Synthesis and characterization of a new vertebroplasty cement based on gold-containing PMMA microspheres.

There are a number of drawbacks to incorporating large concentrations of barium sulfate (BaSO4) as the radiopacifier in PMMA-based bone cements for percutaneous vertebroplasty. These include adverse effects on injectability, viscosity profile, setting time, mechanical properties of the cement and bone resorption. We have synthesized a novel cement that is designed to address some of these drawbacks. Its powder includes PMMA microspheres in which gold particles are embedded and its monomer is the same as that used in commercial cements for vertebroplasty. In comparison to one such commercial cement brand, VertaPlex™, the new cement has longer doughing time, longer injection time, higher compressive strength, higher compressive modulus, and is superior in terms of cytotoxicity. For augmentation of fractured fresh-frozen cadaveric vertebral bodies (T6-L5) using simulated vertebroplasty, results for compressive strength and compressive stiffness of the construct and the percentage of the volume of the vertebral body filled by the cement were comparable for the two cements although the radiopacity of the new cement was significantly lower than that for VertaPlex™. The present results indicate that the new cement warrants further study.

Effect of anti-ApoA-I antibody-coating of stents on neointima formation in a rabbit balloon-injury model.

BACKGROUND AND AIMS: Since high-density lipoprotein (HDL) has pro-endothelial and anti-thrombotic effects, a HDL recruiting stent may prevent restenosis. In the present study we address the functional characteristics of an apolipoprotein A-I (ApoA-I) antibody coating in vitro. Subsequently, we tested its biological performance applied on stents in vivo in rabbits. MATERIALS AND METHODS: The impact of anti ApoA-I- versus apoB-antibody coated stainless steel discs were evaluated in vitro for endothelial cell adhesion, thrombin generation and platelet adhesion. In vivo, response to injury in the iliac artery of New Zealand white rabbits was used as read out comparing apoA-I-coated versus bare metal stents. RESULTS: ApoA-I antibody coated metal discs showed increased endothelial cell adhesion and proliferation and decreased thrombin generation and platelet adhesion, compared to control discs. In vivo, no difference was observed between ApoA-I and BMS stents in lumen stenosis (23.3±13.8% versus 23.3±11.3%, p=0.77) or intima surface area (0.81±0.62 mm2 vs 0.84±0.55 mm2, p=0.85). Immunohistochemistry also revealed no differences in cell proliferation, fibrin deposition, inflammation and endothelialization. CONCLUSION: ApoA-I antibody coating has potent pro-endothelial and anti-thrombotic effects in vitro, but failed to enhance stent performance in a balloon injury rabbit model in vivo.

Adsorption of albumin on flax fibers increases endothelial cell adhesion and blood compatibility in vitro.

The physical and chemical properties of flax (linen) are attractive from the perspective of biomaterials science and engineering. Flax textiles uniquely combine hydrophilicity and strength, with the technical know-how to produce precisely engineered two- and three-dimensional knitted or woven structures. It is, however, extremely difficult to completely remove endotoxins from the flax, and this essentially precludes the use of linen for implant purposes. Herein, the potential utility of flax textiles for blood-contacting applications is investigated, using purified two-dimensional mesh specimens, with and without an albumin surface coating. It was hypothesized that the albumin coating will abolish the effect of adherent endotoxins at the flax's surface. In vitro cell viability assays showed that the flax mesh ± albumin is not cytotoxic. The albumin coating reduced (but not abolished) the effect of surface-exposed endotoxins (Limulus amebocyte lysate test). Under dynamic conditions, the albumin coating favors coverage with endothelial cells. Experiments with fresh human blood plasma (platelet-rich and platelet-free) showed that the albumin coating reduces the thrombogenicity in vitro. Platelets adhered to the albumin-coated flax mesh showed a less flattened structure. Although the results of this work cannot be extrapolated easily to in vivo situations, the data reveal that woven or knitted tubular structures produced from flax fibers may hold promise as implantable blood contacting devices like for instance vascular grafts.

Utilization of flax fibers for biomedical applications.

Over the past decades, a large number of animal-derived materials have been introduced for several biomedical applications. Surprisingly, the use of plant-based materials has lagged behind. To study the feasibility of plant-derived biomedical materials, we chose flax (Linum usitatissimum). Flax fibers possess excellent physical-mechanical properties, are nonbiodegradable, and there is extensive know-how on weaving/knitting of them. One area where they could be useful is as implantable mesh structures in surgery, in particular for the repair of incisional hernias of the abdominal wall. Starting with a bleached flax thread, a prototype mesh was specifically knitted for this study, and its cytocompatibility was studied in vitro and in vivo. The experimental data revealed that application of flax in surgery first requires a robust method to remove endotoxins and purify the flax fiber. Such a method was developed, and purified meshes did not cause loss of cell viability in vitro. In addition, endotoxins determined using limulus amebocyte lysate test were at acceptable levels. In vivo, the flax meshes showed only mild inflammation, comparable to commercial polypropylene meshes. This study revealed that plant-derived biomaterials can provide a new class of implantable materials that could be used as surgical meshes or for other biomedical applications.

Superior in vivo compatibility of hydrophilic polymer coated prosthetic vascular grafts.

PURPOSE: Protein adsorption, cell adhesion and graft patency was compared in hydrophilic versus hydrophobic polymer-coated prosthetic vascular grafts. We hypothesize that in vivo compatibility of hydrophilic polymer-coated prosthetic vascular grafts is superior to in vivo compatibility of hydrophobic grafts. METHODS: A pairwise side-to-side common carotid artery interposition graft was placed eight female landrace goats (mean weight 55 kg). Protein adsorption was assessed using Western Blot in two hydrophilic and two hydrophobic grafts harvested after three days. Graft patency was monitored for 28 days in six goats with continuous wave Doppler ultrasonography. Adherence of endothelial cells, leukocytes and platelets was determined with ELISA and compared between the two graft types after 28 days. RESULTS: After three days, more ApoA-I, albumin and VEGF and less fibrin adsorbed to hydrophilic grafts. After 28 days, compared to hydrophobic grafts, higher numbers of endothelial cells were present on hydrophilic grafts (P=0.016), and less thrombocytes and leukocytes (P=0.012 and 0.024, respectively). Two out of eight hydrophobic grafts lost patency, while none of the hydrophilic grafts failed (P=0.157). CONCLUSIONS: Hydrophilic polymer-coated vascular grafts have superior in vivo compatibility when compared to hydrophobic grafts as characterized by reduced platelet and leukocyte adherence as well as higher endothelialization.

A nontoxic additive to introduce x-ray contrast into poly(lactic acid). Implications for transient medical implants such as bioresorbable coronary vascular scaffolds.

Bioresorbable coronary vascular scaffolds are about to revolutionize the landscape of interventional cardiology. These scaffolds, consisting of a poly(L-lactic acid) interior and a poly(D,L-lactic acid) surface coating, offer a genuine alternative for metallic coronary stents. Perhaps the only remaining drawback is that monitoring during implantation is limited to two X-ray contrast points. Here, a new approach to make the biodegradable scaffolds entirely radiopaque is explored. A new contrast agent is designed and synthesized. This compound is miscible with poly(D,L-lactic acid) matrix, and nontoxic to multiple cell types. Blends of poly(D,L-lactic acid) and the contrast agent are found to be hemocompatible, noncytotoxic, and radiopaque. The data show that it is possible to manufacture fully radiopaque bioresorbable coronary vascular scaffolds. Whole-stent X-ray visibility helps interventionalists ensure that the scaffold deploys completely. This important advantage may translate into improved safety, accuracy, and clinical performance of cardiac stents.

Design, synthesis, imaging, and biomechanics of a softness-gradient hydrogel nucleus pulposus prosthesis in a canine lumbar spine model.

A hydrogel nucleus pulposus prosthesis (NPP) was designed to swell in situ, have intrinsic radiopacity, and restore intervertebral disc height and biomechanical functionality. These features were examined using an ex vivo canine lumbar model. Nine NPPs were implanted in five spines and their visibility was assessed on radiography, computed tomography (CT), and magnetic resonance imaging (MRI). The NPPs were visible on all imaging modalities and 8/9 NPPs stayed intact and in situ. Six other NPPs were tested biomechanically in six canine lumbar spines. Removal of the nucleus pulposus (nuclectomy) caused significant changes in biomechanical parameters. After implantation and swelling of the NPP, values were not significantly different from the native state for range of motion (ROM) of flexion-extension (FE) and lateral bending (LB), the neutral zone (NZ) of all motion directions, and the NZ stiffness (NZS) of FE. Biomechanical restoration by the NPP compared with the nuclectomized state was significant for the ROM of FE and axial rotation, the NZ of FE and LB, and the NZS of FE and LB. Disc height was significantly restored and 6/6 NPPs stayed intact and in situ. In conclusion, the NPPs swell in situ, have intrinsic radiopacity and restored disc height and aforementioned biomechanical properties.

Antimicrobial and anti-thrombogenic features combined in hydrophilic surface coatings for skin-penetrating catheters. Synergy of co-embedded silver particles and heparin.

Percutaneous (skin-penetrating) catheters such as central venous catheters (CVCs), are used ubiquitously in the treatment of critically ill patients, although it is known that the risks for serious complications, particularly bloodstream infection and thromboembolism, are high. Materials science and engineering offer important new perspectives regarding further improvement of CVCs. A promising approach is the use of synthetic biocompatible hydrogel coatings with both silver particles and heparin embedded therein. Such formulations combine the well-known broad-spectrum antimicrobial features of silver with the anticoagulant activity of immobilized heparin. Previous work revealed that heparin augments antimicrobial activity of silver, while maintaining its anticoagulant function. This study set out to investigate the synergy of heparin and silver in more detail. Exit-challenge tests, experiments on bacterial killing and adherence, as well as in vitro challenge tests with three Staphylococcus aureus strains (one reference strain, and two clinical isolates) consistently showed the synergistic effect. In addition, the impact of changing the coating's hydrophilicity, and changing the silver concentration in the coatings, were examined. The experimental results, taken together and combined with data from the literature, point out that synergy of heparin and silver is best explained by binding of Ag(+) ions to heparin within the swollen coating, followed by release of heparin-Ag(+) complexes upon immersion of the coatings in an aqueous environment such as blood. Possible implications of this work regarding the development of improved/safer CVCs are briefly discussed.

A highly radiopaque vertebroplasty cement using tetraiodinated o-carborane additive.

Bone cements for vertebroplasty must have a much better radiocontrast level than cements for knee or hip arthroplasty. This is generally accomplished by adding a relatively large portion of BaSO(4), although this affects the physical-mechanical and biological properties of the cement. This prompted us to develop an alternative radiopaque cement, on the basis of unique highly radiopaque methacrylic microspheres. These contain iodine in two modalities: (i) covalently linked to the methacrylic polymer, and (ii) as constituent of the stable tetraiodocarborane 8,9,10,12-I(4)-1,2-closo-C(2)B(10)H(8). The total iodine content in these particles exceeded 30% by mass. These radiopaque microspheres as well as the cement made thereof were characterized extensively, e.g., by scanning electron microscopy, X-ray contrast measurements, X-ray photoelectron spectroscopy, measurements of compressive strength, infrared spectroscopy, and solid state (11)B{(1)H} NMR spectroscopy. Furthermore, the new cement was subjected to several biocompatibility tests in vitro. The results show that the new bone cement fulfills all physico-chemical criteria for use in vertebroplasty. Further data on the cement's biocompatibility (in vitro), as well as on the handling parameters and doughviscosity, indicate that this material has a potential to become an alternative to vertebroplasty cements with a high BaSO(4) content. The new cement provides two significant advantages: (i) controlled viscosity in the dough phase, which facilitates precise injection during the vertebroplasty procedure; (ii) excellent structural stability, which precludes leaching of contrast post-implantation.

Hydrophilic surface coatings with embedded biocidal silver nanoparticles and sodium heparin for central venous catheters.

Central venous catheters (CVCs) have become indispensable in the treatment of neonates and patients undergoing chemotherapy or hemodialysis. A CVC provides easy access to the patient's circulation, thus enabling facile monitoring of hemodynamic parameters, nutritional support, or administration of (cytostatic) medication. However, complications with CVCs, such as bacterial bloodstream infection or thromboembolism, are common. Bloodstream infections, predominantly caused by Staphylococcus aureus, are notoriously difficult to prevent and treat. Furthermore, patients receiving infusion therapy through a CVC are at risk for deep-vein thrombosis, especially of the upper limbs. Several recent clinical trials have shown that prophylactic anticoagulation (low-molecular-weight heparin or vitamin K antagonists) is not effective. Here, we report on the systematic development of a new bifunctional coating concept that can -uniquely- be applied to make CVC surfaces antimicrobial and antithrombogenic at the same time. The novel coating consists of a moderately hydrophilic synthetic copolymer of N-vinylpyrrollidinone (NVP) and n-butyl methacrylate (BMA), containing embedded silver nanoparticles (AgNPs) and sodium heparin. The work demonstrates that the AgNPs strongly inhibit adhesion of S. aureus (reference strain and clinical isolates). Surprisingly, heparin not only rendered our surfaces practically non-thrombogenic, but also contributed synergistically to their biocidal activity.

Versatile polymer microspheres for injection therapy: aspects of fluoroscopic traceability and biofunctionalization.

Synthesis and characterization of a series of novel microspheres featuring (i) radiopacity (i.e., clear fluoroscopic traceability) and (ii) an outer surface exposing aldehyde groups are reported. The aldehydes allowed us to tether proteins onto the particles' surface under mild conditions, under which the protein conformation and, hence, structural motifs for biorecognition are preserved. Essential monomer building blocks were (i) 4-iodobenzoyl-2-oxo-ethylmethacrylate (4-IEMA) for radiopacity and (ii) propenal for surface tethering of proteins. The particles demonstrated good X-ray visibility and cytocompatibility. Procedures to couple proteins onto the surface were optimized using fluorescent bovine serum albumin (FITC-BSA) or collagen (FITC-collagen). Furthermore, radiopaque microparticles with unlabeled bovine collagen type I were produced. The presence of immobilized collagen was verified with narrow-scan X-ray photoelectron spectroscopy. Fibroblasts readily adhere to and grow on the collagen-modified surfaces, whereas this was much less the case for the unmodified controls. The results led us to suggest that immobilized nondenatured collagen may transform filler particles from passive space-occupying objects to particles that cross-talk with surrounding tissues.

VEGF-E enhances endothelialization and inhibits thrombus formation on polymeric surfaces.

Thrombotic complications of long-term blood-contacting devices can be avoided by formation of an endothelial cell layer on the blood-contacting surface. The endothelial cells form a bioactive boundary between the synthetic surface and blood, regulating haemostasis and inflammation. Biofunctionalization of synthetic blood-contacting surfaces is necessary to accommodate growth of endothelial cells. Vascular endothelial growth factor E (VEGF-E) or collagen I may stimulate endothelialization of a polymeric surface coating of a prototype small diameter vascular prosthesis. VEGF-E was produced in Escherichia coli and could be easily purified in large quantities. Recombinant VEGF-E or purified collagen I was allowed to adsorb onto the polymeric surfaces and enhanced formation of an endothelial cell layer. Adsorption of VEGF-E was increased by the inclusion of the anti-coagulant drug heparin in the polymeric coating. Collagen I adsorption induced rapid thrombin generation and increased platelet adhesion on surfaces with or without heparin. VEGF-E inhibited thrombus formation, and did not interfere with the anti-thrombogenic effect of heparin. Additionally, VEGF-E did not affect platelet adhesion. Adsorption of VEGF-E, especially on heparin containing surfaces, provides an economical strategy to improve endothelialization of cardiovascular implants without disturbing blood-compatibility.

The relationship between the antimicrobial effect of catheter coatings containing silver nanoparticles and the coagulation of contacting blood.

It is well known that surface coatings for medical devices can be made antimicrobial through introduction of silver nanoparticles. By virtue of their extremely large surface-to-volume ratio, the silver particles serve as a depot for sustained release of silver ions, despite the fact that silver is not readily oxidized. Antimicrobial coatings are especially important in connection with indwelling catheters with a high risk of bacterial line infections, such as central venous catheters (CVCs). This study specifically addressed the question what the impact of silver nanoparticles (exposed at the coating's surface) and/or the release of silver ions would be on coagulation of contacting blood. Studies, performed in vitro with fresh platelet-rich blood plasma (PRP) from 5 different healthy volunteer donors, clearly pointed out that: (i) the presence of silver nanoparticles correlates with accelerated thrombin formation upon contact of the coating with PRP; (ii) platelet activation is stronger as a result from the contact with silver nanoparticle-containing coatings as compared to other coatings which are devoid of silver. A series of titration experiments, in which the potential effect of silver ions is mimicked, revealed that the observed activation of blood platelets can be best explained through a collision mechanism. The results suggest that platelets that collide with silver, exposed at the surface, become activated without adhering to the surface. These new results point, rather unexpectedly, at a double effect of the silver nanoparticles in the coating: a strong antimicrobial effect occurs simultaneously with acceleration of the coagulation of contacting blood. This new information is, evidently, most relevant for the development of improved surface coatings for indwelling catheters (such as CVCs) which should combine antimicrobial features and close-to-zero thrombogenicity.

New intrinsically radiopaque hydrophilic microspheres for embolization: synthesis and characterization.

Polymeric particles currently used for embolization procedures have the disadvantage that they are radiolucent, that is, invisible on X-ray images, and consequently the interventional radiologist has to resort to angiography to (indirectly) monitor the fate of the particles. Here, we introduce intrinsically radiopaque hydrophilic microspheres. Since these microspheres can directly be visualized on X-ray images, using these microspheres for embolization purposes will allow superprecise location of the embolic material, both during and after the procedure. The microspheres, which are prepared by suspension polymerization, are based on the radiopaque monomer 2-[4-iodobenzoyl]-oxo-ethylmethacrylate and hydroxyethylmethacrylate (HEMA) and/or 1-vinyl-2-pyrrolidinone (NVP) as hydrophilic component. It has been shown that for clinically relevant X-ray visibility the spheres should contain at least 20 wt % iodine. At this iodine content, copolymerization with HEMA results in spheres that hardly imbibe water (EQ = 1.08). When HEMA is replaced by NVP, the volume swelling ratio can be significantly increased (to 1.33).

Polymers with tunable toxicity: a reference scale for cytotoxicity testing of biomaterial surfaces.

A series of copolymers, with varying ratio di-methylamino-ethylmethacrylate (DMAEMA) and methyl-methacrylate (MMA), was designed as a potential scale for cytotoxicity. These copolymers were characterized for toxicity of their surface. The surfaces of washed copolymers display increasing toxicity with increasing DMAEMA content. The toxicity was observed for three different cell-types, namely mouse fibroblasts, human endothelial cells and human osteoblast-like cells. With an increasing toxic surface, cell growth was inhibited as was indicated by the proliferation marker Ki-67. Staining for F-actin revealed that with increasing DMAEMA, cells adopted a more and more round morphology, resulting in decreased surface-contact area. Immuno-staining for phospho-tyrosine or vinculin demonstrated gradual loss of focal adhesions on increasingly toxic surfaces. Surprisingly loss of focal adhesions coincided with an increase in paxillin and vinculin protein, indicating cells try compensating for loss of adhesion. This series of copolymers may have potential as a cytotoxicity scale. They provoke cellular responses ranging from highly toxic to completely non-toxic, with some showing intermediate toxicity.

Thrombus formation at the surface of guide-wire models: effects of heparin-releasing or heparin-exposing surface coatings.

PURPOSE: This study was conducted to investigate whether thrombus formation at the surface of guide wires occurs, and--if so--whether this can be suppressed or prevented through incorporation of heparin in the surface coating. MATERIALS AND METHODS: Five guide wire models were examined; three had a polymeric hydrophilic surface coating (90/10 guide wire), which was either heparin-free, impregnated with sodium-heparin (Na-hep), or impregnated with benzalkonium heparin (BAK-hep). The other two guide wires had a coating of polytetrafluoroethylene (PTFE), either without heparin, or impregnated with BAK-hep. Release of heparin, exposure of heparin at the surface of the guide wires, thrombogenicity (under static and flow conditions) and their propensity to attract blood platelets were investigated. RESULTS: The guide wire 90/10 Na-hep releases approximately 150-200 mU active heparin per cm coil within the first few minutes after incubation in buffer. The PTFE BAK-hep shows a relatively slow release of 60-70 mU active heparin per cm coil. The 90/10 BAK-hep showed no released heparin but the most exposed heparin. In a static experiment with human full blood excessive thrombus formation occurred at the heparin-free models, whereas the others remained essentially clean. In a thrombin-generation assay under flow the authors observed strong retardation of thrombin formation in the case of the 90/10 Na-hep guide wire. CONCLUSIONS: The static and dynamic in vitro assays, taken together, show that the 90/10 Na-hep provides a coating with an extremely low level of surface thrombogenicity. Use of a guide wire with a hydrophilic distal coating that releases and exposes sodium heparin may contribute to the safety of diagnostic and therapeutic interventional procedures.

New acrylic microspheres for arterial embolization: combining radiopacity for precise localization with immobilized thrombin to trigger local blood coagulation.

Particles currently used in arterial embolization therapy have several disadvantages, most importantly their radiolucency. This means the radiologist cannot precisely asses the fate of embolization particles. Microspheres that combine two additional features have been designed. By incorporating an iodine containing monomer, radiopaque microspheres were obtained that display good visibility under standard X-ray conditions. Incorporation of methacrylic acid makes the surface of the spheres suitable for surface functionalization. Here, thrombin was covalently attached to the surface of the radiopaque microspheres. By induction of a thrombus, improved anchoring of the embolization spheres in the blood vessel can be obtained. The immobilized thrombin induced a biphasic response of the blood namely: (1) fast deposition of fibrin on the surface resulting in sphere aggregation and (2) additional thrombin generation in the surrounding blood and a subsequent local thrombus formation. These microspheres with both intrinsic X-ray visibility and a biofunctionalized surface can potentially improve embolization therapies.

Biocompatibility of a new radiopaque iodine-containing acrylic bone cement.

Radiopacity in the vast majority of the commercially available acrylic bone cements that are used clinically is provided by particles of either BaSO(4) or ZrO(2). Literature reports have shown these agents to have a detrimental effect on some mechanical properties of the cements as well as on its biological response. We, therefore, have developed a new type of bone cement, for which radiopacity results from the presence of an iodine-containing methacrylic copolymer. The focus of the present work was the comparison of the biocompatibility of this new cement and a commercially available cement that contains barium sulfate. In vitro experiments show that both cements are cytocompatible materials, for which no toxic leachables are found. Implantation of the cements in a rabbit for three months resulted in the occasional presence of a thin fibrous tissue at the cement-bone interface, which is common for acrylic bone cements. Consideration of all the results led to the conclusion that the new cement is as biocompatible as the BaSO(4)-containing one.

Radio-opaque and surface-functionalized polymer microparticles: potentially safer biomaterials for different injection therapies.

Injectable polymer particles with a diameter in the range of 30-300 microm find applications as a biomaterial in different clinical fields, such as cosmetic surgery, reconstructive surgery, and urology. However, clinical effects tend to disappear after several months, either due to migration of the particles away from the injection site (caused by weak adherence with the surrounding soft tissues) or due to fibrosis (caused by excessive encapsulation of the particles by fibrous tissue). Little is known about the fate of injected microparticles, due to the fact that they are extremely difficult to trace in a noninvasive manner. Design, synthesis, and characterization of new polymeric microspheres with two additional features that can enhance safety and can help to overcome drawbacks of existing products are reported. First, the new microparticles feature clear radio-opacity (X-ray visibility) as they are prepared on the basis of a reactive methacrylic monomer that contains covalently bound iodine. Model experiments reveal that the level of X-ray contrast is sufficient for clinical monitoring; they can be visualized both during the injection and afterward. The particles feature excellent cytocompatibility in vitro and in vivo. Second, a method is explored to functionalize the surface of the particles, for example, through immobilization of collagen. Other extracellular matrix proteins can also be immobilized, and this provides a mechanism to control anchoring of the particles in soft tissue. The results are briefly discussed in the context of improved biomaterials, contemporary X-ray imaging, and control over biomaterial-soft tissue interactions in vivo.