Blood-contacting Medical Devices Research Articles

In vitro hemocompatibility screening of a slippery liquid impregnated surface coating for extracorporeal organ support applications.

Clot formation, infection, and biofouling are unfortunate but frequent complications associated with the use of blood-contacting medical devices. The challenge of blood-foreign surface interactions is exacerbated during medical device applications involving substantial blood contact area and extended duration of use, such as extracorporeal life support (ECLS). We investigated a novel surface modification, a liquid-impregnated surface (LIS), designed to minimize protein adsorption and thrombus development on medical plastics. The hemocompatibility and efficacy of LIS was investigated first in a low-shear model with LIS applied to the lumen of blood incubation vials and exposed to human whole blood. Additionally, LIS was evaluated in a 6 h ex vivo circulation model with swine blood using full-scale ECLS circuit tubing and centrifugal pumps with clinically relevant flow rate (1.5 L/min) and shear conditions for extracorporeal carbon dioxide removal. Under low-shear, LIS preserved fibrinogen concentration in blood relative to control polymers (+40 ± 6 mg/dL vs polyvinyl chloride, p < .0001), suggesting protein adsorption was minimized. A fibrinogen adhesion assay demonstrated a dramatic reduction in protein adsorption under low shear (87% decrease vs polyvinyl chloride, p = .01). Thrombus deposition and platelet adhesion visualized by scanning electron microscopy were drastically reduced. During the 6 h ex vivo circulation, platelets in blood exposed to LIS tubing did not become significantly activated or procoagulant, as occurred with control tubing; and again, thrombus deposition was visually reduced. A LIS coating demonstrated potential to reduce thrombus formation on medical devices. Further testing is needed specialized to clinical setting and duration of use for specific medical target applications.

Development and In Vitro Whole Blood Hemocompatibility Screening of Endothelium-Mimetic Multifunctional Coatings.

Multifunctional antithrombotic surface modifications for blood-contacting medical devices have emerged as a solution for foreign surface-mediated coagulation disturbance. Herein, we have developed and evaluated an endothelium-inspired strategy to reduce the thrombogenicity of medical plastics by imparting nitric oxide (NO) elution and heparin immobilization on the material surface. This dual-action approach (NO+Hep) was applied to polyethylene terephthalate (PET) blood incubation vials and compared to isolated modifications. Vials were characterized to evaluate NO surface flux as well as heparin density and activity. Hemocompatibility was assessed in vitro using whole blood from human donors. Compared to unmodified surfaces, blood incubated in the NO+Hep vials exhibited reduced platelet aggregation (15% decrease AUC, p = 0.040) and prolonged plasma clotting times (aPTT = 147% increase, p < 0.0001, prothrombin time = 5% increase, p = 0.0002). Prolongation of thromboelastography reaction time and elevated antifactor Xa levels in blood from NO+Hep versus PET vials suggests some heparin leaching from the vial surface, confirmed by post-blood incubation heparin density assessment. Results suggest NO+Hep surface modification is a promising approach for blood-contacting plastics; however, careful tuning of NO flux and heparin stabilization are essential and require assessment using human blood as performed here.

Improved Polymer Hemocompatibility for Blood-Contacting Applications via S-Nitrosoglutathione Impregnation.

Blood-contacting medical devices (BCMDs) are inevitably challenged by thrombi formation, leading to occlusion of flow and device failure. Ideal BCMDs seek to mimic the intrinsic antithrombotic properties of the human vasculature to locally prevent thrombotic complications, negating the need for systemic anticoagulation. An emerging category of BCMD technology utilizes nitric oxide (NO) as a hemocompatible agent, as the vasculature's endothelial layer naturally releases NO to inhibit platelet activation and consumption. In this paper, we report for the first time the novel impregnation of S-nitrosoglutathione (GSNO) into polymeric poly(vinyl chloride) (PVC) tubing via an optimized solvent-swelling method. Material testing revealed an optimized GSNO-PVC material that had adequate GSNO loading to achieve NO flux values within the physiological endothelial NO flux range for a 4 h period. Through in vitro hemocompatibility testing, the optimized material was deemed nonhemolytic (hemolytic index <2%) and capable of reducing platelet activation, suggesting that the material is suitable for contact with whole blood. Furthermore, an in vivo 4 h extracorporeal circulation (ECC) rabbit thrombogenicity model confirmed the blood biocompatibility of the optimized GSNO-PVC. Platelet count remained near 100% for the novel GSNO-impregnated PVC loops (1 h, 91.08 ± 6.27%; 2 h, 95.68 ± 0.61%; 3 h, 97.56 ± 8.59%; 4 h, 95.11 ± 8.30%). In contrast, unmodified PVC ECC loops occluded shortly after the 2 h time point and viable platelet counts quickly diminished (1 h, 85.67 ± 12.62%; 2 h, 54.46 ± 10.53%; 3 h, n/a; 4 h, n/a). The blood clots for GSNO-PVC loops (190.73 ± 72.46 mg) compared to those of unmodified PVC loops (866.50 ± 197.98 mg) were significantly smaller (p < 0.01). The results presented in this paper recommend further investigation in long-term animal models and suggest that GSNO-PVC has the potential to serve as an alternative to systemic anticoagulation in BCMD applications.

Electrospun Chitosan Functionalized with C12, C14 or C16 Tails for Blood-Contacting Medical Devices.

Medical applications stimulate the need for materials with broad potential. Chitosan, the partially deacetylated derivative of chitin, offers many interesting characteristics, such as biocompatibility and chemical derivatization possibility. In the present study, porous scaffolds composed of electrospun interwoven nanometric fibers are produced using chitosan or chitosan functionalized with aliphatic chains of twelve, fourteen or sixteen methylene groups. The scaffolds were thoroughly characterized by SEM and XPS. The length of the aliphatic tail influenced the physico-chemical and dynamic mechanical properties of the functionalized chitosan. The electrospun membranes revealed no interaction of Gram+ or Gram− bacteria, resulting in neither antibacterial nor bactericidal, but constitutively sterile. The electrospun scaffolds demonstrated the absence of cytotoxicity, inflammation response, and eryptosis. These results open the door to their application for blood purification devices, hemodialysis membranes, and vascular grafts.

Strategies for Improving Endothelial Cell Adhesion to Blood-Contacting Medical Devices.

The endothelium is a critical mediator of homeostasis on blood-contacting surfaces in the body, serving as a selective barrier to regulate processes such as clotting, immune cell adhesion, and cellular response to fluid shear stress. Implantable cardiovascular devices, including stents, vascular grafts, heart valves, and left ventricular assist devices, are in direct contact with circulating blood and carry a high risk for platelet activation and thrombosis without a stable endothelial cell (EC) monolayer. Development of a healthy endothelium on the blood-contacting surface of these devices would help ameliorate risks associated with thrombus formation and eliminate the need for long-term antiplatelet or anticoagulation therapy. Although ECs have been seeded onto or recruited to these blood-contacting surfaces, most ECs are lost upon exposure to shear stress due to circulating blood. Many investigators have attempted to generate a stable EC monolayer by improving EC adhesion using surface modifications, material coatings, nanofiber topology, and modifications to the cells. Despite some success with enhanced EC retention in vitro and in animal models, no studies to date have proven efficacious for routinely creating a stable endothelium in the clinical setting. This review summarizes past and present techniques directed at improving the adhesion of ECs to blood-contacting devices. Impact statement Clinical success of blood-contacting devices such as vascular grafts, stents, and heart valves has remained limited by postimplantation problems, including thrombosis and loss of patency. Without a stable endothelial cell (EC) monolayer, blood-contacting devices are at risk for platelet activation and thrombosis. Methods to improve EC adhesion on these devices have not translated to long-term in vivo success, as many ECs are lost after exposure to circulating blood. In this study, we summarize methods to improve EC adhesion and retention. Successful endothelialization of blood-contacting devices may improve patient outcomes after device implantation and limit the need for long-term antiplatelet or anticoagulation therapy.

The porcine abattoir blood model-Evaluation of platelet function for in-vitro hemocompatibility investigations.

The major obstacle of blood-contacting medical devices is insufficient hemocompatibility, particularly thrombogenicity and platelet activation. Pre-clinical in-vitro testing allows for the evaluation of adverse thrombogenicity-related events, but is limited, among others, by the availability and quantity of human blood donations. The use of animal blood is an accepted alternative for several tests; however, animal and particularly abattoir blood might present species-specific differences to human blood as well as elevated blood values, and pre-activated platelets due to stressed animals and non-standardized blood collection. To this end, we investigated porcine abattoir blood in comparison to human donor blood with the focus on platelet pre-activation and remaining activation potential. By means of light transmission aggregometry, aggregation kinetics of platelet rich plasma after stimulation with three different concentrations of each adenosine diphosphate (ADP) (5µM, 10µM, 20µM) and collagen (2.5µg/ml, 5µg/ml, 10µg/ml) were monitored. The activation with collagen revealed no significant differences in platelet behavior of the two species. In contrast, stimulation with ADP resulted in a lower maximum aggregation and a high disaggregation for porcine abattoir blood. The latter is a species-specific phenomenon of porcine platelets. Variations within each study cohort were comparable for human and abattoir pig. The similarities in platelet activation following collagen stimulation and the preservation of the porcine-specific reaction to ADP prove a general functionality of the abattoir blood. This finding provides a first step towards the complete validation of the porcine abattoir blood model.

Discrete responses of erythrocytes, platelets, and von Willebrand factor to shear

Despite decades of technological advancements in blood-contacting medical devices, complications related to shear flow-induced blood trauma are still frequently observed in clinic. Blood trauma includes haemolysis, platelet activation, and degradation of High Molecular Weight von Willebrand Factor (HMW vWF) multimers, all of which are dependent on the exposure time and magnitude of shear stress. Specifically, accumulating evidence supports that when blood is exposed to shear stresses above a certain threshold, blood trauma ensues; however, it remains unclear how various constituents of blood are affected by discrete shears experimentally. The aim of this study was to expose blood to discrete shear stresses and evaluate blood trauma indices that reflect red cell, platelet, and vWF structure. Citrated human whole blood (n = 6) was collected and its haematocrit was adjusted to 30 ± 2% by adding either phosphate buffered saline (PBS) or polyvinylpyrrolidone (PVP). Viscosity of whole blood was adjusted to 3.0, 12.5, 22.5 and 37.5 mPa·s to yield stresses of 3, 6, 9, 12, 50, 90 and 150 Pa in a custom-developed shearing system. Blood samples were exposed to shear for 0, 300, 600 and 900 s. Haemolysis was measured using spectrophotometry, platelet activation using flow cytometry, and HMW vWF multimer degradation was quantified with gel electrophoresis and immunoblotting. For tolerance to 300, 600 and 900 s of exposure time, the critical threshold of haemolysis was reached after blood was exposed to 90 Pa for 600 s (P < 0.05), platelet activation and HMW vWF multimer degradation were 50 Pa for 600 s and 12 Pa for 300 s respectively (P < 0.05). Our experimental results provide simultaneous comparison of blood trauma indices and thus also the relation between shear duration and magnitude required to induce damage to red cells, platelets, and vWF. Our results also demonstrate that near-physiological shear stress (<12 Pa) is needed in order to completely avoid any form of blood trauma. Therefore, there is an urgent need to design low shear-flow medical devices in order to avoid blood trauma in this blood-contacting medical device field.

Oriented homo-epitaxial crystallization of polylactic acid displaying a biomimetic structure and improved blood compatibility.

Epitaxial crystallization and solid hot-drawing technology were employed to fabricate oriented homo-epitaxial crystallization of polylactic acid (PLA) with nano-topography to enhance its blood compatibility and mechanical characteristics as blood-contacting medical devices. The process involved solid hot stretching the PLA plates. A PLA nutrient solution was then used to immerse the oriented plates to dissolve some of the PLA solutes, ensuring plate integrity. Consequently, the drawing process exponentially enhanced the modulus and tensile strength of the PLA. Orientation and epitaxial crystallization could substantially enhance blood compatibility of PLA by prolonging clotting time and decreasing hemolysis rate, protein adsorption, and platelet activation. The oriented homo-epitaxial crystallization of PLA exhibited a nano-topography and fibrous structure similar to the intimal layer of a blood vessel, and this biomimetic structure was advantageous in decreasing the activation and/or adhesion of platelets.

Differences in human and minipig platelet number, volume and activation induced by borosilicate glass beads in a modified chandler loop-system.

The pig is the most widely used large animal model in Europe, with cardiovascular research being one of the main areas of application. Adequate refinement of interventional studies in this field, meeting the requirements of Russel and Burchs' 3 R concept, can only be performed if blood-contacting medical devices are hemocompatible. Because most medical devices for cardiovascular interventional procedures are developed for humans they are tested mostly for compatibility with human blood. The aim of this study was therefore to determine whether there are differences in behavior of porcine and human platelets when they come into contact with glass, which was used as an exemplary thrombogenic material. For this purpose changes of platelet count, platelet volume and platelet expression of the activation markers CD61, CD62P and CD63 were measured using a modified chandler loop-system simulating the fluidic effects of the blood flow. Minipig and human platelets showed significant differences in number and volume, but not in activation after 4-8 min exposure to glass.

Biomaterial Wettability Affects Fibrin Clot Structure and Fibrinolysis.

Thrombosis on blood-contacting medical devices can cause patient fatalities through device failure and unstable thrombi causing embolism. The effect of material wettability on fibrin network formation, structure, and stability is poorly understood. Under static conditions, fibrin fiber network volume and density increase in clots formed on hydrophilic compared to hydrophobic polystyrene surfaces. This correlates with reduced plasma clotting time and increased factor XIIa (FXIIa) activity. These structural differences are consistent up to 50µm away from the material surface and are FXIIa dependent. Fibrin forms fibers immediately at the material interface on hydrophilic surfaces but are incompletely formed in the first 5µm above hydrophobic surfaces. Additionally, fibrin clots on hydrophobic surfaces have increased susceptibility to fibrinolysis compared to clots formed on hydrophilic surfaces. Under low-flow conditions, clots are still denser on hydrophilic surfaces, but only 5µm above the surface, showing the combined effect of the surface wettability and coagulation factor dilution with low flow. Overall, wettability affects fibrin fiber formation at material interfaces, which leads to differences in bulk fibrin clot density and susceptibility to fibrinolysis. These findings have implications for thrombus formed in stagnant or low-flow regions of medical devices and the design of nonthrombogenic materials.

Monolithic polymeric porous superhydrophobic material with pneumatic plastron stabilization for functionally durable drag reduction in blood-contacting biomedical applications

Superhydrophobic (SHP) surfaces can provide substantial reductions in flow drag forces and reduce blood damage in cardiovascular medical devices. However, strategies for functional durability are necessary, as many SHP surfaces have low durability under abrasion or strong fluid jetting or eventually lose their air plastron and slip-flow capabilities due to plastron gas dissolution, high fluid pressure, or fouling. Here, we present a functional material that extends the functional durability of superhydrophobic slip flow. Facile modification of a porous superhydrophobic polytetrafluoroethylene (PTFE, Teflon) foam produced suitable surface structures to enable fluid slip flow and resist protein fouling. Its monolithic nature offered abrasion durability, while its porosity allowed pressurized air to be supplied to resist fluid impalement and to replenish the air plastron lost to the fluid through dissolution. Active pore pressure control could resist high fluid pressures and turbulent flow conditions across a wide range of applied pressures. The pneumatically stabilized material yielded large drag reductions (up to 50%) even with protein fouling, as demonstrated from high-speed water jetting and closed loop pressure drop tests. Coupled with its high hemocompatibility and impaired protein adsorption, this easily fabricated material can be viable for incorporation into blood-contacting medical devices.

Evaluation of blood simulating solvents in extractables and leachables testing for chemical characterization of medical devices based on Abraham general solvation model

Extraction vehicles (simulating solvents) used in chemical characterization (i.e., extractables/leachables testing) of blood-contacting medical devices for biocompatibility assessment per ISO 10993 have been studied by Abraham general solvation models. Chemically equivalent solvents to blood in solvation properties (solubility, partition, extraction, etc.) have been proposed based on Abraham’s organic solvent system coefficients for water and air to condensed organic solvent phases. This evaluation is built upon the conclusion by Abraham, Acree Jr and Cometto-Muñiz that water saturated n-butanol (called wet n-butanol) is chemically corresponding to blood. Although wet n-butanol can be directly used to replace blood in an extraction study (such as a simulated-use extraction study per ISO 10993-18 (2020)), it can be beneficial to have alternate solvents to wet n-butanol (butan-1-ol) when chromatographic interference is practically significant. By comparing Abraham solvent system coefficients for water to condensed organic solvent phases distribution, a five-dimensional space distance (D) between solvents and reference solvent (wet n-butanol) is calculated using Abraham and Martin equation to predict equivalent or similar solvents (to wet n-butanol). It is concluded from the study that ethanol/water (60/40, V/V) and ethanol/water (50/50, V/V) are chemically equivalent or quite similar to wet n-Butanol. Other two less similar solvents are ethylene glycol and ethanol/water (70/30, V/V). Thus, n-butanol, ethanol/water (60/40, V/V) and ethanol/water (50/50, V/V) can be used as blood simulating solvents in chemical characterization study of medical device. In certain situations, ethylene glycol might be desirable as an alternate solvent, as it is not a mixture. As Abraham solvation model is general (solvent system specific, not solute specific), the conclusions from this study are considered as universal.

Ultra-Low Von Willebrand Factor (VWF) Adsorption Under Shear with a Polymer Coating for Blood-Contacting Devices

Introduction. Many human diseases are treated with blood-contacting medical devices. However, they are often compromised because of blood proteins that accumulate on their surfaces. Such accumulation activates both platelets and blood coagulation, and patients supported by blood-contacting devices are at high risk of both bleeding and clotting. Blood passing through medical devices is often subjected to high shear stresses (especially near surfaces) and flow acceleration, conditions that we have shown promote rapid deposition of the large plasma glycoprotein von Willebrand factor (VWF) onto uncoated surfaces. Additionally, acquired Von Willebrand syndrome (AVWS) - a bleeding disorder caused by deficiency of high molecular weight VWF multimers - has been described in patients supported with ECMOs (extracorporeal membrane oxygenators) and LVADs (left ventricular assist devices). Blood in these patients experiences high shear stress when passing through the blood pump in ECMOs and LVADs. Thus, the adsorption of VWF to artificial surfaces under shear is likely to initiate platelet binding and the adsorption of other plasma proteins, including clotting proteins. Here, we show that polymers generated from the sulfobetaine methacrylate (SBMA) when coated onto surfaces are able to render these surfaces resistant to VWF binding (< 5 ng/cm2) under both static conditions and under shear stress.Preliminary results. We synthesized SBMA polymers (pSBMA) from SBMA according to methods described in the literature. Previously, we tested various coating conditions for pSBMA to achieve the lowest VWF adsorption. Under optimal conditions, VWF adsorption to pSBMA-coated polypropylene tubes was 4% of that to uncoated tubes (< 2.6 ng/cm2) after the tubes were exposed to a VWF solution (5 µg/mL) under shear applied with a vortexer (3000 rpm at RT for 90 min). In this work, we prepared a microfluidic device of polydimethylsiloxane that contained a tortuous flow channel to mimic shear stress conditions of blood vessels and tested the ability of pSBMA to prevent VWF deposition and platelet binding under different shear stress conditions.Methods. Purified plasma VWF (2.5 µg/mL) was perfused through the uncoated and pSBMA-coated devices for 1 h with a gravity-driven pump. To image the VWF structure, 5% BSA was perfused through the system to block nonspecific protein adsorption. Then FITC-labeled anti-human VWF antibody was perfused into the chamber system. After incubation at room temperature for 1 h, PBS was introduced to wash the system. The devices were fixed with 3.7% formaldehyde, incubated, and washed with PBS. We also perfused 1 mL of citrated human whole blood containing platelets stained with calcein AM through the devices at 0.05 mL/min with syringe pump to image platelet binding.Results. Under both conditions, VWF and platelet binding to the pSBMA-coated device was much lower than to the uncoated device. On the uncoated device, VWF formed aggregates which could not be washed away. By quantifying fluorescence intensity, we found that most VWF adsorbed along curves compared to the straight regions, the inlet, or the outlet of the chamber, consistent with shear stress simulation results that indicate that VWF deposits in the regions of highest shear and elongational flow. When whole blood was perfused through the chamber, the platelets bound to the entire device surface, with the inlet and outlet having relatively higher levels. In contrast, in the pSBMA-coated device, VWF bound loosely and could be removed almost completely with washing. We then perfused whole blood through uncoated or pSBMA-coated chambers without VWF pre-perfusion. Unlike the findings with VWF pre-perfusion, there was no apparent platelet binding in either device, indicating that the high level of platelet binding on the uncoated devices required time for VWF to deposit on the surface.Conclusions. PSBMA effectively prevented VWF adsorption under shear in two types of devices, and was stable during storage. We expect this coating, or a variation of it to significantly enhance the lifetime of blood-contacting devices and reduce both costs and medical complications of their use. DisclosuresNo relevant conflicts of interest to declare.

Potential of Superhydrophobic Surface for Blood-Contacting Medical Devices.

Medical devices are indispensable in the healthcare setting, ranging from diagnostic tools to therapeutic instruments, and even supporting equipment. However, these medical devices may be associated with life-threatening complications when exposed to blood. To date, medical device-related infections have been a major drawback causing high mortality. Device-induced hemolysis, albeit often neglected, results in negative impacts, including thrombotic events. Various strategies have been approached to overcome these issues, but the outcomes are yet to be considered as successful. Recently, superhydrophobic materials or coatings have been brought to attention in various fields. Superhydrophobic surfaces are proposed to be ideal blood-compatible biomaterials attributed to their beneficial characteristics. Reports have substantiated the blood repellence of a superhydrophobic surface, which helps to prevent damage on blood cells upon cell–surface interaction, thereby alleviating subsequent complications. The anti-biofouling effect of superhydrophobic surfaces is also desired in medical devices as it resists the adhesion of organic substances, such as blood cells and microorganisms. In this review, we will focus on the discussion about the potential contribution of superhydrophobic surfaces on enhancing the hemocompatibility of blood-contacting medical devices.

Mathematical models for shear-induced blood damage based on vortex platform.

Non-physiological shear stress in Ventricular Assist Device (VAD) is considered to be an important trigger of blood damage, which has become the biggest shackle for clinical application. The researches on blood damage in literature were limited to qualitative but did not make much quantitative analysis. The purpose of this study was to investigate the quantitative influence of two flow-dependent parameters: shear stress (rotational speed) and exposure time on the shear-induced damage of red blood cells and von Willebrand Factor (vWF). A vortex blood-shearing platform was constructed to conduct in vitro experiments. Free hemoglobin assay and vWF molecular weight analysis were then performed on the sheared blood samples. MATLAB was used for regression fitting of original experimental data. The quantitative correlations between the hemolysis index, the degradation of high molecular weight vWF and the two flow-dependent parameters were found both following the power law model. The mathematic models indicated that the sensitivity of blood damage on red blood cells and vWF to exposure time was both greater than that of shear stress. Besides, the damage of vWF was more serious than that of red blood cells at the same flow condition. The models could be used to predict blood damage in blood-contacting medical devices, especially for the slow even stagnant blood flow regions in VAD, thus may provide useful guidance for VAD development and improvement. It also indicated that the vortex platform can be used to study the law of blood damage for the simple structure and easy operation.

Mechanical Forces Impacting Cleavage of Von Willebrand Factor in Laminar and Turbulent Blood Flow

Von Willebrand factor (VWF) is a large multimeric hemostatic protein. VWF is critical in arresting platelets in regions of high shear stress found in blood circulation. Excessive cleavage of VWF that leads to reduced VWF multimer size in plasma can cause acquired von Willebrand syndrome, which is a bleeding disorder found in some heart valve diseases and in patients receiving mechanical circulatory support. It has been proposed that hemodynamics (blood flow) found in these environments ultimately leads to VWF cleavage. In the context of experiments reported in the literature, scission theory, developed for polymers, is applied here to provide insight into flow that can produce strong extensional forces on VWF that leads to domain unfolding and exposure of a cryptic site for cleavage through a metalloproteinase. Based on theoretical tensile forces, laminar flow only enables VWF cleavage when shear rate is large enough (&gt;2800 s−1) or when VWF is exposed to constant shear stress for nonphysiological exposure times (&gt;20 min). Predicted forces increase in turbulence, increasing the chance for VWF cleavage. These findings can be used when designing blood-contacting medical devices by providing hemodynamic limits to these devices that can otherwise lead to acquired von Willebrand syndrome.

Hemocompatibility Evaluation of Biomaterials-The Crucial Impact of Analyzed Area.

The hemocompatibility of blood-contacting medical devices remains one of the major challenges in medical device development. A common tool for the analysis of adherent and activated platelets on materials following in vitro tests is microscopy. Currently, most researchers develop their own routines, resulting in numerous different methods that are applied. The majority of those (semi-)manual methods analyze only a very small fraction of the material surface (<1%), which neglects the inhomogeneity of platelet distribution and makes results hardly comparable. Within this study, we examined the relation between the fraction of analyzed sample area and the platelet adhesion result. By means of image segmentation and machine learning algorithms, 103 100 microscopy images were analyzed automatically. We discovered a crucial impact of the analyzed surface fraction and thus a misrepresentation of a surface's platelet adhesion unless up to 40% of the sample surface is analyzed. These findings underline the necessity of standardization in the field of in vitro hemocompatibility tests and analyses in particular and provide a first basis to make future tests more reliable and comparable.

Blood-Compatible Materials: Vascular Endothelium-Mimetic Surfaces that Mitigate Multiple Cell-Material Interactions.

When flowing whole blood contacts medical device surfaces, the most common blood-material interactions result in coagulation, inflammation, and infection. Many new blood-contacting biomaterials have been proposed based on strategies that address just one of these common modes of failure. This study proposes to mitigate unfavorable biological reactions that occur with blood-contacting medical devices by designing multifunctional surfaces, with features optimized to meet multiple performance criteria. These multifunctional surfaces incorporate the release of the small molecule hormone nitric oxide (NO) with surface chemistry and nanotopography that mimic features of the vascular endothelial glycocalyx. These multifunctional surfaces have features that interact with coagulation components, inflammatory cells, and bacterial cells. While a single surface feature alone may not be sufficient to achieve multiple functions, the release of NO from the surfaces along with their modification to mimic the endothelial glycocalyx synergistically improves platelet-, leukocyte-, and bacteria-surface interactions. This work demonstrates that new blood-compatible materials should be designed with multiple features, to better address the multiple modes of failure of blood-contacting medical devices.

Novel Nitric Oxide Releasing Films Based on Inducible Nitric Oxide Synthase and Their Biomedical Applications

Although a tremendous number of biomaterials are labeled as biocompatible for blood-contacting medical devices, such as vascular grafts, stents, heart valves, and catheters, the thrombogenic nature of these materials can cause serious complications in patients, and eventually functional failure. There are two major limiting factors to clinical application of these materials: 1) platelet activation and thrombus formation, and 2) infection. Nitric oxide (NO) has been known to exert anti-thrombotic, anti-proliferative, and anti-inflammatory effects in the vasculature. Several polymers incorporate NO-generating substances in order to improve biocompatibility of blood-contacting medical devices. However, an important drawback for these NO-release materials is the finite stoichiometric amount of embedded NO that they contain, which could impact their function for long-term or semi-permanent types of implants. In addition, controlling the release rates of NO flux that can be emitted at the polymer/blood interface proved to be challenging, and often requires the addition of other components to the polymer. Therefore, we hypothesized that incorporating the enzyme nitric oxide synthase into polymeric films as functional catalytic units that generate NO would result its antithrombotic coatings with potentially unlimited amounts of NO released.In this project, we use layer-by-layer thin film building strategy to form layers of polyethyleneimine (PEI) and recombinant NOS enzymes as NO-releasing coatings. Charge-based layer-by-layer electrostatic adsorption allows for assembly of multi-component protein/PEI films with defined thickness and catalytic properties. When surfaces coated with PEI/NOS multilayered films are exposed to substrate arginine, a source of reducing equivalents, and other ingredients of the NOS reaction, nitric oxide is formed and released. In this work, we characterize the PEI/NOS thin films in terms of structure of NOS within the films as well as the amount of active NOS. Fourier transform infrared (FTIR) spectroscopic analysis was used to characterize structure-activity relationships of these NOS-containing thin films. We used cyclic voltammetry to determine the active enzyme concentration on the modified surfaces. We also examined how this activity relates to enzymatic activity in terms of NO released fluxes from the thin films. Finally, we assessed the functional performance of these films in terms of the extent to which they counteract platelet adhesion on the coated surfaces. To this end, we used platelet adhesion assays to determine how the number of platelets adsorbed on the PEI/NOS films is affected by the amount of NO released from the PEI/NOS thin films.

Automation of hemocompatibility analysis using image segmentation and supervised classification

The hemocompatibility of blood-contacting medical devices remains one of the major challenges in biomedical engineering and makes research in the field of new and improved materials inevitable. However, current in-vitro test and analysis methods are still lacking standardization and comparability, which impedes advances in material design. For example, the optical platelet analysis of material in-vitro hemocompatibility tests is carried out manually or semi-manually by each research group individually.As a step towards standardization, this paper proposes an automation approach for the optical platelet count and analysis. To this end, fluorescence images are segmented using Zach’s convexification of the multiphase-phase piecewise constant Mumford–Shah model. The non-background components then need to be classified as platelet or no platelet. For this purpose, a supervised random forest is applied to feature vectors derived from the components using features like area, perimeter and circularity. With an overall high accuracy (>93%) and low error rates (≤ 5%), the random forest achieves reliable results. This is supported by high areas under the receiver–operator characteristic curve (≥ 0.94) and the prediction–recall curve (≥ 0.77), respectively.We developed a novel method for a fast, user-independent and reproducible analysis of material hemocompatibility tests. The automatized analysis method overcomes the current obstacles in the way of standardized in-vitro material testing and is therefore a unique and powerful tool for advances in biomaterial research.