Micro/nano-patterns for enhancing differentiation of human neural stem cells and fabrication of nerve conduits via soft lithography and 3D printing.

Topographical cues on materials can manipulate cellular fate, particularly for neural cells that respond well to such cues. Utilizing biomaterial surfaces with topographical features can effectively influence neuronal differentiation and promote neurite outgrowth. This is crucial for improving the regeneration of damaged neural tissue after injury. Here, we utilized groove patterns to create neural conduits that promote neural differentiation and axonal growth. We investigated the differentiation of human neural stem cells (NSCs) on silicon dioxide groove patterns with varying height-to-width/spacing ratios. We hypothesize that NSCs can sense the microgrooves with nanoscale depth on different aspect ratio substrates and exhibit different morphologies and differentiation fate. A comprehensive approach was employed, analyzing cell morphology, neurite length, and cell-specific markers. These aspects provided insights into the behavior of the investigated NSCs and their response to the topographical cues. Three groove-pattern models were designed with varying height-to-width/spacing ratios of 80, 42, and 30 for groove pattern widths of 1 µm, 5 µm, and 10 µm and nanoheights of 80 nm, 210 nm, and 280 nm. Smaller groove patterns led to longer neurites and more effective differentiation towards neurons, whereas larger patterns promoted multidimensional differentiation towards both neurons and glia. We transferred these cues onto patterned polycaprolactone (PCL) and PCL-graphene oxide (PCL-GO) composite 'stamps' using simple soft lithography and reproducible extrusion 3D printing methods. The patterned scaffolds elicited a response from NSCs comparable to that of silicon dioxide groove patterns. The smallest pattern stimulated the highest neurite outgrowth, while the middle-sized grooves of PCL-GO induced effective synaptogenesis. We demonstrated the potential for such structures to be wrapped into tubes and used as grafts for peripheral nerve regeneration. Grooved PCL and PCL-GO conduits could be a promising alternative to nerve grafting.

SARS-CoV-2 Virus-like Particles with Plasmonic Au Cores and S1-Spike Protein Coronas.

The COVID-19 pandemic has stimulated the scientific world to intensify virus-related studies aimed at the development of quick and safe ways of detecting viruses in the human body, studying the virus-antibody and virus-cell interactions, and designing nanocarriers for targeted antiviral therapies. However, research on dangerous viruses can only be performed in certified laboratories that follow strict safety procedures. Thus, developing deactivated virus constructs or safe-to-use virus-like objects, which imitate real viruses and allow performing virus-related studies in any research laboratory, constitutes an important scientific challenge. Such species, called virus-like particles (VLPs), contain instead of capsids with viral DNA/RNA empty or synthetic cores with real virus proteins attached to them. We have developed a method for the preparation of VLPs imitating the virus responsible for the COVID-19 disease: the SARS-CoV-2. The particles have Au cores surrounded by "coronas" of S1 domains of the virus's spike protein. Importantly, they are safe to use and specifically interact with SARS-CoV-2 antibodies. Moreover, Au cores exhibit localized surface plasmon resonance (LSPR), which makes the synthesized VLPs suitable for biosensing applications. During the studies, the effect allowed us to visualize the interaction between the VLPs and the antibodies and identify the characteristic vibrational signals. What is more, additional functionalization of the particles with a fluorescent label revealed their potential in studying specific virus-related interactions. Notably, the universal character of the developed synthesis method makes it potentially applicable for fabricating VLPs imitating other life-threatening viruses.

Nanomolar Nitric Oxide Concentrations in Living Cells Measured by Means of Fluorescence Correlation Spectroscopy.

Measurement of the nitric oxide (NO) concentration in living cells in the physiological nanomolar range is crucial in understanding NO biochemical functions, as well as in characterizing the efficiency and kinetics of NO delivery by NO-releasing drugs. Here, we show that fluorescence correlation spectroscopy (FCS) is perfectly suited for these purposes, due to its sensitivity, selectivity, and spatial resolution. Using the fluorescent indicators, diaminofluoresceins (DAFs), and FCS, we measured the NO concentrations in NO-producing living human primary endothelial cells, as well as NO delivery kinetics, by an external NO donor to the immortal human epithelial living cells. Due to the high spatial resolution of FCS, the NO concentration in different parts of the cells were also measured. The detection of nitric oxide by means of diaminofluoresceins is much more efficient and faster in living cells than in PBS solutions, even though the conversion to the fluorescent form is a multi-step reaction.

Novel nanosystems to enhance biological activity of hydroxyapatite against dental caries.

This work aimed to study for the first time to our knowledge the influence of the structure of the dental flosses (DF) coated by hydroxyapatite nanoparticles (HAP NPs) on the biological performance of saliva probiotic bacteria (S. salivarius), and human dermal and osteoblast-like cells. We used three types of HAP@DF composites (based on two unwaxed dental flosses - "fluffy" and "smooth", and one waxed "smooth") with different morphologies. Obtained composites were characterized from the point of view of their structure, morphological characteristics, elemental and chemical composition. We observed that HAP NPs coated "smooth" dental flosses led to an increase of viability and proliferation of oral cavity probiotic bacteria (Streptococcus salivarius) and human cells (dermal fibroblasts and osteoblast-like). In contrast, the highest viability loss of probiotic bacteria (S. salivarius), fibroblasts, and osteoblast-like cells were observed for "fluffy" unwaxed dental flosses due to high cytotoxicity. Our studies showed that HAP NPs significantly improved the biological properties of "fluffy" dental floss. Pristine "smooth" DFs (waxed and unwaxed), as well as all HAP-coated DFs, induced acceptable biocompatibility toward selected human cells.

Milestones and current achievements in development of multifunctional bioscaffolds for medical application.

Tissue engineering (TE) is a rapidly growing interdisciplinary field, which aims to restore or improve lost tissue function. Despite that TE was introduced more than 20 years ago, innovative and more sophisticated trends and technologies point to new challenges and development. Current challenges involve the demand for multifunctional bioscaffolds which can stimulate tissue regrowth by biochemical curves, biomimetic patterns, active agents and proper cell types. For those purposes especially promising are carefully chosen primary cells or stem cells due to its high proliferative and differentiation potential. This review summarized a variety of recently reported advanced bioscaffolds which present new functions by combining polymers, nanomaterials, bioactive agents and cells depending on its desired application. In particular necessity of study biomaterial-cell interactions with in vitro cell culture models, and studies using animals with in vivo systems were discuss to permit the analysis of full material biocompatibility. Although these bioscaffolds have shown a significant therapeutic effect in nervous, cardiovascular and muscle, tissue engineering, there are still many remaining unsolved challenges for scaffolds improvement.

Synthesis, characterization and in vitro cytotoxicity studies of poly-N-isopropyl acrylamide gel nanoparticles and films.

In this work, we show synthesis that leads to thermoreponsive poly-N-isopropyl acrylamide (pNIPAM) nanogels with sizes below 100 nm, irrespectively of the surfactant to crosslinker ratio. We also show that in many environments the temperature induced pNIPAM collapse at Lower Critical Solution Temperature (LCST) of 32.5 °C is accompanied by gel nanoparticles' aggregation. Thus, the proper information on the nanoparticle (NP) structure and deswelling can be obtained only if the routinely measured hydrodynamic radius is supplemented by information on the molecular weight, which can be obtained from the intensity of scattered light. We measured the dynamics and reversibility of the deswelling and subsequent aggregation processes. Furthermore, we show that the highly concentrated pNIPAM gel NPs reversibly form bulk hydrogel networks of varied interconnected porous structure. We show, that in case of drying pNIPAM gel NPs above the LCST, it is possible to obtain films with 20-fold increase in storage modulus (G') compared to hydrogel networks measured at room temperature. They exhibit temperature hysteresis behavior around LCST of 32.5 °C similar to pNIPAM films. Finally, we show that these hydrogel films, lead to extended proliferation of cells across three different types: fibroblast, endothelial and cancer cells. Additionally, none of the films exhibited any cytotoxic effects. Our study brings new insights into physicochemical characterization of pNIPAM gel NPs and networks behavior in realistic conditions of in vitro measurements, especially by means of dynamic light scattering as well as final unique properties of both gel NPs and formed porous films for possible tissue engineering applications.

Unique cellular network formation guided by heterostructures based on reduced graphene oxide - Ti3C2Tx MXene hydrogels.

Two-dimensional (2D) materials remain highly interesting for assembling three-dimensional (3D) structures, amongst others, in the form of macroscopic hydrogels. Herein, we present a novel approach for inducing chemical inter-sheet crosslinks via an ethylenediamine mediated reaction between Ti3C2Tx and graphene oxide in order to obtain a reduced graphene oxide-MXene (rGO-MXene) hydrogel. The composite hydrogels are hydrophilic with a stiffness of ~20 kPa. They also possess a unique inter-connected porous architecture, which led to a hitherto unprecedented ability of human cells across three different types, epithelial adenocarcinoma, neuroblastoma and fibroblasts, to form inter-connected three-dimensional networks. The attachments of the cells to the rGO-MXene hydrogels were superior to those of the sole rGO-control gels. This phenomenon stems from the strong affinity of cellular protrusions (neurites, lamellipodia and filopodia) to grow and connect along architectural network paths within the rGO-MXene hydrogel, which could lead to advanced control over macroscopic formations of cellular networks for technologically relevant bioengineering applications, including tissue engineering and personalized diagnostic networks-on-chip. STATEMENT OF SIGNIFICANCE: Conventional hydrogels are made of interconnected polymeric fibres. Unlike conventional case, we used hydrothermal and chemical approach to form interconnected porous hydrogels made of two-dimensional flakes from graphene oxide and metal carbide from a new family of MXenes (Ti3C2Tx). This way, we formed three-dimensional porous hydrogels with unique porous architecture of well-suited chemical surfaces and stiffness. Cells from three different types cultured on these scaffolds formed extended three-dimensional networks - a feature of extended cellular proliferation and pre-requisite for formation of organoids. Considering the studied 2D materials typically constitute materials exhibiting enhanced supercapacitor performances, our study points towards better understanding of design of tissue engineering materials for the future bioengineering fields including personalized diagnostic networks-on-chip, such as artificial heart actuators.

Fabricating versatile cell supports from nano- and micro-sized graphene oxide flakes.

Micro-sized structures made from graphene oxide (GO) attract high interest for their extensive use in tissue engineering. The fabrication and cytotoxicity of 3D graphene-based scaffolds so far have not been extensively discussed with relation to the flake sizes used. In this work we considered GO flakes of two different lognormal size distributions (GO: 4.9 ± 3.8 µm and GO 1 h: 151.6 ± 1.9 nm) as model flakes for fabrication of 3D graphene-based cell culture supports: paper (i.e. 3D layered film structure) and reduced graphene oxide (rGO) microfiber using hydrothermal methods. We then used two model cell lines of neuronal origin (SH-SY5Y and HEK-293) to study subsequent scaffolds surface-cells interactions. In particular, the adhesion of HEK cells to the formed structures was much higher than for SH-SY5Y cells, as evidenced by various atomic force, electron and optical microscopy techniques. Formed rGO microfibers had more desired nano-topography (surface roughness) for cell adhesion and growth than simple GO paper, making it ideal scaffold for neural tissue engineering. This work provides insights into the fundamental rules for fabrication of graphene oxide-based cell supports and their subsequently differing interactions with malignant and non-malignant cells.

UV cross-linked polyvinylpyrrolidone electrospun fibres as antibacterial surfaces.

Many bacteria become progressively more resistant to antibiotics and it remains a challenging task to control their overall levels. Polymers combined with active biomolecules come to the forefront for the design of antibacterial materials that can address this encounter. In this work, we investigated the photo-crosslinking approach of UV-sensitive benzophenone molecule (BP) with polyvinylpyrrolidone (PVP) polymer within electrospun fibres. The BP and PVP solutions allowed fabricating polymer mats that were subsequently functionalised with antibacterial lysozyme. The physical properties of the crosslinked electrospun fibres were investigated by scanning electron microscopy and atomic force microscopy. The average diameter of the obtained fibres decreased from 290 ± 50 nm to 270 ± 70 nm upon the addition of the crosslinking molecules and then to 240 ± 80 nm and 180 ± 90 nm after subsequent crosslinking reaction at an increasing time: 3 and 5 h, respectively. The peak force quantitative nanomechanical mapping (PF-QNM) indicated the increase of DMT modulus of obtained cross-linked fibres from 4.1 ± 0.8 GPa to 7.2 ± 0.5 GPa. Furthermore, the successful crosslinking reaction of PVP and BP solution into hydrogels was investigated in terms of examining photo-crosslinking mechanism and was confirmed by rheology, Raman, Fourier transform infrared and nuclear magnetic resonance. Finally, lysozyme was successfully encapsulated within cross-linked PVP-BP hydrogels and these were successfully electrospun into mats which were found to be as effective antibacterial agents as pure lysozyme molecules. The dissolution rate of photo cross-linked PVP mats was observed to increase in comparison to pure PVP electrospun mats which opened a potential route for their use as antibacterial, on-demand, dissolvable coatings for various biomedical applications.

Groove-patterned surfaces induce morphological changes in cells of neuronal origin.

It is already known that cells respond strongly to topography and chemistry of 2D surfaces. In this work we study cell-material interactions; in particular, we investigated the attachment and alignment of SH-SY5Y cells of neuronal origin on grooved-patterns made from Silicon (Si) and Gold (Au). The Au-Si groove-pattern stimulated 93% of SH-SY5Y cells to differentiate into neuroblast-like type (N-type) in 2 days and outgrown neurites exhibited strong anisotropy along the grooves with 90% of cells having one or two neurites. In comparison, random distribution of morphology type, neurite number, and alignment were observed on control flat surfaces (Si and Au). We further show that designed Au-Si groove-patterns can be used to form reversed groove patterns on polycarolactone surface via soft lithography approach. Sixty-nine percentage of SH-SY5Y cells aligned along the obtained reversed groove patterns of the same dimensional characteristics to Si-Au grooves. In particular, this work demonstrated that the Au-Si grooves pattern stimulates neurite polarity, elongation, and morphological differentiation of neuroblastoma cells without any exogenous supply of growth factors or stimulants in just 2 days, which can lead to selective procedure of obtaining homologous population of neuron-like cells for future nerve regeneration therapies.

Biomedical Applications of Graphene-Based Structures.

Graphene and graphene oxide (GO) structures and their reduced forms, e.g., GO paper and partially or fully reduced three-dimensional (3D) aerogels, are at the forefront of materials design for extensive biomedical applications that allow for the proliferation and differentiation/maturation of cells, drug delivery, and anticancer therapies. Various viability tests that have been conducted in vitro on human cells and in vivo on mice reveal very promising results, which make graphene-based materials suitable for real-life applications. In this review, we will give an overview of the latest studies that utilize graphene-based structures and their composites in biological applications and show how the biomimetic behavior of these materials can be a step forward in bridging the gap between nature and synthetically designed graphene-based nanomaterials.