Critical-sized bone defects can result from trauma, inflammation, and tumor resection. Such bone defects, often have irregular shapes, resulting in the need for new technologies to produce suitable implants. Bioprinting is an additive manufacturing method to create complex and individualized bone constructs, which can already include vital cells.
In this study, we present extrusion-based printing for the creation of a mechanically stable outer ring with the shape of a cylinder. The digital light processing technology was applied to produce a biologized inner core, which fits into the outer ring. Both scaffolds contained calcium phosphate, which is known to induce osteogenic differentiation of stem cells.
The model with the spongiosa-like structure was created in python based on the signed distance functions. The inner construct (diameter: 3.5 mm, height: 10 mm) and the outer construct in form of a ring (diameter: 20 mm, height: 10 mm) have an irregular, interconnected porous structure with a diameter of 2 mm +/- 0.2 mm standard deviation, which mimic the natural spongiosa structure of bone.
For digital light processing printing technology, the Lumen X (CELLINK, Holecombe, USA) was used. The Photoink polyethylene glycol diacrylate (PEGDA) was modified with calcium phosphate nanopowder (> 150 nm particle size). Before printing, human mesenchymal stem cells (hMSC) (3 x 106 cells/ml) were encapsulated into the bioink. During the layer-by-layer printing process, the bioink was exposed to ultraviolet light (405 nm) to initiate polymerization.
Extrusion-based printing was conducted using the BIO X6 (CELLINK). Polycaprolactone (PCL) (80 kDa) was combined under heating with calcium phosphate nanopowder in a ratio 1:8 (w/w). After printing, 5 x 106 hMSC were seeded on the construct with the help of a rotation incubator.
The viability of encapsulated cells was examined with a live/dead staining using calceinAm and propidium iodide, respectively.
Digital light processing: The encapsulated hMSC inside the printed construct of PEGDA-calcium phosphate construct showed a typical elongated morphology and partly formed cell clusters. The life/dead staining revealed, that hMSC were vital over a time span of 22 days in the printed PEGDA construct.
Extrusion-based printing: We were able to print a highly accurate ring construct with an interconnected pore structure.The PCL combined with calcium phosphate particles resulted in a precise printed construct, which corresponded to the 3D model. The bioink containing calcium phosphate nanoparticles had a higher printing accuracy compared to PCL alone. We found that hMSC cultured on the construct settled in close proximity to the calcium phosphate particles. The hMSC were vital for 22 days on the construct as demonstrated by life/dead staining.
With both printing technologies, it was possible to print spongiosa-like structures. The PCL scaffold offered sufficiently strong mechanics similar to bone. To improve the biological properties of the scaffold, a soft spongiosa-like structure printed with PEGDA could serve as an inner filling. Composite materials mimic bone tissue better than one of the presented materials alone and therefore represents a promising option for the use in regenerative medicine.