(A-31) Melt Electrowriting in Color: Introducing fluorescence to a high precision 3D printing technology

Introduction:: Three-dimensional (3D) printing technologies have become an essential tool for fabricating biomedical materials. Bioresorbable, synthetic polymers are often 3D-printed as scaffolds, and there is a need to increase printing resolutions, achieved here using melt electrowriting (MEW). This increase in resolution together with a thermal requirement for MEW results in highly porous constructs being difficult to track and image during tissue formation. Cycloparaphenylenes (CPPs) are thermally stable yet potent fluorescent molecules with variable emissions based on ring length. This study uses this new class of CPP fluorophores with a commonly used synthetic biomedical polymer, poly(ε-caprolactone) (PCL), and reveals the interaction of multiscale scaffolds with seeded cells at a clarity previously unseen in biofabrication.

Materials and Methods:: Cycloparaphenylene molecules of varying ring lengths (8, 9 or 10 rings) were synthesized using previously described methods (Lovell et al., Chem. Sci., 2019), then dissolved within dichloromethane and mixed into the PCL (Corbion PC-12) also dissolved in the same solvent. Solvent removal was conducted through high vacuum evaporation and subsequent vacuum oven treatment. The cytotoxicity of the CPP-PCL was investigated and followed ISO 10993-5 for protocol design. MEW of CPP-PCL was investigated and used to fabricate fluorescent scaffolds of varying designs and styles. Using a custom-built MEW printer, processing conditions included 6.0 kV, 2.0 bars of pressure, 80 degrees Celsius temperature, a 25 gauge nozzle, and collector distance. Solution electrospinning (SES) was also conducted with a biomaterial ink composed of CPP-PCL dissolved at a concentration of 15 weight % in a 60/40% ratio of dichloromethane and dimethylformamide, and used to fabricate electrospun membranes with various fluorescent profiles.


Results, Conclusions, and Discussions:: Results:
The CPP-PCL and the pure PCL did not demonstrate cytotoxicity. From a fluorescence perspective, the printed materials had excellent uniformity with the CPP well-distributed and without fluorescent crystal “hotspots.” A final CPP concentration of 0.01wt% was considered an optimal level over 0.001 wt%, while 0.1% produced a signal too strong. Despite MEW processing the same CPP-PCL blend for multiple days, there was no observed drop in fluorescence intensity, demonstrating that the CPP retains its florescence while maintaining thermal stability. MEW CPP-PCL scaffolds show stable fluorescent signals with low photobleaching and can be fabricated in succession to generate structures with spatially distributed fluorescent signals.


The emission wavelengths of 533, 494, and 466 nm were observed for the CPPs of ring lengths 8, 9 and 10 respectively. One interesting aspect of the CPPs is that the excitation wavelength is the same for all ring lengths, allowing for single light fluorescence (Figure 1). When comparing 0.01 wt% CPP-PCL with pure PCL, there were no changes in the jet speeds or processing conditions for 3D printing attributed to the CPP molecules. SES of CPP-PCL compared to unprocessed PCL showed that there are also not significant differences in fiber diameter upon the addition of the fluorophore molecules.
Conclusions

CPP fluorophores provide exceptional imaging capabilities when combined with PCL, a common biomedical polymer. Non-toxic and resistant to photobleaching, CPPs can be added at low concentrations to provide robust fluorescent capabilities. This capability is especially pertinent to MEW scaffolds since the high resolution, highly porous scaffolds are sometimes difficult to image. Continued in vitro analysis of the scaffolds showed the usability of scaffolds in relation to immunofluorescence staining and imaging of tissue constructs.



Discussion
Fluorophore-tagging of polymeric scaffolds, which are important biomedical materials, has primarily been conducted through surface functionalization with fluorophore molecules. However, these systems fail when heating is required while many existing fluorophores photobleach. We discovered that blending a new class of fluorophore directly with a commonly polymer provided remarkable fluorescence properties. Such fluorescence enhances both in vitro and in vivo imaging to track the progression of cell and tissue ingrowth into the scaffold.




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