Comparison of Scaffolds Fabricated via 3D Printing and Salt Leaching: In Vivo Imaging, Biodegradation, and Inflammation

Abstract

In this work, we prepared fluorescently labeled poly(ε-caprolactone-ran-lactic acid) (PCLA-F) as a biomaterial to fabricate three-dimensional (3D) scaffolds via salt leaching and 3D printing. The salt-leached PCLA-F scaffold was fabricated using NaCl and methylene chloride, and it had an irregular, interconnected 3D structure. The printed PCLA-F scaffold was fabricated using a fused deposition modeling printer, and it had a layered, orthogonally oriented 3D structure. The printed scaffold fabrication method was clearly more efficient than the salt leaching method in terms of productivity and repeatability. In the in vivo fluorescence imaging of mice and gel permeation chromatography of scaffolds removed from rats, the salt-leached PCLA scaffolds showed slightly faster degradation than the printed PCLA scaffolds. In the inflammation reaction, the printed PCLA scaffolds induced a slightly stronger inflammation reaction due to the slower biodegradation. Collectively, we can conclude that in vivo biodegradability and inflammation of scaffolds were affected by the scaffold fabrication method.

Keywords: biodegradation; in vivo imaging; printing; salt-leaching; scaffold.

Figure 1

Schematic illustration for the comparison of scaffolds fabricated via 3D printing and salt leaching: In vivo imaging, biodegradation, and inflammation (the images were drawn by J.Y.P. and M.S.K. in the Adobe Photoshop 7.0 software).

Figure 2

Schematic illustration of (a) poly(ε-caprolactone-ran-lactic acid) (PCLA), (b) PCLA-fluorescein isothiocyanate (PCLA-F), and (c) PCLA-rhodamine isothiocyanate (PCLA-R) preparation and images of (d) the salt-leached and (e) the printed PCLA-F and PCLA-R scaffolds. Subcutaneous implantation of salt-leached and printed PCLA-F and PCLA-R scaffolds on the dorsal sides of (f) nude mice for fluorescence imaging and (g) Sprague-Dawley (SD) rats to assess biodegradation and inflammation.

Figure 3

In vivo fluorescence images of nude mice recorded over 16 weeks following the implantation of (a) salt-leached and (b) printed PCLA-F and PCLA-R scaffolds.

Figure 4

Time since implantation versus the molecular weights of the degraded scaffolds based on the maximum gel permeation chromatography (GPC) signals (-■-) and green fluorescence intensities (-●-) of (a) salt-leached and (b) printed PCLA-F scaffolds measured over 16 weeks. The tissue volumes were calculated after H&E staining (-▲-).

Figure 5

Cross-sectional SEM images of salt-leached and printed PCLA scaffolds removed from SD rats four, eight, and sixteen weeks after implantation. Scale bars = 100 μm.

Figure 6

(a) Photographs and (b) changes in the GPC spectra of salt-leached and printed PCLA scaffolds removed from SD rats four, eight, and sixteen weeks after implantation.

Figure 7

¹H NMR spectra of a PCLA scaffold (a) before in vivo degradation and (b–d) eight weeks after implantation; (b) the crude mixture; (c) components isolated in n-hexane and ethyl ether; and (d) insoluble components.

Figure 8

H&E staining images of salt-leached and printed PCLA scaffolds excised four, eight, and sixteen weeks after implantation. Blood vessels are indicated by arrows. Scale bars = 200 μm.

Figure 9

(a) Immunofluorescence ED1 staining images and (b) number of ED1-positive cells on salt-leached and printed PCLA scaffolds four, eight, and sixteen weeks after implantation. Scale bars = 100 μm, * p < 0.001.