Development of Printable Natural Cartilage Matrix Bioink for 3D Printing of Irregular Tissue Shape

Abstract

The extracellular matrix (ECM) is known to provide instructive cues for cell attachment, proliferation, differentiation, and ultimately tissue regeneration. The use of decellularized ECM scaffolds for regenerative-medicine approaches is rapidly expanding. In this study, cartilage acellular matrix (CAM)-based bioink was developed to fabricate functional biomolecule-containing scaffolds. The CAM provides an adequate cartilage tissue-favorable environment for chondrogenic differentiation of cells. Conventional manufacturing techniques such as salt leaching, solvent casting, gas forming, and freeze drying when applied to CAM-based scaffolds cannot precisely control the scaffold geometry for mimicking tissue shape. As an alternative to the scaffold fabrication methods, 3D printing was recently introduced in the field of tissue engineering. 3D printing may better control the internal microstructure and external appearance because of the computer-assisted construction process. Hence, applications of the 3D printing technology to tissue engineering are rapidly proliferating. Therefore, printable ECM-based bioink should be developed for 3D structure stratification. The aim of this study was to develop printable natural CAM bioink for 3D printing of a tissue of irregular shape. Silk fibroin was chosen to support the printing of the CAM powder because it can be physically cross-linked and its viscosity can be easily controlled. The newly developed CAM-silk bioink was evaluated regarding printability, cell viability, and tissue differentiation. Moreover, we successfully demonstrated 3D printing of a cartilage-shaped scaffold using only this CAM-silk bioink. Future studies should assess the efficacy of in vivo implantation of 3D-printed cartilage-shaped scaffolds.

Keywords: 3D printing; Cartilage matrix; Extracellular matrix bioink; Silk fibroin; Trochlea.

Fig. 1

Bioink printability testing. A–F Extrusion testing and G measurement of viscosity of CAM-silk bioinks. A 8% silk fibroin–only and B 5% CAM-silk, C 10% CAM-silk, D 15% CAM-silk, E 18% CAM-silk and F 20% CAM-silk bioinks. The silk-only and 5, 10, and 15% CAM-silk bioinks could not be stacked layer by layer. In contrast, 18 and 20% CAM-silk bioinks could be extruded as a fine-resolution filament. Yellow arrows indicate the disconnected extruding lines of bioink. G Viscosity increased gradually with the amount of CAM added into the silk solution (**p < 0.01, ***p < 0.001)

Fig. 2

Gross examination and mechanical-strength analysis. A Morphological features of printed rectangular scaffolds (10 × 10 × 3 mm). Thickness, grid interval, and porosity of the printed scaffolds were quantitatively analyzed in Table 1. B Mechanical-strength analysis of the printed CAM-silk scaffolds. Compressive modulus of the CAM-silk scaffolds was tunable by varying the cross-linking method (*p < 0.05, ***p < 0.001)

Fig. 3

Degradation profiles of 3D-printed scaffolds. A Gross shapes of CAM-silk and PCL scaffolds incubated for 7 d with collagenase. Although non-cross-linked CAM-silk scaffolds collapsed after only 1 d, EDC-M and PCL groups appeared to well maintain the printed 3D structure. B Quantitative analysis of degradation of the 3D-printed scaffolds. EDC-M scaffolds did not degrade for over a week (*p < 0.05, ***p < 0.001)

Fig. 4

Cell compatibility testing. A Cell-seeding efficiency of 3D-printed scaffolds. B Quantification of cell proliferation. Cell growth rates were evaluated by means of the WST-1 kit. C Cell viability analysis by staining. Live and dead cells were stained on the CAM-silk and PCL scaffolds. D Quantitative analysis of cell viability. CAM-silk scaffolds yielded significantly higher cell viability than PCL scaffolds did (*p < 0.05, **p < 0.01)

Fig. 5

Histological analysis of differentiation. Safranin-O staining was performed to confirm that the printed scaffolds could induce the chondrogenesis of rBM-MSCs. CAM-silk printed scaffold appeared to intensively stain for cartilaginous synthesized GAGs under the same culture conditions on Day 21 as compared to printed PCL scaffolds

Fig. 6

Replication of the human articular trochlea cartilage. A Patient’s MRI image, red square indicates the image reconstructing region of 3D modeling for trochlea shape printing. B 3D trochlea modeling on the basis of an MRI image with reproduction of the real 100% scale (curved cartilage). C 3D printed trochlea shape was imitated by the 3D-printing technique using CAM-silk bioink. By means of the CAM-silk bioink, the cartilage trochlea of irregular shape was printed successfully without supporting materials