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Liver fibrosis is generally associated with an over-production and crosslinking of extracellular matrix proteins, causing a progressive increase in both the elastic and viscous properties of the hepatic tissue. We describe a strategy for mimicking and monitoring the mechano-dynamics of the 3D microenvironment associated with liver fibrosis. Cell-laden gelatin hydrogels were crosslinked with microbial transglutaminase using a purpose-designed cytocompatible two-step protocol, which allows for the exposure of cells to a mechanically changing environment during culturing. A bioreactor was re-engineered to monitor the mechanical properties of cell constructs over time. The results showed a shift towards a more elastic (i.e., solid-like) behaviour, which is likely related to an increase in cell stress. The method effectively mimics the time-evolving mechanical microenvironment associated with liver fibrosis and could provide novel insights into pathophysiological processes in which both elastic and viscous properties of tissues change over time. [ABSTRACT FROM AUTHOR]

Metabolic disorders due to over-nutrition are a major global health problem, often associated with obesity and related morbidities. Obesity is peculiar to humans, as it is associated with lifestyle and diet, and so difficult to reproduce in animal models. Here we describe a model of human central adiposity based on a 3-tissue system consisting of a series of interconnected fluidic modules. Given the causal link between obesity and systemic inflammation, we focused primarily on pro-inflammatory markers, examining the similarities and differences between the 3-tissue model and evidence from human studies in the literature. When challenged with high levels of adiposity, the in-vitro system manifests cardiovascular stress through expression of E-selectin and von Willebrand factor as well as systemic inflammation (expressing IL-6 and MCP-1) as observed in humans. Interestingly, most of the responses are dependent on the synergic interaction between adiposity and the presence of multiple tissue types. The set-up has the potential to reduce animal experiments in obesity research and may help unravel specific cellular mechanisms which underlie tissue response to nutritional overload. [ABSTRACT FROM AUTHOR]

The increasing use of multi-walled carbon nanotubes (MWCNTs) in consumer products and their potential to induce adverse lung effects following inhalation has lead to much interest in better understanding the hazard associated with these nanomaterials (NMs). While the current regulatory requirement for substances of concern, such as MWCNTs, in many jurisdictions is a 90-day rodent inhalation test, the monetary, ethical, and scientific concerns associated with this test led an international expert group to convene in Washington, DC, USA, to discuss alternative approaches to evaluate the inhalation toxicity of MWCNTs. Pulmonary fibrosis was identified as a key adverse outcome linked to MWCNT exposure, and recommendations were made on the design of an in vitro assay that is predictive of the fibrotic potential of MWCNTs. While fibrosis takes weeks or months to develop in vivo, an in vitro test system may more rapidly predict fibrogenic potential by monitoring pro-fibrotic mediators (e.g., cytokines and growth factors). Therefore, the workshop discussions focused on the necessary specifications related to the development and evaluation of such an in vitro system. Recommendations were made for designing a system using lung-relevant cells co-cultured at the air-liquid interface to assess the pro-fibrogenic potential of aerosolized MWCNTs, while considering human-relevant dosimetry and NM life cycle transformations. The workshop discussions provided the fundamental design components of an air-liquid interface in vitro test system that will be subsequently expanded to the development of an alternative testing strategy to predict pulmonary toxicity and to generate data that will enable effective risk assessment of NMs. [ABSTRACT FROM AUTHOR]

A fluorescent-photochrome method of quantifying the orientation and surface density of solid phase antibodies is described. The method is based on measurements of quenching and rates of cis–trans photoisomerization and photodestruction of a stilbene-labeled hapten by a quencher in solution. These experimental parameters enable a quantitative description of the order of binding sites of antibodies immobilized on a surface and can be used to characterize the microviscosity and steric hindrance in the vicinity of the binding site. Furthermore, a theoretical method for the determination of the depth of immersion of the fluorescent label in a two-phase system was developed. The model exploits the concept of dynamic interactions and is based on the empirical dependence of parameters of static exchange interactions on distances between exchangeable centers. In the present work, anti-dinitrophenyl (DNP) antibodies and stilbene-labeled DNP were used to investigate three different protein immobilization methods: physical adsorption, covalent binding, and the Langmuir–Blodgett technique. [Copyright &y& Elsevier]

Human-relevant three-dimensional (3D) models of cerebral tissue can be invaluable tools to boost our understanding of the cellular mechanisms underlying brain pathophysiology. Nowadays, the accessibility, isolation and harvesting of human neural cells represents a bottleneck for obtaining reproducible and accurate models and gaining insights in the fields of oncology, neurodegenerative diseases and toxicology. In this scenario, given their low cost, ease of culture and reproducibility, neural cell lines constitute a key tool for developing usable and reliable models of the human brain. Here, we review the most recent advances in 3D constructs laden with neural cell lines, highlighting their advantages and limitations and their possible future applications. [ABSTRACT FROM AUTHOR]

Accurate descriptions of the variability in single-cell oxygen consumption and its size-dependency are key to establishing more robust tissue models. By combining microfabricated devices with multiparameter identification algorithms, we demonstrate that single human hepatocytes exhibit an oxygen level-dependent consumption rate and that their maximal oxygen consumption rate is significantly lower than that of typical hepatic cell cultures. Moreover, we found that clusters of two or more cells competing for a limited oxygen supply reduced their maximal consumption rate, highlighting their ability to adapt to local resource availability and the presence of nearby cells. We used our approach to characterize the covariance of size and oxygen consumption rate within a cell population, showing that size matters, since oxygen metabolism covaries lognormally with cell size. Our study paves the way for linking the metabolic activity of single human hepatocytes to their tissue- or organ-level metabolism and describing its size-related variability through scaling laws. [ABSTRACT FROM AUTHOR]

About two decades ago, West and coworkers established a model which predicts that metabolic rate follows a three quarter power relationship with the mass of an organism, based on the premise that tissues are supplied nutrients through a fractal distribution network. Quarter power scaling is widely considered a universal law of biology and it is generally accepted that were in-vitro cultures to obey allometric metabolic scaling, they would have more predictive potential and could, for instance, provide a viable substitute for animals in research. This paper outlines a theoretical and computational framework for establishing quarter power scaling in three-dimensional spherical constructs in-vitro, starting where fractal distribution ends. Allometric scaling in non-vascular spherical tissue constructs was assessed using models of Michaelis Menten oxygen consumption and diffusion. The models demonstrate that physiological scaling is maintained when about 5 to 60% of the construct is exposed to oxygen concentrations less than the Michaelis Menten constant, with a significant concentration gradient in the sphere. The results have important implications for the design of downscaled in-vitro systems with physiological relevance. [ABSTRACT FROM AUTHOR]

Visceral myopathy is a rare, life-threatening disease linked to identified genetic mutations in 60% of cases. Mostly due to the dearth of knowledge regarding its pathogenesis, effective treatments are lacking. The disease is most commonly diagnosed in children with recurrent or persistent disabling episodes of functional intestinal obstruction, which can be life threatening, often requiring long-term parenteral or specialized enteral nutritional support. Although these interventions are undisputedly life-saving as they allow affected individuals to avoid malnutrition and related complications, they also seriously compromise their quality of life and can carry the risk of sepsis and thrombosis. Animal models for visceral myopathy, which could be crucial for advancing the scientific knowledge of this condition, are scarce. Clearly, a collaborative network is needed to develop research plans to clarify genotype–phenotype correlations and unravel molecular mechanisms to provide targeted therapeutic strategies. This paper represents a summary report of the first 'European Forum on Visceral Myopathy'. This forum was attended by an international interdisciplinary working group that met to better understand visceral myopathy and foster interaction among scientists actively involved in the field and clinicians who specialize in care of people with visceral myopathy. [ABSTRACT FROM AUTHOR]

Engineering materials that recapitulate pathophysiological mechanical properties of native tissues in vitro is of interest for the development of biomimetic organ models. To date, the majority of studies have focused on designing hydrogels for cell cultures which mimic native tissue stiffness or quasi-static elastic moduli through a variety of crosslinking strategies, while their viscoelastic (time-dependent) behavior has been largely ignored. To provide a more complete description of the biomechanical environment felt by cells, we focused on characterizing the micro-mechanical viscoelastic properties of crosslinked hydrogels at typical cell length scales. In particular, gelatin hydrogels crosslinked with different glutaraldehyde (GTA) concentrations were analyzed via nano-indentation tests using the nano-epsilon dot method. The experimental data were fitted to a Maxwell Standard Linear Solid model, showing that increasing GTA concentration results in increased instantaneous and equilibrium elastic moduli and in a higher characteristic relaxation time. Therefore, not only do gelatin hydrogels become stiffer with increasing crosslinker concentration (as reported in the literature), but there is also a concomitant change in their viscoelastic behavior towards a more elastic one. As the degree of crosslinking alters both the elastic and viscous behavior of hydrogels, caution should be taken when attributing cell response merely to substrate stiffness, as the two effects cannot be decoupled. [ABSTRACT FROM AUTHOR]

Stem cell-based regeneration of the heart has focused much scientific and public attention being cardiac diseases the major cause of disability and death in industrialized countries. Innumerable efforts have been taken to unveil the mechanisms undergoing stem cell proliferation and fate, but much remains to be endeavoured for their application in clinical practice. Nevertheless, the discovery of progenitor cells resident within the cardiac tissue has sparked off enthusiasm about the possibility of efficiently and safely engineering them to repair the injured myocardium. Indeed, the early applications of the cardiac progenitor cells, mostly based on simplistic concepts and techniques, have failed highlighting the prerequisite of expanding the knowledge about progenitor cell features and microenvironmental conditioning. In this review, recent information on resident cardiac progenitor cells has been systematically gathered in order to create a valuable instrument to support investigators in their efforts to establish an efficient cardiac cell therapy. J. Cell. Physiol. 224: 590–600, 2010. © 2010 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

A study of antibody immobilisation techniques on quartz and fibre optic surfaces for immunosensors has been carried out. Methods of covalent antibody immobilisation which have not previously been applied to optical fibres were investigated, and compared with classical methods found in the literature. Preliminary experiments on covalent immobilisation methods on planar quartz surfaces were conducted to enable us to choose the most suitable protein immobilisation technique for sensor applications. The immobilisation studies were directed in particular towards obtaining a high density of binding sites for the analyte of interest. Two of the most promising methods, antibody immobilisation on surfaces coated with dextran based hydrogel and F(ab′)-SH fragments bound to silanised glass, which resulted in surface densities of active sites of above 0.45 pmol/cm2, were selected for further experiments on a fibre optic total internal reflection fluorescence immunosensor and gave satisfactory responses to changes in analyte concentrations of the order of 10−8 M. The efficiency of polar organic solvents, such as dimethylsulfoxide, in dissociating the antigen–antibody complex and hence to regenerate the immunosensor surface was also evaluated. [Copyright &y& Elsevier]

Construction of biodegradable, three-dimensional scaffolds for tissue engineering has been previously described using a variety of molding and rapid prototyping techniques. In this study, we report and compare two methods for fabricating poly(dl-lactide-co-glycolide) (PLGA) scaffolds with feature sizes of approximately 10–30 μm. The first technique, the pressure assisted microsyringe, is based on the use of a microsyringe that utilizes a computer-controlled, three-axis micropositioner, which allows the control of motor speeds and position. A PLGA solution is deposited from the needle of a syringe by the application of a constant pressure of 20–300 mm Hg, resulting in a controlled polymer deposition. The second technique is based on ‘soft lithographic’ approaches that utilize a poly(dimethylsiloxane) mold. Three variations of the second technique are presented: polymer casting, microfluidic perfusion, and spin coating. Polymer concentration, solvent composition, and mold dimensions influenced the resulting scaffolds as evaluated by light and electron microscopy. As a proof-of-concept for scaffold utility in tissue engineering applications, multilayer structures were formed by thermal lamination, and scaffolds were rendered porous by particulate leaching. These simple methods for forming PLGA scaffolds with microscale features may serve as useful tools to explore structure/function relationships in tissue engineering. [Copyright &y& Elsevier]

Variations and fluctuations are characteristic features of biological systems and are also manifested in cell cultures. Here, we describe a computational pipeline for identifying the range of three-dimensional (3D) cell-aggregate sizes in which nonisometric scaling emerges in the presence of joint mass and metabolic rate fluctuations. The 3D cell-laden spheroids with size and single-cell metabolic rates described by probability density functions were randomly generated in silico. The distributions of the resulting metabolic rates of the spheroids were computed by modeling oxygen diffusion and reaction. Then, a method for estimating scaling exponents of correlated variables through statistically significant data collapse of joint probability distributions was developed. The method was used to identify a physiologically relevant range of spheroid sizes, where both nonisometric scaling and a minimum oxygen concentration (0.04 mol⋅m-3) is maintained. The in silico pipeline described enables the prediction of the number of experiments needed for an acceptable collapse and, thus, a consistent estimate of scaling parameters. Using the pipeline, we also show that scaling exponents may be significantly different in the presence of joint mass and metabolic-rate variations typically found in cells. Our study highlights the importance of incorporating fluctuations and variability in size and metabolic rates when estimating scaling exponents. It also suggests the need for taking into account their covariations for better understanding and interpreting experimental observations both in vitro and in vivo and brings insights for the design of more predictive and physiologically relevant in vitro models. [ABSTRACT FROM AUTHOR]

The aim of this review is to provide a systematic design guideline to users, particularly engineers interested in developing and deploying lung models, and biologists seeking to identify a suitable platform for conducting in vitro experiments involving pulmonary cells or tissues. We first discuss the state of the art on lung in vitro models, describing the most simplistic and traditional ones. Then, we analyze in further detail the more complex dynamic engineered systems that either provide mechanical cues, or allow for more predictive exposure studies, or in some cases even both. This is followed by a dedicated section on microchips of the lung. Lastly, we present a critical discussion of the different characteristics of each type of system and the criteria which may help researchers select the most appropriate technology according to their specific requirements. Readers are encouraged to refer to the tables accompanying the different sections where comprehensive and quantitative information on the operating parameters and performance of the different systems reported in the literature is provided. [ABSTRACT FROM AUTHOR]

From a mechanical point of view, a native extracellular matrix (ECM) is viscoelastic. It also possesses time-evolving or dynamic behaviour, since pathophysiological processes such as ageing alter their mechanical properties over time. On the other hand, biomaterial research on mechanobiology has focused mainly on the development of substrates with varying stiffness, with a few recent contributions on time- or space-dependent substrate mechanics. This work reports on a new method for engineering dynamic viscoelastic substrates, i.e., substrates in which viscoelastic parameters can change or evolve with time, providing a tool for investigating cell response to the mechanical microenvironment. In particular, a two-step (chemical and enzymatic) crosslinking strategy was implemented to modulate the viscoelastic properties of gelatin hydrogels. First, gels with different glutaraldehyde concentrations were developed to mimic a wide range of soft tissue viscoelastic behaviours. Then their mechanical behaviour was modulated over time using microbial transglutaminase. Typically, enzymatically induced mechanical alterations occurred within the first 24 h of reaction and then the characteristic time constant decreased although the elastic properties were maintained almost constant for up to seven days. Preliminary cell culture tests showed that cells adhered to the gels, and their viability was similar to that of controls. Thus, the strategy proposed in this work is suitable for studying cell response and adaptation to temporal variations of substrate mechanics during culture. [ABSTRACT FROM AUTHOR]

The aim of this review is to provide a systematic design guideline to users, particularly engineers interested in developing and deploying lung models, and biologists seeking to identify a suitable platform for conducting in vitro experiments involving pulmonary cells or tissues. We first discuss the state of the art on lung in vitro models, describing the most simplistic and traditional ones. Then, we analyze in further detail the more complex dynamic engineered systems that either provide mechanical cues, or allow for more predictive exposure studies, or in some cases even both. This is followed by a dedicated section on microchips of the lung. Lastly, we present a critical discussion of the different characteristics of each type of system and the criteria which may help researchers select the most appropriate technology according to their specific requirements. Readers are encouraged to refer to the tables accompanying the different sections where comprehensive and quantitative information on the operating parameters and performance of the different systems reported in the literature is provided. [ABSTRACT FROM AUTHOR]

Three-dimensional in vitro maturation (3D IVM) is a promising approach to improve IVM efficiency as it could prevent cumulus-oocyte complex (COC) flattening and preserve its structural and functional integrity. Methods reported to date have low reproducibility and validation studies are limited. In this study, a bioprinting based production process for generating microbeads containing a COC (COC-microbeads) was optimized and its validity tested in a large animal model (sheep). Alginate microbeads were produced and characterized for size, shape and stability under culture conditions. COC encapsulation had high efficiency and reproducibility and cumulus integrity was preserved. COC-microbeads underwent IVM, with COCs cultured in standard 2D IVM as controls. After IVM, oocytes were analyzed for nuclear chromatin configuration, bioenergetic/oxidative status and transcriptional activity of genes biomarker of mitochondrial activity (TFAM, ATP6, ATP8) and oocyte developmental competence (KHDC3, NLRP5, OOEP and TLE6). The 3D system supported oocyte nuclear maturation more efficiently than the 2D control (P<0.05). Ooplasmic mitochondrial activity and reactive oxygen species (ROS) generation ability were increased (P<0.05). Up-regulation of TFAM, ATP6 and ATP8 and down-regulation of KHDC3, NLRP5 expression were observed in 3D IVM. In conclusion, the new bioprinting method for producing COC-microbeads has high reproducibility and efficiency. Moreover, 3D IVM improves oocyte nuclear maturation and relevant parameters of oocyte cytoplasmic maturation and could be used for clinical and toxicological applications. [ABSTRACT FROM AUTHOR]

Multidrug resistance is still an obstacle for chemotherapeutic treatments. One of the proteins involved in this phenomenon is the P-glycoprotein, P-gp, which is known to be responsible for the efflux of therapeutic substances from the cell cytoplasm. To date, the identification of a drug that can efficiently inhibit P-gp activity remains a challenge, nevertheless some studies have identified natural compounds suitable for that purpose. Amongst them, curcumin has shown an inhibitory effect on the protein in in vitro studies using Caco-2 cells. To understand if flow can modulate the influence of curcumin on the protein’s activity, we studied the uptake of a P-gp substrate under static and dynamic conditions. Caco-2 cells were cultured in bioreactors and in Transwells and the basolateral transport of rhodamine-123 was assessed in the two systems as a function of the P-gp activity. Experiments were performed with and without pre-treatment of the cells with an extract of curcumin or an arylmethyloxy-phenyl derivative to evaluate the inhibitory effect of the natural substance with respect to a synthetic compound. The results indicated that the P-gp activity of the cells cultured in the bioreactors was intrinsically lower, and that the effect of both natural and synthetic inhibitors was up modulated by the presence of flow. Our study underlies the fact that the use of more sophisticated and physiologically relevant in vitro models can bring new insights on the therapeutic effects of natural substances such as curcumin. [ABSTRACT FROM AUTHOR]

• Oxygen concentrators are essential medical devices for basic emergency care, surgery and treatment of respiratory diseases. • Oxygen therapy is little used in low-income countries as there is no supply chain for consumables and spare parts. • Zeolite monoliths were 3D printed to be employed in oxygen concentrators as locally produced spare parts. • Zeolite monoliths were more efficient in terms of specific adsorption and less demanding in terms of power supply. Oxygen concentrators are essential medical devices for basic emergency care, surgery and treatment for chronic and acute respiratory diseases. Despite their reliability and accessibility, they require spare parts which can be difficult to source in low resource settings, where the extreme environmental conditions and the overloads often make failures more frequent. The solution to this shortcoming can be their local production, enabled by 3D printing (3DP) technologies. Here we describe an extrusion-based 3DP process to produce zeolite monolith structures for use in the air adsorption process. The printed structures were physically and mechanically characterized. Compared to the material in pelletized form, the 3D printed zeolite monoliths have better transport properties (30 times increased permeability), comparable adsorption (being comparable with standard pellets), and are easier to handle. They are therefore the more appropriate technology for higher performance, easier maintenance, and lower weight oxygen concentrators in low resource settings. [ABSTRACT FROM AUTHOR]

The functional and structural resemblance of organoids to mammalian organs suggests that they might follow the same allometric scaling rules. However, despite their remarkable likeness to downscaled organs, non-luminal organoids are often reported to possess necrotic cores due to oxygen diffusion limits. To assess their potential as physiologically relevant in vitro models, we determined the range of organoid masses in which quarter power scaling as well as a minimum threshold oxygen concentration is maintained. Using data on brain organoids as a reference, computational models were developed to estimate oxygen consumption and diffusion at different stages of growth. The results show that mature brain (or other non-luminal) organoids generated using current protocols must lie within a narrow range of masses to maintain both quarter power scaling and viable cores. However, micro-fluidic oxygen delivery methods could be designed to widen this range, ensuring a minimum viable oxygen threshold throughout the constructs and mass dependent metabolic scaling. The results provide new insights into the significance of the allometric exponent in systems without a resource-supplying network and may be used to guide the design of more predictive and physiologically relevant in vitro models, providing an effective alternative to animals in research. [ABSTRACT FROM AUTHOR]

Human midbrain-specific organoids (hMOs) serve as an experimental in vitro model for studying the pathogenesis of Parkinson's disease (PD). In hMOs, neuroepithelial stem cells (NESCs) give rise to functional midbrain dopaminergic (mDA) neurons that are selectively degenerating during PD. A limitation of the hMO model is an under-supply of oxygen and nutrients to the densely packed core region, which leads eventually to a “dead core”. To reduce this phenomenon, we applied a millifluidic culture system that ensures media supply by continuous laminar flow. We developed a computational model of oxygen transport and consumption in order to predict oxygen levels within the hMOs. The modelling predicts higher oxygen levels in the hMO core region under millifluidic conditions. In agreement with the computational model, a significantly smaller “dead core” was observed in hMOs cultured in a bioreactor system compared to those ones kept under conventional shaking conditions. Comparing the necrotic core regions in the organoids with those obtained from the model allowed an estimation of the critical oxygen concentration necessary for ensuring cell vitality. Besides the reduced “dead core” size, the differentiation efficiency from NESCs to mDA neurons was elevated in hMOs exposed to medium flow. Increased differentiation involved a metabolic maturation process that was further developed in the millifluidic culture. Overall, bioreactor conditions that improve hMO quality are worth considering in the context of advanced PD modelling. [ABSTRACT FROM AUTHOR]

Maintaining hepatic functional characteristics in-vitro is considered one of the main challenges in engineering liver tissue. As hepatocytes cultured ex-vivo are deprived of their native extracellular matrix (ECM) milieu, developing scaffolds that mimic the biomechanical and physicochemical properties of the native ECM is thought to be a promising approach for successful tissue engineering and regenerative medicine applications. On the basis that the decellularized liver matrix represents the ideal design template for engineering bioinspired hepatic scaffolds, to derive quantitative descriptors of liver ECM architecture, we characterised decellularised liver matrices in terms of their biochemical, viscoelastic and structural features along with porosity, permeability and wettability. Together, these data provide a unique set of quantitative design criteria which can be used to generate guidelines for fabricating biomaterial scaffolds for liver tissue engineering. As proof-of-concept, we investigated hepatic cell response to substrate viscoelasticity. On collagen hydrogels mimicking decellularised liver mechanics, cells showed superior morphology, higher viability and albumin secretion than on stiffer and less viscous substrates. Although scaffold properties are generally inspired by those of native tissues, our results indicate significant differences between the mechano-structural characteristics of untreated and decellularised hepatic tissue. Therefore, we suggest that design rules - such as mechanical properties and swelling behaviour - for engineering biomimetic scaffolds be re-examined through further studies on substrates matching the features of decellularized liver matrices. [ABSTRACT FROM AUTHOR]

Decellularized human livers are considered the perfect extracellular matrix (ECM) surrogate because both three-dimensional architecture and biological features of the hepatic microenvironment are thought to be preserved. However, donor human livers are in chronically short supply, both for transplantation or as decellularized scaffolds, and will become even scarcer as life expectancy increases. It is hence of interest to determine the structural and biochemical properties of human hepatic ECM to derive design criteria for engineering biomimetic scaffolds. The intention of this work was to obtain quantitative design specifications for fabricating scaffolds for hepatic tissue engineering using human livers as a template. To this end, hepatic samples from five patients scheduled for hepatic resection were decellularized using a protocol shown to reproducibly conserve matrix composition and microstructure in porcine livers. The decellularization outcome was evaluated through histological and quantitative image analyses to evaluate cell removal, protein, and glycosaminoglycan content per unit area. Applying the same decellularization protocol to human liver samples obtained from five different patients yielded five different outcomes. Only one liver out of five was completely decellularized, while the other four showed different levels of remaining cells and matrix. Moreover, protein and glycosaminoglycan content per unit area after decellularization were also found to be patient- (or donor-) dependent. This donor-to-donor variability of human livers thus precludes their use as templates for engineering a generic 'one-size fits all' ECM-mimic hepatic scaffold. [ABSTRACT FROM AUTHOR]

Liver organoids (LOs) are of interest in tissue replacement, hepatotoxicity and pathophysiological studies. However, it is still unclear what triggers LO self-assembly and what the optimal environment is for their culture. Hypothesizing that LO formation occurs as a result of a fine balance between cell-substrate adhesion and cell-cell cohesion, we used 3 cell types (hepatocytes, liver sinusoidal endothelial cells and mesenchymal stem cells) to investigate LO self-assembly on different substrates keeping the culture parameters (e.g. culture media, cell types/number) and substrate stiffness constant. As cellular spheroids may suffer from oxygen depletion in the core, we also sought to identify the optimal culture conditions for LOs in order to guarantee an adequate supply of oxygen during proliferation and differentiation. The oxygen consumption characteristics of LOs were measured using an O2 sensor and used to model the O2 concentration gradient in the organoids. We show that no LO formation occurs on highly adhesive hepatic extra-cellular matrix-based substrates, suggesting that cellular aggregation requires an optimal trade-off between the adhesiveness of a substrate and the cohesive forces between cells and that this balance is modulated by substrate mechanics. Thus, in addition to substrate stiffness, physicochemical properties, which are also critical for cell adhesion, play a role in LO self-assembly. [ABSTRACT FROM AUTHOR]

The architecture and dynamic physical environment of tissues can be recreated in-vitro by combining 3D porous scaffolds and bioreactors able to apply controlled mechanical stimuli on cells. In such systems, the entity of the stimuli and the distribution of nutrients within the engineered construct depend on the micro-structure of the scaffolds. In this work, we present a new approach for optimizing computational fluid-dynamics (CFD) models for the investigation of fluid-induced forces generated by cyclic squeeze pressure within a porous construct, coupled with oxygen consumption of cardiomyocytes. A 2D axial symmetric macro-scaled model of a squeeze pressure bioreactor chamber was used as starting point for generating time dependent pressure profiles. Subsequently the fluid movement generated by the pressure fields was coupled with a complete 3D micro-scaled model of a porous protein cryogel. Oxygen transport and consumption inside the scaffold was evaluated considering a homogeneous distribution of cardiomyocytes throughout the structure, as confirmed by preliminary cell culture experiments. The results show that a 3D description of the system, coupling a porous geometry and time dependent pressure driven flow with fluid–structure-interaction provides an accurate and meaningful description of the microenvironment in terms of shear stress and oxygen distribution than simple stationary 2D models. [ABSTRACT FROM AUTHOR]

Permeability studies across biological barriers are of primary importance in drug delivery as well as in toxicology when investigating the absorption and translocation of a substance. The study of nanomaterial interaction with epithelial barriers is of particular interest given their growing use in nanomedicine as well as concerns about their potential hazard. Here we describe the design and fabrication of a new bioreactor with an ultrathin microporous sensing support for the study of nanoparticle toxicity in intestinal epithelial cells in conditions which better recapitulate the physiological environment. Thanks to the integration of 4 electrodes in the microporous membrane, the system allows real-time and continuous sensing of TEER (trans epithelial electrical resistance) during flow without interruption or perturbation of experiments. The TEER bioreactor was tested using Caco-2 cells as an in vitro model of intestinal epithelia. When exposed to silver nanoparticles, which are known to be toxic, the embedded electrodes enabled non-invasive evaluation of barrier impairment over time. This device can be used to study barrier integrity and the kinetics of nanomaterial induced damage to epithelial barriers in physiologically relevant conditions. [ABSTRACT FROM AUTHOR]

Although the detailed structure and function of the claustrum remain enigmatic, its extensive reciprocal connection with the cortex suggests a role in the integration of multisensory information. Claustrum samples, obtained from necropsy of four dogs, were formalin fixed for paraffin embedding. Sections were either stained for morpho-histological analysis or immunostained for parvalbumin (PV). We focused on PV because in cortical and hippocampal areas it is a marker of the fast-spiking interneurons which have an important role in the information transmission and processing. Soma area, perimeter and circularity were considered as morphological parameters to quantitatively group the PV positive somata by k-means clustering. The histological investigation revealed a superior pyramidoid puddle and a posterior puddle characterized by a “cloud” of neurons in its dorso-lateral part. Immunostaining showed positive somata and fibers throughout the rostro-caudal extent of the dog claustrum, localized principally in the dorsal region. k-Means clustering analysis enabled neuron classification according to size, identifying respectively big (radius = 11.42 ± 1.99 μm) and small (radius = 6.33 ± 1.08 μm) cells. No statistical differences in soma shape were observed. The topographical distribution of PV immunoreactivity suggests that the dog dorsal claustrum might be functionally related to the processing of visual inputs. Taken together our findings may help in the understanding the physiology of claustrum when compared with anatomical and functional data obtained in other species. [ABSTRACT FROM AUTHOR]

After passage through biological barriers, nanomaterials inevitably end up in contact with the vascular endothelium and can induce cardiovascular damage. In this study the toxicity and sub-lethal effects of six types of nanoparticle, including four of industrial and biomedical importance, on human endothelial cells were investigated using different in vitro assays. The results show that all the particles investigated induce some level of damage to the cells and that silver particles were most toxic, followed by titanium dioxide. Furthermore, endothelial cells were shown to be more susceptible when exposed to silver nanoparticles under flow conditions in a bioreactor. The study underlines that although simple in vitro tests are useful to screen compounds and to identify the type of effect induced on cells, they may not be sufficient to define safe exposure limits. Therefore, once initial toxicity screening has been conducted on nanomaterials, it is necessary to develop more physiologically relevant in vitro models to better understand how nanomaterials can impact on human health. [ABSTRACT FROM AUTHOR]

To explore whether stent procedure may influence transcriptional response of endothelium, we applied different physical (flow changes) and/or mechanical (stent application) stimuli to human endothelial cells in a laminar flow bioreactor (LFB) system. Gene expression analysis was then evaluated in each experimental condition. Human umbilical vein endothelial cells (HUVECs) were submitted to low and physiological (1 and 10 dyne/cm2) shear stress in absence (AS) or presence (PS) of stent positioning in a LFB system for 24 h. Different expressed genes, coming from Affymetrix results, were identified based on one-way ANOVA analysis with p values <0.01 and a fold changed >3 in modulus. Low shear stress was compared with physiological one in AS and PS conditions. Two major groups include 32 probes commonly expressed in both 1AS versus 10AS and 1PS versus 10PS comparison, and 115 probes consisting of 83 in addition to the previous 32, expressed only in 1PS versus 10PS comparison. Genes related to cytoskeleton, extracellular matrix, and cholesterol transport/metabolism are differently regulated in 1PS versus 10PS condition. Inflammatory and apoptotic mediators seems to be, instead, closely modulated by changes in flow (1 versus 10), independently of stent application. Low shear stress together with stent procedure are the experimental conditions that mainly modulate the highest number of genes in our human endothelial model. Those genes belong to pathways specifically involved in the endothelial dysfunction. [ABSTRACT FROM AUTHOR]

Nutrient balance in the human body is maintained through systemic signaling between different cells and tissues. Breaking down this circuitry to its most basic elements and reconstructing the metabolic network in-vitro provides a systematic method to gain a better understanding of how cross-talk between the organs contributes to the whole body metabolic profile and of the specific role of each different cell type. To this end, a 3-way connected culture of hepatocytes, adipose tissue and endothelial cells representing a simplified model of energetic substrate metabolism in the visceral region was developed. The 3-way culture was shown to maintain glucose and fatty acid homeostasis in-vitro. Subsequently it was challenged with insulin and high glucose concentrations to simulate hyperglycaemia. The aim was to study the capacity of the 3-way culture to maintain or restore normal circulating glucose concentrations in response to insulin and to investigate the effects these conditions on other metabolites involved in glucose and lipid metabolism. The results show that the system's metabolic profile changes dramatically in the presence of high concentrations of glucose, and that these changes are modulated by the presence of insulin. Furthermore, we observed an increase in E-selectin levels in hyperglycaemic conditions and increased IL-6 concentrations in insulin-free-hyperglycaemic conditions, indicating, respectively, endothelial injury and proinflammatory stress in the challenged 3-way system. [ABSTRACT FROM AUTHOR]

Abstract: This report describes the mechanical, thermal and biological characterisation of a solid free form microfabricated carbon nanotube–polycaprolactone composite, in which both the quantity of nanotubes in the matrix as well as the scaffold design were varied in order to tune the mechanical properties of the material. The creep and stress relaxation behaviour of the composite material was analysed to identify an optimal composition for bone tissue engineering. Moreover, the morphology and viability of osteoblast-like cells (MG63) on composite scaffolds were analysed using scanning electron microscopy and MTT assays. Our data demonstrate that by changing the ratio of CNT to PCL, the elastic modulus of the nanocomposite can be varied between 10 and 75MPa. In this range, the geometry of the scaffold can be used to finely tune its stiffness. However our PCL–CNT nanocomposites were able to sustain osteoblast proliferation and modulate cell morphology. Thus we show the potential of custom designed CNT nanocomposites for bone tissue engineering. [Copyright &y& Elsevier]

In this work we describe the fabrication of a biocompatible hydrophilic scaffold composed of cross-linked gelatin that mimics the porous three-dimensional structure of pulmonary tissue as well as its water content and mechanical properties. The lung replica also reproduces the characteristic sonographic signs of pulmonary interstitial syndrome, the B-lines or ultrasound lung comets. [ABSTRACT FROM AUTHOR]

Abstract: The adaptation of inkjet printing technology for the realisation of controlled micro- and nano-scaled biological structures is of great potential in tissue and biomaterial engineering. In this paper we present the Olivetti BioJet system and its applications in tissue engineering and cell printing. BioJet, which employs a thermal inkjet cartridge, was used to print biomolecules and living cells. It is well known that high stresses and forces are developed during the inkjet printing process. When printing living particles (i.e., cell suspensions) the mechanical loading profile can dramatically damage the processed cells. Therefore computational models were developed to predict the velocity profile and the mechanical load acting on a droplet during the printing process. The model was used to investigate the role of the stiffness of the deposition substrate during droplet impact and compared with experimental investigations on cell viability after printing on different materials. The computational model and the experimental results confirm that impact forces are highly dependent on the deposition substrate and that soft and viscous surfaces can reduce the forces acting on the droplet, preventing cell damage. These results have high relevance for cell bioprinting; substrates should be designed to have a good compromise between substrate stiffness to conserve spatial patterning without droplet coalescence but soft enough to absorb the kinetic energy of droplets in order to maintain cell viability. [Copyright &y& Elsevier]

Abstract: Scaffolds are cell adhesive matrices for the realisation of tissue constructs. Here we describe how scaffolds for tissue engineering can also be used as sensors for monitoring cellular activity such as adhesion and spreading. Carbon nanotube polymer composites were fabricated into membranes and scaffolds with electro-conductive properties. Impedance techniques were used to measure the effects of media and cell cultures on composite membranes and the results were analysed using lumped parameter models. We show that protein adhesion can be distinguished from cell adhesion as the impedance changes are much smaller for the latter (5%). In the presence of cells, impedance changes are of the order of 40% and can be correlated with adhesion, spreading and changes in cell density. [Copyright &y& Elsevier]

Abstract: This paper describes the use of a microfluidic gradient maker for the toxicological analysis of some conventional biomolecules such as hydrogen peroxide and a local anaesthetic, lidocaine on different cell cultures, human endothelial cells and myoblasts, respectively. The microfluidic device was designed and simulated using COMSOL Multiphysics® and the concentration gradient in the microfluidic network was analysed through a fluid-dynamic and mass-transport study. Subsequently the device was fabricated with soft lithography, casting PDMS in a master to obtain channels about 250μm deep. Hydrogen peroxide was tested on human endothelial cells, while lidocaine was tested on C2C12 myoblasts and an analysis was performed using propidium iodide staining followed by an imaging processing routine to obtain quantitative dose–response profiles in the gradient maker. The results show that the Gradient Maker (GM) bioreactor is a more sensitive method for detection of cell toxicity, and compared with testing of drug toxicity using microwells with individual cell cultures, allows one shot testing with a single cell culture exposed to a large number of concentrations. Moreover, the Gradient Maker was also modelled in order to realise biological pattern formation using two morphogenes acting as activator and inhibitor with varying diffusion rates. [ABSTRACT FROM AUTHOR]

Abstract: In this work, bioartificial binary blends between poly(ε-caprolactone) (PCL) and a polysaccharide (chitosan (CS) or starch (S)) with different contents of the natural polymer (5–30 wt.%) were produced. Melt-mixing and double-precipitation were the methods used for the obtainment of PCL/S and PCL/CS blends, respectively. Tubular scaffolds were produced from bioartificial blends by melt-extrusion. Physico-chemical characterisation was performed by differential scanning calorimetry analysis (DSC), thermogravimetry (TGA), scanning electron microscopy (SEM), infrared analysis (FTIR-ATR and micro-ATR mapping), atomic force microscopy (AFM) and stress–strain tests. Blends were not miscible, phase-separated systems, showing a homogeneous composition and morphology only at low polysaccharide content (≤10 wt.%). The biocompatibility of bioartificial guides was investigated by culturing NIH-3T3 mouse fibroblasts. Cells response showed the following order: PCL/S>PCL>PCL/CS. For each blend type, biocompatibility increased with decreasing the polysaccharide content. In vitro cell tests using S5Y5 neuroblastoma cells, carried out on the most biocompatible blends, assessed their absence of cytotoxicity towards these model cells of the nervous tissue. Results showed that blends with a low chitosan or starch content (≤10 wt.%) are promising for the regeneration of tissues requiring tubular scaffolds, such as the peripheral nerves. [Copyright &y& Elsevier]

Abstract: A great deal of effort is being dedicated to the development of new devices able to conduct effective in vitro toxicology analyses. This paper describes the use of a microfluidic gradient maker for the toxicological analysis of two conventional local anesthetics, bupivacaine and lidocaine on cell cultures. The microfluidic device was designed and simulated using COMSOL Multiphysics® and the concentration gradient in the microfluidic network was analysed through a fluidodynamic and diffusive study. Subsequently the device was fabricated with soft lithography, casting PDMS in a master to obtain channels about 250μm deep. Both drugs were tested on C2C12 myoblasts and an analysis was performed using propidium iodide staining followed by an imaging processing routine to obtain quantitative dose–response profiles in the gradient maker. The system was critically compared with microwell-based toxicity testing. The results show that the GM is a more sensitive method for detection of cell toxicity, and compared with testing of drug toxicity using microwells with individual cell cultures, allows one shot testing with a single cell culture exposed to a large number of concentrations. However, the flow rates required to obtain a suitable concentration range across the device may damage shear sensitive cells. [Copyright &y& Elsevier]

The optimization of in vitro cell culture for tissue engineering, pharmacological, or metabolic studies requires a large number of experiments to be performed under varying conditions. In this paper, we describe a high-throughput bioreactor system that allows the conduction of parallel experiments in a simulated in vivo-like environment. Our bioreactors consist of tissue-, organ-, or system-specific culture chambers and a mixing device controlled by an embedded system that regulates the insertion of gas in the culture medium in order to control pH and pressure. Each culture chamber and mixing device possesses an autonomous control system that is able to ensure an optimal environment for cells. A computer communicates with the embedded system to acquire data and set up experimental variables. With this apparatus, we can perform a high-throughput experiment controlling several bioreactors working in parallel. In this paper, we discuss the architecture and design of the system, and the results of some experiments which simulate physiological and pathological conditions are presented. [ABSTRACT FROM AUTHOR]

In a previous report on a multicomponent sensor array for electronic tongues, carbon nanotube (CNT) composite sensors were identified as the most reproducible and sensitive. To further elucidate the response mechanisms and optimize the working parameters of CNT composite sensors, we focused on a single polymer CNT combination and tested its response to the 5 tastes in a variety of measurement modes. The results demonstrate that an acquisition frequency of 200 Hz is the most suitable in terms of concentration discrimination, and that this sensor was extremely sensitive to all tastes except sweet, indicating that the recognition is based on charge interactions between the sensor matrix and the analyte. [ABSTRACT FROM AUTHOR]

Abstract: Carbon nanotube (NT) actuators were fabricated using the micromolding method. In order to prevent the nanotubes from aggregating, they were enclosed in a partially cross-linked polyvinylalcohol–polyallylamine matrix. A unimorph matrix bender in the form of a small multi-layered strip, 5 mm×15 mm was fabricated. The resulting composite system: NT/PVA, solid polymer electrolyte and metal contact, was characterised using impedance analysis and cyclic voltammetry. Initial measurements on electro-mechanical transduction show a conversion efficiency of 0.2 μm/mV. An electrochemical model of the impedance of the system was then developed, with which the experimental results agree fairly well. [Copyright &y& Elsevier]

Elevated intraocular pressure may lead to retinal ganglion cell injury and consequent visual deficits. Chronic intraocular pressure increase is a major risk factor for glaucoma, a leading blinding disease, and permanent visual deficits can also occur following acute pressure increments due to trauma, acute glaucoma or refractive surgery. How pressure affects retinal neurons is not firmly established. Mechanical damage at the optic nerve head, reduced blood supply, inflammation and cytotoxic factors have all been called into play. Reasoning that the analysis of retinal neurons soon after pressure elevation would provide useful cues, we imaged individual ganglion cells in isolated rat retinas before and after short hydrostatic pressure increments. We found that slowly rising pressure to peaks observed in trauma, acute glaucoma or refractive surgery (50–90 mmHg) did not damage ganglion cells, whereas a rapid 1 min pulse to 50 mmHg injured 30% of these cells within 1 h. The severity of damage and the number of affected cells increased with stronger or repeated insults. Degrading extracellular ATP or blocking the P2X receptors for ATP prevented acute pressure-induced damage in ganglion cells. Similar effects were observed in vivo. A short intraocular pressure transient increased extracellular ATP levels in the eye fluids and damaged ganglion cells within 1 h. Reducing extracellular ATP in the eye prevented damage to ganglion cells and accelerated recovery of their response to light. These data show that rapid pressure transients induce acute ganglion cell injury and unveil the causal role of extracellular ATP elevation in such injury. [ABSTRACT FROM AUTHOR]

This paper describes a study of surface derivatisation methods applied to two-dimensional polymer matrices microfabricated using the Pressure-Assisted Microsyringe (PAM) technique. A blend of polylactide and polycaprolactone was used as the matrix material, and surface chemistry techniques based on silanes and polyethyleneglycol (PEG) derivatives were employed to render the surface underlying the scaffold anti-adhesive whilst polylysine was covalently coupled to the surface of the polymer matrix to enhance cell adhesion. Prior to cell-adhesion tests, the surfaces and matrices were analysed using physico-chemical techniques, such as surface tension, surface potential and fluorescence. Adhesion of primary endothelial cells was evaluated using cell counting techniques. The results demonstrate that both PEGs and silanes are about 66% efficient at demarcating endothelial cell adhesion in short term experiments and that covalently-bound polylysine to the polymer matrix increases cell adhesion twofold with respect to the adsorbed polypeptide. [ABSTRACT FROM AUTHOR]

A variety of techniques for the manufacture of biodegradable, three-dimensional scaffolds for tissue engineering have been developed in recent years. In this study, we report and compare two simple methods for fabricating poly(dl-lactide-co-glycolide) (PLGA) scaffolds with feature sizes of 10–200 μm, which have been developed in our laboratories.The first technique is based on the use of a microsyringe that makes use of a computer-controlled, three-axis micropositioner, which allows the control of motor speeds and position. A PLGA solution is drawn from the needle of the syringe by the application of a constant pressure of 10–300 mm Hg resulting in controlled polymer deposition of 10–600 μm in diameter. The second technique is based on “soft lithographic” approaches that utilizes a Poly(dimethylsiloxane) (PDMS) mold. The polymer solution is cast on the mold under vacuum. Polymer concentration, solvent composition, and casting conditions influence the integrity and the lateral resolution of the resulting scaffold. Both techniques allow the possibility of constructing three-dimensional architectures that permit the study of cell behaviour in an environment similar to that in vivo, and may provide tools for the construction of engineered tissue. [Copyright &y& Elsevier]

Modeling the physiology of the human placenta is still a challenge, despite the great number of scientific advancements made in the field. Animal models cannot fully replicate the structure and function of the human placenta and pose ethical and financial hurdles. In addition, increasingly stricter animal welfare legislation worldwide is incentivizing the use of 3R (reduction, refinement, replacement) practices. What efforts have been made to develop alternative models for the placenta so far? How effective are they? How can we improve them to make them more predictive of human pathophysiology? To address these questions, this review aims at presenting and discussing the current models used to study phenomena at the placenta level: in vivo, ex vivo, in vitro and in silico. We describe the main achievements and opportunities for improvement of each type of model and critically assess their individual and collective impact on the pursuit of predictive studies of the placenta in line with the 3Rs and European legislation. [ABSTRACT FROM AUTHOR]

The article discusses role of biomedical engineering in ensuring needs-based technology innovation for development of a strong health technology research and development base in Africa. It mentions funding of a project which focuses on developing health technology competencies in forms of rehabilitation engineering to address disabilities caused due to war or late diagnosis of disease, medical devices which are robust to various climatic conditions, and image analysis tools.

Bone extracellular matrix (ECM) is a natural composite made of collagen and mineral hydroxyapatite (HA). Dynamic cell-ECM interactions play a critical role in regulating cell differentiation and function. Understanding the principal ECM cues promoting osteogenic differentiation would be pivotal for both bone tissue engineering and regenerative medicine. Altering the mineral content generally modifies the stiffness as well as other physicochemical cues provided by composite materials, complicating the 'cause-effect' analysis of resultant cell behaviour. To isolate the contribution of mechanical cues from other HA-derived signals, we developed and characterised composite HA/gelatin scaffolds with different mineral contents along with a set of stiffness-matched HA-free gelatin scaffolds. Samples were seeded with human periosteal derived progenitor cells (PDPCs) and cultured over 7 days, analysing their resultant morphology and gene expression. Our results show that both stiffness and HA contribute to directing PDPC osteogenic differentiation, highlighting the role of stiffness in triggering the expression of osteogenic genes and of HA in accelerating the process, particularly at high concentrations. [ABSTRACT FROM AUTHOR]