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DE GRUYTER Current Directions in Biomedical Engineering 2020;6(3): 20203120 Sabine Illner*, Jonathan Ortelt, Daniela Arbeiter, Valeria Khaimov, Katharina Wulf, Stefan Oschatz, Thomas Reske, Volkmar Senz, Klaus-Peter Schmitz and Niels Grabow Opening new paths to tailored polymer properties with optional drug incorporation Abstract: Electrospun fiber nonwoven materials of different are also debated intensively and explored in the field of polymer classes provide promising perspectives in almost all medical engineering . Especially in the cardiovascular fields of application, including medical science. In this paper field, many implant surfaces could benefit from innovative we present the fiber generation of selected biostable fibrous structures, but are also subject to various restrictions polymers (PBT, TPC-ET, PA 6.12 and PVDF) by direct and regulatory barriers. electrospinning, as an extremely powerful tool for The vision of creating adaptive, implant-specific and manufacturing of new superfiber implant materials. This drug-loaded surfaces that are anti-infective, flexible or initial study includes the variation of some relevant process expandable, chemically modifiable and cell-sensitive can be parameters, such as polymer concentrations or electrode achieved relatively straightforward by using modern spacing. The influence on fiber morphology, tensile strength electrospinning or 3D-printing technologies. However, and biocompatibility is shown. The results presented indicate identification of chemically inert, long-term stable and yet that the choice and combination of materials is crucial for the processable materials which are clear for regulatory approval application on load-bearing implants, independent of the appears as an almost unresolvable challenge and has become processing technology and thus of the fiber bonding, an important topic of research worldwide. delamination or fiber strength. This is the background for our endeavors to iteratively introduce extraordinary materials and systematically expand Keywords: electrospinning, nanofiber, polybutylene the material portfolio. In this study we present first terephthalate, polyamide, polyester elastomer, polyvinylidene mechanical, morphological and biological investigations of fluoride. promising polymers for implant coating or covering. The processing procedures have been established and optimized https://doi.org/10.1515/cdbme-2020-3120 for a thermoplastic copolyester elastomer (TPC-ET), poly- vinylidene fluoride (PVDF), polyamide (PA 6.12) and polybutylene terephthalate (PBT). Furthermore, biocompa- 1 Introduction tibility studies and mechanical tests in medium at 37°C were carried out. Electrospun polymeric nanofibers with tailor-made, Each of the selected polymer classes has unique flexible three-dimensional porous structures and a high properties, such as high mechanical strength, thermal stability surface-to-volume ratio offer new solutions in various fields and excellent chemical resistance of PA 6.12 or the rubber- of application such as filtration, desalination, catalysis, tissue like and extremely elastic properties of TPC-ET. Being replacement, nutrient or drug supply and textile industry extremely versatile, PBT combines stiffness and toughness, already today [1,2]. superior electrical insulation properties and exceptional New biomimetic surface structures in the sub micrometer surface finish [4,5]. to nanometer range, both with or without local drug release, Even though all materials have exceptional chemical and physical properties, decisive factors for their use as implant material are often missing, be it long-term stability, ______ availability or fatigue strength. In addition, the mechanical *Corresponding author: Sabine Illner: Institute for Biomedical properties of the individual polymers are often insufficient to Engineering, University Medical Center Rostock, Friedrich- mimic biological materials. Therefore, the combination or Barnewitz-Str. 4, D-18119 Rostock, Germany, sabine.illner@uni- layered structures with tunable local and controllable drug rostock.de Jonathan Ortelt, Daniela Arbeiter, Valeria Khaimov, Katharina depots are indispensable for potential applications in Wulf, Stefan Oschatz, Thomas Reske, Volkmar Senz, Klaus- biomedical engineering. Peter Schmitz, Niels Grabow: Institute for Biomedical Engineering, University Medical Center Rostock, Rostock, Germany Open Access. © 2020 Sabine Illner et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 License. Adaptable Superfibers as Implant Material — 2 2.2 Characterization 2 Materials and Methods Scanning electron microscopy (SEM) was performed on a Quanta FEG 250 (FEI Company, Germany). Samples were 2.1 Electrospinning mounted on aluminum carriers using pyrolytic graphite planchets and Au sputter coated prior to measurements at The following solvents were selected and used as received various magnifications. without further purification for the electrospinning process: Tensile force was measured as a function of sample acetone, dichloromethane (DCM), chloroform (CHCl ), elongation with a Zwick/Roell Z 2.5/TN (Zwick GmbH & trifluoroacetic acid (TFA) and trifluoroethanol (TFE) Co. KG, Ulm, Germany) using a 10 N load cell and a purchased from Fisher Scientific; dimethylformamide (DMF) crosshead speed of 25 mm/min. The tests were performed in supplied by VWR; acetic acid (AA) and formic acid (FA) a 0.9% saline solution at 37°C. According to purchased from Carl Roth. DIN EN ISO 527, all test samples for uniaxial tensile tests All raw polymers used are manufactured under GMP have geometry according to the standard test specimen 1BB. conditions in medical grade. Prior to spinning, each polymer The elongation at break (ε ) and the ultimate tensile strength was dissolved in a specific solvent mixture at 37°C under (σ ) were extracted from the measured curves. moderate agitation. In Table 1 all information with respect to All cell culture reagents were purchased from PAN polymer solution concentration, solvent ratio and electrode Biotech. Cells were maintained at 37°C and 5% CO under spacing are summarized. humidified atmosphere. Human endothelial EA.hy926 cells (ATCC) were cultured in Dulbecco's Modified Eagle's Table 1: Composition of the polymer solution and electrode Medium supplemented with 10% fetal calf serum (FCS) and spacing. penicillin-streptomycin. For cell seeding experiments 10 cells per well were seeded on polymeric scaffolds placed in a Code Polymer c Solvent Electrode polymer 96-well plate 48 h prior to measurement. Cell viability was type [wt%] mixture spacing [mm] determined using the CellQuanti-Blue assay (BioAssay- Systems, USA) according to the manufacturer’s instructions. 1a 12.5 DCM/TFA 160 PBT Briefly, 10% of the total culture medium volume of 1b 12.5 (ratio 1/1) 205 CellQuanti-Blue reagent was added to each well and allowed 2a 5.0 TFE/CHCl 210 to rest for 2 h. Samples were excited at 544 nm and the TPC-ET 2b 7.5 (ratio 4/1) 170 resulting fluorescence was measured at 590 nm using a 3a 14.0 ES/ AS 80 microplate reader (FLUOstar OPTIMA, BMG Labtech, PA 6.12 3b 14.0 (ratio 1/1) 100 Germany). Data was normalized to cells grown on tissue culture-treated polystyrene. 4a 20.0 DMF/ acetone 100 PVDF 4b 20.0 (ratio 6/4) 150 3 Results and Discussion Electrospinning of PBT (DuPont) and PA6.12 (DuPont) as well as PVDF (Sigma-Aldrich) was performed on an in house-constructed spinning device using a needle setup and a 3.1 Surface Characterization rotating collector (AQ 100x100 mm), as well. The applied high voltage varied between 4 to 15 kV and a feed rate of 0.2 To investigate the morphological structure of the nonwovens, to 0.9 mL/h. Electrospinning of TPC-ET (DuPont) was SEM imaging was performed on the materials generated performed on a 4SPIN C4S LAB2 (Contipro, Czech from the different polymers. After process optimization Republic) using a needle setup (E2, G19) and a rotating smooth and consistent fibers were observed for PBT and collector (C3), an applied high voltage of 20-30 kV and a TPC-ET as shown in Figure 1. Moreover, TPC-ET is the feed rate of 200 µL/min. only nonwoven fabric that has a partial fusion on fiber The spinning time for all polymers varied depending on contact points. The manually measured fiber diameters and the polymer concentration in solution, always aiming for a qualitative assessment of fiber bonding are summarized in target layer thickness of at least 100 µm. Table 2. Adaptable Superfibers as Implant Material — 3 Depending on the type of polymer, different fiber (Figure 1: 2a,b). Unfortunately, the spinning process became diameters, fiber arrangements and bonding are clearly visible. unstable and had to be interrupted every 5 minutes. An increase in polymer solution concentration leads to a Table 2: Fiber diameter (n≥10) of the fabricated nonwovens. strong increase in fiber diameter, e.g. in the case of TPC-ET the fiber diameter doubles. The influence on the mechanical Code Polymer Fiber diameter Fiber bonding performance is shown in Figure 2. type [nm] The electrospinning process for PA 6.12 and PVDF still 1a PBT 548.9 ± 79.0 not noticeable require optimization. In a further step, the polymer concentration is slightly adjusted in order to completely 1b 416.0 ± 73.0 not noticeable avoid the slight bead formation. Nevertheless, varying the 2a TPC-ET 558.3 ± 206.5 pronounced electrode distance from 100 to 150 mm resulted in 2b 1315.3 ± 318.5 weakly pronounced agglomeration and inhomogeneous deposition of the fibers. 3a PA 6.12 160.2 ± 22.0 not noticeable Smaller distances are preferable for both PVDF and PA 6.12 3b 195.0 ± 68.5 not noticeable in order to obtain an optically attractive fiber pattern. Overall, it can be seen that the fiber diameters of PA 6.12 and PVDF 4a PVDF 157.6 ± 30.8 not noticeable are smaller by a factor of 4 to 10 compared to PBT or TPC- 4b 329.4 ± 312.0 not noticeable ET; the effects on the physical properties are discussed in the next section. 3.2 Mechanical behavior Tensile testing was performed in 0.9% saline solution at 37°C. Mechanical properties of electrospun TPC-ET, PA 6.12, PBT and PVDF nonwovens are shown in Figure 2. Here, clear differences in tensile strength were observed in the nonwovens produced from the different polymers. Whereas electrospun PBT shows low tensile strength of 6 MPa and elongation at break of 170%, PA6.12 nonwoven have similar tensile strength with lower elongation at break of only 50%. Figure 1: SEM images of electrospun PBT (1a,b), TPC-ET (2a,b), PA 6.12 (3a,b) and PVDF (4a,b) – Influence of the polymer type, concentration and electrode spacing on fiber orientation and diameter. Scale bar equals 10 μm. On closer examination, polymer solution concentration has many different impacts on fiber structure and processing. Increasing in polymer concentration prevents the formation of beaded fibers. The process stability is also improved with higher polymer concentration, but the process window is quite narrow. A weight percentage of the polymer in solution of ± 0.5% often makes a big difference. Exceeding a concentration of 5wt% TPC-ET the nonwovens become Figure 2: Stress-strain curves of electrospun PBT, TPC-ET, much more uniform with increased and more constant fiber PA 6.12 and PVDF, measured in 0.9% saline solution at diameters for increased polymer concentration of 7.5wt% 37°C (screening results with n=1). Adaptable Superfibers as Implant Material — 4 TPC-ET shows analogous tensile strength compared to other In summary, the different surface morphologies and polymers, but a substantially higher elongation at break of mechanical properties of the nonwovens clearly show that the about 700%. PVDF has the lowest tensile strength of all proper choice of polymer or the combination of different polymers analyzed at approx. 2.5 MPa. polymer types is crucial, as is the layer structure with an additional drug depot, which is often essential for biomedical applications [3,6]. Thereby, a combination of different 3.3 Biocompatibility studies materials is an excellent means of merging complementary polymer properties in nonwoven composites, which has To investigate the suitability of the newly established already been shown for one promising material combination nonwovens for biomedical applications, in vitro biocompa- . The results presented here indicate, that in their tibility testing was performed by accessing cell viability upon advantageous combination nonwovens e.g. of PVDF and cultivation on the material (Figure 3). TPC-ET appear very favorable for adaptable superfiber materials, especially in the application on load-bearing implants. Author Statement Acknowledgement: The authors sincerely thank Laurent Hanen (DuPont) for the free samples and of course Katja Hahn, Babette Hummel, Manfred Strotmeier, Martina Nerger and Gabriele Karsten for their skillful work. Research funding: Partial financial support by the Federal Ministry of Education and Research (BMBF) within RESPONSE “Partnership for Innovation in Implant Technology” and by the European Social Fund (ESF) within Figure 3: Relative cell viability of human endothelial EA.hy926 the excellence research program of the state Mecklenburg- cells after 48 h growth on polymeric scaffolds Vorpommern Card-ii-Omics is gratefully acknowledged. (PS: reference material tissue culture-treated polystyrene, Conflict of interest: Authors state no conflict of interest. TETD: treatment with a reference solution of 100 µM Informed consent: Informed consent is not applicable. Ethical tetraethylthiuram disulfide in cell culture medium with a approval: The conducted research is not related to either strong cytotoxic effect; n = 5, MD ± SD) human or animal use. All materials showed good biocompatibility with cell viabilities around 70 to 80%. Noteworthy, viability of the References cells growing on PBT was slightly lower as compared to other polymers. But ultimately, it is not only the  Xue J, Wu T, Dai Y, Xia Y. Electrospinning and Electrospun biocompatibility but also the fatigue strength and polymer Nanofibers: Methods, Materials, and Applications. Chemical reviews 2019; 119: 5298–5415. stability that determine the long-term implant performance.  Wang C, Wang J, Zeng L, et al. Fabrication of Electrospun Polymer Nanofibers with Diverse Morphologies. Molecules (Basel, Switzerland) 2019; 24.  Illner S, Arbeiter D, Teske M, et al. Tissue biomimicry using 4 Summary and Conclusion cross-linked electrospun nonwoven fibre composites. CDBME 2019; 5: 119–122. This parameter study show very obviously that the polymer  DuPont Transportation & Industrial Crastin® Family Page; 2020. Available from: URL:https://www.dupont.com/products/ type is much more decisive for e.g. tensile strength than the crastin.html [cited 2020 Apr 4]. polymer concentration or other process parameters such as  Cho E, Kim C, Park J-Y, et al. Surface modification of the electrode distance. The polymer concentration is crucial electrospun polyvinylidene fluoride nanofiber membrane by for variation of fiber thickness and processability. The plasma treatment for protein detection. JNN 2013; 13: 674– collector-emitter distance should be considered for fine  Illner S, Kohse S, Michaelis C, et al. In vitro study of tuning as this factor determines the fiber bonding and the sirolimus release from nonwoven PLLA matrices. CDBME production of nonwoven layer homogeneity from a 2018; 4: 591–594. macroscopic point of view.
Current Directions in Biomedical Engineering – de Gruyter
Published: Sep 1, 2020
Keywords: electrospinning; nanofiber; polybutylene terephthalate; polyamide; polyester elastomer; polyvinylidene fluoride
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