JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE RESEARCH ARTICLE J Tissue Eng Regen Med 2015; 9: 151–161. Published online 22 October 2012 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/term.1623
PC12 neuron-like cell response to electrospun poly ( 3-hydroxybutyrate) substrates Giada Graziana Genchi1,2*, Gianni Ciofani2**, Alessandro Polini3, Ioannis Liakos4, Donata Iandolo3, Athanassia Athanassiou4, Dario Pisignano3,4,5, Virgilio Mattoli2 and Arianna Menciassi1,2 1
Scuola Superiore Sant’Anna, The BioRobotics Institute, Viale Rinaldo Piaggio 34, 56025 Pontedera, (Pisa), Italy Istituto Italiano di Tecnologia, Center for MicroBioRobotics @SSSA, Viale Rinaldo Piaggio 34, 56025 Pontedera, (Pisa), Italy 3 NNL, National Nanotechnology Laboratory of CNR-Nanoscienze, Via Arnesano 16, 73100 Lecce, Italy 4 Istituto Italiano di Tecnologia, Center for Biomolecular Nanotechnologies, Via Barsanti, 73010 Lecce, Italy 5 Università del Salento, Dipartimento di Matematica e Fisica “Ennio De Giorgi”, Via Arnesano, 73100 Lecce, Italy 2
Abstract In the last decade, the importance of topographic properties of extracellular environments has been shown to be essential to addressing cell response, especially when replacing damaged tissues with functional constructs obtained in vitro. In the current study, densely packed sub-micron poly (3-hydroxybutyrate) (PHB) fibres were electrospun with random and parallel orientations. PC12 pheochromocytoma cells that mimic central dopaminergic neurons and represent a model for neuronal differentiation were cultured on collagen-coated fibres to evaluate cell response dependence on substrate topography. Cell adhesion, viability and proliferation, as well as dopamine production were evaluated after three days since seeding. Cell differentiation was examined in terms of neurite number, orientation and length 6 days after administration of nerve growth factor (NGF). Results showed that proliferating PC12 cells secreted a higher quantity of dopamine on fibres with respect to control cultures and as a result, a possible use of PHB fibres was considered for cell transplantation in the central nervous system when local production of dopamine is impaired. Differentiated PC12 cells were characterized by highly aligned and longer neurites on parallel PHB fibres with respect to random fibres, thereby demonstrating the suitability of parallel PHB fibres for further studies in peripheral nervous system regeneration. Copyright © 2012 John Wiley & Sons, Ltd. Received 21 March 2012; Revised 9 July 2012; Accepted 25 August 2012
Supporting information may be found in the online version of this article. Keywords electrospinning; sub-micron fibres; poly(3-hydroxybutyrate); PC12 cells; dopamine; neurite outgrowth
1. Introduction Structural and functional alterations of nervous tissue usually consist of irreversible degenerative processes that
*Correspondence to: Giada Graziana Genchi, M.Sc., Ph.D. candidate. Scuola Superiore Sant’Anna, The BioRobotics Institute, Viale Rinaldo Piaggio 34, 56025 Pontedera (Pisa), Italy. Tel.: +39050883035; Lab.: +39050883468; Fax: +39050883497. E-mail:
[email protected] **Co-correspondence to: Gianni Ciofani, Ph.D., Istituto Italiano di Tecnologia, Center for MicroBioRobotics @SSSA, Viale Rinaldo Piaggio 34, 56025 Pontedera (Pisa), Italy. Tel.: +39050883019; Lab.: +39050883468. Fax: +39050883497. E-mail: gianni.
[email protected] Copyright © 2012 John Wiley & Sons, Ltd.
result in permanent disability. In individuals affected by Parkinson’s disease, dopamine-producing neurons of substrantia nigra in the central nervous system accumulate large spherical cytoplasmic inclusions termed “Lewy bodies” and exhibit progressive atrophy followed by cell death. Dead cells are not substituted and the decreased amount of dopamine produced by the substrantia nigra determines muscle rigidity, tremor and bradykinesia, as well as other increasing motor and cognitive deficits (Dunnett and Bjorklund, 1999). In subjects affected by severe trauma to the peripheral nervous system, nerve transection activates the coalescence of fibrin molecules and infiltration of glial cells between the nerve stumps with the objective of physically reconnecting the stumps and thereby providing a suitable environment for neurite
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extension. Nonetheless, when stumps are too distant or when immunological reactions occur, the fibrin bridge fails to form or improperly forms, resulting in a partial or complete loss of sensory or motor function (Mukhatyar et al., 2009). Due to the limited self-regenerating ability of nervous tissue, different strategies have been developed with the purpose of recovering structure and function of damaged nerves (Tresco, 2000). Among these strategies, transplantation of dopamine-producing cells for improving parkinsonian symptoms and application of polymeric conduits to support neurite outgrowth among transected nerves have shown highly promising results (Cao et al., 2009; Yoshida et al., 2003). Several studies have demonstrated that the extracellular environment significantly affects cell behaviour both in vivo and in vitro (Ciofani et al., 2007; Ciofani et al., 2009). In particular, topography has been shown to regulate events such as cell migration, proliferation and differentiation (Chang et al., 2011; Von Der Mark et al., 2010). As a result, the possibility of addressing cell behaviour through environmental topographical stimuli represents an important opportunity for recovering the structural and functional integrity of nervous tissue (Hoffman-Kim et al., 2010). Among the different techniques used to produce substrates able to provide cells with different topographical cues in vitro, electrospinning is one of the simplest, least expensive and most versatile (Han and Cheung, 2011; Polini et al., 2011; Sill and von Recum, 2008). Several synthetic polymers have been successfully electrospun with promising results for nervous system regeneration such as polypyrrole/poly(styrene-b-isobutylene-b-styrene) (Liu et al., 2010); poly(3-caprolactone)/gelatin (Alvarex-Perez et al., 2010; Ghasemi-Mobarakeh et al., 2008); poly-L-lactic acid (Koh et al., 2008; Wang et al., 2010), poly(vinylidene fluoride-trifluoroethylene) (Lee et al., 2011); and poly (acrylonitrile-co-methylacrylate) (Kim et al., 2008). Each of these polymers needs to be chemically produced with possible persistent contaminations by chemical catalysts, initiators and solvents that could reduce biological acceptance. Conversely, natural polymers can offer physiologically relevant platforms that cells can easily recognize, populate and ultimately remodel (Sell et al., 2010). Among these polymers, poly(3-hydroxybutyrate) (PHB) is a natural polyester produced in large quantities by many organisms as a carbon storage that can be easily processed and shows excellent biocompatibility and biodegradability properties (Cheng and Wu, 2005; Misra et al., 2006). To date, PHB has been processed according to the following methodologies: melt pressing and solvent evaporation, to produce unidirectional-oriented fibrous sheets suitable for pericardial suture (Malm et al., 1992) and peripheral nerve repair (Ljungberg et al., 1999; Novikova et al., 2008) and solution casting to achieve nanofiber films suitable for neural stem cell attachment, synaptic outgrowth and synaptogenesis (Xu et al., 2010). Other techniques included high-speed melt spinning and spin-drawing, to obtain multifilaments (Schmack et al., 2000) capable of promoting cell migration, proliferation, differentiation and vascularization in bone tissue engineering (Rentsch Copyright © 2012 John Wiley & Sons, Ltd.
et al., 2009) and phase separation to obtain 3D interconnected fibrous networks, suitable for supporting human keratinocyte adhesion and proliferation (Li et al., 2008). PHB has also been successfully electrospun in mats characterized by either randomly oriented fibres with average diameters ranging from 1.6 to 8.8 mm or aligned fibres with average diameters of 2.3 to 4.0 mm (Sombatmankhong et al., 2006). Recent studies have shown that substrate topographic features in the sub-micron scale affect neuronal cell behaviour (Wang et al., 2010; Foley et al., 2005). To the authors’ knowledge, the achievement of PHB sub-micron fibres through electrospinning and their application to neural tissue engineering have to date not been demonstrated. For this reason, PHB fibres were electrospun with diameters in the range of 200-400 nm in the current study. Sub-micron fibres were electrospun with either a random or a parallel orientation to study the effect of the different topography on neuron-like PC12 cell behaviour, thereby obtaining preliminary evidence of the suitability of the developed scaffolds for neural tissue engineering. In particular, we hypothesized that the submicron fibres could enhance some specific cell activities such as dopamine secretion and neurite emission, thus providing useful scaffolds for cell transplantation in the central nervous system and for the support of neurite outgrowth in the peripheral nervous system.Cell viability and extent of proliferation were therefore studied along with quantification of the neurotransmitter dopamine produced by the cells. Neurite emission and orientation were also investigated following administration of nerve growth factor (NGF).
2. Materials and methods 2.1. PHB fibre electrospinning and collagen coating Sub-micron fibres were prepared by spinning a 2% w/w polyhydroxybutyrate (PHB; Sigma-Aldrich S.r.l., Milan, Italy) solution in hexafluoroisopropanol (HFIP; Carlo Erba Reagenti S.p.a., Arese, Milan, Italy). The polymeric solution, which was previously stirred at room temperature for 2 h, was loaded in a plastic syringe with a 27-gauge stainless steel needle and then spun by applying a 4.5 kV voltage through an EL60R0.6-22 high voltage power supply (Glassman Europe Limited UK, Hampshire, UK). Injection flow rate and needle-collector path were respectively set at 0.3 ml/h and 15 cm during electrospinning (1-h deposition time). A grounded disk with a diameter of 8 cm and thickness of 1 cm was rotated at 4,000 rpm through a rotating collector (RT collector, Linari Engineering Srl, Pisa, Italy) to collect parallel fibres. Environmental temperature was ~ 23 C and relative humidity in room air was ~ 40%. Random fibres were produced using a static grounded 10 x 10-cm2 collector and by maintaining constant all other process parameters. PHB samples were left in a vacuum dessicator for one week at room temperature to remove any residual of HFIP and then exposed to a mild, inductively J Tissue Eng Regen Med 2015; 9: 151–161. DOI: 10.1002/term
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coupled air plasma for 60 sec (200 mTorr, 6.8 W) in a PDC32 G Harrick reactor (Harrick Scientific Inc., NY, NY, USA) to increase surface hydrophilicity. PHB samples were then incubated with a 100-mg/ml collagen (Sigma-Aldrich) solution for 90 min to allow collagen adsorption and finally rinsed with phosphate buffer saline (PBS) 1x.
2.2. Surface characterization: SEM imaging Surface features were first investigated through scanning electron microscopy (SEM) imaging. Both native PHB and collagen-coated samples underwent Au-sputtering in a Quorum sputter-coater (Quorum Technologies, Rome, Italy) before imaging with an EVO MA10 Zeiss microscope (Italy Carl Zeiss S.p.A., Arese, Italy). SEM images were also used to quantify fibre orientation with ImageJ software (http://rsbweb.nih.gov/ij/) and fast Fourier transform (FFT) (Ayres et al., 2006). All FFT data were normalized to a baseline value of zero, thereby allowing direct comparison of the results between randomly oriented and parallel oriented fibres.
2.3. Surface characterization: WCA measurements Surface wettability of both native- and collagen-coated samples was assessed by water contact angle (WCA) measurements. Static measurements were performed using an A-100 ramé-hart manual goniometer (ramé-hart Instrument co., Succasunna, NJ, USA) and 2-ml water droplets. Contact angles were measured on both sides of five drops of double distilled water for each sample and then the average value was calculated. On parallel fibres, measurements were done in both parallel (θ//) and perpendicular (θ⊥) directions due to the anisotropic wetting property of the surface (Meng et al., 2011). We focused on θ⊥ values as discussed in the following without specification, whereas θ// measurements are reported in the Supporting Information section.
2.4. Surface characterization: XPS analysis Surface chemistry of both native and collagen-coated samples was quantitatively characterized through X-ray photoelectron spectroscopy (XPS). Measurements were made using a SPECS™ Lab2 electron spectrometer equipped with a monochromatic X-ray source set at 1253 eV with a Has (hemispherical energy analyzer) 3500 Phoibos analyzer (SPECS Surface Nano Analysis GmbH, Berlin, Germany). The applied voltage of the Mg Ka X-ray source was 7.5 kV and applied current was 9.5 mA. The pressure in the analysis chamber was ~ 210-9 mbar. Small area lens mode was used for both wide and narrow scans: for the wide scans, the energy pass was set at 90 eV energy step at 0.5 eV and scan number at 1. The narrow highresolution scan is presented in the Supporting Information Copyright © 2012 John Wiley & Sons, Ltd.
section. All collected spectra were analysed with CasaXPS software (http://www.casaxps.com/).
2.5. Cell cultures Cell adhesion, proliferation and differentiation studies were performed with PC12 cells (CRL-1721, American Tissue Culture Collection) cultured over random PHB, parallel PHB, and collagen-coated cell culture polystyrene (PS) as a control substrate. Derived from a transplantable rat pheochromocytoma, PC12 cells mimic many features of central dopaminergic neurons, including dopamine production. Moreover, they represent a model for neuronal differentiation since they can reversibly respond to the administration of the nerve growth factor (NGF) and express sympathetic neuronal phenotype (Greene and Tischler, 1976). In the current study, PC12 were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% horse serum (HS), 5% fetal bovine serum (FBS), 100 IU/ml penicillin, 100 mg/ml streptomycin and 2 mM L-glutamine. Differentiation was induced by administration of a lowserum (1% FBS) medium supplemented with 50 ng/ml NGF (Sigma- Aldrich). PC12 cells were maintained at 37 C in a 5% CO2 saturated humidity atmosphere.
2.6. Proliferating cultures: Viability and proliferation assessment PC12 cells were seeded at a density of 5000 cells/cm2 on each sample and viability and proliferation were assessed after 72 h of culture. Cell viability was investigated by evaluating membrane integrity and esterase activity with the LIVE/DEADW Viability/Cytotoxicity Kit (Molecular ProbesW; Invitrogen™, Fil. Life Technologies Europe BV, Monza, Italy) following manufacturer instructions. The kit contains calcein AM (4 mM diluted in anhydrous dimethylsulfoxide; DMSO) and ethidium homodimer-1 (EthD-1, 2 mM in DMSO/H2O 1:4 v/v), and it stains live cells in green and dead cells in red. Live cells are characterized by a ubiquitous intracellular esterase activity that determines the enzymatic conversion of the nonfluorescent cell-permeant agent calcein AM to an intensely fluorescent molecule, calcein. Calcein is well retained within live cells, producing an intense uniform green fluorescence. Conversely, EthD-1 enters cells with damaged membranes and undergoes a 40-fold enhancement of fluorescence upon binding to nucleic acids, thereby producing a bright red fluorescence in dead cells. Cell cultures were incubated with a 2-mM calcein AM/4 mM EthD-1 solution in PBS at 37 C for 10 min, whereas nuclei were counterstained in blue with 5 mg/ml Hoechst 33342 (Invitrogen). Cells were then observed with a Nikon TE2000U inverted fluorescence microscope equipped with a DS-Fi1 USB2 CCD camera and NIS Elements imaging software (all from Nikon; Nikon Instruments S.p.a., Calenzano, Florence, Italy). J Tissue Eng Regen Med 2015; 9: 151–161. DOI: 10.1002/term
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Proliferation was quantitatively evaluated by measuring the double-stranded DNA (ds-DNA) content of the cells. Proliferation assays (n = 3) were performed after rinsing cells with PBS 1x and incubating the cultures with appropriate volumes of double distilled (dd)-H2O. Cell lysates were obtained by two freeze/thaw cycles with each cycle performed as follows: 12 h freezing at -80 C and 15 min thawing at 37 C in a Branson 2510 sonication bath. Ds-DNA content in cell lysates was measured with the PicoGreenW kit (Molecular Probes), following manufacturer instructions. PicoGreen dye binds to ds-DNA and the resulting fluorescence intensity is directly proportional to the ds-DNA concentration in solution and therefore to the cell number. Fluorescence intensity was measured on a Victor™ X3 microplate reader (PerkinElmer, Monza, Milan, Italy) using an excitation wavelength of 485 nm and emission wavelength of 535 nm.
2.7. Proliferating cultures: Dopamine production evaluation Dopamine production was quantified using supernatants collected after 72 h of cell culture and cell lysates produced in the proliferation assays (for normalization to cell number). A solid phase enzyme-linked immunosorbent assay (ELISA) based on the sandwich principle was conducted with a Dopamine ELISA kit (GenWay Biotech Inc., San Diego, CA, USA) following manufacturer instructions. Briefly, 20 ml of extracted samples was incubated in wells coated with a goat anti-rabbit antibody directed towards a dopamine epitope for 2 h at room temperature (RT). Next, an enzyme-conjugated secondary antibody directed towards a different region of the dopamine molecule was added and incubated at RT for 1 h. The enzyme-conjugated antibody was provided with substrate for 40 min at RT and the absorbance of the coloured product resulting from the enzyme catalyzed reaction was measured at a wavelength of 405 nm on the microplate reader. Absorbance was proportional to the amount of antigen and allowed direct dopamine quantification by comparison to a standard curve.
2.8. Differentiating cultures: Immunocytochemistry Cellular differentiation was examined on PC12 cells seeded at a density of 5000 cells/cm2. Differentiation was induced with a low serum NGF-supplemented medium 24 h after seeding. It was then evaluated by fluorescent staining of b3-tubulin, an early marker of neuronal differentiation and by analysis of fluorescence microscopy pictures of cells with at least one neurite with a length equal to cell body diameter (Das et al., 2004). Samples were first rinsed with PBS 1x and then fixed in paraformaldehyde (4% in PBS 1x) for 15 min. After three rinses with PBS 1x (5 min each), samples were incubated with sodium borohydryde (1 mg/ml in PBS 1x) for 10 min Copyright © 2012 John Wiley & Sons, Ltd.
to reduce or suppress auto- and aspecific fluorescence. Cellular membranes were permeabilized with Triton X-100 (0.1% in PBS 1x) for 15 min and aspecific binding sites for antibodies were then saturated with goat serum (10% in PBS 1x) for 1 h. A primary polyclonal antibody (rabbit anti-tubulin IgG; Sigma-Aldrich) was diluted 1:75 in 10% goat serum and subsequently added to the samples. After 30 min of incubation at 37 C, samples were rinsed five times (3 min each) with 10% goat serum and a staining solution composed of a secondary antibody (tetramethylrhodamine goat anti-rabbit IgG; Invitrogen) diluted 1:250 in 10% goat serum, was added. After 30 min of incubation at RT, samples were rinsed with high-salt PBS 1x (0.45 M NaCl) for 1 min to remove weakly bound antibodies. After three rinses in PBS 1x (5 min each), samples were observed with the inverted fluorescence microscope. Neurite orientation, number per cell and length distribution were quantified. Neurite orientation on random fibres was quantitatively assessed with respect to an arbitrary axis. Neurite orientation on parallel fibres was quantitatively assessed by overlapping fluorescence microscopy pictures with phase contrast pictures of the same optical fields and by using the “Angle” tool of ImageJ software. Neurite length was manually measured as illustrated in Supporting Figure 3 on isolated cells using ImageJ by three independent experiments. One experiment was used to illustrate a sample distribution of lengths.
2.9. Differentiating cultures: SEM imaging To investigate cell-substrate interactions, cultures were fixed with a 4% paraformaldehyde solution for 30 min at 4 C and with a further incubation in a 2.5% glutaraldehyde solution for 30 min at 4 C. Samples were then dehydrated by an ethanol gradient (10 min in 0, 25, 50, 75 and 100% ethanol in water), overnight dried and Au-sputtered before SEM observation.
2.10. Statistical analyses Analyses of the data was performed by one-way analysis of variance (ANOVA) followed by Tukey’s post-test to test for significance, which was set at 5%. All experiments were performed in triplicate. Results are presented as mean values standard error of the mean.
3. Results 3.1. PHB sample characterization PHB substrate features were investigated by SEM imaging and wettability measurements before and after surface exposure to air plasma and collagen adsorption. As shown in Figure 1, the native substrates are characterized by 200-400 nm wide fibres randomly oriented over different J Tissue Eng Regen Med 2015; 9: 151–161. DOI: 10.1002/term
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Figure 1. Scanning electron microscopy images of random PHB fibres (a, b) and parallel PHB fibres (d, e). Fast Fourier transform outputs are presented as inserts in (a) and (d), whereas pixel intensities are plotted against the angle of acquisition along a circular line (radius: 50 pixels) for rotated images (700 x 700 pixel) of random (c) and parallel fibres (f). The high peak in pixel intensities arises from fibre parallelism
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Water contact angle (°)
overlapping planes (“re-entrant” geometry) in Figure 1a, and parallel oriented in Figure 1d. Fibres are densely packed, in particular when parallel. Collagen adsorption did not affect fibre morphology, as evident for random fibres in Figure 1b and parallel fibres in Figure 1e. Random fibre geometry occasionally seemed to promote a higher deposition of collagen among crossing fibres (arrows in Fig. 1b) as reported by (Polini et al., 2010). SEM imaging also allowed the quantification of fibre orientation upon fast Fourier transform analysis. As shown in Figure 1c, the normalized intensity values of random fibres did not exceed 40 arbitrary units in the whole range 0–180 , whereas parallel fibres were characterized by a high peak exceeding 90 arbitrary units in the range 60 10 (as shown in Figure 1f), thus quantitatively demonstrating the high degree of alignment of the fibres in this kind of substrates. The water contact angles (WCA) reported in Figure 2 clearly show that air plasma and collagen adsorption increased surface wettability for both random and parallel fibres. Specifically, random fibre WCA decreased from 150 3 (indicative of
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Figure 2. Water contact angle (WCA) measurements for both random and parallel fibres before (left) and after (right) exposure to air plasma and collagen incubation. Wettability was increased for both substrates after collagen adsorption
superhydrophobic behaviour) to 56 4 , whereas parallel fibre WCA decreased from 120 2 to 53 3 . XPS analysis confirmed the presence of a collagen coating. As shown in Figures 3a and 3c, the wide scan spectra of J Tissue Eng Regen Med 2015; 9: 151–161. DOI: 10.1002/term
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Figure 3. Wide scan XPS spectra of native (a) and collagen-coated (b) random fibres and native (c) and collagen-coated (d) parallel fibres. (b, d) spectra show the appearance of N1s peak as evidence of collagen adsorption
native fibres are characterized only by the C1s and the O1s peaks, whereas in the wide scan spectra of collagen coated fibres (Figs. 3b, 3d) also the N1s peak occurred, due to amide bonds in the collagen backbone (see also Supporting Figure 2). All survey spectra showed the Si2p peak, which was due to environmental contaminations, as well as the Cl2p peak in Figure 3d. As shown in Table 1, the N1s elemental percentage was slightly higher on random than on parallel fibres (~ 6% vs. 5%, respectively), in conformity to the observation of an apparently higher adsorption of collagen on random fibres by SEM.
3.2. Proliferating PC12 cells As shown in Figure 4, PHB substrates supported cell proliferation to a similar extent: DNA concentration was 0.195 0.030 ng/ml on random fibres whereas it was 0.206 0.013 ng/ml on parallel fibres (p > 0.05). Both
DNA concentrations were higher than on the collagencoated PS, which was 0.169 0.012 ng/ml. DNA concentrations on PHB substrates were therefore statistically different from those achieved on control substrates (p < 0.05). In agreement with these results, both PHB substrates supported high cell viability, which was demonstrated by the diffuse green fluorescent staining in Figures 5a and 5b with < 2% dead cells stained in red. By comparison, Figures 5c and 5d show the position of blue-stained nuclei. As assessed by ELISA, proliferating PC12 cells produced the neurotransmitter dopamine. Dopamine concentrations normalized to cell number were 11.1 2.3 pg/ml/cell and 7.9 1.0 pg/ml/cell in the supernatants of PC12 cells cultured on random and parallel PHB fibres, respectively (Fig. 6). These values were found to be statistically different, with p < 0.05 from dopamine concentration amounting to 3.6 0.4 pg/ml/cell in the supernatants of PC12 cells cultured on control substrates. Dopamine concentrations in the lysates collected after three days of culture on PHB
Table 1. Elemental composition of both random and parallel PHB fibres before and after exposure to air plasma and collagen adsorption. Random fibres show higher N content Elemental composition Native PHB fibres Collagen coated PHB fibres
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Randomly oriented Parallel oriented Randomly oriented Parallel oriented
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77.0 0.8 74.5 0.7 69.3 0.7 76.2 0.8
21.2 0.2 18.3 0.2 16.3 0.2 12.2 0.1
6.1 0.1 4.8 0.0
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Figure 4. DNA concentration in lysates of cells cultured on random and parallel PHB fibres and PS as control. Proliferation was enhanced by fibrous topography
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substrates (35.7 5.6 pg/ml/cell on random fibres and 34.1 2.8 pg/ml/cell on parallel fibres) were alternately found to be statistically non-different from those in the lysates after culture on control substrates (36.9 8.9 pg/ml/cell).
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Cell interaction with the substrates after differentiation induction was first investigated by SEM. Figure 7 shows the cultures at increasing magnifications from top to bottom. At higher magnification, random fibres can be seen underneath spread cells (Fig. 7c) as well as many cell protrusions of the same size(red arrows in Figs. 7c,7f), thereby demonstrating
Control PS
Figure 6. Normalized dopamine concentrations in cell lysates (a) and supernatants (b) of PC12 cultured for 72 h over random and parallel PHB substrates and PS as control
a strong cellular adhesion with the underlying fibres both at cell edges and in correspondence to the cell body.
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Figure 5. LIVE/DEADW and nuclei staining of PC12 cells over random (a, b) and parallel fibres (c, d) after 24 h of culture. Arrows indicate the orientation of parallel fibres Copyright © 2012 John Wiley & Sons, Ltd.
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Figure 7. Scanning electron microscopy images of PC12 cells cultured on random (a, b and c) and parallel (d, e and f) PHB fibres at increasing magnification (from top to bottom). Red arrows show cell protrusions
Cellular differentiation was also evaluated through fluorescent staining of b3-tubulin. As shown in Figures 8a and 8c, cells exhibited long neurites with different orientations based on fibre orientation. Neurite orientation distribution (Fig. 9a) clearly demonstrates that neurites strictly followed fibre orientation. In particular, neurite orientation on parallel fibres was coherent with fibre orientation as assessed by FFT analysis. As quantitatively verified by neurite number per cell counting (Fig. 9b), cells were prevalently bipolar on both substrates. At higher magnification, some branching was observed at the end of the neurites (Supporting Fig. 3). On random PHB fibres, average neurite length was 64 12 mm whereas on parallel fibres it was 108 14 mm (n = 3). Neurite length distribution was representative of a single experiment and is depicted in Figure 9c. Random and parallel fibres displayed a different distribution of neurite length, with an occurrence of neurites > 75% in the range 20-80 mm on random fibres and an occurrence of neurites > 50% in the range 100-200 mm on parallel fibres. Copyright © 2012 John Wiley & Sons, Ltd.
4. Discussion The limited self-regenerative capability of both central and peripheral nervous tissue has required the elaboration of different strategies with the purpose of recovering structure and function of damaged nerves (Tresco, 2000). Some highly promising strategies consist in the transplantation of dopamine secreting cells to improve parkinsonian symptoms and the application of polymeric conduits to direct neurite outgrowth in transected nerves (Cao et al., 2009; Yoshida et al., 2003). Because topographical stimuli provided by the environment have been shown to address neural cell response both in vitro and in vivo, in the present study, sub-micron PHB fibres with either a random or parallel orientation were produced to evaluate their possible applicability in the field of cell transplantation and nervous tissue regeneration. PC12 neuron-like cell response was evaluated in terms of viability and dopamine production during proliferation as well as neurite J Tissue Eng Regen Med 2015; 9: 151–161. DOI: 10.1002/term
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Figure 8. Immunofluorescent staining of b3-tubulin as an early marker of PC12 differentiation on PHB random (a, b) and parallel fibres (c, d). Low magnification images are on top row whereas high magnification images are on bottom row. Parallel fibre axis is oriented as yellow arrows
orientation and length after differentiation induction with NGF. Prior to PC12 cell seeding, PHB fibres were exposed to air plasma and coated with collagen to enhance cell adhesion. SEM imaging showed that the substrate topography was not affected by the coating procedure, whereas WCA measurements and elemental composition analysis by XPS confirmed the effectiveness of collagen adsorption. Indeed, WCA was significantly decreased on both random- and parallel-oriented fibres, whereas the N1s peak appeared in the XPS spectra only after the coating procedure, due to the amide bonds in the collagen backbone (Supporting Information). Moreover, XPS elemental analysis showed that random fibres promoted the adsorption of a slightly higher amount of protein (increase in N content) with respect to parallel fibres. This result can be easily explained by the “re-entrant” geometry of random fibres, which was also responsible for the higher hydrophobicity of native fibres (Ma et al., 2008; Tuteja et al., 2008). After 72 h of culture in proliferation medium, cells were found highly viable by LIVE/DEADW staining coherently to other authors’ findings (Chang et al., 2011; Sombatmankhong et al., 2006), proliferating to a significantly higher extent on fibrous substrates in comparison to flat substrates, thereby demonstrating that fibrous topography enhanced cell proliferation. As assessed by ELISA assay, surface structuring also increased dopamine secretion in the supernatants with respect to flat polystyrene substrates, whereas the intracellular content of the neurotransmitter was found comparable in the cells cultured on all the substrate typologies. Copyright © 2012 John Wiley & Sons, Ltd.
In 2003, Yoshida et al. reported that ~ 5105 PC12 cells encapsulated in polysulfone hollow fibres were able to produce a dopamine mean value of 0.46 pmol/15 min two weeks after explantation from Japanese monkey brains and 9 capsules were suggested as sufficient to improve symptoms in patients affected by Parkinson’s disease (Yoshida et al., 2003). Based on calculations, it can be determined that each cell in Yoshida’ capsules secreted ~ 910-7 pmol/15 min. Considering that dopamine rapidly auto-oxidizes and that its measured values closely reflect the amount immediately secreted with very little accumulation (Bethea and Kozak, 1984), we calculated that cell secreted dopamine amounted to 3.6010-2 pmol/cell on random PHB fibres and 2.6010-2 pmol/cell on parallel PHB fibres (p > 0.05). Therefore, we found that these values were four orders of magnitude higher than those suggested by Yoshida et al. as effective for improving parkinsonian symptoms. This encourages further studies on the encapsulation of PC12 cells seeded on PHB fibres in Parkinson’s disease treatment. After 6 days in differentiation medium, PC12 cells exhibited a strong adhesion to the substrates, as shown by SEM imaging. At high magnification, SEM imaging showed cellular protrusions of the same size as the substrate fibres. In addition to results from the immunofluorescent staining of b3-tubulin, these results could be considered preliminary evidence of the appropriateness of the 200-400 nm diameter range for promoting neurite outgrowth and orientation. Fluorescently stained neurites, in fact, strictly followed fibre orientation. Moreover, they exhibited greater lengths on parallel PHB fibres. J Tissue Eng Regen Med 2015; 9: 151–161. DOI: 10.1002/term
G. G. Genchi et al.
160
b
100
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80
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120
60 40 20
n=120
n=147
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2 1
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25 20 15 10 5
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Neurite length (um) Figure 9. Neurite orientation distribution (a), number per cell (b) and length distribution (c) on random and parallel PHB fibres. “n” is the considered cell number. Neurite orientation depended on fibre orientation whereas cells were prevalently bipolar on both substrates. Neurite length was affected by fibre orientation
More than 75% of neurites occurred in the length range of 20-80 mm on random fibres, whereas more than of 50% neurites occurred in the range of 100-200 mm on parallel fibres. Consistent with other studies (Koh et al., 2008; Wang et al., 2010; Yang et al., 2005), it is suggested that anisotropic fibres with diameters in the 200-400 nm range could promote neurite guidance and a faster growth over longer distances, all valuable conditions to a possible application of parallel PHB fibres as nerve conduits for peripheral nervous system regeneration.
5. Conclusions Sub-micron poly(hydroxybutyrate) fibres were successfully electrospun in two different experimental conditions, achieving densely packed fibres with either random or parallel orientation. PC12 neuron-like cell response to different topographies was examined during both proliferation and differentiation. We demonstrated that proliferating cells were highly viable and released a greater amount of dopamine after culture on sub-micron fibres with respect to flat standard substrates. Parallel fibres, Copyright © 2012 John Wiley & Sons, Ltd.
however, supported the outgrowth of longer neurites after differentiation induction with nerve growth factor. Neurite orientation was found to be dependent on fibre orientation: neurites were randomly- and parallel-oriented on random and parallel fibres, respectively. These results are therefore highly promising for the use of electrospun polyhydroxybutyrate fibres in the field of nervous tissue regeneration, where both dopamine production and neurite guidance represent fundamental requisites for a functional recovery in different pathological conditions. Additional studies on these kinds of substrates could help elucidate the relevance of sub-micron topography in addressing cellular activities of specific neuronal populations, with possible beneficial outcomes for other pathological conditions of the nervous tissue.
Acknowledgements The authors gratefully acknowledge Mr. Valfredo Zolesi (Kayser Italia S.r.l.) for his economical support, Dr. Barbara Mazzolai (IIT-CMBR) for her kind support to the research line, and Dr. Antonella Milella (University of Bari) for her kind contribution to the WCA measurements. Mr. Carlo Filippeschi is acknowledged J Tissue Eng Regen Med 2015; 9: 151–161. DOI: 10.1002/term
161
PC12 neuron-like cell response to electrospun poly(3-hydroxybutyrate) substrates for his technical assistance during sample sputtering. The NNL group acknowledges the support from the Italian Minister of University and Research by the FIRB Contracts RBIP068JL9 and RBNE08BNL7 (MERIT Program).
Conflict of interest No conflicts of interest were declared by the authors.
References Alvarex-Perez MA, Guarino V, Cirillo V et al. 2010; Influence of gelatin cues in PCL electrospun membranes on nerve outgrowth. Biomacromolecules 11: 2238–2246. Ayres C, Bowlin GL, Henderson SC V et al. 2006; Modulation of anisotropy in electrospun tissue-engineering scaffolds: Analysis of fiber alignment by the fast Fourier transform. Biomaterials 27: 5524–5534. Bethea CL, Kozak SL. 1984; Effect of extracellular matrix on PC12 cell shape and dopamine processing. Mol Cell Endocrinol 37: 319–329. Cao H, Liu T, Chew SY. 2009; The application of nanofibrous scaffolds in neural tissue engineering. Adv Drug Deliver Rev 61: 1055–1064. Chang SF, Yang CP, Tai CC et al. 2011; Gene expression and behavior analysis of pc12 cells grown on biodegradable nano-fibrous membranes. Curr Nanosci 7: 886–892. Cheng GQ, Wu Q. 2005; Polyhydroxyalkanoates as tissue engineering materials. Biomaterials 26: 6565–6578. Ciofani G, Raffa V, Menciassi A et al. 2009; Magnetic alginate microspheres: system for the position controlled delivery of nerve growth factor. Biomed Microdev 11: 517–527. Ciofani G, Raffa V, Menciassi A et al. 2007; A drug delivery system based on alginate microspheres: mass-transport test and in vitro validation. Biomed Microdev 9: 395–403. Das KP, Freudenrich TM, Mundy WR. 2004; Assessment of PC12 cell differentiation and neurite growth: a comparison of morphological and neurochemical measures. Neurotoxicol Teratol 26: 397–406. Dunnett SB, Bjorklund A. 1999; Prospects for new restorative and neuroprotective treatments in Parkinson’s disease. Nature 399: 32–39. Foley JD, Grunwald EW, Nealey PF et al. 2005; Cooperative modulation of neuritogenesis by PC12 cells by topography and nerve growth factor. Biomaterials 27: 3639–3644. Ghasemi-Mobarakeh L, Prabhakaran MP, Morshed M et al. 2008; Electrospun poly (3-caprolactone)/gelatin nanofibrous scaffolds for nerve tissue engineering. Biomaterials 29: 4532–4539. Greene LA, Tischler AS. 1976; Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci USA 73: 2424–2428. Han D, Cheung KC. 2011; Biodegradable cell-seeded nanofiber scaffolds for neural repair. Polymers 3: 1684–1733.
Copyright © 2012 John Wiley & Sons, Ltd.
Hoffman-Kim D, Mitchel JA, Bellamkonda RV. 2010; Topography, cell response, and nerve regeneration. Annu Rev Biomed Eng 12: 203–231. Kim YT, Haftel VK, Kumar S et al. 2008; The role of aligned polymer fiber-based constructs in the bridging of long peripheral nerve gaps. Biomaterials 29: 3117–3127. Koh HS, Yong T, Chan CK et al. 2008; Enhancement of neurite outgrowth using nano-structured scaffolds coupled with laminin. Biomaterials 29: 3574–3582. Lee YS, Collins G, Livingston AT. 2011; Neurite extension of primary neurons on electrospun piezoelectric scaffolds. Acta Biomater 7: 3877–3886. Li XT, Zhang Y, Chen QG. 2008; Nanofibrous polyhydroxyalkanoate matrices as cell growth supporting materials. Biomaterials 29: 3720–3728. Liu X, Chen J, Gilmore KJ et al. 2010; Guidance of neurite outgrowth on aligned electrospun polypyrrole/poly(styrene-b-isobutylene-bstyrene) fiber platforms. J Biomed Mat Res Part A 94A: 1004–1011. Ljungberg C, Johansson-Ruden G, Junemo Boström K et al. 1999; Neuronal survival using a resorbable synthetic conduit as an alternative to primary nerve repair. Microsurg 19: 259–264. Ma M, Hill RM, Rutledge GC. 2008; A review of recent results on superhydrophobic materials based on micro- and nanofibers. J Adhes Sci Technol 22: 1799–1817. Malm T, Bowald S, Karacagil S et al. 1992; A new biodegradable patch for closure of atrial septal defect. An experimental study. Scand J Thorac Card 26: 9–14. Meng XL, Wan LS, Xu ZK. 2011; Insights into the static and advancing water contact angles on surfaces anisotropised with aligned fibers: Experiments and modelling. Colloid Surface A 389: 213–221. Misra SK, Valappil SP, Roy I et al. 2006; Polyhydroxyalkanoate (PHA)/inorganic phase composites for tissue engineering applications. Biomacromolecules 7: 2249–2258. Mukhatyar V, Karumbaiah L, Yeh J et al. 2009; Tissue engineering strategies designed to realize the endogenous regenerative potential of peripheral nerves. Adv Mat 21: 4670–4679. Novikova LN, Pettersson J, Brohlin M et al. 2008; Biodegradable poly-b-hydroxybutyrate scaffold seeded with Schwann cells to promote spinal cord repair. Biomaterials 29: 1198–1206. Polini A, Pisignano D, Parodi M et al. 2011; Osteoinduction of human mesenchymal stem cells by bioactive composite scaffolds
without supplemental osteogenic growth factors. PLoS One 6: e26211. Polini A, Pagliara S, Stabile R et al. 2010; Collagen-functionalised electrospun polymer fibers for bioengineering applications. Soft Matter 6: 1668–1674. Rentsch C, Rentsch B, Breier A et al. 2009; Evaluation of the osteogenic potential and vascularization of 3D poly(3)hydroxybutyrate scaffolds subcutaneously implanted in nude rats. J Biomed Mat Res Part A 92A: 185–195. Schmack G, Jehnichen D, Vogel R et al. 2000; Biodegradable fibres of Poly (3-hydroxybutyrate) produced by high-speed melt spinning and spin drawing. J Pol Sci Phy 38: 2841–2850. Sell SA, Wolfe PS, Garg K et al. 2010; The use of natural polymers in tissue engineering: A focus on electrospun extracellular matrix analogues. Polymers 2: 522–553. Sill TJ, von Recum HA. 2008; Electrospinning: Applications in drug delivery and tissue engineering. Biomaterials 29: 1989–2006. Sombatmankhong K, Suwantong O, Waleetorncheepsawat S et al. 2006; Electrospun fiber mats of poly(3-hydroxybutyrate), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and their blends. J Pol Sci Pol Phy 44: 2923–2933. Tresco PA. 2000; Tissue engineering strategies for nervous system repair. Prog Brain Res 128: 349–363. Tuteja A, Choi W, McKinley GH et al. 2008; Design parameters for superhydrophobicity and superoleophobicity. MRS Bull 33: 752–758. Von Der Mark K, Park J, Bauer S et al. 2010; Nanoscale engineering of biomimetic surfaces: Cues from the extracellular matrix. Cell Tissue Res 339: 131–153. Wang HB, Mullins ME, Cregg JM et al. 2010; Varying the diameter of aligned electrospun fibers alters neurite outgrowth and Schwann cell migration. Acta Biomater 6: 2970–2978. Xu X, Li X, Peng S, Xiao J et al. 2010; The behaviour of neural stem cells on polyhydroxyalkanoate nanofiber scaffolds. Biomaterials 31: 3967–3975. Yang F, Murugan R, Wang S, Ramakrishna S. 2005; Electrospinning of nano/micro scale poly(l-lactic acid) aligned fibers and their potential in neural tissue engineering. Biomaterials 26: 2603–2610. Yoshida H, Date I, Shingo T et al. 2003; Stereotactic transplantation of a dopamineproducing capsule into the striatum for the treatment of Parkinson disease: a preclinical primate study. J Neurosurg 98: 874–881.
J Tissue Eng Regen Med 2015; 9: 151–161. DOI: 10.1002/term
