4D Bioprinting of Self-Bending Scaffolds for Articular Cartilage Tissue Engineering Applications


Díaz-Payno, P.J. (Department of Biomechanical Engineering, Faculty of Mechanical, Maritime and Materials Engineering, Delft University of Technology / Department of Orthopedics and Sports Medicine, Erasmus MC University Medical Center )


"Introduction: Articular cartilage (AC) defects remain a significant clinical challenge[1]. This is partially due to the challenging nature of recapitulating the complex layered structure observed in the naturally curved AC tissue. While three-dimensional (3D) bioprinting appeared as a promising Tissue Engineering (TE) approach, it has serious limitations in the fabrication of curved constructs[2]. This has motivated the development of four-dimensional (4D) bioprinting as the next generation of biofabrication technologies, combining 3D-bioprinting with time-dependent shape transformation, and introducing time as the fourth dimension[3]. 4D-bioprinting allows for the fabrication of self-bending scaffolds and shape-transforming constructs. In this study, we report an advanced 4D-biofabrication method based on the differential swelling of a multi-material smart bioink.

Methods: Two biomaterial ink formulations with different swelling properties were selected: tyramine-functionalized hyaluronan (HAT, high-swelling) and alginate with HAT (AHAT, low-swelling). Firstly, the inks were characterized with an MCR-501 rheometer (AntonPaar) to measure their storage/elastic modulus, loss/viscous modulus, shear-thinning, and viscosity. BioX-bioprinter (Cellink) was used to fabricate a bilayered scaffold. The bottom zone was made of HAT and the top zone of AHAT. After printing, the bilayered scaffold was crosslinked in 200 mM CaCl2, and then submerged in saline or DMEM medium. Finally, human bone-marrow derived cells (hMSC) were incorporated into AHAT (top zone) before 4D-bioprinting the bilayered scaffolds. The scaffolds, cultured in chondrogenic medium for 28 days, were analyzed by live/dead and histology.

Results: Rheological characterization demonstrated that both HAT and AHAT inks had i) similar elastic, gel-like behaviors, as their elastic modulus was 8x higher than their viscous modulus; ii) shear-thinning behavior, and iii) relatively fast recovery reaching 100% and 65% (respectively) of the storage modulus. After 3D printing, AHAT showed a higher compression modulus than HAT (6.7 vs. 2.1 kPa). Upon 24 h submersion in saline HAT absorbed 2x more liquid than AHAT. The inks were 3D printed into a bilayer. After time (4D), the differential swelling between the two zones led to the scaffold’s self-bending behavior. Different scaffold designs were used to characterize the degree of curvature. The live/dead results demonstrated high cell-viability in the 4D-bioprinted scaffolds. After 28 days, the curvature was still evident, with no delamination observed, and histology suggested an increase in sGAG production.

Discussion and conclusion: A proof of concept of the recently emerged technology of 4D-bioprinting with a specific application for articular cartilage tissue engineering was achieved. We fabricated smart cell-laden scaffolds with self-bending properties for the design of curved structures mimicking the native AC tissue architecture in specific regions. This approach allowed for the fabrication of a curved bilayer made from two biocompatible and commonly used hydrogel-based materials in TE. Further studies should focus on increasing the mechanical properties of the scaffold, as well as improving the tissue formation through incorporation of tissue-specific biological cues.

Acknowledgments: Dutch Medical Delta (RegMed4D) and TUD-EMC Convergence Initiative Health & Technology.

References: [1]DeNiese P.J. et al, Arthrosc Surg Sport Med 1, 16, 2020. [2]Giannopoulos A. et al, Nat rev, 13, 701, 2016. [3]An J. et al, Int J Biop, 2, 3, 2016."


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