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Cell Adhesion on Nanofibrous Polytetrafluoroethylene (nPTFE) Kristy M. Ainslie,† Eric M. Bachelder,‡,§ Sachin Borkar,| Alisar S. Zahr,† Ayusman Sen,*,| John V. Badding,*,| and Michael V. Pishko*,†,|,⊥ Department of Chemical Engineering, Department of Chemistry, and Department of Materials Science and Engineering, The PennsylVania State UniVersity, UniVersity Park, PennsylVania 16802, National Institutes of Health, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892, and Department of Chemical and Biomolecular Engineering, UniVersity of Nebraska, Lincoln, Nebraska 68588 ReceiVed April 7, 2006. In Final Form: September 26, 2006 Here, we described the in vitro biocompatibility of a novel nanostructured surface composed of PTFE as a potential polymer for the prevention of adverse host reactions to implanted devices. The foreign body response is characterized at the tissue-material interface by several layers of macrophages and large multinucleated cells known as foreign body giant cells (FBGC), and a fibrous capsule. The nanofibers of nanofibrous PTFE (nPTFE) range in size from 20 to 30 nm in width and 3-4 mm in length. Glass surfaces coated with nPTFE (produced by jet-blowing of PTFE 601A) were tested under in vitro conditions to characterize the amount of protein adsorption, cell adhesion, and cell viability. We have shown that nPTFE adsorbs 495 ( 100 ng of bovine serum albumin (BSA) per cm2. This level was considerably higher than planar PTFE, most likely due to the increase in hydrophobicity and available surface area, both a result of the nanoarchitecture. Endothelial cells and macrophages were used to determine the degree of cell adsorption on the surface of the nanostructured polymer. Both cell types were significantly more round and occupied less area on nPTFE as compared to tissue culture polystyrene (TCPS). Furthermore, a larger majority of the cells on the nPTFE were dead compared to TCPS, at dead-to-live ratios of 778 ( 271 to 1 and 23 ( 5.6 to 1, respectively. Since there was a high amount of cell death (due to either apoptosis or necrosis), and the foreign body response is a form of chronic inflammation, an 18 cytokine Luminex panel was performed on the supernatant from macrophages adherent on nPTFE and TCPS. As a positive control for inflammation, lipopolysaccharide (LPS) was added to macrophages on TCPS to estimate the maximum inflammation response of the macrophages. From the data presented with respect to IL-1, TNF-R, IFN-γ, and IL-5, we concluded that nPTFE is nonimmunogenic and should not yield a huge inflammatory response in vivo, and cell death observed on the surface of nPTFE was likely due to apoptosis resulting from the inability of cells to spread on these surface. On the basis of the production of IL-1, IL-6, IL-4, and GM-CSF, we concluded that FBGC formation on nPTFE may be decreased as compared to materials known to elicit FBGC formation in vivo.
Introduction As an implant is introduced into soft tissue, the first event at the tissue-material interface is the adsorption of proteins onto the surface of the material.1 The adsorbed proteins serve as an anchor to which cells and additional proteins adhere and mediate the host response to the implant.1 The results of this host response can be detrimental to both the host and the function of the implant.2 The foreign body response is characterized at the tissue-material interface by several layers of macrophages and large multinucleated cells known as foreign body giant cells (FBGC). These FBGCs are generally composed of several fused macrophages and are surrounded by collagen and other extracellular matrix (ECM) proteins.3 ECM proteins also serve as anchors for these giant cells to form.4,5 These giant cells not only serve as a mass transfer barrier if the implant is a sensor, but they also have been * E-mail addresses:
[email protected],
[email protected],
[email protected]. † Department of Chemical Engineering, The Pennsylvania State University. ‡ National Institutes of Health. § University of Nebraska. | Department of Chemistry, The Pennsylvania State University. ⊥ Department of Materials Science and Engineering, The Pennsylvania State University. (1) Balasubramanian, V.; Grusin, N. K.; Bucher, R. W.; Turitto, V. T.; Slack, S. M. Residence-time dependent changes in fibrinogen adsorbed to polymeric biomaterials. J. Biomed. Mater. Res. 1999, 44 (3), 253-60. (2) Padera, R.; Colton, C. Time course of membrane microarchitecture-driven neovascularization. Biomaterials 1996, 17 (3), 277-284. (3) Anderson, J. M. Inflammation and the foreign body response. Probl. Gen. Surg. 1994, 11, 147-160. (4) Rosales, C.; Juliano, R. L. Signal transduction by cell adhesion receptors in leukocytes. J. Leukocyte Biol. 1995, 57 (2), 189-98.
shown to release lysosomal enzymes and reactive oxygen intermediates, which can degrade the surface of the implant.6-8 In addition, it has been shown that FBGC formation is linked to autoimmune disease.9 Therefore, biofouling and FBGC formation can compromise the implant and may make the host susceptible to autoimmune diseases. Here, we describe protein and cell adhesion to nanostructured PTFE produced via jet-blowing. Materials produced from poly(tetrafluoroethylene) (PTFE) are used in numerous areas of biotechnology, including mostly cardiovascular science,10-12 but (5) Ruoslahti, E.; Pierschbacher, M. D. New perspectives in cell adhesion: RGD and integrins. Science 1987, 238 (4826), 491-7. (6) Abramson, S. L.; Gallin, J. I. IL-4 inhibits superoxide production by human mononuclear phagocytes. J. Immunol. 1990, 144 (2), 625-30. (7) Adams, D. O.; Hamilton, T. A. Macrophages as destructive cells in host defense. In Inflamation: Basic Principles and Clinical Correlates, 2nd ed.; Gallin, J. I., Goldstein, I. M., Snyderman, R., Eds.; Raven Presse: New York, 1992; pp 637-662. (8) Zhao, Q.; Agger, M. P.; Fitzpatrick, M.; Anderson, J. M.; Hiltner, A.; Stokes, K.; Urbanski, P. Cellular interactions with biomaterials: in vivo cracking of pre-stressed Pellethane 2363-80A. J. Biomed. Mater. Res. 1990, 24 (5), 62137. (9) Shoaib, B. O.; Patten, B. M.; Calkins, D. S. Adjuvant breast disease: an evaluation of 100 symptomatic women with breast implants or silicone fluid injections. Keio J. Med. 1994, 43 (2), 79-87. (10) Chandy, T.; Das, G. S.; Wilson, R. F.; Rao, G. H. Use of plasma glow for surface-engineering biomolecules to enhance bloodcompatibility of Dacron and PTFE vascular prosthesis. Biomaterials 2000, 21 (7), 699-712. (11) Roy-Chaudhury, P.; Kelly, B. S.; Miller, M. A.; Reaves, A.; Armstrong, J.; Nanayakkara, N.; Heffelfinger, S. C. Venous neointimal hyperplasia in polytetrafluoroethylene dialysis grafts. Kidney Int. 2001, 59 (6), 2325-2334. (12) Muller-Hulsbeck, S.; Walluscheck, K. P.; Priebe, M.; Grimm, J.; Cremer, J.; Heller, M., Experience on endothelial cell adhesion on vascular stents and stent-grafts - First in vitro results. InVest. Radiol. 2002, 37 (6), 314-320.
10.1021/la060948s CCC: $37.00 © 2007 American Chemical Society Published on Web 12/19/2006
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Figure 1. SEM images of jet-blown nPTFE fibrous mats with nanofibrous morphologies.74
also neurology,13 plastic surgery,14,15 and peridontics.16,17 PTFE’s desirable properties include the polymer’s inertness, limited toxicity, thermal stability, and low coefficient of friction. We have hypothesized, as have others, that the nanomorphology of a material may significantly influence protein and cell adhesion on the material and hence influence its biocompatibility. Protein adhesion to nanoparticles,18-25 nanotubes,26-29 nanowires,30 and (13) Miloro, M.; Halkias, L. E.; Mallery, S.; Travers, S.; Rashid, R. G. Lowlevel laser effect on neural regeneration in Gore-Tex tubes. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endodont. 2002, 93 (1), 27-34. (14) Maas, C. S.; Eriksson, T.; McCalmont, T.; Mabry, M.; Cooke, D.; Schindler, R. Evaluation of expanded polytetrafluoroethylene as a soft-tissue filling substance: An analysis of design-related implant behavior using the porcine skin model. Plast. Reconstr. Surg. 1998, 101 (5), 1307-1314. (15) Drubaix, I.; Legeais, J. M.; MalekChehire, N.; Savoldelli, M.; Menasche, M.; Robert, L.; Renard, G.; Pouliquen, Y. Collagen synthesized in fluorocarbon polymer implant in the rabbit cornea. Exp. Eye Res. 1996, 62 (4), 367-376. (16) Zardeneta, G.; Mukai, H.; Marker, V.; Milam, S. B. Protein interactions with particulate Teflon: Implications for the foreign body response. J. Oral Maxillofac. Surg. 1996, 54 (7), 873-878. (17) Haas, R.; Baron, M.; Dortbudak, O.; Watzek, G. Lethal photosensitization, autogenous bone, and e-PTFE membrane for the treatment of peri-implantitis: Preliminary results. International Journal of Oral and Maxillofacial Implants 2000, 15 (3), 374-382. (18) Weng, L.; Van Riemsdijk, W. H.; Hiemstra, T. Adsorption free energy of variable-charge nanoparticles to a charged surface in relation to the change of the average chemical state of the particles. Langmuir 2006, 22 (1), 389-97. (19) Owens, D. E., III; Peppas, N. A. Opsonization, biodistribution, and pharmacokinetics of polymeric nanoparticles. Int. J. Pharm. 2006, 307 (1), 93102. (20) Goppert, T. M.; Muller, R. H. Adsorption kinetics of plasma proteins on solid lipid nanoparticles for drug targeting. Int. J. Pharm. 2005, 302 (1-2), 17286. (21) Vertegel, A. A.; Siegel, R. W.; Dordick, J. S. Silica nanoparticle size influences the structure and enzymatic activity of adsorbed lysozyme. Langmuir 2004, 20 (16), 6800-7. (22) Gupta, A. K.; Curtis, A. S. Surface modified superparamagnetic nanoparticles for drug delivery: interaction studies with human fibroblasts in culture. J. Mater. Sci. Mater. Med. 2004, 15 (4), 493-6. (23) Lvov, Y.; Caruso, F. Biocolloids with ordered urease multilayer shells as enzymatic reactors. Anal. Chem 2001, 73 (17), 4212-7. (24) Gref, R.; Luck, M.; Quellec, P.; Marchand, M.; Dellacherie, E.; Harnisch, S.; Blunk, T.; Muller, R. “Stealth” corona-core nanoparticles surface modified by polyethylene glycol (PEG): influences of the corona (PEG chain length and surface density) and of the core composition on phagocytic uptake and plasma protein adsorption. Colloids Surf., B 2000, 18, 301-313. (25) Lundqvist, M.; Sethson, I.; Jonsson, B. H. Transient interaction with nanoparticles “freezes” a protein in an ensemble of metastable near-native conformations. Biochemistry 2005, 44 (30), 10093-9. (26) Karajanagi, S. S.; Vertegel, A. A.; Kane, R. S.; Dordick, J. S., Structure and function of enzymes adsorbed onto single-walled carbon nanotubes. Langmuir 2004, 20 (26), 11594-9. (27) in het Panhuis, M.; Salvador-Morales, C.; Franklin, E.; Chambers, G.; Fonseca, A.; Nagy, J. B.; Blau, W. J.; Minett, A. I. Characterization of an interaction between functionalized carbon nanotubes and an enzyme. J. Nanosci. Nanotechnol. 2003, 3 (3), 209-13. (28) Salvador-Morales, C.; Flahaut, E.; Sim, E.; Sloan, J.; Green, M. L.; Sim, R. B. Complement activation and protein adsorption by carbon nanotubes. Mol. Immunol. 2006, 43 (3), 193-201. (29) Ding, H. M.; Shao, L.; Liu, R. J.; Xiao, Q. G.; Chen, J. F. Silica nanotubes for lysozyme immobilization. J. Colloid Interface Sci. 2005, 290 (1), 102-6. (30) Ainslie, K. M.; Sharma, G.; Dyer, M. A.; Grimes, C. A.; Pishko, M. V. Attenuation of protein adsorption on static and oscillating magnetostrictive nanowires. Nano Lett. 2005, 5 (9), 1852-6.
nanostructured materials31-33 has been studied for a number of years for a wide variety of materials, though not nanostructured PTFE. For example, Reidel and colleagues explored the adsorption of protein on germanium nanoislands on Si(100), finding that albumin adsorption increased dramatically due to the presence of these nanoislands and that adherent monocytes released increased amounts of IL-1β and TNF-R.31 Peppas and colleagues also found that proteins adhered quickly to “nonstealth” nanoparticles (i.e., particles unmodified with poly(ethylene glycol)), resulting in their opsonization and clearance by the mononuclear phagocyte system in vivo.19 Studies have begun to shed light on protein adsorption phenomena on nanoparticles and other nanostructures. For example, Vertegel and colleagues examined how lysozyme adsorbed to silica nanoparticles and found that adsorbed protein levels decreased with decreasing nanoparticle size.21 They hypothesized that the increased radius of curvature for small nanoparticles, which are approaching the size of proteins, resulted in less protein denaturation on these surfaces, preserving native protein conformations. Supporting this notion using 2-D NMR, Lindqvist and colleagues demonstrated that carbonic anhydrase is “frozen” in a nearly native conformation when adsorbed on 9-nm silica.25 The protein returned to its native active conformation when the nanoparticles were removed. Thus, one can see from the literature that proteins do adhere to nanostructures, but it is difficult to develop broad generalizations regarding the strength of adhesion and whether proteins will be denatured on these surfaces. As such, one cannot say a priori how proteins will interact with nPTFE with different nanomorphologies and how these surfaces will subsequently influence cell adhesion and behavior. However, given what has been discovered for other nanomaterials, nPTFE may perform quite differently than ePTFE. A nanofibrous PTFE (nPTFE) has been developed to explore the effects of nanoarchitecture on cell and protein adhesion over traditional PTFE and expanded PTFE (ePTFE). nPTFE is produced in an environmentally friendly, single-step, solventfree technique in which pure ultrahigh molecular weight PTFE, at temperatures below the melting point, is jet-blown with gases such as nitrogen and argon under high pressure into nanofibers. Jet-blown nPTFE is produced at a rapid rate and can adhere to many different materials, resulting in dense nanofibrous mats and facile fabrication of surface modifying coatings. Individual nanofibers can range in size from 20 to 30 nm in width and 3-4 (31) Riedel, M.; Muller, B.; Wintermantel, E. Protein adsorption and monocyte activation on germanium nanopyramids. Biomaterials 2001, 22 (16), 2307-16. (32) Wei, G.; Ma, P. X. Structure and properties of nano-hydroxyapatite/ polymer composite scaffolds for bone tissue engineering. Biomaterials 2004, 25 (19), 4749-57. (33) Rosenbloom, A. J.; Sipe, D. M.; Shishkin, Y.; Ke, Y.; Devaty, R. P.; Choyke, W. J. Nanoporous SiC: a candidate semi-permeable material for biomedical applications. Biomed. MicrodeVices 2004, 6 (4), 261-7.
Cell Adhesion to Nanofibrous PTFE
mm in length (Figure 1). These dense nPTFE mats exhibit substantial molecular orientation with respect to fiber alignment, resulting in a novel nanoarchitecture structure that incorporates the beneficial chemical properties of PTFE. To explore the suitability of nPTFE as a material for implanted medical devices, we have performed studies which quantify the biocompatibility of unmodified nPTFE. Glass surfaces coated with nPTFE (produced by jet-blowing of PTFE 601A) were challenged with the ubiquitous blood protein serum albumin, to determine protein adsorption characteristics. Adhesion of endothelial cells and macrophages on nPTFE surfaces was also characterized. Finally, to determine if the nPTFE was an immunostimulant, cytokines released from macrophages adherent to nPTFE were studied. The rejection rate of proteins and cells on nPTFE is compared to poly(ethylene glycol) (PEG), so as to relate the surface into a well-studied biomaterial frame of reference. Experimental Methods Bovine serum albumin (BSA), Dulbecco’s Modified Eagle Medium (DMEM), antibiotic-antimycotic solution, trypsin-EDTA solution 0.25%, and phosphate buffered saline (PBS; 0.15 M NaCl, 0.001 M KHB2BPOB4B, and 0.002 M NaB2BHPOB4B; pH 7.4) were purchased from Sigma-Aldrich, St. Louis, MO. 3B-11 mouse endothelial cells and RAW 264.7 mouse macrophages were purchased from ATCC (Manasas, VA). Fetal bovine serum (FBS) was purchased from Hyclone (Logan, UT). Cell Tracker Green CMFDA was purchased from Molecular Probes (Eugene, OR). Iodinated bovine serum albumin ([P125PI]-BSA) was purchased from PerkinElmer (Wellesley, MA). PTFE 601A was purchased from DuPont. Scanning Electron Microscopy. Low-magnification SEM imaging was performed with an environmental SEM (FEI) on uncoated fibers in the presence of several mmHg of water vapor to prevent charging. High-resolution images were obtained with a field-emission SEM (JEOL) after coating the samples with gold. X-ray Photoelectron Spectroscopy. X-ray photoelectron spectroscopy (XPS) analysis was performed using a Kratos Analytical Axis Ultra instrument. The X-ray source was monochromatic aluminum (1486.6 eV), and it was powered at 280 W. A survey spectrum was collected at a takeoff angle of 90° with respect to the sample plane. Survey spectra were collected from 0 to 1250 eV with pass energy of 80 eV. Relative sensitivity factors were derived from polymer standards: poly(ethyleneimine), PEI; poly(ethylene terephthalate), PET; and poly(tetrafluoroethylene), PTFE. Quantification of relative atomic concentrations was performed from the spectra collected at pass energy of 80 eV. Sample preparation involved placing each Teflon sample directly onto double-sided carbon tape, which was then mounted onto the sample rod. Cell Culture. For both cell types, cell culture media was composed of 10% FBS and 1% antibiotic-antimycotic in DMEM. The cells were maintained in T-75 polystyrene culture flasks at 5% CO2 and 37 °C and subcultured with trypsin-EDTA solution. For each subculture, cells were assigned a passage number. For all experiments presented here, passages 4-9 were used. Cell Imaging. Cells were seeded (50 000 cells/mL of DMEM with 10% FBS) on tissue culture polystyrene (TCPS) or nPTFE surfaces and imaged at discrete time points with fluorescent dye Cell Tracker Green CMFDA. Cell tracker solution was prepared and incubated with cells in accordance with manufacturer’s directions. Samples were washed in PBS prior to imaging. The fluorophoreloaded cells were imaged with a fluorescein isothiocyanate (FITC) filter on a Zeiss Axiovert 200M. The number of cells and their morphology (area and length of the long axis if spread) was calculated by manually tracing or measuring the cell via AxioVision LE version 4.3 (Zeiss). Morphology of adherent cells was further characterized by determining their circularity as calculated by eq 1.
Langmuir, Vol. 23, No. 2, 2007 749 Circularity )
Area L2 π 4
(1)
where area is the area of the cell and L is the long axis length of the cell. Viability Assay. To determine the number of dead and live cells on the surface of interest, two fluorescent dyes were used. Cell Tracker was used to image live cells. To image dead cells, SYTOX (Molecular Probes, Eugene, OR) was used. SYTOX is a nucleic acid stain that easily penetrates the compromised plasma membranes of dead cells, but will not cross the membranes of live cells. All dyes were used in accordance with manufacturer directions. Monitoring of Protein Adsorption Using [125I]-BSA. Iodinated bovine serum albumin, ([P125PI]-BSA) was purchased from PerkinElmer in the amount of 500 µCi. An initial stock solution of [P125PI]-BSA was created of 125 µCi/mL with PBS (10 µCi per 4 mg BSA/mL).34 Dilutions were made of the stock solution to create a 1 mg/mL stock solution of BSA spiked with radio-labeled BSA. After protein adsorption for 15 min, the raw count of emitted γ rays was detected on a Wallac 1470 Wizard Automatic Gamma Counter (PerkinElmer). Cytokine Profiles. CytokineProfiler Testing Service from Upstate, Inc. (Lake Placid, NY) was contracted to measure a panel of 18 cytokines (Table 1) using a Luminex 100 instrument from the Luminex Corporation (Austin, Texas). For these measurements, the cytokine profile of lipopolysaccharide-activated macrophages served as controls to determine if the cytokine profile of nPTFE material was consistent with an inflammatory response. Lipopolysaccharide (LPS) is a major constituent of the cell wall of gram-negative bacteria, is highly immunogenic, and is one of the best activators of macrophages.35 LPS was added at 1 µg/mL to macrophages seeded on TCPS to obtain the maximum production of inflammatory cytokines. The immunostimulatory nature of nPTFE was determined by comparing the amount and type of cytokines released by the LPS-activated macrophages to those released by macrophages cultured on nPTFE.
Results and Discussion Surface Analysis. The nPTFE surface was extensively characterized in a prior publication which also describes in detail the preparation of nPTFE.36 Here, X-ray photoelectron spectroscopy (XPS) analysis was performed on three samples, indicating a composition of 67 ( 1% F, 32 ( 1% C, and 0.13 ( 0.2% O. These results were in very good agreement with the results of other XPS analysis performed on different forms of PTFE.37,38 The results of this XPS analysis also agreed with the nPTFE surface characterized by Gu et al.36 They also noted that the contact angle of nPTFE was greater (147°) then that of flat PTFE (113°).39 Several studies have also concluded that the presence of surface nanostructures leads to increase surface hydrophobicity, potentially through the entrapment of air within these structures.40-42 (34) Hardin, J.; Kimm, M.; Wirasinghe, M.; Gall, D. Macromolecular transport across the rabbit proximal and distal colon. Gut 1999, 44 (2), 215-18. (35) Paul, W. E. Fundamental Immunology, 4th ed.; Raven Press: New York, 1994. (36) Gu, B.; Borkar, S.; Sen, A.; Jackson, B.; Badding, J. Polytetrafluoroethylene Nano/Microfibers Formed by Jet Blowing. Polymer 2006, 47, 8337-8343. (37) Pu, F. R.; Williams, R. L.; Markkula, T. K.; Hunt, J. A. Effects of plasma treated PET and PTFE on expression of adhesion molecules by human endothelial cells in vitro. Biomaterials 2002, 23 (11), 2411-2428. (38) Levesque, S.; Thibault, J.; Castonguay, M.; C-Gaudreault, R.; Laroche, G. Modification of lipid transport through a microporous PTFE membrane wall grafted with poly(ethylene glycol). Colloids Surf., B 2002, 25 (3), 205-217. (39) Extrand, C. W. A thermodynamic model for contact angle hysteresis. J. Colloid Interface Sci. 1998, 207 (1), 11-19. (40) Barthlott, W.; Neinhuis, C. The lotus effect: A self-cleaning surface based on a model taken from nature. Tekstil 2001, 50 (9), 461-465. (41) Oner, D.; McCarthy, T. Ultrahydrophobic Surfaces. Effects of Topography Length Scales on Wettability. Langmuir 2000, 16, 7777-7782.
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Table 1. Cytokine Definitions for Inflammation Panel Results in Table 335,75 role(s) GM-CSF
granulocyte/macrophage colony-stimulating factor
IL-1
Interleukin-1
IL-10
Interleukin-10
IL-12
Interleukin-12
IL-13
Interleukin-13
IL-17
Interleukin-17
IL-2
Interleukin-2
IL-3 IL-4
Interleukin-3 Interleukin-4
IL-5
Interleukin-5
IL-6
Interleukin-6
IL-8
Interleukin-8
IFN-γ
Interferon-γ
KC MCP-1
KC Monocyte chemotactic protein 1 Regulated upon activation, normal T-cell expressed and secreted Tumor necrosis factor
RANTES TNF
A hematopoietic growth factor with a regulatory effect on the transformation of immature macrophages into multinucleated giant cells (MNGC). Plays a role in antibody stimulation. Aids in the formation of giant macrophages. Major inflammatory mediator produced by activated macrophages. Increases local blood flow, fever, release of other cytokines and enhanced expression of adhesion molecules. Produced by macrophages and cytotoxic T-cells. Inhibits the production of pro-inflammatory cytokines and interferences with macrophage-mediated antigen presentation. Product of macrophages and B-cells that enhances the synthesis of IFN-γ and stimulates proliferation of NK and T-cells. Produced by macrophages and cytotoxic T-cells. Inhibits the production of pro-inflammatory cytokines and interferences with macrophage-mediated antigen presentation. Aids in the differentiation of B-cells. Pro-inflammatory cytokine which induces stromal cell pro-inflammatory responses and the production of hematopoietic cytokines. Produced by T-cells in response to antigenic or mitogenic stimulation. Also termed a T-cell growth factor. Increases the growth and activity of macrophages, B-cells, NK cells, and other white blood cells. Produced in NK and T-cells to target mast and stem cells for growth and histamine release. Participates in cell fusion related to granulomas and is anti-inflammatory. Also related to allergic inflammation including stimulation of basophil development, eosinophil chemotaxis, and expression of IgE receptors on B cells. Aids in the formation of giant macrophages. Produced in T-helper cells and activates B-cells and the antibody response. Proliferates and differentiates B-cells. Produced by T-cells, endothelial cells, monocytes and fibroblasts. Promotes monocytes differentiations, increased number of platelets, and synthesis of fibrinogen in T- and B-cells and macrophages. Involved in the downregulation of neutrophil superoxide production. Produced by monocytes, lymphocyte, and Europhiles when stimulated with IL-1R, IL-1β, or TNF. In neutrophils, enhances chemotactic and degranulation response. Induces an increase in the expression of cell surface adhesion molecules. Product of T- and natural killer (NK) cells. Increases generation of highly reactive oxygen species (e.g. superoxide anion and hydrogen peroxide) and alters the cell surface antigens of macrophages permitting them to engulf pathogens. Potent neutrophil activator. Plays an important role in inflammation. Produced by most non-lymphocytes and plays roles in chronic inflammation, activation of macrophages, humoral response, and histamine release. Produced in T-cells, endothelial cells and platelets. A T-cell product that promotes mononuclear infiltrating (e.g., migration of macrophages into cancerous growth) Derived from activated macrophages. Is associated with the production of a fever and promotes the stimulation of most other pro-inflammatory mediators. It is primarily associated with the induction of cellular apoptosis.
Protein Adsorption. Using radiolabeled (IP125P) BSA, the amount of protein adsorption per square centimeter (true surface area as determined by BET surface area analysis; 0.3 m2/g for nPTFE) of nPTFE was measured at 495 ( 100 ng of BSA. Jenney et al.43 reported 70 ng/cm2 of human serum albumin on flat PTFE with competitive adsorption of other serum proteins. Increased protein adsorption on nPTFE was not unexpected, given the increase in protein adsorption observed for other nanomaterials.31 Cell Number and Shape. Table 2 shows the number of cells per square centimeter of surface. The number of adherent macrophages on nPTFE and tissue culture polystyrene (TCPS) per square centimeter is shown in the top part of Table 2. The bottom part shows the number of endothelial cells on nPTFE and TCPS per square centimeter. The area the cells occupied on the surface, given as percent area in Table 2, was determined by (42) Feng, L.; Li, S. H.; Li, H. J.; Zhai, J.; Song, Y. L.; Jiang, L.; Zhu, D. B. Super-hydrophobic surface of aligned polyacrylonitrile nanofibers. Angew. Chem., Int. Ed. 2002, 41 (7), 1221-+. (43) Jenney, C. R.; Anderson, J. M., Adsorbed serum proteins responsible for surface dependent human macrophage behavior. J. Biomed. Mater. Res. 1999, 49 (4), 435-447.
multiplying the number of cells times the average cell area and then normalizing by the area in which the cells were counted. The average area occupied by the two cell types on the surfaces of interest are shown in Figure 2. On average, macrophages occupied per cell 147.8 ( 19.0 µm2 on nPTFE and 229.2 ( 27.5 µm2 on TCPS after 48 h. Similarly, Figure 2b shows that endothelial cells occupied 93.2 ( 52.4 µm2 on nPTFE and 658.5 ( 80.6 µm2 on TCPS after 6 h. The area occupied by adherent cells is related to the circularity of the cells. Figure 3a shows the circularity of macrophages as 0.96 ( 0.07 and 0.38 ( 0.09 for the nPTFE and TCPS surfaces after 48 h, respectively. As seen in Figure 3b, the circularities of endothelial cells were 0.84 ( 0.11 and 0.47 ( 0.07 for the nPTFE and TCPS surfaces, respectively. The area and circularity results demonstrated that cells attached to the surface of nPTFE were considerably smaller and more circular than cells on TCPS, demonstrating that the cells on the nPTFE surface did not spread and were not thriving. Previous research showed that adhesion of anchorage-dependent cells on hydrophobic PTFE was also low.10,12,37,44 The results in Table 2 agreed with these findings; however, the number of cells on the surface was reduced in comparison to previous studies
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Table 2. Cell Number per Square Centimeter of Macrophages and Endothelial Cellsa 3 hr macrophages on nPTFE macrophages on TCPS
6 hr
24 hr
value
area %
value
area %
value
area %
value
area %
60 ( 32 570 ( 43
0.07% 10.1%
9(3 513 ( 17
0.02% 10.5%
64 ( 35 2150 ( 1020
1.5% 74.5%
45 ( 30 2375 ( 437
0.7% 54.4%
1 hr endothlial cells on nPTFE endothlial cells on TCPS
48 hr
4 hr
6 hr
value
area %
value
area %
value
area %
13.7 ( 15 229 ( 80
0.28% 6.1%
11 ( 11 297 ( 56
0.31% 17.3%
8 ( 16 279 ( 24
0.1% 18.4%
a Area % is the percentage of area live cells occupy per square centimeter. This value is calculated by multiplying the number of cells by the average area given in Figure 2 and normalizing it with respect to the area the cells were counted (1 square centimeter). Data are presented as average plus or minus a 95% confidence interval. Tissue culture polystyrene is represented as TCPS and nPTFE represents nanostructured poly(tetrafluoroethylene).
Figure 2. Cell area in square microns of (a) macrophages and (b) endothelial cells. Data are presented as average cell area plus or minus a 95% confidence interval. Tissue culture polystyrene is represented as TCPS, and nPTFE stands for nanostructured poly(tetrafluoroethylene).
on flat PTFE. Gumpenberger et al. and Pu et al. reported approximately 20 000 cells/cm2 and 2000 cells/cm2, respectively, with different seeding densities of endothelial cells after 1 day of adhesion time. Furthermore, Lohbach et al. reported 4.5 ( 2.2% of the surface covered with endothelial cells44 after 1 day. The values reported here, presented in Figure 2 and Table 2, were roughly half of those previously seen by other researchers. Although the time course of these endothelial cell experiments was slightly shorter than values reported by other researchers, it can be seen in Figure 2b that the number of cells on the surface decreased significantly with time. In addition to PTFE, low levels of cell attachment on other hydrophobic surfaces37,45-47 and hydrophilic polymer PEG48-53 were also observed. After 8 h of contact time, Lan et al. reported macrophage attachment to be approximately 4 cells per square centimeter of PEG-coated silicon.52 On gold-based PEG selfassembled monolayers (SAMs) Ostuni et al. reported 10 bovine (44) Lohbach, C.; Bakowsky, U.; Kneuer, C.; Jahn, D.; Graeter, T.; Schafers, H. J.; Lehr, C. M. Wet chemical modification of PTFE implant surfaces with a specific cell adhesion molecule. Chem. Commun. 2002, 21, 2568-2569.
Figure 3. Circularity of (a) macrophages and (b) endothelial cells. Circularity is represented as a calculation of area divided by π multiplied by the square of one-half the cell length. A value close to 1 represents a perfect circle, and a rectangle would be 0.57 or less. Data are presented as average plus or minus a 95% confidence interval. Tissue culture polystyrene is represented as TCPS, and nPTFE stands for nanostructured PTFE.
capillary endothelial cells per square centimeter after 1 day.51 The number of cells observed on nPTFE appears to be in good agreement with the number of cells found on PEG-modified surfaces. Cell shape is important in determining the viability of a cell on a surface, as “rounded” cells may undergo apoptosis.49 This study examined cell spreading on nPTFE in two ways: first by monitoring its area of spreading and second by noting the shape of the cell and how circular it is. For both endothelial cells and macrophages, the area was less on nPTFE than on the tissue culture polystyrene. In addition, the cells were significantly more circular on nPTFE than on the TCPS. One may conclude that, because cells were spreading less on nPTFE than on TCPS, cells may die through lysis or apoptosis. Figure 4 shows that indeed there was a significantly greater amount of dead cells compared (45) KottkeMarchant, K.; Veenstra, A. A.; Marchant, R. E. Human endothelial cell growth and coagulant function varies with respect to interfacial properties of polymeric substrates. J. Biomed. Mater. Res. 1996, 30 (2), 209-220. (46) Pratt, K. J.; Williams, S. K.; Jarrell, B. E. Enhanced Adherence of Human Adult Endothelial-Cells to Plasma Discharge Modified Polyethylene Terephthalate. J. Biomed. Mater. Res. 1989, 23 (10), 1131-1147.
752 Langmuir, Vol. 23, No. 2, 2007
Figure 4. Live/dead assay results for macrophages on nPTFE and TCPS. The percentage of dead cells is presented here with 95% confidence intervals. Tissue culture polystyrene is represented as TCPS, and nPTFE stands for nanostructured poly(tetrafluoroethylene).
to live cells on nPTFE, with a viability (defined as the ratio of live to dead cells) of 11.3% on nPTFE. The shape and viability studies demonstrated that cells attached to the surface of nPTFE were fewer, less spread, and more apt to be dead than on tissue culture polystyrene. Inflammation Cytokines. The expression of cytokines related to inflammatory response for macrophage cultures on an nPTFE surface was examined for two reasons. First, the immune system can become activated when there is a high amount of cell death on the surface of a biomaterial.54 As seen in Figure 4, there was a significant amount of cell death among the cells cultured on nPTFE, which in and of itself may induce an immune response. The second reason the immune response was examined was that the in vivo foreign body response is generally characterized as a chronic inflammation, with many cells at the tissue-material interface being host inflammatory cells.2 To measure the inflammatory cytokines expressed by macrophages cultured on nPTFE, a Luminex cytokine panel was performed. A description of the cytokine panel can be seen in Table 1. Cytokines released by macrophages may either stimulate (e.g., IL-1, IL-12, MCP-1, or TNF-R) or inhibit (e.g., IL-10 and IL-13) the inflammatory response. Tissue culture polystyrene (TCPS) served as a negative control surface, while macrophages cultured on TCPS and stimulated by lipopolysaccharide (LPS) served as a positive control for an inflammatory response. Cytokine expression by immune cells on the surface of implants has previously been studied, including PTFE.55-57 Of particular interest in the cytokine plan measured here are IL-1, TNF-R, (47) Ertel, S. I.; Ratner, B. D.; Horbett, T. A. Radiofrequency Plasma Deposition of Oxygen-Containing Films on Polystyrene and Poly(Ethylene-Terephthalate) Substrates Improves Endothelial-Cell Growth. J. Biomed. Mater. Res. 1990, 24 (12), 1637-1659. (48) Jenney, C.; Anderson, J. Effects of surface-couples polyethylene oxide on human macrophage adhesion and foreign body giant cell formation in vitro. J. Biomed. Mater. Res. 1999, 44 (2), 206-216. (49) Faucheux, N.; Schweiss, R.; Lutzow, K.; Werner, C.; Groth, T. Selfassembled monolayers with different terminating groups as model substrates for cell adhesion studies. Biomaterials 2004, 25 (14), 2721-2730. (50) Drumheller, P. D.; Hubbell, J. A., Densely Cross-Linked Polymer Networks of Poly(Ethylene Glycol) in Trimethylolpropane Triacrylate for Cell-AdhesionResistant Surfaces. J. Biomed. Mater. Res. 1995, 29 (2), 207-215. (51) Ostuni, E.; Chapman, R. G.; Liang, M. N.; Meluleni, G.; Pier, G.; Ingber, D. E.; Whitesides, G. M. Self-assembled monolayers that resist the adsorption of proteins and the adhesion of bacterial and mammalian cells. Langmuir 2001, 17 (20), 6336-6343. (52) Lan, S.; Veiseh, M.; Zhang, M. Surface modification of silicon and goldpatterned silicon surfaces for improved biocompatibility and cell patterning selectivity. Biosens. Bioelectron. 2005, 20 (9), 1697-708. (53) Zhang, M. Q.; Desai, T.; Ferrari, M. Proteins and cells on PEG immobilized silicon surfaces. Biomaterials 1998, 19 (10), 953-960. (54) Matzinger, P., The danger model: a renewed sense of self. Science 2002, 296 (5566), 301-5. (55) Lu, H. K.; Ko, M. T.; Wu, M. F. Comparison of Th1/Th2 cytokine profiles of initial wound healing of rats induced by PDCM and e-PTFE. J. Biomed. Mater. Res., Part B 2004, 68 (1), 75-80.
Ainslie et al.
RANTES, MCP-1, and IL-10. In the case of IL-1, elevated levels of IL-1 have been found to circulate in hosts receiving implanted materials,57-60 indicating a chronic inflammatory response against the implant. Expression levels of IL-1β also increased as compared to TCPS and the positive LPS control at the 24 h time point; however, this elevation subsided by 48 h. From this, one can conclude that the nPTFE did not create a chronic up-regulation of IL-1β in macrophages (Table 3). In addition, IL-1R was significantly less on nPTFE and TCPS surfaces compared to LPS at both time points. Tumor necrosis factor R (TNF-R) is another cytokine important to the host response to biomaterials.61-68 For example, Martinesi et al. showed that Ti-6Al-4V, a common implant alloy, caused a substantial secretion of TNF-R in vitro which will result in elevated chronic inflammation at the implant site.35,69 On nPTFE, TNF-R expression by macrophages cultured for 24 and 48 h was undetectable, as was TNF-R expression on TCPS. The positive inflammation control (macrophages stimulated with LPS) resulted in 1000 pg/mL of TNF-R in 24 and 48 h. On the basis of TNF-R expression, nPTFE appeared not to significantly stimulate an inflammatory response. RANTES and MCP-1 are both chemokines, a group of molecules that are secreted by macrophages to recruit macrophages and other immune cells to the site of inflammation. As seen in Table 3, RANTES and MCP-1 expression was very low for macrophages cultured on nPTFE when compared to LPSstimulated macrophages (positive inflammation control). Thus, because of this low chemokine expression, one may anticipate that macrophage and other inflammatory cell recruitment would be low to the site of nPTFE implantation. (56) Wikesjo, U. M.; Xiropaidis, A. V.; Thomson, R. C.; Cook, A. D.; Selvig, K. A.; Hardwick, W. R. Periodontal repair in dogs: space-providing ePTFE devices increase rhBMP-2/ACS-induced bone formation. J. Clin. Periodontol. 2003, 30 (8), 715-25. (57) Schachtrupp, A.; Klinge, U.; Junge, K.; Rosch, R.; Bhardwaj, R. S.; Schumpelick, V. Individual inflammatory response of human blood monocytes to mesh biomaterials. Br. J. Surg. 2003, 90 (1), 114-20. (58) Ojo-Amaize, E. A.; Lawless, O. J.; Peter, J. B. Elevated concentrations of interleukin-1 beta and interleukin-1 receptor antagonist in plasma of women with silicone breast implants. Clin. Diagn. Lab. Immunol. 1996, 3 (3), 257-9. (59) Suska, F.; Gretzer, C.; Esposito, M.; Emanuelsson, L.; Wennerberg, A.; Tengvall, P.; Thomsen, P. In vivo cytokine secretion and NF-kappaB activation around titanium and copper implants. Biomaterials 2005, 26 (5), 519-27. (60) Gollwitzer, H.; Thomas, P.; Diehl, P.; Steinhauser, E.; Summer, B.; Barnstorf, S.; Gerdesmeyer, L.; Mittelmeier, W.; Stemberger, A. Biomechanical and allergological characteristics of a biodegradable poly(D,L-lactic acid) coating for orthopaedic implants. J. Orthop. Res. 2005, 23 (4), 802-9. (61) Epstein, N. J.; Bragg, W. E.; Ma, T.; Spanogle, J.; Smith, R. L.; Goodman, S. B. UHMWPE wear debris upregulates mononuclear cell proinflammatory gene expression in a novel murine model of intramedullary particle disease. Acta Orthop. 2005, 76 (3), 412-20. (62) Suska, F.; Gretzer, C.; Esposito, M.; Tengvall, P.; Thomsen, P. Monocyte viability on titanium and copper coated titanium. Biomaterials 2005, 26 (30), 5942-50. (63) Bailey, L. O.; Lippiatt, S.; Biancanello, F. S.; Ridder, S. D.; Washburn, N. R. The quantification of cellular viability and inflammatory response to stainless steel alloys. Biomaterials 2005, 26 (26), 5296-302. (64) Hwang, J. J.; Jelacic, S.; Samuel, N. T.; Maier, R. V.; Campbell, C. T.; Castner, D. G.; Hoffman, A. S.; Stayton, P. S. Monocyte activation on polyelectrolyte multilayers. J. Biomater. Sci., Polym. Ed. 2005, 16 (2), 237-51. (65) Refai, A. K.; Textor, M.; Brunette, D. M.; Waterfield, J. D. Effect of titanium surface topography on macrophage activation and secretion of proinflammatory cytokines and chemokines. J. Biomed. Mater. Res., Part A 2004, 70 (2), 194-205. (66) Steinberg, B. M.; Grossi, E. A.; Schwartz, D. S.; McLoughlin, D. E.; Aguinaga, M.; Bizekis, C.; Greenwald, J.; Flisser, A.; Spencer, F.,C.; Galloway, A. C.; Colvin, S. B. Heparin bonding of bypass circuits reduces cytokine release during cardiopulmonary bypass. Ann. Thorac. Surg. 1995, 60 (3), 525-9. (67) Kim, B.; Peppas, N. Poly(ethylene glycol)-containing hydrogels for oral protein delivery applications. Biomed. MicrodeVices 2003, 5 (4), 333-341. (68) Schulz, C. M.; Pritisanac, A.; Schutz, A.; Kilger, E.; Platzer, H.; Reichart, B.; Wildhirt, S. M. Effects of phospholipid-coated extracorporeal circuits on clinical outcome parameters and systemic inflammatory response in coronary artery bypass graft patients. Heart Surg. Forum 2002, 6 (1), 47-52. (69) Martinesi, M.; Bruni, S.; Stio, M.; Treves, C.; Borgioli, F. In vitro interaction between surface-treated Ti-6Al-4V titanium alloy and human peripheral blood mononuclear cells. J. Biomed. Mater. Res., Part A 2005, 74 (2), 197-207.
Cell Adhesion to Nanofibrous PTFE
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Table 3. Inflammation Panel as Measured with Luminex of 15 Cytokinesa Part A: After 24 Hours GM-CSF IFN-γ IL-10 IL-12p40 IL-12p70 IL-13 IL-17 IL-1R IL-1β IL-2 IL-3 IL-4 IL-5 IL-6 KC MCP-1 RANTES TNFR
nPTFE
TCPS
LPS
DMEM
202 ( 43 69 ( 30 104 ( 14
