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Haemocompatibility of blood contacting devices; Material Design Strategies

Sinad Lyons- 0640093-University of Limerick, Biomaterials 2009 Abstract In biomedical engineering, haemocompatibility is the compatibility of a material with blood. It is an important consideration when designing devices that contact blood. Events that determine haemocompatibility often occur at the molecular level. Haemocompatibility can influence inflammatory processes. The use of biomaterials contacting blood is challenged by blood coagulation and immune attacks. These blood reactions are initiated through characteristics of the surface of biomaterials whichdespite being nontoxic and inertinduce protein adsorption and blood cell adhesion patterns often provoking the activation of the body defence systems. Procoagulant processes still form the major barrier in a variety of demanding applications [1]. In that context, extensive investigations on the surface modification of biomaterials have been undertaken with the prospect of attaining haemocompatibility characteristics similar to that of the healthy endothelium. Intact endothelial cells constitute an anticoagulant lining of the vessel wall with essential synthetic and metabolic functions concerning the haemostatic system [2, 3]. After injury the damage of blood vessels exposes blood to tissue factor produced constitutively by cells beneath the endothelium [4]. Tissue factor can also be produced through vascular endothelial cells and monocytes, triggered through endotoxin [5, 6]. It binds to factor VII or VIIa and promotes the formation of thrombin. The use of polymers, metals, ceramics and composite materials in the formulation of medical devices dates back centuries, however today these materials are designed to maximise haemocompatibility and the bulk and surface properties of the biomaterial are designed to aim to control the dynamic interactions that take place at the tissue/blood-implant interface.

Introduction The use of metallic biomaterials in permanent contact with blood has been permanently increased, especially since the first clinical application of coronary stents in the mid eighties [9]. Modern medical and surgical practise has increasingly come to rely upon indwelling devices of various kinds. Such devices may be utilised briefly or intermittently (e.g. intravenous catheters), for months to years (intra-uterine devices) or permanently (prosthetic heart valves and hips). Indwelling medical devices have certainly been instrumental in saving patients lives and have enhanced the quality of life of many more people. As with all forms of therapeutic intervention, however, adverse side effects are to be anticipated. In practise, one of the most frequent and serious complications of the use of these devices has been the development of infections. One of the most serious forms of intravascular device-related infections can occur when thrombus surrounding cannula becomes infected, producing septic thrombophlebitis with peripheral venous cannulas or septic thrombosis of a great central vein with centrally placed catheters. The magnitude of problems posed by infections and haemocompatibility issues associated with implant devices has grown in line with the proliferation of the devices themselves. Materials implanted for many months or years must meet several criteria. First, they must have specific mechanical properties, to replace the function of defective body tissues (10). Second, they must be accepted and integrated by the host in a controlled and predictable way (10). No

implanted artificial material can be considered totally inert in the body, as suggested by Hench and Wilson (11), who described four major categories of host responses: (i) the material releases some toxic compounds leading to surrounding tissue death, (ii) the nontoxic material is gradually resorbed and replaced by the surrounding tissue that is under repair, (iii) the material is nontoxic and biologically inactive but cannot be degraded by the host, which reacts by encapsulation, and (iv) the material is non toxic and biologically inactive but is highly interactive with the surrounding tissue forming chemical bonds (12). Materials coming in contact with the flowing blood must fulfil additional requirements: they must not damage blood cells or encourage formation of blood clots (13). When blood contacts foreign materials, there is an immediate reaction leading to some degree of protein and blood cell deposition on the material (13). If this process continues, clotting and thrombosis may result, leading to severe complications (13). Blood- Biomaterial Interactions Coagulation of blood regulated is by platelet activation, intrinsic pathways, extrinsic pathways, common pathways and the control systems involved in thrombus inhibition and fibrinolysis. The interaction between blood and biomaterials however is different, (a) unlike the endothelium biomaterials cannot perform an active role in thromboresistance, (b) biomaterials, unlike the endothelium cannot provide a non attractive surface. The haemocompatibility of a biomaterial is determined by the protein absorption at the interface. Uptake of protein at

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the blood-biomaterial interface is affected by: the surface charge, the hydrophobicity/hydrophilicity, the surface energy, the microstructure- microdomains of hydrophobic or hydrophilic regions potentially inhibit protein uptake, finally the adherence and release of proteins from the surface leads to a change in protein structure, which may cause an immune response or interfere with the blood clotting system. Protein adsorption is the first process which occurs at the bloodmaterial interface. The subsequent reactions are determined by the interactions with the absorbed protein layer. In production of viable biomaterials development has focused on albumen and fibrinogen proteins as albumen is present in largest amounts in blood and fibrinogen promotes platelet activation. Thrombosis The circulatory system must be selfsealing; otherwise continued blood loss from even the smallest injury would be life threatening. Normally, all but the most catastrophic bleeding

embolus is formed [14]. Thrombosis is the result of the blood coagulation (clotting) cascade which is activated in the body in response to damaged vessel walls and tissue. This damage can be caused by the interaction of foreign body materials implant materials- with surrounding tissues at the implant site. Thrombus on the artificial surface results in an interaction between platelets and the intrinsic pathway. Initiation of intrinsic coagulation may he induce by thromboplastins liberated from the platelets or factor XII activation caused by platelets stimulated by the release of adenosine diphosphate. Coagulation is a complex process by which blood forms clots. It is an important part of haemostasis (the cessation of blood loss from a damaged vessel), wherein a damaged blood vessel wall is covered by a platelet and fibrincontaining clot to stop bleeding and begin repair of the damaged vessel. Disorders of coagulation can lead to an increased risk of bleeding (haemorrhage) or clotting (thrombosis). Coagulation begins almost instantly after an injury to the blood vessel has damaged the endothelium (lining of the vessel), this releases phospholipid components called tissue factor and Fibrinogen that initiate a chain reaction. Platelets immediately form a plug at the site of injury; this is called primary haemostasis. Secondary haemostasis occurs simultaneously: Proteins in the blood plasma, called coagulation factors or clotting factors, respond in a complex cascade to form fibrin strands, which strengthen the platelet plug. Platelet activation
Figure 1 Platelet activation is an important early step in the pathophysiology of atherothrombosis. Platelet activation involves: 1) a shape change in which the platelet membrane surface area is greatly increased; 2) the secretion of pro-inflammatory, prothrombotic, adhesive, and chemo tactic mediators (release reaction), that propagate, amplify, and sustain the atherothrombotic process; and 3) the activation of the glycoprotein (GP) IIb/IIIa receptor from its inactive form. Multiple agonists including thromboxane A2 (TXA2), adenosine diphosphate (ADP), thrombin, serotonin, epinephrine, and collagen, can activate the platelet and thus contribute toward establishing the environmental conditions necessary for atherothrombosis to occur. Antithrombins such as unfractionated or low-molecular-weight heparin, hirudin, or bivalirudin are important in interfering with both thrombin-induced platelet activation and coagulation. The GP IIb/IIIa receptor antagonists act at a later step in the process by preventing fibrinogen mediated cross-linking of platelets, which have already become activated. ATP = adenosine

is rapidly stopped, by a process known as Coagulation, through several sequential processes. Coagulation involves the formation of a blood clot (thrombus) that prevents further blood loss from damaged tissues, blood vessels or organs. This is a complicated process with a cellular system comprised of cells called platelets that circulate in the blood and serve to form a platelet plug over damaged vessels and a second system based upon the actions of multiple proteins (called clotting factors) that act in concert to produce a Fibrin clot. Thrombosis is the formation of a blood clot (thrombus) inside a blood vessel, obstructing the flow of blood through the circulatory system. When a blood vessel is injured, the body uses platelets and fibrin to form a blood clot, because the first step in repairing it (haemostasis) is to prevent loss of blood. If that mechanism causes too much clotting, and the clot breaks free, an

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triphosphate; PAI = plasminogen activator inhibitor; PDGF = platelet-derived growth factor; vWF = von Willebrand factor. [11]

Blood contact with an artificial surface generally results in inducing adhesion and aggregation of platelets, with the absorbed protein layer playing a controlling factor in the platelet response. Adhesion of the platelets to the biomaterial surface is succeeded by the platelet release reaction in the adhered platelets followed by platelet aggregation on the surface. Damage to blood vessel walls by implanted biomaterials can expose subendothelium proteins, most notably von Willebrand factor (vWF), present under the endothelium. vWF is a protein secreted by healthy endothelium, forming a layer between the endothelium and underlying basement membrane. When the endothelium is damaged, the normallyisolated, underlying vWF is exposed to blood and recruits Factor VIII, collagen, and other clotting factors. Circulating platelets bind to collagen with surface collagen-specific glycoprotein Ia/IIa receptors. This adhesion is strengthened further by additional circulating proteins vWF), which forms additional links between the platelets glycoprotein Ib/IX/V and the collagen fibrils. These adhesions activate the platelets. Activated platelets release the contents of stored granules into the blood plasma. The granules include ADP, serotonin, plateletactivating factor (PAF), vWF, platelet factor 4, and thromboxane A2 (TXA2), which, in turn, activate additional platelets. The granules' contents activate a Gq-linked protein receptor cascade, resulting in increased calcium concentration in the platelets' cytosol. The calcium activates protein kinase C, which, in turn, activates phospholipase A2 (PLA2). PLA2 then modifies the integrin membrane glycoprotein IIb/IIIa, increasing its affinity to bind fibrinogen. The activated platelets change shape from spherical to stellate, and the fibrinogen crosslinks with glycoprotein IIb/IIIa aid in aggregation of adjacent platelets.

Figure 2: the Coagulation Cascade: Cambridge University Press (2004)[12]

Key Factors in Blood Clotting: 1. Multifactor system 2. Biochemical Amplification 3. Localisation of surface dependent steps on exposed phospholipid 4. Vitamin K dependent synthesis of factors VII,IX, X and prothrombin 5. Calcium requirement for activation of factors VII, IX, X and prothrombin 6. Ordered branch polymer formation 7. Covalent cross linking The coagulation cascade The coagulation cascade of secondary haemostasis has two pathways, the contact activation pathway (formerly known as the intrinsic pathway), and the tissue factor pathway (formerly known as the extrinsic pathway), which lead to fibrin formation. It was previously thought that the coagulation cascade consisted of two pathways of equal importance joined to a common pathway. It is now known that the primary pathway for the initiation of blood coagulation is the tissue factor pathway. The pathways are a series of reactions, in which a zymogen (inactive enzyme precursor) of a serine protease and its glycoprotein co-factor are activated to become active components that then catalyze the next reaction in the cascade, ultimately resulting in cross-linked fibrin. Coagulation factors are generally indicated by Roman numerals, with a lowercase a appended to indicate an active form. The coagulation factors are generally serine proteases (enzymes). There are some exceptions. For example, FVIII and FV are glycoproteins, and Factor XIII is a transglutaminase. Serine proteases act by

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cleaving other proteins at specific sites. The coagulation factors circulate as inactive zymogens. The coagulation cascade is classically divided into three pathways. The tissue factor and contact activation pathways both activate the "final common pathway" of factor X, thrombin

and fibrin. [22] Tissue factor pathway (extrinsic) Eventually the Intrinsic pathway activates FX , a process that can also be brought about by the Extrinsic pathway, an alternative route for the activation of the clotting cascade. It provides a very rapid response to tissue injury, generating FX a almost instantaneously, compared to the seconds or even minutes required for the Intrinsic pathway to activate FX . The main function of the Extrinsic pathway is to augment the activity of the Intrinsic pathway. The Extrinsic pathway takes place at the site of a break in a blood vessel that has the platelet aggregate. There are two components unique to the Extrinsic pathway: TF/FIII (Factor-III), and FVII. TF and FVIIa activate FX, forming FXa. TF is present in most human cells bound to the cell membrane. Once activated, TF binds rapidly to FVII which is then activated to form a complex of TF ,FVIIa , Calcium, and a Phospholipid, and this complex then rapidly activates FX The main role of the tissue factor pathway is to generate a "thrombin burst," a process by which thrombin, the most important constituent of the coagulation cascade in terms of its feedback activation roles, is released instantaneously. FVIIa circulates in a

higher amount than any other activated coagulation factor. [20&21]. Following damage to the blood vessel, FVII leaves the circulation and comes into contact with tissue factor (TF) expressed on tissue-factorbearing cells (stromal fibroblasts and leukocytes), forming an activated complex (TF-FVIIa). TF-FVIIa activates FIX and FX. FVII is itself activated by thrombin, FXIa, plasmin, FXII and FXa. The activation of FXa by TF-FVIIa is almost immediately inhibited by tissue factor pathway inhibitor (TFPI). FXa and its co-factor FVa form the prothrombinase complex, which activates prothrombin to thrombin. Thrombin then activates other components of the coagulation cascade, including FV and FVIII (which activates FXI, which, in turn, activates FIX), and activates and releases FVIII from being bound to vWF. FVIIIa is the co-factor of FIXa, and together they form the "tenase" complex, which activates FX; and so the cycle continues. ("Tenase" is a contraction of "ten" and the suffix "-ase" used for enzymes.) [20&21] Figure 3: Intrinsic and Extrinsic Pathways [13]
Diagram showing intrinsic and extrinsic pathways of blood coagulation. The coagulation cascade is initiated via the extrinsic pathway as a result of tissue damage and the exposure of blood to tissue factor (TF). The two pathways converge when factor X is activated. The active forms of the serine proteases and of the two cofactors V and VIII are indicated by a lower case a; X denotes the zymogen factor X, and Xa the active enzyme factor Xa. The activation of factors V and VIII by thrombin and by factor Xa is denoted, as well as the initiation of the intrinsic pathway by thrombin-mediated activation of factor XI. Thrombin also activates factor XIII and protein C of the protein C anticoagulant system. PL = phospholipid.

[Source: Neurosurgoen Focus 2004 American Association of Neurological Surgeons]

Contact activation pathway (intrinsic) The Intrinsic pathway is activated when blood comes into contact with sub-endothelial connective tissues or with negatively charged surface that are exposed as a result of tissue damage. Quantitatively it is more important of the

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two pathways, but is slower to cleave Fibrin than the extrinsic pathway. The Hageman factor/FXII (Factor-XII),FXI (Factor-XI), PKlk (Prekallikrein),and HK (High Molecular Weight Kininogen) are involved in the activation of this pathway. The first step is the binding of FXII to a sub-endothelial surface exposed by an injury. A complex of PK and HK also interacts with the exposed surface in close proximity to the bound FXII ,which becomes activated. During activation, the single chain protein of the native Hageman factor is cleaved into two chains (50 and 28 kea),that remain linked by a disulphide bond. The light chain (28kDa) contains the active site and the molecule is referred to as activated Hageman factor/ FXII a (Activated Factor-XII). There is evidence that the FXII can auto activate and FXII a can also activate PK, thus making the pathway self-amplifying, once triggered. The Klk (Kallikrein) produced can then cleave FXII ,and a further amplification mechanism is triggered. The FXII a remains in close contact with the activating surface, such that it can activate FXI (Factor-XI), the next step in the Intrinsic pathway which, to proceed efficiently, requires Ca2+ (Calcium). Also involved at this stage is HK, which binds to FXI and facilitates the activation process. Activated factors FXIa , FXIIa, and Kallikrein are all serine proteases. In the presence of Ca2+,FXIa activates FIX to FIXa. F9 is a proenzyme that contains vitamin K-dependent Gamma-Carboxyglutamate residues, whose serine protease activity is activated following Ca2+ binding to these Gamma-Carboxyglutamate residues. FXIa converts FX to FXa; to do this it requires: Calcium ions, FVIIIa (Factor-VIIIa), and a negatively charged surface; in vivo, this is the surface of platelets, i.e., PL (Phospholipids) [15& 16]. Final common pathway The Intrinsic and Extrinsic systems converge at FX, to a single Common pathway which is ultimately responsible for the production of Thrombin (Factor-IIa). Thrombin has a large array of functions. Its primary role is the conversion of fibrinogen to fibrin, the building block of a haemostatic plug. In addition, it activates Factors VIII and V and FXI in a

feedback manner (their inhibitor protein C in the presence of thrombomodulin), and it activates Factor XIII, which forms covalent bonds that crosslink the fibrin polymers that form from activated monomers. Thrombin converts Fibrinogen (Factor-I) to Fibrin (Factor-Ia). In addition, Thrombin also acts to enhance the activation of factor: FV,F8. Fibrinogen is a dimer soluble in plasma. The exposure of Fibrinogen to Thrombin results in its rapid proteolysis that results in the release of Fibrinopeptide-A. The loss of small Peptide-A is not sufficient to render the resulting Fibrin molecule insoluble, a process that is required for clot formation, but it tends to form complexes with adjacent Fibrin and Fibrinogen molecules. A second peptide, Fibrinopeptide-B, is then cleaved by Thrombin, and the Fibrin monomers formed by this second proteolytic cleavage polymerize spontaneously to form an insoluble gel. The polymerized Fibrin, held together by non-covalent and electrostatic forces, is stabilized by the transamidating enzyme FXIIIa (Factor-XIIIa), produced by the action of Thrombin on FXIII . These insoluble Fibrin aggregates (Clots), together with aggregated platelets (Thrombus), block the damaged blood vessel and prevent further bleeding [17, 18 &19]. Following activation by the contact factor or tissue factor pathways, the coagulation cascade is maintained in a prothrombotic state by the continued activation of FVIII and FIX to form the tenase complex, until it is down-regulated by the anticoagulant pathways. Table1. Coagulation factors Factor Name I Fibrinogen II Prothrombin III Thromboplastin IV Calcium ion V Proaccelerin VI Not assigned VII Proconvertin VIII Antihaemophilic factor XI Christmas factor X Stuart factor XI Plasma thromboplastin antecedent XII Hageman factor XIII Fibrin stabilizing factor

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Summary of the Coagulation Cascade and Platelet Activation in response to Tissue Damage Fi gu re 4:

The Blood Coagulation Cascade: Ambion, Inc. GeneAssist Pathway Atlas 14-3-3 Induced
Apoptosis Pathway. "Caspase Protein Gene Information". Retrieved 26 Nov 2007. http://www.ambion.com/tools/pathway/protein_list_grp.php?pathway=14-3 3%20Induced %20Apoptosis&pname=Caspase3[38]

Material Design Strategies

Biomaterials which are in contact with flowing blood must meet several criteria, they

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must not cause adverse reactions or release leachable components into the bloodstream, they must not suffer alterations or generate any debris, and no chronic or acute inflammatory response should be induced by the material. Therefore materials should not be thrombogenic, a stimulant of haemolysis or an activator of the complement system. These properties are controlled by bulk and surface chemistry and physics, surface morphology and extrinsic factors like; implant site, function, duration of implant, and the local haemodynamics. Ideal blood compatible materials (material used in contact with blood) do not activate the intrinsic blood coagulation system or attract or alter platelets or leukocytes. Blood compatibility of any material is dependent on surface charge, surface free energy, chemical group distribution, heterogeneity, surface texture, porosity, smoothness and flow conditions. International efforts continue toward production of materials that prevent thrombosis, surface modification to enhance blood compatibility and use of therapeutic substances that prevent deposition of fibrin. Some of the commonly used blood compatible materials are polydimethyl

siloxane, cellulose acetate, polyacrylonitrile, PTFE, nylon, polycarbonate, polyurethane, poly (methyl methacrylate) (PMMA), pyrolytic and low-temperature isotropic (LTI) carbons, etc. When any material comes in contact with blood, instant adsorption of proteins takes place with a certain selectivity, rate and concentration. The interaction involves intermolecular forces, which develop at the interface, e.g., London dispersion forces, hydrogen bonds, dipole-dipole interactions, acid-base interactions, etc. Albumincoated surfaces do not seem to attract platelets [23], whereas -globulin and fibrinogen coatings cause not only platelet adhesion but also aggregation and release of platelet constituents [24]. The processing parameter also can change the nature of the adsorbed proteins due to variations in microstructure, for example, the glass side and air side of cast polyurethane urea with polypropylene glycol (MW 1025) are found to be different as the air-side domain size is 80 120 with 5080 domains in 10001000 2 area, and the glass-side size 3050 [25].

Figure 5: Complement Activation, Platelet Activation & Protein Absorption in the coagulation cascade [39]

All synthetic foreign materials are blood compatible in the sense that they do not have the intrinsic capacity of inducing the specific, evolution-selected mechanisms that lead to the thrombus formation. These mechanisms occur directly only with natural materials such as collagen and tissue factors. In contrast, artificial materials acquire thrombogenicity only after first interacting with blood, and the event responsible for the transformation of the inert polymer to an active surface is the interaction of plasma proteins with the surface that then initiates foreign-surface thrombosis. Although these statements pose an oversimplification, they suggest a critical role for

protein adsorption in thrombosis on foreign materials. Processes such as protein adsorption, platelet and complement activation, and fibrin polymerization are all surface-induced phenomena and depend to a large extent on the properties and composition of the outer few atomic layers of a material. The successful design of materials for use as blood-contacting devices thus demands both advanced techniques for surface analysis and adequate criteria for surface property determination, with the intent of correlating the molecular events occurring at the interface with the chemistry and the haemocompatibility of the biomaterial. The

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successful design of materials for use as bloodcontacting devices thus demands both advanced techniques for surface analysis and adequate criteria for surface property determination, with the intent of correlating the molecular events occurring at the interface with the chemistry and the haemocompatibility of the biomaterial. Fundamental progress in design of new materials with improved haemocompatibility will, moreover, be determined by a deeper understanding of the nature of interfacial phenomena, mainly protein and platelet interactions with surfaces. Surface Modifications to Improve biomaterial Haemocompatibility Various strategies have been followed for enhancing material haemocompatibility with most of them related to surface modification approaches; [26] in particular: 1) strategies for creating inert materials and 2) strategies for developing active materials. Inert Materials Much of the effort in biomaterials research over the past 35 yr has been directed toward the development of materials that do not react with platelets and coagulation factors. A number of approaches, often conflicting, have been developed and researchers have primarily focused their efforts on modifying surfaces of existing polymeric materials. This approach is reasonable as it is only the surface chemistry of the material that should dictate its biological responses. Despite the success in reducing protein and cellular deposits on some materials, a truly hem compatible surface has not been achieved. A popular method to improve the blood compatibility of materials is to increase their surface hydrophilicity by surface-incorporation of hydrogels, which permit the retention of large amounts of water providing the surface with minimal interfacial energy similar to biological tissues. Although the surface-incorporation of hydrogels produces protein-resistant surfaces, a large ex vivo study of a variety of materials, containing hydrogels incorporated at their surface, concluded that hydrogels do not posses low thrombogenicity.[26] Surfaces have also been coated with hydrophilic polymers such as poly(vinyl pyrrolidone) (PVP). The PVP is generally used to increase lubricity and ease catheter insertion, but its utilization also provided beneficial effects on thrombogenicity and bacterial adhesion. [26]

Immobilization of the water-soluble synthetic polymer poly (ethylene glycol) (PEG) is a very popular means of making a biomaterial surface more protein and cell resistant. The protein-surface interactions have theoretically been modelled in the presence of PEG, concluding that steric repulsion by surface-bound PEG was largely responsible for the prevention of protein adsorption. The PEG and block copolymers poly (ethylene oxide)/ poly (propylene oxide) (PEO/PPO) have been inserted into surfaces [26] or combined to other strategies [27] in an attempt to increase surface haemocompatibility. While the in vitro results have looked very promising, the lack of correlation between in vitro and ex vivo studies is of concern. More recent efforts with plasmadeposits tetraglyme [28] have led to surfaces which adsorb very low amount of fibrinogen, suggesting that previous attempts at using PEG modification have not been successful because of the inability to achieve the desired ultralow levels of adsorbed protein. Based on the hypothesis that a surface similar to the external phospholipid membrane of cells should be blood compatible, various approaches have been pursued to incorporate phospholipids into surfaces, including phosphorylcholine coated or blended materials. In vivo observations proved that platelet adhesion was significantly reduced on phosphorylcholinecontaining surfaces. [29] The mechanism of action of such materials is however unclear, although the low protein adsorption observed on these surfaces has been attributed to their high free-water fraction. The blending of a copolymer, consisting of polar and non polar blocks, to a base polymer has been considered a useful means of improving haemocompatibility of materials. Copolymers with an amphipathic structure, added in low concentration, demonstrated the ability of migrating to the base polymer surface during and after fabrication. The polycaprolactone polysiloxane block copolymers have been used to lower the thrombogenicity of cardiopulmonary bypass and haemodialysis components by using surface-modified additives (SMA) - blended polymers or SMA-coated surfaces. A clinical evaluation of the effect of SMA on cardiopulmonary bypass circuits demonstrated a reduction in platelet interactions with no effect on complement activation. [23] The incorporation of fluorine into materials is another investigated strategy to improve blood compatibility. Fluorine groups are believed to inhibit protein adsorption

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and platelet adhesion because of their low surface energy. Fluor alkyl groups as chain extender have been used to realize non thrombogenic polyurethane, and a number of fluorinated SMA have been developed. [26] Plasma treatment is another surface modification technique, which results in a covalently attached coating on the surface of a substrate. Various materials were treated with a variety of monomers in plasma reactors, but the comparison of many plasma-modified surfaces did not allow to identify a modification chemistry that was superior to the base material in terms of platelet or leukocyte activation.[24] A number of synthetic polymers have been synthesized or modified[25] to prepare polymers with heparinlike activities and then incorporated into polymer matrices or surfaces to enhance their haemocompatibility.[25] Self-assembled surface layers have been envisioned as useful templates to nucleate or organize ordered, designed biomaterials.[30] Self-assembled monolayers (SAMs) of alkylsilanes on poly(dimethylsiloxane) (PDMS) have been used as model system.[31] They have been demonstrated to lower platelet and fibrinogen deposition than surfaces composed of hydrophilic head groups in a canine ex vivo arteriovenus series shunt model. Active Materials The limited successes of the previously discussed surface treatments for enhancing haemocompatibility have encouraged researchers to pursue also other strategies. A significant part of current research for more haemocompatible materials is focused on the surface modification of already existing materials with satisfying mechanical properties rather than the development of new materials. Surface modification methods in order to improve the haemocompatibility include chemical treatment (surface oxidation, fluorination, introduction of reactive groups, glow plasma discharge, surface grafting, etc.).More recently new surface modification techniques have been developed which include immobilization of specific biological molecules on the materials surface, mostly heparin and albumin. The most popular and the one that started the field of bioactive materials is the heparinisation of surfaces, although the incorporation of other antithrombotic and anti-platelet agents into material surfaces is gaining popularity. Heparin, an often-used anticoagulant is an anionic, highly sulphated mucopolysaccharide not having a

uniform molecular structure. In an effort to counteract thrombogenicity, while preventing bleeding caused by direct administration of heparin, many researchers tried to develop methods of attaching and binding heparin on biomaterials surface. Early work on anti thrombogenic agents was done by Gott et al. [20]. The method comprised treating a graphited surface first with Zephiran (benzalkonium chloride) and then with heparin providing a good non-thrombogenicity. Since then a lot of research has been done on this particular item so that a few years later review articles and books on this subject where published [21 & 22] Generally, heparinised materials are of two major types:
Bulk heparinised materials, where heparin is

included in or under a bio stable or biodegradable polymeric material (depending on the application) throughout which it is progressively released at the bloodmaterial interface. The efficacy of these materials depends on the duration of heparin release, which in vivo can range from weeks to months. And Heparin coated materials having a surface layer of bound heparin which is thought to be capable of complexing in situ with AT III and exerting its anticoagulant effect. Such heparin-coated materials can also be divided into three main categories: a) Materials formed simply by mixing heparin (normally in the form of heparin sodium) into a material. Such materials have the disadvantage that, while in contact with the blood, the heparin is washed out into the blood and the antithrombotic properties disappear in a short time. b) Materials in which ionic bonding is effected between ionic residual groups in the heparin (COO, SO42, NHSO3) and cationic residual groups in the material (fe. using ionic-bonded complexes of heparin and a polymerisable basic compound having a quaternary ammonium salt). Such a material has the disadvantage that the setting of conditions for polymerization of the ionicbonded complex is difficult and discoloration of the material tends to occur, so obtaining an antithrombotic medical material of uniform product quality is difficult. Also attention must be paid that the ionic association of the cation and heparin anion must be sufficiently high to preclude blood from exchanging with the cationheparin association and removing heparin from the surface of the polymer.

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c) Materials in which covalent bonding is performed with the material, utilizing the hydroxyl, carboxyl or amino groups of the heparin. Usually, covalent immobilization of heparin to a biomaterial consists of activating the material in such a way that coupling between the biomaterial and functional groups on the heparin (COOH, OH, NH2) can be achieved.

Depending on its molecular weight and structure, heparin is able to inhibit, in association with its cofactor, antithrombin III, the serine proteases: thrombin and factor X. Heparinised surfaces should also help to minimize platelet activation. Heparin has been covalently, ionically, and physically incorporated to various substrates utilizing a number of chemistries. The main concern has been that once immobilized, the heparin should be able to assume its native conformation and interact with antithrombin III. If heparin is bound ionically to the surface, then it can be slowly released over time. Selected techniques to produce materials that release heparin at biologically significant rates have been investigated. [26] The thromboresistance of materials based on controlled release appears to be because of the microenvironment of heparin in solution at the bloodmaterial interface. A commercial procedure to ionically bind heparin (Duraflo II) has been used to coat cardiopulmonary bypass circuits and other devices. While the heparin coating is able to reduce thrombus formation, during clinical trials, its effect on complement, contact activation, and inflammation has been contradictory. [26] To make the activity of immobilized heparin longer than that reached with ionic linkages, the molecule has been covalently immobilized to material surfaces. The conformation of the attached heparin and the point of attachment are critical factors determining the catalytic efficiency of the immobilized bio molecule. Heparin has been covalently bound to surfaces by different methods.[26] The most popular chemistry for covalently binding heparin to surfaces of plastics, glass, and steel is the Carmeda method[32] The technique used provided highly stable coatings with low thrombogenicity that have been demonstrated to retain good efficacy in vivo. In this case, a beneficial effect on in vivo bacterial colonization of treated polyurethane central venous catheters was also observed.[26] Recently, a comparison of heparinised materials obtained by the Duraflo and Carmeda methods showed no dramatic differences between the two

surfaces in clinical trials.[32] Bioline coating is a newer surface-heparinisation method that is used to cover components of extracorporeal perfusion circuits.[33] The effectiveness of this surface coating is associated with platelet preservation, amelioration of inflammatory response, and reduction of fibrinolytic activity during cardiopulmonary bypass. Surface heparinisation limited to the oxygenator and the arterial filter had similar results as totally surface-heparinised circuits. [34] Agents other than heparin have been surface immobilized to inhibit thrombin, and thereby enhance material haemocompatibility. Among these, hirudin has some advantages compared to heparin, because it has no influence on platelet function, has no immune mediated platelet-activating activity, and, most importantly, does not require the presence of endogenous cofactors such as antithrombin III. Thus, hirudin has been covalently bound to different surfaces with the aim of improving their blood compatibility and its controlled release has been exploited. [26] Another approach involves the immobilization of thrombomodulin, an endothelial cell-associated protein that inhibits thrombin by the activation of protein C. Thrombomodulin has been immobilized on different surfaces and both its anticoagulant activity and the ability to reduce platelet adhesion have been demonstrated. [26] The inevitable presence of platelets on biomaterials dictated the interest to incorporate anti-platelet agents into materials. Prostaglandins, prostacyclins, and their conjugates with heparin have been incorporated into various material surfaces.[26] Anti-platelet agents based on inhibiting fibrinogen binding to activated platelet GPIIb/IIIa receptors have been used to reduce surface thrombogenicity via drug release. A novel approach was to exploit endogenous Snitrosoproteins in plasma to produce NO from immobilized cysteine to minimize platelet adhesion on polyurethane and poly (tetrafluoroethylene) (PTFE). [35] Surface thrombogenicity can also be lowered by the lysis of fibrin clots on artificial surfaces. Fibronolysis can be promoted by inducing surface generation of plasmin. Plasminogen has been directly immobilized, or selectively adsorbed by the immobilized lysine, onto surfaces and then converted to plasmin, so that the surfaces acquire fibrinolytic activity. [26] It is intuitive to believe that the ideal surface for vascular grafts/artificial hearts will consist of an intact luminal endothelial cell layer. Dacron [poly

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ethylene terephthalate (PET)] and PTFE have been seeded with endothelial cells in a preliminary clotting step, [36] and encouraging results have been obtained in vivo experiments with dogs. [36] Several attempts have been made to modify materials to give them a higher affinity for endothelial cells. For example endothelial cells have been seeded on CO2 plasma-treated polystyrene pre-coated with substances able to promote endothelial cell adhesion. [36] Seeding this surface with endothelial cells (ECs) significantly reduced the number of adhered platelets. Moreover, polystyrene and PTFE surfaces modified by NH3 plasma facilitated the attachment of ECs. [36] Surfaces precoated with fibrinogen or fibronectin or containing Arg-GlyAsp (RGD) peptides [36] resulted in higher number of adhered cells. The recent development of stents for maintaining patency in coronary arteries after angioplasty introduces metallic surfaces to the circulatory system. In lowering metal thrombogenicity, the interest was firstly directed toward heparin-coated stents. These have been then supplanted by rapamycin-eluting stents that have the effect of eliminating in-stent restenosis for at least six months after deployment.[37] Other strategies have been used to enhance the stent haemocompatibility by coating the metal surface with polyurethane and parylene, or hyaluronic acid, or anti-platelet agents to reduce platelet adhesion.[26] Study A study carried out in 2002 in the Biomedical Engineering Laboratory, Department of Mechanical Engineering and Aeronautics, University of Patras, Greece, compared the haemocompatibility improvement of four polymeric biomaterials by two heparinisation techniques. The two heparinisation techniques applied, one indirect and one direct, were selected to covalently bind heparin molecules on the biomaterials surface, were first introduced by Bamford and Al-Lamee and Seifert and Groth, respectively. 1. The first heparinisation method was executed in three stages:

Hydroxylation of the surface: the polymers were treated with a 10% aqueous solution the oxidising agent potassium peroxydisulfate for 1 h at 70C dynamically, using a Harvard Apparatus syringe pump at a shear rate of 200 s1 infusionwithdrawal mode. Subsequently the polymers were washed with hot water and vacuum dried. Grafting to functionalized polymers: the polymers were then submitted to a grafting reaction in an aqueous solution utilising the ceric ion technique [27] with acrylamide (10% w/v) in nitric acid and ceric ammonium solution (0.04 and 0.002 respectively) at 50C for 1 h under a stream of nitrogen. The materials were washed with 0.05 NaOH and hot water. The Heparin was immobilised on the treated materials surface using 1% w/v heparin solution at pH 4.5 containing; 0.1% w/v carcodiimide hydrochloride (EDC-Sigma), which acts as a coupling agent. The reaction was continued for 48 h at ambient temperature under static conditions. In the second method heparin immobilization was performed using heparin sodium with glutaraldehyde (as a coupling agent). A mixture of 12% aqueous solution of heparin and 6% aqueous solution of glutaraldehyde adjusted to pH 5.2 was incubated in the tubes for 2 h under static conditions. The amount of immobilized heparin was estimated by a spectrophotometric assay described. Both heparinisation techniques did not give a good yield of surface immobilized heparin compared to the amount of heparin initially provided, the indirect technique giving the more satisfactory results in terms of heparin immobilization. However, in terms of the amount of immobilized heparin per surface area, the results are satisfactory compared to the ones mentioned in the literature by various methods. Quite different values of immobilized heparin are reported depending on the starting polymer and the heparinisation method. (Table 2)[42]

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Table 2: Herparin Immobilisation Yield [42] In order to design biomaterials with desirable levels of haemocompatibility it is essential to understand the blood-material interactions which lead to thrombosis, inflammation and other tissue and blood implant associated/ induced injury at the implantation site. The complexity of the interactions between materials and blood makes the development of truly haemocompatible surfaces very difficult. Biomaterials research is currently focusing on the development of materials which reduce the risks and increase the benefits of the specific biomaterial, but which have yet to eliminate or provide a solution to the issue of haemocompatibility. Stents that actively prevent restenosis are a great example of how surface modification of metals can result in a dramatic clinical and effect. Inert materials are protein and platelet resistant, but they do not prevent thromboembolic reactions when implanted at sites in the body. On the other hand however, the incorporation of anticoagulants in the surfaces of biomaterials reduces thrombin production, but this may not be sufficient to prevent platelet adhesion and activation. Thus, which approach will ultimately be successful is impossible to predict. Perhaps, the failure to produce an ideal blood-compatible surface merely reflects our limited understanding of the complex blood-materials interaction. Progress in designing haemocompatible materials will be then determined by our deeper understanding of both the surface and bulk properties of the materials and the interfacial phenomena and reactions originating at the bloodmaterial interface which induces the thrombogenic and immune response.

Conclusion

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