Geometric engineering of organoid culture for enhanced organogenesis in a dish.

Here, we introduce a facile, scalable engineering approach to enable long-term development and maturation of organoids. We have redesigned the configuration of conventional organoid culture to develop a platform that converts single injections of stem cell suspensions to radial arrays of organoids that can be maintained for extended periods without the need for passaging. Using this system, we demonstrate accelerated production of intestinal organoids with significantly enhanced structural and functional maturity, and their continuous development for over 4 weeks. Furthermore, we present a patient-derived organoid model of inflammatory bowel disease (IBD) and its interrogation using single-cell RNA sequencing to demonstrate its ability to reproduce key pathological features of IBD. Finally, we describe the extension of our approach to engineer vascularized, perfusable human enteroids, which can be used to model innate immune responses in IBD. This work provides an immediately deployable platform technology toward engineering more realistic organ-like structures in a dish.

Inducible gene expression of IκB-kinase ε (IKKε) is dependent on nuclear factor-κB (NF-κB) in human pulmonary epithelial cells.

While IkB-kinase-ε (IKKε) induces immunomodulatory genes following viral stimuli, its upregulation by inflammatory cytokines remains under-explored. Since airway epithelial cells respond to airborne insults and potentiate inflammation, IKKε expression was characterized in pulmonary epithelial cell lines (A549, BEAS-2B) and primary human bronchial epithelial cells (pHBECs) grown as submersion or differentiated air-liquid interface (ALI) cultures. IKKε expression was upregulated by the pro-inflammatory cytokines, IL-1ß and TNF⍺. Thus, mechanistic interrogations in A549 cells were used to demonstrate the NF-κB dependence of cytokine-induced IKKε. Furthermore, chromatin immunoprecipitation in A549 and BEAS-2B cells revealed robust recruitment of the NF-κB subunit, p65, to one 5' and two intronic regions within the IKKε locus (IKBKE). In addition, IL-1ß and TNFα induced strong RNA polymerase 2 recruitment to the 5' region, the first intron, and the transcription start site. Stable transfection of the p65-binding regions into A549 cells revealed IL-1ß- and TNFα-inducible reporter activity that required NF-κB, but was not repressed by glucocorticoid. While critical NF-κB motifs were identified in the 5' and downstream intronic regions, the first intronic region did not contain functional NF-κB motifs. Thus, IL-1ß- and TNFα-induced IKKε expression involves three NF-κB-binding regions, containing multiple functional NF-κB motifs, and potentially other mechanisms of p65 binding through non-classical NF-κB binding motifs. By enhancing IKKε expression, IL-1ß may prime, or potentiate, responses to alternative stimuli, as modeled by IKKε phosphorylation induced by phorbol 12-myristate 13-acetate. However, since IKKε expression was only partially repressed by glucocorticoid, IKKε-dependent responses could contribute to glucocorticoid-resistant disease.

In Vitro Evaluation of Gastric Acid and Toothbrushing Effect on the Surface State of Different Types of Composite Resins.

Background and Objectives: The aim of this in vitro study was to evaluate the effect of gastric acid associated with the effect of toothbrushing on the surface roughness of different types of composite resin used for direct restorations. Materials and Methods: The materials used in this study were two microhybrid (Filtek Z250, Herculite XRV) and two nanohybrid (Filtek Z550, Herculite XRV Ultra) composite resins. Two hundred and forty cylindrical samples with a height of 2 mm and a diameter of 6 mm were divided into four groups (groups A, B, C and D) corresponding to each tested material (n = 60). Each group was divided in two subgroups: subgroup I-the samples were submersed in hydrochloric acid and immediately submitted to toothbrushing; subgroup II-the samples were submitted only to toothbrushing. The simulation of the acid attack was performed by immersing the samples in a 0.01 M hydrochloric acid solution for 90 min. This procedure was followed immediately by toothbrushing simulation with 10,000 cycles. The acid attack and toothbrushing simulation were performed for two times. The surface roughness evaluation was performed with a Proscan 2100 profilometer. Repeated Measures ANOVA and Bonferroni post-hoc tests were used to perform the statistical analysis. Results: Simulation of one year of toothbrushing associated or not to hydrochloric acid exposure increases the surface roughness of microhybrid and nanohybrid composite resins. Six months of toothbrushing associated to six months of hydrochloric acid exposure increase the surface roughness of nanohybrid composite resins. Conclusions: Microhybrid composite resins surface becomes rougher after toothbrush and acid submersion when comparing to nanohybrid composite resins.

Generalized On-Demand Production of Nanoparticle Monolayers on Arbitrary Solid Surfaces via Capillarity-Mediated Inverse Transfer.

Century-old Langmuir monolayer deposition still represents the most convenient approach to the production of monolayers of colloidal nanoparticles on solid substrates for practical biological and chemical-sensing applications. However, this approach simply yields arbitrarily shaped large monolayers on a flat surface and is strongly limited by substrate topography and interfacial energy. Here, we describe a generalized and facile method of rapidly producing uniform monolayers of various colloidal nanoparticles on arbitrary solid substrates by using an ordinary capillary tube. Our method is based on an interesting finding of inversion phenomenon of a nanoparticle-laden air-water interface by flowing through a capillary tube in a manner that prevents the particles from adhesion to the capillary sidewall, thereby presenting the nanoparticles face-first at the tube's opposite end for direct and one-step deposition onto a substrate. We show that our method not only allows the placement of a nanoparticle monolayer at target locations of solid substrates regardless of their surface geometry and adhesion but also enables the production of monolayers containing nanoparticles with different size, shape, surface charge, and composition. To explore the potential of our approach, we demonstrate the facile integration of gold nanoparticle monolayers into microfluidic devices for the real-time monitoring of molecular Raman signals under dynamic flow conditions. Moreover, we successfully extend the use of our method to developing on-demand Raman sensors that can be built directly on the surface of consumer products for practical chemical sensing and fingerprinting. Specifically, we achieve both the pinpoint deposition of gold nanoparticle monolayers and sensitive molecular detection from the deposited region on clothing fabric for the detection of illegal drug substances, a single grain of rice and an orange for pesticide monitoring, and a $100 bill as a potential anti-counterfeit measure, respectively. We believe that our method will provide unique opportunities to expand the utility of colloidal nanoparticles and to greatly improve the accessibility of nanoparticle-based sensing technologies.

Events-Affect-Personality: A Daily Diary Investigation of the Mediating Effects of Affect on the Events-Personality Relationship.

Our 10-day diary investigation anchored in dynamic personality theories, such as Whole Trait Theory examined (a) whether within-person variability in two broad personality traits Extraversion and Neuroticism is consistently predicted by daily events, (b) whether positive and negative affect, respectively partly mediate this relationship and (c) the lagged relationships between events, and next day variations in affect and personality. Results revealed that personality exhibited significant within-person variability, that positive and negative affect partly mediate the relationship between events and personality, affect accounting for up to 60% of the effects of events on personality. Additionally, we identified that event-affect congruency was accountable for larger effects compared to event-affect non-congruency.

The Effect of Three Desensitizing Toothpastes on Dentinal Tubules Occlusion and on Dentin Hardness.

There are two main methods used for dentin hypersensitivity (DH) treatment: dentinal tubule occlusion and blockage of nerve activity. Dentifrices are the most common vehicles for active ingredients used for DH treatment. The aim of this study was to evaluate the efficacy of three toothpastes on dentinal tubule occlusion, mineral acquisition, and dentin hardness. Forty human dentin disks were submerged in 40% citric acid for 30 s and then exposed to tooth brushing for 2 min twice a day for 14 days using three toothpastes: Dontodent Sensitive (group 1), Dr. Wolff's Biorepair (group 2), and Sensodyne Repair and Protect (group 3). In the control group (group 4), the samples were brushed with water. All of the samples were evaluated using scanning electron microscopy (SEM), energy-dispersive X-ray (EDX), and Vickers dentin hardness determination. On SEM images, the degree of dentinal tubule occlusion was assessed using a five-grade scale. The mean score values in groups 1-4 were 3.60 ± 0.69, 2.20 ± 0.91, 2.30 ± 1.16, and 5.00 ± 0.00, significantly higher in study groups when compared to the control group (Kruskal Wallis test p < 0.05). EDX evaluation showed significantly higher calcium and phosphorus concentrations in groups 1 and 3 when compared to control group d. The mean values of Vickers dentin hardness numbers in groups 1-4 were 243.03 ± 10.014, 327.38 ± 56.65, 260.29 ± 37.69, and 225.83 ± 29.93, respectively. No statistically significant results were obtained when comparing the hardness mean values in groups (Kruskal-Wallis statistical test, p = 0.372 > 0.05). All three toothpastes tested demonstrated significant occlusion of dentinal tubules. Dontodent Sensitive and Sensodyne Repair and Protect toothpastes enhanced the calcium and phosphorus content of the dentin surface. None of the toothpastes increased dentin hardness as a result of mineral acquisition.

Differential regulation of BIRC2 and BIRC3 expression by inflammatory cytokines and glucocorticoids in pulmonary epithelial cells.

Roles for the baculoviral inhibitor of apoptosis repeat-containing (BIRC) genes, BIRC2 and BIRC3, may include signaling to the inflammatory transcription factor, nuclear factor-κB (NF-κB) and protection from cell death. However, distinct functions for each BIRC are not well-delineated. Given roles for the epithelium in barrier function and host defence, BIRC2 and BIRC3 expression was characterized in pulmonary epithelial cell lines and primary human bronchial epithelial cells (pHBECs) grown as undifferentiated cells in submersion culture (SC) or as highly differentiated cells at air-liquid interface (ALI). In A549 cells, interleukin-1ß (IL1B) and tumor necrosis factor α (TNF) induced BIRC3 mRNA (~20-50-fold), with maximal protein expression from 6-24 h. Similar effects occurred in BEAS-2B and Calu-3 cells, as well as SC and ALI pHBECs. BIRC2 protein was readily detected in unstimulated cells, but was not markedly modulated by IL1B or TNF. Glucocorticoids (dexamethasone, budesonide) modestly increased BIRC3 mRNA and protein, but showed little effect on BIRC2 expression. In A549 cells, BIRC3 mRNA induced by IL1B was unchanged by glucocorticoids and showed supra-additivity with TNF-plus-glucocorticoid. Supra-additivity was also evident for IL1B-plus-budesonide induced-BIRC3 in SC and ALI pHBECs. Using A549 cells, IL1B- and TNF-induced BIRC3 expression, and to a lesser extent, BIRC2, was prevented by NF-κB inhibition. Glucocorticoid-induced BIRC3 expression was prevented by silencing and antagonism of the glucocorticoid receptor. Whereas TNF, but not IL1B, induced degradation of basal BIRC2 and BIRC3 protein, IL1B- and TNF-induced BIRC3 protein remained stable. Differential regulation by cytokines and glucocorticoids shows BIRC2 protein expression to be consistent with roles in rapid signaling events, whereas cytokine-induced BIRC3 may be more important in later effects. While TNF-induced degradation of both BIRCs may restrict their activity, cytokine-enhanced BIRC3 expression could prime for its function. Finally, shielding from glucocorticoid repression, or further enhancement by glucocorticoid, may indicate a key protective role for BIRC3.

Severe Acute Pancreatitis Treated with Negative Pressure Wound Therapy System: Influence of Laboratory Markers.

(1) Background: An open abdomen is a serious medical condition that requires prompt and effective treatment to prevent complications and improve patient outcomes. Negative pressure therapy (NPT) has emerged as a viable therapeutic option for temporary closure of the abdomen, offering several benefits over traditional methods. (2) Methods: We included 15 patients with pancreatitis who were hospitalized in the I-II Surgery Clinic of the Emergency County Hospital "St. Spiridon" from Iasi, Romania, between 2011-2018 and received NPT. (3) Results: Preoperatively, the mean IAP level was 28.62 mmHg, decreasing significantly postoperatively to 21.31 mmHg. The mean level of the highest IAP value recorded in pancreatitis patients treated with VAC did not differ significantly by lethality (30.31 vs. 28.50; p = 0.810). In vacuum-treated pancreatitis patients with a IAP level > 12, the probability of survival dropped below 50% during the first 7 days of stay in the ICU, so that after 20 days the probability of survival was approximately 20%. IAP enters the determinism of surgery with a sensitivity of 92.3% and a specificity of 99%, the cut-off value of IAP being 15 mmHg. (4) Conclusions: The timing of surgical decompression in abdominal compartment syndrome is very important. Consequently, it is vital to identify a parameter, easy to measure, within the reach of any clinician, so that the indication for surgical intervention can be made judiciously and without delay.

A Model of Human Small Airway on a Chip for Studies of Subacute Effects of Inhalation Toxicants.

Testing for acute inhalation hazards is conducted in animals; however, a number of robust in vitro human cell-based alternatives have been developed and tested. These models range in complexity from cultures of cell lines or primary cells in air-liquid interface on Transwells, to more complex and physiologically relevant flow- and mechanical stimulation-enabled tissue chips. Although the former models are relatively straightforward to establish and can be tested in medium/high throughput, the latter require specialized equipment and lack in throughput. In this study, we developed a device that can be easily manufactured while allowing for the production of a differentiated lung tissue. This multilayered microfluidic device enables coculture of primary human small airway epithelial cells and lung microvascular endothelial cells under physiological conditions for up to 18 days and recreates the parenchymal-vascular interface in the distal lung. To explore the potential of this airway on a chip for applications in inhalation toxicology, we also devised a system that allows for direct gas/aerosol exposures of the engineered airway epithelium to noxious stimuli known to cause adverse respiratory effects, including dry flowing air, lipopolysaccharide, particulate matter, and iodomethane. This study generated quantitative, high-content data that were indicative of aberrant changes in biochemical (lactate dehydrogenase), barrier (dextran permeability), functional (ciliary beating), and molecular (imaging for various markers) phenotypes of the small airway epithelium due to inhalational exposures. This study is significant because it established an in vitro model of human small airway on a chip that can be used in medium/high-throughput studies of subacute effects of inhalation toxicants.

Organoids-on-a-chip.

Recent studies have demonstrated an array of stem cell-derived, self-organizing miniature organs, termed organoids, that replicate the key structural and functional characteristics of their in vivo counterparts. As organoid technology opens up new frontiers of research in biomedicine, there is an emerging need for innovative engineering approaches for the production, control, and analysis of organoids and their microenvironment. In this Review, we explore organ-on-a-chip technology as a platform to fulfill this need and examine how this technology may be leveraged to address major technical challenges in organoid research. We also discuss emerging opportunities and future obstacles for the development and application of organoid-on-a-chip technology.

Polydopamine-Based Interfacial Engineering of Extracellular Matrix Hydrogels for the Construction and Long-Term Maintenance of Living Three-Dimensional Tissues.

Diverse biological processes in the body rely on the ability of cells to exert contractile forces on their extracellular matrix (ECM). In three-dimensional (3D) cell culture, however, this intrinsic cellular property can cause unregulated contraction of ECM hydrogel scaffolds, leading to a loss of surface anchorage and the resultant structural failure of in vitro tissue constructs. Despite advances in the 3D culture technology, this issue remains a significant challenge in the development and long-term maintenance of physiological 3D in vitro models. Here, we present a simple yet highly effective and accessible solution to this problem. We leveraged a single-step surface functionalization technique based on polydopamine to drastically increase the strength of adhesion between hydrogel scaffolds and cell culture substrates. Our method is compatible with different types of ECM and polymeric surfaces and also permits prolonged shelf storage of functionalized culture substrates. The proof-of-principle of this technique was demonstrated by the stable long-term (1 month) 3D culture of human lung fibroblasts. Furthermore, we showed the robustness and advanced application of the method by constructing a dynamic cell stretching system and performing over 100 000 cycles of mechanical loading on 3D multicellular constructs for visualization and quantitative analysis of stretch-induced tissue alignment. Finally, we demonstrated the potential of our technique for the development of microphysiological in vitro models by establishing microfluidic 3D co-culture of vascular endothelial cells and fibroblasts to engineer self-assembled, perfusable 3D microvascular beds.

iPreP is a three-dimensional nanofibrillar cellulose hydrogel platform for long-term ex vivo preservation of human islets.

Islet transplantation is an effective therapy for achieving and maintaining normoglycemia in patients with type 1 diabetes mellitus. However, the supply of transplantable human islets is limited. Upon removal from the pancreas, islets rapidly disintegrate and lose function, resulting in a short interval for studies of islet biology and pretransplantation assessment. Here, we developed a biomimetic platform that can sustain human islet physiology for a prolonged period ex vivo. Our approach involved the creation of a multichannel perifusion system to monitor dynamic insulin secretion and intracellular calcium flux simultaneously, enabling the systematic evaluation of glucose-stimulated insulin secretion under multiple conditions. Using this tool, we developed a nanofibrillar cellulose hydrogel-based islet-preserving platform (iPreP) that can preserve islet viability, morphology, and function for nearly 12 weeks ex vivo, and with the ability to ameliorate glucose levels upon transplantation into diabetic hosts. Our platform has potential applications in the prolonged maintenance of human islets, providing an expanded time window for pretransplantation assessment and islet studies.

Multiscale reverse engineering of the human ocular surface.

Here we present a miniaturized analog of a blinking human eye to reverse engineer the complexity of the interface between the ocular system and the external environment. Our model comprises human cells and provides unique capabilities to replicate multiscale structural organization, biological phenotypes and dynamically regulated environmental homeostasis of the human ocular surface. Using this biomimetic system, we discovered new biological effects of blink-induced mechanical forces. Furthermore, we developed a specialized in vitro model of evaporative dry-eye disease for high-content drug screening. This work advances our ability to emulate how human physiological systems interface with the external world, and may contribute to the future development of novel screening platforms for biopharmaceutical and environmental applications.

Placental Drug Transport-on-a-Chip: A Microengineered In Vitro Model of Transporter-Mediated Drug Efflux in the Human Placental Barrier.

The current lack of knowledge about the effect of maternally administered drugs on the developing fetus is a major public health concern worldwide. The first critical step toward predicting the safety of medications in pregnancy is to screen drug compounds for their ability to cross the placenta. However, this type of preclinical study has been hampered by the limited capacity of existing in vitro and ex vivo models to mimic physiological drug transport across the maternal-fetal interface in the human placenta. Here the proof-of-principle for utilizing a microengineered model of the human placental barrier to simulate and investigate drug transfer from the maternal to the fetal circulation is demonstrated. Using the gestational diabetes drug glyburide as a model compound, it is shown that the microphysiological system is capable of reconstituting efflux transporter-mediated active transport function of the human placental barrier to limit fetal exposure to maternally administered drugs. The data provide evidence that the placenta-on-a-chip may serve as a new screening platform to enable more accurate prediction of drug transport in the human placenta.