Using integrated meta-omics to appreciate the role of the gut microbiota in epilepsy.

The way the human microbiota may modulate neurological pathologies is a fascinating matter of research. Epilepsy is a common neurological disorder, which has been largely investigated in correlation with microbiota health and function. However, the mechanisms that regulate this apparent connection are scarcely defined, and extensive effort has been conducted to understand the role of microbiota in preventing and reducing epileptic seizures. Intestinal bacteria seem to modulate the seizure frequency mainly by releasing neurotransmitters and inflammatory mediators. In order to elucidate the complex microbial contribution to epilepsy pathophysiology, integrated meta-omics could be pivotal. In fact, the combination of two or more meta-omics approaches allows a multifactorial study of microbial activity within the frame of disease or drug treatments. In this review, we provide information depicting and supporting the use of multi-omics to study the microbiota-epilepsy connection. We described different meta-omics analyses (metagenomics, metatranscriptomics, metaproteomics and metabolomics), focusing on current technical challenges in stool collection procedures, sample extraction methods and data processing. We further discussed the current advantages and limitations of using the integrative approach of multi-omics in epilepsy investigations.

Technological tools and strategies for culturing human gut microbiota in engineered in vitro models.

The gut microbiota directly impacts the pathophysiology of different human body districts. Consequently, microbiota investigation is an hot topic of research and its in vitro culture has gained extreme interest in different fields. However, the high sensitivity of microbiota to external stimuli, such as sampling procedure, and the physicochemical complexity of the gut environment make its in vitro culture a challenging task. New engineered microfluidic gut-on-a-chip devices have the potential to model some important features of the intestinal structure, but they are usually unable to sustain culture of microbiota over an extended period of time. The integration of gut-on-a-chip devices with bioreactors for continuous bacterial culture would lead to fast advances in the study of microbiota-host crosstalk. In this review, we summarize the main technologies for the continuous culture of microbiota as upstream systems to be coupled with microfluidic devices to study bacteria-host cells communication. The engineering of integrated microfluidic platforms, capable of sustaining both anaerobic and aerobic cultures, would be the starting point to unveil complex biological phenomena proper of the microbiota-host crosstalks, paving to way to multiple research and technological applications.

Influence of the static magnetic field on cell response in a miniaturized optically accessible bioreactor for 3D cell culture.

Hydraulic sealing is a crucial condition for the maintenance of sterility during long term operation of microfluidic bioreactors. We developed a miniaturized optically accessible bioreactor (MOAB) allowing perfused culture of 3D cellularised constructs. In the MOAB, the culture chambers are sealed by magnets that generate a weak static magnetic field (SMF). Here, we predicted computationally the exact level of SMF to which cells are subjected during culture in the MOAB and we assessed its influence on the viability, metabolic activity and gene expression of neuroblastoma-derived cells cultured up to seven days. The predicted SMF ranged from 0.32 to 0.57 T using an axial-symmetric model of a single chamber, whereas it ranged from 0.35 to 0.62 T using a 3D model of the complete device. Cell function was evaluated in SH-SY5Y neuroblastoma cells at 2 and 7 days of culture in the MOAB, compared to 2D monolayer, 3D non-perfused constructs, and 3D perfused constructs cultured in a modified MOAB with magnet-free sealing. We measured the cell metabolic activity normalized by the DNA content and the expression levels of heat-shock protein 70 (Hsp-70), Bcl-2 and Bax. We found that the level of SMF applied to cells in the MOAB did not influence their metabolic activity and exerted a stressful effect in 2D monolayer, not confirmed in 3D conditions, neither static not perfused. Instead, the magnets provided a significantly greater hydraulic sealing in long-term culture, thus the MOAB might be potentially exploitable for the development of reliable in vitro models of neurodegeneration.

Recombinant human Tat-Hsp70-2: A tool for neuroprotection.

Human Hsp70-2 is a chaperone expressed mainly in the nervous system. Up to now, no study has reported on the recombinant expression of this important human chaperone. Herein, we describe the successful purification and characterization of recombinant human Hsp70-2 in Escherichia coli in both the full-length and the chimeric protein containing the protein transduction domain corresponding to the trans-activator of transcription (Tat) from HIV. Under optimized conditions, the Tat-Hsp70-2 was expressed in a soluble form and purified by two chromatographic steps (in a 3.6 mg/L fermentation broth yield): recombinant Tat-Hsp70-2 was folded and showed ATPase activity. In contrast, the full-length recombinant protein was only expressed in the form of inclusion bodies and thus was purified following a refolding procedure. The refolded Hsp70-2 protein was inactive and the protein conformation slightly altered as compared to the corresponding Tat-fused variant. The Tat-Hsp70-2 protein (100 nM), when added to human neuroblastoma SH-SY5Y cells subjected to hydrogen peroxide or 6-hydroxydopamine stress, partially protected from the deleterious effect of these treatments. This work describes an approach for the functional expression of human Tat-Hsp70-2 that provides sufficient material for detailed structure-function studies and for testing its ability to protect neuroblastoma cells from oxidative stress.

Cross-linked poly(acrylic acids) microgels and agarose as semi-interpenetrating networks for resveratrol release.

Carbomers, cross-linked poly(acrylic acid) microgels, have been widely used in pharmaceutical formulations as swollen hydrogels. Agarose, whose thermoreversibility may be exploited for drug loading, forms a gel with a mechanism involving coil-helix transition at about 36 °C. In this work carbomer microgels were combined with agarose networks in a semi-interpenetrating polymer network structure, aiming at obtaining suitable delivery systems for the loading and release of molecules with poor bioavailability but high therapeutic interest, like resveratrol. The rheological properties of the formulations and their in vitro cytocompatibility were studied and optimized acting on the neutralizing agent (triethylamine (N,N-diethylethanamine), triethanolamine (tris(2-hydroxyethyl)amine) and sodium hydroxide) and amount of OH donors (1,2-propanediol and glycerol). As a preparation method, autoclaving was introduced to simultaneously obtain heating and sterilising. Among the different neutralizing agents, NaOH was chosen to avoid the use of amines, considering the final application. Without the addition of alcohols as typical OH donors to induce Carbomer gelification, gels with appropriate rheological properties and stability were produced. For this formulation, the release of resveratrol after 7 days was about 80 % of the loaded mass, suggesting it is an interesting approach to be exploited for the development of innovative resveratrol delivery systems.

Hydrogel-based nanocomposites and mesenchymal stem cells: a promising synergistic strategy for neurodegenerative disorders therapy.

Hydrogel-based materials are widely employed in the biomedical field. With regard to central nervous system (CNS) neurodegenerative disorders, the design of injectable nanocomposite hydrogels for in situ drug or cell release represents an interesting and minimally invasive solution that might play a key role in the development of successful treatments. In particular, biocompatible and biodegradable hydrogels can be designed as specific injectable tools and loaded with nanoparticles (NPs), to improve and to tailor their viscoelastic properties upon injection and release profile. An intriguing application is hydrogel loading with mesenchymal stem cells (MSCs) that are a very promising therapeutic tool for neurodegenerative or traumatic disorders of the CNS. This multidisciplinary review will focus on the basic concepts to design acellular and cell-loaded materials with specific and tunable rheological and functional properties. The use of hydrogel-based nanocomposites and mesenchymal stem cells as a synergistic strategy for nervous tissue applications will be then discussed.

Development of an Induced Pluripotent Stem Cell-Based Liver-on-a-Chip Assessed with an Alzheimer's Disease Drug.

Liver-related drug metabolism is a key aspect of pharmacokinetics and possible toxicity. From this perspective, the availability of advanced in vitro models for drug testing is still an open need, also to the end of reducing the burden of in vivo experiments. In this scenario, organ-on-a-chip is gaining attention as it couples a state-of-the art in vitro approach to the recapitulation of key in vivo physiological features such as fluidodynamics and a tri-dimensional cytoarchitecture. We implemented a novel liver-on-a-chip (LoC) device based on an innovative dynamic device (MINERVA 2.0) where functional hepatocytes (iHep) have been encapsulated into a 3D hydrogel matrix interfaced through a porous membrane with endothelial cells (iEndo)]. Both lines were derived from human-induced pluripotent stem cells (iPSCs), and the LoC was functionally assessed with donepezil, a drug approved for Alzheimer's disease therapy. The presence of iEndo and a 3D microenvironment enhanced the expression of liver-specific physiologic functions as in iHep, after 7 day perfusion, we noticed an increase of albumin, urea production, and cytochrome CYP3A4 expression compared to the iHep static culture. In particular, for donepezil kinetics, a computational fluid dynamic study conducted to assess the amount of donepezil diffused into the LoC indicated that the molecule should be able to pass through the iEndo and reach the target iHep construct. Then, we performed experiments of donepezil kinetics that confirmed the numerical simulations. Overall, our iPSC-based LoC reproduced the in vivo physiological microenvironment of the liver and was suitable for potential hepatotoxic screening studies.

Human gut epithelium features recapitulated in MINERVA 2.0 millifluidic organ-on-a-chip device.

We developed an innovative millifluidic organ-on-a-chip device, named MINERVA 2.0, that is optically accessible and suitable to serial connection. In the present work, we evaluated MINERVA 2.0 as millifluidic gut epithelium-on-a-chip by using computational modeling and biological assessment. We also tested MINERVA 2.0 in a serially connected configuration prodromal to address the complexity of multiorgan interaction. Once cultured under perfusion in our device, human gut immortalized Caco-2 epithelial cells were able to survive at least up to 7 days and form a three-dimensional layer with detectable tight junctions (occludin and zonulin-1 positive). Functional layer development was supported by measurable trans-epithelial resistance and FITC-dextran permeability regulation, together with mucin-2 expression. The dynamic culturing led to a specific transcriptomic profile, assessed by RNASeq, with a total of 524 dysregulated transcripts (191 upregulated and 333 downregulated) between static and dynamic condition. Overall, the collected results suggest that our gut-on-a-chip millifluidic model displays key gut epithelium features and, thanks to its modular design, may be the basis to build a customizable multiorgan-on-a-chip platform.

Induced pluripotent stem cell-based organ-on-a-chip as personalized drug screening tools: A focus on neurodegenerative disorders.

The Organ-on-a-Chip (OoC) technology shows great potential to revolutionize the drugs development pipeline by mimicking the physiological environment and functions of human organs. The translational value of OoC is further enhanced when combined with patient-specific induced pluripotent stem cells (iPSCs) to develop more realistic disease models, paving the way for the development of a new generation of patient-on-a-chip devices. iPSCs differentiation capacity leads to invaluable improvements in personalized medicine. Moreover, the connection of single-OoC into multi-OoC or body-on-a-chip allows to investigate drug pharmacodynamic and pharmacokinetics through the study of multi-organs cross-talks. The need of a breakthrough thanks to this technology is particularly relevant within the field of neurodegenerative diseases, where the number of patients is increasing and the successful rate in drug discovery is worryingly low. In this review we discuss current iPSC-based OoC as drug screening models and their implication in development of new therapies for neurodegenerative disorders.

The microbiota-gut-brain axis and epilepsy from a multidisciplinary perspective: Clinical evidence and technological solutions for improvement of in vitro preclinical models.

Epilepsy is a common neurological disease characterized by the enduring predisposition of the brain to generate seizures. Among the recognized causes, a role played by the gut microbiota in epilepsy has been hypothesized and supported by new investigative approaches. To dissect the microbiota-gut-brain (MGB) axis involvement in epilepsy, in vitro modeling approaches arouse interest among researchers in the field. This review summarizes, first of all, the evidence of a role of the MGB axis in epilepsy by providing an overview of the recent clinical and preclinical studies and showing how dietary modification, microbiome supplementations, and hence, microbiota alterations may have an impact on seizures. Subsequently, the currently available strategies to study epilepsy on animal and in vitro models are described, focusing attention on these latter and the technological challenges for integration with already existing MGB axis models. Finally, the implementation of existing epilepsy in vitro systems is discussed, offering a complete overview of the available technological tools which may improve reliability and clinical translation of the results towards the development of innovative therapeutic approaches, taking advantage of complementary technologies.

Efficacy of zosteric acid sodium salt on the yeast biofilm model Candida albicans.

Candida albicans is the most notorious and the most widely studied yeast biofilm former. Design of experiments (DoE) showed that 10 mg/L zosteric acid sodium salt reduced C. albicans adhesion and the subsequent biofilm formation by at least 70%, on both hydrophilic and hydrophobic surfaces of 96-well plates. Indeed, biofilm imaging revealed the dramatic impact of zosteric acid sodium salt on biofilm thickness and morphology, due to the inability of the cells to form filamentous structures while remaining metabolically active. In the same way, 10 mg/L zosteric acid sodium salt inhibited C. albicans biofilm formation when added after the adhesion phase. Contrary to zosteric acid sodium salt, methyl zosterate did not affect yeast biofilm. In addition, zosteric acid sodium salt enhanced sensitivity to chlorhexidine, chlorine, hydrogen peroxide, and cis-2-decenoic acid, with a reduction of 0.5 to 8 log units. Preliminary in vitro studies using suitable primary cell based models revealed that zosteric acid sodium salt did not compromise the cellular activity, adhesion, proliferation or morphology of either the murine fibroblast line L929 or the human osteosarcoma line MG-63. Thus the use of zosteric acid sodium salt could provide a suitable, innovative, preventive, and integrative approach to preventing yeast biofilm formation.

Microbiota-Host Immunity Communication in Neurodegenerative Disorders: Bioengineering Challenges for In Vitro Modeling.

Human microbiota communicates with its host by secreting signaling metabolites, enzymes, or structural components. Its homeostasis strongly influences the modulation of human tissue barriers and immune system. Dysbiosis-induced peripheral immunity response can propagate bacterial and pro-inflammatory signals to the whole body, including the brain. This immune-mediated communication may contribute to several neurodegenerative disorders, as Alzheimer's disease. In fact, neurodegeneration is associated with dysbiosis and neuroinflammation. The interplay between the microbial communities and the brain is complex and bidirectional, and a great deal of interest is emerging to define the exact mechanisms. This review focuses on microbiota-immunity-central nervous system (CNS) communication and shows how gut and oral microbiota populations trigger immune cells, propagating inflammation from the periphery to the cerebral parenchyma, thus contributing to the onset and progression of neurodegeneration. Moreover, an overview of the technological challenges with in vitro modeling of the microbiota-immunity-CNS axis, offering interesting technological hints about the most advanced solutions and current technologies is provided.

Microbiological-Chemical Sourced Chondroitin Sulfates Protect Neuroblastoma SH-SY5Y Cells against Oxidative Stress and Are Suitable for Hydrogel-Based Controlled Release.

Chondroitin sulfates (CS) are a class of sulfated glycosaminoglycans involved in many biological processes. Several studies reported their protective effect against neurodegenerative conditions like Alzheimer's disease. CS are commonly derived from animal sources, but ethical concerns, the risk of contamination with animal proteins, and the difficulty in controlling the sulfation pattern have prompted research towards non-animal sources. Here we exploited two microbiological-chemical sourced CS (i.e., CS-A,C and CS-A,C,K,L) and Carbopol 974P NF/agarose semi-interpenetrating polymer networks (i.e., P.NaOH.0 and P.Ethanol.0) to set up a release system, and tested the neuroprotective role of released CS against H2O2-induced oxidative stress. After assessing that our CS (1-100 µM) require a 3 h pre-treatment for neuroprotection with SH-SY5Y cells, we evaluated whether the autoclave type (i.e., N- or B-type) affects hydrogel viscoelastic properties. We selected B-type autoclaves and repeated the study after loading CS (1 or 0.1 mg CS/0.5 mL gel). After loading 1 mg CS/0.5 mL gel, we evaluated CS release up to 7 days by 1,9-dimethylmethylene blue (DMMB) assay and verified the neuroprotective role of CS-A,C (1 µM) in the supernatants. We observed that CS-A,C exhibits a broader neuroprotective effect than CS-A,C,K,L. Moreover, sulfation pattern affects not only neuroprotection, but also drug release.

3D brain tissue physiological model with co-cultured primary neurons and glial cells in hydrogels.

Recently, researchers have focused on the role of gut microbiota on human health and reported the existence of a bidirectional relationship between intestinal microbiota and the brain, referred to as microbiota-gut-brain axis (MGBA). In this context, the development of an organ-on-a-chip platform recapitulating the main players of the MGBA would help in the investigations of the biochemical mechanisms involved. In this work, we focused on the development of a new, hydrogel-based, 3D brain-like tissue model to be hosted in the brain compartment of the aforementioned platform. We previously cultured primary mouse microglial cells, cortical neurons and astrocytes independently, once embedded or covered by a millimeter layer of two selected collagen-based hydrogels. We evaluated cell metabolic activity up to 21 days, cell morphology, spatial distribution and synapse formation. Then, we exploited the best performing culturing condition and developed a more complex brain-like tissue model based on the co-culture of cortical neurons and glial cells in physiological conditions. The obtained results indicate that our 3D hydrogel-based brain tissue model is suitable to recapitulate in vitro the key biochemical parameters of brain tissue.

A miniaturized hydrogel-based in vitro model for dynamic culturing of human cells overexpressing beta-amyloid precursor protein.

Recent findings have highlighted an interconnection between intestinal microbiota and the brain, referred to as microbiota-gut-brain axis, and suggested that alterations in microbiota composition might affect brain functioning, also in Alzheimer's disease. To investigate microbiota-gut-brain axis biochemical pathways, in this work we developed an innovative device to be used as modular unit in an engineered multi-organ-on-a-chip platform recapitulating in vitro the main players of the microbiota-gut-brain axis, and an innovative three-dimensional model of brain cells based on collagen/hyaluronic acid or collagen/poly(ethylene glycol) semi-interpenetrating polymer networks and ß-amyloid precursor protein-Swedish mutant-expressing H4 cells, to simulate the pathological scenario of Alzheimer's disease. We set up the culturing conditions, assessed cell response, scaled down the three-dimensional models to be hosted in the organ-on-a-chip device, and cultured them both in static and in dynamic conditions. The results suggest that the device and three-dimensional models are exploitable for advanced engineered models representing brain features also in Alzheimer's disease scenario.

Human Gut-Microbiota Interaction in Neurodegenerative Disorders and Current Engineered Tools for Its Modeling.

The steady increase in life-expectancy of world population, coupled to many genetic and environmental factors (for instance, pre- and post-natal exposures to environmental neurotoxins), predispose to the onset of neurodegenerative diseases, whose prevalence is expected to increase dramatically in the next years. Recent studies have proposed links between the gut microbiota and neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. Human body is a complex structure where bacterial and human cells are almost equal in numbers, and most microbes are metabolically active in the gut, where they potentially influence other target organs, including the brain. The role of gut microbiota in the development and pathophysiology of the human brain is an area of growing interest for the scientific community. Several microbial-derived neurochemicals involved in the gut-microbiota-brain crosstalk seem implicated in the biological and physiological basis of neurodevelopment and neurodegeneration. Evidence supporting these connections has come from model systems, but there are still unsolved issues due to several limitations of available research tools. New technologies are recently born to help understanding the causative role of gut microbes in neurodegeneration. This review aims to make an overview of recent advances in the study of the microbiota-gut-brain axis in the field of neurodegenerative disorders by: (a) identifying specific microbial pathological signaling pathways; (b) characterizing new, advanced engineered tools to study the interactions between human cells and gut bacteria.

New aliphatic glycerophosphoryl-containing polyurethanes: synthesis, platelet adhesion and elution cytotoxicity studies.

in this study new poly(ether)urethanes (PeUs) based on aliphatic diisocyanates were synthesized with phospholipid-like residues as chain extenders. The primary objective was to prepare new polyurethanes from diisocyanates that are less toxic than the aromatic ones widely used in medical-grade polyurethanes, in order to investigate the effect of the different aromatic or aliphatic hard segment content on the final properties of the materials. Some glycerophospho residues were simultaneously introduced to enhance the hemocompatibility of these materials. Polymers were prepared by a conventional two-step solution polymerization procedure using hexamethylene diisocyanate (HDi) and dodecametilendiisocyanate (DDi) and poly(1,4-butanediol) with molecular weight 1000 to form prepolymers, which were subsequently polymerized with 1-glycerophosphorylcholine (1-GPC) or glycerophosphorylserine (GPS) to act as chain extenders. The reference polymers bearing 1,4-butandiol (BD) were also synthesized. The polymers obtained were characterized by fourier transform infrared spectroscopy (fT-iR), nuclear magnetic resonance (1H nmR), and differential scanning calorimetry (DSC). The hemocompatibility of synthesized segmented polyurethanes was preliminarily investigated by platelet-rich plasma contact studies and related scanning electron microscopy (Sem) photographs as well as by cell viability assay after cell exposure to material elutions to assess the effect of any toxic leachables coming out from the samples. Two of the polymers gave interesting results, suggesting the desirability of further investigation into their possible use in biomedical devices.

Osteogenic differentiation of human mesenchymal stromal cells on surface-modified titanium alloys for orthopedic and dental implants.

PURPOSE: Surface properties of titanium alloys, used for orthopedic and dental applications, are known to affect implant interactions with host tissues. Osteointegration, bone growth and remodeling in the area surrounding the implants can be implemented by specific biomimetic treatments; these allow the preparation of micro/nanostructured titanium surfaces with a thickened oxide layer, doped with calcium and phosphorus ions. We have challenged these experimental titanium alloys with primary human bone marrow stromal cells to compare the osteogenic differentiation outcomes of the cells once they are seeded onto the modified surfaces, thus simulating a prosthetic device-biological interface of clinical relevance. METHODS: A specific anodic spark discharge was the biomimetic treatment of choice, providing experimental titanium disks treated with different alkali etching approaches. The disks, checked by electron microscopy and spectroscopy, were subsequently used as substrates for the proliferation and osteogenic differentiation of human cells. Expression of markers of the osteogenic lineage was assessed by means of qualitative and quantitative PCR, by cytochemistry, immunohistochemistry, Western blot and matrix metalloprotease activity analyses. RESULTS: Metal surfaces were initially less permissive for cell growth. Untreated control substrates were less efficient in sustaining mineralized matrix deposition upon osteogenic induction of the cells. Interestingly, bone sialo protein and matrix metalloprotease 2 levels were enhanced on experimental metals compared to control surfaces, particularly for titanium oxide coatings etched with KOH. DISCUSSION: As a whole, the KOH-modification of titanium surfaces seems to allow the best osteogenic differentiation of human mesenchymal stromal cells, representing a possible plus for future clinical prosthetic applications.

Multidisciplinary perspectives for Alzheimer's and Parkinson's diseases: hydrogels for protein delivery and cell-based drug delivery as therapeutic strategies.

This review presents two intriguing multidisciplinary strategies that might make the difference in the treatment of neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. The first proposed strategy is based on the controlled delivery of recombinant proteins known to play a key role in these neurodegenerative disorders that are released in situ by optimized polymer-based systems. The second strategy is the use of engineered cells, encapsulated and delivered in situ by suitable polymer-based systems, that act as drug reservoirs and allow the delivery of selected molecules to be used in the treatment of Alzheimer's and Parkinson's diseases. In both these scenarios, the design and development of optimized polymer-based drug delivery and cell housing systems for central nervous system applications represent a key requirement. Materials science provides suitable hydrogel-based tools to be optimized together with suitably designed recombinant proteins or drug delivering-cells that, once in situ, can provide an effective treatment for these neurodegenerative disorders. In this scenario, only interdisciplinary research that fully integrates biology, biochemistry, medicine and materials science can provide a springboard for the development of suitable therapeutic tools, not only for the treatment of Alzheimer's and Parkinson's diseases but also, prospectively, for a wide range of severe neurodegenerative disorders.

An Organ-On-A-Chip Engineered Platform to Study the Microbiota-Gut-Brain Axis in Neurodegeneration.

After decades of research, the etiology of neurodegenerative disorders such as Alzheimer's or Parkinson's disease is still mostly unknown. Recent findings indicate that the microorganisms in the human gut might be involved in neurodegenerative pathways. Here, we discuss an innovative groundbreaking bioengineering approach that could make a difference in this intriguing scenario.