Hydrogels Based on Recombinant Spidroin Stimulate Proliferation and Migration of Human Corneal Cells.

The effect of recombinant spidroin (RS) hydrogel (HG) on anterior epithelial cells and keratocytes of the human cornea was studied in vitro. Corneal injuries are highly prevalent in developing countries according to the World Health Organization. Various technologies have recently been proposed to restore the damaged surface of the cornea. Use of biodegradable silk-based materials, including recombinant analogs of the spider silk protein spidroin, is an important avenue of research in the field of wound healing and corneal regeneration. Spidroins are well known for their optimal balance of strength and elasticity. Given their biological compatibility, lack of immunogenicity, and biodegradability, spidroins provide a biomaterial for tissue engineering and regenerative medicine. HGs based on RS rS2/12-RGDS were therefore tested for cytotoxicity toward isolated corneal epithelial cells and keratocytes with regard to possible changes in cell phenotype and migratory activity. A promising outlook and therapeutic potential were demonstrated for RS-based HGs.

The effect of biodegradable silk fibroin-based scaffolds containing glial cell line-derived neurotrophic factor (GDNF) on the corneal regeneration process.

Corneal injury due to ocular trauma or infection is one of the most challenging vision impairing pathologies. The aim of the work was to study the effect of biodegradable silk fibroin-based scaffolds containing GDNF on the corneal regeneration process. During cultivate the highest keratocytes proliferative activity was registered with scaffolds containing 250 ng/ml and 500 ng/ml GDNF. In mice with an experimental model of epithelial-stromal damage to the cornea, silk fibroin-based scaffolds containing GDNF in various concentrations were used (in groups 1, 2 and 3 silk fibroin-based scaffolds containing GDNF in a concentration of 50 ng/ml, 250 ng/ml and 500 ng/ml, respectively; in group 4 - silk fibroin-based scaffolds without GDNF; in group 5 - a solution of GDNF with concentration of 500 ng/ml; group 6- control). The area of the corneal epithelial defect in groups 2, 3, and 5 was less than in the other groups. The most pronounced positive immunohistochemical reaction with antibodies to Bcl2, Bax, phosphoERK1/2 and phospho-JNK1/2, Ki67, Gap43 was observed in groups 2 and 3. Thus, silk fibroin-based scaffolds with GDNF stimulate the epithelialization process, proliferative activity of epithelial cells and keratocytes, accelerate the formation of the stromal nerve plexus and exhibit anti-apoptotic activity.

[Morphological changes in rabbit cornea after implantation of a new keratoprosthesis supporting plate]. / Morfologicheskie izmeneniya rogovitsy krolika pri implantatsii novoi modeli opornoi plastinki keratoproteza.

PURPOSE: To evaluate biocompatibility of the new keratoprosthesis supporting plates (KSP) in rabbits in vivo. MATERIAL AND METHODS: The study included 15 chinchilla rabbits. In the first group (5 rabbit eyes) KSP made of hydrophobic acryl with square penetrating holes of 220×220 micron (model 1) were inserted into rabbits' corneas. In the second group (5 eyes), KSP made of hydrophobic acryl were used that had trapezoidal fenestrations with size (from 170×130 micron to 180×70 microns) gradually changing from periphery to the center of KSP (model 2). The control group rabbits (5 eyes) had 1/2 of Fyodorov-Zuev KSP made of titanium implanted. All animals were observed for up to 3 months with biomicroscopy and optical coherence tomography of the anterior segment. The animals were then euthanized and had their corneo-scleral discs excised and then examined with optical microscopy and scanning electron microscopy (SEM). RESULTS: After 3 months, there was only one case of KSP protrusion in the first group. In the second group, thinning of the corneal layers above the central part of KSP occurred in one case. The presence of polymer KSP (of both models) in the corneal stroma was found not to cause formation of rough fibrotic tissue. At the same time, adhered cellular and fibrous elements were discovered on the surface and inside the holes of the polymer KSP, while on the surface of the titanium plate cellular elements were absent. CONCLUSION: Supporting plates made of hydrophobic acrylic material can potentially serve as a foundation for the new keratoprosthesis design.

[Study of the antiviral activity of the liquid corneal storage medium in relation to the herpes simplex virus in vitro].

The aim of the study was to assess the antiviral activity of the 7 types of liquid corneal storage medium on an experimental model of herpesvirus infection in vitro. MATERIAL AND METHODS: The study of antiviral activity of 7 samples of liquid corneal storage medium on a model of herpesvirus infection caused by the herpes simplex virus type 1 in Vero-cell using virological and statistical research methods was carried out. RESULTS AND DISCUSSION: All the studied images of the corneal storage medium, including the Borzenka-Moroz base medium, did not have a cytotoxic effect on Vero cell culture. Out of of 7 samples of liquid corneal storage medium, 4 samples had reliable antiviral activity against HSV-1 when used under the therapeutic regimen (1 hour after infection) and under the preventive regimen (2 hours before infection). Antiviral activity was established in 2 samples containing the interferon inducer cycloferon at a concentration of 10 mg/kg and 30 mg/kg (sample 2, 3), in a sample containing the interferon inducer gamapren 15 mg/kg (sample 5), and in a sample containing a combination of drugs - 10 mg/kg cycloferon and an acyclic nucleoside analog-acyclovir 10 mg/kg (sample 6). According to the results of 2 test regimens, the maximum statistically significant inhibitory effect in relation to HSV-1 was detected in sample 6, containing a combination of drugs. Against the background of sample 6, the infectious activity of the test virus decreased by an average of 3.2 lg, the inhibition coefficient was 54.5%. CONCLUSION: The results of the study indicate the prospects of using types of media with antiviral activity (samples 2, 3, 5, 6) for storing donor corneas in order to increase the effectiveness of keratoplasty in patients with ophthalmic herpes.

[Exophthalmometry with computed tomography]. / Metod ékzoftal'mometrii s ispol'zovaniem komp'iuternoi tomografii.

Bone abnormalities of orbit seen in patients with trauma or radiolesion are accompanied by enophthalmos or exophthalmos. Conventional measurements of eyeball protrusion with Hertel's exophthalmometer or with computed tomography do not provide accurate and reliable data, especially in patients with asymmetry of lateral orbital rims. PURPOSE: To develop a method of computed exophthalmometry that provides accurate and reliable measurements in patients with various orbital conditions. MATERIAL AND METHODS: Medical records and computerized axial tomography scans of 25 patients' orbits without false enophthalmos or exophthalmos were analyzed posthoc. First group included 13 patients with trauma or radiolesion of the orbit at the different stages of plastic-reconstructive treatment. Second group consisted of 12 patients with lacrimal duct obstruction and without any orbital bone abnormalities. Eyeball protrusion was measured from a line joining styloid processes of temporal bones according to the developed method. RESULTS: Comparison of the results of three independent measurements showed that in group 1 mean value varied from 0.40 mm to 10.13 mm and in group 2 - from 0.10 mm to 0.87 mm. Standard deviation (0.00-0.29 mm) and standard error in mean (0.00-0.17 mm) was the same in both groups. CONCLUSION: The newly developed method of exophthalmometry with the use of computed tomography provides accurate and reliable data in patients with various orbital conditions including asymmetry of lateral orbital rims. Eyeball protrusion with asymmetry not exceeding 0.9 mm does not lead to functional and esthetical abnormalities and may be considered normal. The developed method is easy to setup and use, it can be applied in medical practice for diagnostics, surgery planning and evaluation of postoperative results in patients with various orbital conditions.

In Vitro Modeling of Co-Transplantation of Multipotent Stromal Mesenchymal Cells from Orbital Fat Pad and Lipoaspirate of Human Subcutaneous Adipose Tissue in Organ Culture in Collagen Gel.

The interplay of multipotent stromal cells derived from the orbital fat pads and cells of the lipoaspirate from the subcutaneous adipose tissue was studied using in vitro co-transplantation model in an organ culture in a collagen gel. Microscopy findings and intensity of apoptosis and cell proliferation in cultures of lipoaspirate with and without multipotent stromal cells showed that the cells maintained their viability, proliferation capacity, and cytokine secretion activity. Higher proliferatitive activity of cells in cocultures promotes renewal of fat transplant cells and can help to maintain its stable volume in delayed terms after transplantation.

Comparison of Morphology of Adipose Body of the Orbit and Subcutaneous Fat in Humans.

We compared histological structure of the orbital and subcutaneous adipose tissue. Quantitative morphometry showed that the mean diameter of adipocytes and their volume density in the orbital adipose tissue were significantly lower than in the subcutaneous fat, while volume density of vessels and elements of the connective tissue in the orbital adipose tissue was higher. Previously known and revealed here differences in the structure of orbital and subcutaneous adipose tissue do not answer the question, whether subcutaneous adipose tissue can be used for replacement of the orbital tissue deficit.

The fluorescence lifetime of lipofuscin granule fluorophores contained in the retinal pigment epithelium cells from human cadaver eyes in normal state and in the case of visualized pathology.

A comparative analysis of fluorescence lifetime of lipofuscin granule fluorophores contained in the retinal pigment epithelium cells from human cadaver eyes in normal state and in the case of visualized pathology was carried out. Measurements of fluorescence lifetimes of bis-retinoids and their photooxidation and photodegradation products were carried out using the method of counting time-correlated photons. Comparative analysis showed that, in the case of visualized pathology, the contribution of photooxidation and photodegradation products of bis-retinoids to the total fluorescence of the retinal pigment epithelium cell suspension increases in comparison with the norm.

Estimation of fluorescence lifetime of lipofuscin fluorophores contained in lipofuscin granules of retinal pigment epithelium of human cadaver eyes without signs of pathology.

The fluorescence lifetimes of lipofuscin fluorophores contained in chloroform extracts from retinal pigment epithelium (RPE) of human cadaver eyes without signs of pathology were evaluated by single photon counting. The comparison of fluorescence lifetimes of N-retinylidene-N-retinylethanolamine (A2E) and its photooxidation and photodegradation products has been carried out. It was shown that the contribution of A2E to the total fluorescence of chloroform extract from lipofuscin granules is not major. The results are important for the improvement of noninvasive diagnostic method of degenerative diseases of the retina and RPE-fundus autofluorescence (FAF).

The technology of obtaining multipotent spheroids from limbal mesenchymal stromal cells for reparation of injured eye tissues.

It is known that stem and progenitor cells open new possibilities for restoring injured eye tissues. Limbal eye zone, formed mainly by derivatives of neural crest, is the main source of stem cells for regeneration. The current study considers development of innovative technology for obtaining 3D spheroids from L-MMSC. It was shown that under 3D conditions L-MMSC due to compactization and mesenchymal-epithelial transition self-organize into cellular reparative modules. Formed L-MMSC spheroids retain and promote undifferentiated population of stem and progenitor limbal cells, as supported by expression of pluripotency markers - Oct4, Sox2, Nanog. Extracellular matrix synthetized by cells in spheroids allows retaining the functional potential of L-MMSC that are involved in regeneration of both anterior and, probably, posterior eye segment.

[Orbital Adipose Tissue: Just a Fat Pad or Terra Incognita in Ophthalmology].

Our understanding of the role of adipose tissue has been completely changed during the past decades. The knowledge of its contribution to endocrine and immune pathways opened the new insights on the pathogenesis and therapy of many diseases and new perspectives for the regenerative medicine. The further researches should be provided to study anatomy and functions of local fat depots in more details. Of the most interest is the orbital adipose tissue due to its origin from the neural crest. This review represents the current data about anatomy, structure, cell composition and biochemistry of orbital fat. The main attention is put to such cell types as adipocytes and adipose derived mesenchymal stem cells. The foreign authors' findings on such characteristics of stem cells from orbital adipose tissue as CD markers and differential capacity are reviewed. The found evidences of interaction between orbital adipose tissue, eyeball and associated structures allow us to hypothesize that this fat depot may contribute to various ocular pathology. In this paper, we outlined the possible directions for further investigation and clinical application of orbital fat and cells its composing in ophthalmology, reconstructive and plastic surgery and regenerative medicine.

Regulation of Human Adenovirus Replication by RNA Interference.

Adenoviruses cause a wide variety of human infectious diseases. Adenoviral conjunctivitis and epidemic keratoconjunctivitis are commonly associated with human species D adenoviruses. Currently, there is no sufficient or appropriate treatment to counteract these adenovirus infections. Thus, there is an urgent need for new etiology-directed therapies with selective activity against human adenoviruses. To address this problem, the adenoviral early genes E1A and E2B (viral DNA polymerase) seem to be promising targets. Here, we propose an effective approach to downregulate the replication of human species D adenoviruses by means of RNA interference. We generated E1A expressing model cell lines enabling fast evaluation of the RNA interference potential. Small interfering RNAs complementary to the E1A mRNA sequences of human species D adenoviruses mediate significant suppression of the E1A expression in model cells. Furthermore, we observed a strong downregulation of replication of human adenoviruses type D8 and D37 by small hairpin RNAs complementary to the E1A or E2B mRNA sequences in primary human limbal cells. We believe that our results will contribute to the development of efficient anti-adenoviral therapy.

[Justification of the use of particular biopolymers as matrix materials for artificial corneas].

This article reviews distinctive features, advantages, and drawbacks of different biopolymers used to construct the 3D matrices of artificial corneas. Modern requirements for matrices are provided.

[Experience of culturing anterior epithelial corneal cells from human eye ball].

Adult corneal epithelium is often exposed to environmental stress, injured and repaired by limbal stem cells. Injury of corneal epithelial layer leads to reduction of visual clarity and loss of vision. Recently it was shown that epithelial layer also contains stem cells. Obtaining cell culture of corneal epithelium will allow understanding mechanisms of cell behavior and differentiation, their metabolism and reaction on environmental stress in health and disease. Moreover, cultured corneal epithelial cells can be considered as a promising material for constructing bioartificial cornea. The aim of this study was to isolate cells of anterior corneal epithelium from human donor cornea and to study their morphological and functional characteristics in vitro. The results of our study showed the possibility of culturing epithelial cells in vitro. The observed changes in cell morphology, their flow growth character as well as active proliferation and up-regulation of mesenchymal markers expression, indicate, in our opinion, epithelial-mesenchymal transition taking place in long-lasting culture of human anterior corneal epithelial cells. The obtained cultures can be used for further studies of pathological processes taking place in cells during drugs testing or controlling the phototoxic effect of different types of emission.

[Experimental study of safety in application of the Iris Medical IQ 810 diode laser in clinical treatment of age-related macular degeneration].

During the study of diode laser radiation effect in micropulse mode on culture cells of human retinal pigment epithelium it was revealed that the quota of dead cells was a minimum. Besides, a certain conformity between dead cells quota and parameter characteristics of laser radiation. Based on the performed experimental study it was revealed that for a work using the Iris Medical IQ 810 diode laser in the micropulse mode following parameters: duration of pulse set--300ms, duration of function--9.1%, power--750mW are safe for retinal pigment epithelium cells. Rationales of safety in application of the infrared diode laser radiation in micropulse mode in clinic for treatment of age-related macular degeneration (AMD) exampled by cell culture of human retinal pigment epithelium.

[Achievements of S. N. Fyodorov's school in the field of keratoplasty and cornea replacement].

Reduction in corneal transparence leading to the loss of sight may be caused by different factors, such as eye burns and injuries, keratitis, corneal ulcers, primary and secondary dystrophy, keratoconus, and keratoglobus. In many cases, keratoplasty presents an effective treatment. S. N. Fyodorov made a great contribution to the development of cornea replacement. This was him who placed the entire ophthalmology, including keratoplasty, at the microsurgical level, suggested the use of large-diameter grafts, and was the initiator of establishing the first Eye Tissue Bank. Perfection of microsurgical technique, selection and protection of the graft, as well as new approaches to pre- and postoperation therapy have provided transparent engrafting in 35 to 95%. Presently, Eye Microsurgery Complex is the only Russian institution where the problem of cornea replacement is being worked out as a scientific and practical issue. Three models of keratoprosthesis have been developed: Fyodorov-Zuyev model for implantation into a burn leukoma, Moroz-Glazko model for implantation into a dystrophic leukoma, and a "grid" for implantation into a leukoma of any etiology.