Differential sensitivity of assays for determining vein endothelial cell senescence.

In vivo endothelialization of polymer-based cardiovascular implant materials is a promising strategy to reduce the risk of platelet adherence and the subsequent thrombus formation and implant failure. However, endothelial cells from elderly patients are likely to exhibit a senescent phenotype that may counteract endothelialization. The senescence status of cells should therefore be investigated prior to implantation of devices designed to be integrated in the blood vessel wall. Here, human umbilical vein endothelial cells (HUVEC) were cultivated up to passage (P) 4, 10 and 26/27 to determine the population doubling time and the senescence status by four different methods. Determination of the senescence-associated ß-galactosidase activity (SA-ß-Gal) was carried out by colorimetric staining and microscopy (i), as well as by photometric quantification (ii), and the expression of senescence-associated nuclear proteins p16 and p21 as well as the proliferation marker Ki67 was assessed by immunostaining (iii), and by flow cytometry (iv). The population doubling time of P27-cells was remarkably greater (103±65 h) compared to P4-cells (24±3 h) and P10-cell (37±15 h). Among the four different methods tested, the photometric SA-ß-Gal activity assay and the flow cytometric determination of p16 and Ki67 were most effective in discriminating P27-cells from P4- and P10-cells. These methods combined with functional endothelial cell analyses might aid predictions on the performance of implant endothelialization in vivo.

Immunocompatibility and non-thrombogenicity of gelatin-based hydrogels.

Immunocompatibility and non-thrombogenicity are important requirements for biomedical applications such as vascular grafts. Here, gelatin-based hydrogels formed by reaction of porcine gelatin with increasing amounts of lysine diisocyanate ethyl ester were investigated in vitro in this regard. In addition, potential adverse effects of the hydrogels were determined using the "Hen's egg test on chorioallantoic membrane" (HET-CAM) test and a mouse model.The study revealed that the hydrogels were immunocompatible, since complement activation was absent and a substantial induction of reactive oxygen species generating monocytes and neutrophils could not be observed in whole human blood. The density as well as the activation state of adherent thrombocytes was comparable to medical grade polydimethylsiloxane, which was used as reference material. The HET-CAM test confirmed the compatibility of the hydrogels with vessel functionality since no bleedings, thrombotic events, or vessel destructions were observed. Only for the samples synthesized with the highest LDI amount the number of growing blood vessels in the CAM was comparable to controls and significantly higher than for the softer materials. Implantation into mice showed the absence of adverse or toxic effects in spleen, liver, or kidney, and only a mild lymphocytic activation in the form of a follicular hyperplasia in draining lymph nodes (slightly increased after the implantation of the material prepared with the lowest LDI content). These results imply that candidate materials prepared with mid to high amounts of LDI are suitable for the coating of the blood contacting surface of cardiovascular implants.

The influence of different rewetting procedures on the thrombogenicity of nanoporous poly(ether imide) microparticles.

Nanoporous microparticles prepared from poly(ether imide) (PEI) are discussed as candidate adsorber materials for the removal of uremic toxins during apheresis. Polymers exhibiting such porosity can induce the formation of micro-gas/air pockets when exposed to fluids. Such air presenting material surfaces are reported to induce platelet activation and thrombus formation. Physical or chemical treatments prior to implantation are discussed to reduce the formation of such gas nuclei. Here, we report about the influence of different rewetting procedures - as chemical treatments with solvents - on the thrombogenicity of hydrophobic PEI microparticles and PEI microparticles hydrophilized by covalent attachment of poly(vinyl pyrrolidone) (PVP) of two different chain lengths.Autoclaved dry PEI particles of all types with a diameter range of 200 - 250 µm and a porosity of about 84% ±2% were either rewetted directly with phosphate buffered saline (24 h) or after immersion in an ethanol-series. Thrombogenicity of the particles was studied in vitro upon contact with human sodium citrated whole blood for 60 min at 5 rpm vertical rotation. Numbers of non-adherent platelets were quantified, and adhesion of blood cells was qualitatively analyzed by bright field microscopy. Platelet activation (percentage of CD62P positive platelets and amounts of soluble P-Selectin) and platelet function (PFA100 closure times) were analysed.Retention of blood platelets on the particles was similar for all particle types and both rewetting procedures. Non-adherent platelets were less activated after contact with ethanol-treated particles of all types compared to those rewetted with phosphate buffered saline as assessed by a reduced number of CD62P-positive platelets and reduced amounts of secreted P-Selectin (P < 0.05 each). Interestingly, the hydrophilic surfaces significantly increased the number of activated platelets compared to hydrophobic PEI regardless of the rewetting agent. This suggests that, apart from wettability, other material properties might be more important to regulate platelet activation. PFA100 closure times were reduced and within the reference ranges in the ethanol group, however, significantly increased in the saline group. No substantial difference was detected between the tested surface modifications. In summary, rewetting with ethanol resulted in a reduced thrombogenicity of all studied microparticles regardless of their wettability, most likely resulting from the evacuation of air from the nanoporous particles.

Age-related morphology and function of human arterial endothelial cells.

Endothelialization of cardiovascular implants is regarded as a promising strategy for long-term compatibility. While umbilical vein endothelial cells are typically applied in research, human arterial endothelial cells (HAEC) from elderly donors would be the obvious source for autologous cellularization strategies.In our approach, HAEC from 16 donors of varying age (16-63 years) were divided into two groups (<30 years and >30 years) and analyzed regarding morphology, viability, proliferation, function and senescence status.No age-related differences were found regarding morphology, viability, density, prostacyclin and nitrite secretion or collagen and laminin production. However, the metabolic activity was slightly decreased (p = 0.0374) and the membrane integrity marginally impaired (p = 0.0404) in cells from older donors. Two out of three senescence assays detected more senescence markers in cells from older donors.According to the assays applied here, HAEC from young and elderly donors up to the age of 63 years could be judged equally suitable for autologous cellularization strategies. However, this finding should be regarded with caution due to the extremely large variability between individual donors. Further studies comprising a larger sample size are necessary to investigate this issue more thoroughly.

Effects of acrolein in comparison to its prodrug cyclophosphamide on human primary endothelial cells in vitro.

Cyclophosphamide (CPA) is one of the most successful anticancer prodrugs that becomes effective after biotransformation in the liver resulting in the toxic metabolite acrolein. Cancer is often accompanied by thromboembolic events, which might be a result of dysfunctional endothelial cells due to CPA treatment. Here, the effect of 1 mM CPA or acrolein (10/50/100/500 µM) on human umbilical vein endothelial cells (HUVECs) was analyzed after two days of treatment. The addition of CPA or 10 µM acrolein did not affect HUVECs. However, concentrations of 100 µM and 500 µM acrolein significantly reduced the number of adherent cells by 86 ±â€¯13% and 99 ±â€¯1% and cell viability by 51 ±â€¯29% and 93 ±â€¯8% compared to the control. Moreover, pronounced stress fibers as well as multiple nuclei were observed and von Willebrand factor (vWF) was completely released. Lactate dehydrogenase was 8.5 ±â€¯7.0-fold and 252.9 ±â€¯42.9-fold increased showing a loss of cell membrane integrity. The prostacyclin and thromboxane secretion was significantly increased by the addition of 500 µM acrolein (43.1 ±â€¯17.6-fold and 246.4 ±â€¯106.3-fold) indicating cell activation/pertubation. High doses of acrolein led to HUVEC death and loss of vWF production. This effect might be associated with the increased incidence of thromboembolic events in cancer patients treated with high doses of CPA.

Spontaneous and induced platelet aggregation in apparently healthy subjects in relation to age.

Thrombotic disorders remain the leading cause of mortality and morbidity, despite the fact that anti-platelet therapies and vascular implants are successfully used today. As life expectancy is increasing in western societies, the specific knowledge about processes leading to thrombosis in elderly is essential for an adequate therapeutic management of platelet dysfunction and for tailoring blood contacting implants. This study addresses the limited available data on platelet function in apparently healthy subjects in relation to age, particularly in view of subjects of old age (80-98 years). Apparently healthy subjects between 20 and 98 years were included in this study. Platelet function was assessed by light transmission aggregometry and comprised experiments on spontaneous as well as ristocetin-, ADP- and collagen-induced platelet aggregation. The data of this study revealed a non-linear increase in the maximum spontaneous platelet aggregation (from 3.3% ±3.3% to 10.9% ±5.9%). The maximum induced aggregation decreased with age for ristocetin (from 85.8% ±7.2% to 75.0% ±7.8%), ADP (from 88.5% ±4.6% to 64.8% ±7.3%) and collagen (from 89.5% ±3.0% to 64.0% ±4.0%) in a non-linear manner (linear regression analysis). These observations indicate that during aging, circulating platelets become increasingly activated but lose their full aggregatory potential, a phenomenon that was earlier termed "platelet exhaustion". In this study we extended the limited existing data for spontaneous and induced platelet aggregation of apparently healthy donors above the age of 75 years. The presented data indicate that the extrapolation of data from a middle age group does not necessarily predict platelet function in apparently healthy subjects of old age. It emphasizes the need for respective studies to improve our understanding of thrombotic processes in elderly humans.

Albumin solder covalently bound to a polymer membrane: New approach to improve binding strength in laser tissue soldering in-vitro.

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.

Evidence for cytostatic effect of cyclophosphamide on human vein endothelial cells in cancer therapy: Preliminary in vitro results.

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.

Influence of different surface treatments of poly(n-butyl acrylate) networks on fibroblasts adhesion, morphology and viability.

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 < 0.05, each), while lowest values were observed after the combined surface treatment (OPT+FIB). The latter differed significantly from the single OPT and FIB. The number of adherent fibroblasts and their adherence behavior differed on both pristine cPnBA networks. The fibroblast density on cPnBA04 was 743±434 cells·mm-2, was about 6.5 times higher than on cPnBA73 with 115±73 cells·mm-2. On cPnBA04 about 20% of the cells were visible as very small, round and buckled cells while all other cells were in a migrating status. On cPnBA73, nearly 50% of fibroblasts were visible as very small, round and buckled cells. The surface functionalization either using oxygen plasma treatment or fibrinogen coating led to a significant increase of adherent fibroblasts, particularly the combination of both techniques, for both cPnBA networks. It is noteworthy to mention that the fibrinogen coating overruled the characteristics of the pristine surfaces; here, the fibroblast densities after seeding were identical for both cPnBA networks. Thus, the binding rather depended on the fibrinogen coating than on the substrate characteristics anymore. While the integrity of the fibroblasts membrane was comparable for both polymers, the MTS tests showed a decreased metabolic activity of the fibroblasts on cPnBA. CONCLUSION: The applied surface treatments of cPnBA successfully improved the adhesion of viable fibroblasts. Under resting conditions as well as after shearing the highest fibroblast densities were found on surfaces with combined post-treatment.

Effects of Tacrolimus or Sirolimus on the adhesion of vascular wall cells: Controlled in-vitro comparison study.

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.

Bone regeneration induced by a 3D architectured hydrogel in a rat critical-size calvarial defect.

Bone regeneration can be stimulated by implantation of biomaterials, which is especially important for larger bone defects. Here, healing potency of the porous ArcGel was evaluated in a critical-size calvarial bone defect in rats in comparison with clinical standard autologous bone and Bio-Oss® Collagen (BioOss), a bone graft material frequently used in clinics. Bone healing and metabolic processes involved were monitored longitudinally by [18F]-fluoride and [18F]-FDG µ-PET/CT 1d, 3d, 3w, 6w, and 12w post implantation. Differences in quality of bone healing were assessed by ex vivo µ-CT, mechanical tests and histomorphometry. The amount of bone formed after implantation of ArcGel was comparable to autologous bone and superior to BioOss (histomorphometry). Furthermore, microarchitecture of newly formed bone was more physiological and better functional in case of ArcGel (push-out tests). [18F]-FDG uptake increased until 3d after implantation, and decreased until 12w for both ArcGel and BioOss. [18F]-fluoride uptake increased until 3w post implantation for all materials, but persisted significantly longer at higher levels for BioOss, which indicates a prolonged remodelling phase. The study demonstrates the potential of ArcGel to induce restitutio ad integrum comparable with clinical standard autologous bone and better bone regeneration in large defects compared to a commercial state-of-the-art biomaterial.

Strategy for the hemocompatibility testing of microparticles.

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: θadv = 112±10° PEI-KOH θadv = 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.

Effect of temperature on platelet adherence.

BACKGROUND: Thrombogenicity is one of the main parameters tested in vitro to evaluate the hemocompatibility of artificial surfaces. While the influence of the temperature on platelet aggregation has been addressed by several studies, the temperature influence on the adherence of platelets to body foreign surfaces as an important aspect of biomedical device handling has not yet been explored. Therefore, we analyzed the influence of two typically applied incubation-temperatures (22°C and 37°C) on the adhesion of platelets to biomaterials. MATERIAL AND METHODS: Thrombogenicity of three different polymers - medical grade poly(dimethyl siloxane) (PDMS), polytetrafluoroethylene (PTFE) and polyethylene terephthalate (PET) - were studied in an in vitro static test. Platelet adhesion was studied with stringently characterized blood from apparently healthy subjects. Collection of whole blood and preparation of platelet rich plasma (PRP) was carried out at room temperature (22°C). PRP was incubated with the polymers either at 22°C or 37°C. Surface adherent platelets were fixed, fluorescently labelled and assessed by an image-based approach. RESULTS AND DISCUSSION: Differences in the density of adherent platelets after incubation at 22°C and 37°C occurred on PDMS and PET. Similar levels of adherent platelets were observed on the very thrombogenic PTFE. The covered surface areas per single platelet were analyzed to measure the state of platelet activation and revealed no differences between the two incubation temperatures for any of the analyzed polymers. Irrespective of the observed differences between the low and medium thrombogenic PDMS and PET and the higher variability at 22°C, the thrombogenicity of the three investigated polymers was evaluated being comparable at both incubation temperatures.

Adhesion and activation of platelets from subjects with coronary artery disease and apparently healthy individuals on biomaterials.

On the basis of the clinical studies in patients with coronary artery disease (CAD) presenting an increased percentage of activated platelets, we hypothesized that hemocompatibility testing utilizing platelets from healthy individuals may result in an underestimation of the materials' thrombogenicity. Therefore, we investigated the interaction of polymer-based biomaterials with platelets from CAD patients in comparison to platelets from apparently healthy individuals. In vitro static thrombogenicity tests revealed that adherent platelet densities and total platelet covered areas were significantly increased for the low (polydimethylsiloxane, PDMS) and medium (Collagen) thrombogenic surfaces in the CAD group compared to the healthy subjects group. The area per single platelet-indicating the spreading and activation of the platelets-was markedly increased on PDMS treated with PRP from CAD subjects. This could not be observed for collagen or polytetrafluoroethylene (PTFE). For the latter material, platelet adhesion and surface coverage did not differ between the two groups. Irrespective of the substrate, the variability of these parameters was increased for CAD patients compared to healthy subjects. This indicates a higher reactivity of platelets from CAD patients compared to the healthy individuals. Our results revealed, for the first time, that utilizing platelets from apparently healthy donors bears the risk of underestimating the thrombogenicity of polymer-based biomaterials.

Mechanical characterization of oligo(ethylene glycol)-based hydrogels by dynamic nanoindentation experiments.

Oligo(ethylene glycol)-based (OEG) hydrogel samples of varying cross-link densities and degrees of swelling were characterized through dynamic nanoindentation testing. Experiments were performed using a non-standard nanoindentation method, which was validated on a standard polystyrene sample. This method maximizes the capability of the instrument to measure the stiffness and damping of highly compliant, viscoelastic materials. Experiments were performed over the frequency range of 1 to 50 Hz, using a 1mm diameter flat punch indenter. A hydration method was adopted to avoid sample dehydration during testing. Values of storage modulus (E') ranged from 3.5 to 8.9 MPa for the different OEG-hydrogel samples investigated. Samples with higher OEG concentrations showed greater scatter in the modulus measurements and it is attributed to inhomogeneities in these materials. The (E') values did not show a strong variation over frequency for any of the samples. Values of loss modulus (E") were two orders of magnitude lower than the storage modulus, resulting in very low values of loss factor (E"/E'<0.1). These are characteristics of strong gels, which present negligible viscous properties.

Changes in platelet morphology and function during 24 hours of storage.

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.

Triple-shape effect in polymer-based composites by cleverly matching geometry of active component with heating method.

A triple-shape effect is created for a segmented device consisting of an active component encapsulated in a highly flexible polymer network. Segments with the same composition but different interface areas can be recovered independently either at specific field strengths (Hsw ) during inductive heating, at a specific time during environmentally heating, or at different airflow during inductive heating at constant H. Herein the type of heating method regulates the sequence order.

Interaction of human umbilical vein endothelial cells (HUVEC) with platelets in vitro: Influence of platelet concentration and reactivity.

Endothelialisation of polymer-based cardiovascular implants is one strategy to render biomaterials hemocompatible. The evaluation of the functionality and the confluence of an endothelial cell (EC) monolayer in vitro is therefore of crucial importance, because a non-functional or non-confluent EC monolayer can contribute to the failure of vascular grafts. Moreover, the comparison of different potential biomaterials regarding their ability to induce the formation of a functional confluent EC monolayer is of great value. Most of the currently reported in vitro studies focus on direct or indirect markers of EC behaviour. However, these studies still lack the final proof that the EC monolayer, which can be developed on polymers is confluent and functional. In this study, we investigated the suitability of an in vitro co-culture of human umbilical vein endothelial cells (HUVEC) with platelets to predict the functionality of an EC monolayer. The interaction of platelets with HUVEC was evaluated depending on the concentration of the platelets in the added plasma and of the reactivity of the platelets to pharmacological stimuli. For this purpose, HUVEC were seeded in a 24 well plate. After three days of cultivation, platelets were added to the HUVEC cell culture medium to final concentrations of 200, 2,000 or 20,000 platelets/µl (n = 7 each). The platelets were processed immediately after blood collection and added to the HUVEC culture after a 30 minutes resting period. As a first control, an EC monolayer just cultured with EC medium was used. As a second control EC supplemented with plasma without platelets were applied. The HUVEC monolayer was investigated microscopically after 1 hour of platelet exposition. The addition of thrombocytes to EC affected the EC adherence dependent on the initial cell seeding number of HUVEC, the platelet concentration and also on the reactivity of platelets added. In both controls no significant EC detachment was detected. The results demonstrated a significant influence of platelet concentration and reactivity on the adherence of EC in a static model.

Influence of physically crosslinked gelatins on the vasculature in the avian chorioallantoic membrane.

Gelatins functionalized with desaminotyrosine (DAT) or desaminotyrosyl tyrosine (DATT) form physically crosslinked hydrogels, due to the interactions between the introduced aromatic moieties and gelatin triple helices, whose extent depends on the thermal treatment of the material. The G-modulus of these hydrogels can be tailored to the range of the natural extracellular matrix by adjusting the degree of crosslinking. While these gelatin-based materials have been shown to be not angiogenic, the aim of the study was to evaluate whether these biomaterials influence the regulation of blood vessels when positioned on the chorionallantoic membrane (CAM) of fertilized eggs. The results clearly indicate that the DAT-functionalized gelatin led to an increase of the diameter of the blood vessels in the CAM, which at the same time is probably associated with an increased blood flow in these CAM vessels. The vessel diameters of the four groups (DAT-functionalized gelatin, DATT-functionalized gelatin, plain gelatin, control group without gelatin, each n = 10) differed significantly (p < 0.0001). Vessels in the CAM exposed to the DAT-functionalized gelatin showed with 36.4 µm ± 3.4 µm the largest mean diameters compared to the mean diameters of the samples exposed to DATT gelatin (16.0 µm ± 0.8 µm; p < 0.05) and the plain gelatin (21.2 µm ± 1.0 µm; p < 0.05), which both did not differ significantly from the vessels of the control group. The biocompatibility of the materials in vitro motivates the exploration of their application as matrix in local drug-release systems with short half-life times (one hour up to several days).

Local drug delivery by personalized, intraoperative custom-made implant coating.

Local administration of drugs can enhance regeneration, prevent infection, or treat postsurgical pain. If used in conjunction with implants, coating strategies should allow the choice of a drug or combination of drugs, their doses, localization, and release due to intraoperative considerations. Current coating technologies lack the ability for personalized medicine strategies. Here, we describe a new intraoperative strategy for drug delivery that allows a personalized approach as local drug delivery by implant coating. A polyvinylalcohol (PVA) patch provides rapid attachment to implant surfaces by cyanoacrylate (CA) adhesives. The CA polymerization was initiated by water uptake of the patch due to exposure to a humid environment. The coating strength depended on the type of the CA, the time of external pressing load and humidification, the properties of the patch and the implant surface. The CA adhesive penetrated and polymerized within the patch without impeding the bioactivity of the embedded molecules or strongly altering the protein release pattern after attachment to the implant surface. The use of CA in combination with the PVA patch proved to be noncytotoxic in vitro. This technology platform opens the possibility for personalized medicine to locally administer drugs due to intraoperative requirements.