A Novel Plasma-Based Bioink Stimulates Cell Proliferation and Differentiation in Bioprinted, Mineralized Constructs

Abstract

Extrusion-based bioprinting, also known as 3D bioplotting, is a powerful tool for the fabrication of tissue equivalents with spatially defined cell distribution. Even though considerable progress has been made in recent years, there is still a lack of bioinks which enable a tissue-like cell response and are plottable at the same time with good shape fidelity. Herein, we report on the development of a bioink which includes fresh frozen plasma from full human blood and thus a donor/patient-specific protein mixture. By blending of the plasma with 3 w/v% alginate and 9 w/v% methylcellulose, a pasty bioink (plasma-alg-mc) was achieved, which could be plotted with high accuracy and furthermore allowed bioplotted mesenchymal stromal cells (MSC) and primary osteoprogenitor cells to spread within the bioink. In a second step, the novel plasma-based bioink was combined with a plottable self-setting calcium phosphate cement (CPC) to fabricate bone-like tissue constructs. The CPC/plasmaalg-mc biphasic constructs revealed open porosity over the entire time of cell culture (35 d), which is crucial for bone tissue engineered grafts. The biphasic structures could be plotted in volumetric and clinically relevant dimensions and complex shapes could be also generated, as demonstrated for a scaphoid bone model. The plasma bioink potentiated that bioplotted MSC were not harmed by the setting process of the CPC. Latest after 7 days, MSC migrated from the hydrogel to the CPC surface, where they proliferated to 20-fold of the initial cell number covering the entire plotted constructs with a dense cell layer. For bioplotted and osteogenically stimulated osteoprogenitor cells, a significantly increased alkaline phosphatase activity was observed in CPC/plasmaalg-mc constructs in comparison to plasma-free controls. In conclusion, the novel plasma-alg-mc bioink is a promising new ink for several forms of bioprinted tissue equivalents and especially gainful for the combination with CPC for enhanced, biofabricated bonelike constructs.

Methylcellulose – a versatile printing material that enables biofabrication of tissue equivalents with high shape fidelity

“…Due to this reason, commercially available mc usually is characterized by its viscosity (of a 2% solution at 20°C) and the molecular weight is a recalculated value, which does not allow drawing conclusions for the distribution of the molecular weight. We found that most studies [32][33][34][35][36][37][38][39][40][41] used an mc with a given viscosity of 4000 mPa s (M n ≈ 86 kDa); 30 these studies have in common to have achieved printing of multiple layers and only limited collapse of predesigned macropores. Other studies 42,43 reported about the use of mc with a given viscosity of 15 mPa s (M n ≈ 14 kDa) and found significant improvements of the printed shape fidelity in presence of mc compared to mc-free controls, but those structures lacked the evidence of multiple layer stacking.…”

“…Whereas the good printability of the blend combination was maintained ( printing of centimetre-scaled complexly shaped constructs with more than 50 layers was demonstrated), the proteins of the blood plasma significantly increased the cell viability and allowed spreading of osteoprogenitor cells within the bioink. 36 Beside alginate, other (bio-)polymers were blended with mc obtaining improved bioinks providing high shape fidelity and good cytocompatibility. Already in 2012, the printability of 2% hyaluronic acid (HA) blended with 7% mc was described as significantly improved compared to a range of other polymers.…”

Formulation and Characterization of Alginate Dialdehyde, Gelatin, and Platelet-Rich Plasma-Based Bioink for Bioprinting Applications

“…In a recent study, Tilman et al, showed that a plasma-alginate-based bioink promotes cell growth and proliferation. Plasma contains growth factors and proteins, which helps cellular adhesion; these proteins also play an important role in cell-matrix interactions [27]. Hemocompatibility of a bioink is an important aspect, as 3D scaffolds materials are used to treat wounds and injuries to the patients, thus having a short-or long-term exposure of the material to the blood.…”

“…In a recent study, Tilman et al showed that a plasma-alginate-based bioink promotes cell growth and proliferation. Plasma contains growth factors and proteins, which helps cellular adhesion; these proteins also play an important role in cell-matrix interactions [27]. showed ≥100% cell viability, and diluted phenol (positive cytotoxic control) showed <10% cell viability, as anticipated (100% extracts: undiluted culture medium with extracts of the hydrogels, at 50%, 25%, and 12.5%; v/v extracts: undiluted culture medium with extracts of the hydrogels mixed with fresh culture medium in 1:2, 1:4, and 1:8 v/v ratios, respectively).…”

3D Printed Calcium Phosphate Cement (CPC) Scaffolds for Anti-Cancer Drug Delivery

“…Similarly, Ahlfeld et al, 2020, investigated a two-fold study in which a novel plasma-based bioink was combined with a printable self-setting CPC to fabricate bone-like tissue constructs. The authors concluded that their developed novel bioink was a promising platform for tissue engineering applications supplemented with the combination of CPC for enhanced bioprinted bone-like constructs [13]. Trombetta et al 2020, reported 3D printing of bioresorbable CPC scaffolds for sustained antimicrobial drug release and investigated its efficacy of femoral implant-associated osteomyelitis in vivo.…”