Introduction

Biomaterials with tunable mechanical properties that can be processed in various sizes and shapes are potentially very interesting for various clinical applications. Porous biocompatible scaffolds may carry drugs and ensure their slow release, or they can be seeded with stem cells and enable regeneration of the desired tissue. The scaffolds need to mimic the mechanical properties of the target tissue and be degradable in a chosen time frame. In this study, we tested 3D-printed gelatin-based scaffolds with an implemented ¹⁹F MRI tracer for their monitoring in vivo using ¹⁹F magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI). While ¹⁹F MRI provides specific hotspot imaging, which can be easily colocalized with standard anatomical ¹H MRI, MRS enables quantification for estimation of the scaffold dissolution speed. Combination of ¹H and ¹⁹F MRI supplemented by ¹⁹F MRS represents a tool applicable even in future clinical practice.

Materials and Methods

Gelatin-based scaffolds with implemented ¹⁹F MRI tracer were prepared. Amine moieties of the starting material (gelatin-based hydrogel) were converted into methacrylamide moieties (methacrylated portion was ≈ 100% - G1, ≈ 70% - G2, ≈ 30% - G3)¹. Subsequently, the gels were crosslinked with N-(2,2‑difluoroethyl)acrylamide (DFEA) monomers, which enabled scaffold detection by ¹⁹F MRI/MRS and controlled modification of mechanical properties and biodegradability. Scaffolds G1 – G3 were 3D printed, samples with different sizes (50 – 400 mm³) were scanned in vitro by ¹H/¹⁹F MRI to estimate the detection limit. In vivo animal experiments included implantation of the scaffolds subcutaneously through an incision in the abdominal area and MR scanning of mice immediately after the implantation, 1, 2, 7, 14, 28 days after and then monthly up to nine months. Each scaffold type was tested on 7 mice. Both in vitro and in vivo MR experiments were performed using an animal 7 T scanner with a ¹H/¹⁹F rat whole-body volume coil. ¹H MR images were obtained using a gradient echo sequence (echo/repetition time TE/TR = 3.1/200 ms, flip angle FA = 50°, number of acquisitions NA = 4, matrix 256×256, field of view FOV = 30×30 mm² in axial, and 512×256, FOV = 60×30 mm² in coronal directions, slice thickness 1 mm). ¹⁹F MR images were measured using both turbospin echo (TE/TR = 8/800 ms, turbofactor 8, NA = 128, matrix 64×64, FOV = 30×30 mm² in axial, and matrix 128×64, FOV = 60×30 mm² in coronal directions, slice thickness 10 mm) and gradient echo (TE/TR = 2.5/120 ms, same geometry) sequences. ¹H and ¹⁹F images were interpolated to the same image matrix, color coded (¹H – grayscale, ¹⁹F – red scale) and merged using ImageJ software². Unlocalized ¹⁹F MR spectra for signal quantification were acquired using an FID sequence (FA = 90°, TE/TR = 3.1/1000 ms, NA = 128) and processed using a home-made script written in Matlab³.

Results and Discussion

The incorporation of DFEA monomer into the reaction mixture increased the fluorine content of the cross-linked hydrogels, which enabled reliable detection by ¹⁹F MRI/MRS and decreased hydrophilicity.
Suitable fluorine relaxation times (T₁ ≈ 400 ms, T₂ ≈ 120 ms) enabled scaffold visualization by both gradient and turbospin echo sequences within acceptable scanning times (15 minutes). In vitro MRI (Fig. 1) confirmed visibility of scaffolds down to 50 mm³ of all tested samples (G1, G2, G3), nevertheless, we consider this volume as a detection of limit for the sample with the lowest ¹⁹F content (G3).
In vivo imaging (Fig. 2) confirmed that the implanted scaffolds can be reliably visualized and monitored in time by ¹⁹F MRI. The scaffolds degraded very slowly. Dissolution depended on the level of crosslinking, as was proved by ¹⁹F MRS (Fig. 3). Signal decrease corresponding to dissolution of the scaffolds was observed 4 months after implantation in the case of G3, while G1 and G2 implants remained stable for up to 9 months. The implants caused no adverse reaction (inflammation) in experimental animals.

Conclusion

Fluorinated hydrogel scaffolds are biocompatible and suitable for regenerative medicine either as drug carriers or as matrices for cell therapies. Mechanical properties and biodegradability can be fine-tuned by controlled crosslinking. Their fate after implantation can be easily monitored in vivo by ¹⁹F MRI/MRS, which enables their visualization and quantification.

Acknowledgements

The facility infrastructure was supported by European Regional Development Fund No. CZ.02.1.01/0.0/0.0/18_046/0016045 (OPVVV project) and by Ministry of Education, Youth and Sports of the Czech Republic (Large RI Project LM2023050 Czech-BioImaging), financial support was provided by Research Foundation – Flanders (FWO, Fonds Wetenschappelijk Onderzoek – Vlaanderen, project no. 1229422N), and by European Union’s Horizon 2020 research and innovation program under grant agreement no. 828835.