Acta Biomaterialia 10 (2014) 2423–2433
Contents lists available at ScienceDirect
Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat
Schwann cell response on polypyrrole substrates upon electrical stimulation Leandro Forciniti b, Jose Ybarra III a, Muhammad H. Zaman c, Christine E. Schmidt d,⇑ a
Department of Biomedical Engineering, The University of Texas at Austin, 1 University Station, MC C0800, Austin, TX 78712, USA Department of Chemical Engineering, The University of Texas at Austin, 1 University Station, MC C0400, Austin, TX 78712, USA c Department of Biomedical Engineering, Boston University, 38 Cummington Street, Boston, MA 02215, USA d J. Crayton Pruitt Family Department of Biomedical Engineering, The University of Florida, 1275 Center Drive, Gainesville, FL 32611, USA b
a r t i c l e
i n f o
Article history: Received 29 October 2013 Received in revised form 21 December 2013 Accepted 28 January 2014 Available online 8 February 2014 Keywords: Schwann cell migration Polypyrrole Protein adsorption Electrically mediated cell migration
a b s t r a c t Current injury models suggest that Schwann cell (SC) migration and guidance are necessary for successful regeneration and synaptic reconnection after peripheral nerve injury. The ability of conducting polymers such as polypyrrole (PPy) to exhibit chemical, contact and electrical stimuli for cells has led to much interest in their use for neural conduits. Despite this interest, there has been very little research on the effect that electrical stimulation (ES) using PPy has on SC behavior. Here we investigate the mechanism by which SCs interact with PPy in the presence of an electric field. Additionally, we explored the effect that the adsorption of different serum proteins on PPy upon the application of an electric field has on SC migration. The results indicate an increase in average displacement of the SC with ES, resulting in a net anodic migration. Moreover, indirect effects of protein adsorption due to the oxidation of the film upon the application of ES were shown to have a larger effect on migration speed than on migration directionality. These results suggest that SC migration speed is governed by an integrin- or receptor-mediated mechanism, whereas SC migration directionality is governed by electrically mediated phenomena. These data will prove invaluable in optimizing conducting polymers for their different biomedical applications such as nerve repair. Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Currently it is believed that Schwann cell (SC) migration precedes and enhances axonal repair in the peripheral nervous system [1,2]. Migration from both the distal and proximal ends of injury sites have been observed in vivo [2]. Furthermore these migratory cells have been shown to guide axon reinnervation [2], control synaptic formation [3] and induce faster axon regeneration [1]. This has led to interest in understanding how different external cues presented by biomaterials impact SC migration. Soluble chemical factors affect SC migration at several different levels. Numerous growth factors are known to mitigate SC migration. These include growth factors that promote migration such as nerve growth factor (NGF) [4], glial growth factor II [3,5], insulinlike growth factor 1 [6], and b neuregulin as well as growth factors that inhibit migration such as brain-derived neurotrophic factor (BDNF) [7]. In addition to growth factors, complex sugars and proteoglycans have been shown to promote SC migration [8].
⇑ Corresponding author. Tel.: +1 352 273 9222. E-mail address: [email protected]fl.edu (C.E. Schmidt). http://dx.doi.org/10.1016/j.actbio.2014.01.030 1742-7061/Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
Contact guidance and force mitigated mechano-transduction also have an important role in SC migration and maturation. Laminin 1, laminin 2 (merosin) and fibronectin (FN) all interact with integrins to promote migration. Specifically, Milner et al. showed that laminin 1 and 2 promote SC migration through b1 integrins, whereas FN promotes migration through a5 integrins [9]. Furthermore, aligned collagen gels [10] and collagen:poly((epsilon)-caprolactone) gels [11] have been shown to promote and orient migration. Mechanistically, this contact guidance is thought to occur through mechano-transduction. Specifically, Chew et al. [11] used microarray analysis to demonstrate that aligned SCs cultured on patterned substrates down-regulate the expression of neurotrophins and neurotrophic receptors while up-regulating the expression of myelin specific gene (P0). Furthermore, Rosner et al. [10] showed that by introducing transforming growth factor b1 into cell culture medium, SCs could more accurately detect the aligned collagen fibrils through the up-regulation of b1 integrins. SCs have multiple means by which they interact with electrical cues. Of these the most basic is their interaction with electrical cues through voltage-activated ion channels. SCs contain several types of voltage-activated ion channels, including sodium channels, two types of calcium channels (HVA Ca2+; LVA Ca2+), four
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types of potassium channels (Kir, KD, KA, Kca) and chloride channels. Ransom et al. [12] showed that chloride channels play an important role in glioma cell migration, and studies by Lascola and Kraig [13] indicated that for glial cells, Cl channels are intrinsically connected to the cell’s cytoskeleton. Furthermore, because calcium ions act as ubiquitous secondary messengers, they may influence SC migration; however, this is controversial as Ca2+ seems to be absent from SCs unless co-cultured with dorsal root ganglions SC migration is affected by several different types of extracellular cues. Therefore, a better understanding of how these cues integrate and interact to dictate SC behavior on scaffolds that contain multiple stimuli is essential in engineering superior biomaterials and scaffolds for future use. Polypyrrole (PPy), a conducting polymer that is easily synthesized both chemically and electrochemically, has been investigated for use in neural probes [14] and as a scaffold to promote axonal elongation for use in nerve guidance channels [15]. Moreover, this biomaterial inherently contains all three types of stimuli (i.e., electrical, physical and chemical) described above. The extent to which these stimuli are present has been well characterized for different synthesis paradigms [15–17]. It was found that PPy doped with poly(styrenesulfonate) (PSS) resulted in the optimal properties for SC culture [18]. Despite the wealth of information available on the characterization of electrically conductive PPy materials, there has been little effort made to link the effect of electrical stimulation (ES) through these electroconductive materials on SC motility. Recent studies show that SC migrations from dorsal root ganglions are affected by ES on PPy:poly(lactic-co-glygolic) acid wetspun hybrid fibers [19]. In addition, another study has shown that ES through a PPy:chitosan blend up-regulates SC secretion of NGF and BDNF [20]. However, both studies used hybrid materials and/ or co-cultures that simultaneously exhibit chemical, contact and cellular stimuli, thus making it difficult to assess the direct effect of ES via PPy on SC behavior. Furthermore, it has been shown that increased adsorption of proteins on PPy surfaces occurs upon the application of ES [21]. However, a parametric study evaluating the effect different electrical fields transmitted through PPy and the indirect effect of protein adsorption on PPy has on SC behavior has yet to be accomplished. As such, we believe that a detailed understanding of SC migration on PPy is warranted.
2. Materials and methods 2.1. Polymer synthesis and substrate assembly PPy was synthesized electrochemically (DVSCE = 0.72 V) on indium tin oxide (ITO)-coated unpolished float glass slides (Delta Technologies, Stillwater, MN) in an aqueous media containing pyrrole monomer (0.1 M, Sigma–Aldrich, St Louis, MO) and PSS (Sigma–Aldrich, St Louis, MO) anions as dopants (0.1 M) using a three-electrode cell (Fig. 1). The accessible surface area of the ITO working electrode was restricted to 12.5 cm2. The passage of charge (135.68 mC) was used to monitor the polymerization reaction to control film thickness (109 nm) and roughness (10 nm). Synthesized films were rinsed with double deionized water and allowed to dry overnight. To serve as electrodes during stimulation, copper tape was placed 25 mm apart on the PPy films. The films were then affixed using polydimethysiloxane (PDMS, Sylgard 184, Dow Corning, Midland, MI) to the bottom of previously cut 10 cm diameter Petri dishes, resulting in a total culture area of 78.5 cm2 (Fig. 2). The PDMS was then allowed to cure for 12 h before sterilization and subsequent cell culture was performed. For sterilization, a 20 min ultraviolet exposure and two 10 min washes with 70% filtered (0.22 lm) ethanol were performed.
Fig. 1. Three-electrode cell. A three-electrode cell was used to electrochemically synthesize PPy:poly(styrene-4-sulfonate) films (black rectangle) on indium tin oxide electrodes. A platinum mesh was used as a counter electrode and a saturated c calomel electrode was used as a reference electrode.
Collagen has been shown to promote adhesion while having little effect on migration (9) and as such PPy films coated with collagen were used as negative controls. To make the negative controls films were rinsed with sterile phosphate buffered saline (PBS) and incubated with 10 lg ml 1 collagen at 37 °C for 2 h. 2.2. SC isolation and culture Cells were isolated from P4 neonatal rat sciatic nerves using a modified Brockes method [21] to obtain 95% cell purity as measured by S-100 immunostaining. SCs were maintained in high glucose Dulbecco’s modified Eagle medium (Gibco DMEM, Life Technologies, Carlsbad, CA) with 10% fetal bovine serum (FBS, Sigma–Aldrich, St Louis, MO), 3 lg ml–1 of bovine pituitary extract (Invitrogen, Carlsbad, CA) and 2 lM forskolin (Sigma–Aldrich, St Louis, MO) on 10 cm diameter tissue culture dishes coated with poly-l-lysine (2 lg cm 2, Fisher Scientific, Pittsburgh, PA). Cells were cultured to 70–80% confluence, then detached using 0.25% trypsin–EDTA (Gibco, Life Technologies, Carlsbad, CA) and seeded at 10,000 cells per PPy substrate in serum–free medium. Cultures grown on PPy were cultured with the addition of 1% penicillin–streptomycin (Sigma–Aldrich, St Louis, MO) to the cell medium. 2.3. Blocking by protein adsorption and stimulation condition PPy films were divided into two main treatment groups: one in which proteins were adsorbed with ES prior to seeding and one in which proteins were adsorbed without stimulation (US) prior to seeding. Within the first treatment group, films were further divided by the voltage at which protein would be adsorbed and cells would be stimulated (i.e. 0.1 V, 0.5 V, and 1.0 V). Films were then adsorbed with protein at a particular voltage for 2 h with one of three proteins of interest (FN (Sigma–Aldrich, St Louis MO), laminin (LN, Sigma–Aldrich, St Louis, MO), NGF (Sigma–Aldrich, St Louis MO)) present in concentrations representing high and low concentration of proteins which have been shown to stimulate SC migration [9]. Additionally, a control film which was adsorbed with collagen was utilized for each voltage. These conditions can be seen in Table 1.
L. Forciniti et al. / Acta Biomaterialia 10 (2014) 2423–2433
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Fig. 2. PPy substrate used for Schwann cell time-lapse experiments. A poly(styrene4-sulfonate)-doped PPy film was affixed using PDMS to the bottom of a 10 cm Petri dish with a 20 mm hole. Copper tape served as working and counter electrodes for ES pre-cell incubation and during cell incubation.
Table 1 Single electrode stimulation conditions. 0V
0.1 V
0.5 V
1V
Fibronectin (lg/mL)
10 100
10 100
10 100
10 100
NGF (ng/mL)
50 100
50 100
50 100
50 100
Laminin (lg/mL)
10 100
10 100
10 100
10 100
0
0
0
0
Control (lg/mL)
The second treatment group was divided into those receiving ES after cells are introduced and those which will remain US to serve as a control group. All films were adsorbed with one protein of interest in either high or low concentration, US for 2 h. Controls to be later stimulated were adsorbed with collagen. Data from various conditions were selected from within and across groups to serve as indicative comparisons to test questions of interest. 2.4. Time-lapse imaging and SC stimulation In all cases, 800 cell cm–2 were allowed to adhere for 30 min to the PPy film in an incubator at 37 °C, 5% CO2, after which, the remaining 9 ml of medium was added to the dish and the entire dish was placed in a time-lapse chamber (37 °C, 5% CO2) and connected to a potentiostat (CH Instruments). Cell adhesion was then monitored with an Olympus IX70 microscope (10 magnification) in conjunction with the SensiCam plug-in from Image J (NIH). Images were taken at 10 min intervals using phase contrast optics for 2 h. Next, cells were subjugated to a 2 h ES (or not, if in a control group which was to receive no stimulation); migration both during stimulation and 16 h post-stimulation was monitored. After the 20 h incubation, cell medium was saved for postanalysis and cells were fixed using 4% paraformaldehyde (Sigma– Aldrich, St Louis, MO) (30 min, 37 °C). Immunohistochemistry for SC specific S-100 as well as DAPI nuclear stain was performed. S-100/DAPI ratios were calculated to verify that culture purity was maintained at above 95. 2.5. Calculation of SC displacement and speed Time-lapse movies were analyzed for average displacement, migration speed and directionality. For each time-lapse movie, individual cells were translated to the origin and the resultant net displacement vector (i.e., average displacement vector) (Fig. 4b) was drawn from the origin. On average 15–25 cells were imaged per experimental trial. The resultant angle between this
vector and the anode was then calculated for each cell track. All cell tracks for each substrate condition were then binned into four equally sized quadrants (i.e., working electrode, counter-electrode, right or left). Instantaneous velocity of a cell was calculated using the distance a cell moves from one frame to the next and the elapsed time. The average of instantaneous velocities for a cell’s track was then calculated, and the average of the distribution of average instantaneous velocities for all cells within a trial was calculated and reported here as cell migration speed for a trial (Fig. 4a). To determine migration directionality, an arrow was drawn on the bottom of the ITO slide to mark the location of the anode. This arrow was imaged prior to the time-lapse recording. 2.6. Protein adsorption assays PPy slides were synthesized and substrate assembled as described above. Protein solutions (i.e., 10 lg ml 1 FN, 10 lg ml 1 LN, 50 ng ml 1 NGF, or 10% FBS growth medium) were prepared. Slides were then placed in an incubation chamber to minimize evaporation. For each solution one slide was used and three 40 ll dots were deposited on the PPy slide. In addition each slide had one Dulbecco’s PBS (DPBS, Life Technologies, Carlsbad, CA) dot as a control. The incubation chamber was placed inside an incubator (37 °C) for 2 h. The subsequent slides were washed three times with DPBS. Samples were then incubated with their corresponding antibodies in 3% goat serum (Sigma–Aldrich, St Louis, MO) overnight at 4 °C. The primary antibodies were for FN monoclonal mouse anti-FN (1:100, Sigma–Aldrich, St Louis, MO), for LN rabbit anti-LN (1:100, Millipore, Billerica, MA) and for NGF rabbit antimouse NGF (1:200, Sigma–Aldrich, St Louis, MO). For the 10% FBS growth medium all three antibodies were used. Following the primary antibody incubation, a secondary antibody incubation was performed for 1 h at room temperature. The secondary antibodies and their dilutions were Alexa 488-conjugated anti-mouse IgG (1:600, Molecular Probes, Carlsbad, CA), Alexa 568-conjugated anti-rabbit IgG (1:300, Molecular Probes, Carlsbad, CA) and Alexa 568-conjugated anti-rabbit IgG (1:200, Molecular Probes, Carlsbad, CA) for FN, LN and NGF, respectively. After the 1 h incubation, three washes with DPBS were performed. Samples were then mounted with 1:1 glycerol:PBS solution, inverted and imaged using an Olympus IX70 fluorescence microscope (20 magnification). For those films that were electrically stimulated, two strips of copper tape were placed 25 mm apart to serve as working and counter-electrodes before affixing the substrate to the bottom of previously cut 10 cm diameter Petri dishes, resulting in a total culture area of 78.5 cm2. Each substrate was placed into an incubator and connected to a potentiostat before protein dots (40 ll) were added. For each protein solution (i.e., LN, FN, NGF, 10% FBS) three dots on one slide were used and incubated for 2 h at 37 °C while an electrical field (0.1 V) was administered. Primary, secondary
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Fig. 3. Representative phase contrast micrographs of time-lapse experiments. (a) Neonatal P5 Schwann cells cultured on PPy:PSS substrates pre-incubated in 10 lg ml 1 FN solutions with ES (0.5 V, 2 h) during time-lapse; the first image is 1 h before ES, and the second image is just after ES. For image analysis images were taken 10 min apart. Hours post-stimulation are indicated by (+ # Hr ES) for each image. Arrow indicates the direction of the current. Scale bars = 250 lm.
Fig. 4. Representative diagram explaining the difference between average displacement and speed. (a) Speed is defined as ensemble average of the instantaneous speed of each cell (i.e., the distance the cells travels divide by the time it took to get there) in the field of view over the entire sampling window after 5.5 h of incubation. (b) The average displacement is defined as the ensemble average of each cell’s root mean square displacement over the entire viewing window.
antibody incubation and imaging were performed as described above. Each sample was imaged using an Olympus IX70 fluorescence microscope. For each dot, nine images were acquired such that the entire dot was spanned. In addition, an image was taken of the PPy with no protein and PPy incubated with DPBS for controls. The exposure for each condition was kept constant at 200 ms. The experiment was repeated three times.
2.7. Sodium dodecyl sulfate (SDS) elution and micro-BCA Pre-adsorbed protein was eluted from the PPy films using 4% SDS (Sigma–Aldrich, St Louis, MO) in DPBS. The aforementioned PPy synthesis and stimulation set-up was used. Cell medium containing 10% FBS was incubated (37 °C) on top of the film for 2 h for the non-stimulated controls. For stimulated films, a voltage of 0.1 V was applied across the films while they were being
L. Forciniti et al. / Acta Biomaterialia 10 (2014) 2423–2433
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incubated. Afterwards, films were washed twice with DPBS with each wash taking 5 min. This was done to remove non-specifically bound proteins. Finally, 1 ml of 4% SDS was incubated at 37 °C for 1 h on top of a 25 mm2 PPy substrate. 150 ll of solution was then used to run the micro-BCA (Thermo Scientific, Rockford, IL) assay according to the manufacturer’s protocol.
2.8. Statistical analysis In all calculations, the general relation of propagation of uncertainty was used. To determine statistical significance, an n of three repeated experiments was used. Standard deviation was reported for all relevant figures. Data comparisons with a p-value
Contents lists available at ScienceDirect
Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat
Schwann cell response on polypyrrole substrates upon electrical stimulation Leandro Forciniti b, Jose Ybarra III a, Muhammad H. Zaman c, Christine E. Schmidt d,⇑ a
Department of Biomedical Engineering, The University of Texas at Austin, 1 University Station, MC C0800, Austin, TX 78712, USA Department of Chemical Engineering, The University of Texas at Austin, 1 University Station, MC C0400, Austin, TX 78712, USA c Department of Biomedical Engineering, Boston University, 38 Cummington Street, Boston, MA 02215, USA d J. Crayton Pruitt Family Department of Biomedical Engineering, The University of Florida, 1275 Center Drive, Gainesville, FL 32611, USA b
a r t i c l e
i n f o
Article history: Received 29 October 2013 Received in revised form 21 December 2013 Accepted 28 January 2014 Available online 8 February 2014 Keywords: Schwann cell migration Polypyrrole Protein adsorption Electrically mediated cell migration
a b s t r a c t Current injury models suggest that Schwann cell (SC) migration and guidance are necessary for successful regeneration and synaptic reconnection after peripheral nerve injury. The ability of conducting polymers such as polypyrrole (PPy) to exhibit chemical, contact and electrical stimuli for cells has led to much interest in their use for neural conduits. Despite this interest, there has been very little research on the effect that electrical stimulation (ES) using PPy has on SC behavior. Here we investigate the mechanism by which SCs interact with PPy in the presence of an electric field. Additionally, we explored the effect that the adsorption of different serum proteins on PPy upon the application of an electric field has on SC migration. The results indicate an increase in average displacement of the SC with ES, resulting in a net anodic migration. Moreover, indirect effects of protein adsorption due to the oxidation of the film upon the application of ES were shown to have a larger effect on migration speed than on migration directionality. These results suggest that SC migration speed is governed by an integrin- or receptor-mediated mechanism, whereas SC migration directionality is governed by electrically mediated phenomena. These data will prove invaluable in optimizing conducting polymers for their different biomedical applications such as nerve repair. Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Currently it is believed that Schwann cell (SC) migration precedes and enhances axonal repair in the peripheral nervous system [1,2]. Migration from both the distal and proximal ends of injury sites have been observed in vivo [2]. Furthermore these migratory cells have been shown to guide axon reinnervation [2], control synaptic formation [3] and induce faster axon regeneration [1]. This has led to interest in understanding how different external cues presented by biomaterials impact SC migration. Soluble chemical factors affect SC migration at several different levels. Numerous growth factors are known to mitigate SC migration. These include growth factors that promote migration such as nerve growth factor (NGF) [4], glial growth factor II [3,5], insulinlike growth factor 1 [6], and b neuregulin as well as growth factors that inhibit migration such as brain-derived neurotrophic factor (BDNF) [7]. In addition to growth factors, complex sugars and proteoglycans have been shown to promote SC migration [8].
⇑ Corresponding author. Tel.: +1 352 273 9222. E-mail address: [email protected]fl.edu (C.E. Schmidt). http://dx.doi.org/10.1016/j.actbio.2014.01.030 1742-7061/Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
Contact guidance and force mitigated mechano-transduction also have an important role in SC migration and maturation. Laminin 1, laminin 2 (merosin) and fibronectin (FN) all interact with integrins to promote migration. Specifically, Milner et al. showed that laminin 1 and 2 promote SC migration through b1 integrins, whereas FN promotes migration through a5 integrins [9]. Furthermore, aligned collagen gels [10] and collagen:poly((epsilon)-caprolactone) gels [11] have been shown to promote and orient migration. Mechanistically, this contact guidance is thought to occur through mechano-transduction. Specifically, Chew et al. [11] used microarray analysis to demonstrate that aligned SCs cultured on patterned substrates down-regulate the expression of neurotrophins and neurotrophic receptors while up-regulating the expression of myelin specific gene (P0). Furthermore, Rosner et al. [10] showed that by introducing transforming growth factor b1 into cell culture medium, SCs could more accurately detect the aligned collagen fibrils through the up-regulation of b1 integrins. SCs have multiple means by which they interact with electrical cues. Of these the most basic is their interaction with electrical cues through voltage-activated ion channels. SCs contain several types of voltage-activated ion channels, including sodium channels, two types of calcium channels (HVA Ca2+; LVA Ca2+), four
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types of potassium channels (Kir, KD, KA, Kca) and chloride channels. Ransom et al. [12] showed that chloride channels play an important role in glioma cell migration, and studies by Lascola and Kraig [13] indicated that for glial cells, Cl channels are intrinsically connected to the cell’s cytoskeleton. Furthermore, because calcium ions act as ubiquitous secondary messengers, they may influence SC migration; however, this is controversial as Ca2+ seems to be absent from SCs unless co-cultured with dorsal root ganglions SC migration is affected by several different types of extracellular cues. Therefore, a better understanding of how these cues integrate and interact to dictate SC behavior on scaffolds that contain multiple stimuli is essential in engineering superior biomaterials and scaffolds for future use. Polypyrrole (PPy), a conducting polymer that is easily synthesized both chemically and electrochemically, has been investigated for use in neural probes [14] and as a scaffold to promote axonal elongation for use in nerve guidance channels [15]. Moreover, this biomaterial inherently contains all three types of stimuli (i.e., electrical, physical and chemical) described above. The extent to which these stimuli are present has been well characterized for different synthesis paradigms [15–17]. It was found that PPy doped with poly(styrenesulfonate) (PSS) resulted in the optimal properties for SC culture [18]. Despite the wealth of information available on the characterization of electrically conductive PPy materials, there has been little effort made to link the effect of electrical stimulation (ES) through these electroconductive materials on SC motility. Recent studies show that SC migrations from dorsal root ganglions are affected by ES on PPy:poly(lactic-co-glygolic) acid wetspun hybrid fibers [19]. In addition, another study has shown that ES through a PPy:chitosan blend up-regulates SC secretion of NGF and BDNF [20]. However, both studies used hybrid materials and/ or co-cultures that simultaneously exhibit chemical, contact and cellular stimuli, thus making it difficult to assess the direct effect of ES via PPy on SC behavior. Furthermore, it has been shown that increased adsorption of proteins on PPy surfaces occurs upon the application of ES [21]. However, a parametric study evaluating the effect different electrical fields transmitted through PPy and the indirect effect of protein adsorption on PPy has on SC behavior has yet to be accomplished. As such, we believe that a detailed understanding of SC migration on PPy is warranted.
2. Materials and methods 2.1. Polymer synthesis and substrate assembly PPy was synthesized electrochemically (DVSCE = 0.72 V) on indium tin oxide (ITO)-coated unpolished float glass slides (Delta Technologies, Stillwater, MN) in an aqueous media containing pyrrole monomer (0.1 M, Sigma–Aldrich, St Louis, MO) and PSS (Sigma–Aldrich, St Louis, MO) anions as dopants (0.1 M) using a three-electrode cell (Fig. 1). The accessible surface area of the ITO working electrode was restricted to 12.5 cm2. The passage of charge (135.68 mC) was used to monitor the polymerization reaction to control film thickness (109 nm) and roughness (10 nm). Synthesized films were rinsed with double deionized water and allowed to dry overnight. To serve as electrodes during stimulation, copper tape was placed 25 mm apart on the PPy films. The films were then affixed using polydimethysiloxane (PDMS, Sylgard 184, Dow Corning, Midland, MI) to the bottom of previously cut 10 cm diameter Petri dishes, resulting in a total culture area of 78.5 cm2 (Fig. 2). The PDMS was then allowed to cure for 12 h before sterilization and subsequent cell culture was performed. For sterilization, a 20 min ultraviolet exposure and two 10 min washes with 70% filtered (0.22 lm) ethanol were performed.
Fig. 1. Three-electrode cell. A three-electrode cell was used to electrochemically synthesize PPy:poly(styrene-4-sulfonate) films (black rectangle) on indium tin oxide electrodes. A platinum mesh was used as a counter electrode and a saturated c calomel electrode was used as a reference electrode.
Collagen has been shown to promote adhesion while having little effect on migration (9) and as such PPy films coated with collagen were used as negative controls. To make the negative controls films were rinsed with sterile phosphate buffered saline (PBS) and incubated with 10 lg ml 1 collagen at 37 °C for 2 h. 2.2. SC isolation and culture Cells were isolated from P4 neonatal rat sciatic nerves using a modified Brockes method [21] to obtain 95% cell purity as measured by S-100 immunostaining. SCs were maintained in high glucose Dulbecco’s modified Eagle medium (Gibco DMEM, Life Technologies, Carlsbad, CA) with 10% fetal bovine serum (FBS, Sigma–Aldrich, St Louis, MO), 3 lg ml–1 of bovine pituitary extract (Invitrogen, Carlsbad, CA) and 2 lM forskolin (Sigma–Aldrich, St Louis, MO) on 10 cm diameter tissue culture dishes coated with poly-l-lysine (2 lg cm 2, Fisher Scientific, Pittsburgh, PA). Cells were cultured to 70–80% confluence, then detached using 0.25% trypsin–EDTA (Gibco, Life Technologies, Carlsbad, CA) and seeded at 10,000 cells per PPy substrate in serum–free medium. Cultures grown on PPy were cultured with the addition of 1% penicillin–streptomycin (Sigma–Aldrich, St Louis, MO) to the cell medium. 2.3. Blocking by protein adsorption and stimulation condition PPy films were divided into two main treatment groups: one in which proteins were adsorbed with ES prior to seeding and one in which proteins were adsorbed without stimulation (US) prior to seeding. Within the first treatment group, films were further divided by the voltage at which protein would be adsorbed and cells would be stimulated (i.e. 0.1 V, 0.5 V, and 1.0 V). Films were then adsorbed with protein at a particular voltage for 2 h with one of three proteins of interest (FN (Sigma–Aldrich, St Louis MO), laminin (LN, Sigma–Aldrich, St Louis, MO), NGF (Sigma–Aldrich, St Louis MO)) present in concentrations representing high and low concentration of proteins which have been shown to stimulate SC migration [9]. Additionally, a control film which was adsorbed with collagen was utilized for each voltage. These conditions can be seen in Table 1.
L. Forciniti et al. / Acta Biomaterialia 10 (2014) 2423–2433
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Fig. 2. PPy substrate used for Schwann cell time-lapse experiments. A poly(styrene4-sulfonate)-doped PPy film was affixed using PDMS to the bottom of a 10 cm Petri dish with a 20 mm hole. Copper tape served as working and counter electrodes for ES pre-cell incubation and during cell incubation.
Table 1 Single electrode stimulation conditions. 0V
0.1 V
0.5 V
1V
Fibronectin (lg/mL)
10 100
10 100
10 100
10 100
NGF (ng/mL)
50 100
50 100
50 100
50 100
Laminin (lg/mL)
10 100
10 100
10 100
10 100
0
0
0
0
Control (lg/mL)
The second treatment group was divided into those receiving ES after cells are introduced and those which will remain US to serve as a control group. All films were adsorbed with one protein of interest in either high or low concentration, US for 2 h. Controls to be later stimulated were adsorbed with collagen. Data from various conditions were selected from within and across groups to serve as indicative comparisons to test questions of interest. 2.4. Time-lapse imaging and SC stimulation In all cases, 800 cell cm–2 were allowed to adhere for 30 min to the PPy film in an incubator at 37 °C, 5% CO2, after which, the remaining 9 ml of medium was added to the dish and the entire dish was placed in a time-lapse chamber (37 °C, 5% CO2) and connected to a potentiostat (CH Instruments). Cell adhesion was then monitored with an Olympus IX70 microscope (10 magnification) in conjunction with the SensiCam plug-in from Image J (NIH). Images were taken at 10 min intervals using phase contrast optics for 2 h. Next, cells were subjugated to a 2 h ES (or not, if in a control group which was to receive no stimulation); migration both during stimulation and 16 h post-stimulation was monitored. After the 20 h incubation, cell medium was saved for postanalysis and cells were fixed using 4% paraformaldehyde (Sigma– Aldrich, St Louis, MO) (30 min, 37 °C). Immunohistochemistry for SC specific S-100 as well as DAPI nuclear stain was performed. S-100/DAPI ratios were calculated to verify that culture purity was maintained at above 95. 2.5. Calculation of SC displacement and speed Time-lapse movies were analyzed for average displacement, migration speed and directionality. For each time-lapse movie, individual cells were translated to the origin and the resultant net displacement vector (i.e., average displacement vector) (Fig. 4b) was drawn from the origin. On average 15–25 cells were imaged per experimental trial. The resultant angle between this
vector and the anode was then calculated for each cell track. All cell tracks for each substrate condition were then binned into four equally sized quadrants (i.e., working electrode, counter-electrode, right or left). Instantaneous velocity of a cell was calculated using the distance a cell moves from one frame to the next and the elapsed time. The average of instantaneous velocities for a cell’s track was then calculated, and the average of the distribution of average instantaneous velocities for all cells within a trial was calculated and reported here as cell migration speed for a trial (Fig. 4a). To determine migration directionality, an arrow was drawn on the bottom of the ITO slide to mark the location of the anode. This arrow was imaged prior to the time-lapse recording. 2.6. Protein adsorption assays PPy slides were synthesized and substrate assembled as described above. Protein solutions (i.e., 10 lg ml 1 FN, 10 lg ml 1 LN, 50 ng ml 1 NGF, or 10% FBS growth medium) were prepared. Slides were then placed in an incubation chamber to minimize evaporation. For each solution one slide was used and three 40 ll dots were deposited on the PPy slide. In addition each slide had one Dulbecco’s PBS (DPBS, Life Technologies, Carlsbad, CA) dot as a control. The incubation chamber was placed inside an incubator (37 °C) for 2 h. The subsequent slides were washed three times with DPBS. Samples were then incubated with their corresponding antibodies in 3% goat serum (Sigma–Aldrich, St Louis, MO) overnight at 4 °C. The primary antibodies were for FN monoclonal mouse anti-FN (1:100, Sigma–Aldrich, St Louis, MO), for LN rabbit anti-LN (1:100, Millipore, Billerica, MA) and for NGF rabbit antimouse NGF (1:200, Sigma–Aldrich, St Louis, MO). For the 10% FBS growth medium all three antibodies were used. Following the primary antibody incubation, a secondary antibody incubation was performed for 1 h at room temperature. The secondary antibodies and their dilutions were Alexa 488-conjugated anti-mouse IgG (1:600, Molecular Probes, Carlsbad, CA), Alexa 568-conjugated anti-rabbit IgG (1:300, Molecular Probes, Carlsbad, CA) and Alexa 568-conjugated anti-rabbit IgG (1:200, Molecular Probes, Carlsbad, CA) for FN, LN and NGF, respectively. After the 1 h incubation, three washes with DPBS were performed. Samples were then mounted with 1:1 glycerol:PBS solution, inverted and imaged using an Olympus IX70 fluorescence microscope (20 magnification). For those films that were electrically stimulated, two strips of copper tape were placed 25 mm apart to serve as working and counter-electrodes before affixing the substrate to the bottom of previously cut 10 cm diameter Petri dishes, resulting in a total culture area of 78.5 cm2. Each substrate was placed into an incubator and connected to a potentiostat before protein dots (40 ll) were added. For each protein solution (i.e., LN, FN, NGF, 10% FBS) three dots on one slide were used and incubated for 2 h at 37 °C while an electrical field (0.1 V) was administered. Primary, secondary
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Fig. 3. Representative phase contrast micrographs of time-lapse experiments. (a) Neonatal P5 Schwann cells cultured on PPy:PSS substrates pre-incubated in 10 lg ml 1 FN solutions with ES (0.5 V, 2 h) during time-lapse; the first image is 1 h before ES, and the second image is just after ES. For image analysis images were taken 10 min apart. Hours post-stimulation are indicated by (+ # Hr ES) for each image. Arrow indicates the direction of the current. Scale bars = 250 lm.
Fig. 4. Representative diagram explaining the difference between average displacement and speed. (a) Speed is defined as ensemble average of the instantaneous speed of each cell (i.e., the distance the cells travels divide by the time it took to get there) in the field of view over the entire sampling window after 5.5 h of incubation. (b) The average displacement is defined as the ensemble average of each cell’s root mean square displacement over the entire viewing window.
antibody incubation and imaging were performed as described above. Each sample was imaged using an Olympus IX70 fluorescence microscope. For each dot, nine images were acquired such that the entire dot was spanned. In addition, an image was taken of the PPy with no protein and PPy incubated with DPBS for controls. The exposure for each condition was kept constant at 200 ms. The experiment was repeated three times.
2.7. Sodium dodecyl sulfate (SDS) elution and micro-BCA Pre-adsorbed protein was eluted from the PPy films using 4% SDS (Sigma–Aldrich, St Louis, MO) in DPBS. The aforementioned PPy synthesis and stimulation set-up was used. Cell medium containing 10% FBS was incubated (37 °C) on top of the film for 2 h for the non-stimulated controls. For stimulated films, a voltage of 0.1 V was applied across the films while they were being
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incubated. Afterwards, films were washed twice with DPBS with each wash taking 5 min. This was done to remove non-specifically bound proteins. Finally, 1 ml of 4% SDS was incubated at 37 °C for 1 h on top of a 25 mm2 PPy substrate. 150 ll of solution was then used to run the micro-BCA (Thermo Scientific, Rockford, IL) assay according to the manufacturer’s protocol.
2.8. Statistical analysis In all calculations, the general relation of propagation of uncertainty was used. To determine statistical significance, an n of three repeated experiments was used. Standard deviation was reported for all relevant figures. Data comparisons with a p-value
