Bioinspired Bioinks for the Fabrication of Chemomechanically Relevant Standalone Disease Models of Hepatic Steatosis.

Hepatotoxicity-related issues are poorly predicted during preclinical experimentation, as its relevance is limited by the inadequacy to screen all the non-physiological subclasses of the population. These pitfalls can be solved by implementing complex in vitro models of hepatic physiology and pathologies in the preclinical phase. To produce these platforms, extrusion-based bioprinting is focused on, since it allows to manufacture tridimensional cell-laden constructs with controlled geometries, in a high-throughput manner. Different bioinks, whose formulation is tailored to mimic the chemomechanical environment of hepatic steatosis, the most prevalent hepatic disorder worldwide, are proposed. Internally crosslinked alginate hydrogels are chosen as structural components of the inks. Their viscoelastic properties (G' = 512-730 Pa and G″ = 94-276 Pa, depending on frequency) are tuned to mimic those of steatotic liver tissue. Porcine hepatic ECM is introduced as a relevant biochemical cue. Sodium oleate is added to recall the accumulation of lipids in the tissue. Downstream analyses on 14-layered bioprinted structures cultured for 10 days reveal the establishment of steatotic-like features (intracellular lipid vesicles, viability decrease up to ≈50%) without needing external conditionings. The presented bioinks are thus suitable to fabricate complex models of hepatic steatosis to be implemented in a high-throughput experimental frame.

Development of a multi-component gastroretentive expandable drug delivery system (GREDDS) for personalized administration of metformin.

OBJECTIVES: Efficacy and compliance of type II diabetes treatment would greatly benefit from dosage forms providing controlled release of metformin in the upper gastrointestinal tract. In this respect, the feasibility of a new system ensuring stomach-retention and personalized release of this drug at its absorption window for multiple days was investigated. METHODS: The system proposed comprised of a drug-containing core and a viscoelastic umbrella-like skeleton, which were manufactured by melt-casting and 3D printing. Prototypes, alone or upon assembly and insertion into commercially-available capsules, were characterized for key parameters: thermo-mechanical properties, accelerated stability, degradation, drug release, deployment performance, and resistance to simulated gastric contractions. RESULTS: Each part of the system was successfully manufactured using purposely-selected materials and the performance of final prototypes matched the desired one. This included: i) easy folding of the skeleton against the core in the collapsed administered shape, ii) rapid recovery of the cumbersome configuration at the target site, even upon storage, and iii) prolonged release of metformin. CONCLUSIONS: Composition, geometry, and performance of the system developed in this work were deemed acceptable for stomach-retention and prolonged as well as customizable release of metformin in its absorption window, laying promising bases for further development steps.

Electrospinning of pullulan-based orodispersible films containing sildenafil.

Feasibility of electrospinning in the manufacturing of sildenafil-containing orodispersible films (ODFs) intended to enhance oxygenation and to reduce pulmonary arterial pressure in pediatric patients was evaluated. Given the targeted subjects, the simplest and safest formulation was chosen, using water as the only solvent and pullulan, a natural polymer, as the sole fiber-forming agent. A systematic characterization in terms of shear and extensional viscosity as well as surface tension of solutions containing different amounts of pullulan and sildenafil was carried out. Accordingly, electrospinning parameters enabling the continuous production, at the highest possible rate, of defect-free fibers with uniform diameter in the nanometer range were assessed. Morphology, microstructure, drug content and relevant solid state as well as ability of the resulting non-woven films to interact with aqueous fluids were evaluated. To better define the role of the fibrous nanostructure on the performance of ODFs, analogous films were produced by spin- and blade-coating and tested. Interestingly, the disintegration process of electrospun products turned out to be the fastest (i.e. occurring within few s) and compliant with Ph. Eur. and USP limits, making relevant ODFs particularly promising for increasing sildenafil bioavailability, thus lowering its dosages.

Internally crosslinked alginate-based bioinks for the fabrication ofin vitrohepatic tissue models.

Bioprinting is a key technique to fabricate cell-laden volumetric constructs with controlled geometry. It can be used not only to replicate the architecture of a target organ but also to produce shapes that allow for the mimicry,in vitro,of specific desired features. Among the various materials suitable to be processed with this technique, sodium alginate is currently considered one of the most appealing because of its versatility. To date, the most widespread strategies to print alginate-based bioinks exploit external gelation as a primary process, by directly extruding the hydrogel-precursor solution into a crosslinking bath or within a sacrificial crosslinking hydrogel, where the gelation takes place. In this work, we describe the print optimization and the processing of Hep3Gel: an internally crosslinked alginate and ECM-based bioink for the production of volumetric hepatic tissue models. We adopted an unconventional strategy, by moving from the reproduction of the geometry and the architecture of liver tissue to the use of bioprinting to fabricate structures that can promote a high degree of oxygenation, as is the case with hepatic tissue. To this end, the design of structures was optimized by employing computational methods. The printability of the bioink was then studied and optimized through a combination of differenta priorianda posteriorianalyses. We produced 14-layered constructs, thus highlighting the possibility to exploit internal gelation alone to directly print self-standing structures with finely controlled viscoelastic properties. Constructs loaded with HepG2 cells were successfully printed and cultured in static conditions for up to 12 d, underlining the suitability of Hep3Gel to support mid/long-term cultures.

Hep3Gel: A Shape-Shifting Extracellular Matrix-Based, Three-Dimensional Liver Model Adaptable to Different Culture Systems.

Drug-induced hepatotoxicity is a leading cause of clinical trial withdrawal. Therefore, in vitro modeling the hepatic behavior and functionalities is not only crucial to better understand physiological and pathological processes but also to support drug development with reliable high-throughput platforms. Different physiological and pathological models are currently under development and are commonly implemented both within platforms for standard 2D cultures and within tailor-made chambers. This paper introduces Hep3Gel: a hybrid alginate-extracellular matrix (ECM) hydrogel to produce 3D in vitro models of the liver, aiming to reproduce the hepatic chemomechanical niche, with the possibility of adapting its shape to different manufacturing techniques. The ECM, extracted and powdered from porcine livers by a specifically set-up procedure, preserved its crucial biological macromolecules and was embedded within alginate hydrogels prior to crosslinking. The viscoelastic behavior of Hep3Gel was tuned, reproducing the properties of a physiological organ, according to the available knowledge about hepatic biomechanics. By finely tuning the crosslinking kinetics of Hep3Gel, its dualistic nature can be exploited either by self-spreading or adapting its shape to different culture supports or retaining the imposed fiber shape during an extrusion-based 3D-bioprinting process, thus being a shape-shifter hydrogel. The self-spreading ability of Hep3Gel was characterized by combining empirical and numerical procedures, while its use as a bioink was experimentally characterized through rheological a priori printability evaluations and 3D printing tests. The effect of the addition of the ECM was evident after 4 days, doubling the survival rate of cells embedded within control hydrogels. This study represents a proof of concept of the applicability of Hep3Gel as a tool to develop 3D in vitro models of the liver.

Expandable Drug Delivery Systems Based on Shape Memory Polymers: Impact of Film Coating on Mechanical Properties and Release and Recovery Performance.

Retentive drug delivery systems (DDSs) are intended for prolonged residence and release inside hollow muscular organs, to achieve either local or systemic therapeutic goals. Recently, formulations based on shape memory polymers (SMPs) have gained attention in view of their special ability to recover a shape with greater spatial encumbrance at the target organ (e.g., urinary bladder or stomach), triggered by contact with biological fluids at body temperature. In this work, poly(vinyl alcohol) (PVA), a pharmaceutical-grade SMP previously shown to be an interesting 4D printing candidate, was employed to fabricate expandable organ-retentive prototypes by hot melt extrusion. With the aim of improving the mechanical resistance of the expandable DDS and slowing down relevant drug release, the application of insoluble permeable coatings based on either Eudragit® RS/RL or Eudragit® NE was evaluated using simple I-shaped specimens. The impact of the composition and thickness of the coating on the shape memory, swelling, and release behavior as well as on the mechanical properties of these specimens was thoroughly investigated and the effectiveness of the proposed strategy was demonstrated by the results obtained.

Toward 3D-Bioprinted Models of the Liver to Boost Drug Development.

The main problems in drug development are connected to enormous costs related to the paltry success rate. The current situation empowered the development of high-throughput and reliable instruments, in addition to the current golden standards, able to predict the failures in the early preclinical phase. Being hepatotoxicity responsible for the failure of 30% of clinical trials, and the 21% of withdrawal of marketed drugs, the development of complex in vitro models (CIVMs) of liver is currently one of the hottest topics in the field. Among the different fabrication techniques, 3D-bioprinting is emerging as a powerful ally for their production, allowing the manufacture of three-dimensional constructs characterized by computer-controlled and customized geometry, and inter-batches reproducibility. Thanks to these, it is possible to rapidly produce tailored cell-laden constructs, to be cultured within static and dynamic systems, thus reaching a further degree of personalization when designing in vitro models. This review highlights and prioritizes the most recent advances related to the development of CIVMs of the hepatic environment to be specifically applied to pharmaceutical research, with a special focus on 3D-bioprinting, since the liver is primarily involved in the metabolism of drugs.

Thermal expansion of free volume in "classic" and regulated dimethacrylates: photocured directly and via a mask to study pillar formation.

The temperature dependence of free volume in dimethacrylates (poly2M), cured by direct irradiation (poly2M-A) or via a mask (poly2M-B), and in a thiol-based 2M sample (poly2M-co-EDDT), was investigated by positron annihilation lifetime spectroscopy (PALS) and dilatometry (DIL) to study the influence of thiol regulation on the microstructure via free volume characteristics. It was found that the free volume fraction as determined from experimental data by using the standard spherical approach for the hole shapes showed systematic differences from the analogous quantity as evaluated from the lattice-hole theory. Much better results were obtained for cylindrical holes, which expand 'anisotropically' in poly2M samples and 'isotropically' in the poly2M-co-EDDT resin. In addition, the hydrogen bond changes and the conversion of monomers in cured samples studied by near infra-red spectroscopy (NIR) revealed spectrum-structure correlations for the final cured thermosets.

Pectin-based bioinks for 3D models of neural tissue produced by a pH-controlled kinetics.

Introduction: In the view of 3D-bioprinting with cell models representative of neural cells, we produced inks to mimic the basic viscoelastic properties of brain tissue. Moving from the concept that rheology provides useful information to predict ink printability, this study improves and expands the potential of the previously published 3D-reactive printing approach by introducing pH as a key parameter to be controlled, together with printing time. Methods: The viscoelastic properties, printability, and microstructure of pectin gels crosslinked with CaCO3 were investigated and their composition was optimized (i.e., by including cell culture medium, HEPES buffer, and collagen). Different cell models representative of the major brain cell populations (i.e., neurons, astrocytes, microglial cells, and oligodendrocytes) were considered. Results and Discussion: The outcomes of this study propose a highly controllable method to optimize the printability of internally crosslinked polysaccharides, without the need for additives or post-printing treatments. By introducing pH as a further parameter to be controlled, it is possible to have multiple (pH-dependent) crosslinking kinetics, without varying hydrogel composition. In addition, the results indicate that not only cells survive and proliferate following 3D-bioprinting, but they can also interact and reorganize hydrogel microstructure. Taken together, the results suggest that pectin-based hydrogels could be successfully applied for neural cell culture.

3D-Reactive printing of engineered alginate inks.

Alginate is a common component of bioinks due to its well-described ionic crosslinking mechanism and tunable viscoelastic properties. Extrusion-based 3D-printing of alginate inks requires additives, such as gelatin and Pluronic, pre- or post-printing crosslinking processes and/or coextrusion with crosslinkers. In this work, we aim to develop a different printing approach for alginate-based inks, introducing 3D-reactive printing. Indeed, the control over the crosslinking kinetics and the printing time allowed printing different inks while maintaining their final composition unaltered to identify a suitable formulation in terms of printability. Alginate solutions were crosslinked with insoluble calcium salts (CaCO3) inducing a dynamic modification of their microstructure and viscoelastic properties over time. The monitoring of fiber printability and internal microstructure, at different time points of ink gelation, was performed by means of a well-defined set of rheological tests to obtain a priori ink properties for the a posteriori 3D-printing process. This new perspective allowed 3D-reactive printing of alginate fibers with predetermined properties, without involving post-extrusion crosslinking steps and additives.

Bioprinting of Matrigel Scaffolds for Cancer Research.

Cancer is one of the most life-threatening diseases worldwide. Despite the huge efforts, the failure rate of therapies remains high due to cells heterogeneity, so physiologically relevant models are strictly necessary. Bioprinting is a technology able to form highly complex 3D tissue models and enables the creation of large-scale constructs. In cancer research, Matrigel® is the most widely used matrix, but it is hardly bioprinted pure, without the use of any other bioink as reinforcement. Its complex rheological behavior makes the control with a standard bioprinting process nearly impossible. In this work, we present a customized bioprinting strategy to produce pure Matrigel® scaffolds with good shape fidelity. To this aim, we realized a custom-made volumetric dispensing system and performed printability evaluations. To determine optimal printing parameters, we analyzed fibers spreading ratio on simple serpentines. After identifying an optimal flow rate of 86.68 ± 5.77 µL/min and a printing speed of 10 mm/min, we moved forward to evaluate printing accuracy, structural integrity and other key parameters on single and multi-layer grids. Results demonstrated that Matrigel® was able to maintain its structure in both simple and complex designs, as well as in single and multilayer structures, even if it does not possess high mechanical strength. In conclusion, the use of volumetric dispensing allowed printing pure Matrigel® constructs with a certain degree of shape fidelity on both single and multiple layers.

The Chronotopic™ System for Pulsatile and Colonic Delivery of Active Molecules in the Era of Precision Medicine: Feasibility by 3D Printing via Fused Deposition Modeling (FDM).

The pulsatile-release Chronotopic™ system was conceived of as a drug-containing core surrounded by a coat made of swellable/soluble hydrophilic polymers, the latter being able to provide a programmable lag phase prior to drug liberation. This system was also proposed in a colon-targeting configuration, entailing a gastroresistant film to prevent early interaction of the inner coat with gastric fluids and enabling the attainment of a lag phase matching the small intestinal transit time. Over the years, various multiple-step manufacturing processes have been tested for the fabrication of the Chronotopic™ system in both its configurations. This work focused on the evaluation of 3D printing by fused deposition modeling in view of its potential towards product personalization, on demand one-step manufacturing and efficient scale down of batches. The feasibility of each part of the Chronotopic™ system was independently investigated starting from in-house made filaments, characterizing the resulting specimens for physico-technological and performance characteristics. The printing parameters identified as suitable during the set-up phase were then used to fabricate prototypes either in a single step for the pulsatile configuration or following two different fabrication approaches for the colon-targeting one.

Engineered modular microphysiological models of the human airway clearance phenomena.

Mucociliary clearance is a crucial mechanism that supports the elimination of inhaled particles, bacteria, pollution, and hazardous agents from the human airways, and it also limits the diffusion of aerosolized drugs into the airway epithelium. In spite of its relevance, few in vitro models sufficiently address the cumulative effect of the steric and interactive barrier function of mucus on the one hand, and the dynamic mucus transport imposed by ciliary mucus propulsion on the other hand. Here, ad hoc mucus models of physiological and pathological mucus are combined with magnetic artificial cilia to model mucociliary transport in both physiological and pathological states. The modular concept adopted in this study enables the development of mucociliary clearance models with high versatility since these can be easily modified to reproduce phenomena characteristic of healthy and diseased human airways while allowing to determine the effect of each parameter and/or structure separately on the overall mucociliary transport. These modular airway models can be available off-the-shelf because they are exclusively made of readily available materials, thus ensuring reproducibility across different laboratories.

Quality considerations on the pharmaceutical applications of fused deposition modeling 3D printing.

3D printing, and particularly fused deposition modeling (FDM), has rapidly brought the possibility of personalizing drug therapies to the forefront of pharmaceutical research and media attention. Applications for this technology, described in published articles, are expected to grow significantly in 2020. Where are we on this path, and what needs to be done to develop a FDM 2.0 process and make personalized medicines available to patients? Based on literature analysis, this manuscript aims to answer these questions and highlight the critical technical aspects of FDM as an emerging technology for manufacturing safe, high-quality personalized oral drug products. In this collaborative paper, experts from different fields contribute strategies for ensuring the quality of starting materials and discuss the design phase, printer hardware and software, the process, the environment and the resulting products, from the perspectives of both patients and operators.

Properties and role of interfaces in multimaterial 3D printed composites.

In polyjet printing photopolymer droplets are deposited on a build tray, leveled off by a roller and cured by UV light. This technique is attractive to fabricate heterogeneous architectures combining compliant and stiff constituents. Considering the layer-by-layer nature, interfaces between different photopolymers can be formed either before or after UV curing. We analyzed the properties of interfaces in 3D printed composites combining experiments with computer simulations. To investigate photopolymer blending, we characterized the mechanical properties of the so-called digital materials, obtained by mixing compliant and stiff voxels according to different volume fractions. We then used nanoindentation to measure the spatial variation in mechanical properties across bimaterial interfaces at the micrometer level. Finally, to characterize the impact of finite-size interfaces, we fabricated and tested composites having compliant and stiff layers alternating along different directions. We found that interfaces formed by deposition after curing were sharp whereas those formed before curing showed blending of the two materials over a length scale bigger than individual droplet size. We found structural and functional differences of the layered composites depending on the printing orientation and corresponding interface characteristics, which influenced deformation mechanisms. With the wide dissemination of 3D printing techniques, our results should be considered in the development of architectured materials with tailored interfaces between building blocks.

Expandable drug delivery system for gastric retention based on shape memory polymers: Development via 4D printing and extrusion.

Several diseases would benefit from prolonged drug release provided by systems retained in the stomach for extended time periods. Expandable gastroretentive devices are administered in a collapsed configuration enabling swallowing and regain in situ their native shape having larger spatial encumbrance, thus hindering voidance through the wide open pylorus. An expandable system for gastric retention was here proposed relying on the shape memory behavior of pharmaceutical-grade poly(vinyl alcohol). Different original configurations to be recovered upon exposure to aqueous fluids at 37 °C, potentially enabling gastric retention, were conceived. Prototypes containing allopurinol were directly manufactured by fused deposition modeling or shaped by purposely-designed templates from hot melt extruded rods immediately after production. Various temporary shapes, in principle suitable for administration, were programmed by manual deformation of samples by means of specific templates, under defined temperature conditions. In 0.1 N hydrochloric solution at 37 °C, the prototypes recovered their original shape, reaching the desired spatial encumbrance within few minutes. Release from the samples, although of relatively short duration, was independent of the original shape and processing undergone, and was noticeably slowed down by application of Eudragit® RS/RL-based coatings.

Disassembling the complexity of mucus barriers to develop a fast screening tool for early drug discovery.

Mucus is a natural barrier with a protective role that hinders drug diffusion, representing a steric and interactive barrier to overcome for an effective drug delivery to target sites. In diseases like cystic fibrosis (CF), pulmonary mucus exhibits altered features, which hamper clearance mechanisms and drug diffusion, ultimately leading to lung failure. Effectively modelling the passage through mucus still represents an unmet challenge. An airway CF mucus model is herein proposed to disassemble the complexity of the mucus barrier following a modular approach. A hydrogel, mainly composed of mucin in an alginate (Alg) network, is proposed to specifically model the chemical-physical properties of CF mucus. The steric retention of pathological mucus was reproduced by targeting its mesh size (approximately 50 nm) and viscoelastic properties. The interactive barrier was reproduced by a composition inspired from the CF mucus. Optimized mucus models, composed of 3 mg ml-1 Alg and 25 mg ml-1 mucin, exhibited a G' increasing from ∼21.2 to 55.2 Pa and a G'' ranging from ∼5.26 to 28.8 Pa in the frequency range of 0.1 to 20 Hz. Drug diffusion was tested using three model drugs. The proposed mucus model was able to discriminate between the mucin-drug interaction and the steric barrier of a mucus layer with respect to the parallel artificial membrane permeability (PAMPA) that models the phospholipidic cell membrane, the state-of-the-art screening tool for passive drug diffusion. The mucus model can be proposed as an in vitro tool for early drug discovery, representing a step forward to model the mucus layer. Additionally, the proposed methodology allows to easily include other molecules present within mucus, as relevant proteins, lipids and DNA.

Effect of Polyethylene Glycol Content and Molar Mass on Injection Molding of Hydroxypropyl Methylcellulose Acetate Succinate-Based Gastroresistant Capsular Devices for Oral Drug Delivery.

Capsular devices for oral drug delivery were recently proposed and manufactured by injection molding (IM) as an evolution of traditional reservoir systems comprising a core and a functional coating. IM allowed the fabrication of capsule shells with release-controlling features based on the employed materials and the design characteristics. These features are independent of the drug, with significant savings in development time and costs. In previous work, IM was used to produce enteric-soluble capsules from blends of hydroxypropyl methylcellulose acetate succinate, with polyethylene glycol (PEG) as the plasticizer. In this work, the range of plasticizer concentrations and molar mass was broadened to evaluate in-depth how those parameters affect material processability and capsule performance over time. As expected, increasing the amount of the low molar mass plasticizer decreased the viscosity and modulus of the material. This simplified the molding process and enhanced the mechanical resistance of the shell, as observed during assembly. However, some samples turned out translucent, depending on several factors including storage conditions. This was attributed to plasticizer migration issues. Such results indicate that higher molar mass PEGs, while not significantly impacting on processability, lead to capsular devices with consistent performance in the investigated time lapse.

Modular genetic design of multi-domain functional amyloids: insights into self-assembly and functional properties.

Engineering functional amyloids through a modular genetic strategy represents new opportunities for creating multifunctional molecular materials with tailored structures and performance. Despite important advances, how fusion modules affect the self-assembly and functional properties of amyloids remains elusive. Here, using Escherichia coli curli as a model system, we systematically studied the effect of flanking domains on the structures, assembly kinetics and functions of amyloids. The designed amyloids were composed of E. coli biofilm protein CsgA (as amyloidogenic cores) and one or two flanking domains, consisting of chitin-binding domains (CBDs) from Bacillus circulans chitinase, and/or mussel foot proteins (Mfps). Incorporation of fusion domains did not disrupt the typical ß-sheet structures, but indeed affected assembly rate, morphology, and stiffness of resultant fibrils. Consequently, the CsgA-fusion fibrils, particularly those containing three domains, were much shorter than the CsgA-only fibrils. Furthermore, the stiffness of the resultant fibrils was heavily affected by the structural feature of fusion domains, with ß-sheet-containing domains tending to increase the Young's modulus while random coil domains decreasing the Young's modulus. In addition, fibrils containing CBD domains showed higher chitin-binding activity compared to their CBD-free counterparts. The CBD-CsgA-Mfp3 construct exhibited significantly lower binding activity than Mfp5-CsgA-CBD due to inappropriate folding of the CBD domain in the former construct, in agreement with results based upon molecular dynamics modeling. Our study provides new insights into the assembly and functional properties of designer amyloid proteins with increasing complex domain structures and lays the foundation for the future design of functional amyloid-based structures and molecular materials.

Towards bioinspired in vitro models of intestinal mucus.

Intestinal mucus is a biological structure that acts as a barrier between the external environment and the epithelium. It actively selects nutrient and drug intake, regulates the symbiosis with the intestinal microbiota and keeps the epithelium protected from the attack of pathogens. All these functions are closely connected to the chemical and structural complexity of this biological material, on which its viscoelastic and diffusive properties depend. Many models have been proposed to replicate these characteristics using glycoproteins in solution and possibly the addition of other mucus components, such as lipids and other proteins. In the field of mucus modelling, an overall view of the mucus as a material, having its own viscous, rheological and diffusive characteristics, has been undersized with respect to a pure biological-functional analysis. In this review, we propose a description of the mucus as a biomaterial, including a presentation of its chemical and structural complexity, and of its main viscoelastic-diffusive properties, in order to provide a synthesis of the characteristics necessary for the engineering of more advanced mucus models.