Electrospun Methacrylated Gelatin/Poly(L-Lactic Acid) Nanofibrous Hydrogel Scaffolds for Potential Wound Dressing Application

Electrospun nanofiber mats have attracted intense attention as advanced wound dressing materials. The objective of this study was to fabricate methacrylated gelatin (MeGel)/poly(L-lactic acid) (PLLA) hybrid nanofiber mats with an extracellular matrix (ECM) mimicking nanofibrous structure and hydrogel-like properties for potential use as wound dressing materials. MeGel was first synthesized via the methacryloyl substitution of gelatin (Gel), a series of MeGel and PLLA blends with various mass ratios were electrospun into nanofiber mats, and a UV crosslinking process was subsequently utilized to stabilize the MeGel components in the nanofibers. All the as-crosslinked nanofiber mats exhibited smooth and bead-free fiber morphologies. The MeGel-containing and crosslinked nanofiber mats presented significantly improved hydrophilic properties (water contact angle = 0°; 100% wettability) compared to the pure PLLA nanofiber mats (~127°). The swelling ratio of crosslinked nanofiber mats notably increased with the increase of MeGel (143.6 ± 7.4% for PLLA mats vs. 875.0 ± 17.1% for crosslinked 1:1 MeGel/PLLA mats vs. 1135.2 ± 16.0% for crosslinked MeGel mats). The UV crosslinking process was demonstrated to significantly improve the structural stability and mechanical properties of MeGel/PLLA nanofiber mats. The Young’s modulus and ultimate strength of the crosslinked nanofiber mats were demonstrated to obviously decrease when more MeGel was introduced in both dry and wet conditions. The biological tests showed that all the crosslinked nanofiber mats presented great biocompatibility, but the crosslinked nanofiber mats with more MeGel were able to notably promote the attachment, growth, and proliferation of human dermal fibroblasts. Overall, this study demonstrates that our MeGel/PLLA blend nanofiber mats are attractive candidates for wound dressing material research and application.

Molecular and cellular mechanisms of ultraviolet corneal crosslinking

In the presented mainly experimental work, the regularities and mechanisms of ultrastructural transformation and changes in corneal metabolism under ultraviolet exposure are established. High-quality saturation of the stroma with riboflavin is necessary to perform safe and effective UV crosslinking of the cornea, which can be achieved mainly due to a sufficient area of de-epithelization (at least 8-9 mm). It was found that UV crosslinking of the cornea with the use of developed riboflavin solutions (Dextralink, Ribolink and Khitolink) as photosensitizers increases its strength properties associated with changes in the structural state of collagen fibrils. It is proved that the riboflavin-UV effect on the optical shell of the eyeball is caused by a short-term local increase in free radical processes, a decrease in the overall antioxidant status, and the absence of necrotic and significant inflammatory reactions. It is shown that the lack of riboflavin in the stroma forms a cascade of pathomorphological events of an irreversible nature, leading to the loss of the native structure of the cornea. On the basis of the conducted studies, the necessity of a differentiated approach to the use of riboflavin-containing solutions during ultraviolet crosslinking of the cornea is justified. Key words: ultraviolet corneal crosslinking, molecular-cellular mechanisms, photosensitizers, riboflavin.

Ten years of experience in applying an original algorithm for surgical treatment of patients with the initial stages of keratoconus

Purpose. Analyze of the long-term results of treatment of patients with progressive keratoconus initial stages performed according to the developed algorithm. Material and methods. Clinical studies were carried out on the basis of a retrospective analysis of three comparison groups, depending on the tactics of surgery. Group 1 consisted of 843 patients (1186 eyes) with progression of keratoconus who underwent crosslinking. Group 2 consisted of 1185 patients (1343 eyes) who underwent intrastromal keratoplasty with implantation of corneal segments (ISKP with implantation of RS) with a mechanical (1007 eyes) or femtolaser (336 eyes) method. Group 3 consisted of 912 patients (1270 eyes), these patients underwent combined treatment – ISKP+UV crosslinking. The observation was carried out for 10 years. Results. As a result of the observation of patients for 10 years from the moment of UV crosslinking, an increase in UCVA and BCVA was revealed 10 years after surgery by 0.33±0.05 and 0.2±0.04, respectively. There was a significant increase in the central thickness of the cornea, on average by 12.75±0.08µm after 10 years. When analyzing the results of the observation of group 2, an increase in UCVA and BCVA was observed by 3–6 months after MS implantation. Keratometry indices (Kmax) decreased by an average of 3.5±1.3 diopters after 10 years. There was an increase in the central corneal thickness by 19.25±2.2µm after 10 years. As a result of the observation of patients for 15 months after the combined treatment (UV-crosslinking+ISCP), an increase in UCVA and BCVA was revealed by 0.17±0.10 and 0.25±15, respectively. Keratometry indices (Kmax) decreased by an average of 4.1±0.9 diopters after 15 months. There was an increase in the central corneal thickness by an average of 15±6.2µm after 15 months. Conclusion. Our research has shown that after 6 years there is no need to repeat UV crosslinking. With the correct location of the corneal segments, the progression of ectasia stops, and the UCVA and BCVA increase. Differences were revealed in the dynamics of the postoperative course in patients with combined UV crosslinking and implantation of corneal segments, which is obviously associated with their mutual effect on the ectasized cornea. In order to improve the quality of life of patients with the initial stages of keratoconus, these methods of treating keratectasias can be used in wide clinical practice without the threat of a decrease in clinical and functional results over a long period of time. Key words: keratoconus, classification, algorithm, crosslinking, corneal segment

An improved iCLIP protocol

Crosslinking and Immunoprecipitation (CLIP) is a powerful technique to obtain transcriptome-wide maps of in vivo protein-RNA interactions, which are important to understand the post-transcriptional mechanisms mediated by RNA binding proteins (RBPs). Many variant CLIP protocols have been developed to improve the efficiency and convenience of cDNA library preparation. Here we describe an improved individual nucleotide resolution CLIP protocol (iiCLIP), which can be completed within 4 days from UV crosslinking to libraries for sequencing. For benchmarking, we directly compared PTBP1 iiCLIP libraries with the iCLIP2 protocol produced under standardised conditions, and with public eCLIP and iCLIP PTBP1 data. We visualised enriched motifs surrounding the identified crosslink positions and RNA maps of these crosslinks around the alternative exons regulated by PTBP1. Notably, motif enrichment was higher in iiCLIP and iCLIP2 in comparison to public eCLIP and iCLIP, and we show how this impacts the specificity of RNA maps. In conclusion, iiCLIP is technically convenient and efficient, and enables production of highly specific datasets for identifying RBP binding sites.

Systematic mapping of small nucleolar RNA targets in human cells

Altered expression of box C/D small nucleolar RNAs (snoRNAs) is implicated in human diseases, including cancer. Box C/D snoRNAs canonically direct site-specific, 2′-O-methylation but the extent to which they participate in other functions remains unclear. To identify RNA targets of box C/D snoRNAs in human cells, we applied two techniques based on UV crosslinking, proximity ligation and sequencing of RNA hybrids (CLASH and FLASH). These identified hundreds of novel snoRNA interactions with rRNA, snoRNAs and mRNAs. We developed an informatic pipeline to rigorously call interactions predicted to direct methylation. Multiple snoRNA-rRNA interactions identified were not predicted to direct RNA methylation. These potentially modulate methylation efficiency and/or contribute to folding dynamics. snoRNA-mRNA hybrids included 1,300 interactions between 117 snoRNA families and 940 mRNAs. Human U3 is substantially more abundant than other snoRNAs and represented about 50% of snoRNA-mRNA hybrids. The distribution of U3 interactions across mRNAs also differed from other snoRNAs. Following U3 depletion, mRNAs showing altered abundance were strongly enriched for U3 CLASH targets. Most human snoRNAs are excised from pre-mRNA introns. Enrichment for snoRNA association with branch point regions of introns that contain snoRNA genes was common, suggesting widespread regulation of snoRNA maturation.

Fabrication of Nanoscale Oxide Textured Surfaces on Polymers

Nanoscale textured surfaces play an important role in creating antibacterial surfaces, broadband anti-reflective properties, and super-hydrophobicity in many technological systems. Creating nanoscale oxide textures on polymer substrates for applications such as ophthalmic lenses and flexible electronics imposes additional challenges over conventional nanofabrication processes since polymer substrates are typically temperature-sensitive and chemically reactive. In this study, we investigated and developed nanofabrication methodologies to create highly ordered oxide nanostructures on top of polymer substrates without any lithography process. We developed suitable block copolymer self-assembly, sequential infiltration synthesis (SIS), and reactive ion etching (RIE) for processes on polymer substrates. Importantly, to prevent damage to the temperature-sensitive polymer and polymer/oxide interface, we developed the process to be entirely performed at low temperatures, that is, below 80 °C, using a combination of UV crosslinking, solvent annealing, and modified SIS and RIE processes. In addition, we developed a substrate passivation process to overcome reactivity between the polymer substrate and the SIS precursors as well as a high precision RIE process to enable deep etching into the thermally insulated substrate. These methodologies widen the possibilities of nanofabrication on polymers.

Application of the method of accelerated ultraviolet crosslinking of corneal collagen using a customized protective contact lens without an ultraviolet filter in a patient with secondary keratoectasia after Lasik (a clinical case)

Purpose. To evaluate the clinical and functional results of the technique of ultraviolet crosslinking on the thin cornea using a customized soft contact lens without an ultraviolet filter in a patient with secondary corneal ectasia. Material and methods. Under observation was patient S., 32 years old, diagnosed with secondary keratoectasia of the left eye after LASIK surgery, performed in 2008 for moderate myopia. To stabilize the keratectatic process, complicated by the presence of a thin cornea with a thickness of less than 400 microns, an operation was performed: ultraviolet crosslinking of corneal collagen using a customized protective lens without an ultraviolet filter. Results. There were no complications during and after the operation. According to keratorefractometry and keratotopography, there was no progression of the disease after surgery, and no loss of endothelial cells was detected. Conclusion. Thus, conducting ultraviolet crosslinking of corneal collagen using a protective customized lens without an ultraviolet filter is a promising technique that allows you to achieve stabilization of the keratectatic process. Key words: corneal ectasia, thin cornea, UV crosslinking, customized lens.

Keratitis induced by soft contact lens

A clinical case demonstrates that improper use of soft contact lenses can induce serious changes in the cornea, followed by long-term treatment, which in turn may be ineffective due to the developed antibiotic resistance. In similar cases, the use of UV crosslinking provides an additional antimicrobial effect, which ultimately allows the inflammatory process to be resolved. Key words: keratitis, soft contact lens, crosslinking.