Volume-by-volume bioprinting of chondrocytes-alginate bioinks in high temperature thermoplastic scaffolds for cartilage regeneration

Abstract

3D bioprinting represents a novel advance in the area of regenerative biomedicine and tissue engineering for the treatment of different pathologies, among which are those related to cartilage. Currently, the use of different thermoplastic polymers, such as PLA or PCL, for bioprinting processes presents an important limitation: the high temperatures that are required for extrusion affect the cell viability and the final characteristics of the construct. In this work, we present a novel bioprinting process called volume-by-volume (VbV) that allows us to preserve cell viability after bioprinting. This procedure allows cell injection at a safe thermoplastic temperature, and also allows the cells to be deposited in the desired areas of the construct, without the limitations caused by high temperatures. The VbV process could make it easier to bring 3D bioprinting into the clinic, allowing the generation of tissue constructs with polymers that are currently approved for clinical use.

Keywords: Bioprinting; additive manufacturing; cartilage; engineering; regenerative medicine; scaffold.

Figure 1.

Bioprinter characteristics, hardware and software of the bioprinting system. Process flow of bioprinting system (a). System and bioprinter images (b). (A color version of this figure is available in the online journal.)

Figure 2.

Bioprinter configuration. Layout of the Designer GUI (a) and scaffold parameter configuration (b). Example of VbV configuration process selecting the layer in which VbV will take place, the volume to be injected and the infill model (c). (A color version of this figure is available in the online journal.)

Figure 3.

Bioprinter system. Configuration of the printing head, with the FDM unit (right), a syringe with a pink needle (T0) and two syringes with a blue tip (T1,T2) (left) (a). Injection volume filling after a few layers in a cylindrical shape (b). Representative examples of shapes with different mesh structures that can be printed (c). (A color version of this figure is available in the online journal.)

Figure 4.

Comparison between conventional FDM deposition and VbV printing procedure. Schematic representation of the printing procedures found in the literature with restricted geometries to avoid contact of the cell-laden material (pink) with the high temperature parts of the printed thermoplastics (grey). FDM deposition of the first layer and filling the spaces with the cell-laden materials. Zoom of the space filling, FDM of next layer, and restricted multimaterial scaffold (a).Working process diagram represented by a schematic representation of the VbV printing procedure (b). FDM deposition of the x layers without restrictions in the mesh geometry. Zoom of the volume to be filled with the cell-loaded bioink, and injection in the N points selected by the software (c). (A color version of this figure is available in the online journal.)

Figure 5.

IVF printing process. Schematic images of the bioprinter with the FDM extruder (right) and the three syringes (T0,T1 and T2) (a) and the bioprinting process with the FDM deposition of four layers of PLA by the nozzle, and the injection of alginate with chondrocytes (T0) and calcium chloride (T1) in the selected points (b). Images of representative 3D printed scaffolds with cells embedded in alginate and cultured during seven days (c). (A color version of this figure is available in the online journal.)

Figure 6.

Effect of the VbV bioprinting process on cell viability. Characterization of freshly isolated chondrocytes that displayed a typical polygonal shape, high expression of collagen 2 (Col2) and proteoglycans. Original magnification: 10× (a). Cell viability values of chondrocytes manually printed with alginate (ALG) and bioprinted using the VbV method with alginate and alginate in combination with PLA. *P < 0.05 compared with control cells before the printing process (b). Cell proliferation using Alamar blue assay at different time points (c). Confocal laser scanning microscopy images of bioprinted human chondrocytes stained with CTG (green) and DAPI (blue) after 24 h and seven days. Scale bars indicate 100 µm (d). (A color version of this figure is available in the online journal.)