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Octopamine is a well-established invertebrate neurotransmitter involved in fight or flight responses. In mammals, its function was replaced by epinephrine. Nevertheless, it is present at trace amounts and can modulate the release of monoamine neurotransmitters by a yet unidentified mechanism. Here, through a multidisciplinary approach utilizing in vitro and in vivo models of α-synucleinopathy, we uncovered an unprecedented role for octopamine in driving the conversion from toxic to neuroprotective astrocytes in the cerebral cortex by fostering aerobic glycolysis. Physiological levels of neuron-derived octopamine act on astrocytes via a trace amine-associated receptor 1–Orai1–Ca 2+ –calcineurin-mediated signaling pathway to stimulate lactate secretion. Lactate uptake in neurons via the monocarboxylase transporter 2–calcineurin-dependent pathway increases ATP and prevents neurodegeneration. Pathological increases of octopamine caused by α-synuclein halt lactate production in astrocytes and short-circuits the metabolic communication to neurons. Our work provides a unique function of octopamine as a modulator of astrocyte metabolism and subsequent neuroprotection with implications to α-synucleinopathies.

—The key aspects of the modification of biocompatible materials in a medium of supercritical carbon dioxide (SC–CO2) and the main trends in the application of the described method to the fabrication of three-dimensional scaffolds, sterilization of medical devices, decellularization of mammalian tissues, and impregnation of materials with biologically active molecules are described. Due to the nondestructive action of the medium of SC–CO2 on the architectonics of an extracellular matrix, the preservation of the mechanical properties and structure of the materials being treated, and an absence of the need for the postprocessing and purification of the formed matrices, the presented method can be considered as a versatile approach to the solution of urgent problems in biomedical materials science.

One of the leading trends in the modern tissue engineering is the development of new effective methods of decellularization aimed at the removal of cellular components from a donor tissue, reducing its immunogenicity and the risk of rejection. Supercritical CO2 (scCO2)-assisted processing has been proposed to improve the outcome of decellularization, reduce contamination and time costs. The resulting products can serve as personalized tools for tissue-engineering therapy of various somatic pathologies. However, the decellularization of heterogeneous 3D structures, such as the aortic root, requires optimization of the parameters, including preconditioning medium composition, the type of co-solvent, values of pressure and temperature inside the scCO2 reactor, etc. In our work, using an ovine aortic root model, we performed a comparative analysis of the effectiveness of decellularization approaches based on various combinations of these parameters. The protocols were based on the combinations of treatments in alkaline, ethanol or detergent solutions with scCO2-assisted processing at different modes. Histological analysis demonstrated favorable effects of the preconditioning in a detergent solution. Following processing in scCO2 medium provided a high decellularization degree, reduced cytotoxicity, and increased ultimate tensile strength and Young&rsquo, s modulus of the aortic valve leaflets, while the integrity of the extracellular matrix was preserved.

One of the leading trends in the modern tissue engineering is the development of new effective methods of decellularization aimed at the removal of cellular components from donor tissue reducing its immunogenicity and the risk of rejection. Supercritical CO2 (scCO2) extraction can significantly improve the outcome of decellularization, reduce contamination and time costs. The resulting products can serve as personalized tools for tissue-engineering therapy of various somatic pathologies. However, decellularization of complex 3D structures, such as the aortic root, requires optimization of the parameters, including preconditioning medium composition, type of co-solvent, values of pressure and temperature insight the scCO2 reactor, etc. In our work, using an ovine aortic root model, we performed a comparative analysis of the efficiency of decellularization using approaches based on various combinations of these parameters. The protocols were based on combinations of treatment in alkaline, ethanol or detergent solutions with scCO2 extraction at different modes. Based on a histological analysis, we have selected an optimal protocol for the decellularization of ovine aortic root employing preconditioning in a detergent solution. The positive effects of scCO2 on the decellularization extent, cytotoxicity and histoarchitecture of the tissue were demonstrated.

Neural activity depends on the maintenance of ionic and osmotic homeostasis. Under these conditions, the cell volume must be regulated to maintain optimal neural function. A disturbance in the neuronal volume regulation often occurs in pathological conditions such as glutamate excitotoxicity. The cell volume, mechanical properties, and actin cytoskeleton structure are tightly connected to achieve the cell homeostasis. Here, we studied the effects of glutamate-induced excitotoxicity, external osmotic pressure, and inhibition of actin polymerization on the viscoelastic properties and volume of neurons. Atomic force microscopy was used to map the viscoelastic properties of neurons in time-series experiments to observe the dynamical changes and a possible recovery. The data obtained on cultured rat cortical neurons were compared with the data obtained on rat fibroblasts. The neurons were found to be more responsive to the osmotic challenges but less sensitive to the inhibition of actin polymerization than fibroblasts. The alterations of the viscoelastic properties caused by glutamate excitotoxicity were similar to those induced by the hypoosmotic stress, but, in contrast to the latter, they did not recover after the glutamate removal. These data were consistent with the dynamic volume changes estimated using ratiometric fluorescent dyes. The recovery after the glutamate-induced excitotoxicity was slow or absent because of a steady increase in intracellular calcium and sodium concentrations. The viscoelastic parameters and their changes were related to such parameters as the actin cortex stiffness, tension, and cytoplasmic viscosity.

Biomeshes based on decellularized bovine pericardium (DBP) are widely used in reconstructive surgery due to their wide availability and the attractive biomechanical properties. However, their efficacy in clinical applications is often affected by the uncontrolled immunogenicity and proteolytic degradation. To address this issue, we present here in vivo multiparametric imaging analysis of epoxy crosslinked DBPs to reveal their fate after implantation. We first analyzed the structure of the crosslinked DBP using scanning electron microscopy and evaluated proteolytic stability and cytotoxicity. Next, using combination of fluorescence and hypoxia imaging, X-ray computed microtomography and histology techniques we studied the fate of DBPs after subcutaneous implantation in animals. Our approach revealed high resistance to biodegradation, gradual remodeling of a surrounding tissue forming the connective tissue capsule and calcification of crosslinked DBPs. These changes were concomitant to the development of hypoxia in the samples within 3 weeks after implantation and subsequent induction of angiogenesis and vascularization. Collectively, presented approach provides new insights on the transplantation of the epoxy crosslinked biomeshes, the risks associated with its applications in soft-tissue reconstruction and can be transferred to studies of other types of implants.

Recent years have witnessed the development of an enormous variety of hydrogel-based systems for neuroregeneration. Formed from hydrophilic polymers and comprised of up to 90% of water, these three-dimensional networks are promising tools for brain tissue regeneration. They can assist structural and functional restoration of damaged tissues by providing mechanical support and navigating cell fate. Hydrogels also show the potential for brain injury therapy due to their broadly tunable physical, chemical, and biological properties. Hydrogel polymers, which have been extensively implemented in recent brain injury repair studies, include hyaluronic acid, collagen type I, alginate, chitosan, methylcellulose, Matrigel, fibrin, gellan gum, self-assembling peptides and proteins, poly(ethylene glycol), methacrylates, and methacrylamides. When viewed as tools for neuroregeneration, hydrogels can be divided into: (1) hydrogels suitable for brain injury therapy, (2) hydrogels that do not meet basic therapeutic requirements and (3) promising hydrogels which meet the criteria for further investigations. Our analysis shows that fibrin, collagen I and self-assembling peptide-based hydrogels display very attractive properties for neuroregeneration., Graphical abstract Unlabelled Image

In this paper, terahertz (THz) pulsed spectroscopy and solid immersion microscopy were applied to study interactions between water vapor and tissue scaffolds–the decellularized bovine pericardium (DBP) collagen matrices, in intact form, cross-linked with the glutaraldehyde or treated by plasma. The water-absorbing properties of biomaterials are prognostic for future cell-mediated reactions of the recipient tissue with the scaffold. Complex dielectric permittivity of DBPs was measured in the 0.4–2.0 THz frequency range, while the samples were first dehydrated and then exposed to water vapor atmosphere with 80.0 ± 5.0% relative humidity. These THz dielectric measurements of DBPs and the results of their weighting allowed to estimate the adsorption time constants, an increase of tissue mass, as well as dispersion of these parameters. During the adsorption process, changes in the DBPs’ dielectric permittivity feature an exponential character, with the typical time constant of =8–10 min, the transient process saturation at =30 min, and the tissue mass improvement by =1–3%. No statistically-relevant differences between the measured properties of the intact and treated DBPs were observed. Then, contact angles of wettability were measured for the considered DBPs using a recumbent drop method, while the observed results showed that treatments of DBP somewhat affects their surface energies, polarity, and hydrophilicity. Thus, our studies revealed that glutaraldehyde and plasma treatment overall impact the DBP–water interactions, but the resultant effects appear to be quite complex and comparable to the natural variability of the tissue properties. Such a variability was attributed to the natural heterogeneity of tissues, which was confirmed by the THz microscopy data. Our findings are important for further optimization of the scaffolds’ preparation and treatment technologies. They pave the way for THz technology use as a non-invasive diagnosis tool in tissue engineering and regenerative medicine.

We report of surface modification approaches of nanoparticles with anti-Stokes luminescence, known as upconversion nanoparicles (UCNPs), comprised of inorganic host NaYF4 codoped with Yb3+ and Er3+or Tm3+. These approaches enabled the facile, lossless preparation of hybrid polymer-encapsulated UCNPs suitable for bioassays. These probes inherited UCNP properties, such as excellent photoluminescence under excitation with NIR light from the biotissue “transparency window”, as well as they were dispersible in aqueous media and physiological buffers, exhibiting chemical stability. The feasibility of the hybrid UCNPs was demonstrated for in vitro bioassay and in vivo optical whole animal imaging using a home-built epiluminescence imaging system.

The cytotoxicity and non-specific cellular uptake of the most popular composition of upconversion nanoparticle (UCNP), NaYF4:Yb3+:Er3+, is reported using normal human skin cells, including dermal fibroblasts and immortalized human epidermal linear keratinocytes (HaCaT). A new hydrophilization reaction of as-synthesized UCNPs based on tetramethylammonium hydroxide (TMAH) enabled evaluation of the intrinsic cytotoxicity of bare UCNPs. The cytotoxicity effects of the UCNP surface-coating and polystyrene host were investigated over the concentration range 62.5–125 μg/mL with 24-h incubation, using a MTT test and optical microscopy. The fibroblast viability was not compromised by UCNPs, whereas the viability of keratinocytes varied from 52% ± 4% to 100% ± 10% than the control group, depending on the surface modification. Bare UCNPs reduced the keratinocyte viability to 76% ± 3%, while exhibiting profound non-specific cellular uptake. Hydrophilic poly(D,L-lactide)- and poly(maleic anhydride-alt-1-octadecene)-coated UCNPs were found to be least cytotoxic among the polymer-coated UCNPs, and were readily internalized by human skin cells. Polystyrene microbeads impregnated with UCNPs remained nontoxic. Surprisingly, no correlation was found between UCNP cytotoxicity and the internalization level in cells, although the latter ranged broadly from 0.03% to 59%, benchmarked against 100% uptake level of TMAH-UCNPs.

Innovative luminescent nanomaterials, termed upconversion nanoparticles (UCNPs), have demonstrated considerable promise as molecular probes for high-contrast optical imaging in cells and small animals. The fea- sibility study of optical diagnostics in humans is reported here based on experimental and theoretical modeling of optical imaging of an UCNP-labeled breast cancer lesion. UCNPs synthesized in-house were surface-capped with an amphiphilic polymer to achieve good colloidal stability in aqueous buffer solutions. The scFv4D5 mini-anti- bodies were grafted onto the UCNPs via a high-affinity molecular linker barstar:barnase (Bs:Bn) to allow their spe- cific binding to the human epidermal growth factor receptor HER2/neu, which is overexpressed in human breast adenocarcinoma cells SK-BR-3. UCNP-Bs:Bn-scFv4D5 biocomplexes exhibited high-specific immobilization on the SK-BR-3 cells with the optical contrast as high as 10∶1 benchmarked against a negative control cell line. Breast cancer optical diagnostics was experimentally modeled by means of epi-luminescence imaging of a mono- layer of the UCNP-labeled SK-BR-3 cells buried under a breast tissue mimicking optical phantom. The experimental results were analyzed theoretically and projected to in vivo detection of early-stage breast cancer. The model pre- dicts that the UCNP-assisted cancer detection is feasible up to 4 mm in tissue depth, showing considerable potential for diagnostic and image-guided surgery applications. © The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI. (DOI: 10 .1117/1.JBO.18.7.076004)

The unique luminescent properties of new-generation synthetic nanomaterials, upconversion nanoparticles (UCNPs), enabled high-contrast optical biomedical imaging by suppressing the crowded background of biological tissue autofluorescence and evading high tissue absorption. This raised high expectations on the UCNP utilities for intracellular and deep tissue imaging, such as whole animal imaging. At the same time, the critical nonlinear dependence of the UCNP luminescence on the excitation intensity results in dramatic signal reduction at (similar to 1 cm) depth in biological tissue. Here, we report on the experimental and theoretical investigation of this trade-off aiming at the identification of optimal application niches of UCNPs e. g. biological liquids and subsurface tissue layers. As an example of such applications, we report on single UCNP imaging through a layer of hemolyzed blood. To extend this result towards in vivo applications, we quantified the optical properties of single UCNPs and theoretically analyzed the prospects of single-particle detectability in live scattering and absorbing bio-tissue using a human skin model. The model predicts that a single 70-nm UCNP would be detectable at skin depths up to 400 mm, unlike a hardly detectable single fluorescent (fluorescein) dye molecule. UCNP-assisted imaging in the ballistic regime thus allows for excellent applications niches, where high sensitivity is the key requirement

The development of targeted constructs on the basis of photoluminescent nanoparticles with a high photo- and chemical stability and absorption/emission spectra in the transparency window of biological tissues is an important focus area of present-day medical diagnostics. In this work, a targeted two-component construct on the basis of upconversion nanophosphors (UCNPs) and anti-tumor 4D5 scFv was developed for selective labeling of tumor cells overexpressing the HER2 tumor marker characteristic of a number of human malignant tumors. A high affinity barnase : barstar (Bn : Bs) protein pair, which exhibits high stability in a wide range of pH and temperatures, was exploited as a molecular adapter providing self-assembly of the two-component construct. High selectivity for the binding of the two-component 4D5 scFv-Bn : UCNP-Bs construct to human breast adenocarcinoma SK-BR-3 cells overexpressing HER2 was demonstrated. This approach provides an opportunity to produce similar constructs for the visualization of different specific markers in pathogenic tissues, including malignant tumors.