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Effect of Surface Nanotopography on Blood-Biomaterial Interactions
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Surface Biotechnology. (Nano-biomaterial research and Tissue Culturing)
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Biologically inspired materials are being developed with the aim of improving the integration of medical implants and minimizing non-desirable host reactions. A promising strategy is the design of topographically patterned surfaces that resemble those found in the extracellular environment.

Nanoporous alumina has been recognized as a potential biomaterial and as an important template for the fabrication of nanostructures.

In this thesis in vitro studies were done to elucidate the role of alumina nanoporosity on the inflammatory response. Specifically, by comparing alumina membranes with two pore sizes (20 and 200 nm in diameter). Complement and platelet activation were evaluated as well as monocyte/macrophage behaviour.

Whole blood was incubated with the alumina membranes and thereafter the biomaterial surfaces were evaluated in terms of protein and platelet adhesion as well as procoagulant properties. The fluid phase was analyzed for complement activation products and platelet activation markers. Besides, human mononuclear cells were cultured on the alumina membranes and cell adhesion, viability, morphology and release of pro-inflammatory cytokines were evaluated.

The results indicated that nanoporous alumina with 200 nm pores promotes higher complement activation than alumina with 20 nm pores.

In addition, platelet response to nanoporous alumina was found to be highly dependent on the material porosity, as reflected by differences in adhesion, PMP generation and procoagulant characteristics.

A clear difference in monocyte/macrophage adhesion and activation was found between the two pore size alumina membranes. Few but highly activated cells adhered to the 200 nm membrane in contrast to many but less activated monocytes/macrophages on the 20 nm surface.

The outcome of this work emphasizes that nanotopography plays an important role in the host response to biomaterials.

Better understanding of molecular interactions on nano-level will undoubtedly play a significant role in biomaterial implant development and will contribute to design strategies for controlling specific biological events.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2010. , p. 74
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 693
Keywords [en]
nanoporous alumina, nanotopography, biomaterial, platelets, complement system, macrophages, whole blood, inflammatory response
National Category
Other Basic Medicine Immunology in the medical area
Research subject
Immunology; Materials Science
Identifiers
URN: urn:nbn:se:uu:diva-110614ISBN: 978-91-554-7668-7 (print)OAI: oai:DiVA.org:uu-110614DiVA, id: diva2:278318
Public defence
2010-01-20, B21, BMC, Husarg. 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2009-12-16 Created: 2009-11-18 Last updated: 2018-01-12Bibliographically approved
List of papers
1. Nanoporesize affects complement activation
Open this publication in new window or tab >>Nanoporesize affects complement activation
2008 (English)In: Journal of biomedical materials research. Part A, ISSN 1552-4965, Vol. 87, no 3, p. 575-81Article in journal (Refereed) Published
Abstract [en]

In the present study, we have shown the vast importance of biomaterial nanotexture when evaluating inflammatory response. For the first time in an in vitro whole blood system, we have proven that a small increase in nanoporesize, specifically 180 nm (from 20 to 200 nm), has a huge effect on the complement system. The study was done using nanoporous aluminiumoxide, a material that previously has been evaluated for potential implant use, showing good biocompatibility. This material can easily be manufactured with different pore sizes making it an excellent candidate to govern specific protein and cellular events at the tissue-material interface. We performed whole blood studies, looking at complement activation after blood contact with two pore size alumina membranes (pore diameters, 20 and 200 nm). The fluid phase was analyzed for complement soluble components, C3a and sC5b-9. In addition, surface adsorbed proteins were eluted and dot blots were performed to detect IgG, IgM, C1q, and C3. All results point to the fact that 200 nm pore size membranes are more complement activating. Significantly, higher values of complement soluble components were found after whole blood contact with 200 nm alumina and all studied proteins adsorbed more readily to this membrane than to the 20 nm pore size membrane. We hypothesize that the difference in complement activation between our two test materials is caused by the type and the amount of adsorbed proteins, as well as their conformation and orientation. The different protein patterns created on the two alumina membranes are most likely a consequence of the material topography.

Keywords
nanotopography, nanoporous alumina, complement, whole blood, protein adsorption
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-88357 (URN)10.1002/jbm.a.31818 (DOI)000260984800002 ()18186072 (PubMedID)
Available from: 2009-01-30 Created: 2009-01-29 Last updated: 2009-07-13Bibliographically approved
2. Influence of nanoporesize on platelet adhesion and activation
Open this publication in new window or tab >>Influence of nanoporesize on platelet adhesion and activation
2008 (English)In: Journal of materials science. Materials in medicine, ISSN 0957-4530, E-ISSN 1573-4838, Vol. 19, no 9, p. 3115-21Article in journal (Refereed) Published
Abstract [en]

In this study we have evaluated the influence of biomaterial nano-topography on platelet adhesion and activation. Nano-porous alumina membranes with pore diameters of 20 and 200 nm were incubated with whole blood and platelet rich plasma. Platelet number, adhesion and activation were determined by using a coulter hematology analyzer, scanning electron microscopy, immunocytochemical staining in combination with light microscopy and by enzyme immunoassay. Special attention was paid to cell morphology, microparticle generation, P-selectin expression and beta-TG production. Very few platelets were found on the 200 nm alumina as compared to the 20 nm membrane. The platelets found on the 20 nm membrane showed signs of activation such as spread morphology and protruding filipodia as well as P-selectin expression. However no microparticles were detected on this surface. Despite the fact that very few platelets were found on the 200 nm alumina in contrast to the 20 nm membrane many microparticles were detected on this surface. Interestingly, all microparticles were found inside circular shaped areas of approximately 3 mum in diameter. Since this is the approximate size of a platelet we speculate that this is evidence of transient, non-adherent platelet contact with the surface, which has triggered platelet microparticle generation. To the authors knowledge, this is the first study that demonstrates how nanotexture can influence platelet microparticle generation. The study highlights the importance of understanding molecular and cellular events on nano-level when designing new biomaterials.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-88354 (URN)10.1007/s10856-008-3449-7 (DOI)000256964300016 ()18414999 (PubMedID)
Available from: 2009-01-30 Created: 2009-01-29 Last updated: 2017-12-14Bibliographically approved
3. Procoagulant behavior and platelet microparticle generation on nanoporous alumina
Open this publication in new window or tab >>Procoagulant behavior and platelet microparticle generation on nanoporous alumina
2010 (English)In: Journal of biomaterials applications, ISSN 0885-3282, E-ISSN 1530-8022, Vol. 24, no 8, p. 675-692Article in journal (Refereed) Published
Abstract [en]

In the present work, we have investigated platelet microparticle(PMP) generation in whole blood after contact with nanoporous alumina.Alumina membranes with pore sizes of 20 and 200nm in diameter were incubated with whole blood and the number of PMP in the fluid phase was determined by flow cytometry. The role of the complement system in PMP generation was investigated using an analog of the potent complement inhibitor compstatin. Moreover, the procoagulant activity of the two pore size membranes were compared by measuring thrombin formation. Results indicated that PMP were not present in the fluid phase after whole blood contact with either of the alumina membranes. However, scanning electron microscope micrographs clearly showed the presence of PMP clusters on the 200nm pore size alumina, while PMP were practically absent on the 20nm membrane. We probed no influence of complement activation in PMP generation and adhesion and we hypothesize that other specific material-related protein–platelet interactions are taking place. A clear difference in procoagulant activity between the membranes could also be seen, 20nm alumina showed 100% higher procoagulant activity than 200nm membrane. By combining surface evaluation and flow cytometry analyses of the fluid phase, we are able to conclude that 200nm pore size alumina promotes PMP generation and adhesion while the 20nm membrane does not appreciably cause any release or adhesion of PMP, thus indicating a direct connection between PMP generation and nanoporosity.

Place, publisher, year, edition, pages
SAGE, 2010
Keywords
nanoporous alumina, nanotopography, platelets, platelet microparticles, procoagulant activity, compstatin
National Category
Chemical Sciences
Research subject
Immunology; Materials Science
Identifiers
urn:nbn:se:uu:diva-130496 (URN)10.1177/0885328209338639 (DOI)000277806100001 ()19581322 (PubMedID)
Available from: 2010-09-08 Created: 2010-09-08 Last updated: 2017-12-12Bibliographically approved
4. Time sequence of blood activation by nanoporous alumina: Studies on platelets and complement system
Open this publication in new window or tab >>Time sequence of blood activation by nanoporous alumina: Studies on platelets and complement system
2010 (English)In: Microscopy research and technique (Print), ISSN 1059-910X, E-ISSN 1097-0029, Vol. 73, no 12, p. 1101-1109Article in journal (Refereed) Published
Abstract [en]

In the present work the time sequence of blood activation by alumina membranes with different porosities (20 and 200 nm in diameter) was studied. The membranes were incubated with whole blood from 2 min to 4 h. Platelet adhesion and activation in addition to complement activation were monitored at different time points. Evaluation of platelet adhesion and activation was done by determining the change in platelet number and the levels of thrombospondin-1 in the fluid phase. Scanning electron microscopy studies were done to further evaluate platelet adhesion and morphology. Immunocytochemical staining was used to evaluate the presence of CD41 and CD62P antigens on the material surface. Complement activation was monitored by measuring C3a and sC5b-9 in plasma samples by means of enzyme immunoassays. Both alumina membranes displayed similar complement activation time profiles, with levels of C3a and sC5b-9 increasing with incubation time. A statistically significant difference between the membranes was found after 60 min of incubation. Platelet activation characteristics and time profile were different between the two membranes. Platelet adhesion increased over time for the 20 nm surface, while the clusters of microparticles on the 200 nm surface did not appreciably change during the course of the experiment. The release of thrombospondin-1 increased with time for both membranes, however much later for the 200 nm alumina (240 min) as compared to the 20 nm membrane (60 min). The surface topography of the alumina most probably influence protein transition rate, which in turn affects material-platelet activation kinetics.

Place, publisher, year, edition, pages
Wiley-Liss Inc., 2010
Keywords
nanotopography, biomaterial, whole blood, thrombospondin-1, platelet microparticles
National Category
Other Basic Medicine
Research subject
Immunology; Materials Science
Identifiers
urn:nbn:se:uu:diva-110620 (URN)10.1002/jemt.20854 (DOI)000284063800005 ()
Available from: 2009-11-18 Created: 2009-11-18 Last updated: 2018-01-12Bibliographically approved
5. Nanoporosity of alumina surfaces induces different patterns of activation in adhering monocytes/macrophages
Open this publication in new window or tab >>Nanoporosity of alumina surfaces induces different patterns of activation in adhering monocytes/macrophages
2010 (English)In: International Journal of Biomaterials, ISSN 1687-8787, E-ISSN 1687-8795, Vol. 2010, p. 402715-Article in journal (Refereed) Published
Abstract [en]

The present study shows that alumina nanotopography affects monocyte/macrophage behaviour. Human mononuclear cells cultured on alumina membranes with pore diameters of 20 and 200 nm were evaluated in terms of cell adhesion, viability, morphology and release of pro-inflammatory cytokines. After 24 hours, cell adhesion was assessed by means of light microscopy and cell viability by measuring LDH release. The inflammatory response was evaluated by quantifying interleukin-1ß and tumour necrosis factor-α. Finally, scanning electron microscopy was used to study cell morphology. Results showed pronounced differences in cell number, morphology and cytokine release depending on the nanoporosity. Few but highly activated cells were found on the 200 nm porous alumina, while relatively larger number of cells was found on the 20 nm porous surface. However, despite their larger number, the cells adhering on the 20 nm surface exhibited reduced pro-inflammatory activity. It can be speculated that the difference in surface topography may lead to distinct protein adsorption patterns and therefore to different degree of cell activation. The data of this paper emphasize the role played by the material nanotexture in dictating cell responses and implies that nanotopography could be exploited for controlling the inflammatory response to implants.

Keywords
macrophages, nanoporous alumina, biomaterials, nanotopography, inflammatory response
National Category
Other Basic Medicine
Research subject
Immunology
Identifiers
urn:nbn:se:uu:diva-110623 (URN)10.1155/2010/402715 (DOI)21234322 (PubMedID)
Available from: 2009-11-18 Created: 2009-11-18 Last updated: 2018-01-12Bibliographically approved

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