An ex vivo study of infections of vascular grafts and endografts with scanning electron microscopy.

OBJECTIVE: Vascular graft and endograft infections (VGEIs) are complicated by high morbidity, mortality, and recurrence rates, notably due to biofilm formation on the graft surface, hardly dislodgeable by the sole anti-infectious treatment. The characteristics of this biofilm are still poorly documented. The aim of this study was to evaluate ex vivo biofilm on removed infected vascular grafts and endografts (VGEs). METHODS: Explanted VGEs were prospectively collected from 2019 to 2022 at Bordeaux University Hospital, France. Two samples per graft were used for scanning electron microscopy imaging; one was sonicated, and both grafts' sides were imaged. RESULTS: A total of 26 patients were included, 18 with VGEI, eight without any infection (endoleak and/or thrombosis), and 29 VGEs were collected. Microbial documentation was obtained in 83% of VGEIs. A thick layer of fibrin was visible on almost all grafts, mixed with a dense biofilm matrix on infected grafts visible as early as 1 month after the onset of infection. Bacteria were not always visualized on infected grafts' surface (80% on outer side and 85% on luminal side) but were surprisingly present on one-third of non-infected grafts. There was no significant difference between biofilm, fibrin, and microorganisms' distribution between the two grafts' sides. However, there were clear differences between infected and non-infected grafts, since immune cells, bacteria and biofilm were more frequently visualized on both sides of infected grafts (P < .05). Bacteria and immune cells although still visible, were significantly less present after sonication; the number of other elements including biofilm was not significantly different. CONCLUSIONS: The persistence of a thick layer of fibrin and biofilm embedding microorganisms on both sides of infected VGE even after 1 month of infection could be the explanation for the low success rates of conservative management and the usual need for graft removal to treat VGEIs.

Staphylococcus aureus Adhesion and Biofilm Formation on Vascular Polyester Grafts are Inhibited In Vitro by Triclosan.

OBJECTIVE: This study evaluated Staphylococcus aureus adhesion and biofilm formation on vascular grafts, which has seldom been investigated. METHODS: Adhesion and biofilm formation capabilities of three methicillin susceptible S. aureus strains (one biofilm forming reference strain and two clinical isolates) on five different vascular biomaterials were evaluated in vitro, including polyester (P), P + gelatin (PG), P + collagen (PC), PC + silver (PCS), and PCS + triclosan (PCST). Staphylococcus aureus adhesion on grafts was evaluated after one hour of culture and biofilm formation after 24 hours of culture by four different methods: spectrophotometry after crystal violet staining; sonicate fluid culture; metabolic assay; and scanning electron microscopy (SEM). Optical density was compared using Mann-Whitney pairwise test, and bacterial counts using Wilcoxon pairwise test. RESULTS: PCST grafts were most efficient in preventing S. aureus adhesion and biofilm formation, regardless of the method used. Bacterial counts and metabolic activity were significantly lower on PCST grafts after 24 hours (5.65 vs. 9.24 [PCS], 8.99 [PC], 8.82 [PG], and 10.44 log10 CFU/mL [P]; p < .015), and only PCST grafts were bactericidal. Biofilm formation was significantly diminished on PCST grafts compared with all other grafts (p < .001). Bacterial viability and metabolic activity after 24 hours were more impaired on PG compared with PC graft, and were surprisingly higher on PCS compared with PC grafts. Biofilm biomass formed after exposure to P, PG, PC, and PCS grafts was also reduced after 24 hours of incubation with PCST grafts (p < .001). After 24 hours, few bacteria were visible by SEM on PCST grafts, whereas bacterial biofilm colonies were clearly identified on other graft surfaces. CONCLUSION: Triclosan impregnated PCST grafts appeared to interfere with S. aureus adhesion from early stages of biofilm formation in vitro. Silver impregnation was not efficient in preventing biofilm formation, and collagen coating promoted S. aureus biofilm formation more than gelatin coating.

Preservation of exopolymeric substances in estuarine sediments.

The surface of intertidal estuarine sediments is covered with diatom biofilms excreting exopolymeric substances (EPSs) through photosynthesis. These EPSs are highly reactive and increase sediment cohesiveness notably through organo-mineral interactions. In most sedimentary environments, EPSs are partly to fully degraded by heterotrophic bacteria in the uppermost millimeters of the sediment and so they are thought to be virtually absent deeper in the sedimentary column. Here, we present the first evidence of the preservation of EPSs and EPS-mineral aggregates in a 6-m-long sedimentary core obtained from an estuarine point bar in the Gironde Estuary. EPSs were extracted from 18 depth intervals along the core, and their physicochemical properties were characterized by (i) wet chemical assays to measure the concentrations of polysaccharides and proteins, and EPS deprotonation of functional groups, (ii) acid-base titrations, and (iii) Fourier transform infrared spectroscopy. EPS-sediment complexes were also imaged using cryo-scanning electron microscopy. EPS results were analyzed in the context of sediment properties including facies, grain size, and total organic carbon, and of metabolic and enzymatic activities. Our results showed a predictable decrease in EPS concentrations (proteins and polysaccharides) and reactivity from the surface biofilm to a depth of 0.5 m, possibly linked to heterotrophic degradation. Concentrations remained relatively low down to ca. 4.3 m deep. Surprisingly, at that depth EPSs abundance was comparable to the surface and showed a downward decrease to 6.08 m. cryo-scanning electron microscopy (Cryo-SEM) showed that the EPS complexes with sediment were abundant at all studied depth and potentially protected EPSs from degradation. EPS composition did not change substantially from the surface to the bottom of the core. EPS concentrations and acidity were anti-correlated with metabolic activity, but showed no statistical correlation with grain size, TOC, depth or enzymatic activity. Maximum EPS concentrations were found at the top of tide-dominated sedimentary sequences, and very low concentrations were found in river flood-dominated sedimentary sequences. Based on this observation, we propose a scenario where biofilm development and EPS production are maximal when (i) the point bar and the intertidal areas were the most extensive, i.e., tide-dominated sequences and (ii) the tide-dominated deposit were succeeded by rapid burial beneath sediments, potentially decreasing the probability of encounter between bacterial cells and EPSs.

Development of a vascular substitute produced by weaving yarn made from human amniotic membrane.

Because synthetic vascular prostheses perform poorly in small-diameter revascularization, biological vascular substitutes are being developed as an alternative. Although theirin vivoresults are promising, their production involves long, complex, and expensive tissue engineering methods. To overcome these limitations, we propose an innovative approach that combines the human amniotic membrane (HAM), which is a widely available and cost-effective biological raw material, with a rapid and robust textile-inspired assembly strategy. Fetal membranes were collected after cesarean deliveries at term. Once isolated by dissection, HAM sheets were cut into ribbons that could be further processed by twisting into threads. Characterization of the HAM yarns (both ribbons and threads) showed that their physical and mechanical properties could be easily tuned. Since our clinical strategy will be to provide an off-the-shelf allogeneic implant, we studied the effects of decellularization and/or gamma sterilization on the histological, mechanical, and biological properties of HAM ribbons. Gamma irradiation of hydrated HAMs, with or without decellularization, did not interfere with the ability of the matrix to support endothelium formationin vitro. Finally, our HAM-based, woven tissue-engineered vascular grafts (TEVGs) exhibited clinically relevant mechanical properties. Thus, this study demonstrates that human, completely biological, allogeneic, small-diameter TEVGs can be produced from HAM, thereby avoiding costly cell culture and bioreactors.

Nucleoside-Derived Low-Molecular-Weight Gelators as a Synthetic Microenvironment for 3D Cell Culture.

For the last few decades, many efforts have been made in developing cell culture methods in order to overcome the biological limitations of the conventional two-dimensional culture. This paradigm shift is driven by a large amount of new hydrogel-based systems for three-dimensional culture, among other systems, since they are known to mimic some living tissue properties. One class of hydrogel precursors has received interest in the field of biomaterials, low-molecular-weight gelators (LMWGs). In comparison to polymer gels, LMWG gels are formed by weak interactions upon an external trigger between the molecular subunits, giving them the ability to reverse the gelation, thus showing potential for many applications of practical interest. This study presents the use of the nucleoside derivative subclass of LMWGs, which are glyco-nucleo-bola-amphiphiles, as a proof of concept of a 3D cell culture scaffold. Physicochemical characterization was performed in order to reach the optimal features to fulfill the requirements of the cell culture microenvironment, in terms of the mechanical properties, architecture, molecular diffusion, porosity, and experimental practicality. The retained conditions were tested by culturing glioblastoma cells for over a month. The cell viability, proliferation, and spatial organization showed during the experiments demonstrate the proof of concept of nucleoside-derived LMWGs as a soft 3D cell culture scaffold. One of the hydrogels tested permits cell proliferation and spheroidal organization over the entire culture time. These systems offer many advantages as they consume very few matters within the optimal range of viscoelasticity for cell culture, and the thermoreversibility of these hydrogels permits their use with few instruments. The LMWG-based scaffold for the 3D cell culture presented in this study unlocked the ability to grow spheroids from patient cells to reach personalized therapies by dramatically reducing the variability of the lattice used.

Biological, geological and chemical effects of oxygen injection in underground gas storage aquifers in the setting of biomethane deployment.

The last few years have seen the proliferation of anaerobic digestion plants to produce biomethane. Oxygen (O2) traces added to biogas during the desulfurization process are co-injected in the gas network and can be stored in Underground Gas Storage (UGS). However, there are no data available for the undesirable effects of O2 on these anoxic environments, especially on deep aquifers. In addition to mineral alteration, O2 can have an impact on the anaerobic autochthonous microbial life. In our study, the storage conditions of an UGS aquifer were reproduced in a high-pressure reactor and bio-geo-chemical interactions between the aqueous, gas and solid phases were studied. Sulfate was depleted from the liquid phase for three consecutive times during the first 130 days of incubation reproducing the storage conditions (36 °C, 60 bar, methane with 1% CO2). Sulfate-reducers, such as Desulfovibrionaceae, were identified from the high-pressure system. Simulations with PHREEQC were used to determine the thermodynamic equilibrium to confirm any gas consumption. CO2 quantities decreased in the gas phase, suggesting its use as carbon source by microbial life. Benzene and toluene, hydrocarbons found in traces and known to be biodegradable in storages, were monitored and a decrease of toluene was revealed and associated to the Peptococcaceae family. Afterwards, O2 was added as 1% of the gas phase, corresponding to the maximum quantity found in biomethane after desulfurization process. Re-oxidation of sulfide to sulfate was observed along with the end of sulfate reducing activity and toluene biodegradation and the disappearance of most of the community. H2 surprisingly appeared and accumulated as soon as hydrogenotrophic sulfate-reducers decreased. H2 would be produced via the necromass fermentation accomplished by microorganisms able to resist the oxic conditions of 4.42·10-4 mol.Kgw-1 of O2. The solid phase composed essentially of quartz, presented no remarkable changes.

Hydrogel based lipid-oligonucleotides: a new route to self-delivery of therapeutic sequences.

Synthetic OligoNucleotides (ON) provide promising therapeutic tools for controlling specifically genetic expression in a broad range of diseases from cancers to viral infections. Beside their chemical stability and intracellular delivery, the controlled release of therapeutic sequences remains an important challenge for successful clinical applications. In this work, Lipid-OligoNucleotide (LON) conjugates stabilizing hydrogels are reported and characterized by rheology and cryo-scanning electron microscopy (cryo-SEM). These studies revealed that lipid conjugation of antisense oligonucleotides featuring partial self-complementarity resulted in entangled pearl-necklace networks, which were obtained through micelle-micelle interaction driven by duplex formation. Owing to these properties, the Lipid AntiSense Oligonucleotide (LASO) sequences exhibited a prolonged release after subcutaneous administration compared to the non-lipidic antisense (ASO) one. The LASO self-assembly based hydrogels obtained without adjuvant represent an innovative approach for the sustained self-delivery of therapeutic oligonucleotides.

Human textiles: A cell-synthesized yarn as a truly "bio" material for tissue engineering applications.

In the field of tissue engineering, many groups have come to rely on the extracellular matrix produced by cells as the scaffold that provides structure and strength to the engineered tissue. We have previously shown that sheets of Cell-Assembled extracellular Matrix (CAM), which are entirely biological yet robust, can be mass-produced for clinical applications using normal, adult, human fibroblasts. In this article, we demonstrate that CAM yarns can be generated with a range of physical and mechanical properties. We show that this material can be used as a simple suture to close a wound or can be assembled into fully biological, human, tissue-engineered vascular grafts (TEVGs) that have high mechanical strength and are implantable. By combining this truly "bio" material with a textile-based assembly, this original tissue engineering approach is highly versatile and can produce a variety of strong human textiles that can be readily integrated in the body. STATEMENT OF SIGNIFICANCE: Yarn of synthetic biomaterials have been turned into textiles for decades because braiding, knitting and weaving machines can mass-produce medical devices with a wide range of shapes and mechanical properties. Here, we show that robust, completely biological, and human yarn can be produced by normal cells in vitro. This yarn can be used as a simple suture material or to produce the first human textiles. For example, we produced a woven tissue-engineered vascular grafts with burst pressure, suture retention strength and transmural permeability that surpassed clinical requirements. This novel strategy holds the promise of a next generation of medical textiles that will be mechanically strong without any foreign scaffolding, and will have the ability to truly integrate into the host's body.

Isolated straight hair nevus in a White child.

We report a 6-month-old girl born with a fronto-parietal patch of hair straighter than the remainder of the scalp hairs. We took a biopsy to rule out a congenital melanocytic nevus. We concluded after additional scanning electron microscopy study of the hair shafts that the lesion corresponds to a possible local mosaicism causing an isolated straight hair nevus phenotype.