Glycerol-based sustainably sourced resin for volumetric printing.

Volumetric Additive Manufacturing (VAM) represents a revolutionary advancement in the field of Additive Manufacturing, as it allows for the creation of objects in a single, cohesive process, rather than in a layer-by-layer approach. This innovative technique offers unparalleled design freedom and significantly reduces printing times. A current limitation of VAM is the availability of suitable resins with the required photoreactive chemistry and from sustainable sources. To support the application of this technology, we have developed a sustainable resin based on polyglycerol, a bioderived (e.g., vegetable origin), colourless, and easily functionisable oligomer produced from glycerol. To transform polyglycerol-6 into an acrylate photo-printable resin we adopted a simple, one-step, and scalable synthesis route. Polyglycerol-6-acrylate fulfils all the necessary criteria for volumetric printing (transparency, photo-reactivity, viscosity) and was successfully used to print a variety of models with intricate geometries and good resolution. The waste resin was found to be reusable with minimal performance issues, improving resin utilisation and minimising waste material. Furthermore, by incorporating dopants such as poly(glycerol) adipate acrylate (PGA-A) and 10,12-pentacosadyinoic acid (PCDA), we demonstrated the ability to print objects with a diverse range of functionalities, including temperature sensing probes and a polyester excipient, highlighting the potential applications of these new resins.

An effective route to the additive manufacturing of a mechanically gradient supramolecular polymer nanocomposite structure.

3D Printing techniques are additive methods of fabricating parts directly from computer-aided designs. Whilst the clearest benefit is the realisation of geometrical freedom, multi-material printing allows the introduction of compositional variation and highly tailored product functionality. The paper reports a proof-of-concept additive manufacturing study to deposit a supramolecular polymer and a complementary organic filler to form composites with gradient composition to enable spatial distribution of mechanical properties and functionality by tuning the number of supramolecular interactions. We use a dual-feed extrusion 3D printing process, with feed stocks based on the supramolecular polymer and its organic composite, delivered at ratios predetermined. This allows for production of a graded specimen with varying filler concentration that dictates the mechanical properties. The printed specimen was inspected under dynamic load in a tensile test using digital image correlation to produce full-field deformation maps, which showed clear differences in deformation in regions with varying compositions, corresponding to the designed-in variations. This approach affords a novel method for printing material with graded mechanical properties which are not currently commercially available or easily accessible, however, the method can potentially be directly translated to the generation of biomaterial-based composites featuring gradients of mechanical properties.

A potential alternative to fungicides using actives-free (meth)acrylate polymers for protection of wheat crops from fungal attachment and infection.

Fungicidal compounds are actives widely used for crop protection from fungal infection, but they can also kill beneficial organisms, enter the food chain and promote resistant pathogen strains from overuse. Here we report the first field crop trial of homopolymer materials that prevent fungal attachment, showing successful crop protection via an actives-free approach. In the trial, formulations containing two candidate polymers were applied to young wheat plants that were subject to natural infection with the wheat pathogen Zymoseptoria tritici. A formulation containing one of the candidate polymers, poly(di(ethylene glycol) ethyl ether acrylate) (abbreviated DEGEEA), produced a significant reduction (26%) in infection of the crop by Z. tritici, delivering protection against fungal infection that compared favourably with three different commercially established fungicide programmes tested in parallel. Furthermore, the sprayed polymers did not negatively affect wheat growth. The two lead polymer candidates were initially identified by bio-performance testing using in vitro microplate- and leaf-based assays and were taken forward successfully into a programme to optimize and scale-up their synthesis and compound them into a spray formulation. Therefore, the positive field trial outcome has also established the validity of the smaller-scale, laboratory-based bioassay data and scale-up methodologies used. Because fungal attachment to plant surfaces is a first step in many crop infections, this non-eluting polymer: (i) now offers significant potential to deliver protection against fungal attack, while (ii) addressing the fourth and aligning with the eleventh principles of green chemistry by using chemical products designed to preserve efficacy of function while reducing toxicity. A future focus should be to develop the material properties for this and other applications including other fungal pathogens.

Hydrogels and Bioprinting in Bone Tissue Engineering: Creating Artificial Stem-Cell Niches for In Vitro Models.

Advances in bioprinting have enabled the fabrication of complex tissue constructs with high speed and resolution. However, there remains significant structural and biological complexity within tissues that bioprinting is unable to recapitulate. Bone, for example, has a hierarchical organization ranging from the molecular to whole organ level. Current bioprinting techniques and the materials employed have imposed limits on the scale, speed, and resolution that can be achieved, rendering the technique unable to reproduce the structural hierarchies and cell-matrix interactions that are observed in bone. The shift toward biomimetic approaches in bone tissue engineering, where hydrogels provide biophysical and biochemical cues to encapsulated cells, is a promising approach to enhancing the biological function and development of tissues for in vitro modeling. A major focus in bioprinting of bone tissue for in vitro modeling is creating dynamic microenvironmental niches to support, stimulate, and direct the cellular processes for bone formation and remodeling. Hydrogels are ideal materials for imitating the extracellular matrix since they can be engineered to present various cues whilst allowing bioprinting. Here, recent advances in hydrogels and 3D bioprinting toward creating a microenvironmental niche that is conducive to tissue engineering of in vitro models of bone are reviewed.

Predictive Molecular Design and Structure-Property Validation of Novel Terpene-Based, Sustainably Sourced Bacterial Biofilm-Resistant Materials.

Presented in this work is the use of a molecular descriptor, termed the α parameter, to aid in the design of a series of novel, terpene-based, and sustainable polymers that were resistant to biofilm formation by the model bacterial pathogen Pseudomonas aeruginosa. To achieve this, the potential of a range of recently reported, terpene-derived monomers to deliver biofilm resistance when polymerized was both predicted and ranked by the application of the α parameter to key features in their molecular structures. These monomers were derived from commercially available terpenes (i.e., α-pinene, ß-pinene, and carvone), and the prediction of the biofilm resistance properties of the resultant novel (meth)acrylate polymers was confirmed using a combination of high-throughput polymerization screening (in a microarray format) and in vitro testing. Furthermore, monomers, which both exhibited the highest predicted biofilm anti-biofilm behavior and required less than two synthetic stages to be generated, were scaled-up and successfully printed using an inkjet "valve-based" 3D printer. Also, these materials were used to produce polymeric surfactants that were successfully used in microfluidic processing to create microparticles that possessed bio-instructive surfaces. As part of the up-scaling process, a novel rearrangement was observed in a proposed single-step synthesis of α-terpinyl methacrylate via methacryloxylation, which resulted in isolation of an isobornyl-bornyl methacrylate monomer mixture, and the resultant copolymer was also shown to be bacterial attachment-resistant. As there has been great interest in the current literature upon the adoption of these novel terpene-based polymers as green replacements for petrochemical-derived plastics, these observations have significant potential to produce new bio-resistant coatings, packaging materials, fibers, medical devices, etc.

Application of microfluidic systems in modelling impacts of environmental structure on stress-sensing by individual microbial cells.

Environmental structure describes physical structure that can determine heterogenous spatial distribution of biotic and abiotic (nutrients, stressors etc.) components of a microorganism's microenvironment. This study investigated the impact of micrometre-scale structure on microbial stress sensing, using yeast cells exposed to copper in microfluidic devices comprising either complex soil-like architectures or simplified environmental structures. In the soil micromodels, the responses of individual cells to inflowing medium supplemented with high copper (using cells expressing a copper-responsive pCUP1-reporter fusion) could be described neither by spatial metrics developed to quantify proximity to environmental structures and surrounding space, nor by computational modelling of fluid flow in the systems. In contrast, the proximities of cells to structures did correlate with their responses to elevated copper in microfluidic chambers that contained simplified environmental structure. Here, cells within more open spaces showed the stronger responses to the copper-supplemented inflow. These insights highlight not only the importance of structure for microbial responses to their chemical environment, but also how predictive modelling of these interactions can depend on complexity of the system, even when deploying controlled laboratory conditions and microfluidics.

Ink-jet 3D printing as a strategy for developing bespoke non-eluting biofilm resistant medical devices.

Chronic infection as a result of bacterial biofilm formation on implanted medical devices is a major global healthcare problem requiring new biocompatible, biofilm-resistant materials. Here we demonstrate how bespoke devices can be manufactured through ink-jet-based 3D printing using bacterial biofilm inhibiting formulations without the need for eluting antibiotics or coatings. Candidate monomers were formulated and their processability and reliability demonstrated. Formulations for in vivo evaluation of the 3D printed structures were selected on the basis of their in vitro bacterial biofilm inhibitory properties and lack of mammalian cell cytotoxicity. In vivo in a mouse implant infection model, Pseudomonas aeruginosa biofilm formation on poly-TCDMDA was reduced by ∼99% when compared with medical grade silicone. Whole mouse bioluminescence imaging and tissue immunohistochemistry revealed the ability of the printed device to modulate host immune responses as well as preventing biofilm formation on the device and infection of the surrounding tissues. Since 3D printing can be used to manufacture devices for both prototyping and clinical use, the versatility of ink-jet based 3D-printing to create personalised functional medical devices is demonstrated by the biofilm resistance of both a finger joint prosthetic and a prostatic stent printed in poly-TCDMDA towards P. aeruginosa and Staphylococcus aureus.

Customisable Tablet Printing: The Development of Multimaterial Hot Melt Inkjet 3D Printing to Produce Complex and Personalised Dosage Forms.

One of the most striking characteristics of 3D printing is its capability to produce multi-material objects with complex geometry. In pharmaceutics this translates to the possibility of dosage forms with multi-drug loading, tailored dosing and release. We have developed a novel dual material hot-melt inkjet 3D printing system which allows for precisely controlled multi-material solvent free inkjet printing. This reduces the need for time-consuming exchanges of printable inks and expensive post processing steps. With this printer, we show the potential for design of printed dosage forms for tailored drug release, including single and multi-material complex 3D patterns with defined localised drug loading where a drug-free ink is used as a release-retarding barrier. For this, we used Compritol HD5 ATO (matrix material) and Fenofibrate (model drug) to prepare both drug-free and drug-loaded inks with drug concentrations varying between 5% and 30% (w/w). The printed constructs demonstrated the required physical properties and displayed immediate, extended, delayed and pulsatile drug release depending on drug localisation inside of the printed formulations. For the first time, this paper demonstrates that a commonly used pharmaceutical lipid, Compritol HD5 ATO, can be printed via hot-melt inkjet printing as single ink material, or in combination with a drug, without the need for additional solvents. Concurrently, this paper demonstrates the capabilities of dual material hot-melt inkjet 3D printing system to produce multi-material personalised solid dosage forms.

Microbial Metal Resistance within Structured Environments Is Inversely Related to Environmental Pore Size.

The physical environments in which microorganisms naturally reside rarely have homogeneous structure, and changes in their porous architecture may have effects on microbial activities that are not typically captured in conventional laboratory studies. In this study, to investigate the influence of environmental structure on microbial responses to stress, we constructed structured environments with different pore properties (determined by X-ray computed tomography). First, using glass beads in different arrangements and inoculated with the soil yeast Saitozyma podzolica, increases in the average equivalent spherical diameters (ESD) of a structure's porous architecture led to decreased survival of the yeast under a toxic metal challenge with lead nitrate. This relationship was reproduced when yeasts were introduced into additively manufactured lattice structures, comprising regular arrays with ESDs comparable to those of the bead structures. The pore ESD dependency of metal resistance was not attributable to differences in cell density in microenvironments delimited by different pore sizes, supporting the inference that pore size specifically was the important parameter in determining survival of stress. These findings highlight the importance of the physical architecture of an organism's immediate environment for its response to environmental perturbation, while offering new tools for investigating these interactions in the laboratory. IMPORTANCE Interactions between cells and their structured environments are poorly understood but have significant implications for organismal success in both natural and nonnatural settings. This work used a multidisciplinary approach to develop laboratory models with which the influence of a key parameter of environmental structure-pore size-on cell activities can be dissected. Using these new methods in tandem with additive manufacturing, we demonstrated that resistance of yeast soil isolates to stress (from a common metal pollutant) is inversely related to pore size of their environment. This has important ramifications for understanding how microorganisms respond to stress in different environments. The findings also establish new pathways for resolving the effects of physical environment on microbial activity, enabling important understanding that is not readily attainable with traditional bulk sampling and analysis approaches.

Bespoke 3D-Printed Polydrug Implants Created via Microstructural Control of Oligomers.

Controlling the microstructure of materials by means of phase separation is a versatile tool for optimizing material properties. Phase separation has been exploited to fabricate intricate microstructures in many fields including cell biology, tissue engineering, optics, and electronics. The aim of this study was to use phase separation to tailor the spatial location of drugs and thereby generate release profiles of drug payload over periods ranging from 1 week to months by exploiting different mechanisms: polymer degradation, polymer diluent dissolution, and control of microstructure. To achieve this, we used drop-on-demand inkjet three-dimensional (3D) printing. We predicted the microstructure resulting from phase separation using high-throughput screening combined with a model based on the Flory-Huggins interaction parameter and were able to show that drug release from 3D-printed objects can be predicted from observations based on single drops of mixtures. We demonstrated for the first time that inkjet 3D printing yields controllable phase separation using picoliter droplets of blended photoreactive oligomers/monomers. This new understanding gives us hierarchical compositional control, from droplet to device, allowing release to be "dialled up" without manipulation of device geometry. We exemplify this approach by fabricating a biodegradable, long-term, multiactive drug delivery subdermal implant ("polyimplant") for combination therapy and personalized treatment of coronary heart disease. This is an important advance for implants that need to be delivered by cannula, where the shape is highly constrained and thus the usual geometrical freedoms associated with 3D printing cannot be easily exploited, which brings a hitherto unseen level of understanding to emergent material properties of 3D printing.

Generation and Characterization of a Library of Novel Biologically Active Functional Surfactants (Surfmers) Using Combined High-Throughput Methods.

We report the first successful combination of three distinct high-throughput techniques to deliver the accelerated design, synthesis, and property screening of a library of novel, bio-instructive, polymeric, comb-graft surfactants. These three-dimensional, surface-active materials were successfully used to control the surface properties of particles by forming a unimolecular deep layer on the surface of the particles via microfluidic processing. This strategy deliberately utilizes the surfactant to both create the stable particles and deliver a desired cell-instructive behavior. Therefore, these specifically designed, highly functional surfactants are critical to promoting a desired cell response. This library contained surfactants constructed from 20 molecularly distinct (meth)acrylic monomers, which had been pre-identified by HT screening to exhibit specific, varied, and desirable bacterial biofilm inhibitory responses. The surfactant's self-assembly properties in water were assessed by developing a novel, fully automated, HT method to determine the critical aggregation concentration. These values were used as the input data to a computational-based evaluation of the key molecular descriptors that dictated aggregation behavior. Thus, this combination of HT techniques facilitated the rapid design, generation, and evaluation of further novel, highly functional, cell-instructive surfaces by application of designed surfactants possessing complex molecular architectures.

Inkjet 3D Printing of Polymers Resistant to Fungal Attachment.

Inkjet 3D printing is an additive manufacturing method that allows the user to produce a small batch of customized devices for comparative study versus commercial products. Here, we describe the use of a commercial 2D ink development system (Dimatix material printing) to manufacture small batches of 3D medical or other devices using a recently characterized fungal anti-attachment material. Such printed devices may resist problems that beset commercial medical products due to colonization by the fungal pathogen Candida albicans. By sequentially introducing the cross-section bitmaps of the product's CAD model and elevating the print head height using the auto-clicking script, we were able to create complex self-support geometries with the 2D ink development system. The use of this protocol allows researchers to produce a small batch of specimens for characterization from only a few grams of raw material. Additionally, we describe the testing of manufactured specimens for fungal anti-attachment. In comparison with most commercial AM systems, which require at least a few hundred grams of ink for printing trials, our protocol is well suited for smaller-scale production in material studies.

Exploiting Generative Design for 3D Printing of Bacterial Biofilm Resistant Composite Devices.

As the understanding of disease grows, so does the opportunity for personalization of therapies targeted to the needs of the individual. To bring about a step change in the personalization of medical devices it is shown that multi-material inkjet-based 3D printing can meet this demand by combining functional materials, voxelated manufacturing, and algorithmic design. In this paper composite structures designed with both controlled deformation and reduced biofilm formation are manufactured using two formulations that are deposited selectively and separately. The bacterial biofilm coverage of the resulting composites is reduced by up to 75% compared to commonly used silicone rubbers, without the need for incorporating bioactives. Meanwhile, the composites can be tuned to meet user defined mechanical performance with ±10% deviation. Device manufacture is coupled to finite element modelling and a genetic algorithm that takes the user-specified mechanical deformation and computes the distribution of materials needed to meet this under given load constraints through a generative design process. Manufactured products are assessed against the mechanical and bacterial cell-instructive specifications and illustrate how multifunctional personalization can be achieved using generative design driven multi-material inkjet based 3D printing.

Development of Conductive Gelatine-Methacrylate Inks for Two-Photon Polymerisation.

Conductive hydrogel-based materials are attracting considerable interest for bioelectronic applications due to their ability to act as more compatible soft interfaces between biological and electrical systems. Despite significant advances that are being achieved in the manufacture of hydrogels, precise control over the topographies and architectures remains challenging. In this work, we present for the first time a strategy to manufacture structures with resolutions in the micro-/nanoscale based on hydrogels with enhanced electrical properties. Gelatine methacrylate (GelMa)-based inks were formulated for two-photon polymerisation (2PP). The electrical properties of this material were improved, compared to pristine GelMa, by dispersion of multi-walled carbon nanotubes (MWCNTs) acting as conductive nanofillers, which was confirmed by electrochemical impedance spectroscopy and cyclic voltammetry. This material was also confirmed to support human induced pluripotent stem cell-derived cardiomyocyte (hPSC-CMs) viability and growth. Ultra-thin film structures of 10 µm thickness and scaffolds were manufactured by 2PP, demonstrating the potential of this method in areas spanning tissue engineering and bioelectronics. Though further developments in the instrumentation are required to manufacture more complex structures, this work presents an innovative approach to the manufacture of conductive hydrogels in extremely low resolution.

3D printed polymeric drug-eluting implants.

An extrusion-based 3D printer has been used for the manufacturing of sustained drug release poly(ε-caprolactone) (PCL) implants. Such implants can address issues of reduced patient compliance due to the necessary frequent administration of conventional drug delivery systems, such as tablets, capsules and solutions. The selected model drug for this study was lidocaine. Polycaprolactone core-shell implants, as well as polymeric implants with no barrier shell were printed with different drug loading, without the addition of solvents or further excipients. Scanning Electron Microscopy (SEM) analysis revealed the structural integrity of the printed formulations, while Differential Scanning Calorimetry (DSC), X-Ray Diffraction (XRD) and Attenuated Total Reflectance Fourier Transform Infrared spectroscopy (ATR-FTIR) were used to detect potential chemical interactions or modifications. Raman spectroscopy was also used to study material distribution in the prints. The drug release rate of the differently printed formulations was evaluated using a USP4 flow-through cell apparatus. All printed implants demonstrated sustained lidocaine release and the effectiveness of the PCL barrier in this regard. The Korsmeyer-Peppas model was suggested as the best fit to drug release profiles for all the produced implants. This work demonstrates that hot-melt extrusion-based 3D printing is a robust and promising technology for the production of personalisable drug-eluting implants.

Challenges and approaches in assessing the interplay between microorganisms and their physical micro-environments.

Spatial structure over scales ranging from nanometres to centimetres (and beyond) varies markedly in diverse habitats and the industry-relevant settings that support microbial activity. Developing an understanding of the interplay between a structured environment and the associated microbial processes and ecology is fundamental, but challenging. Several novel approaches have recently been developed and implemented to help address key questions for the field: from the use of imaging tools such as X-ray Computed Tomography to explore microbial growth in soils, to the fabrication of scratched materials to examine microbial-surface interactions, to the design of microfluidic devices to track microbial biofilm formation and the metabolic processes therein. This review discusses new approaches and challenges for incorporating structured elements into the study of microbial processes across different scales. We highlight how such methods can be pivotal for furthering our understanding of microbial interactions with their environments.

Discovery of (meth)acrylate polymers that resist colonization by fungi associated with pathogenesis and biodeterioration.

Fungi have major, negative socioeconomic impacts, but control with bioactive agents is increasingly restricted, while resistance is growing. Here, we describe an alternative fungal control strategy via materials operating passively (i.e., no killing effect). We screened hundreds of (meth)acrylate polymers in high throughput, identifying several that reduce attachment of the human pathogen Candida albicans, the crop pathogen Botrytis cinerea, and other fungi. Specific polymer functional groups were associated with weak attachment. Low fungal colonization materials were not toxic, supporting their passive, anti-attachment utility. We developed a candidate monomer formulation for inkjet-based 3D printing. Printed voice prosthesis components showed up to 100% reduction in C. albicans biofilm versus commercial materials. Furthermore, spray-coated leaf surfaces resisted fungal infection, with no plant toxicity. This is the first high-throughput study of polymer chemistries resisting fungal attachment. These materials are ready for incorporation in products to counteract fungal deterioration of goods, food security, and health.

Making tablets for delivery of poorly soluble drugs using photoinitiated 3D inkjet printing.

In this study, we investigate the viability of three-dimensional (3D) inkjet printing with UV curing to produce solid dosage forms containing a known poorly soluble drug, carvedilol. The formulation consists of 10 wt% carvedilol, Irgacure 2959, and a photocurable N-vinyl-2-pyrrolidone (NVP) and poly(ethylene glycol) diacrylate matrix, with the intention of forming an amorphous solid solution for release of carvedilol. Characterization of the printed tablets showed that the drug is an amorphous state and indicated hydrogen bonding interactions between the drug and cross-linked matrix. Several simple geometries (ring, mesh, cylinder, thin film) were printed, and the surface area to volume ratio of the prints was estimated. Over 80% carvedilol release was observed for all printed tablet geometries within ten hours. The release behaviour of carvedilol was fastest for the thin films, followed by the ring and mesh geometries, and slowest in the cylindrical forms. More rapid release was correlated to an increased surface area to volume ratio. This is the first study to implement 3D UV inkjet to make solid dispersion tablets suitable for poorly soluble drugs. Results also demonstrate that high drug-loaded tablets with a variety of release profiles can successfully be accessed with the same UV-curable inkjet formulation by varying the tablet geometry.

Simultaneous Tracking of Pseudomonas aeruginosa Motility in Liquid and at the Solid-Liquid Interface Reveals Differential Roles for the Flagellar Stators.

Bacteria sense chemicals, surfaces, and other cells and move toward some and away from others. Studying how single bacterial cells in a population move requires sophisticated tracking and imaging techniques. We have established quantitative methodology for label-free imaging and tracking of individual bacterial cells simultaneously within the bulk liquid and at solid-liquid interfaces by utilizing the imaging modes of digital holographic microscopy (DHM) in three dimensions (3D), differential interference contrast (DIC), and total internal reflectance microscopy (TIRM) in two dimensions (2D) combined with analysis protocols employing bespoke software. To exemplify and validate this methodology, we investigated the swimming behavior of a Pseudomonas aeruginosa wild-type strain and isogenic flagellar stator mutants (motAB and motCD) within the bulk liquid and at the surface at the single-cell and population levels. Multiple motile behaviors were observed that could be differentiated by speed and directionality. Both stator mutants swam slower and were unable to adjust to the near-surface environment as effectively as the wild type, highlighting differential roles for the stators in adapting to near-surface environments. A significant reduction in run speed was observed for the P. aeruginosa mot mutants, which decreased further on entering the near-surface environment. These results are consistent with the mot stators playing key roles in responding to the near-surface environment.IMPORTANCE We have established a methodology to enable the movement of individual bacterial cells to be followed within a 3D space without requiring any labeling. Such an approach is important to observe and understand how bacteria interact with surfaces and form biofilm. We investigated the swimming behavior of Pseudomonas aeruginosa, which has two flagellar stators that drive its swimming motion. Mutants that had only either one of the two stators swam slower and were unable to adjust to the near-surface environment as effectively as the wild type. These results are consistent with the mot stators playing key roles in responding to the near-surface environment and could be used by bacteria to sense via their flagella when they are near a surface.