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In traditional tissue engineering, synthetic or natural scaffolds are usually used as removable temporal support, which involves some biotechnology limitations. The concept of "scaffield" approach utilizing the physical fields instead of biomaterial scaffold has been proposed recently. In particular, a combination of intense magnetic and acoustic fields can enable rapid levitational bioassembly of complex-shaped 3D tissue constructs from tissue spheroids at low concentration of paramagnetic agent (gadolinium salt) in the medium. In the current study, the tissue spheroids from human bladder smooth muscle cells (myospheres) are used as building blocks for assembling the tubular 3D constructs. Levitational assembly is accomplished at low concentrations of gadolinium salts in the high magnetic field at 9.5 T. The biofabricated smooth muscle constructs demonstrate contraction after the addition of vasoconstrictive agent endothelin-1. Thus, hybrid magnetoacoustic levitational bioassembly is considered as a new technology platform in the emerging field of formative biofabrication. This novel technology of scaffold-free, nozzle-free, and label-free bioassembly opens a unique opportunity for rapid biofabrication of 3D tissue and organ constructs with complex geometry.

We present a 3D study of nanostructural features of a bioprinted tissue spheroid interacting with polyurethane dual-scale biocompatible scaffold manufactured by three-dimensional printing and electrospinning. Three-dimensional analysis of fibroblasts interacting with electrospun polyurethane fibers was conducted using scanning probe nanotomography with an experimental setup combining ultramicrotome and a scanning probe microscope. Three-dimensional reconstruction demonstrates direct visualization of cell membrane protrusions and coherent cell-fiber interfaces, the formation of which is a prerequisite for an efficient tissue engineered implant. Analysis of obtained 3D data allows for quantitative calculation of the important morphological parameters of adhered cells, scaffolds, and cell-scaffold interfaces. The proposed method may be successfully applied to investigate 3D cell-scaffold constructs at nanoscale.

We have tested for iodine sorption properties a variety of samples with ZIF-8 structure and various Zn/Co ratios (100/0, 95/5, 75/25, 50/50 and 0/100 in mol%). For iodine saturation, an excess of sublimated iodine crystals was used. We have observed that the prevailing trend for all compositions is adsorption of most of the iodine molecules by the internal surface and retaining them up to framework collapse. However, Co-doping results in a small decrease in the thermal stability of the samples after iodine saturation. Moreover, in the samples with high cobalt content, the part of iodine adsorbed by the external surface is higher than in zinc analogs. Combination of TGA and FTIR data evaluated the impact of lower flexibility of Co-linker bonds on the transfer of iodine molecules from the external surface of the crystals to internal pores.

Cytoskeleton systems, actin microfilaments, microtubules (MTs) and intermediate filaments (IFs) provide the biomechanical stability and spatial organization in cells. To understand the specific contributions of each cytoskeleton systems to intrinsic properties of spheroids, we've scrutinized the effects of the cytoskeleton perturbants, cytochalasin D (Cyto D), nocodazole (Noc) and withaferin A (WFA) on fusion, spreading on adhesive surface, morphology and biomechanics of chondrospheres (CSs). We confirmed that treatment with Cyto D but not with Noc or WFA severely affected CSs fusion and spreading dynamics and significantly reduced biomechanical properties of cell aggregates. Noc treatment affected spheroids spreading but not the fusion and surprisingly enhanced their stiffness. Vimentin intermediate filaments (VIFs) reorganization affected CSs spreading only. The analysis of all three cytoskeleton systems contribution to spheroids intrinsic properties was performed for the first time.

The review focuses on techniques for scalable standardized tissue spheroids biofabrication, on tissue spheroids biological properties and the methods for its morphology evaluation. The comparative analysis of existing approaches provided here will guide to the optimal protocol for tissue spheroids fabrication and characterization. Key words: tissue spheroids, biofabrication, bioprinting, drug discovery, morphology evaluation

Collagen is one of the most promising materials for 3D bioprinting because of its distinguished biocompatibility. Cell-laden constructs made of pure collagen with or without incorporated growth supplements support engineered constructs persistence in culture and are perfectly suitable for grafting. The limiting factor for direct 3D collagen printing was poor printability of collagen solutions, especially admixed with cells or tissue spheroids. In our study, we showed that concentrated solutions of native collagen branded Viscoll were effective as bioinks with high fidelity performance. Viscoll containing 20, 30, or 40 mg/ml collagen were used for direct extrusion 3D bioprinting to form scaffolds appropriate to support spatial arrangement of tissue spheroids into rigid patterns with resolution of 0.5 mm in details. Incorporated cells demonstrated sufficient viability. Associated rheological study showed that good printability of the collagen solutions correlates with their increased storage modulus value, notably exceeding the loss modulus value. The proper combination of these physical parameters could become technological criteria for manufacturing various collagen bioinks for 3D bioprinting.

This paper presents a study of nanostructural features of contacts of bioprinted tissue spheroids with polyurethane dual scale biocompatible scaffold made by three-dimensional printing and electrospinning. Analysis of nanostructural features of cell contacts was carryed out by scanning probe microscopy with use of experimental setup combining ultramicrotome and scanning probe microscope. Measured mean cell volume is 460 ± 104 μm3, mean contact area of cells with scaffold fibers–104.8 μm2 per cell (16.7% of total cell area). Maximum distance of migrating cells from spheroid border at 48 h. is ~200 μm, what corresponds to mean velocity of cell migration more than 4 μm/h. Obtained quantitative characteristics of micro- and nanostructure of human fibroblast cell contacts with elecrospun polyurethane scaffold secure high efficacy of tissue regeneration with its usage for implanted bioprinted dual scale tissue-engineered scaffolds.

Tissue spheroids formed from fibroblasts using a micromolded non-adhesive hydrogel were located using a three-dimensional (3D) bioprinter on the surface of a nanofibrous polyurethane matrix produced by electrospinning. It was shown that the tissue spheroids attach to the matrix surface within a few hours and completely flatten after several days, indicating high biocompatibility of the matrix used. Tissue structures formed by the attachment and spreading of tissue spheroids on an electrospun matrix are a new technological platform for biofabrication and 3D bioprinting of tissues and organs.

A patient-specific auricular prosthesis made of biocompatible non-biodegradable polyurethane with biomimetic mechanical properties was developed and printed using a 3D printer. A three-point bending study of the mechanical properties of printed samples of this material showed that the printed prosthesis is similar in its material properties to natural human aural cartilage. After subcutaneous implantation into mice the auricular prosthesis maintained its initial shape and size. Thus, 3D printing enables creation of a patient-specific biocompatible auricular prosthesis with biomimetic material properties and the ability to maintain the original shape and size after in vivo implantation.

This study investigates the impact of iodine, chlorine and bromine adsorption on the structure of bimetallic Co/Zn zeolitic imidazolate frameworks (ZIFs). The experimental Co and Zn K-edges XANES (X-ray Absorption Near Edge Structure) spectra of Zn0.5Co0.5C8H10N4 synthesized by microwave synthesis were measured before and after halogens treatment.

Objective Chondrospheres represent a variant of tissue spheroids biofabricated from chondrocytes. They are already being used in clinical trials for cartilage repair; however, their biomechanical properties have not been systematically investigated yet. The aim of our study was to characterize chondrospheres in long-term in vitro culture conditions for morphometric changes, biomechanical integrity, and their fusion and spreading kinetics. Results It has been demonstrated that the increase in chondrospheres secant modulus of elasticity is strongly associated with the synthesis and accumulation of extracellular matrix. Additionally, significant interplay has been found between biomechanical properties of tissue spheroids and their fusion kinetics in contrast to their spreading kinetics. Conclusions Extracellular matrix is one of the main structural determinants of chondrospheres biomechanical properties during chondrogenic maturation in vitro. The estimation of tissue spheroids’ physical behavior in vitro prior to operative treatment can be used to predict and potentially control fusogenic self-assembly process after implantation in vivo.

This paper presents a study of three-dimensional micro- and nanosctucture of polyurethane dual scale biocompatible scaffold made by three-dimensional printing and electrospinning. The three-dimensional structure of the scaffold was analyzed by scanning probe nanotomography with use of an experimental setup combining an ultramicrotome and a scanning probe microscope. We performed a quantitative analysis of microporosity, nanoroughness, and three-dimensional morphology parameters of the scaffold. The electrospun scaffold consists of a network of microfibers with diameter ranging from 1.7 to 6.0 μm. The measured mean microfiber diameter is 3.54 ± 1.23 μm. The volume porosity of the electrospun scaffold is 72.5%, while mean surface area to volume ration is 0.28 μm–1 and mean nanoroughness of microfiber surface is 22.1 ± 3.0 nm. The quantitative characteristics of the micro- and nanostructure of elecrospun polyurethane matrices secure the high efficacy of its usage for increasing the biocompatibility of dual-scale hybrid bioengineered scaffolds for regenerative medicine tasks. The use of scanning probe nanotomography for analyzing threedimensional morphology characteristics and the topology of electrospun microfiber systems enables us to improve the efficiency of development of new bioengineered products.

A focused library of heterocyclic compounds including a 2-aminomethyl-1H-benzimidazole (1 – 19), 2-aminomethylindole (20 – 83), benzofuran-2-ylmethylamine (84 – 92), or 2-piperazin-1-ylmethylbenzoxazole (93) fragment was screened for the ability to inhibit in vitro hepatitis C virus (HCV). The synthetic methods were described. The antiviral activity and cytotoxicity data were presented. Most of the compounds carrying a benzoxazol-2-ylmethylamine fragment inhibited Huh7.3 human hepatoma cells infected in vitro with HCV with nanomolar potency but were inactive against the HCV RNA-replicon. The only exception was 9-methyl-N(6)-(3-nitrophenyl)-2,3,4,9-tetrahydro-1H-carbazole-1,6-diamine (67), which demonstrated nanomolar potency against HCV in both models. The most active and selective compounds were (piperazin-1-yl)-[(1Hindol-2-ylmethyl)piperidin-4-yl]-ketones (EC50 0.31 – 2.2 μM, CC50 10.2-110 μM) and 2-(1,2,3a,4,5,6-hexahydropyrazino[3,2,1-jk]carbazol-3-yl)acetamide (EC50 1.69 ± 0.5 μM, CC50 114 ± 42 μM). The two most selective inhibitors (28, TI50 = 52 and 77, TI50 = 68) were selected for further preclinical trials.

Novel substituted 5-hydroxy-2-aminomethyl-1H-indole-3-carboxylic acids and their derivatives were synthesized. The antiviral properties of these compounds were investigated in relation to bovine viral diarrhea virus (BVDV), hepatitis C virus (HCV), and influenza A/Aichi/2/69 (H3N2) virus. Of the compounds synthesized here, only the 5-hydroxy-2-(dimethylaminomethyl)-1-methyl-6-pyridin-3-yl- and 5-hydroxy-2-(dimethylaminomethyl)-1-methyl-6-fluoro-1H-indole-3-carboxylic acid ethyl ester hydrochlorides had significant activity against these viruses, these agents not only suppressing the replication of influenza A/Aichi/2/69 (H3N2) virus in cell cultures at micromolar concentrations, but also demonstrating high efficacy, greater than that of Arbidol, in a model of influenza pneumonia in mice infected with influenza A/Aichi/2/69 (H3N2) virus, when given at a dose of 25 mg/kg/day.

The bimetallic Zn/Co-ZIF MOFs with different Zn:Co ratio were produced by microwave-assisted solvothermal synthesis. The morphological, structural and electronic properties of the obtained materials were probed with different experimental techniques (XRD, TEM, TGA, BET, UV−Vis, IR, Co and Zn K-edge XANES). The result of the overall characterization study evidences that: (i) for all Zn:Co ratio Zn/Co-ZIFs are isostructural to ZIF-8; (ii) Zn and Co occupy the same crystallographic site having the same local environment; (iii) increasing Co content results in the decrease of solvent and iodine amount adsorbed on the surface of the crystals, while the amount adsorbed inside the pores is almost constant. The Zn/Co-ZIF MOFs were tested for iodine and chlorine sorption.

Bioprinting can be defined as additive biofabrication of three-dimensional (3D) tissues and organ constructs using tissue spheroids, capable of self-assembly, as building blocks. The thyroid gland, a relatively simple endocrine organ, is suitable for testing the proposed bioprinting technology. Here we report the bioprinting of a functional vascularized mouse thyroid gland construct from embryonic tissue spheroids as a proof of concept. Based on the self-assembly principle, we generated thyroid tissue starting from thyroid spheroids (TS) and allantoic spheroids (AS) as a source of thyrocytes and endothelial cells (EC), respectively. Inspired by mathematical modeling of spheroid fusion, we used an original 3D bioprinter to print TS in close association with AS within a collagen hydrogel. During the culture, closely placed embryonic tissue spheroids fused into a single integral construct, EC from AS invaded and vascularized TS, and epithelial cells from the TS progressively formed follicles. In this experimental setting, we observed formation of a capillary network around follicular cells, as observed during in utero thyroid development when thyroid epithelium controls the recruitment, invasion and expansion of EC around follicles. To prove that EC from AS are responsible for vascularization of the thyroid gland construct, we depleted endogenous EC from TS before bioprinting. EC from AS completely revascularized depleted thyroid tissue. The cultured bioprinted construct was functional as it could normalize blood thyroxine levels and body temperature after grafting under the kidney capsule of hypothyroid mice. Bioprinting of functional vascularized mouse thyroid gland construct represents a further advance in bioprinting technology, exploring the self-assembling properties of tissue spheroids.

Reproducible, scalable, and cost effective fabrication and versatile characterization of tissue spheroids (TS) is highly demanded by 3D bioprinting and drug discovery. Consistent geometry, defined mechanical properties, optimal viability, appropriate extracellular matrix/cell organization are required for cell aggregates aimed for application in these fields. A straightforward procedure for fabrication and systematic multiparametric characterization of TS with defined properties and uniform predictable geometry employing non-adhesive technology is suggested. Applying immortalized and primary cells, the reproducibility of spheroid generation, the strong correlation of ultimate spheroid diameter, and growth pattern with cell type and initial seeding concentration are demonstrated. Spheroids viability and mechanical properties are governed by cell derivation. In this study, a new decision procedure to apply for any cell type one starts to work with to prepare and typify TS meeting high quality standards in biofabrication and drug discovery is suggested.

New substituted ethyl esters of 2,4-bis-aminomethyl-5-hydroxy-1H-indole-3-carboxylic acids, 8-aminomethyl-2-methyl-2,3-dihydro-1H,7H-[1,3]-oxazino-[5,6-e]indole-9-carboxylic acids, and members of the previously unknown 4,5-dihydro-1H-pyrrolo[4,3,2-de]isoquinolin-3-ones of 1,4-dihydropyrrolo[4,3,2-de]isoquinolin-3,6-diones were synthesized. Their cytotoxicities and antiviral activities against bovine viral diarrhea virus (BVDV), hepatitis C virus (HCV), and A/Aichi/2/69 (H3N2) were studied in vitro. These compounds were found not to be active against these viruses. The only exceptions were the hydrochlorides of the ethyl esters of 5-hydroxy-2-(dimethylaminomethyl)-1-methyl-6-pyridine-3-yl- and 5-hydroxy-2-(dimethylaminomethyl)-1-methyl-6-fluoro-1H-indole-3-carboxylic acids, which at micromolar concentrations not only produced effective suppression of influenza A/Aichi/2/69 (H3N2) virus replication in cell cultures, but also showed high in vivo efficacy in a model of influenza pneumonia in mice infected with influenza A/Aichi/2/69 (H3N2) virus at a dose of 25 mg/kg/day.

New substituted ethyl 5-hydroxy-1,2-dimethyl-1H-indole-3-carboxylates and 7,8-dimethyl-1,2,3,7-tetrahydro[1,3]oxazino[5,6-e]indole-9-carboxylates including arbidol analogs in addition to 6-hydroxy-1-methyl-7-pyridin-3-yl-4,5-dihydropyrrolo[4,3,2-de]isoquinolin-3(1H)-ones and 1,4-dimethyl-7-pyridin-3-yl-2-(phenylsulfonylmethyl)-1,4-dihydropyrrolo[4,3,2-de]isoquinoline-3,6-dione were synthesized. Their antiviral activity against influenza A/New Caledonia/20/99 virus (H1N1), bovine viral diarrhea virus (BVDV), and hepatitis C virus (HCV) was studied. It was found that the synthesized compounds were not noticeably active against these viruses. The exceptions were only ethyl 5-hydroxy-4-(dimethylaminomethyl)-1-methyl-6-pyridin-3-yl-2-(phenylsulfinylmethyl)-1H-indole-3-carboxylate and 5-hydroxy-1,2-dimethyl-6-fluoro-1H-indole-3-carboxylate hydrochlorides, which exhibited micromolar activities EC50 = 6.6 and 9.8 iM, respectively, against a human hepatoma cell line (Huh7.3) with increased sensitivity to HCV infection (strain JFH-1, genotype 2a).

In this paper we present, for the first time, a simple hydrothermal recipe for the synthesis of ZIF-8 Metal-Organic Framework (MOF) with a large specific surface area (1340 m 2 /g by BET). An important feature of the method is that the product forms in aqueous medium under standard hydrothermal conditions without DMF and great excess of linker with the use of TEA as structure directing agent. The ZIF-8 crystal phase of the product was confirmed by XRD; this technique has been also exploited to check the crystallinity and to follow the changes in the MOF structure induced by heating. TGA and temperature dependent XRD testify the high thermal stability of the material (470 °C in N 2 and at 400 °C in air). The IR spectral profile of the material provides a complete picture of vibrations assigned to the linker and the metal center. The systematic investigation of the products obtained by increasing the TEA amount in the reacting medium from 0 to 25.5 mol equivalent Zn 2+ , allowed us to understand its role and to find 2.6 mol equivalent Zn 2+ as the minimum amount needed to obtain a single phase ZIF-8 material with the high standard reported above. The stability of the material under severe basic conditions makes it a promising candidate in heterogeneous catalysis. The material has shown high capacity in I 2 uptake, making it interesting also for selective molecular adsorption.

The local atomic structure in bimetallic Co/Zn zeolitic imidazolate frameworks (ZIFs) was studied using X-ray Absorption Fine Structure (XAFS) spectroscopy and theoretical calculations. The experimental Co K-edge and Zn K-edge XANES (X-ray Absorption Near Edge Structure) spectra of Zn1-xCoxC8H10N4 samples (x = 0.05, 0.25, 0.75) synthesized by microwave synthesis were compared with the data for the ZIF-67 (x=1) and ZIF-8 (x=0). Theoretical XANES spectra for the bimetallic ZIFs were calculated. It was shown that in bimetallic ZIFs the Co and Zn atoms have the similar local environment.

The synthesis, molecular docking, and biological testing of a series of new heteroaryl-substituted oxadiazole-5-carboxamide inhibitors of glycogen synthase kinase 3β (GSK-3β) are described. The synthesis includes several stages and is based on the advanced liquid-phase combinatorial approach. Molecular docking was used for the rational selection of synthesized compounds for the subsequent biological testing. It is established that the inhibitory activity of the synthesized compounds strongly depends on the character of substituents in the phenyl ring and the nature of terminal heterocyclic fragments. The most active compounds inhibit GSK-3β at IC50 in the micromolar range and can be considered as potential drug candidates.

Synthesis, biological evaluation, and SAR dependencies for a series of novel aryl and heteroaryl substituted N-[3-(4-phenylpiperazin-1-yl)propyl]-1,2,4-oxadiazole-5-carboxamide inhibitors of GSK-3beta kinase are described. The inhibitory activity of the synthesized compounds is highly dependent on the character of substituents in the phenyl ring and the nature of terminal heterocyclic fragment of the core molecular scaffold. The most potent compounds from this series contain 3,4-di-methyl or 2-methoxy substituents within the phenyl ring and 3-pyridine fragment connected to the 1,2,4-oxadiazole heterocycle. These compounds selectively inhibit GSK-3beta kinase with IC(50) value of 0.35 and 0.41 microM, respectively.

Organ printing is a computer-aided additive biofabrication of functional three-dimensional human tissue and organ constructs according to digital model using the tissue spheroids as building blocks. The fundamental biological principle of organ printing technology is a phenomenon of tissue fusion. Closely placed tissue spheroids undergo tissue fusion driven by surface tension forces. In order to ensure tissue fusion in the course of post-printing, tissue spheroids must be placed and maintained close to each other. We report here that tissue spheroids biofabricated from primary human fibroblasts could be placed and maintained on the surface of biocompatible electrospun polyurethane matrix using 3D bioprinter according to desirable pattern. The patterned tissue spheroids attach to polyurethane matrix during several hours and became completely spread during several days. Tissue constructions biofabricated by spreading of patterned tissue spheroids on the biocompatible electrospun polyurethane matrix is a novel technological platform for 3D bioprinting of human tissue and organs.

Recent studies suggest that progesterone may possess anti-tumorigenic properties. However, a growth-modulatory role of progestins in human cancer cells remains obscure. With the discovery of a new class of membrane progesterone receptors (mPRs) belonging to the progestin and adipoQ receptor gene family, it becomes important to study the effect of this hormone on proliferation of tumor cells that do not express classical nuclear progesterone receptors (nPRs). To identify a cell line expressing high levels of mPRs and lacking nPRs, we examined mRNA levels of nPRs and three forms of mPRs in sixteen human tumor cell lines of different origin. High expression of mPR mRNA has been found in pancreatic adenocarcinoma BxPC3 cells, while nPR mRNA has not been detected in these cells. Western blot analysis confirmed these findings at the protein level. We revealed specific binding of labeled progesterone in these cells with affinity constant similar to that of human mPR expressed in yeast cells. Progesterone at high concentration of 20 μM significantly reduced the mRNA levels of proliferation markers Ki67 and PCNA, as well as of cyclin D1, and increased the mRNA levels of cyclin dependent kinase inhibitors p21 and p27. Progesterone (1 μM and 20 μM) significantly inhibited proliferative activity of BxPC3 cells. These results point to anti-proliferative effects of the progesterone high concentrations on BxPC3 cells and suggest that activation of mPRs may mediate this action. Our data are a starting point for further investigations regarding the application of progesterone in pancreatic cancer.

A series of next in class small-molecule hepatitis C virus (HCV) NS5A inhibitors with picomolar potency containing 2-pyrrolidin-2-yl-5-{4-[4-(2-pyrrolidin-2-yl-1H-imidazol-5-yl)buta-1,3-diynyl]phenyl}-1H-imidazole cores was designed based on the SAR studies available for the reported NS5A inhibitors. Compound 13a (AV4025), with (S,S,S,S)-stereochemistry (EC50 = 3.4 ± 0.2 pM, HCV replicon genotype 1b), was dramatically more active than were the compounds with two (S)- and two (R)-chiral centers. Human serum did not significantly reduce the antiviral activity (4-fold). Relatively favorable pharmacokinetic features and good oral bioavailability were observed during animal studies. Compound 13a was well tolerated in rodents (in mice, LD50 = 2326 mg/kg or higher), providing a relatively high therapeutic index. During safety, pharmacology and subchronic toxicity studies in rats and dogs, it was not associated with any significant pathological or clinical findings. This compound is currently being evaluated in phase I/II clinical trials for the treatment of HCV infection.

A new fast (15 min) microwave-assisted solvothermal synthesis of ZIF-8 with a high specific surface area (1419 m 2 g– 1 ) is proposed, and the molar ratio between zinc salt and linker is optimized.

Afilopodium protrudes by elongation of bundled actin filaments in its core. However, the mechanism of filopodia initiation remains unknown. Using live-cell imaging with GFP-tagged proteins and correlative electron microscopy, we performed a kinetic-structural analysis of filopodial initiation in B16F1 melanoma cells. Filopodial bundles arose not by a specific nucleation event, but by reorganization of the lamellipodial dendritic network analogous to fusion of established filopodia but occurring at the level of individual filaments. Subsets of independently nucleated lamellipodial filaments elongated and gradually associated with each other at their barbed ends, leading to formation of cone-shaped structures that we term Λ-precursors. An early marker of initiation was the gradual coalescence of GFP-vasodilator-stimulated phosphoprotein (GFP-VASP) fluorescence at the leading edge into discrete foci. The GFP-VASP foci were associated with Λ-precursors, whereas Arp2/3 was not. Subsequent recruitment of fascin to the clustered barbed ends of Λ-precursors initiated filament bundling and completed formation of the nascent filopodium. We propose a convergent elongation model of filopodia initiation, stipulating that filaments within the lamellipodial dendritic network acquire privileged status by binding a set of molecules (including VASP) to their barbed ends, which protect them from capping and mediate association of barbed ends with each other.