Effects of nanoporous alumina on inflammatory cell response Shiuli Pujari,1 Andreas Hoess,1 Jinhui Shen,2 Annika Thormann,3 Andreas Heilmann,3 Liping Tang,2 Marjam Karlsson-Ott1 € m Laboratory, Uppsala University, Applied Material Science, Department of Engineering Sciences, The A˚ngstro SE-751 21 Uppsala, Sweden 2 Department of Bioengineering, University of Texas at Arlington, Arlington, Texas 76019-0138 3 Fraunhofer Institute for Mechanics of Materials IWM, 06120 Halle, Germany 1
Received 7 October 2013; revised 2 November 2013; accepted 20 November 2013 Published online 9 December 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35048 Abstract: The present study focuses on the effects of nanoscale porosity on inflammatory response in vitro and in vivo. Nanoporous alumina membranes with different pore sizes, 20 and 200 nm in diameter, were used. We first evaluated cell/alumina interactions in vitro by observing adhesion, proliferation, and activation of a murine fibroblast and a macrophage cell line. To investigate the chronic inflammatory response, the membranes were implanted subcutaneously in mice for 2 weeks. Cell recruitment to the site of implantation was determined by histology and the production of cytokines was measured by protein array analysis. Both in vitro and in vivo studies showed that 200 nm pores induced a stronger
inflammatory response as compared to the alumina with 20 nm pores. This was observed by an increase in macrophage activation in vitro as well as higher cell recruitment and generation of proinflammatory cytokines around the alumina with 200 nm pores, in vivo. Our results suggest that nanofeatures can be modulated in order to control the inflammatory C 2013 Wiley Periodicals, Inc. J Biomed response to implants. V Mater Res Part A: 102A: 3773–3780, 2014.
Key Words: nanoporous alumina, nanofeatures, inflammation, in vivo
How to cite this article: Pujari S, Hoess A, Shen J, Thormann A, Heilmann A, Tang L, Karlsson-Ott M. 2014. Effects of nanoporous alumina on inflammatory cell response. J Biomed Mater Res Part A 2014:102A:3773–3780.
INTRODUCTION
Biomedical implants have become increasingly important in our society, as the current population continues to age. Implanted biomaterials can successfully improve the quality and length of life for patients suffering from, for example, cardiac, orthopedic, and dental diseases.1 Nanostructured materials offer many advantages over traditional biomaterials making them increasingly attractive. They can, for example, be manufactured2 to closely mimic the native architecture of the extracellular matrix in order to regulate specific cell functions.3 Nanotopographical features, such as grooves, ridges, and pores, have been shown to affect cell response in terms of adhesion, morphology, phagocytic activity, and cytokine production.4,5 Despite the widespread use of nanostructured materials, chronic inflammation associated with implantation is poorly characterized. The cell response that nanostructured materials could potentially cause should thus be addressed before implementation for biomedical applications. Nanoporous alumina is a well-characterized material and is recognized as an important template to create complex nanostructures.6 Anodic oxidation of aluminum in poly-
protic acids produces an ordered nanoporous structure. The pore diameter is controlled by the applied voltage and can give rise to varying nanoporous structures, ranging from 10 to 450 nm.7,8 The narrow pore size distribution, therefore, makes this material an appealing template for the fabrication of ordered nanostructures. Nanoporous alumina has been evaluated for various biomedical applications such as bone implant coatings,9 stent coatings for drug delivery,10 cocultivation of cells,11 and immunoisolation devices.12 It can also direct differentiation of mesenchymal stem cells and progenitor cells toward an osteogenic path, thus providing an added benefit for the potential use in orthopedic applications.13,14 Previous studies on nanoporous alumina have focused on the acute (2 were considered as upregulation. No cytokine expression was observed below 0.5. The expression of BLC, CD30L, Fas Ligand, GM-CSF, IL-13, IL-1b, IL-6, IL-9, KC, Leptin, Lymphotactin, RANTES, SDF-1, TCA-3, TECK, and IL-2 were higher in tissue surrounding the 200nm alumina membranes (>2) as compared to the 20 nm membranes. For IL-1a, IL-3, IL-4, IL-17, LIX, MCSF, sTNF RII, TIMP-2, sTNF RI, MIP-1g, and Eotaxin-2 there was
EFFECTS OF NANOPOROUS ALUMINA ON INFLAMMATORY CELL RESPONSE
ORIGINAL ARTICLE
FIGURE 3. A RAW264.7 generated ROS. Generated ROS (measured at an absorbance of 595 nm) was normalized to total cell number (absorbance of 570 nm) giving a unitless measurement of Abs 595/570 nm. Groups were found to be significantly different at p < 0.05. B: SEM micrographs of RAW264.7 cell morphology grown on 20 nm (top) and 200 nm (bottom) after 3 days (scale bar at 10 lm). Cells on the 20 nm membranes conformed to a rounded morphology (B, top), while cells on the 200 nm membranes exhibited a more flattened morphology (B, bottom).
however no difference in expression between the 20 and 200 nm alumina (Table I).
DISCUSSION
Recent studies have shown that nanotopographical features and surface roughness can have a lasting effect on cell response.22–26 Nanosurface topography is able to modulate cell behavior through the influence of spatial organization of cytoskeletal linked membrane receptors, further modulating several cell functions.23 In the present study, fibroblast and macrophage cell adhesion and proliferation on the nanoporous alumina membranes were however found to be the same despite of porosity. On the other hand, the pore size did have an effect on macrophage activation. For all evaluated time points, ranging from 1 to 7 days, we observed that macrophages cultured on the 200-nm alumina membranes produced higher levels of ROS, thus exhibiting a higher degree of activation, as compared to the 20 nm membranes. The amount of ROS produced is a reliable indicator of the amount of phagocytic activity present.27 The production of ROS has widely been linked to signaling pathways that cause inflammation.28 Recent studies suggest that ROS generation is needed in order to activate the inflammasome, a cytosolic molecular complex that is involved in promoting the maturation of proinflammatory cytokines.29,30 Nanofeatures can have diverse effects on macrophage behavior such as spreading,
migration, cell orientation, and phagocytic activity.2,31–35 It has also been shown that macrophages grown on 200-nm alumina membranes expressed increased rough plasmalemma and filopodial extensions, two signs of activation.36 The observed flattened and spread morphology, seen on macrophages cultured on the 200 nm pores additionally, confirm the higher levels of ROS generated on the 200 nm as compared to the 20 nm membranes. Furthermore, it was shown that macrophages secrete increased amounts of proinflammatory cytokines when cultured on 200 nm alumina versus 20 nm membranes,36 thus in agreement with the present work. Histological evaluation confirmed higher cellular recruitment in the fibrotic capsule surrounding the 200-nm alumina membranes as compared to the 20 nm membranes, 2 weeks after implantation. Previous studies have evaluated the effect of nanoporous alumina on the acute immune response and found that, 200-nm alumina membranes prompted higher immune cell recruitment to the peritoneal cavity after 16 h of implantation as compared to the 20 nm membranes.15 Our results reflect this same trend. Using a whole blood in vitro system, previous investigations have also shown higher levels of soluble complement activation products after incubation with 200 nm versus 20 nm membranes.35 The activation of the complement system influences the subsequent activation of platelets and leukocytes, thereby affecting recruitment of other inflammatory cells to the site of implantation. This could also explain the
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FIGURE 4. Foreign body response analysis on nanoporous alumina membranes after 2 weeks of subcutaneous implantation in Balb/C mice. Masson trichrome collagen staining (A) and H&E staining (B) were performed to assess the extent of implant-associated fibrotic tissue responses, by evaluating the thickness (C), collagen content (D), and cell density (E) of the fibrotic capsule around the implants. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
difference in cell recruitment we see on the membranes after the subcutaneous implantation. Furthermore, the inflammatory protein array analysis demonstrated that 200 nm alumina membranes induced higher expression of many potent proinflammatory cytokines such as IL-13, IL1b, IL-6, IL-9, IL-2, leptin, and fas ligand. Upregulation of these cytokines directs the innate response by recruiting and activating immune cells, and subsequently initiating a fibrotic tissue response. The increased levels of IL-1b and IL-6 suggest higher macrophage activation in agreement with the elevated ROS production on the 200 nm membrane, found in vitro. IL-13 has been implicated in fibroblast proliferation and collagen production while fas ligand is involved in biomaterial-induced fibrosis.36 Taken together, the increased cytokine expression promoted by the 200 nm pores suggests that they have an increased potential to elicit an inflammatory response as compared to 20 nm pores.
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There are many ways in which nanofeatures can influence cell behavior. Nanosurface topography can change the native conformation and orientation of adhered proteins by inducing local regions of hydrophobicity, electrostatic attraction or by stabilizing a normally unstable conformation of an adhered protein.37,38 Studies have also shown that protein adsorption to surfaces can increase the level of phagocytic activity in macrophages.34,39 For the present study, It can be speculated that large proteins are able to diffuse into the 200 nm pores, while proteins instead adsorb on the 20 nm surface. It is also likely that proteins adopt different conformational states due to differing porosity thus revealing different cell surface receptors, subsequently causing varying inflammatory response. Two proteins specifically known for their role in inflammation, fibrinogen, and von Willebrand factor, have found to be affected by nanoscale topography due to size and shape of the proteins itself.40–42
EFFECTS OF NANOPOROUS ALUMINA ON INFLAMMATORY CELL RESPONSE
ORIGINAL ARTICLE
TABLE I. Production of Inflammatory Cytokines in Tissue Sections Surrounding the Nanoporous Alumina Membranes Elevated Cytokines BLC CD30 L Fas ligand GM-CSF IL-13 IL-1b IL-6 IL-9 KC Leptin Lymphotactin RANTES SDF-1 TCA-3 TECK IL-2 Cytokines with no significant change IL-1a, IL-3, IL-4, IL-17, LIX, MCSF, sTNF RII, TIMP-2, sTNF RI, MIP-1g, Eotaxin-2
2.524 2.293 2.291 2.437 2.289 2.415 2.095 2.131 2.113 2.269 2.029 2.095 2.182 2.230 2.137 3.029
Data represents the ratio of expression for the 200 nm membranes to the 20 nm membranes (n 5 2). Protein expression is considered significantly elevated or lowered if the ratio of expression (200 nm/ 20 nm) is >2 or
Received 7 October 2013; revised 2 November 2013; accepted 20 November 2013 Published online 9 December 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35048 Abstract: The present study focuses on the effects of nanoscale porosity on inflammatory response in vitro and in vivo. Nanoporous alumina membranes with different pore sizes, 20 and 200 nm in diameter, were used. We first evaluated cell/alumina interactions in vitro by observing adhesion, proliferation, and activation of a murine fibroblast and a macrophage cell line. To investigate the chronic inflammatory response, the membranes were implanted subcutaneously in mice for 2 weeks. Cell recruitment to the site of implantation was determined by histology and the production of cytokines was measured by protein array analysis. Both in vitro and in vivo studies showed that 200 nm pores induced a stronger
inflammatory response as compared to the alumina with 20 nm pores. This was observed by an increase in macrophage activation in vitro as well as higher cell recruitment and generation of proinflammatory cytokines around the alumina with 200 nm pores, in vivo. Our results suggest that nanofeatures can be modulated in order to control the inflammatory C 2013 Wiley Periodicals, Inc. J Biomed response to implants. V Mater Res Part A: 102A: 3773–3780, 2014.
Key Words: nanoporous alumina, nanofeatures, inflammation, in vivo
How to cite this article: Pujari S, Hoess A, Shen J, Thormann A, Heilmann A, Tang L, Karlsson-Ott M. 2014. Effects of nanoporous alumina on inflammatory cell response. J Biomed Mater Res Part A 2014:102A:3773–3780.
INTRODUCTION
Biomedical implants have become increasingly important in our society, as the current population continues to age. Implanted biomaterials can successfully improve the quality and length of life for patients suffering from, for example, cardiac, orthopedic, and dental diseases.1 Nanostructured materials offer many advantages over traditional biomaterials making them increasingly attractive. They can, for example, be manufactured2 to closely mimic the native architecture of the extracellular matrix in order to regulate specific cell functions.3 Nanotopographical features, such as grooves, ridges, and pores, have been shown to affect cell response in terms of adhesion, morphology, phagocytic activity, and cytokine production.4,5 Despite the widespread use of nanostructured materials, chronic inflammation associated with implantation is poorly characterized. The cell response that nanostructured materials could potentially cause should thus be addressed before implementation for biomedical applications. Nanoporous alumina is a well-characterized material and is recognized as an important template to create complex nanostructures.6 Anodic oxidation of aluminum in poly-
protic acids produces an ordered nanoporous structure. The pore diameter is controlled by the applied voltage and can give rise to varying nanoporous structures, ranging from 10 to 450 nm.7,8 The narrow pore size distribution, therefore, makes this material an appealing template for the fabrication of ordered nanostructures. Nanoporous alumina has been evaluated for various biomedical applications such as bone implant coatings,9 stent coatings for drug delivery,10 cocultivation of cells,11 and immunoisolation devices.12 It can also direct differentiation of mesenchymal stem cells and progenitor cells toward an osteogenic path, thus providing an added benefit for the potential use in orthopedic applications.13,14 Previous studies on nanoporous alumina have focused on the acute (2 were considered as upregulation. No cytokine expression was observed below 0.5. The expression of BLC, CD30L, Fas Ligand, GM-CSF, IL-13, IL-1b, IL-6, IL-9, KC, Leptin, Lymphotactin, RANTES, SDF-1, TCA-3, TECK, and IL-2 were higher in tissue surrounding the 200nm alumina membranes (>2) as compared to the 20 nm membranes. For IL-1a, IL-3, IL-4, IL-17, LIX, MCSF, sTNF RII, TIMP-2, sTNF RI, MIP-1g, and Eotaxin-2 there was
EFFECTS OF NANOPOROUS ALUMINA ON INFLAMMATORY CELL RESPONSE
ORIGINAL ARTICLE
FIGURE 3. A RAW264.7 generated ROS. Generated ROS (measured at an absorbance of 595 nm) was normalized to total cell number (absorbance of 570 nm) giving a unitless measurement of Abs 595/570 nm. Groups were found to be significantly different at p < 0.05. B: SEM micrographs of RAW264.7 cell morphology grown on 20 nm (top) and 200 nm (bottom) after 3 days (scale bar at 10 lm). Cells on the 20 nm membranes conformed to a rounded morphology (B, top), while cells on the 200 nm membranes exhibited a more flattened morphology (B, bottom).
however no difference in expression between the 20 and 200 nm alumina (Table I).
DISCUSSION
Recent studies have shown that nanotopographical features and surface roughness can have a lasting effect on cell response.22–26 Nanosurface topography is able to modulate cell behavior through the influence of spatial organization of cytoskeletal linked membrane receptors, further modulating several cell functions.23 In the present study, fibroblast and macrophage cell adhesion and proliferation on the nanoporous alumina membranes were however found to be the same despite of porosity. On the other hand, the pore size did have an effect on macrophage activation. For all evaluated time points, ranging from 1 to 7 days, we observed that macrophages cultured on the 200-nm alumina membranes produced higher levels of ROS, thus exhibiting a higher degree of activation, as compared to the 20 nm membranes. The amount of ROS produced is a reliable indicator of the amount of phagocytic activity present.27 The production of ROS has widely been linked to signaling pathways that cause inflammation.28 Recent studies suggest that ROS generation is needed in order to activate the inflammasome, a cytosolic molecular complex that is involved in promoting the maturation of proinflammatory cytokines.29,30 Nanofeatures can have diverse effects on macrophage behavior such as spreading,
migration, cell orientation, and phagocytic activity.2,31–35 It has also been shown that macrophages grown on 200-nm alumina membranes expressed increased rough plasmalemma and filopodial extensions, two signs of activation.36 The observed flattened and spread morphology, seen on macrophages cultured on the 200 nm pores additionally, confirm the higher levels of ROS generated on the 200 nm as compared to the 20 nm membranes. Furthermore, it was shown that macrophages secrete increased amounts of proinflammatory cytokines when cultured on 200 nm alumina versus 20 nm membranes,36 thus in agreement with the present work. Histological evaluation confirmed higher cellular recruitment in the fibrotic capsule surrounding the 200-nm alumina membranes as compared to the 20 nm membranes, 2 weeks after implantation. Previous studies have evaluated the effect of nanoporous alumina on the acute immune response and found that, 200-nm alumina membranes prompted higher immune cell recruitment to the peritoneal cavity after 16 h of implantation as compared to the 20 nm membranes.15 Our results reflect this same trend. Using a whole blood in vitro system, previous investigations have also shown higher levels of soluble complement activation products after incubation with 200 nm versus 20 nm membranes.35 The activation of the complement system influences the subsequent activation of platelets and leukocytes, thereby affecting recruitment of other inflammatory cells to the site of implantation. This could also explain the
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | NOV 2014 VOL 102A, ISSUE 11
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FIGURE 4. Foreign body response analysis on nanoporous alumina membranes after 2 weeks of subcutaneous implantation in Balb/C mice. Masson trichrome collagen staining (A) and H&E staining (B) were performed to assess the extent of implant-associated fibrotic tissue responses, by evaluating the thickness (C), collagen content (D), and cell density (E) of the fibrotic capsule around the implants. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
difference in cell recruitment we see on the membranes after the subcutaneous implantation. Furthermore, the inflammatory protein array analysis demonstrated that 200 nm alumina membranes induced higher expression of many potent proinflammatory cytokines such as IL-13, IL1b, IL-6, IL-9, IL-2, leptin, and fas ligand. Upregulation of these cytokines directs the innate response by recruiting and activating immune cells, and subsequently initiating a fibrotic tissue response. The increased levels of IL-1b and IL-6 suggest higher macrophage activation in agreement with the elevated ROS production on the 200 nm membrane, found in vitro. IL-13 has been implicated in fibroblast proliferation and collagen production while fas ligand is involved in biomaterial-induced fibrosis.36 Taken together, the increased cytokine expression promoted by the 200 nm pores suggests that they have an increased potential to elicit an inflammatory response as compared to 20 nm pores.
3778
PUJARI ET AL.
There are many ways in which nanofeatures can influence cell behavior. Nanosurface topography can change the native conformation and orientation of adhered proteins by inducing local regions of hydrophobicity, electrostatic attraction or by stabilizing a normally unstable conformation of an adhered protein.37,38 Studies have also shown that protein adsorption to surfaces can increase the level of phagocytic activity in macrophages.34,39 For the present study, It can be speculated that large proteins are able to diffuse into the 200 nm pores, while proteins instead adsorb on the 20 nm surface. It is also likely that proteins adopt different conformational states due to differing porosity thus revealing different cell surface receptors, subsequently causing varying inflammatory response. Two proteins specifically known for their role in inflammation, fibrinogen, and von Willebrand factor, have found to be affected by nanoscale topography due to size and shape of the proteins itself.40–42
EFFECTS OF NANOPOROUS ALUMINA ON INFLAMMATORY CELL RESPONSE
ORIGINAL ARTICLE
TABLE I. Production of Inflammatory Cytokines in Tissue Sections Surrounding the Nanoporous Alumina Membranes Elevated Cytokines BLC CD30 L Fas ligand GM-CSF IL-13 IL-1b IL-6 IL-9 KC Leptin Lymphotactin RANTES SDF-1 TCA-3 TECK IL-2 Cytokines with no significant change IL-1a, IL-3, IL-4, IL-17, LIX, MCSF, sTNF RII, TIMP-2, sTNF RI, MIP-1g, Eotaxin-2
2.524 2.293 2.291 2.437 2.289 2.415 2.095 2.131 2.113 2.269 2.029 2.095 2.182 2.230 2.137 3.029
Data represents the ratio of expression for the 200 nm membranes to the 20 nm membranes (n 5 2). Protein expression is considered significantly elevated or lowered if the ratio of expression (200 nm/ 20 nm) is >2 or
