Improved haemocompatibility of cysteine-modified polymers via endogenous nitric oxide.

A novel method for improving the haemocompatibility of biomedical materials through endogenous nitric oxide (NO) is presented. L-cysteine was covalently immobilized onto two biomedical polymers: polyurethane (PU) and polyethylene terephthalate (PET). The L-cysteine content on the polymers was approximately 5-8 nmol/cm2 as quantified via a chemiluminescence-based assay. The haemocompatibility of the modified polymers was evaluated in terms of the number of adhered platelets when exposed to a platelet suspension labeled with Cr51. Platelet adherence on the L-cysteine-modified polymers was reduced more than 50% as compared to the control (glycine-modified polymers) when the platelet suspension contained plasma constituents. No difference in platelet adhesion was observed in the absence of plasma constituents. Further experiments demonstrated that NO was easily transferred to the L-cysteine-modified polymers from S-nitroso-albumin in PBS buffer. The NO was then released from the polymer. NO transfer or release was not observed for the control. The results suggest that L-cysteine-modified polymers are effective in reducing platelet adhesion via the transfer of NO from endogenous S-nitrosoproteins in plasma to the polymer followed by the subsequent release of NO. Thus, exploiting endogenous NO is a viable option for improving the haemocompatibility of biomaterials.     

Improved hemocompatibility of poly(ethylene terephthalate) modified with various thiol-containing groups

Thiol groups were attached to polyethylene terephthalate (PET) to promote the transfer of a known platelet inhibitor, nitric oxide (NO), from nitrosated thiols naturally found in the body to PET, followed by the release of NO from PET to prevent platelet adhesion. In order to immobilize the most thiols on the modified polymer, the processing parameters used to attach the following three thiol containing groups were assessed: L-cysteine, 2-iminothiolane, and a cysteine polypeptide. When comparing the immobilized concentrations of thiol groups from each of the optimized processes the amount of immobilized thiol groups increased in order with the following groups: cysteine polypeptide <2-iminothiolane 相似文献   

Polymeric coatings that mimic the endothelium: combining nitric oxide release with surface-bound active thrombomodulin and heparin

Multi-functional bilayer polymeric coatings are prepared with both controlled nitric oxide (NO) release and surface-bound active thrombomodulin (TM) alone or in combination with immobilized heparin. The outer-layer is made of CarboSil, a commercially available copolymer of silicone rubber (SR) and polyurethane (PU). The CarboSil is either carboxylated or aminated via an allophanate reaction with a diisocyanate compound followed by a urea-forming reaction between the generated isocyanate group of the polymer and the amine group of an amino acid (glycine), an oligopeptide (triglycine) or a diamine. The carboxylated CarboSil can then be used to immobilize TM through the formation of an amide bond between the surface carboxylic acid groups and the lysine residues of TM. Aminated CarboSil can also be employed to initially couple heparin to the surface, and then the carboxylic acid groups on heparin can be further used to anchor TM. Both surface-bound TM and heparin's activity are evaluated by chromogenic assays and found to be at clinically significant levels. The underlying NO release layer is made with another commercial SR-PU copolymer (PurSil) mixed with a lipophilic NO donor (N-diazeniumdiolated dibutylhexanediamine (DBHD/N(2)O(2))). The NO release rate can be tuned by changing the thickness of top coatings, and the duration of NO release at physiologically relevant levels can be as long as 2 weeks. The combination of controlled NO release as well as immobilized active TM and heparin from/on the same polymeric surface mimics the highly thromboresistant endothelium layer. Hence, such multifunctional polymer coatings should provide more blood-compatible surfaces for biomedical devices.     

Preparation and characterization of polymeric coatings with combined nitric oxide release and immobilized active heparin

A new dual acting polymeric coating is described that combines nitric oxide (NO) release with surface-bound active heparin, with the aim of mimicking the nonthrombogenic properties of the endothelial cell (EC) layer that lines the inner wall of healthy blood vessels. A trilayer membrane configuration is employed to create the proposed blood compatible coating. A given polymeric substrate (e.g., the outer surface of a catheter sleeve, etc.) is first coated with a dense polymer layer, followed by a plasticized poly(vinyl chloride) (PVC) or polyurethane (PU) layer doped with a lipophilic N-diazeniumdiolate as the NO donor species. Finally, an outer aminated polymer layer is applied. Porcine heparin is then covalently linked to the outer layer via formation of amide bonds. The surface-bound heparin is shown to possess anti-coagulant activity in the range of 4.80-6.39 mIU/cm2 as determined by a chromogenic anti-Factor Xa assay. Further, the surface NO flux from the underlying polymer layer containing the diazeniumdiolate species can be controlled and maintained at various levels (from 0.5 to 60 x 10(-10) mol cm(-2)min(-1)) for at least 24 h and up to 1 week (depending on the flux level desired) by changing the chemical/polymer composition of the NO release layer. The proposed polymeric coatings are capable of functioning by two complementary anti-thrombotic mechanisms, one based on the potent anti-platelet activity of NO, and the other the result of the ability of immobilized heparin to inhibit Factor Xa and thrombin (Factor IIa). Thus, the proposed polymeric coatings are expected to exhibit greatly enhanced thromboresistivity compared to polymers that utilize either immobilized heparin or NO release alone.     

A comparison of seven methods to analyze heparin in biomaterials: quantification, location, and anticoagulant activity

Glycosaminoglycans, like heparin, are frequently incorporated in biomaterials because of their capacity to bind and store growth factors and because of their hydrating properties. Heparin is also often used in biomaterials for its anticoagulant activity. Analysis of biomaterial-bound heparin is challenging because most assays are based on heparin in solution. In this study, seven different methods were probed to analyze heparin covalently attached to collagen scaffolds. For each method, the basic mechanism and the advantages and disadvantages are given. An analysis by the factor Xa assay and the Farndale assay clearly indicated that the amount of immobilized heparin cannot be determined correctly when the scaffold is intact. Scaffolds had to be proteolytically digested or acid treated to obtain reliable measurements. Methods used to quantify the amount of bound heparin included a hexosamine assay, an uronic acid assay, a Farndale assay, agarose gel electrophoresis, and immuno-dot blot analysis. Location and semiquantification of heparin were accomplished by immunofluorescence. Although all assays had their advantages and disadvantages, the hexosamine assay turned out to be the most robust and is recommended as the preferred assay to quantify the amount of heparin bound to scaffolds. It is applicable to all scaffolds that are acid hydrolyzable. This study may allow researchers in the field to select the most appropriate method to analyze glycosaminoglycans in biomaterials.     

Ligand density characterization of peptide-modified biomaterials

A simple fluorescence based characterization method was developed to assess ligand density on peptide-modified biomaterials. The method exploits the exquisite sensitivity of proteolysis for the purpose of liberating a fluorescently labeled probe fragment from an immobilized peptide. The released fragment can then be detected in solution using high-throughput fluorometry. In silico screening tools identified the enzyme chymotrypsin as a promising candidate for releasing a detectable probe fragment from the fluorescently labeled peptide, Ac-CGGNGEPRGDTYRAYK(FITC)GG-NH(2). After chymotrypsin digestion of the peptide in solution was first characterized using mass spectrometry and HPLC, a basic enzyme mediated release protocol was developed and implemented to generate peptide-binding isotherms on various peptide-modified biomaterials. The new method is sensitive, has good signal-to-noise ratio (S/N), and is easily standardized. Furthermore, the technique can be applied independent of material chemistry and geometry, making it a suitable alternative to radiolabeling for a wide range of biomaterial applications.     

Preparation of a protein micro-array using a photo-reactive polymer for a cell-adhesion assay

A protein micro-array, called a "cell chip" was constructed by using a photo-reactive polymer for a cell-adhesion assay. Various amounts of albumin or fibronectin were covalently immobilized on a polystyrene dish using a micro-spotter with a dip pen. First, poly(acrylic acid) carrying azidophenyl groups was synthesized as the photo-reactive polymer. Secondly, the aqueous solution of a photo-reactive polymer (several nanoliters) was cast using the dip pen of the micro-spotter and dried in air. Subsequently, aqueous solutions of proteins were cast on the same place using the micro-spotter. After drying, the dish was irradiated with ultraviolet light. Finally, the immobilization was confirmed by staining with a dye. The immobilization was stable even after washing with Tween-20. The protein-immobilized area depended on the manipulation of the micro-spotter and the size of the dip pen. Subsequently, cell adhesion on the photo-immobilized protein micro-array was investigated. The adhesion behavior of cells depended on the kind of immobilized proteins and the kind of cells. The protein micro-array will be useful for cell diagnosis and for the selection of biomaterials to regulate cell behavior.     

Photo-reactive polyvinylalcohol for photo-immobilized microarray

A new photo-reactive polymer, polyvinylalcohol modified with phenylazido groups, was synthesized as a microarray matrix. The polymer is soluble in water and spin-coated onto glass plate. Aqueous solutions of proteins were micro-spotted onto the coated glass and were fixed by ultraviolet light irradiation. Subsequently, cell adhesion on the photo-immobilized protein microarray was investigated. Non-specific adhesion of cells onto non-protein-spotted regions was reduced in comparison with the previously prepared microarray chip (Biomaterials 24 (2003) 3021). The adhesion behavior of cells depended on the kind of immobilized proteins and the type of cells. The microarray will be useful for cell diagnosis and for the selection of biomaterials to regulate cell behavior.     

Quantitative evaluation of interaction force of fibrinogen at well-defined surfaces with various structures

The effects of functional groups and structures at the surface of biomaterials on protein adsorption were examined using direct interaction force measurements. Three kinds of surface structures were evaluated: polymer brushes, self-assembled monolayers with low molecular weight compounds, and surfaces with conventional polymer coatings. These surfaces had various functional groups including phosphorylcholine (PC) group. The surface characterization demonstrated that surface wettability and flexibility depended on both the structure of the surface and the functional groups at the surface. The interactions of protein with these surfaces were evaluated by a force vs. distance curve using an atomic force microscope (AFM). We used fibrinogen as the protein, and the fibrinogen was immobilized on the surface of the AFM cantilever by a conventional technique. It was observed that the interaction force of fibrinogen was strongly related to surface hydrophobic nature and flexibility. That is, the interaction force increased with the increasing hydrophobic nature of the surface. The relationship between the amount of fibrinogen adsorbed on the surface and the interaction force showed good correlation in the range of fibrinogen adsorption from 0 to 250?ng/cm², that is, in a monolayered adsorption region. The interaction force decreased with increasing surface viscoelasticity. The most effective surface for preventing fibrinogen adsorption was the polymer brush surface with phosphorylcholine (PC) groups, that is, poly(2-methacryloyloxyethyl phosphorylcholine) brush. The interaction force of this sample was less than 0.1?nN and the amount of fibrinogen adsorbed on the surface was minimal. It was found that the evaluation of protein adsorption based on the interaction force measurement is useful for low-protein adsorption surfaces. It was demonstrated that an extremely hydrophilic and flexible surface could weaken the protein interactions at the surface, resulting in greater resistance to protein adsorption.     

RGD-Functionalized polymer brushes as substrates for the integrin specific adhesion of human umbilical vein endothelial cells

This report demonstrates the feasibility of surface-initiated atom transfer radical polymerization to prepare thin polymer layers ("brushes") that can be functionalized with short peptide ligands and which may be of use as coatings to promote endothelialization of blood-contacting biomaterials. The brushes are composed of poly(2-hydroxyethyl methacrylate) (PHEMA) or poly(poly(ethylene glycol) methacrylate) (PPEGMA), which do not only suppress non-specific adhesion of proteins and cells but also contain hydroxyl groups that can be used to introduce small peptide ligands. A protocol has been developed that allows functionalization of the brushes with RGD containing peptide ligands resulting in surface concentrations ranging from approximately 0.5-12 pmol/cm(2). At peptide surface concentrations >1-5.3 pmol/cm(2), human umbilical vascular endothelial cells (HUVECs) were found to adhere and spread rapidly. A difference in size and morphology of focal adhesions between HUVECs immobilized on PHEMA and PPEGMA brushes was observed. It is proposed that this is due to the increased ethylene glycol spacer length and hydrophilicity of the PPEGMA brushes, which may lead to increased ligand mobility and reduced ligand-integrin affinity. HUVECs immobilized on the polymer brushes were also found to be able to retain homeostasis when exposed to shear stresses that simulated arterial blood flow.     

Combinatorial protein display for the cell-based screening of biomaterials that direct neural stem cell differentiation

Neural stem cell (NSC) has emerged as a potential source for cell replacement therapy following traumatic injuries and degenerative diseases of the central nervous system. However, clinical applications of NSC further require technological advances especially for controlling differentiation of NSC. This study aimed at developing biomaterials that serve to expand undifferentiated NSC or to induce cells with specific phenotypes. Our approach is to construct composite biomaterials that consist of extracellular matrix components and growth factors. In order to optimize matrix-growth factor combinations, we conducted the parallel and rapid screening of composite biomaterials through assays using cell-based arrays. The photo-assisted patterning of an alkanethiol self-assembled monolayer was employed to achieve site-addressable combinatorial immobilization of natural and synthetic matrices incorporated with growth factors including epidermal growth factor (EGF), ciliary neurotrophic factor (CNTF), nerve growth factor (NGF), and neurotrophin-3 (NT-3). NSC obtained from the rat embryonic striatum was cultured directly on the array to screen for cell adhesion, proliferation, and promotion of neuronal and glial specification. The results showed that the significant number of cells adhered to laminin-1, fibronectin, ProNectin, and poly(ethyleneimine). It was found that cells proliferated most extensively on a spot with immobilized EGF among the spots with different matrix-growth factor combinations. The results also showed that neuronal differentiation was promoted on the spots with immobilized NGF or NT-3, and astroglial differentiation with CNTF. Importantly, observed effects of growth factors were frequently altered depending on the type of co-immobilized matrices, suggesting synergic effects of adhesion and growth factor signals.     

Modeling and Optimization of High-Sensitivity,Low-Volume Microfluidic-Based Surface Immunoassays

Microfluidics are emerging as a promising technology for miniaturizing biological assays for applications in diagnostics and research in life sciences because they enable the parallel analysis of multiple analytes with economy of samples and in short time. We have previously developed microfluidic networks for surface immunoassays where antibodies that are immobilized on one wall of a microchannel capture analytes flowing in the microchannel. This technology is capable of detecting analytes with picomolar sensitivity and from sub-microliter volume of sample within 45 min. This paper presents the theoretical modeling of these immunoassays where a finite difference algorithm is applied to delineate the role of the transport of analyte molecules in the microchannel (convection and diffusion), the kinetics of binding between the analyte and the capture antibodies, and the surface density of the capture antibody on the assay. The model shows that assays can be greatly optimized by varying the flow velocity of the solution of analyte in the microchannels. The model also shows how much the analyte-antibody binding constant and the surface density of the capture antibodies influence the performance of the assay. We then derive strategies to optimize assays toward maximal sensitivity, minimal sample volume requirement or fast performance, which we think will allow further development of microfluidic networks for immunoassay applications.     

D,L-聚乳酸基形状记忆聚合物的生物安全性和细胞相容性

背景：课题组前期实验研制了输卵管避孕器材料D, L-聚乳酸基形状记忆聚合物,依据国内《生物材料和医疗器材生物学评价技术要求》规定,植入体内的组织工程材料必须进行生物安全评价和细胞相容性实验。 目的：观察D, L-聚乳酸基形状记忆聚合物的生物安全性。 方法：①内毒素实验：在鲎试剂中分别加入聚合物浸提液、内毒素工作标准品溶液和细菌内毒素检查用水。②致敏实验：在昆明小鼠肩胛骨内侧分别注射聚合物浸提液+弗氏完全佐剂+生理盐水、弗氏完全佐剂+生理盐水,通过皮内诱导、局部诱导和激发阶段,观察动物激发部位皮肤红斑和水肿反应程度。③急性毒性实验：分别在昆明小鼠腹腔注射100%,50%,25%聚合物浸提液及生理盐水。④细胞增殖MTT实验：直接法为将人脐静脉内皮细胞分别接种于聚合物膜、聚乳酸与玻璃片上;间接法为将人脐静脉内皮细胞分别接种于聚合物浸提液、丙烯酰胺溶液及1640培养液。 结果与结论：D, L-聚乳酸基形状记忆聚合物材料无细菌污染状况,符合生物安全标准,无致敏性及毒性,并且具有较好的细胞相容性。     

Influences of protein films on antibacterial or bacteria-repellent surface coatings in a model system using silicon wafers

Immobilization of defined chemical functionalities to biomaterial surfaces is employed to optimize them not only for tissue compatibility but also for prevention of bacterial infection. Grafting surfaces with chains of poly(ethylene glycol) (PEG) results in bacterial repellence whereas modification with cationic groups conveys them with bactericidal properties. Since biomaterials in situ will become exposed to a protein-rich environment, it is necessary to investigate the influence of prior protein adsorption on the antibacterial activity of this type of chemical surface modification. In the present study, we immobilized short-chain PEG and two pyridinium group-containing methacrylate monomers, 12-methacryloyloxydodecylpyridinium bromide (MDPB) and 6-methacryloyloxyhexylpyridinium chloride (MHPC), to silicon wafer model surfaces to investigate the influence of prior protein adsorption on the bactericidal activity of the surface coating towards subsequently attached bacteria. Adsorbed amounts of human serum albumin and salivary proteins were found to be two times higher on cationic compared to PEG-modified surfaces. An analogous tendency was found for attachment of Streptococcus gordonii and Streptococcus mutans to the same surfaces without prior protein exposure. However, most bacteria attached to cationic surfaces were found to be dead. Prior exposure of cationic surfaces to protein solutions drastically altered bacterial attachment dependent on the type of protein solution and bacterial species employed. Significantly, the original bactericidal activity of pyridinium-coated surfaces was found greatly reduced upon adsorption of a protein film. As a conclusion we propose that future approaches should combine the protein- and bacteria-repellent properties of PEG-coatings with the bactericidal function of charged cationic groups.     

Electrospun scaffolds of self-assembling peptides with poly(ethylene oxide) for bone tissue engineering

Structural, mechanical and biochemical properties have to be considered when searching for suitable extracellular matrix substitutes. Fibrous structures of synthetic or natural polymers have received increasing interest as three-dimensional scaffolds for tissue engineering applications as they can be easily produced by electrospinning with different topographical features by changing the process parameters. On the other hand, the nanobiotechnology approach suggests mimicking molecular architectures in nature through self-assembly. In particular, self-assembling peptide-based biomaterials have been successfully used as scaffolds for cell growth. In order to amalgamate these two strategies nanofibrous electrospun scaffolds of hybrid polymer were designed and obtained by mixing poly(ethylene oxide) and self-assembling peptides in aqueous solution. The results of in vitro osteoblast adhesion and proliferation assays on the electrospun scaffolds obtained using different self-assembling peptide sequences are discussed.     

Surfactant polymers designed to suppress bacterial (Staphylococcus epidermidis) adhesion on biomaterials

We describe a series of surfactant polymers designed as surface-modifying agents for the suppression of bacterial adhesion on biomaterials. The surfactant polymers consist of a poly(vinyl amine) backbone with hydrophilic poly(ethylene oxide) (PEO) and hydrophobic hexanal (Hex) side chains (PVAm/PEO:Hex). Surface modification is accomplished by simple dip coating from aqueous solution, from which surfactant polymers undergo spontaneous surface-induced assembly on hydrophobic biomaterials. The stability of PVAm/PEO:Hex on pyrolytic graphite (HOPG) and polyethylene (PE) was demonstrated by the absence of detectable desorption under flow conditions of pure water over a 24-h period. PEO surfactant polymers with four different PEO:Hex ratios (1:1.4, 1:2.5, 1:4.6, and 1:10.7) and a dextran surfactant polymer were compared with respect to S. epidermidis adhesion under dynamic flow conditions. Suppression of S. epidermidis adhesion was achieved for all modified surfaces over the shear range 0-15 dyn/cm(2). The effectiveness depended on the surfactant polymer composition such that S. epidermidis adhesion to modified surfaces decreased significantly with increasing PEO packing density. Modified HOPG was more effective in reducing bacterial adhesion compared with the corresponding modification on PE, which we attribute to the presence of defects in surfactant polymer assembly on PE. Our results are discussed from the perspective of critical factors, such as optimal PEO packing density and hydration thickness, that contribute to the effectiveness of surfactant polymers to shield a biomaterial from adhesive bacterial interactions.     

Nitric oxide-generating polymers reduce platelet adhesion and smooth muscle cell proliferation

We have developed polymeric biomaterials capable of providing localized and sustained production of nitric oxide (NO) for the prevention of thrombosis and restenosis. In the current study, we have characterized the kinetics of NO production by these materials and investigated their efficacy in reducing platelet adhesion and smooth muscle cell proliferation in vitro. Three nitric oxide donors with different half-lives were covalently incorporated into photopolymerized polyethylene glycol hydrogels. Under physiological conditions, NO was produced by these hydrogels over periods ranging from hours to months, depending upon the polymer formulation. NO production was inhibited at acidic pH, which may be useful for storage of the materials. The NO-releasing materials successfully inhibited smooth muscle cell growth in culture. Platelet adhesion to collagen-coated surfaces was also inhibited following exposure of whole blood to NO-producing hydrogels. The effects of NO production by these hydrogels on platelet adhesion and the proliferation of smooth muscle cells suggest that these materials could reduce thrombosis and restenosis following procedures such as balloon angioplasty.     

Catalytic generation of nitric oxide from nitrite at the interface of polymeric films doped with lipophilic CuII-complex: a potential route to the preparation of thromboresistant coatings

A novel approach potentially useful for the development of more thromboresistant polymeric materials is examined. The method is based on the catalytic generation of nitric oxide (NO) via Cu(I) mediated reduction of nitrite ions. Preliminary solution phase studies demonstrate that ascorbate or thiolate anions can generate Cu(I) from Cu(II) with subsequent catalytic conversion of any nitrite ions present to NO by the unstable Cu(I) species. Incorporation of this same chemistry within a hydrophobic polymeric material requires immobilizing Cu(II) ions into a polymeric phase via use of a lipophilic Cu(II) chelating ligand (dibenzo [e,k]-2,3,8,9-tetraphenyl-1,4,7,10-tetraaza-cyclododeca-1,3,7,9-tetraene (DTTCT)). It is shown that this complex can be reduced to its Cu(I) form by appropriate reducing equivalents present in the bathing solution. The resulting Cu(I) complex can then reduce nitrite to NO with the NO generation occurring at the polymer/solution interface at physiological pH. Data from chemiluminescence experiments indicate that the flux of NO at the polymer surface is comparable to that of endothelial cells (>/=1x10(-10)mol/cm(2)min) when 0.5mM nitrite/1mM ascorbate are present in the bathing solution. Potentially more useful NO generation can be achieved by doping the polymer film with the Cu(II) complex along with a lipophilic quaternary ammonium nitrite salt. In this case reducing equivalents within the aqueous phase enable the nitrite derived from the polymer to be converted into NO by the Cu(II/I) ligand complex. Films of this type are shown to generate NO for at least 6h in PBS buffer with fluxes on the order of 1.5x10(-10)mol/cm(2)min. Physiologically relevant levels of NO release are also shown to exist at the polymer interface when films are soaked in fresh plasma as well as undiluted whole blood, indicating that endogenous reducing equivalents present in blood can efficiently reduce the Cu(II)-ligand within the polymer film. The prospects of using these new NO releasing films to devise more biocompatible polymeric coatings for biomedical applications are discussed.     

Development and application of immunochromatographic tests for the detection of staphylococcal enterotoxin E

Two rapid immunochromatographic assay formats for the detection of Staphylococcus aureus enterotoxin serotype E (SEE) were developed. For both tests, polyclonal antibodies against SEE were immobilized on a membrane strip representing the test zone, while second anti‐SEE antibodies conjugated to dyed latex particles were employed as markers in sandwich immunoassay. In a two‐step test format, sample and labelled antibodies were preincubated prior to immunochromatography, whereas in a one‐step test the labelled antibodies were integrated in the test pad and activated by addition of the sample solution. In the presence of SEE in a sample solution, colour development at the antibody‐coated zone occured within 20 min. The visual detection limits for SEE in buffer solution were at 5 ng ml^?1 (two‐step test) and at 10 ng ml^?1 (one‐step test), respectively. Both assays were specific for SEE and showed no cross‐reaction with serotype A, B, C, and D enterotoxins. When the two‐step test format was used for the identification of SEE in S. aureus culture broth, the detection limit was found to be 10 ng ml^?1. Culture supernatants of 10 enterotoxigenic strains of S. aureus were analysed with the immunochromatographic two‐step test and with a microtitre plate enzyme immunoassay (EIA) test kit for enterotoxins A‐E. Results obtained by the immunochromatographic test (presence or absence of SEE) were in excellent agreement with those of the microtitre EIA.     

Surface immobilized bisphosphonate improves stainless-steel screw fixation in rats

An increase in the mechanical fixation in bone of metallic biomaterials is considered advantageous in joint replacement and fracture surgery. Different approaches to improve fixation may be e.g. surface roughening, Ca-mineral coating or surface immobilization of growth factors or drugs. In the present work, bisphosphonate, a class of drugs that inhibit bone resorption, was immobilized onto stainless-steel screws.The screws were first roughened and coated with immobilized and cross-linked fibrinogen. Subsequently, an N-bisphosphonate, pamidronate, was immobilized onto fibrinogen, and another N-bisphosphonate, ibandronate, adsorbed on top of this. The so coated screws were inserted into the tibiae of eight male Sprague-Dawley rats. Another eight rats received screws prepared in the same way, but without the bisphosphonate coating. Pullout strength tests were performed after 2 weeks of implantation.The results showed a 28% (p=0.0009) higher pullout force and 90% increased pullout energy for the bisphosphonate coated screws, and support the idea that surface immobilized bisphosphonates can be used to improve biomaterials fixation in bone.