An in vitro intestinal model captures immunomodulatory properties of the microbiota in inflammation.

Considerable effort has been put forth to understand mechanisms by which the microbiota modulates and responds to inflammation. Here, we explored whether oxidation metabolites produced by the host during inflammation, sodium nitrate and trimethylamine oxide, impact the composition of a human stool bacterial population in a gut simulator. We then assessed whether an immune-competent in vitro intestinal model responded differently to spent medium from bacteria exposed to these cues compared to spent medium from a control bacterial population. The host-derived oxidation products were found to decrease levels of Bacteroidaceae and overall microbiota metabolic potential, while increasing levels of proinflammatory Enterobacteriaceae and lipopolysaccharide in bacterial cultures, reflecting shifts that occur in vivo in inflammation. Spent microbiota media induced elevated intracellular mucin levels and reduced intestinal monolayer integrity as reflected in transepithelial electrical resistance relative to fresh medium controls. However, multiplexed cytokine analysis revealed markedly different cytokine signatures from intestinal cultures exposed to spent medium with added oxidation products relative to spent control medium, while cytokine signatures of cultures exposed to fresh media were similar regardless of addition of host-derived cues. Further, the presence of immune cells in the intestinal model was required for this differentiation of cytokine signatures. This study indicates that simple in vitro immune-competent intestinal models can capture bacterial-mammalian cross-talk in response to host-derived oxidation products and supports utility of these systems for mechanistic studies of interactions between the gut microbiome and host in inflammation.

Efficient gene knockout in primary human and murine myeloid cells by non-viral delivery of CRISPR-Cas9.

Myeloid cells play critical and diverse roles in mammalian physiology, including tissue development and repair, innate defense against pathogens, and generation of adaptive immunity. As cells that show prolonged recruitment to sites of injury or pathology, myeloid cells represent therapeutic targets for a broad range of diseases. However, few approaches have been developed for gene editing of these cell types, likely owing to their sensitivity to foreign genetic material or virus-based manipulation. Here we describe optimized strategies for gene disruption in primary myeloid cells of human and murine origin. Using nucleofection-based delivery of Cas9-ribonuclear proteins (RNPs), we achieved near population-level genetic knockout of single and multiple targets in a range of cell types without selection or enrichment. Importantly, we show that cellular fitness and response to immunological stimuli is not significantly impacted by the gene editing process. This provides a significant advance in the study of myeloid cell biology, thus enabling pathway discovery and drug target validation across species in the field of innate immunity.

Impact of Developmental Age, Necrotizing Enterocolitis Associated Stress, and Oral Therapeutic Intervention on Mucus Barrier Properties.

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease of incompletely understood pathophysiology predominantly affecting premature infants. While NEC is associated with microbial invasion of intestinal tissues, and mucus modulates interactions between microbes and underlying tissues, variations in mucus barrier properties with NEC-associated risk factors have not been investigated. This study explored differences in mucus composition (total protein, DNA, mucin content, sialic acid, and immunoregulatory proteins), as well as structural and transport properties, assessed by tracking of particles and bacteria (E. coli and E. cloacae) with developmental age and exposure to NEC stressors in Sprague Dawley rats. Early developmental age (5 day old) was characterized by a more permeable mucus layer relative to 21 day old pups, suggesting immaturity may contribute to exposure of the epithelium to microbes. Exposure to NEC stressors was associated with reduced mucus permeability, which may aid in survival. Feeding with breastmilk as opposed to formula reduces incidence of NEC. Thus, NEC-stressed (N-S) rat pups were orally dosed with breastmilk components lysozyme (N-S-LYS) or docosahexaenoic acid (N-S-DHA). N-S-LYS and N-S-DHA pups had a less permeable mucus barrier relative to N-S pups, which suggests the potential of these factors to strengthen the mucus barrier and thus protect against disease.

Intestinal mucus is capable of stabilizing supersaturation of poorly water-soluble drugs.

The utilization of polymers to stabilize drug supersaturation and enhance oral drug absorption has recently garnered considerable interest. The potential role of intestinal mucus in stabilizing drug supersaturation, however, has not been previously explored. The ability for intestinal mucus to stabilize drug supersaturation and delay drug precipitation is potentially useful in enhancing the absorption of orally dosed compounds from drug delivery systems that generate supersaturation within the gastrointestinal tract (e.g., solid dispersions, lipid-based drug delivery systems). This work aims to evaluate the precipitation-delaying abilities of intestinal mucus using carvedilol (CVDL) and piroxicam (PXM) as model drugs. In supersaturation-precipitation (S-P) experiments, CVDL and PXM supersaturation were induced in test media (0, 0.1, 0.2, 0.4%w/v mucin and 8%w/v native pig intestinal mucus (PIM)) via the solvent-shift method at supersaturation ratios (SSR) of 5 and 6, respectively. Time to drug precipitation was assessed using ion-selective electrodes and HPLC. The S-P experiments showed that increasing mucin concentration led to increasingly delayed CVDL precipitation, while PXM precipitation was prevented at all mucin concentrations studied. The ability of mucus-stabilized CVDL supersaturation to translate into enhanced CVDL absorption was evaluated in transport experiments using mucus-producing (90% Caco-2:10% HT29-MTX-E12 co-cultures) vs. non-mucus-producing intestinal monolayers (100% Caco-2 cultures). The absorption enhancement of CVDL (SSR = 5 relative to SSR = 1) was higher across mucus-producing than non-mucus-producing intestinal monolayers. This work demonstrates the potential for intestinal mucus to delay the precipitation and enhance the absorption of poorly water-soluble compounds, suggesting that drug supersaturation can be stabilized in close proximity to the absorptive site, thereby presenting a possible novel approach for targeted supersaturating drug delivery systems.

Lipids alter microbial transport through intestinal mucus.

Mucus constitutes a protective layer which coats the gastrointestinal tract, controlling interactions of both commensal and pathogenic microbes with underlying tissues. Changes to the mucus barrier, for example due to altered mucin expression or external stimuli, may impact interactions with microbes and thus potentially contribute to altered gut homeostasis, onset of inflammation, or pathogen invasion. Food-associated stimuli, including lipids, have been shown to change mucus barrier properties and reduce transport of model drug carriers through mucus. Here, we explore the impact of lipids, specifically triglycerides in a model intestinal medium mimicking a fed state, on Escherichia coli (E. coli) transport through mucus by directly imaging swimming patterns and analyzing associated changes in mucus structure. Lipids in model fed state intestinal contents reduced E. coli speed and track linearity within mucus. These changes may be due in part to changes in molecular interactions within the mucus network as well as crowding of the mucus network by lipid emulsion droplets, which visibly stay intact in the mucus gel. In addition, observed physical interactions between bacteria and lipid structures may impact microbial speed and trajectories. As lipids are normal food components and thus represent safe, mild stimuli, these results support exploration of lipid-based strategies to alter the mucus barrier to control interactions with microbes and potentially prevent microbial invasion of underlying epithelium.

Acute Exposure to Commonly Ingested Emulsifiers Alters Intestinal Mucus Structure and Transport Properties.

The consumption of generally regarded as safe emulsifiers has increased, and has been associated with an increased prevalence of inflammatory bowel and metabolic diseases, as well as an altered microbiome. The mucus barrier, which selectively controls the transport of particulates and microorganisms to the underlying epithelial layer, has been previously shown to be altered by dietary salts and lipids. However, the potential impact of emulsifiers on the protective mucus barrier, its permeability, and associated structural changes are not clear. In this study, we analyzed changes in the mucus barrier to both passively diffusing nanoparticles and actively swimming E. coli upon exposure to two emulsifiers, carboxymethylcellulose (CMC) and polysorbate 80 (Tween). When exposed to CMC, mucus pore size decreased, which resulted in significantly slower E. coli speed and particle diffusion rates through mucus. Tween exposure minimally impacted mucus microstructure and particle diffusion, but increased E. coli speed in mucus. Moreover, both emulsifiers appeared to alter mucus amount and thickness in rat intestinal tissue and mucus-producing cell cultures. These results indicate that acute exposure to emulsifiers impacts barrier and structural properties of intestinal mucus, modulating interactions between intestinal lumen contents, microbes, and underlying tissue, which may contribute to development of intestinal inflammation.

Mucus models to evaluate the diffusion of drugs and particles.

Mucus is a complex hydrogel that acts as a natural barrier to drug delivery at different mucosal surfaces including the respiratory, gastrointestinal, and vaginal tracts. To elucidate the role mucus plays in drug delivery, different in vitro, in vivo, and ex vivo mucus models and techniques have been utilized. Drug and drug carrier diffusion can be studied using various techniques in either isolated mucus gels or mucus present on cell cultures and tissues. The species, age, and potential disease state of the animal from which mucus is derived can all impact mucus composition and structure, and therefore impact drug and drug carrier diffusion. This review provides an overview of the techniques used to characterize drug and drug carrier diffusion, and discusses the advantages and disadvantages of the different models available to highlight the information they can afford.

Emulation of Colonic Oxygen Gradients in a Microdevice.

Gut-on-a-chip in vitro modeling is an emerging field, as the human gut epithelium and gut microbiome have been recently identified as novel drug targets for a wide variety of diseases. Realistic in vitro gut models require a variety of precise environmental cues, such as chemical and gas gradients, in combination with substrates like mucus that support the growth of microbial communities. This technical brief describes a microfluidic architecture capable of developing a physiologically relevant oxygen gradient that emulates the oxygen profile proximal to the epithelial inner lining of the human colon. The device generates stable and repeatable defined oxygen gradients from 0% to 4 % partial pressure O2 over a length scale of hundreds of microns, and was applied to study the effects of oxygenation on the structure of native mucus that lines the colon wall. Using simulation as a design tool for hybrid gas-liquid microfluidic devices enables on-chip creation of defined, physiologically oxygen gradients. These microfluidic architectures have powerful potential applications for gut physiology, including providing optimal oxygenation conditions for the culture of mammalian epithelial cells in the gut lining, as well as creating a realistic mimic of the oxygen gradient found in the intestinal lumen for complex microbiome cultures.

Magnetic Nanocomposite Hydrogel for Potential Cartilage Tissue Engineering: Synthesis, Characterization, and Cytocompatibility with Bone Marrow Derived Mesenchymal Stem Cells.

Hydrogels possess high water content and closely mimic the microenvironment of extracellular matrix. In this study, we created a hybrid hydrogel containing type II collagen, hyaluronic acid (HA), and polyethylene glycol (PEG) and incorporated magnetic nanoparticles into the hybrid hydrogels of type II collagen-HA-PEG to produce a magnetic nanocomposite hydrogel (MagGel) for cartilage tissue engineering. The results showed that both the MagGel and hybrid gel (Gel) were successfully cross-linked and the MagGel responded to an external magnet while maintaining structural integrity. That is, the MagGel could travel to the tissue defect sites in physiological fluids under remote magnetic guidance. The adhesion density of bone marrow derived mesenchymal stem cells (BMSCs) on the MagGel group in vitro was similar to the control group and greater than the Gel group. The morphology of BMSCs was normal and consistent in all groups. We also found that BMSCs engulfed magnetic nanoparticles in culture and the presence of magnetic nanoparticles did not affect BMSC adhesion and morphology. We hypothesized that the ingested nanoparticles may be eventually broken down by lysosome and excreted through exocytosis; further studies are necessary to confirm this. This study reports a promising magnetic responsive nanocomposite hydrogel for potential cartilage tissue engineering applications, which should be further studied for its effects on cell functions when combined with electromagnetic stimulation.

Degradation and antibacterial properties of magnesium alloys in artificial urine for potential resorbable ureteral stent applications.

This article presents an investigation on the effectiveness of magnesium and its alloys as a novel class of antibacterial and biodegradable materials for ureteral stent applications. Magnesium is a lightweight and biodegradable metallic material with beneficial properties for use in medical devices. Ureteral stent is one such example of a medical device that is widely used to treat ureteral canal blockages clinically. The bacterial colony formation coupled with the encrustation on the stent surface from extended use often leads to clinical complications and contributes to the failure of indwelling medical devices. We demonstrated that magnesium alloys decreased Escherichia coli viability and reduced the colony forming units over a 3-day incubation period in an artificial urine (AU) solution when compared with currently used commercial polyurethane stent. Moreover, the magnesium degradation resulted in alkaline pH and increased magnesium ion concentration in the AU solution. The antibacterial and degradation properties support the potential use of magnesium-based materials for next-generation ureteral stents. Further studies are needed for clinical translation of biodegradable metallic ureteral stents.

Development and evaluation of a magnesium-zinc-strontium alloy for biomedical applications--alloy processing, microstructure, mechanical properties, and biodegradation.

A new biodegradable magnesium-zinc-strontium (Mg-Zn-Sr) alloy was developed and studied for medical implant applications. This first study investigated the alloy processing (casting, rolling, and heat treatment), microstructures, mechanical properties, and degradation properties in simulated body fluid (SBF). Aging treatment of the ZSr41 alloy at 175 °C for 8h improved the mechanical properties when compared to those of the as-cast alloy. Specifically, the aged ZSr41 alloy had an ultimate tensile strength of 270 MPa, Vickers hardness of 71.5 HV, and elongation at failure of 12.8%. The mechanical properties of the ZSr41 alloy were superior as compared with those of pure magnesium and met the requirements for load-bearing medical implants. Furthermore, the immersion of the ZSr41 alloy in SBF showed a degradation mode that progressed cyclically, alternating between pitting and localized corrosion. The steady-state average degradation rate of the aged ZSr41 alloy in SBF was 0.96 g/(m(2)·hr), while the pH of SBF immersion solution increased. The corrosion current density of the ZSr41 alloy in SBF solution was 0.41 mA/mm(2), which was much lower than 1.67 mA/mm(2) for pure Mg under the same conditions. In summary, compared to pure Mg, the mechanical properties of the new ZSr41 alloy improved while the degradation rate decreased due to the addition of Zn and Sr alloying elements and specific processing conditions. The superior mechanical properties and corrosion resistance of the new ZSr41 alloy make it a promising alloy for next-generation implant applications.

Nanophase hydroxyapatite and poly(lactide-co-glycolide) composites promote human mesenchymal stem cell adhesion and osteogenic differentiation in vitro.

Human mesenchymal stem cells (hMSCs) typically range in size from 10 to 50 µm and proteins that mediate hMSC adhesion and differentiation usually have a size of a few nanometers. Nanomaterials with a feature size smaller than 100 nm have demonstrated the unique capability of promoting osteoblast (bone forming cell) adhesion and long-term functions, leading to more effective bone tissue regeneration. For new bone deposition, MSCs have to be recruited to the injury or disease sites and then differentiate into osteoblasts. Therefore, designing novel nanomaterials that are capable of attracting MSCs and directing their differentiation is of great interest to many clinical applications. This in vitro study investigated the effects of nanophase hydroxyapatite (nano-HA), nano-HA/poly(lactide-co-glycolide) (PLGA) composites and a bone morphogenetic protein (BMP-7) derived short peptide on osteogenic differentiation of hMSCs. The short peptide was loaded by physical adsorption to nano-HA or by dispersion in nanocomposites and in PLGA to determine their effects on hMSC adhesion and differentiation. The results showed that the nano-HA/PLGA composites promoted hMSC adhesion as compared to the PLGA controls. Moreover, nano-HA/PLGA composites promoted osteogenic differentiation of hMSCs to a similar extent with or without the presence of osteogenic factors in the media. In the MSC growth media without the osteogenic factors, the nanocomposites supported greater calcium-containing bone mineral deposition by hMSC than the BMP-derived short peptide alone. The nanocomposites provided promising alternatives in controlling the adhesion and differentiation of hMSCs without osteogenic factors from the culture media, and, thus, should be further studied for clinical translation and the development of novel nanocomposite-guided stem cell therapies.

Antimicrobial properties of biodegradable magnesium for next generation ureteral stent applications.

Bacterial infection often causes clinical complications and failure of indwelling medical devices. This is a major problem of current ureteral stents, which are used clinically to treat the blockage of ureteral canals. This study investigates the effectiveness and applicability of magnesium as a novel biodegradable ureteral stent material that has inherent antimicrobial properties. Incubating Escherichia coli with the magnesium samples showed a decrease in the bacterial cell density as compared with the currently used commercial polyurethane stent. Magnesium degradation in the immersion solutions (artificial urine, luria bertani broth, and deionized water) resulted in an alkaline pH shift. Antimicrobial and biodegradation properties of magnesium make it an attractive alternative as next-generation ureteral stent material.

Improved bone marrow stromal cell adhesion on micropatterned titanium surfaces.

Implant longevity is desired for all bone replacements and fixatives. Titanium (Ti) implants fail due to lack of juxtaposed bone formation, resulting in implant loosening. Implant surface modifications have shown to affect the interactions between the implant and bone. In clinical applications, it is crucial to improve osseointegration and implant fixation at the implant and bone interface. Moreover, bone marrow derived cells play a significant role for implant and tissue integration. Therefore, the objective of this study is to investigate how surface micropatterning on Ti influences its interactions with bone marrow derived cells containing mesenchymal and hematopoietic stem cells. Bone marrow derived mesenchymal stem cells (BMSC) have the capability of differentiating into osteoblasts that contribute to bone growth, and therefore implant/bone integration. Hematopoietic stem cell derivatives are precursor cells that contribute to inflammatory response. By using all three cells naturally contained within bone marrow, we mimic the physiological environment to which an implant is exposed. Primary rat bone marrow derived cells were seeded onto Ti with surfaces composed of arrays of grooves of equal width and spacing ranging from 0.5 to 50 µm, fabricated using a novel plasma-based dry etching technique. Results demonstrated enhanced total cell adhesion on smaller micrometer-scale Ti patterns compared with larger micrometer-scale Ti patterns, after 24-hr culture. Further studies are needed to determine bone marrow derived cell proliferation and osteogenic differentiation potential on micropatterned Ti, and eventually nanopatterned Ti.

Nanomaterials enhance osteogenic differentiation of human mesenchymal stem cells similar to a short peptide of BMP-7.

BACKGROUND: Nanomaterials have unique advantages in controlling stem cell function due to their biomimetic characteristics and special biological and mechanical properties. Controlling adhesion and differentiation of stem cells is critical for tissue regeneration. METHODS: This in vitro study investigated the effects of nano-hydroxyapatite, nano-hydroxyapatite-polylactide- co-glycolide (PLGA) composites, and a bone morphogenetic protein (BMP-7)- derived short peptide (DIF-7c) on osteogenic differentiation of human mesenchymal stem cells (MSC). The peptide was chemically functionalized onto nano-hydroxyapatite, incorporated into a nanophase hydroxyapatite-PLGA composite or PLGA control, or directly injected into culture media. RESULTS: Unlike the PLGA control, the nano-hydroxyapatite-PLGA composites promoted adhesion of human MSC. Importantly, nano-hydroxyapatite and nano-hydroxyapatite-PLGA composites promoted osteogenic differentiation of human MSCs, comparable with direct injection of the DIF-7c peptide into culture media. CONCLUSION: Nano-hydroxyapatite and nano-hydroxyapatite-PLGA composites provide a promising alternative in directing the adhesion and differentiation of human MSC. These nanocomposites should be studied further to clarify their effects on MSC functions and bone remodeling in vivo, eventually translating to clinical applications.