3D-Printed Collagen-Based Waveform Microfibrous Scaffold for Periodontal Ligament Reconstruction

Reconstruction of the periodontal ligament (PDL) to fulfill functional requirement remains a challenge. This study sought to develop a biomimetic microfibrous system capable of withstanding the functional load to assist PDL regeneration. Collagen-based straight and waveform microfibers to guide PDL cell growth were prepared using an extrusion-based bioprinter, and a laminar flow-based bioreactor was used to generate fluidic shear stress. PDL cells were seeded on the respective microfibers with 0 or 6 dynes/cm2 fluidic shear stress for 1–4 h. The viability, morphology, adhesion pattern, and gene expression levels of PDL cells were assessed. The results revealed that upon bioprinting optimization, collagen-based microfibers were successfully fabricated. The straight microfibers were 189.9 ± 11.44 μm wide and the waveform microfibers were 235.9 ± 11.22 μm wide. Under 6 dynes/cm2 shear stress, PDL cells were successfully seeded, and cytoskeleton expansion, adhesion, and viability were greater. Cyclin D, E-cadherin, and periostin were upregulated on the waveform microfibers. In conclusion, 3D-printed collagen-based waveform microfibers preserved PDL cell viability and exhibited an enhanced tendency to promote healing and regeneration under shear stress. This approach is promising for the development of a guiding scaffold for PDL regeneration.

Natural and synthetic antimicrobials reduce adherence of enteroaggregative and enterohemorrhagic Escherichia coli to epithelial cells

Adherence of bacteria to the human intestinal mucosa can facilitate their internalization and the development of pathological processes. Escherichia coli O104:H4 is considered a hybrid strain (enteroaggregative hemorrhagic E. coli [EAHEC]), sharing virulence factors found in enterohemorrhagic (EHEC), and enteroaggregative (EAEC) E. coli pathotypes. The objective of this study was to analyze the effects of natural and synthetic antimicrobials (carvacrol, oregano extract, brazilin, palo de Brasil extract, and rifaximin) on the adherence of EHEC O157:H7, EAEC 042, and EAHEC O104:H4 to HEp-2 cells and to assess the expression of various genes involved in this process. Two concentrations of each antimicrobial that did not affect (p≤0.05) bacterial viability or damage the bacterial membrane integrity were used. Assays were conducted to determine whether the antimicrobials alter adhesion by affecting bacteria and/or alter adhesion by affecting the HEp-2 cells, whether the antimicrobials could detach bacteria previously adhered to HEp-2 cells, and whether the antimicrobials could modify the adherence ability exhibited by the bacteria for several cycles of adhesion assays. Giemsa stain and qPCR were used to assess the adhesion pattern and gene expression, respectively. The results showed that the antimicrobials affected the adherence abilities of the bacteria, with carvacrol, oregano extract, and rifaximin reducing up to 65% (p≤0.05) of E. coli adhered to HEp-2 cells. Carvacrol (10 mg/ml) was the most active compound against EHAEC O104:H4, even altering its aggregative adhesion pattern. There were changes in the expression of adhesion-related genes (aggR, pic, aap, aggA, and eae) in the bacteria and oxidative stress-related genes (SOD1, SOD2, CAT, and GPx) in the HEp-2 cells. In general, we demonstrated that carvacrol, oregano extract, and rifaximin at sub-minimal bactericidal concentrations interfere with target sites in E. coli, reducing the adhesion efficiency.

Acto-myosin network geometry defines centrosome position

The centrosome is the main organizer of microtubules and as such, its position is a key determinant of polarized cell functions. As the name says, the default position of the centrosome is considered to be the cell geometrical center. However, the mechanism regulating centrosome positioning is still unclear and often confused with the mechanism regulating the position of the nucleus to which it is linked. Here we used enucleated cells plated on adhesive micropatterns to impose regular and precise geometrical conditions to centrosome-microtubule networks. Although frequently observed there, the equilibrium position of the centrosome is not systematically at the cell geometrical center and can be close to cell edge. Centrosome positioning appears to respond accurately to the architecture and anisotropy of the actin network, which constitutes, rather than cell shape, the actual spatial boundary conditions the microtubule network is sensitive to. We found that the contraction of the actin network defines a peripheral margin, in which microtubules appeared bent by compressive forces. The disassembly of the actin network away from the cell edges defines an inner zone where actin bundles were absent and microtubules were more radially organized. The production of dynein-based forces on microtubules places the centrosome at the center of this inner zone. Cell adhesion pattern and contractile forces define the shape and position of the inner zone in which the centrosome-microtubule network is centered.

Effect of Mucin13 gene polymorphism on diarrhoeagenic <i>E. coli</i> adhesion pattern and its expression analysis in native Indian pigs

We identified genetic polymorphism in Mucin13 gene affecting E. coli adhesion patterns using (local isolate) diarrhoeagenic E. coli in Indian desi pigs. Five SNPs and one indel previously reported to be associated with enterotoxigenic E. coli (ETEC) F4ab/ac adhesion pattern were examined by designing PCR-RFLP protocol. The genotypic frequencies of only one SNP (g.22304A > G) differed significantly (at P≤0.05) in adhesive, non-adhesive and weakly adhesive population. The AA (306 sbp, 231 bp), AG (306, 231, 108, 198 bp) and GG (231, 198 bp, 108 b) genotypes of g.22304A > G locus were observed with frequencies 50.0 %, 21.25 % and 28.75 %, respectively and AG genotype was significantly (P≤0.05) associated with a non-adhesive pattern. The polymorphism information content of SNPs ranged from 17.67 (g.22124T > C) to 37.36 % (g.21471C > T) loci. Three loci (g.21471C > T, g.22124T > C and g.22304A > G) were significantly departed from Hardy–Weinberg equilibrium. The linkage disequilibrium analysis revealed locus g.22124T > C and g.22304A > G were significantly (P≤0.05) associated with each other. Expression profiling of target gene in jejuna of animals having AA, AG and GG genotypes revealed differences in various genotypes with the highest in the AA, moderate in the GG and low levels in the AG genotype, although they were statistically non-significant (at P≤0.05). The absence of significant effect of genotypes on MUC13 mRNA expression indicates no direct functional role, although the structural role can not be ignored as the putative receptor gene is located within targeted genomic region. Further, reports of same SNP association with an ETEC F4ab/ac adhesion pattern indicate the target gene's role in diarrhoea even caused by other strains of E. coli which is not ETEC.

A massive reduction of dust particle adhesion in a cyclone by the introduction of a wedge

Particle adhesion in a cyclone, such as a cyclonic vacuum cleaner, can significantly reduce its efficiency. An investigation is presented here on the particle adhesion in a cyclone from a vacuum cleaner that consists of a primary separation stage (a cylindrical chamber) and a secondary separation stage (14 cyclones). The flow direction in the primary separation stage was modified by the use of a wedge of 40 mm × 40 mm × 6 mm at the inlet of the primary separation stage, which affected the particle trajectory in the primary separation stage and the particle inlet position in the cyclone while keeping the air flow direction and velocity (without particles being loaded), the Hamaker constant, particle size and the particle charge unaffected. The particle inlet position in the cyclone was varied from the lower portion (without wedge) to the upper portion (with wedge). Without the wedge, a spiral pattern of particle (plaster particles, average size 1.13 μm) adhesion onto the inner wall of the cyclone was found and a thicker deposited layer of particles at the cyclone tip region was observed. With the introduction of the wedge, the spiral particle adhesion pattern was not observed and a reduction of particle adhered to the inner wall by up to 94% was achieved, although there was an increase in the amount of particles entering the cyclone. This demonstrates almost a complete elimination of particle adhesion onto the cyclone wall, without compromising separation efficiencies.

Bacterial attachment on poly[acrylonitrile-co-(2-methyl-2-propene-1-sulfonic acid)] surfaces

ABSTRACTThe influence of material properties on bacterial attachment to surfaces needs to be understood when applying polymer-based biomaterials. Positively charged materials can kill adhered bacteria when the charge density is sufficiently high [1] but such materials initially increase the adherence of some bacteria such asEscherichia coli[2]. On the other hand, negatively charged materials have been shown to inhibit initial bacterial adhesion [3], but this effect has only been demonstrated in relatively few biomaterial classes and needs to be evaluated using additional systems. Gradients in surface charge can impact bacterial adhesion and this was tested in our experimental setup.Moreover, the evaluation of bacterial adhesion to biomaterials is required to assess their potential for biological applications. Here, we studied the bacterial adhesion ofE. coliandBacillus subtilison the surfaces of acrylonitrile-based copolymer samples with different amounts of 2-methyl-2-propene-1-sulfonic acid sodium salt (NaMAS) comonomer. The content related to NaMAS based repeating units nNaMASvaried in the range from 0.9 to 1.5 mol%.We found a reduced colonized area ofE. colifor NaMAS containing copolymers in comparison to pure PAN materials, whereby the bacterial colonization was similar for copolymers with different nNaMASamounts. A different adhesion behavior was obtained for the second tested organismB. subtilis, where the implementation of negative charges into PAN did not change the overall adhesion pattern. Furthermore, it was observed thatB. subtilisadhesion was significantly increased on copolymer samples that exhibited a more irregular surface roughness.