Tailoring the pore design of embroidered structures by melt electrowriting to enhance the cell alignment in scaffold-based tendon reconstruction.

Tissue engineering of ligaments and tendons aims to reproduce the complex and hierarchical tissue structure while meeting the biomechanical and biological requirements. For the first time, the additive manufacturing methods of embroidery technology and melt electrowriting (MEW) were combined to mimic these properties closely. The mechanical benefits of embroidered structures were paired with a superficial micro-scale structure to provide a guide pattern for directional cell growth. An evaluation of several previously reported MEW fiber architectures was performed. The designs with the highest cell orientation of primary dermal fibroblasts were then applied to embroidery structures and subsequently evaluated using human adipose-derived stem cells (AT-MSC). The addition of MEW fibers resulted in the formation of a mechanically robust layer on the embroidered scaffolds, leading to composite structures with mechanical properties comparable to those of the anterior cruciate ligament. Furthermore, the combination of embroidered and MEW structures supports a higher cell orientation of AT-MSC compared to embroidered structures alone. Collagen coating further promoted cell attachment. Thus, these investigations provide a sound basis for the fabrication of heterogeneous and hierarchical synthetic tendon and ligament substitutes.

Scale-up of an amoeba-based process for the production of the cannabinoid precursor olivetolic acid.

BACKGROUND: The availability of new biological platform organisms to get access to innovative products and processes is fundamental for the progress in biotechnology and bioeconomy. The amoeba Dictyostelium discoideum represents a novel host system that has recently been employed for both the discovery of new natural products and as a cell factory for the production of bioactive compounds such as phytochemicals. However, an essential parameter to evaluate the potential of a new host system is the demonstration of its scalability to allow industrial applicability. Here, we aimed to develop a bioprocess for the production of olivetolic acid, the main precursor of cannabinoids synthesized by a recently engineered D. discoideum strain. RESULTS: In this study, a sophisticated approach is described to scale-up an amoeba-based polyketide production process in stirred tank bioreactors. Due to the shear sensitivity of the cell wall lacking amoebae, the maximum local energy dissipation rate (εmax) was selected as a measure for the hydromechanical stress level among different scales. By performing 1.6-L scale batch fermentations with different stress conditions, we determined a maximum tolerable εmax of 3.9 W/kg for D. discoideum. Further, we used this parameter as scale-up criterion to develop a bioprocess for olivetolic acid production starting from a 7-L stirred tank reactor to the industrially relevant 300-L scale with a product concentration of 4.8 µg/L, a productivity of 0.04 µg/L/h and a yield of 0.56 µg/g glucose. CONCLUSION: We developed a robust and reliable scale-up strategy for amoeba-based bioprocesses and evaluated its applicability for the production of the cannabinoid precursor olivetolic acid. By determining the maximum tolerable hydromechanical stress level for D. discoideum, we were able to scale-up the process from shake flasks to the 300-L stirred tank reactor without any yield reduction from cell shearing. Hence, we showed the scalability and biotechnological exploitation of amoeba-based processes that can provide a reasonable alternative to chemical syntheses or extractions of phytochemicals from plant biomass.