after blood collection, no debris or platelet aggregates could be visualized microscopically. After four hours, first debris and very small aggregates occurred. After 24 hours, platelet aggregates and also debris progressively increased. In accordance to this, the CASY system revealed an increase of platelet aggregates (up to 90 μm diameter) with increasing storage time. The percentage of CD62P positive platelets and PF4 increased significantly with storage time in resting PRP. When soluble ADP was added to stored PRP samples, the number of activatable platelets decreased significantly over storage time. The present study reveals the importance of a consequent standardization in the preparation of WB and PRP. Platelet morphology and function, particularly platelet reactivity to adherent or soluble agonists in their surrounding milieu, changed rapidly outside the vascular system. This knowledge is of crucial interest, particularly in the field of biomaterial development for cardiovascular applications, and may help to define common standards in the in vitro hemocompatibility testing of biomaterials.

Background The technique of flap-prefabrication has been successfully established in tissue engineering: missing intrinsic vascularisation of engineered tissue can be generated in vivo by microsurgical vesselloop construction. It is possible to move engineered tissue into a defect with microsurgery. In the literature, the combination of engineered tissue covered with skin is not widely reported. Objective Aim of this study was to establish a model to investigate scaffold prefabrication with full thickness skin graft coverage with subsequent free tissue transfer. Methods 8 Wistar rats were operated in 2 separate steps: 1) after creating an arteriovenous loop with the femoral vessels, a porous scaffold was placed on the loop and covered with an inguinally based skin flap. A control was implanted without loop into the contralateral groin. 2) 6 weeks later the prefabricated composite flaps were microsurgically transferred to the cervical region. Skin-island monitoring was performed with Laser Doppler-scanner after the transfer. Results Continuous loss of the skin islands was observed within 72 hours. Complications included wound-dehiscence, thrombosis and death from anaesthesia; in spite of consistent loop viability. Conclusion Evaluation showed that modifications are necessary to maintain the skin-island cove.

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Linear, side-chain methylated oligoglycerols (OGMe) were recently reported as potential surface passivating molecules for improving the protein resistance of cardiovascular application relevant poly(ether imide) (PEI) membranes. A previously reported in vitro screening under static test conditions allowed an end-point evaluation of the adhesion and activation of adherent thrombocytes performed on the material surfaces and revealed similar levels of thrombogenicity on PEI membranes, functionalized with OGMe and oligo(ethylene glycol) (OEG) of similar molecular weight (Mn = 1,300 g·mol−1 - 1,800 g·mol−1). In the present study, we investigated the hemocompatibility of these materials in a dynamic closed loop system, in order to study time-dependent thrombocyte material interactions also of the circulating thrombocytes by mimicking in vivo relevant flow conditions in a dynamic test system with multiple material contacts. Activation and aggregation of circulating thrombocytes as well as complement activation and plasmatic coagulation were evaluated after 40 circulations of thrombocyte rich plasma in the closed loop system. The results of the dynamic tests revealed no differences between the OGMe and OEG functionalized PEI membranes. Furthermore, no differences were observed between the latter and a PEI membrane treated under the conditions of functionalization at pH 11 (PEI-pH11) without an oligoether being present. Blood plasma protein adsorption, as well as activation, and adherence of circulating thrombocytes occurred in a comparable, but minor manner on all investigated PEI membranes. From this we conclude that the OGMe and OEG surface functionalization did not lead to an improvement of the already good hemocompatibility of the PEI-pH11 membrane.

Polymers exhibiting cell-selective effects represent an extensive research field with high relevance for biomedical applications e.g. in the cardiovascular field supporting re-endothelialization while suppressing smooth muscle cell overgrowth. Such an endothelial cell-selective effect could be recently demonstrated for a copolyetheresterurethane (PDC) containing biodegradable poly(p-dioxanone) and poly(epsilon-caprolactone) segments, which selectively enhanced the adhesion of human umbilical vein endothelial cells (HUVEC) while suppressing the attachment of smooth muscle cells (SMC). In this study we investigated the influence of the fibre orientation (random and aligned) and fibre diameter (2 mu m and 500 nm) of electrospun PDC scaffolds on the adhesion, proliferation and apoptosis of HUVEC and SMC. Adhesion, viability and proliferation of HUVEC was diminished when the fibre diameter was reduced to a submicron scale, while the orientation of the microfibres did only slightly influence the cellular behaviour. In contrast, a submicron fibre diameter improved SMC viability. In conclusion, PDC scaffolds with micron-sized single fibres could be promising candidate materials for cell-selective stent coatings.

Back scattered Laser Doppler (LD) signals are composed of two different individual signals. The number of the moving particles and the speed of the particles in the measured tissue volume determine the frequency shift and the band width of the Doppler signal. The dependence of the Laser Doppler flux on the number of scattering particles is highly nonlinear: at very low hematocrit and high speed the axial migration of the cells to the centre of the blood vessels is very strong, so that in these cases - because of the parabolic flow profile - the Doppler flux measurement overestimates the mean real blood flow (up to two- or three-fold). The opposite is the case when the hematocrit is very high, then the blood flow might be underestimated (due to the increased amounts of blood cells near the vessel wall). In addition, a very change in number of moving particles - as can occur during the postprandial phase or during therapy - can change the signal also at a constant cell number. Also, it must be mentioned that the LD signal possibly is not only reflected by moving blood cells in the different skin layers but also by blood cells flowing in tissues below the skin (particularly below atrophied skin areas of older patients) so that in such cases the LD Flux signal reflects not exclusively the skin blood flow. Therefore, LD flux at rest may still be within the normal range even in advanced states of disease, since the scattered light is sampled from a tissue volume which may contain also non-nutritive shunt vessels. This critical analysis of the LD signals of course shall not lead to an overall rejection of the application of laser Doppler systems. Actual progress only can, however, be obtained under the exact consideration of anatomical conditions, technical restrictions and when generalizations are avoided.

The formation of a functionally-confluent and shear-resistant endothelial cell (EC) monolayer on cardiovascular implants is a promising strategy to prevent thrombogenic processes after implantation. On the basis of existing studies with arterial endothelial cells adhering after two hours on gelatin-based hydrogels in marked higher numbers compared to tissue culture plates, we hypothesized that also venous endothelial cells (HUVEC) should be able to adhere and form an endothelial monolayer on these hydrogels after days. Furthermore, variation of the hydrogel composition, which slightly influences the materials elasticity and even more the degradation behaviour, should have no considerable effect on HUVEC. Therefore, the monolayer formation and shear resistance of HUVEC were explored on two gelatin-based hydrogels differing in their elasticity (Young's moduli between 35 and 55 kPa) in comparison to a positive control (HUVEC on glass cover slips) and a negative control (HUVEC on glass cover slips activated with interleukin-1β) after 9 days of culturing. HUVEC density after 9 days of culturing under static conditions was lower on the hydrogels compared to both controls (p

There is a widely recognized need to improve the performance of vascular implants and external medical devices that come into contact with blood by reducing adverse reactions they cause, such as thrombosis and inflammation. These reactions lead to major adverse cardiovascular events such as heart attacks and strokes. Currently, they are managed therapeutically. This need remains unmet by the biomaterials research community. Recognized stagnation of the blood-biomaterial interface research translates into waning interest from clinicians, funding agencies, and practitioners of adjacent fields. The purpose of this contribution is to stir things up. It follows the 2014 BloodSurf meeting (74th International IUVSTA Workshop on Blood-Biomaterial Interactions), offers reflections on the situation in the field, and a three-pronged strategy integrating different perspectives on the biological mechanisms underlying blood-biomaterial interactions. The success of this strategy depends on reengaging clinicians and on the renewed cooperation of the funding agencies to support long-term efforts.