Introduction: The ability to resolve pulpal inflammation to achieve predictable regeneration of the dentin-pulp complex has remained elusive and presents a challenge for clinicians and researchers. ...Although the dentin-pulp complex can react naturally to injury by forming a bridge of reparative dentin that protects the pulp from further damage, this process is significantly impaired if inflammation persists. Because the secretion of inflammatory cytokines by injured pulpal cells causes significant pain and discomfort to patients, it is critical to resolve pulpal inflammation in a timely manner so as to create a microenvironment conducive for pulpal healing and reparative dentin formation. The emergent field of regenerative endodontics has encouraged the development and application of biologically driven therapies that take advantage of the intrinsic healing capacities of host cells within dental pulp and the periapical complex. Methods: These studies were designed to test the hypothesis that exposure to hypoxic conditions can modulate the production of inflammatory cytokines/factors by mesenchymal cells in vitro. A multi-domain peptide hydrogel system that is highly conducive for the growth and differentiation of tooth-derived stem cells was used for these studies. Stem cells from human exfoliated deciduous teeth (SHEDs) were first cultured within 3-dimensional hydrogel constructs and then challenged with hypoxic stresses via addition of H2O2. Results: MDP constructs were successfully generated, challenged with H2O2, decellularized and lyophilized, forming a potential biomaterial containing hypoxia induced repair molecules. The ability of cell-derived factors to convert the phenotype of lipopolysaccharide-primed macrophages from a proinflammatory to a pro-resolving state was examined in the presence of the lyophilized SHED cell constructs. Conclusions: Our data suggest that hypoxia induced SHED cell products can be captured within the hydrogel system and may be useful in the resolution of pulpal inflammation to create a favorable microenvironment for regeneration of the dentin-pulp complex.
The dominance of national culture in organisational environments has been widely acknowledged. This study aims to explore the impact of national culture on information security awareness by examining ...cultural characteristics in the public sector. Drawing on Hofstede's model, a literature review was conducted to analyze existing knowledge and identify cultural traits in selected countries. Empirical data were collected through semi-structured interviews conducted in Sweden, France, and Tunisia. The data were analyzed using the Human Aspects of Information Security (HAIS) model, revealing distinct patterns and emphasising the role of culture in shaping behaviours. The findings underscore the importance of involving Swedish employees in the policy design process to effectively enhance information security awareness. For French employees, it was found that learning from past experiences and respecting hierarchical structures are crucial factors in raising awareness. Conversely, in Tunisia, information security initiatives should take into account the social and relational aspects to promote awareness effectively. These insights provide valuable guidance to managers in multinational organisations, enabling them to comprehend the underlying forces and dynamics in countries with similar characteristics.
Autism spectrum disorder (ASD) is a neurodevelopmental disorder with unclear etiology and imprecise genetic causes. The main goal of this work was to investigate neuronal connectivity and the ...interplay between neurons and astrocytes from individuals with nonsyndromic ASD using induced pluripotent stem cells.
Induced pluripotent stem cells were derived from a clinically well-characterized cohort of three individuals with nonsyndromic ASD sharing common behaviors and three control subjects, two clones each. We generated mixed neural cultures analyzing synaptogenesis and neuronal activity using a multielectrode array platform. Furthermore, using an enriched astrocyte population, we investigated their role in neuronal maintenance.
ASD-derived neurons had a significant decrease in synaptic gene expression and protein levels, glutamate neurotransmitter release, and, consequently, reduced spontaneous firing rate. Based on co-culture experiments, we observed that ASD-derived astrocytes interfered with proper neuronal development. In contrast, control-derived astrocytes rescued the morphological neuronal phenotype and synaptogenesis defects from ASD neuronal co-cultures. Furthermore, after identifying interleukin-6 secretion from astrocytes in individuals with ASD as a possible culprit for neural defects, we were able to increase synaptogenesis by blocking interleukin-6 levels.
Our findings reveal the contribution of astrocytes to neuronal phenotype and confirm previous studies linking interleukin-6 and autism, suggesting potential novel therapeutic pathways for a subtype of individuals with ASD. This is the first report demonstrating that glial dysfunctions could contribute to nonsyndromic autism pathophysiology using induced pluripotent stem cells modeling disease technology.
Acne is one of the most widespread skin diseases. The acne mechanism is intricate, involving interactions between different types of cells (i.e., sebocytes and macrophages). One of the challenges in ...studying the mechanism of acne is that current in vitro culture methods cannot reflect the 3D cellular environment in the tissue, including inflammatory stimuli and cellular interactions especially the interactions between sebocytes and immune cells. To solve this issue, we generated an in vitro acne disease model consisting of 3D artificial sebocyte glands and macrophages through the inertial focusing effect method. Using this model, we produced a controllable inflammatory environment similar to the acne pathogenetic process in the skin. The 3D artificial sebocyte glands and macrophages can be separated for analyzing each cell type, assisting the in‐depth understanding of the acne mechanism. This study indicates that proinflammatory macrophages promote lipid accumulation and induce oxidative stress in sebocyte glands. Additionally, in an inflammatory environment, sebocyte glands induce macrophage polarization into the M1 phenotype. Employing this model for drug screening, we also demonstrated that, cannabidiol (CBD), a clinically investigated drug, is effective in restoring lipid accumulation, oxidative stress, inflammatory cytokines and macrophage polarization in the acne disease.
Graphical and Lay Summary
An in vitro acne disease model consisting of 3D artificial sebocyte glands and macrophages was developed through the inertial focusing effect method. This disease model was adopted to investigate the cellular and immune interactions between sebocytes and macrophages, as well as drug screening, in acne.
Endoscopic treatment of Barrett's oesophagus often leads to further damage of healthy tissue causing fibrotic tissue formation termed as strictures. This study shows that synthetic, self‐assembling ...peptide hydrogels (PeptiGelDesign) support the activity and function of primary oesophageal cells, leading to epithelialization and stratification during in vitro 3D co‐culture. Following buffering in culture media, rat oesophageal stromal fibroblasts (rOSFs) are incorporated into a library of peptide hydrogels, whereas mouse oesophageal epithelial cells (mOECs) are seeded on the surface. Optimal hydrogels (PGD‐AlphaProC and PGD‐CGD2) support mOEC viability (>95%), typical cell morphology (cobblestone‐like), and slower migration over a shorter distance compared to a collagen control, at 48 h. Positive expression of typical epithelial markers (ZO‐1 and cytokeratins) is detected using immunocytochemistry at day 3 in culture. Furthermore, optimal hydrogels are identified which support rOSF viability (>95%) with homogeneous distribution when incorporated into the hydrogels and also promote the secretion of collagen type I detected using an enzyme linked immunosorbent assay (ELISA), at day 7. A 3D co‐culture model using optimal hydrogels for both cell types supports a stratified epithelial layer (expressing involucrin and AE1/AE3 markers). Findings from this study could lead to the use of peptide hydrogels as a minimally invasive endoscopic therapy to manage oesophageal strictures.
Synthetic, peptide hydrogels support the activity and function of primary oesophageal epithelial cells and stromal fibroblasts during in vitro 3D co‐culture. Epithelial markers were positively detected (at day 3) as well as the secretion of collagen I by stromal fibroblasts (day 7 and 14). Peptide hydrogels can be delivered endoscopically to manage oesophageal strictures.
The aim of this study was to assess the anti‐inflammatory effects of acai berries in a Caco‐2 and RAW 264.7 macrophage co‐culture model. The acai berry extract (ABE) was prepared using 70% ethanol, ...and total anthocyanin, polyphenol, and flavonoid contents in ABE were analyzed. To the antioxidant activity of ABE, we measured radical scavenging activity as well as ferric reducing antioxidant power values. Prior to inducing inflammation, Caco‐2 cells were co‐cultured with RAW 264.7. Inflammation was induced using lipopolysaccharides (LPS) in RAW 264.7 cells. The transepithelial electrical resistance value was significantly recovered and the mRNA level of tight junction proteins, including ZO‐1, JAM‐1, and claudin‐4, tended to increase compared with that in the LPS group. LPS‐induced interleukin (IL)‐6, IL‐8, and prostaglandin E2 levels reduced significantly following treatment with the highest ABE concentration. In the highest ABE concentration, the phosphorylation of p65, p38 mitogen‐activated protein kinase, and c‐Jun N‐terminal kinase was downregulated compared with the LPS group. The phosphorylation of extracellular signal‐regulated kinase showed a decreased tendency. These results suggest that acai berry may improve gastrointestinal health.
Practical applications
Acai berry is known to have abundant anthocyanin, which has many biological activities, including anti‐inflammatory, antioxidant, antihypertensive, and anticytotoxic/cytoprotective activities. This study demonstrated the anti‐inflammatory effects of acai berry extracts via TEER value, expression of tight junction protein, and production of inflammatory mediators and cytokines in the Caco‐2 and RAW 264.7 co‐culture model. Therefore, acai berry has the potential to prevent intestinal inflammatory diseases.
The aim of this study was to assess the anti‐inflammatory effects of acai berries in a Caco‐2 and RAW 264.7 macrophage co‐culture model. Acai berry extract (ABE) was significantly recovered transepithelial electrical resistance value and mRNA level of tight junction proteins. LPS‐induced interleukin (IL)‐6, IL‐8, and prostaglandin E2 level significantly reduced and, the phosphorylation of p65, p38 mitogen‐activated protein kinase, and c‐Jun N‐terminal kinase was downregulated in the ABE‐treated group. These results suggest that acai berry may improve gastrointestinal health.
The traditional two-dimensional (2D) in vitro cell culture system (on a flat support) has long been used in cancer research. However, this system cannot be fully translated into clinical trials to ...ideally represent physiological conditions. This culture cannot mimic the natural tumor microenvironment due to the lack of cellular communication (cell-cell) and interaction (cell-cell and cell-matrix). To overcome these limitations, three-dimensional (3D) culture systems are increasingly developed in research and have become essential for tumor research, tissue engineering, and basic biology research. 3D culture has received much attention in the field of biomedicine due to its ability to mimic tissue structure and function. The 3D matrix presents a highly dynamic framework where its components are deposited, degraded, or modified to delineate functions and provide a platform where cells attach to perform their specific functions, including adhesion, proliferation, communication, and apoptosis. So far, various types of models belong to this culture: either the culture based on natural or synthetic adherent matrices used to design 3D scaffolds as biomaterials to form a 3D matrix or based on non-adherent and/or matrix-free matrices to form the spheroids. In this review, we first summarize a comparison between 2D and 3D cultures. Then, we focus on the different components of the natural extracellular matrix that can be used as supports in 3D culture. Then we detail different types of natural supports such as matrigel, hydrogels, hard supports, and different synthetic strategies of 3D matrices such as lyophilization, electrospiding, stereolithography, microfluid by citing the advantages and disadvantages of each of them. Finally, we summarize the different methods of generating normal and tumor spheroids, citing their respective advantages and disadvantages in order to obtain an ideal 3D model (matrix) that retains the following characteristics: better biocompatibility, good mechanical properties corresponding to the tumor tissue, degradability, controllable microstructure and chemical components like the tumor tissue, favorable nutrient exchange and easy separation of the cells from the matrix.
Multiple myeloma (MM) remains to be incurable despite recent therapeutic advances. CD47, an immune checkpoint known as the "don't eat me" signal, is highly expressed on the surface of various ...cancers, allowing cancer cells to send inhibitory signals to macrophages and impede phagocytosis and immune response. In this study, we hypothesized that blocking the "don't eat me" signaling using an anti-CD47 monoclonal antibody will induce killing of MM cells. We report that CD47 expression was directly correlated with stage of the disease, from normal to MGUS to MM. Moreover, MM cells had remarkably higher CD47 expression than other cell populations in the bone marrow. These findings indicate that CD47 is specifically expressed on MM and can be used as a potential therapeutic target. Further, blocking of CD47 using an anti-CD47 antibody induced immediate activation of macrophages, which resulted in induction of phagocytosis and killing of MM cells in the 3D-tissue engineered bone marrow model, as early as 4 hours. These results suggest that macrophage checkpoint immunotherapy by blocking the CD47 "don't eat me" signal is a novel and promising strategy for the treatment of MM, providing a basis for additional studies to validate these effects in vivo and in patients.
Hepatic in vitro platforms ranging from multi‐well cultures to bioreactors and microscale systems have been developed as tools to recapitulate cellular function and responses to aid in drug screening ...and disease model development. Recent developments in microfabrication techniques and cellular materials enabled fabrication of next‐generation, advanced microphysiological systems (MPSs) that aim to capture the cellular complexity and dynamic nature of the organ presenting highly controlled extracellular cues to cells in a physiologically relevant context. Historically, MPSs have heavily relied on elastomeric materials in their manufacture, with unfavorable material characteristics (such as lack of structural rigidity) limiting their use in high‐throughput systems. Herein, we aim to create a microfluidic bilayer model (microfluidic MPS) using thermoplastic materials to allow hepatic cell stabilization and culture, retaining hepatic functional phenotype and capturing cellular interactions. The microfluidic MPS consists of two overlapping microfluidic channels separated by a porous tissue‐culture membrane that acts as a surface for cellular attachment and nutrient exchange; and an oxygen permeable material to stabilize and sustain primary human hepatocyte (PHH) culture. Within the microfluidic MPS, PHHs are cultured in the top channel in a collagen sandwich gel format with media exchange accomplished through the bottom channel. We demonstrate PHH culture for 7 days, exhibiting measures of hepatocyte stabilization, secretory and metabolic functions. In addition, the microfluidic MPS dimensions provide a reduced media‐to‐cell ratio in comparison with multi‐well tissue culture systems, minimizing dilution and enabling capture of cellular interactions and responses in a hepatocyte‐Kupffer coculture model under an inflammatory stimulus. Utilization of thermoplastic materials in the model and ability to incorporate multiple hepatic cells within the system is our initial step towards the development of a thermoplastic‐based high‐throughput microfluidic MPS platform for hepatic culture. We envision the platform to find utility in development and interrogation of disease models of the liver, multi‐cellular interactions and therapeutic responses.
Design, development and integration of high‐throughput microfluidic models in drug‐screening processes is hindered by the lack of scalability and appropriate microenvironment to sustain primary cell culture. In this work, we have developed a novel, thermoplastic microfluidic bilayer device with an oxygen‐permeable membrane to sustain primary human hepatocyte culture. This microfluidic MPS is an integral part of the overall platform to allow long‐term hepatic cultures for disease modeling, and drug screening applications.
Understanding the effects of antiepileptic drugs on glial cells and glia-mediated inflammation is a new approach to future treatment of epilepsy. Little is known about direct effects of the ...antiepileptic drug lacosamide (LCM) on glial cells. Therefore, we aimed to study the LCM effects on glial viability, microglial activation, expression of gap-junctional (GJ) protein Cx43 as well as intercellular communication in an in vitro astrocyte-microglia co-culture model of inflammation.
Primary rat astrocytes co-cultures containing 5% (M5, “physiological” conditions) or 30% (M30, “pathological inflammatory” conditions) of microglia were treated with different concentrations of LCM 5, 15, 30, and 90 μg/ml for 24 h. Glial cell viability was measured by MTT assay. Immunocytochemistry was performed to analyze the microglial activation state. Western blot analysis was used to quantify the astroglial Cx43 expression. The GJ cell communication was studied via Scrape Loading.
A concentration-dependent incubation with LCM did not affect the glial cell viability both under physiological and pathological conditions. LCM induced a significant concentration-dependent decrease of activated microglia with parallel increase of ramified microglia under pathological inflammatory conditions. This correlated with an increase in astroglial Cx43 expression. Nevertheless, the functional coupling via GJs was significantly reduced after incubation with LCM.
LCM has not shown effects on the glial cell viability. The reduced GJ coupling by LCM could be related to its anti-epileptic activity. The anti-inflammatory glial features of LCM with inhibition of microglial activation under inflammatory conditions support beneficial role in epilepsy associated with neuroinflammation.
•Anti-inflammatory glial features of lacosamide with inhibition of microglial activation under inflammatory conditions.•Lacosamide has not shown effects on the glial cell viability.•The reduced gap-junctional coupling by lacosamide can be related to its anti-epileptic activity.•Astrocytes-microglia co-culture model of inflammation allows to study the inflammatory reaction under antiepileptic drugs.
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