Surface potential controlled cells proliferation and collagen

Subscriber access provided by University of Winnipeg Library

Bio-interactions and Biocompatibility

Surface potential controlled cells proliferation and collagen mineralization on electrospun polyvinylidene fluoride (PVDF) fibers scaffolds for bone regeneration Piotr K Szewczyk, Sara Metwally, Joanna E Karbowniczek, Mateusz Marek Marzec, E Stodolak, Adam Gruszczynski, Andrzej Bernasik, and Urszula Stachewicz ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.8b01108 • Publication Date (Web): 20 Dec 2018 Downloaded from http://pubs.acs.org on December 23, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Biomaterials Science & Engineering

Surface potential controlled cells proliferation and collagen mineralization on electrospun polyvinylidene fluoride (PVDF) fibers scaffolds for bone regeneration Piotr K. Szewczyk1, Sara Metwally1, Joanna E. Karbowniczek1, Mateusz M. Marzec2, Ewa StodolakZych3, Adam Gruszczyński1, Andrzej Bernasik2, 4, Urszula Stachewicz1* 1International

Centre of Electron Microscopy for Materials Science, Faculty of Metals Engineering and Industrial Computer Science, AGH University of Science and Technology, Poland 2Academic Centre for Materials and Nanotechnology, AGH University of Science and Technology, Poland 3Department of Biomaterials and Composite Materials, Faculty of Materials Science and Ceramics, AGH University of Science and Technology, Poland 4Faculty of Physics and Applied Computer Science, AGH University of Science and Technology, Poland AGH University of Science and Technology, Al. A. Mickiewicza 30, 30-059 Kraków, Poland *Corresponding author: Urszula Stachewicz, Email: [email protected], Phone: +48 12 617 5230 Keywords: fibers, PVDF, cell, surface potential, filopodia, proliferation, electrospinning, collagen mineralization

Abstract This study represents the unique analysis of the electrospun scaffolds with the controlled and stable surface potential without any additional biochemical modifications for bone tissue regeneration. We controlled surface potential of polyvinylidene fluoride (PVDF) fibers with applied positive and negative voltage polarities during electrospinning, to obtain two types of scaffolds PVDF(+) and, PVDF(-). The cells’ attachments to PVDF scaffolds were imaged in great details with advanced scanning electron microscopy (SEM) and 3D tomography based on focus ion beam (FIB-SEM). We presented the distinct variations in cells shapes and in filopodia and lamellipodia formation according to the surface potential of PVDF fibers that was verified with Kelvin probe force microscopy (KPFM). Notable, cells usually reach their maximum spread area through increased proliferation, suggesting the stronger adhesion, which was indeed double for PVDF(-) scaffolds having surface potential of -90 mV. Moreover, by tuning surface potential of PVDF fibers we were able to enhance collagen mineralization for possible use in bone regeneration. The scaffolds build of PVDF(-) fibers demonstrated the greater potential for bone regeneration than PVDF(+), showing after 7 days in osteoblasts culture produce well-mineralized 1 ACS Paragon Plus Environment

ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 30

osteoid required for bone nodules. The collagen mineralization was confirmed with energy dispersive x-ray spectroscopy (EDX) and Sirius Red staining, additionally the cells proliferation with fluorescence microscopy and Alamar Blue assays. The scaffolds made of PVDF fibers with the similar surface potential to the cell membranes promoting bone growth for next-generation tissue scaffolds, which are on a high demand in bone regenerative medicine.

1. Introduction One of the most important properties of biomaterials is surface potential together with wellstudied roughness, porosity, chemical composition, wetting and surface free energy.1–4 All materials used as scaffolds for tissue engineering need to promote and felicitate cell adhesion and proliferation, that are accompanied by focal adhesion formation and filopodia development.5 Cells spreading and cells integration with a matrix depends strongly on the adhesive bonding which consists of electrostatic and chemicals bonds, van der Waals and dipole-dipole interactions.6 In the electrostatic interaction between matrix and cells, surface potential plays the major role, showing increased cells attachment and proliferation on positively charged biomaterials surfaces, as cell membrane is negatively charged.7– 12 Various types of cells exhibit negative surface potential from -95 mV to -10 mV.8,13–15 One of the most

popular culture lines is osteoblast-like cells MG63 that we used also in our studies was characterized by – 60 mV membrane potential.16 The concept of restoring physiological electric microenvironment is becoming more often included in the research of materials for bone regeneration.17 Bone is known for its piezoelectric properties18 therefore many approaches in regenerative medicine consider using materials that can produce similar bioelectrical signals to natural tissue.19 The engineered bone scaffolds should be osteo-conductive so osteoblasts can proliferate and adhere, subsequently building new tissue. One of the piezoelectric and biocompatible polymers is polyvinylidene fluoride (PVDF) that has been used in the various biomedical application including bone tissue regeneration.8,20,21 Although PVDF is negatively charged considering surface potential,22 is often used as a biomaterial for example was used as vascular grafts, ligament and artificial cornea.23,24 It was verified that modified β-PVDF films and its composites with positive surface potential promote higher osteoblast adhesion, proliferation25,26 differentiation and bone tissue formation.17,27–29. Also mechanically stimulated PVDF, with induced transient surface charge, enhanced cells proliferation and differentiation.30 In bone regeneration, the sufficient adherence and anchoring of cell to scaffolds, generate the extracellular matrix ECM conditions to promote osteointegration and new bone formation by the mineralization of collagen produced by cells. The mineralization process31 should lead finally to

2 ACS Paragon Plus Environment

Page 3 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


hydroxyapatite (HA) formation preceded by accumulation of calcium and phosphate ions.32 The early osteogenic responses include the formation of amorphous calcium phosphate and carbonated apatite in the first few days of cell culture and transient mineral phase such as octa calcium phosphate over two days later.33 Mineralized collagen is the fundamental element of the bone matrix,34 therefore surface chemistry and geometry has a tremendous effect on stress fiber formation via extended pseudopodia and collagen mineralization for bone regeneration.35 Electrospun fibers have been proved to be an excellent support for tissue engineering36 due to the geometrical similarities with ECM. In many cases surface properties of fibers were modified37 with proteins38, peptides39 or roughness40 to increase the bioactivity. Other studies on electrospun scaffolds, focused on a shape, porosity and mechanical properties, to verify their suitability in cell culture studies for bone tissue regeneration.41–43 The method itself becomes very interesting for a wide range of studies as in electrospinning the high voltage is applied to the needle containing the polymer solution,26,27 where liquid jet stretching and whipping under the electric field, while the solvent evaporates, produces solid fibers. Typically, the positive voltage polarity is applied to the nozzle during electrospinning. However, previous studies showed the possibility of tailoring the chemical composition of fibers surfaces by applying negative voltage polarities during electrospinning.44 Voltage polarity defines the charge accumulated on the surface of the liquid jet and the surface of the fibers. Positive polarity attracts negatively charged groups to fibers’ surface, whereas negative polarity moves the negatively charged functional groups away from the surface. This way we can control the surface chemistry and additionally surface potential of electrospun fibers. Noticing a lack of investigation of the direct influence of electrospun fibers surface potential on cells behavior we performed the electrospinning of PVDF with positive and negative voltage polarity at the nozzle to control surface chemistry of fibers, by reorienting F- at their surface. In our study, we showed the new approach of the direct measurement of surface potential on electrospun fibers using Kelvin probe force microscopy (KPFM) in line with surface chemistry analysis based on X-ray photoelectron spectroscopy (XPS). Moreover, we verify the impact of the surface potential of PVDF fibers on cells anchoring to scaffolds using electron microscopy and 3D tomography based on focus ion beam and scanning electron microscopy (FIB-SEM). We showed the distinct variations in cell shape, filopodia and lamellipodia formation and attachment according to the measured surface potential of electrospun fibers produced with positive and negative voltage polarities. Importantly, the surface potential enhanced mineralization as that occurred within a week time of cells culture. It is a unique report showing so fast mineralization that can happen at the fibrous scaffold with tailored potential adjusted to the potential of the cell membrane.



Page 4 of 30

2. Materials and Methods 2.1 Scaffolds fabrication Polyvinylidene fluoride (PVDF, Mm= 275000 g mol-1, Sigma Aldrich, UK) was dissolved in a solution of acetone (analytical standard, Avantor, Poland) and dimethylacetamide (DMAc, analytical standard, Avantor, Poland) in the ratio of 1:1 to produce a concentration of 22 wt. %. The solution was stirred on a hot plate (IKA RCT basic, Germany) at a constant speed of 700 rpm at 50oC over 4 h. Production of PVDF fibers was carried via electrospinning apparatus EC–DIG with climate chamber system (IME Technologies, The Netherlands) at a temperature of 25 °C and relative humidity of 60 %. The constant voltages of 15 kV with positive and negative polarities were applied to the stainless needle with an inner diameter of 0.8 mm with the 18 cm distance to the grounded collector. The flow rate of the polymer solution was 1.5 ml·h-1. The thickness of PVDF scaffolds was kept the same for the two types of scaffolds PVDF(+) and PVDF(-) with respect to the applied voltage polarities. PVDF fibers were deposited on an aluminum foil for SEM imaging and Alamar Blue assay study, on glass slides (15x15 mm) for surface profilometry and cell culture studies, moreover for surface potential measurement and chemical analysis on Si wafers (15x15 mm) that were coated with 10 nm gold layer using rotary-pumped sputter coating (Q150RS, Quorum Technologies, UK). Membrane used for Alamar Blue study was detached from the aluminum foil prior to the experiments. All samples were stored in polystyrene Petri dishes and placed in the exicator to avoid surface contamination. 2.2 Scaffolds characterization 2.2.1 Fibers morphology and diameter Fibers’ morphology was characterized using SEM (Merlin Gemini II, Zeiss, Germany) at a current of 120 pA and voltage of 3 kV, after gold coating using rotary-pumped sputter coating (Q150RS, Quorum Technologies, UK). The fibers diameters were measured from SEM micrographs using Fiji LifeLine Version, (2015 December 22, USA), for 100 fibers per sample. 2.2.2 Roughness and Contact Angle Measurements The average roughness parameter Ra, of PVDF fibers, was measured using laser microscopy (Olympus OLS4000, Japan). The area of 640 × 640 μm was scanned to calculate the Ra with the digital approximation equation: 1

M N 𝑅a = MN∑j = 1∑i = 1|Zij|

(1)


Page 5 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


where: M and N are a number of data points in X, Y directions, Z is the surface height relative to the reference mean plate. The Ra is given, as an average of 10 measurements, with the error based on standard deviation. Advancing contact angles on electrospun fibers were measured using deionized water (DI water, Spring 5UV purification system - Hydrolab, Poland), Phosphate-Buffered Saline (PBS), Dulbecco's Modified Eagle's Medium (DMEM) and medium used for cell culture. Droplets of 3 μL volume were pipetted onto the samples and immediately after depositing each droplet the image was taken using Canon EOS 700D camera with EF-S 60mm f/2.8 Macro USM zoom lens. Experiments were carried out at 21°C and a humidity of 50 %. The receding contact angles of 10 droplets were measured using MB Ruler (MB-Software solutions, Germany) with the standard deviation analysis. 2.2.3

Surface potential and chemistry KPFM measurements were done with Agilent 5500 apparatus (USA) working in amplitude

modulation and intermittent-contact mode in which surface topography and CPD (contact potential difference/surface potential) maps were recorded simultaneously. Gold covered monolithic silicon cantilevers (NanoSensors, Switzerland) with a spring constant of 2.5 Nm-1, the quality factor of about 100 and the resonance frequency of 75 kHz, were used. Maximum scan size range was 45×45 μm. The KPFM DC and AC voltage with a frequency of 10 kHz were applied to the cantilever. All measurements were performed under ambient conditions. XPS analyses were performed using scanning XPS system (PHI VersaProbe II, USA) equipped with monochromatic Al Kα (1486.6 eV) radiation. X-rays were focused to a 100 µm spot and scanned over the surface area of 400 x 400 µm2. The photoelectron take-off angles were set to 45° and the pass energy in the analyzer was set to 23.50 eV to obtain high energy resolution spectra for the C 1s and F 1s regions. The depth of chemical analysis in electrospun PVDF was approximately 5 nm from the fiber surface. A dual beam charge compensation with 7 eV Ar+ ions and 1 eV electrons were used to maintain a constant sample surface potential. The operating pressure in the analytical chamber was less than 2x10-9 mbar. Deconvolution of spectra was carried out using PHI MultiPak software and in the obtained spectra, backgrounds were subtracted using the Shirley method. 2.3 Cell culture analysis Human osteoblast-like cell line MG63 (ECACC) were cultured on PVDF scaffolds. All materials were sterilized by wash its in 96% ethyl alcohol or UV irradiation for 30 min. Cells media was: DMEM supplemented with 10% FBS, 1% amino acids, 1% glutamine, 2% penicillin-streptomycin (Sigma Aldrich, UK). Cells seeding density was 2×104 cell/cm2, they were cultured on samples for 1, 3 and 7 days in a 5 ACS Paragon Plus Environment


Page 6 of 30

temperature of 37°C, humidity of 95% and 5% CO2 atmosphere. At each time point after removing media samples were washed with PBS and fixed: for fluorescence microscopy and SEM with 2.5 % glutaraldehyde (Sigma Aldrich, UK) for 1 hour in 4°C; for collagen staining with 3.5% formaldehyde (Sigma Aldrich, UK) for 30 minutes. 2.3.1 Cells proliferation assay Cells proliferation and viability were evaluated after 1, 3 and 7 days with Alamar Blue assay (Sigma Aldrich, UK). At each time point, complete culture media was replaced with media containing 10% of Alamar Blue and incubated for 4 h. Subsequently, 100 μl of media from each sample was transferred to a 48-well microplate (Nunclon, Thermo Fisher Scientific) in 6 replicates. Fluorescence was measured at excitation at 530 nm and emission 590 nm with a microplate reader (FLUOstar Omega, BMG labtech, Germany). Tissue culture polystyrene (TCPS) was used as a control. The assay measurement is based on the reduction of non-fluorescent resazurin (oxidized form) to highly fluorescent resorufin (reduced form) by the reducing environment of the cells. A percentage of reduction of Alamar Blue calculated according to the following formula: 𝑝𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑟𝑒𝑑𝑢𝑐𝑡𝑖𝑜𝑛 𝑜𝑓 𝐴𝑙𝑎𝑚𝑎𝑟 𝐵𝑙𝑢𝑒 =

𝑆𝑥 ― 𝑆𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑆100% 𝑟𝑒𝑑𝑢𝑐𝑒𝑑 ― 𝑆𝑐𝑜𝑛𝑡𝑟𝑜𝑙

(2)

Results from Alamar Blue are presented considering the surface area for cells growth, in the case of PVDF samples it was 0.75 cm2 and for TCPS it was 1.9 cm2. 2.3.2. Fluorescence microscopy The fixed samples after cell culture were washed with PBS and the cell’s nucleus was stained with 0.5 μg·mL-1 HOECHST solution (Sigma Aldrich, UK) for 30 min and then rinsed in PBS 3 times. Inverted light microscope IB-100 with fluorescence lamp (Delta Optical, Poland) was used for the fluorescence imaging. The average cell number was calculated from 10 random images. Prior imaging, we ensured the observation of cells attached to fibrous scaffolds avoiding this way imaging any glass slides surfaces. TCPS was used as a reference material also in the assay analysis. 2.3.3 Collagen staining and light microscopy The solution for collagen staining was prepared by dissolving 50 mg of Sirius Red 80 in 50 ml of saturated picric acid (both Sigma Aldrich, UK). For staining 2 ml of Sirius Red solution were added per sample and incubated for 1 hour. Samples were rinsed with distilled water, subsequently washed with 100% ethanol and air dried. Imaging was performed using inverted light microscope IB-100 (Delta Optical, Poland) in phase contrast mode at magnification 20x and Axio Imager M1m (Zeiss, Germany) 6 ACS Paragon Plus Environment

Page 7 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


in the bright field at a magnification 50x with extended Focus (z-Stack) mode from 20 subsequent images taken with 1 μm steps. The images used for optical density estimation of collagen were acquired at the same settings and light conditions. The quantification of collagen was performed using ImageJ by color deconvolution from the images with stained collagen by splitting from RGB images. The integrated densities of the deconvoluted Sirius Red images from 50 cells (taken from 5 images from different sample regions) were then converted to optical density (OD) using the following equation: 255

(3)

𝑂𝐷 = log (𝑖𝑛𝑡𝑒𝑔𝑟𝑎𝑡𝑒𝑑 𝑑𝑒𝑛𝑠𝑖𝑡𝑦)

Additionally, the light microscopy images with Sirius Red staining were used to calculate the number of cells from 5 images for each of samples after 1, 3 and 7 days per surfaces area, see examples of images in Figure 6S. 2.3.4 Scanning electron microscopy, energy dispersive spectroscopy and 3D tomography The fixed samples after 1, 3 and 7 days in cell culture were washed with PBS and stained with 1% OsO4 (Sigma Aldrich, UK) for 30 minutes and dehydrated in a series of ethanol solutions: 50%, 70%, 96% and ~99,9 % (analytical standard, Avantor, Poland), 3 times for 3 min. in each solution and finally in hexamethyldisilazane (HMDS, Sigma Aldrich, UK) overnight in the fume hood according to the previously used protocols.36 In a similar way as electrospun scaffolds, all samples with cells were Au coated and imaged with SEM (Merlin Gemini II, Zeiss, Germany) at 3 kV and 50 pA, at the working distance from 5 to 10 mm. To investigate early mineralization on osteoblasts, the collagen fibers diameter and length were measured from SEM micrographs using Fiji software. Additionally, the energy-dispersive X-ray spectroscopy (EDX) spectra of mineralized collagen were obtained from the samples after 7 days in cell culture, with point analysis using Quantax 800 EDS detector (Bruker, Germany) in SEM at 10 kV, 480 pA with a working distance of 8 mm. 5 points were analyzed for each sample. The depth of chemical analysis was approximately 2 µm, thus considered taken form bulk of the measured point, which was, 1-cell, 2-mineralized spheroids, 3-PVDF fibers, as indicated in Figure 8. Moreover, for scaffolds produced with positive voltage polarities, PVDF (+) and negative voltage polarities PVDF (-) after 3 days of cell culture, we performed slice and view procedure using focus ion beam (FIB) at 30 kV and 200 pA. Data stacks obtained from FIB-SEM tomography were used to create 3D reconstructions of cells attached via filopodia to PVDF fibers using Avizo Fire (6.3 edition, USA). The voxel size was 10x10x40 nm for PVDF (-) sample and 12.5x12.5x50 nm for PVDF (+).



Page 8 of 30

2.4 Statistical analysis The statistical analyses of the results from Alamar Blue assay and fluorescence microscopy were performed using OriginPro (v2017 SR2, OriginLab, USA) software according to the analysis of variance (ANOVA) with Tukey test. The significance for all the tests was set at p > 0.05. All the measurements from SEM images were performed at random points. The data from fiber diameter measurements, collagen fibril length and contact angle measurements were expressed as the arithmetic average ± standard deviation (SD). For collagen intensity measurements from 50 cells at various images were measured using ImageJ and OD values were expressed as the arithmetic average ± SD.

3. Results 3.1 Scaffolds morphology, roughness and wetting properties Two types of PVDF scaffolds were investigated, one with fibers produced with positive PVDF(+) and the second with negative voltage polarity PVDF(-). In both cases, as shown in Figure 1S in the Supporting Information, the electrospun PVDF fibers were wrinkled with similar fiber size distribution.45 The average fiber diameter for PVDF(+) was 1.39 ± 0.58 μm and for PVDF(-) was 1.37 ± 0.57 μm. Surface roughness measurements resulted in similar Ra values of 4.2 ± 0.5 μm for PVDF(+) and 3.1 ± 0.3 μm and PVDF(-), proving we had very similar geometry for two types of PVDF scaffolds. The initial scaffolds thickness was also similar for both cases. The contact angle measured with water, PBS, DMEM and cell culture medium on PVDF scaffolds are summarized in Table 1S in the Supporting Information. The lowest contact angles were for PVDF(+) with culture medium, 133.8°, while for water it was 144.5°. In case of PVDF(-) the highest contact angle was for PBS, 145.6° and the lowest also for culture medium, 139°. 3.2 Surface potential and chemistry The surface potential of PVDF scaffolds produced with positive and negative voltage polarities was examined using KPFM. The surface topography of PVDF fibers presented in Figure 1 a) and d) was measured simultaneously with a map of surface potential, see Figure 1 b) and e). The CPD showed values of - 173 mV for PVDF (+) fibers and was almost two times higher for PVDF (-) fibers showing the value of -95 mV, see Figure 1 c) and f). In Table 1, the ARXPS results showed higher F(1s) on the PVDF(+) fibers. The ratio F/C at % values for PVDF (-) were 0.77 comparing to the higher value for PVDF(+) reaching 1. The obtained surface chemistry of samples was found in good correlation with KPFM results, showing increased negative charge on PVDF fibers surface of the samples with increased


Page 9 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


fluorine content at the surface. The PVDF fibers were analyzed again with KPFM after 2 months showing very similar potential values.

Figure 1. KPFM results for PVDF(+) and PVDF(-) showing: a),d) surface topography, b),e) surface potential, c),f) contact potential difference, respectively. Table 1. The XPS results for PVDF fibers produced with positive PVDF(+) and negative PVDF(-) voltage polarities, indicating at. % of C(1s), F(1s) and F/C ratio. Fibers

C (1s) [%]

F (1s) [%]

F/C [%]

PVDF(+)

50.0

50.0

1.00

PVDF(-)

56.47

43.53

0.77

3.3 Cells proliferation: Alamar Blue assay and fluorescence microscopy Since the first day of cells incubation we observed the increased viability and proliferation of cells on PVDF(-) scaffolds with higher surface potential (-95 mV) in comparison to PVDF(+) with surface potential reaching (-173 mV), see Figure 2 a). From day 3 to 7 the cells density was increased more than twice on PVDF(-). We also used the images from fluorescence microscopy to calculate a number of cells growing on PVDF(+), PVDF(-) samples and TCPS control after 1, 3 and 7 days, see Figure 2b). After 1 day 35% higher cells number was observed on PVDF(-) in comparison to PVDF(+) scaffold. After 3 days PVDF(-) and TCPS control shown almost the same cell number, that was 46% higher than on PVDF(+) scaffold. After 7 days still 40% higher cell number was observed on PVDF(-) compared to 9 ACS Paragon Plus Environment


Page 10 of 30

PVDF(+) fibers, confirming the Alamar Blue results that surface potential significantly (p

Surface potential controlled cells proliferation and collagen

Recommend Documents