Derivation of Human Corneal Keratocytes from ReLEx SMILE Lenticules for Cell Therapy and Tissue Engineering.

Fibroblasts isolated and expanded from ReLEx SMILE lenticules can be a source of human keratocytes. Since corneal keratocytes are quiescent cells, it is difficult to expand them in vitro in suitable numbers for clinical and experimental use. In the present study, this problem was solved by isolating and growing corneal fibroblasts (CFs) with a high proliferative potential and their reversion to keratocytes in a selective serum-free medium. Fibroblasts reversed into keratocytes (rCFs) had a dendritic morphology and ultrastructural signs of activation of protein synthesis and metabolism. The cultivation of CFs in a medium with 10% FCS and their reversion into keratocytes was not accompanied by the induction of myofibroblasts. After reversion, the cells spontaneously formed spheroids and expressed keratocan and lumican markers, but not mesenchymal ones. The rCFs had low proliferative and migratory activity, and their conditioned medium contained a low level of VEGF. CF reversion was not accompanied by a change with the levels of IGF-1, TNF-alpha, SDF-1a, and sICAM-1. In the present study, it has been demonstrated that fibroblasts from ReLEx SMILE lenticules reverse into keratocytes in serum-free KGM, maintaining the morphology and functional properties of primary keratocytes. These keratocytes have a potential for tissue engineering and cell therapy of various corneal pathologies.

Effect of the Nanorough Surface of TiO2 Thin Films on the Compatibility with Endothelial Cells.

The cytocompatibility of titanium oxides (TiO2) and oxynitrides (N-TiO2, TiOxNy) thin films depends heavily on the surface topography. Considering that the initial relief of the substrate and the coating are summed up in the final topography of the surface, it can be expected that the same sputtering modes result in different surface topography if the substrate differs. Here, we investigated the problem by examining 16 groups of samples differing in surface topography; 8 of them were hand-abraded and 8 were machine-polished. Magnetron sputtering was performed in a reaction gas medium with various N2:O2 ratios and bias voltages. Abraded and polished uncoated samples served as controls. The surfaces were studied using atomic force microscopy (AFM). The cytocompatibility of coatings was evaluated in terms of cytotoxicity, adhesion, viability, and NO production. It has been shown that the cytocompatibility of thin films largely depends on the surface nanostructure. Both excessively low and excessively high density of peaks, high and low kurtosis of height distribution (Sku), and low rates of mean summit curvature (Ssc) have a negative effect. Optimal cytocompatibility was demonstrated by abraded surface with a TiOxNy thin film sputtered at N2:O2 = 1:1 and Ub = 0 V. The nanopeaks of this surface had a maximum height, a density of about 0.5 per 1 µm2, Sku from 4 to 5, and an Ssc greater than 0.6. We believe that the excessive sharpness of surface nanostructures formed during magnetron sputtering of TiO2 and N-TiO2 films, especially at a high density of these structures, prevents both adhesion of endothelial cells, and their further proliferation and functioning. This effect is apparently due to damage to the cell membrane. At low height, kurtosis, and peak density, the main factor affecting the cell/surface interface is inefficient cell adhesion.

Integral Algorithms to Evaluate TiO2 and N-TiO2 Thin Films' Cytocompatibility.

Titanium oxide (TiO2) and oxynitride (N-TiO2) coatings can increase nitinol stents' cytocompatibility with endothelial cells. Methods of TiO2 and N-TiO2 sputtering and cytocompatibility assessments vary significantly among different research groups, making it difficult to compare results. The aim of this work was to develop an integral cytocompatibility index (ICI) and a decision tree algorithm (DTA) using the "EA.hy926 cell/TiO2 or N-TiO2 coating" model and to determine the optimal cytocompatible coating. Magnetron sputtering was performed in a reaction gas medium with various N2:O2 ratios and bias voltages. The samples' morphology was studied by scanning electron microscopy (SEM) and Raman spectroscopy. The cytocompatibility of the coatings was evaluated in terms of their cytotoxicity, adhesion, viability, and NO production. The ICI and DTA were developed to assess the cytocompatibility of the samples. Both algorithms demonstrated the best cytocompatibility for the sample sputtered at Ubias = 0 V and a gas ratio of N2:O2 = 2:1, in which the rutile phase dominated. The DTA provided more detailed information about the cytocompatibility, which depended on the sputtering mode, surface morphology, and crystalline phase. The proposed mathematical models relate the cytocompatibility and the studied physical characteristics.

New data on the fine structure of Deuteramoeba mycophaga CCAP 1586/1 (Amoebozoa, Tubulinea).

The genus Deuteramoeba is one of the six amoebae genera belonging to the best-known amoeba family - Amoebidae (Amoebozoa, Tubulinea), containing such a popular species as Amoeba proteus. However, members of other genera of the family Amoebidae are much less known, and most of the studies of their morphology and ultrastructure date back to the 1970s and 1980s. Since these "classical" species are believed to be "well studied", their morphology and fine structure rarely become a subject of re-investigation. The absence of modern morphological data may be critical when molecular data of the type strain are not available, and the only way to identify a species is by morphological comparison. For this paper, we performed an ultrastructural study of the strain CCAP 1586/1 - the type strain of the species Deuteramoeba mycophaga. Our study revealed new details of the nuclear structure, including a peripheral layer of filaments and a heterogeneous nucleolus, and provided new data on the cytoplasmic inclusions of this species. We performed a whole-genome amplification of the DNA from a single amoeba cell followed by NGS sequencing and searched for genetic evidence for the presence of a putative nuclear parasite detected in 2017, but found no evidence for the presence of Opisthosporidia.

Conformational Analysis of N-Alkyl-N-[2-(diphenylphosphoryl)ethyl]amides of Diphenylphosphorylacetic Acid: Dipole Moments, IR Spectroscopy, DFT Study.

Experimental and theoretical conformational analysis of N-methyl-N-[2-(diphenylphosphoryl)ethyl]diphenylphosphorylacetamide, N-butyl-N-[2-(diphenylphosphoryl)ethyl]diphenylphosphorylacetamide, and N-octyl-N-[2-(diphenylphosphoryl)ethyl]diphenylphosphorylacetamide was carried out by the methods of dipole moments, IR spectroscopy, and Density Functional Theory (DFT) B3PW91/6-311++G(df,p) calculations. In solution, these N,N-dialkyl substituted bisphosphorylated acetamides exist as a conformational equilibrium of several forms divided into two groups-with Z- or E-configuration of the carbonyl group and alkyl substituent, and syn or anti arrangement of the phosphoryl-containing fragments relative to the amide plane. The substituents at the phosphorus atoms have eclipsed cis- or staggered gauche-orientation relative to the P=O groups, and cis orientation of the substituents is due to the presence of intramolecular H-contacts P=O...H-Cphenyl or p,π conjugation between the phosphoryl group and the phenyl ring. Preferred conformers of acetamides molecules are additionally stabilized by various intramolecular hydrogen contacts with the participation of oxygen atoms of the P=O or C=O groups and hydrogen atoms of the methylene and ethylene bridges, alkyl substituents, and phenyl rings. However, steric factors, such as a flat amide fragment, the bulky phenyl groups, and the configuration of alkyl bridges, make a significant contribution to the realization of preferred conformers.