Engineering considerations on extrusion-based bioprinting: interactions of material behavior, mechanical forces and cells in the printing needle.

Systematic analysis of the extrusion process in 3D bioprinting is mandatory for process optimization concerning production speed, shape fidelity of the 3D construct and cell viability. In this study, we applied numerical and analytical modeling to describe the fluid flow inside the printing head based on a Herschel-Bulkley model. The presented analytical calculation method nicely reproduces the results of Computational Fluid Dynamics simulation concerning pressure drop over the printing head and maximal shear parameters at the outlet. An approach with dimensionless flow parameter enables the user to adapt rheological characteristics of a bioink, the printing pressure and needle diameter with regard to processing time, shear sensitivity of the integrated cells, shape fidelity and strand dimension. Bioinks consist of a blend of polymers and cells, which lead to a complex fluid behavior. In the present study, a bioink containing alginate, methylcellulose and agarose (AMA) was used as experimental model to compare the calculated with the experimental pressure gradient. With cultures of an immortalized human mesenchymal stem cell line and plant cells (basil) it was tested how cells influence the flow and how mechanical forces inside the printing needle affect cell viability. Influences on both sides increased with cell (aggregation) size as well as a less spherical shape. This study contributes to a systematic description of the extrusion-based bioprinting process and introduces a general strategy for process design, transferable to other bioinks.

LED-enhanced biosynthesis of biologically active ingredients in callus cultures of Ocimum basilicum.

Ocimum basilicum is a medicinal plant with multiple health benefits including cardiovascular, cancer and diabetics. In the present study, the influences of light emitting diodes (LEDs) were investigated on the accumulation of biologically active ingredients in callus cultures of Ocimum basilicum. Among the various tested treatments optimum levels of Total phenolic content (TPC) was noted in callus culture grown under blue lights as compared to control, while maximum accumulation of Total flavonoid content (TFC) was noted in callus culture grown under red light as compared to control. HPLC analyses showed that highest concentrations of Rosmarinic acid (96.0 mg/g DW) and Eugenol (0.273 mg/g DW) were accumulated in blue light which was 2.46 and 2.25 times greater than control (39.0 mg/g DW, 0.171 mg/g DW), respectively. Chicoric acid (81.40 mg/g DW) optimum accumulation was noted in callus grown under the continuous white light, which was almost 4.52 times greater than control. Anthocyanins content were also analyzed, the highest amount of Peonidin (0.127 mg/g DW) and cyanidin (0.1216 mg/g DW) were found in callus culture grown under red light. These findings suggest that application of LED's is a promising strategy for enhancing production of biologically active ingredients in callus cultures Ocimum basilicum.

Spearmint R2R3-MYB transcription factor MsMYB negatively regulates monoterpene production and suppresses the expression of geranyl diphosphate synthase large subunit (MsGPPS.LSU).

Many aromatic plants, such as spearmint, produce valuable essential oils in specialized structures called peltate glandular trichomes (PGTs). Understanding the regulatory mechanisms behind the production of these important secondary metabolites will help design new approaches to engineer them. Here, we identified a PGT-specific R2R3-MYB gene, MsMYB, from comparative RNA-Seq data of spearmint and functionally characterized it. Analysis of MsMYB-RNAi transgenic lines showed increased levels of monoterpenes, and MsMYB-overexpressing lines exhibited decreased levels of monoterpenes. These results suggest that MsMYB is a novel negative regulator of monoterpene biosynthesis. Ectopic expression of MsMYB, in sweet basil and tobacco, perturbed sesquiterpene- and diterpene-derived metabolite production. In addition, we found that MsMYB binds to cis-elements of MsGPPS.LSU and suppresses its expression. Phylogenetic analysis placed MsMYB in subgroup 7 of R2R3-MYBs whose members govern phenylpropanoid pathway and are regulated by miR858. Analysis of transgenic lines showed that MsMYB is more specific to terpene biosynthesis as it did not affect metabolites derived from phenylpropanoid pathway. Further, our results indicate that MsMYB is probably not regulated by miR858, like other members of subgroup 7.