‎3D Bioprinting of Complex Hydrogel ‎Structures by ‎‏‎the Aid of Nanoclay‎-‎Hydrogel ‎Composite ‎Support‎-‎Bath

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Bioprinting created revolutionary perspectives not only limited to fabrication of ‌‎structures geometrically similar to the native tissues, but also in providing biochemically similar ‎complex microenvironment for cells to induce and guide functionalities in tissue regeneration. In this respect, hydrogels showed great potential in ‎providing a favorable ‎microenvironment mimicking the natural three-‎dimensional (3D) extracellular matrix. The challenges arise with bioprinting process for the hydrogel, which needs to ‎have a particular sol-gel transition state without clogging the extrusion nozzle and to keep its ‎shape fidelity after deposition. In addition, it is difficult to keep the structure of such a low ‎mechanical strength of printed biomaterials in overhanging structures.

3D bioprinting in a sacrificial support-bath, is an emerging solution for ‎fabrication of complex hydrogel-based overhanging structures. The support-‎bath material must possess a rigid matrix that yields by a passing nozzle and rapidly ‎recovers itself after nozzle motion. Meanwhile it needs to hold bioprinted structure by providing necessary gelation ‎to the extruded hydrogel before spreading into support-bath and to allow the ‎integration of the subsequent layers without clogging the nozzle. ‎‎A number of materials have been implemented as a sacrificial support-bath in direct free form ‎‎writing of ‎‎hydrogels‎. ‎However, ‎their applications are limited for several reasons including ‎‎ionic sensitivity, ‎‎working temperature range and possible reactions with the extruded ‎‎hydrogel‎. ‎Pluronic (PF) ‎is one of ‎‎the biocompatible materials which has been employed as ‎‏fugitive bioink and support ‎bath due to its ‎‎thermoreversible sol-gel phase transition property. ‎However, due to its mechanical weakness and ‎‎tendency of quick dissolving ‎in physiological ‎‏conditions‎, ‎it might not be able to provide enough support ‎‎required for long‎-‎lasting printing ‎‏processes‎. ‎Moreover‎, ‎pure PF‎ ‎as a sacrificial ‎support-bath, ‎‎did not show promising ‎properties regarding harvesting of the printed structure.

Laponite, a synthetic biocompatible nanoclay mostly known as a rheology-modifier with ‎enhanced ‎thixotropic behavior, has been utilized as a support-bath material. However, ‎instability in the rheological ‎properties does not allow printing of low-viscosity hydrogels in ‎Laponite support bath, which is ‎unfavorable for the encapsulated cells and hence low ‎efficiency. ‎ Despite their unique properties, PF and Laponite RDS have various drawbacks ‎when they are ‎employed as a support bath individually‎. ‎In the work we described in the recent ‏Scientific Reports ‎publication, a blend of PF and nanoclay (Laponite RDS) in ‎the presence of calcium chloride (CaCl2) was utilized with the aim of benefit the distinct characteristics of ‎each, namely the ‎thermoresponsive gelation of PF and the thixotropic behavior of Laponite‎.‎‏ ‏To have optimum rheological properties for printing in a long time printing and recovery ‎process of the structure, various concentrations of PF-Laponite RDS and CaCl2 were evaluated and, an ‎optimum rheological properties were achieved for support-bath. Optimized printing ‎parameters allowed printing of computer aided design (CAD) hollow and overhanging structures ‎from low viscous sodium alginate solution in a long-time process. The homogeneous ‎distribution of cells with high viability revealed a cell friendly bioprinting process and to be ‎further used for various tissue engineering applications. We believe that PF-Laponite RDS composite ‎support-bath will be further modified for other types of hydrogels by providing appropriate ‎crosslinking mechanism.

‎(a and c) CAD models, (b and d) digital images of 3D printed structures after recovery from ‎support-bath for star and nose shape, respectively.(a) Bioprinted tubular cell-laden alginate structure after recovery from support-bath. (b) ‎Confocal microscopy image of live/dead cells of bioprinted structure after 3 days of incubation
(a) Bioprinted tubular cell-laden alginate structure after recovery from support-bath. (b) ‎Confocal microscopy image of live/dead cells of bioprinted structure after 3 days of incubation

Fast forward 3D printing video of a vascular-like structure and the leakage test which a dye passes through its lumen are available here: Fast forward printing demo and Leakage test

You can access the full article from the link below:

Scientific Reports

Members of 3D Bioprinting Lab in Sabanci University (Ferdows Afghah, Dr. Mine Altunbek and Caner Dikyol) have contributed in this research under supervision of Prof. Bahattin Koc.

Ferdows Afghah

PhD Candidate, Sabanci University