mSLAb - An open-source masked stereolithography (mSLA) bioprinter.

3D bioprinting is a tissue engineering approach using additive manufacturing to fabricate tissue equivalents for regenerative medicine or medical drug testing. For this purpose, biomaterials that provide the essential microenvironment to support the viability of cells integrated directly or seeded after printing are processed into three-dimensional (3D) structures. Compared to extrusion-based 3D printing, which is most commonly used in bioprinting, stereolithography (SLA) offers a higher printing resolution and faster processing speeds with a wide range of cell-friendly materials such as gelatin- or collagen-based hydrogels and SLA is, therefore, well suited to generate 3D tissue constructs. While there have been numerous publications of conversions and upgrades for extrusion-based printers, this is not the case for state-of-the-art SLA technology in bioprinting. The high cost of proprietary printers severely limits teaching and research in SLA bioprinting. With mSLAb, we present a low-cost and open-source high-resolution 3D bioprinter based on masked SLA (mSLA). mSLAb is based on an entry-level (€350) desktop mSLA printer (Phrozen Sonic Mini 4 K), equipped with temperature control and humidification of the printing chamber to enable the processing of cell-friendly hydrogels. Additionally, the build platform was redesigned for easy sample handling and microscopic analysis of the printed constructs. All modifications were done with off-the-shelf hardware and in-house designed 3D printed components, printed with the same printer that was being modified. We validated the system by printing macroscopic porous scaffolds as well as hollow channels from gelatin-based hydrogels as representative structures needed in tissue engineering.

Synthetic cell-based materials extract positional information from morphogen gradients.

Biomaterials composed of synthetic cells have the potential to adapt and differentiate guided by physicochemical environmental cues. Inspired by biological systems in development, which extract positional information (PI) from morphogen gradients in the presence of uncertainties, we here investigate how well synthetic cells can determine their position within a multicellular structure. To calculate PI, we created and analyzed a large number of synthetic cellular assemblies composed of emulsion droplets connected via lipid bilayer membranes. These droplets contained cell-free feedback gene circuits that responded to gradients of a genetic inducer acting as a morphogen. PI is found to be limited by gene expression noise and affected by the temporal evolution of the morphogen gradient and the cell-free expression system itself. The generation of PI can be rationalized by computational modeling of the system. We scale our approach using three-dimensional printing and demonstrate morphogen-based differentiation in larger tissue-like assemblies.

Pilus-1 Backbone Protein RrgB of Streptococcus pneumoniae Binds Collagen I in a Force-Dependent Way.

Attachment to host tissue is a prerequisite for successful host colonization and invasion of pathogens. Many pathogenic bacteria use surface appendices, called pili, to bind and firmly attach to host tissue surfaces. Although it has been speculated that the laterally positioned D3 domain of the pilus-1 backbone protein RrgB of Streptococcus pneumoniae may promote bacterial-host interaction, via adhesion to extracellular matrix molecules, such as collagen, earlier studies showed no affinity of RrgB to collagen I. Using atomic force microscopy-based single molecule force spectroscopy combined with lateral force microscopy, we show that under mechanical load, RrgB in fact binds to human collagen I in a force-dependent manner. We observe exceptionally strong interactions, with interaction forces reaching as much as 1500 pN, and we show that high force loading and shearing rates enhance and further strengthen the interaction. In addition, the affinity of RrgB to collagen I under mechanical load not only depends on the orientation of the D3 domain but also on the orientation of the collagen fibrils, relative to the pulling direction. Both exceptionally high binding forces and force-induced bond strengthening resemble the behavior of so-called catch bonds, which have recently been observed in bacterial adhesins, but have not been reported for multimeric backbone subunits of virulence related pili.