Evaluation of Alginate-Based Bioinks for 3D Bioprinting, Mesenchymal Stromal Cell Osteogenesis, and Application for Patient-Specific Bone Grafts

Abstract

To realize the promise of 3D bioprinting, it is imperative to develop bioinks that possess the biological and rheological characteristics needed for the printing of cell-laden tissue grafts. Alginate is widely used as a bioink because its rheological properties can be modified through pre-crosslinking or the addition of thickening agents to increase printing resolution. However, modification of alginate's physicochemical characteristics using common crosslinking agents can affect its cytocompatibility. Therefore, we evaluated the printability, physicochemical properties, and osteogenic potential of four common alginate bioinks: alginate-CaCl2 (alg-CaCl2), alginate-CaSO4 (alg-CaSO4), alginate-gelatin (alg-gel) and alginate-nanocellulose (alg-ncel) for the 3D bioprinting of patient-specific osteogenic grafts. While all bioinks possessed similar viscosity, printing fidelity was lower in the pre-crosslinked bioinks. When used to print geometrically defined constructs, alg-CaSO4 and alg-ncel exhibited higher mechanical properties and lower mesh size than those printed with alg-CaCl2 or alg-gel. The physical properties of these constructs affected the biological performance of encapsulated bone marrow-derived mesenchymal stromal cells (MSCs). Cell-laden constructs printed using alg-CaSO4 and alg-ncel exhibited greater cell apoptosis and contained fewer living cells 7 days post-printing. In addition, effective cell-matrix interactions were only observed in alg-CaCl2 printed constructs. When cultured in osteogenic media, MSCs in alg-CaCl2 constructs exhibited increased osteogenic differentiation compared to the other three bioinks. This bioink was then used to 3D print anatomically accurate cell-laden scaphoid bones that were capable of partial mineralization after 14 days of in vitro culture. These results highlight the importance of bioink properties to modulate cell behavior and the biofabrication of clinically relevant bone tissues.

3D Printed Osteoblast–Alginate/Collagen Hydrogels Promote Survival, Proliferation and Mineralization at Low Doses of Strontium Calcium Polyphosphate

“…The presence of the carboxylic acids in the alginate polymeric structure allows for the interaction between divalent cations, permitting ionic crosslinking [ 33 , 34 ]. Previous studies involving bioprinted alginate have used CaCl 2 –Alginate inks that were crosslinked prior to 3D printing at low concentrations [ 35 ]. Unfortunately, the addition of SCPP to the alginate prior to printing resulted in compact gelation within the printing cartridges that were unable to be extruded; hence, the SCPP and CaCl 2 were added after printing.…”

Solvent types used for the preparation of hydrogels determine their mechanical properties and influence cell viability through gelatine and calcium ions release

“…However, one should remember that an increase in rigidity can lead to a decrease in hydrogel permeability and thus cell survival. [76][77][78] The rigidity of a hydrogel material that is intended to mimic the extracellular matrix is one of the major factors regulating many cellular activities, including proliferation, apoptosis, and differentiation, among others. The permeability of such material to nutrients and oxygen is a second important factor in maintaining the viability and homeostasis of the cells that reside within such a material 76 and mainly depends on the material's porosity.…”

“…In the case of hydrogels, it is very common for research to be conducted to improve mechanical properties ‐ most notably increasing rigidity. However, one should remember that an increase in rigidity can lead to a decrease in hydrogel permeability and thus cell survival 76–78 . The rigidity of a hydrogel material that is intended to mimic the extracellular matrix is one of the major factors regulating many cellular activities, including proliferation, apoptosis, and differentiation, among others.…”

Bioprinting of Stem Cells in Multimaterial Scaffolds and Their Applications in Bone Tissue Engineering

“…After 2 weeks, calcification was present with positive alizarin red staining. Despite the increase in calcification, deposition was not homogenous and was focused near the peripheral edges [111]. Future applications of cell-laden bone fabrication should be explored with a focus on vascularization and improved mechanical properties as alginate concentration may impact nutrient and waste diffusion [112].…”