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Biomaterials and Hemocompatibility
Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences. (Kristina Nilsson Ekdahl)
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Biomaterials are commonly used in the medical clinic today; however, artificial materials can activate the cascade systems in the blood (complement-, coagulation-, contact- and fibrinolytic systems) as well as the platelets to various degrees. When an artificial surface comes in contact with blood, plasma proteins will be adsorbed to the surface within seconds. The composition of the layer of proteins differs between materials and is crucial for the hemocompatibility of the material.

This thesis includes five projects.

In Paper I the anticoagulants heparin and the thrombin inhibitor hirudin were evaluated in a whole blood model. Hirudin was found to be superior to low dose heparin since it did not affect the activation of the complement system nor the leukocytes. The most interesting observation was that expression of TF was seen on surface-attached monocytes in hirudin- treated blood but not heparin blood.

In Paper II peptides from the streptococcal M-protein, which has affinity for the human complement inhibitor C4BP, were attached to a polymeric surface. When being exposed to blood the endogenous complement regulator was enriched at the surface of the material, via the M-peptides. With this new approach we created a self-regulatory surface, showing significant lowered material-induced complement activation.

In Paper III apyrase, an enzyme which hydrolyzes nucleoside ATP and ADP, was immobilized on a polymer surface. Lower platelet activation and platelet-induced coagulation activation was seen for the apyrase-coated surface compared to control surfaces after exposure to whole human blood, due to the enzymes capability to degrade ADP released from activated platelets.

In Paper IV and V we synthesized an array of polymeric materials which were characterized regarding physical-chemical properties, adsorption of plasma proteins, and hemocompatibility. The polymers showed widely heterogeneous protein adsorption. Furthermore, when the polymers were exposed to whole blood, two of the materials showed superior hemocompatibility (monitored as complement- and coagulation activation), compared to the reference poly(vinyl chloride).

Place, publisher, year, edition, pages
Kalmar, Växjö: Linnaeus University Press , 2010. , p. 167
Series
Linnaeus University Dissertations ; 2/2010
Keywords [en]
Complement, Coagulation, Whole blood, Biomaterials, Polymers and Hemocompatibility
National Category
Immunology in the medical area
Research subject
Natural Science, Biomedical Sciences
Identifiers
URN: urn:nbn:se:lnu:diva-5437ISBN: 978-91-86491-01-7 (print)OAI: oai:DiVA.org:lnu-5437DiVA, id: diva2:315766
Public defence
2010-01-29, N2007, Smålandsgatan 26, Kalmar, 09:30 (English)
Opponent
Supervisors
Available from: 2010-04-30 Created: 2010-04-29 Last updated: 2018-04-19Bibliographically approved
List of papers
1. Hirudin versus heparin for use in whole blood in vitro biocompatibility models
Open this publication in new window or tab >>Hirudin versus heparin for use in whole blood in vitro biocompatibility models
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2009 (English)In: Journal of Biomedical Materials Research. Part A, ISSN 1549-3296, E-ISSN 1552-4965, Vol. 89A, no 4, p. 951-959Article in journal (Refereed) Published
Abstract [en]

Background: Heparin has traditionally been a widely used anticoagulant in blood research, but has been shown to be inappropriate for work with the complement system because of its complement-interacting properties. In this work, we have compared the effects of heparin with those of the specific thrombin inhibitor hirudin on complement and blood cells in vitro.

Methods: Whole blood collected in the presence of hirudin (50 µg/mL) or heparin (1 IU/mL) was incubated in the slide chamber model. The plasma was analyzed for complement activation markers C3a and sC5b-9, and the polyvinylchloride test slides were stained for adhering cells. The integrity of the complement system was tested by incubating serum and hirudin-treated plasma in the presence of various activating agents.

Results: In contrast to heparin, the addition of hirudin generally preserved the complement reactivity, and complement activation in hirudin plasma closely resembled that in normal serum. Importantly, immunochemical staining of surface-bound cells demonstrated the inducible expression of tissue factor on bound monocytes from hirudin-treated blood, an effect that was completely abolished in heparin-treated blood.

Conclusion: Our results indicate that hirudin as an anticoagulant produces more physiological conditions than heparin, making hirudin well-suited for in vitro studies, especially those addressing the regulation of cellular processes.

Place, publisher, year, edition, pages
Hoboken, NJ, US: John Wiley & Sons Inc, 2009
Keywords
biocompatibility, complement, heparin, hirudin, whole blood models
National Category
Immunology in the medical area
Research subject
Natural Science, Biomedical Sciences
Identifiers
urn:nbn:se:hik:diva-2786 (URN)10.1002/jbm.a.32034 (DOI)
Available from: 2010-04-07 Created: 2010-02-22 Last updated: 2018-01-12Bibliographically approved
2. Inhibition of complement activation on a model biomaterial surface by streptococcal M protein-derived peptides
Open this publication in new window or tab >>Inhibition of complement activation on a model biomaterial surface by streptococcal M protein-derived peptides
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2009 (English)In: Biomaterials, ISSN 0142-9612, E-ISSN 1878-5905, Vol. 30, no 13, p. 2653-2659Article in journal (Refereed) Published
Abstract [en]

The aim of this study was to evaluate a new approach to inhibit complement activation triggered by biomaterial surfaces in contact with blood. In order to inhibit complement activation initiated by the classical pathway (CP), we used streptococcal M protein-derived peptides that specifically bind human C4BP, an inhibitor of the CP. The peptides were used to coat polystyrene microtiter wells which served as a model biomaterial. The ability of coated peptides to bind C4BP and to attenuate complement activation via the CP (monitored as generation of fluid-phase C3a and binding of fragments of C3 and C4 to the surface) was investigated using diluted normal human serum, where complement activation by the AP is minimal, as well as serum from a patient lacking alternative pathway activation. Complement activation (all parameters) was significantly decreased in serum incubated in well surfaces coated with peptides. Total inhibition of complement activation was obtained at peptide coating concentrations as low as 1-5 mu g/mL. Successful use of Streptococcus-derived peptides shows that it is feasible to control complement activation at a model biomaterial surface by capturing autologous complement regulatory molecules from plasma. (C) 2009 Elsevier Ltd. All rights reserved.

Place, publisher, year, edition, pages
Elsevier, 2009
Keywords
Blood compatibility, Complement, C4b-binding protein (C4BP), In vitro test, Regulator of complement activation (RCA), Streptococcal M proteins
National Category
Immunology
Research subject
Biomedical Sciences, Immunology
Identifiers
urn:nbn:se:lnu:diva-73088 (URN)10.1016/j.biomaterials.2009.01.001 (DOI)000264691200026 ()19171378 (PubMedID)
Available from: 2018-04-19 Created: 2018-04-19 Last updated: 2018-04-19Bibliographically approved
3. IMMOBILIZATION OF APYRASE CREATES AN ANTITHROMBOTIC BIOMATERIAL SURFACE
Open this publication in new window or tab >>IMMOBILIZATION OF APYRASE CREATES AN ANTITHROMBOTIC BIOMATERIAL SURFACE
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Blood incompatibility reactions caused by surfaces often involve platelet activation and subsequent platelet-initiated activation of the coagulation and complement cascades. The goal of this proof-of-principle study was to immobilize apyrase on a biomaterial surface in order to develop an enzymatically active surface that would have the capacity to inhibit platelet activation by degradating ADP. We were able to immobilize apyrase on a polystyrene surface with preservation of the enzymatic activity. We then analyzed the hemocompatibility of the apyrase surface and of control surfaces (serum albumin, avidin, polystyrene, and glass) by incubation with platelet-rich plasma (PRP) or whole blood. Monitoring of markers of platelet, coagulation, and complement activation and staining of the surfaces revealed decreased levels of platelet and coagulation activation parameters on the apyrase surface. The level of complex formation between antithrombin and thrombin or factor XIa and the extent of the platelet loss were significantly lower on the apyrase surface than on any of the control surfaces. No significant differences were seen in complement activation (C3a levels). Staining of the apyrase surface revealed low platelet adherence and no formation of granulocyte-platelet complexes. These results demonstrate that it is possible to create an anti-thrombotic surface targeting the ADP amplification of platelet activation by immobilizing apyrase.

Keywords
Hemocompatibility, biomaterials, platelets, coagulation
National Category
Immunology in the medical area
Research subject
Natural Science, Biomedical Sciences
Identifiers
urn:nbn:se:hik:diva-2790 (URN)
Available from: 2010-04-07 Created: 2010-02-22 Last updated: 2018-01-12Bibliographically approved
4. BLOOD PROTEIN-POLYMER ADSORPTION FINGERPRINTING:IMPLICATIONS FOR UNDERSTANDING HEMOCOMPATIBILITYAND FOR BIOMATERIAL DESIGN
Open this publication in new window or tab >>BLOOD PROTEIN-POLYMER ADSORPTION FINGERPRINTING:IMPLICATIONS FOR UNDERSTANDING HEMOCOMPATIBILITYAND FOR BIOMATERIAL DESIGN
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Within seconds after an artificial material has been implanted into the blood thesurface will be covered by adsorbed plasma proteins. The composition of theprotein layer is determined by the physical-chemical properties of the surface. Asthe layer itself will become the new interface between the material and blood, itis of major importance for the hemocompatibility. In this project we have studiedthe adsorption of proteins to a model material (polystyrene, PS) with the peptidemass fingerprint technique (PMF) analyzed on a Matrix Assisted LaserDesorption/Ionization Time-of-Flight (MALDI-TOF). To further be able to studythe adsorption of plasma proteins to polymer surfaces, we have synthesized 33new polymer compositions with variable surface properties. Six of thosepolymers were selected and their protein binding ability was determined as wellas quantification of adsorption of 20 plasma proteins to the surface of thepolymers. Our results showed that fourteen high abundant plasma proteins werepositively identified on the PS-surface by MALDI-TOF. Further, the resultsshowed that the synthesized polymers had very distinctive adsorption patterns,with enrichment of different proteins after incubation in plasma and serum. Oneof the polymers was shown to adsorb large amounts of the complementactivating recognition protein C1q, which makes this polymer to a potentialactivating surface. Two of the polymers showed a clear enrichment of thecomplement regulating protein vitronectin as well as two apolipoproteins (AI andAIV) to the surface of the polymers, while some of the polymers bound proteinsapproximately in correlation to the concentration found in plasma.

Keywords
Polymers, biomaterials, plasma proteins, protein adsorption hemocompatibility
National Category
Immunology in the medical area
Research subject
Natural Science, Biomedical Sciences
Identifiers
urn:nbn:se:hik:diva-2791 (URN)
Available from: 2010-03-22 Created: 2010-02-22 Last updated: 2018-01-12Bibliographically approved
5. EVALUATION OF THE HEMOCOMPATIBILITY OF NOVEL POLYMERIC MATERIALS
Open this publication in new window or tab >>EVALUATION OF THE HEMOCOMPATIBILITY OF NOVEL POLYMERIC MATERIALS
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

When a biomaterial surface comes in contact with blood an immediate adsorption of plasma proteins to the surface will occur, and the cascade systems in the blood, such as the complement, coagulation and contact system, will be activated to various degrees. The intensity of this reaction will determine the hemocompatibility of the materials. Here we present an evaluation of the link between the composition, the physico-chemical properties and the protein adsorption properties of six newly synthesized polymers (P1-P6) and the hemocompatibility.The hemocompatibility of the polymeric surfaces was evaluated in human blood plasma and whole blood. Commercially available polyvinylchloride (PVC) was used as reference material. The hemocompatibility of the polymeric surfaces was evaluated with regard to complement activation (C3a and sC5-9 generation) and coagulation activation (platelet loss and TAT-formation) and cytokine productions (27 analytes in multiplex assay) after contact with whole blood. Contact activation was quantified by analyses of FXIIa-C1INH, FXIa-C1INH, and kallikrein-C1INH complexes.Polymers P2 (p<0.05 for C3a), P3, P5 and P6 showed less complement activation, and polymers P1 and P4 (p<0.05 for platelet loss), as well as P5 and P6 showed less coagulation activation compared with reference PVC. Polymers P1-P3 induced activation of the contact system, P3 being the most potent. Secretion of 17 cytokines including chemokines and growth factors were differentially influenced by the polymers, P1 and P3 being significantly (p<0.05) more compatible for five of the analytes.Collectively these data demonstrate that the composition of the polymers clearly leads to different biological properties as a consequence of distinctive physico-chemical properties and protein adsorption patterns.1

Keywords
Polymers, biomaterials, whole blood, complement, coagulation, contact activation, cytokines, hemocompatibility
National Category
Immunology in the medical area
Research subject
Natural Science, Biomedical Sciences
Identifiers
urn:nbn:se:hik:diva-2792 (URN)
Available from: 2010-02-22 Created: 2010-02-22 Last updated: 2018-01-12Bibliographically approved

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