Coarse-grained modeling and dynamics tracking of nanoparticles diffusion in human gut mucus.

The gastrointestinal (GI) tract's mucus layer serves as a critical barrier and a mediator in drug nanoparticle delivery. The mucus layer's diverse molecular structures and spatial complexity complicates the mechanistic study of the diffusion dynamics of particulate materials. In response, we developed a bi-component coarse-grained mucus model, specifically tailored for the colorectal cancer environment, that contained the two most abundant glycoproteins in GI mucus: Muc2 and Muc5AC. This model demonstrated the effects of molecular composition and concentration on mucus pore size, a key determinant in the permeability of nanoparticles. Using this computational model, we investigated the diffusion rate of polyethylene glycol (PEG) coated nanoparticles, a widely used muco-penetrating nanoparticle. We validated our model with experimentally characterized mucus pore sizes and the diffusional coefficients of PEG-coated nanoparticles in the mucus collected from cultured human colorectal goblet cells. Machine learning fingerprints were then employed to provide a mechanistic understanding of nanoparticle diffusional behavior. We found that larger nanoparticles tended to be trapped in mucus over longer durations but exhibited more ballistic diffusion over shorter time spans. Through these discoveries, our model provides a promising platform to study pharmacokinetics in the GI mucus layer.

Biophysical determinants of biofilm formation in the gut.

The gastrointestinal (GI) tract harbors the most complex microbial ecosystem in the human body. The mucosal layer that covers the GI tract serves as a polymer-based defensive barrier that prevents the microbiome from reaching the epithelium and disseminating inside the body. Colonization of the mucus may result in the formation of structured polymicrobial communities or biofilms, a hallmark in pathologies such as colorectal cancer, inflammatory bowel disease, and chronic gut wounds. However, the mechanisms by which multispecies biofilms establish on the gut mucosa is unknown. Whether mucus-associated biofilms exist as part of a healthy mucosal barrier is still debated. Here, we discuss the impact that diet and microbial-derived polymers has on mucus structure and microcolony formation and highlight relevant biophysical forces that further shape nascent biofilms.

Escherichia coli Adhesion and Biofilm Formation on Polydimethylsiloxane are Independent of Substrate Stiffness.

Bacterial adhesion and biofilm formation on the surface of biomedical devices are detrimental processes that compromise patient safety and material functionality. Several physicochemical factors are involved in biofilm growth, including the surface properties. Among these, material stiffness has recently been suggested to influence microbial adhesion and biofilm growth in a variety of polymers and hydrogels. However, no clear consensus exists about the role of material stiffness in biofilm initiation and whether very compliant substrates are deleterious to bacterial cell adhesion. Here, by systematically tuning substrate topography and stiffness while keeping the surface free energy of polydimethylsiloxane substrates constant, we show that topographical patterns at the micron and submicron scale impart unique properties to the surface which are independent of the material stiffness. The current work provides a better understanding of the role of material stiffness in bacterial physiology and may constitute a cost-effective and simple strategy to reduce bacterial attachment and biofilm growth even in very compliant and hydrophobic polymers.

Bacterial Envelope Damage Inflicted by Bioinspired Nanostructures Grown in a Hydrogel.

Surface-associated bacterial communities, known as biofilms, are responsible for a broad spectrum of infections in humans. Recent studies have indicated that surfaces containing nanoscale protrusions, like those in dragonfly wings, create a hostile niche for bacterial colonization and biofilm growth. This functionality has been mimicked on metals and semiconductors by creating nanopillars and other high aspect ratio nanostructures at the interface of these materials. However, bactericidal topographies have not been reported on clinically relevant hydrogels and highly compliant polymers, mostly because of the complexity of fabricating nanopatterns in hydrogels with precise control of the size that can also resist aqueous immersion. Here, we report the fabrication of bioinspired bactericidal nanostructures in bacterial cellulose (BC) hydrogels using low-energy ion beam irradiation. By challenging the currently accepted view, we show that the nanostructures grown in BC affect preferentially stiff membranes like those of the Gram-positive bacteria Bacillus subtilis in a time-dependent manner and, to a lesser extent, the more deformable and softer membrane of Escherichia coli. Moreover, the nanostructures in BC did not affect the viability of murine preosteoblasts. Using single-cell analysis, we demonstrate that indeed B. subtilis requires less force than E. coli to be penetrated by nanoprobes with dimensions comparable to those of the nanostructured BC, providing the first direct experimental evidence validating a mechanical model of membrane rupture via a tension-induced mechanism within the activation energy theory. Our findings bridge the gap between mechano-bactericidal surfaces and low-dimensional materials, including single-walled carbon nanotubes and graphene nanosheets, in which a higher bactericidal activity toward Gram-positive bacteria has been extensively reported. Our results also demonstrate the ability to confer bactericidal properties to a hydrogel by only altering its topography at the nanoscale and contribute to a better understanding of the bacterial mechanobiology, which is fundamental for the rational design bactericidal topographies.

Designing Nanostructured Ti6Al4V Bioactive Interfaces with Directed Irradiation Synthesis toward Cell Stimulation to Promote Host-Tissue-Implant Integration.

A new generation of biomaterials are evolving from being biologically inert toward bioactive surfaces, which can further interact with biological components at the nanoscale. Here, we present directed irradiation synthesis (DIS) as a novel technology to selectively apply plasma ions to bombard any type of biomaterial and tailor the nanofeatures needed for in vitro growth stimulation. In this work, we demonstrate for the first time, the influence of physiochemical cues (e.g., self-organized topography at nanoscale) of medical grade Ti6Al4V results in control of cell shape, adhesion, and proliferation of human aortic smooth muscle stem cells. The control of surface nanostructures was found to be correlated to ion-beam incidence angle linked to a surface diffusive regime during irradiation synthesis with argon ions at energies below 1 keV and a fluence of 2.5 × 1017 cm-2. Cell viability and cytoskeleton morphology were evaluated at 24 h, observing an advance cell attachment state on post-DIS surfaces. These modified surfaces showed 84% of cell biocompatibility and an increase in cytoplasmatic protusions ensuring a higher cell adhesion state. Filopodia density was promoted by a 3-fold change for oblique incidence angle DIS treatment compared to controls (e.g., no patterning) and lamellipodia structures were increased more than a factor of 2, which are indicators of cell attachment stimulation due to DIS modification. In addition, the morphology of the nanofeatures were tailored, with high fidelity control of the main DIS parameters that control diffusive and erosive regimes of self-organization. We have correlated the morphology and the influence in cell behavior, where nanoripple formation is the most active morphology for cell stimulation.

Balancing biofunctional and biomechanical properties using porous titanium reinforced by carbon nanotubes.

Despite the well-known advantages of the titanium-based implant systems, they still lack an optimal balance between biofunctionality and mechanical strength, especially regarding the modulation of cellular response and a desired implant osseointegration. In this work, we fabricated a nanocomposite based on porous commercially pure grade 4 titanium (c.p. Ti) reinforced with carbon nanotubes (CNT) at 5% and 10% w/w, with the aim of obtaining a nanocomposite with lower stiffness compared to traditional titanium-based implants and with the mechanical strength and bioactivity owed by the CNT. Results obtained by scanning electron microscopy, X-ray photoelectron spectroscopy, and atomic force microscopy characterization showed that the CNT dispersed and incorporated into the porous c.p. Ti matrix. Interestingly, CNT were associated with a higher twining within neighbor Ti grains, which was indeed consistent with an increased in nano-hardness. Biological evaluation by MTT and Comet assay revealed that the nanocomposites did not induce genotoxicity and cytotoxicity on two different cells lines despite the presence of nickel at the surface. Accordingly, a purification step would be required before these CNT can be used for biomedical applications. Our results indicate that incorporation of CNT into porous c.p. Ti is a promising avenue to achieve an adequate balance between biofunctionality and mechanical strength in Ti-based scaffolds for tissue replacement. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 719-731, 2019.

Magnetic targeting of smooth muscle cells in vitro using a magnetic bacterial cellulose to improve cell retention in tissue-engineering vascular grafts.

Tissue-engineered vascular grafts (TEVG) use biologically-active cells with or without supporting scaffolds to achieve tissue remodeling and regrowth of injured blood vessels. However, this process may take several weeks because the high hemodynamic shear stress at the damaged site causes cellular denudation and impairs tissue regrowth. We hypothesize that a material with magnetic properties can provide the force required to speed up re-endothelization at the vascular defect by facilitating high cell density coverage, especially during the first 24 h after implantation. To test our hypothesis, we designed a magnetic bacterial cellulose (MBC) to locally target cells in vitro under a pulsatile fluid flow (0.514 dynes cm-2). This strategy can potentially increase cell homing at TEVG, without the need of blood cessation. The MBC was synthesized by an in situ precipitation method of Fe3+ and Fe2+ iron salts into bacterial cellulose (BC) pellicles to form Fe3O4 nanoparticles along the BC's fibrils, followed by the application of dextran coating to protect the embedded nanoparticles from oxidation. The iron salt concentration used in the synthesis of the MBC was tuned to balance the magnetic properties and cytocompatibility of the magnetic hydrogel. Our results showed a satisfactory MBC magnetization of up to 10 emu/g, which is above the value considered relevant for tissue engineering applications (0.05 emu/g). The MBC captured magnetically-functionalized cells under dynamic flow conditions in vitro. MBC magnetic properties and cytocompatibility indicated a dependence on the initial iron oxide nanoparticle concentration. STATEMENT OF SIGNIFICANCE: Magnetic hydrogels represent a new class of functional materials with great potential in TVEG because they offer a platform to (1) release drugs on demand, (2) speed up tissue regrowth, and (3) provide mechanical cues to cells by its deformability capabilities. Here, we showed that a magnetic hydrogel, the MBC, was able to capture and retain magnetically-functionalized smooth muscle cells under pulsatile flow conditions in vitro. A magnetic hydrogel with this feature can be used to obtain high-density cell coverage on sites that are aggressive for cell survival such as the luminal face of vascular grafts, whereas simultaneously can support the formation of a biologically-active cell layer that protects the material from restenosis and inflammation.

Bacterial Nanocellulose Magnetically Functionalized for Neuro-Endovascular Treatment.

Current treatments for brain aneurysms are invasive, traumatic, and not suitable in most patients with increased risks. A new alternative method is using scaffold stents to create a local and focal attraction force of cells for an in situ reconstruction of the tunica media. For this purpose, a nanostructured bioactive coating is designed to render an asymmetric region of the stent scaffold magnetic and biomimetic, which utilizes bacterial nanocellulose (BNC) as a platform for both magnetic and cell attraction as well as proliferation. The magnetization of the BNC is realized through the reaction of Fe III and II, precipitating superparamagnetic iron oxide nanoparticles (SPION). Subsequently, magnetic bacterial nanocellulose (MBNC) is coated with polyethylene glycol to improve its biocompatibility. Cytotoxicity and biocompatibility are evaluated using porcine aortic smooth muscle cells. Preliminary cellular migration assays demonstrate the behavior between MBNC and cells labeled with SPION. In this work, (1) synthesis of BNC impregnated with magnetic nanoparticles is successfully demonstrated; (2) a viable, resilient, and biocompatible hydrogel membrane is tested for neuroendovascular application using a stent scaffold; (3) cell viability and minimal cytotoxicity is achieved; (4) cell migration tests and examination of cellular magnetic attraction confirm the viability of MBNC as a multifunctional coating.

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles.

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide nanoparticles (IONP) (Fe3O4) to yield a magnetic bacterial nanocellulose (MBNC). The synthesis of MBNC is a precise and specifically designed multi-step process. Briefly, bacterial nanocellulose (BNC) pellicles are formed from preserved G. xylinus strain according to our experimental requirements of size and morphology. A solution of iron(III) chloride hexahydrate (FeCl3·6H2O) and iron(II) chloride tetrahydrate (FeCl2·4H2O) with a 2:1 molar ratio is prepared and diluted in deoxygenated high purity water. A BNC pellicle is then introduced in the vessel with the reactants. This mixture is stirred and heated at 80 °C in a silicon oil bath and ammonium hydroxide (14%) is then added by dropping to precipitate the ferrous ions into the BNC mesh. This last step allows forming in situ magnetite nanoparticles (Fe3O4) inside the bacterial nanocellulose mesh to confer magnetic properties to BNC pellicle. A toxicological assay was used to evaluate the biocompatibility of the BNC-IONP pellicle. Polyethylene glycol (PEG) was used to cover the IONPs in order to improve their biocompatibility. Scanning electron microscopy (SEM) images showed that the IONP were located preferentially in the fibril interlacing spaces of the BNC matrix, but some of them were also found along the BNC ribbons. Magnetic force microscope measurements performed on the MBNC detected the presence magnetic domains with high and weak intensity magnetic field, confirming the magnetic nature of the MBNC pellicle. Young's modulus values obtained in this work are also in a reasonable agreement with those reported for several blood vessels in previous studies.