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Polymers are increasingly employed in implant materials. To reduce the incidence of complications, which in the case of vascular grafts include incorrect placement and restenosis, materials are needed which allow for image-guided implantation, as well as for accurate and efficient postoperative implant imaging. We here describe amorphous fluorinated polymers based on thermoplastic polyurethane (19F-TPU), and show that are useful starting materials for developing tissue-engineered vascular grafts which can be detected using 19F MRI.
Non-invasive imaging plays an increasingly important role in regenerative medicine. The direct visualization of implants using magnetic resonance imaging (MRI) can provide longitudinal feedback on the position, functionality, resorption and remodeling of the material. It also helps to identify collapsed or damaged regions in vascular grafts, and facilitates decision-making with respect to pharmacological or surgical interventions.
Besides the advantages of standard 1H MRI, which include high spatial resolution, very good soft-tissue contrast and lack of ionizing radiation, 19F MRI offers additional advantages.1,2 First and foremost, since there are hardly any endogenous fluorine atoms in the body (high levels are only found in teeth), the background MRI signal is very low, enabling 'hot spot' imaging and highly specific implant detection. The absence of background signals also allows for accurate and efficient quantification. A further advantage of 19F MRI (over labeling with superparamagnetic iron oxide nanoparticles and standard 1H MR imaging of implants3,4) is the absence of susceptibility artifacts, which can disturb the analysis of adjacent tissues and of remodeling processes within the vascular graft. Fluorinated scaffold materials are consequently considered to be interesting starting materials for developing image-guided tissue-engineered vascular grafts.
Commonly used fluorinated polymers, such as polytetrafluoro-ethylene (PTFE; Teflon; GoreTex) and polyvinylidene fluoride (PVDF) cannot be employed for 19F MRI, because their high crystallinity compromises the molecular mobility of the signal-producing fluorine atoms. As a consequence, even at ultra-short echo times, hardly any MRI signals are generated and the signals decay very fast, leading to poor sensitivity and prohibiting image acquisition (Figure 1). To enable 19F MR imaging of scaffold materials, we generated a novel fluorinated polymer based on thermoplastic polyurethane (19F-TPU) which possesses distinct properties rendering it suitable for fluorine-based MRI: (i) it possesses a large number of magnetically equivalent 19F atoms, yielding strong resonance peaks; (ii) it has an amorphous structure, resulting in high mobility of the incorporated 19F atoms; and (iii) it has favorable relaxation properties to achieve relatively long echo times.
The 19F-TPU is synthesized from a perfluorinated aromatic diisocyanate and butanediol. After polymerization, the polyurethane is N-alkylated to obtain a non-crystalline material with partially fluorinated alkyl side chains (Figure 2A and Scheme S1). The average size of the final polymeric material was 7.4*103 Da (Mn; Mw=1.5*104), and the polydispersity 2.0. It is known that PVDF exhibits high crystallinity (see DSC and XRD spectra in Figure S1 and S2). In order to model a spinning process, PVDF was heated to T = 210 °C, which is above Tm (~180 °C as determined by DSC in Figure S1), followed by subsequent cooling to room temperature. After this thermal treatment, PVDF retains its high crystallinity, which was verified using DSC and wide-angle X-ray scattering (WAXS) analysis (Figure 2D). Conversely, 19F-TPU remained largely amorphous after the same thermal treatment (Figure 2C and S1).
Due to their amorphous nature, 19F-TPU polymers have a much higher chain mobility than crystalline PVDF polymers. Therefore, the fluorine atoms in 19F-TPU are less confined and can more efficiently tumble within the amorphous structure of the polymer. As a result, the fluorine atoms in 19F-TPU generate two resonance peaks at -83.6 ppm and -123.5 ppm with favorable relaxation times (T1= 225±6 ms; T2= 0.76±0.04 ms; Figure 3A-C). The separation of both resonances enables undisturbed excitation and subsequent image readout of a single resonance, preventing the occurrence of artifacts. The generation of strong and specific 19F MRI signals with slow signal decay permits background-free imaging of 19F-TPU at clinically relevant settings, i.e. in a 3T patient scanner, at a relatively short acquisition time (<3 min), with a good signal-to-noise ratio (>10) and with a relatively high image resolution (<1 mm isotropic voxel size) (Figure 3D).
To provide proof-of-principle for the usefulness of 19F-TPU for developing MRI-detectable scaffold materials, it was processed into monofilaments via melt spinning. Therefore, 19F-TPU pellets were introduced into a twin-screw extruder and spun into monofilaments at a process temperature of 225-230°C. Subsequently, a vascular graft with a diamond braid and twelve monofilaments was produced. The braided structure had a braiding angle of 33° and its inner diameter was 6 mm. The resulting scaffold was imaged in a clinical 3T MRI scanner using a dual-tuned (19F/1H) volume coil and ultra-short echo time (UTE) sequences with an echo/repetition time of 0.12/100 ms and a flip angle of 67°. Afterwards, the 19F imaging data was 3D-rendered and overlaid with a 1H image to demonstrate ‘hot spot’ MR imaging (Figure 4).
These findings indicate that 19F-TPU can be utilized to develop MR imageable cardiovascular implants, enabling a detailed visualization of the architecture of vascular grafts and their unambiguous identification upon in vivo implantation. In recent years, several other fluorine-containing materials have been developed for 19F MRI, but thus far only for functional and molecular imaging applications6–11, for monitoring drug delivery12, for cell tracking13,14 and for visualizing encapsulated cellular therapeutics15,16. We here for the first time provide proof-of-principle for the generation of 19F MRI-detectable polymeric scaffolds, which can be used as a starting material to develop imageable vascular grafts, and which may serve as a basis for theranostic tissue engineering.
Supporting information is available online. This includes Materials and Methods, plus 1 additional scheme and 2 figures.
The authors gratefully acknowledge Rainer Haas (DWI) for performing the GPC analysis. This work is financially supported by the NRW/EU-Ziel 2-Programm (EFRE) 2007-2013: “Entwicklung und Bildgebung patientenoptimierter Implantate”, by the Helmholtz-Society Portfolio Grant “Technologie und Medizin: Multimodale Bildgebung zur Aufklärung des In-vivo-Verhaltens von polymeren Biomaterialien”, by the European Research Council (ERC Starting Grant 309495: NeoNaNo), and by the ERS Boost Fund Program at RWTH Aachen University.