3D Printed Alginate Hydrogels with Stiffness-Gradient Structure in a Carbomer Supporting Bath by Controlled Ca²⁺ Diffusion

Manufacturing biocompatible materials with higher-order structure has great significance because they can mimic the extracellular medium of the human organism and are a novel strategy for tissue regeneration. In this study, a device with stiffness-gradient characteristics based on two biocompatible materials, alginate with presolidification and photocurable acrylamide-containing supporting bath, was designed and constructed by the 3D printing technique. The presolidification can avoid rapid diffusion of alginate in aqueous solutions, improve mechanical properties without the introduction of heterogeneous gel precursor, and endow gradient stiffness by the controlled diffusion of calcium ions. Besides, a photocurable supporting bath was combined to manufacture a device with a dual-gradient structure by a 4-step procedure, including 3D printing, removal of the inner hydrogel, solidification of alginate, and curing of the supporting bath. A cylinder-like container was manufactured as the template, and the wall of the resultant container with two types of gradient structures showed parabola-like stiffness changes (open upward), resulting from calcium ion diffusion-controlled gradient solidification and alginate diffusion-controlled gradient photocuring. Moreover, the resultant device exhibited lower cytotoxicity to both adherent and suspension cells than containers manufactured with alginate. Because of the high water uptake of the photocured supporting bath, the removal of toxic metabolic products together with cell culture medium from the container leads to better cell compatibility. This diffusion-controlled device is also applicable to other additive manufacturers with biomedical significance

Bonding Preference of Carbon, Nitrogen, and Oxygen in Niobium-Based Rock-Salt Structures

Carbon, nitrogen, and oxygen are essential components in solid-state materials. However, understanding their preference on the bonding to metals has not been straightforward. Here, niobium carbide, nitride, and oxide with simple rock-salt-based structures were analyzed by first-principles calculations and synchrotron X-ray diffraction. We found that an increase in the atomic number from carbon to oxygen formed fewer and shorter bonds to metals with better hybridization of atomic orbitals. This can provide a simple guiding principle for understanding the bonding and designing carbides, nitrides, oxides, and mixed-anion compounds

H₂O₂ produced by <i>S</i>. <i>sanguinis</i> induced release of NETs from infected neutrophils.

<p>Neutrophils were infected with the <i>S</i>. <i>sanguinis</i> strains at an MOI of 10. Exogenously added H₂O₂ (1 mM) or PMA (200 nM) served as a control. Following incubation for 3 h, cells were reacted with SYTOX Green (50 nM), then observed with a fluorescent microscope. Bar, 20 μm.</p

Bacterial association with human neutrophils.

<p>Human neutrophils were pre-incubated for 30 min, then infected with the WT, KO, or Wr strain at an MOI of 10. Following incubation for 0.5 or 1 h, cells were fixed with methanol and visualized by Giemsa staining. Using phase-contrast microscopy, 10 random fields (x 1000) were observed and the amount of bacteria demonstrating phagocytosis by neutrophils was determined by counting bacterial chains. (A) Representative images. (B) The numbers of bacterial chains associated with neutrophils at 0.5 and 1 h after infection are shown as the mean ± SD of 5 independent experiments. Statistically significant differences were evaluated using one-way ANOVA and Tukey’s multiple comparison test. *<i>p</i><0.01, **<i>p</i><0.05.</p

Extracellular DNA from neutrophils increased by streptococcal H₂O₂.

<p>Neutrophils were infected with the <i>S</i>. <i>sanguinis</i> strains at an MOI of 10 with or without catalase (100 units/ml), and cultured for 1–3 h. Neutrophils treated with 200 nM of PMA served as a control. Cells were reacted with SYTOX Green and fluorescent intensity was determined. Data are presented as the mean ± SD of triplicate samples from 3 independent experiments. Statistically significant differences were evaluated using two-way ANOVA and Tukey’s multiple comparison test. *<i>p</i><0.01.</p

H₂O₂ produced by <i>S</i>. <i>sanguinis</i> showed cytotoxicity against neutrophils.

<p>Human neutrophils were mixed with the <i>S</i>. <i>sanguinis</i> WT, KO, or Wr strain at an MOI of 10. As a positive control, PMA was added at concentrations of 200 nM. Following incubation for 1 or 3 h, LDH released into culture supernatant was examined. Cytotoxicity was determined by relative absorbance, with the absorbance of cells lysed with PBS containing 0.25% Triton X-100 set at 100%. Data are presented as the mean ± SD from 3 independent experiments. Statistically significant differences were evaluated using one-way ANOVA and Tukey’s multiple comparison test. *<i>p</i><0.01.</p

Effect of <i>spxB</i> deletion on H₂O₂ production by <i>S</i>. <i>sanguinis</i>.

<p>(A) H₂O₂ production by <i>S</i>. <i>sanguinis</i> strains was examined using Prussian blue agar plates. Overnight cultures were spotted on the plates and incubated for 24 h. BHI medium was used as a negative control. Bar, 5 mm. (B) After 24 h of incubation, the area of blue halos around colonies was determined using Image J software. Data are shown as the mean ± SD of triplicate samples from 3 independent experiments. Statistically significant differences were evaluated using one-way ANOVA and Tukey’s multiple comparison test. *<i>p</i><0.01. (C) Concentrations of H₂O₂ in culture supernatants of <i>S</i>. <i>sanguinis</i> strains grown overnight were quantified using peroxidase reactions. Data are shown as the mean ± SD from 3 (B) or 4 independent experiments. Statistically significant differences were evaluated using two-way ANOVA and Tukey’s multiple comparison test. *<i>p</i><0.01.</p

rSWAN cleaves NET DNA.

<p>Neutrophils were isolated from heparinized human blood and NETs were induced with PMA. NETs were incubated with or without rSWAN (40 µg/ml) at 37°C for 1 h. After fixation and permealization, neutrophil elastase was labeled with rabbit anti-human elastase IgG and an Alexa Fluor 594-conjugated secondary antibody, and DNA was stained with DAPI. Neutrophils without treatment with PMA and rSWAN were utilized as a control. The slides were observed using fluorescent microscopy. Bar, 20 µm.</p

DNA digestion around growing colonies of streptococcal species.

<p>The DNase activities of oral streptococcus strains were examined using BHI agar plates containing salmon sperm DNA. The plates were incubated for 72-digested DNA. Halos seen around colonies reflect DNA digestion. The following strains were tested: <i>S. sanguinis</i>, SK36; <i>S. oralis</i>, NCTC 11427T/SK23; <i>S. mutans</i>, MT8148; <i>S. salivarius</i>, HHT; <i>S. parasanguinis</i>, ATCC 903; <i>S. sobrinus</i>, MT10186. Bar, 1 cm.</p

Efficient DNase activity of rSWAN requires Ca²⁺ and Mg²⁺.

<p>(A) λDNA (0.3 µg) was incubated with recombinant SWAN (rSWAN, 0.1 µg) with or without CaCl₂ (1 mM) and MgCl₂ (1 mM) at 37°C for 1 h. Following electrophoresis, DNA was stained with ethidium bromide and visualized under UV light. The sizes of the λDNA <i>Hin</i>d III digest markers are indicated on the left. (B) λDNA (0.3 µg) was incubated with rSWAN (0.1 µg) with varying concentrations of either Ca²⁺ (red circles) or Mg²⁺ (blue circles) at 37°C for 30 min. Using gel images and Image J software, densitometric analyses were performed to calculate % DNA cleavage. Values shown represent the average ± SE of 3 independent experiments.</p