Dynamische Biomaterialien

Most (bio)resins for volumetric printing (VP) are based on (meth)acrylated polymer precursors mixed with gelatine or Pluronic as thermoreversible thickening agents. Here we investigate if fast photocrosslinking reactions, like the thiol-ene, allow VP of low viscosity bioresins without the need of viscosity modifiers. We used a 4% w/v hyaluronic acid norbornene (HA-NB) resin with viscosity <0.1 Pa s, a frequently used matrix for encapsulation of cells. Using light doses in the range of 150–200 mJ cm−2, stable 8 × 5 × 2 mm3 crosslinked scaffolds with internal bifurcated channels down to ∼200 µm dimensions were printed in <30 s. At this concentration, HA methacrylate (HA-MA) resins with comparable viscosity and functionalization degree did not form stable structures. RGD-functionalized HA-NB scaffolds were printed in the presence of fibroblasts and showed homogeneous cell distribution across the scaffold, with cell viability >90%. Encapsulated cells spread and showed fibroblast-like morphology in 7 d cultures. We demonstrate volumetric bioprinting of low molecular weight HA-based bioresins without viscosity modifiers and in the absence of post-printing steps.

Living Therapeutic Materials (LTMs) are a promising alternative to polymeric drug carriers for long term release of biotherapeutics. LTMs contain living drug biofactories that produce the drug using energy sources from the body fluids. To clarify their application potential, it is fundamental to adapt biocompatibility and cytotoxicity assays applied from non-living biomaterials and therapeutics to evaluate how LTMs interact with host cells. Here, we have established a first step in this direction, by developing a practical workflow to parallelize in vitro assessment of minimal safety and cytocompatibility properties of bacterial LTMs. It allows systematic monitoring and quantification of the dynamic evolution of the bacterial population (growth, metabolic activity) in parallel to quantify the response of different mammalian cells to LTM supernatants with regards to cytotoxicity and release of pro-inflammatory cytokines over a period of 7 days using a maximum of 10 samples. The protocol was tested with a Pluronic-based thin film containing ClearColi. The results show no cytotoxic effects of ClearColi containing hydrogels in three mammalian cell lines, and no induction of pro-inflammatory cytokines under the tested conditions. This workflow represents a first step in establishing a roadmap for the safety assessment of LTMs, and investigation of biocompatibility potential of future living therapeutic devices.

Side-emitting optical fibers allow light to be deliberately outcoupled along the fiber. Introducing a customized side-emission profile requires modulation of the guiding and emitting properties along the fiber length, which is a particular challenge in continuous processing of soft waveguides. In this work, it is demonstrated that multimaterial extrusion printing can generate hydrogel optical fibers with tailored segments for light-side emission. The fibers are based on diacrylated Pluronic F-127 (PluDA). 1 mm diameter fibers are printed with segments of different optical properties by switching between a PluDA waveguiding ink and a PluDA scattering ink containing nanoparticles. The method allows the fabrication of fibers with segment lengths below 500 microns in a continuous process. The length of the segments is tailored by varying the switching time between inks during printing. Fibers with customized side-emission profiles along their length are presented. The functionality of the printed fibers is demonstrated by exciting fluorescence inside a surrounding 3D hydrogel. The presented technology and material combination allow unprecedented flexibility for designing soft optical fibers with customizable optical properties using simple processes and a medical material. This approach can be of interest to improve illumination inside tissues for photodynamic therapy (PDT).

Engineered living materials (ELMs), which usually comprise bacteria, fungi, or animal cells entrapped in polymeric matrices, offer limitless possibilities in fields like drug delivery or biosensing. Determining the conditions that sustain ELM performance while ensuring compatibility with ELM hosts is essential before testing them in vivo. This is critical to reduce animal experimentation and can be achieved through in vitro investigations. Currently, there are no standards that ensure ELM compatibility with host tissues. Towards this goal, we designed a 96-well plate-based screening method to streamline ELM growth across culture conditions and determine their compatibility potential in vitro. We showed proliferation of three bacterial species encapsulated in hydrogels over time and screened six different cell culture media. We fabricated ELMs in bilayer and monolayer formats and tracked bacterial leakage as a measure of ELM biocontainment. After screening, an appropriate medium was selected that sustained growth of an ELM, and it was used to study cytocompatibility in vitro. ELM cytotoxicity on murine fibroblasts and human monocytes was studied by adding ELM supernatants and measuring cell membrane integrity and live/dead staining, respectively, proving ELM cytocompatibility. Our work illustrates a simple setup to streamline the screening of compatible environmental conditions of ELMs with the host.

Photocrosslinkable formulations based on the radical thiol-ene reaction are considered better alternatives than methacrylated counterparts for light-based fabrication processes. This study quantifies differences between thiol-ene and methacrylated crosslinked hydrogels in terms of precursors stability, the control of the crosslinking process, and the resolution of printed features particularized for hyaluronic acid (HA) inks at concentrations relevant for bioprinting. First, the synthesis of HA functionalized with norbornene, allyl ether, or methacrylate groups with the same molecular weight and comparable degrees of functionalization is presented. The thiol-ene hydrogel precursors show storage stability over 15 months, 3.8 times higher than the methacrylated derivative. Photorheology experiments demonstrate up to 4.7-times faster photocrosslinking. Network formation in photoinitiated thiol-ene HA crosslinking allows higher temporal control than in methacrylated HA, which shows long post-illumination hardening. Using digital light processing, 4% w/v HA hydrogels crosslinked with a dithiol allowed printing of 13.5 × 4 × 1 mm3 layers with holes of 100 µm resolution within 2 s. This is the smallest feature size demonstrated in DLP printing with HA-based thiol-ene hydrogels. The results are important to estimate the extent to which the synthetic effort of introducing –ene functions can pay off in the printing step.

The potential of bioactive coatings as an innovative biotechnology to overcome the mass-transfer limitations of conventional technologies when treating air pollutants, especially hydrophobic volatile organic compounds, was herein assessed. Bioactive coatings consist of active microorganisms entrapped in a polymer matrix, which needs to be porous to facilitate an effective gas pollutant exchange. To increase porosity, two additives, sucrose and glycerol mixtures (Suc/Gly) and halloysite nanotubes (HNTs), were included in the bioactive coatings at two concentration levels. The toluene removals of the different bioactive coatings were studied in batch mode at low (∼300 mg m−3) and high (∼3000 mg m−3) toluene concentrations. Overall, low HNTs concentration coatings supported optimum toluene removals (>95 %), comparable to biofilm controls at both toluene concentrations. High HNTs concentration coatings and low Suc/Gly concentration coatings achieved toluene removals over 95 % after 7 toluene injections at low toluene concentration. At high toluene concentrations, these coatings eventually outperformed the biofilm controls. High Suc/Gly concentration coatings supported a limited toluene removal (4 and 1 injection at low and high toluene concentrations, respectively), attributed to a preferential consumption of sucrose over toluene. These findings were corroborated by ESEM/conventional SEM imaging, revealing porosity in the HNTs bioactive coatings, visible at both the surface and internal levels. On the contrary, more homogeneous surfaces were observed in the Suc/Gly bioactive coatings, where total polymer coalescence was partially hindered by the addition of Suc/Gly. These results paved the way towards the implementation of bioactive coating in larger bioreactors for real-life air purification.

The persistent accumulation of plastic waste, particularly polystyrene (PS), poses significant environmental challenges because of its extensive use and low recycling rates. Addressing these challenges necessitates innovative and sustainable solutions. This study presents a strategy to upcycle PS waste into valuable chemical products, including adipic acid, hexanediol, hexamethylenediamine, and nylon-6,6, using metabolically engineered Pseudomonas putida KT2440. This process involves the photolytic degradation of PS into benzoic acid, followed by microbial conversion into cis,cis-muconate (MA) and chemical synthesis of the final products. The engineered strains withstood 30 mM concentrations of PS-derived aromatics and converted them stoichiometrically into MA in the presence of glucose as a growth substrate. 13C metabolic flux analysis revealed energy and redox limitations in the presence of 25 mM benzoate and 300 mM MA. The cells responded to stress by enhancing the flux for periplasmic glucose oxidation and fluxes through the NADPH-forming dehydrogenases; this process caused more than 40 % glucose‑carbon loss into byproducts. Fine-tuned dynamic glucose and benzoate feeding enabled high-level MA production. Energy-optimized genome-reduced strains were used to increase carbon efficiency. A final MA titer of over 65 g L−1 was achieved in fed-batch fermentation. This process was demonstrated using the glucose derived from a viscose textile waste blend as the growth substrate and resulted in fully waste-based products. The resulting adipic acid and hexamethylenediamine were polymerized into nylon-6,6 with properties comparable to those of petrochemical-derived polymers, revealing a sustainable pathway for PS upcycling. This research provides a proof-of-concept for bacterial upgrading of PS-derived substrates and a viable method for managing plastic waste and producing valuable chemical products.

Focal adhesions (FAs) are dynamic structures central to cell migration, serving as mechanotransduction sites linking the extracellular matrix (ECM) to intracellular signaling pathways such as FA kinase (FAK). How FAK becomes activated in response to cell-ECM adhesive forces at single FAs to facilitate directional motion is poorly understood. Using micropillar-based force microscopy and FA-targeted FRET biosensors, we monitored real-time traction forces and FAK activity at individual FAs during assembly and disassembly. Our results demonstrate oscillatory temporal coupling of traction force and FAK activity in high-tension FAs before FA disassembly. Cross-correlation analyses revealed that force precedes FAK activation, guiding FA turnover. Atomistic molecular simulations unveiled a force-induced mechanism where traction forces disrupt autoinhibitory FERM-kinase interactions in FAK, enabling catalytic activity without structural unfolding. Our findings provide mechanistic insights into the spatiotemporal integration of mechanical forces and biochemical signaling in cell migration.

Pluronic (Plu) hydrogels mixed with variable fractions of Pluronic diacrylate (PluDA) have become popular matrices to encapsulate bacteria and control their growth in engineered living materials. Here we study the rheological response of 30 wt.% Plu/PluDA hydrogels with PluDA fraction between 0 and 1. We quantify the range of viscoelastic properties that can be covered in this system by varying in the PluDA fraction. We present stress relaxation and creep-recovery experiments and describe the variation of the critical yield strain/stress, relaxation and recovery parameters of Plu/PluDA hydrogels as function of the covalent crosslinking degree using the Burgers and Weilbull models. The analyzed hydrogels present two stress relaxations with different timescales which can be tuned with the covalent crosslinking degree. We expect this study to help users of Plu/PluDA hydrogels to estimate the mechanical properties of their systems, and to correlate them with the behaviour of bacteria in future Plu/PluDA devices of similar composition.

The hierarchical design of the toe pad surface in geckos and its reversible adhesiveness have inspired material scientists for many years. Micro- and nano-patterned surfaces with impressive adhesive performance have been developed to mimic gecko's properties. While the adhesive performance achieved in some examples has surpassed living counterparts, the durability of the fabricated surfaces is limited and the capability to self-renew and restore function—inherent to biological systems—is unimaginable. Here the morphogenesis of gecko setae using skin samples from the Bibron´s gecko (Chondrodactylus bibronii) is studied. Gecko setae develop as specialized apical differentiation structures at a distinct cell–cell layer interface within the skin epidermis. A primary role for F-actin and microtubules as templating structural elements is necessary for the development of setae's hierarchical morphology, and a stabilization role of keratins and corneus beta proteins is identified. Setae grow from single cells in a bottom layer protruding into four neighboring cells in the upper layer. The resulting multicellular junction can play a role during shedding by facilitating fracture of the cell–cell interface and release of the high aspect ratio setae. The results contribute to the understanding of setae regeneration and may inspire future concepts to bioengineer self-renewable patterned adhesive surfaces.