Startseite Cell adhesion and viability of human endothelial cells on electrospun polymer scaffolds
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Cell adhesion and viability of human endothelial cells on electrospun polymer scaffolds

  • Claudia Matschegewski EMAIL logo , Jörn-Bo Matthies , Niels Grabow und Klaus-Peter Schmitz
Veröffentlicht/Copyright: 30. September 2016
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Current Directions in Biomedical Engineering
Aus der Zeitschrift Current Directions in Biomedical Engineering Band 2 Heft 1

Abstract

The usage of electrospun polymer scaffolds is a promising approach for artificial heart valve design. This study aims at the evaluation of biological performance of nanofibrous polymer scaffolds poly(L-lactide) PLLA L210, PLLA L214 and polyamide-6 fabricated by electrospinning via analyzing viability, adhesion and morphology of human umbilical vein endothelial cells (EA.hy926). Nanofibrous surface topography was shown to influence cell phenotype and cell viability according to the observation of diminished cell spreading accompanied with reduced cell viability on nonwovens. Among those, highest biocompatibility was assessed for PLLA L214, although being generally low when compared to the planar control surface. Electrospinning was demonstrated as an innovative technique for the fabrication of advanced biomaterials aiming at guided cellular behavior as well as the design of novel implant platforms. A better understanding of cell–biomaterial interactions is desired to further improve implant development.

Keywords: biocompatibility; cell morphology; electrospinning; endothelial cells; polymeric nanofiber nonwovens; prosthetic heart valve

1 Introduction

Structural heart diseases are of increasing incidence and research on artificial heart valves has been steadily intensified [1]. In contrast to mechanical or bioprosthetic heart valves, heart valve design using polymeric materials is a promising approach towards the development of advanced cardiovascular prosthetics. Major disadvantages of bioprosthetic heart valves, most commonly being xenografts of porcine aortic valves or calf pericardium, are structural valve deterioration often accompanied with subsequent valve thickening and calcification as well as limited durability [2]. The application of polymeric scaffolds holds great potential to overcome these limitations and thus, is promising for future prosthetic heart valve design seeking e.g. longer durability, enhanced cell compatibility, cell guidance as well as reduced risk of infection and thrombosis and optimized anticoagulant therapy. Therefore innovative techniques, such as electrospinning, will support the development of novel scaffold designs for cardiovascular implants. Electrospinning enables the fabrication of fused fiber biomaterial scaffolds from several natural and synthetic polymers with random or aligned fibers of tunable fiber diameters ranging up to a minima of 5 nm [3], including the range of feature sizes known to facilitate cellular contact guidance [4].

Beyond the physico-mechanical parameters that have to be fulfilled to ensure mechanical integrity and functionality of the implant, biological properties of the biomaterials are of crucial relevance for implant’s success. Implants have to ensure biocompatibility as well as additional features such as anti-infectiveness or anti-thrombogenicity, the latter especially demanded for cardiovascular devices. Moreover, implants that hold the capability to induce directed cellular response are highly demanded.

Cell–biomaterial interactions are strongly influenced by both, the surface topography and the chemistry of a biomaterial [4], [5]. The adhesion of cells to a surface triggers a signaling cascade subsequently regulating diverse cell functions, i.e. viability, proliferation and activation of structural and signaling proteins [6]. The knowledge about the complexity of cell physiology in dependence of the characteristics of biomaterials is of elementary clinical relevance regarding the development of optimal implant designs.

This study aims at the investigation of the effect of chemical and topographical characteristics of polymeric nonwovens on adhesion, morphology and viability of human endothelial EA.hy926 cells. The resulting evaluation of the biological performance of nanofibrous polymer scaffolds will be of great benefit for the determination of biomaterial’s potential as innovative biomaterials for heart valve design finally leading to the development of novel or improved for cardiovascular applications and a better understanding of cell–biomaterial interactions.

2 Material and methods

2.1 Fabrication of polymeric nanofiber nonwovens by electrospinning

Nanofibrous nonwovens were fabricated of polymers poly-(L-lactide) Resomer L210 S (PLLA L210) and poly-(L-lactide) Resomer L214 (PLLA L214) (Evonik Industries AG, Darmstadt, Germany) and polyamide-6 (PA-6) (BASF Ludwigshafen, Germany) by free-surface electrospinning. Solvents used were: chloroform (CHCl3, J.T. Baker, Deventer, Netherlands) for PLLA L210 and CHCl3 added with Tetrahydrofuran (THF) for PLLA L214. Polymer concentrations were 2% (w/w) and 5% (w/w) for PLLA L210 and PLLA L214, respectively. PA-6 was 16% (w/w) in solution of formic acid and acetic acid (1:2). Free-surface electrospinning was performed with Nanospider LAB 200 (ELMARCO, Liberec, Czech Republic) under high voltage at 50–65 kV at ambient temperature and ambient humidity with a distance of 160 mm between the wire electrode and the substrate. The generated polymeric nonwoven mats were dried for 12 h at 40°C using the vacuum oven VO 200 (Memmert GmbH & Co., Schwabach, Germany).

2.2 Cell culture

Human umbilical endothelial cells EA.hy926 (ATCC) were cultured in DMEM (GIBCO) supplemented with 10% fetal calf serum (FCS, PAN Biotech, Aidenbach, Germany) at 37°C and 5% CO2 under humidified atmosphere. For the experiments, cells were seeded at a density of 1.5 × 104 cells/cm2 onto the polymeric scaffolds and incubated for 48 h.

2.3 Cell viability assay

Cell viability was determined by using the CellQuanti-Blue™ assay (BioAssaySystems, Hayward, CA, USA) according to the manufacturer’s instructions. Briefly, cell viability was assessed via measuring the metabolic activity by the reduction of the substrate resazurin to resorufin by cellular reductases. Cells were grown on the polymeric surfaces for 48 h and incubated with CellQuanti-Blue™ as 10% of the culture medium volume for 2 h. The resulting fluorescence of resorufin was measured at an emission wavelength of 590 nm with an excitation wavelength of 544 nm using a microplate reader (FLUOstar OPTIMA, BMG Labtech, Offenburg, Germany). Four independent biological replications were performed. Data was normalized to cells grown on plane polystyrene control surface (NC).

2.4 Cell morphology analysis by scanning electron microscopy

Cell morphology of human endothelial EA.hy926 cells was analyzed by scanning electron microscopy. After 48 h cultivation period on the polymeric surfaces, cells were fixed with 25% glutaraldehyde and 0.2 M sodium cacodylate in PBS for 30 min. Samples were then washed with sodium phosphate buffer, dehydrated in a graded series of ethanol (50%, 75%, 90% and 100%) and dried with CO2 in a critical point dryer (CPD 7501, Quorum Technologies Ltd., Laughton, Lewes, East Sussex, UK). Samples were sputter-coated with gold by Agar Sputter Coater (Canemco Inc., Quebec, Canada) and image acquisition was performed with the scanning electron microscope Quanta™ FEG 250 (FEI Company, Hillsboro, OR, USA).

2.5 Statistical analysis

Data were reported as mean value + standard deviation and were analyzed by one-way ANOVA carried out with GraphPad© Prism 5 software (La Jolla, CA, USA). Statistical significance was assumed at p < 0.05.

3 Results

3.1 Cell adhesion and morphology

Cell morphology analysis by scanning electron microscopy revealed phenotypic differences of human endothelial EA.hy926 cells grown on polymeric nanofiber nonwovens compared to those that were grown on the plane control surface (Figure 1A–D). On all polymeric nanofiber nonwovens, comprising PLLA L210, PLLA L214 and PA-6, only moderate spreading of EA.hy926 cells could be observed (Figure 1B–D). In particular, cells that were grown on electrospun polymer scaffolds mainly exhibited spherical phenotypes. Especially on PLLA L210 nonwovens, EA.hy926 cells were shown to circum-grow the scaffold nanofibers (Figure 1C). In contrast, on the plane control surface cells were well attached to the surface while demonstrating a flattened phenotype (Figure 1A).

Figure 1: SEM images of human endothelial EA.hy926 cells grown on polymeric nanofiber nonwovens PA-6 (B), PLLA L210 (C) and PLLA L214 (D) and planar control surface (NC) (A) for 48 h.
Figure 1:

SEM images of human endothelial EA.hy926 cells grown on polymeric nanofiber nonwovens PA-6 (B), PLLA L210 (C) and PLLA L214 (D) and planar control surface (NC) (A) for 48 h.

3.2 Cell viability

Cell viability of human endothelial EA.hy926 cells grown on polymeric nanofiber nonwovens PLLA L210, PLLA L214 and PA-6 for 48 h was significantly reduced (p < 0.001) when compared to the control surface (NC) (Figure 2). Among the electrospun polymer scaffolds, relative viability of human endothelial EA.hy926 cells was highest on PLLA L214 (51.43 ± 2.97%), followed by PLLA L210 (40.97 ± 9.73%), while being lowest on PA-6 (40.40 ± 0.70%). Thus, cell viability was nearly similar among the nanofibrous scaffolds PLLA L210 and PA-6. However, observed differences in cell viability between the three polymeric nonwovens were not significant.

Figure 2: Relative cell viability of human endothelial EA.hy926 cells after 48 h cultivation on polymeric nanofiber nonwovens PLLA L210, PLLA L214 and PA-6 compared to the planar control surface (NC) (n = 4, mean + SD, ***p < 0.001).
Figure 2:

Relative cell viability of human endothelial EA.hy926 cells after 48 h cultivation on polymeric nanofiber nonwovens PLLA L210, PLLA L214 and PA-6 compared to the planar control surface (NC) (n = 4, mean + SD, ***p < 0.001).

4 Discussion

Nanofibrous polymer scaffolds were successfully processed by electrospinning technology which makes them applicable for a variety of biomedical applications, e.g. heart valve prosthetics or novel drug-eluting stent platforms.

The biological evaluation of electrospun polymeric nonwovens by assessing cellular behavior of human endothelial EA.hy926 cells showed altered cell physiology in response to the physico-chemical properties of the nanofibrous polymer scaffolds. Human endothelial EA.hy926 cells grown on PLLA L210-, PLLA L214- and PA-6-nonwovens showed lower cell adhesion and reduced spreading in comparison to cells grown on the plane control surface. This might probably be a result of the distinct nanofibrous topography of the polymeric scaffolds since Badami et al. [7] already reported smaller projected areas of osteoblastic cells grown on, i.e. PLLA-fibers than for cells grown on planar surfaces but similarly a higher cell aspect ratio on the fibers. Thus, topographical factors designed into biomaterial scaffolds can regulate cellular functions like cell adhesion and spreading. Moreover, in the present study, it seems most likely that surface chemistry might be subordinated to surface topography because endothelial EA.hy926 cells exhibited similar phenotypes on all polymeric nonwovens, regardless of their distinct chemical composition, i.e. PLLA vs. PA-6. Regarding the differentiation between the relative influence of topographical and chemical effects, Cousin [8] showed that changes in the morphology of fibroblasts could be predominantly attributed to the surface topography of nanoparticulated coatings, irrespective of the surface chemistry, which is in consistence with the results of the present study.

Similarly, also cell viability of human endothelial EA.hy926 cells was not significantly different among the polymeric nonwovens, which additionally supports the superordinate effect of surface topography. Moreover, results obtained from cell viability study were consistent with those from the cell morphology analysis since altered cell morphology, expressed by lower spreading, was accompanied with altered cell function, expressed by lower cell viability. Similar cell architecture–cell function dependencies are already described in other studies [6].

In order to improve biocompatibility and biological integrity of the nanofibrous scaffolds, surface modifications, by either chemical or biological surface activation, might be of additional benefit. Among them, plasma-functionalization or the immobilization of biological molecules have already been described as promising methods to optimize biomaterials towards a better cell compatibility and implant integrity. Rudolph et al. [9] showed that NH3-plasma treatment of different polymer surfaces enhanced cell viability as well as cell adhesion of human endothelial cells. In addition, also biofunctionalization of biomaterial surfaces, e.g. with vascular endothelial growth factor (VEGF) can improve biocompatibility of implant surfaces [10].

5 Conclusion

Nanofibrous scaffolds of PLLA L210, PLLA L214 and PA-6 were successfully fabricated by electrospinning enabling its usage as appropriate matrices for endothelialization whereat surface topography-dependent influences on cell physiology were detected. Together with the finely tunable fabrication properties of the electrospinning technique, polymer nonvovens are promising for directing cellular response and several cardiovascular applications. Systematic modulation of biomaterial’s physico-chemical properties will support the elucidation of key cell–biomaterial interactions to further improve implant technology.

Acknowledgement

The authors acknowledge technical assistance of Babette Hummel, Martina Nerger and Martina Schröder and Nathalie Böttcher.

Author’s Statement

Research funding: Partial financial support by the European Regional Development Fund (ERDF) and the European Social Fund (ESF) within the collaborative research between economy and science of the state Mecklenburg-Vorpommern and by the Federal Ministry of Education and Research (BMBF) within RESPONSE “Partnership for Innovation in Implant Technology” is gratefully acknowledged. Conflict of interest: Authors state no conflict of interest. Material and Methods: Informed consent: Informed consent is not applicable. Ethical approval: The conducted research is not related to either human or animal use.

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Published Online: 2016-9-30
Published in Print: 2016-9-1

©2016 Claudia Matschegewski et al., licensee De Gruyter.

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

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https://doi.org/10.1515/cdbme-2016-0006
Schlagwörter für diesen Artikel
biocompatibility; cell morphology; electrospinning; endothelial cells; polymeric nanofiber nonwovens; prosthetic heart valve
Creative Commons
BY-NC-ND 4.0

Artikel in diesem Heft

  1. Synthesis and characterization of PIL/pNIPAAm hybrid hydrogels
  2. Novel blood protein based scaffolds for cardiovascular tissue engineering
  3. Cell adhesion and viability of human endothelial cells on electrospun polymer scaffolds
  4. Effects of heat treatment and welding process on superelastic behaviour and microstructure of micro electron beam welded NiTi
  5. Long-term stable modifications of silicone elastomer for improved hemocompatibility
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  8. Protection of active implant electronics with organosilicon open air plasma coating for plastic overmolding
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  59. Artificial blood circulatory and special Ultrasound Doppler probes for detecting and sizing gaseous embolism
  60. Detection of microsleep events in a car driving simulation study using electrocardiographic features
  61. A method to determine the kink resistance of stents and stent delivery systems according to international standards
  62. Comparison of stented bifurcation and straight vessel 3D-simulation with a prior simulated velocity profile inlet
  63. Transient Euler-Lagrange/DEM simulation of stent thrombosis
  64. Automated control of the laser welding process of heart valve scaffolds
  65. Automation of a test bench for accessing the bendability of electrospun vascular grafts
  66. Influence of storage conditions on the release of growth factors in platelet-rich blood derivatives
  67. Cryopreservation of cells using defined serum-free cryoprotective agents
  68. New bioreactor vessel for tissue engineering of human nasal septal chondrocytes
  69. Determination of the membrane hydraulic permeability of MSCs
  70. Climate retainment in carbon dioxide incubators
  71. Multiple factors influencing OR ventilation system effectiveness
  72. Evaluation of an app-based stress protocol
  73. Medication process in Styrian hospitals
  74. Control tower to surgical theater
  75. Development of a skull phantom for the assessment of implant X-ray visibility
  76. Surgical navigation with QR codes
  77. Investigation of the pressure gradient of embolic protection devices
  78. Computer assistance in femoral derotation osteotomy: a bottom-up approach
  79. Automatic depth scanning system for 3D infrared thermography
  80. A service for monitoring the quality of intraoperative cone beam CT images
  81. Resectoscope with an easy to use twist mechanism for improved handling
  82. In vitro simulation of distribution processes following intramuscular injection
  83. Adjusting inkjet printhead parameters to deposit drugs into micro-sized reservoirs
  84. A flexible standalone system with integrated sensor feedback for multi-pad electrode FES of the hand
  85. Smart control for functional electrical stimulation with optimal pulse intensity
  86. Tactile display on the remaining hand for unilateral hand amputees
  87. Effects of sustained electrical stimulation on spasticity assessed by the pendulum test
  88. An improved tracking framework for ultrasound probe localization in image-guided radiosurgery
  89. Improvement of a subviral particle tracker by the use of a LAP-Kalman-algorithm
  90. Learning discriminative classification models for grading anal intraepithelial neoplasia
  91. Regularization of EIT reconstruction based on multi-scales wavelet transforms
  92. Assessing MRI susceptibility artefact through an indicator of image distortion
  93. EyeGuidance – a computer controlled system to guide eye movements
  94. A framework for feedback-based segmentation of 3D image stacks
  95. Doppler optical coherence tomography as a promising tool for detecting fluid in the human middle ear
  96. 3D Local in vivo Environment (LivE) imaging for single cell protein analysis of bone tissue
  97. Inside-Out access strategy using new trans-vascular catheter approach
  98. US/MRI fusion with new optical tracking and marker approach for interventional procedures inside the MRI suite
  99. Impact of different registration methods in MEG source analysis
  100. 3D segmentation of thyroid ultrasound images using active contours
  101. Designing a compact MRI motion phantom
  102. Cerebral cortex classification by conditional random fields applied to intraoperative thermal imaging
  103. Classification of indirect immunofluorescence images using thresholded local binary count features
  104. Analysis of muscle fatigue conditions using time-frequency images and GLCM features
  105. Numerical evaluation of image parameters of ETR-1
  106. Fabrication of a compliant phantom of the human aortic arch for use in Particle Image Velocimetry (PIV) experimentation
  107. Effect of the number of electrodes on the reconstructed lung shape in electrical impedance tomography
  108. Hardware dependencies of GPU-accelerated beamformer performances for microwave breast cancer detection
  109. Computer assisted assessment of progressing osteoradionecrosis of the jaw for clinical diagnosis and treatment
  110. Evaluation of reconstruction parameters of electrical impedance tomography on aorta detection during saline bolus injection
  111. Evaluation of open-source software for the lung segmentation
  112. Automatic determination of lung features of CF patients in CT scans
  113. Image analysis of self-organized multicellular patterns
  114. Effect of key parameters on synthesis of superparamagnetic nanoparticles (SPIONs)
  115. Radiopacity assessment of neurovascular implants
  116. Development of a desiccant based dielectric for monitoring humidity conditions in miniaturized hermetic implantable packages
  117. Development of an artifact-free aneurysm clip
  118. Enhancing the regeneration of bone defects by alkalizing the peri-implant zone – an in vitro approach
  119. Rapid prototyping of replica knee implants for in vitro testing
  120. Protecting ultra- and hyperhydrophilic implant surfaces in dry state from loss of wettability
  121. Advanced wettability analysis of implant surfaces
  122. Patient-specific hip prostheses designed by surgeons
  123. Plasma treatment on novel carbon fiber reinforced PEEK cages to enhance bioactivity
  124. Wear of a total intervertebral disc prosthesis
  125. Digital health and digital biomarkers – enabling value chains on health data
  126. Usability in the lifecycle of medical software development
  127. Influence of different test gases in a non-destructive 100% quality control system for medical devices
  128. Device development guided by user satisfaction survey on auricular vagus nerve stimulation
  129. Empirical assessment of the time course of innovation in biomedical engineering: first results of a comparative approach
  130. Effect of left atrial hypertrophy on P-wave morphology in a computational model
  131. Simulation of intracardiac electrograms around acute ablation lesions
  132. Parametrization of activation based cardiac electrophysiology models using bidomain model simulations
  133. Assessment of nasal resistance using computational fluid dynamics
  134. Resistance in a non-linear autoregressive model of pulmonary mechanics
  135. Inspiratory and expiratory elastance in a non-linear autoregressive model of pulmonary mechanics
  136. Determination of regional lung function in cystic fibrosis using electrical impedance tomography
  137. Development of parietal bone surrogates for parietal graft lift training
  138. Numerical simulation of mechanically stimulated bone remodelling
  139. Conversion of engineering stresses to Cauchy stresses in tensile and compression tests of thermoplastic polymers
  140. Numerical examinations of simplified spondylodesis models concerning energy absorption in magnetic resonance imaging
  141. Principle study on the signal connection at transabdominal fetal pulse oximetry
  142. Influence of Siluron® insertion on model drug distribution in the simulated vitreous body
  143. Evaluating different approaches to identify a three parameter gas exchange model
  144. Effects of fibrosis on the extracellular potential based on 3D reconstructions from histological sections of heart tissue
  145. From imaging to hemodynamics – how reconstruction kernels influence the blood flow predictions in intracranial aneurysms
  146. Flow optimised design of a novel point-of-care diagnostic device for the detection of disease specific biomarkers
  147. Improved FPGA controlled artificial vascular system for plethysmographic measurements
  148. Minimally spaced electrode positions for multi-functional chest sensors: ECG and respiratory signal estimation
  149. Automated detection of alveolar arches for nasoalveolar molding in cleft lip and palate treatment
  150. Control scheme selection in human-machine- interfaces by analysis of activity signals
  151. Event-based sampling for reducing communication load in realtime human motion analysis by wireless inertial sensor networks
  152. Automatic pairing of inertial sensors to lower limb segments – a plug-and-play approach
  153. Contactless respiratory monitoring system for magnetic resonance imaging applications using a laser range sensor
  154. Interactive monitoring system for visual respiratory biofeedback
  155. Development of a low-cost senor based aid for visually impaired people
  156. Patient assistive system for the shoulder joint
  157. A passive beating heart setup for interventional cardiology training
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Heruntergeladen am 7.9.2025 von https://www.degruyterbrill.com/document/doi/10.1515/cdbme-2016-0006/html
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