Reversed Corneal Fibroblasts Therapy Restores Transparency of Mouse Cornea after Injury.

Cell-based therapies using corneal stromal stem cells (CSSC), corneal keratocytes, or a combination of both suppress corneal scarring. The number of quiescent keratocytes in the cornea is small; it is difficult to expand them in vitro in quantities suitable for transplantation. This study examined the therapeutic effect of corneal fibroblasts reversed into keratocytes (rCF) in a mouse model of mechanical corneal injury. The therapeutic effect of rCF was studied in vivo (slit lamp, optical coherence tomography) and ex vivo (transmission electron microscopy and immunofluorescence staining). Injection of rCF into the injured cornea was accompanied by recovery of corneal thickness, improvement of corneal transparency, reduction of type III collagen in the stroma, absence of myofibroblasts, and the improvement in the structural organization of collagen fibers. TEM results showed that 2 months after intrastromal injection of cells, there was a decrease in the fibril density and an increase in the fibril diameter and the average distance between collagen fibrils. The fibrils were well ordered and maintained the short-range order and the number of nearest-neighbor fibrils, although the averaged distance between them increased. Our results demonstrated that the cell therapy of rCF from ReLEx SMILe lenticules promotes the recovery of transparent corneal stroma after injury.

Which Gelatin and Antibiotic Should Be Chosen to Seal a Woven Vascular Graft?

Among the vascular prostheses used for aortic replacement, 95% are woven or knitted grafts from polyester fibers. Such grafts require sealing, for which gelatin (Gel) is most often used. Sometimes antibiotics are added to the sealant. We used gelatin type A (GelA) or type B (GelB), containing one of the three antibiotics (Rifampicin, Ceftriaxone, or Vancomycin) in the sealant films. Our goal was to study the effect of these combinations on the mechanical and antibacterial properties and the cytocompatibility of the grafts. The mechanical characteristics were evaluated using water permeability and kinking radius. Antibacterial properties were studied using the disk diffusion method. Cytocompatibility with EA.hy926 endothelial cells was assessed via indirect cytotoxicity, cell adhesion, and viability upon direct contact with the samples (3, 7, and 14 days). Scanning electron microscopy (SEM) and energy dispersive spectrometry (EDS) were used to visualize the cells in the deep layers of the graft wall. "GelA + Vancomycin" and "GelB + vancomycin" grafts showed similar good mechanical characteristics (permeability~10 mL/min/cm2, kinking radius 21 mm) and antibacterial properties (inhibition zones for Staphilococcus aureus~15 mm, for Enterococcus faecalis~12 mm). The other samples did not exhibit any antibacterial properties. The cytocompatibility was good in all the tested groups, but endothelial cells exhibited the ability to self-organize capillary-like structures only when interacting with the "GelB + antibiotics" coatings. Based on the results obtained, we consider "GelB + vancomycin" sealant to be the most promising.

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.

Endothelial Cell Behavior and Nitric Oxide Production on a-C:H:SiOx-Coated Ti-6Al-4V Substrate.

This paper focuses on the surface modification of the Ti-6Al-4V alloy substrate via a-C:H:SiOx coating deposition. Research results concern the a-C:H:SiOx coating structure, investigated using transmission electron microscopy and in vitro endothelization to study the coating. Based on the analysis of the atomic radial distribution function, a model is proposed for the atomic short-range order structure of the a-C:H:SiOx coating, and chemical bonds (C-O, C-C, Si-C, Si-O, and Si-Si) are identified. It is shown that the a-C:H:SiOx coating does not possess prolonged cytotoxicity in relation to EA.hy926 endothelial cells. In vitro investigations showed that the adhesion, cell number, and nitric oxide production by EA.hy926 endothelial cells on the a-C:H:SiOx-coated Ti-6Al-4V substrate are significantly lower than those on the uncoated surface. The findings suggest that the a-C:H:SiOx coating can reduce the risk of endothelial cell hyperproliferation on implants and medical devices, including mechanical prosthetic heart valves, endovascular stents, and mechanical circulatory support devices.

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.