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.

Recombinant Spidroin Films Attenuate Individual Markers of Glucose Induced Aging in NIH 3T3 Fibroblasts.

The effect of bioresorbable materials on aging in cultured mouse NIH 3T3 fibroblasts treated with elevated glucose concentration was investigated. The cells were grown on films produced from the silkworm fibroin and rS1/9, a recombinant analog of Nephila clavipes spidroin 1. Exposure to 50 mM glucose of the cells grown on uncoated glass support resulted in the cell growth retardation. The average areas of the cells and nuclei and the percentage of apoptotic cells increased, whereas the amount of soluble collagen decreased. In contrast, on the fibroin and spidroin films, the cell density and the percentage of 5-bromo-2'-deoxyuridine (BrdU)-positive cells were higher vs. the cells grown on the glass support. The films protected NIH 3T3 fibroblasts from the glucose-induced death. The most prominent effects on the cell density, BrdU incorporation, and apoptosis prevention were observed in the cells cultured on spidroin films. Unlike the cells grown on glass support (decrease in the soluble collagen production) or fibroin (no effect), production of soluble collagen by the cells grown on spidroin films increased after cell exposure to 50 mM glucose. Molecular analysis demonstrated that 50 mM glucose upregulated phosphorylation of the NFκB heterodimer p65 subunit in the cells grown on the glass support. The treatment of cells grown on fibroin films with 5.5 mM or 50 mM glucose had no effect on p65 phosphorylation. The same treatment decreased p65 phosphorylation in the cells on the spidroin films. These results demonstrate the anti-aging efficacy of biomaterials derived from the silk proteins and suggest that spidroin is more advantageous for tissue engineering and therapy than fibroin.

Recombinant 1F9 spidroin microgels for murine full-thickness wound repairing.

The study of the stimulating effect of the microgels (MGs) based on recombinant 1F9 spidroin on the regeneration of the deep skin wound in mice was carried out. The use of spidroin MGs was shown to increase significantly the quality of healing compared to the control. The introduction of the MG in the wound edges led to recovery of all the structural elements of the skin: the epidermis, the dermis, including vascular and nervous network, in the periphery of the wound underlying muscles, and skin appendages (sebaceous and sweat glands and hair follicles) was revealed.

Novel 3D-microcarriers from recombinant spidroin for regenerative medicine.

Microcarriers generated from recombinant spidroin 1F9 are suitable for use as an injection material. The microcarriers were a heterogeneous mixture of microgel particles ranging from 50 to 300 µm in size with the predominance of particles of 50-150 µm. The surface of these microparticles had a complex topography and ensured efficient cultivation of primary and immortalized fibroblasts. Intradermal injections of microgel suspensions into the area of full-thickness skin wounds did not lead to the development of acute inflammation in mice; instead, they accelerated the recovery of skin tissue and stimulated neurogenesis and angiogenesis.

[Quaternary structure formation by recombinant analogues of spider silk].

A study has been conducted on the morphology of artificial spider silk fibers, prepared from recombinant analogues of spiridons 1 and 2. It has been shown that by stretching out the "as spun" fiber, a reorganization of its spongy matrix occurs, which leads to the formation of microfibrills, followed by a reduction of the diameter of the fiber. The durability of an artificial fiber depends on the degree of stretching and on the substructure of the microfibrills. The model process of artificial fibers preparation reproduces to the great detail the natural process of spider web spinning. Future applications of this model include production of biomaterials with unique properties.

[Left helix of polyproline II type and genesis of beta-structures in spidroins 1 and 2 and their recombinant analogs].

The distribution of secondary structures along the polypeptide chains of spider proteins spidroins 1 and 2 and their recombinant analogs has been studied by statistical methods. It was found that these proteins in the monomolecular form contain only trace amounts of beta-structures. At the same time, the regions of the sequence including Ala and Gly are predicted as helical-containing (with alpha-helices and left-helices of polyproline II type). An analysis of literature data and our data obtained in this study shows that the main conformation of the polypeptide chain solutions of spidroins 1 and 2 and their recombinant analogs in water solutions is the left-helix of polyproline II type with some contaminations of alpha-helices and a very small share of beta-structures. The transition to the state with extended conformations, which are peculiar to mature filaments of spider webs, requires the dehydration of the polypeptide chain backbone. Thus, the genesis of beta-structure in spider web proteins is determined by the conditions of conformation transitions between the main regular conformations of the polypeptide chain backbone.

[The problem of osteopenia in a iodine-deficit area].

The aim of the study was to evaluate the condition of bone system in 335 subjects with thyroid gland dysfunction (TGD), residing in iodine deficit areas. Among the subjects there were 116 individuals with thyrotoxicosis (TT), and 219--with hypothyroidism (HT). The control group included 200 practically healthy people of comparable age and sex. The examination included: evaluation of pain syndrome intensity using a 4-point scale; revealing of atraumatic fracture background; measurement of serum levels of thyroid stimulating hormone (TSH) andfree thyroxin by immune-enzyme assay; measurement of bone mineral density (BMD) by dual-energy x-ray absorptiometry using a Dexa-Scan DX-10 device (Direx, Israel) with evaluation of the results according to 1994 WHO recommendations. 79.3% of TT patients and 68% of HT patients suffered from pain syndrome. The difference in atraumatic fracture rate between the groups was insignificant. The mean BMD values in subjects with TGD dysfunction corresponded to osteopenia, while in the control group they were within normal limits. The difference in osteopenic syndrome rate between TT and HTpatients was insignificant, although osteopenia was revealed in 25% of TT patients. The frequency of BMD decrease grew with age; however, this parameter was higher in all groups of patients with TGD vs. control. The most prone to osteopenia are postmenopausal women regardless of thyroid dysfunction character; difference in BMD decrease frequency between men and women with preserved menstrual function, was insignificant. Osteopenic syndrome was more frequent in men with TGD then in healthy men. Thus, in residents of iodine deficit areas TGD is a factor of BTMD decrease.

[Features of osteopenic syndrome in diffuse toxic goiter].

AIM: To characterize action of thyroid hormones overproduction on bone mineral density. MATERIAL AND METHODS: Pain intensity was graded according to 4-score scale, atraumatic fractures in the past were recorded, mineral bone density (MBD) was studied, TTH and T4 levels and biochemical indices of calcium-phosphorus and bone metabolism were studied, thyroid volume was calculated by J. Brunn (1981) and USI data in 116 patients with diffuse toxic goiter aged 20-65 years compared to 200 healthy controls matched by gender and age. RESULTS: Pain in bones was registered in 79% examinees (1.8 +/- 0.2 scores, on the average). Atraumatic bone fractures were in 9.5% (8% in the controls). MBD reduction was revealed at densitometry in 71% patients with thyrotoxicosis. It was most frequent and severe (in UD--1.78 +/- 0.1 SD, in MD--2.1 +/- 0.1 SD) in patients with severe disease associated with intensive bone remodeling. Loss of MBD does not depend on DTG duration. Mild thyrotoxicosis had no negative effect on bone tissue. Significant differences between patients with thyrotoxicosis and drug euthyrosis by rate and severity of osteopenia were not observed. CONCLUSION: Patients with DTG, especially in severe DTG, demand prophylactic measures concerning osteopenia immediately after diagnosis of thyrotoxicosis.

[Prevalence of cough as a predictor and symptom of chronic bronchitis among male population of Kharkov]. / Rasprostranennost' kashlia kak prediktora i simptoma khronicheskogo bronkhita sredi muzhskogo naseleniia Khar'kova.

A questionnaire survey using WHO-recommended unified questionnaires was performed in Kharkov to elucidate the role of short-term (less than 3 months yearly) and long-term (greater than 3 months, per 2 years) cough in prediction and diagnosis of chronic bronchitis. Cough prevalence was investigated in 5-year age groups (40-44, 45-49, 50-54, 55-59) and correlated to education level and risk of chronic noninfectious diseases. The study covered a population of 7901 males at the mean age of 49 +/- 2.3 years. A group of active prophylaxis and two reference groups comprised 2001 males from an open population. The trial revealed an increase in cough incidence for smokers, low-educated and aged examinees. It is suggested that social and economic status dependent on educational level may represent a risk of chronic bronchitis not less than occupation, smoking, etc.

[Effectiveness of the cooperative program of multifactorial prevention of ischemic heart disease (results of the 3-year study)]. / Effektivnost' kooperativnoi programmy mnogofaktornoi profilaktiki ishemicheskoi bolezni serdtsa (rezul'taty 3-letnego nabliudeniia).

The paper provides the results obtained in the course of the cooperative All-Union programme on multifactor prevention of coronary heart disease (CHD), which has been implemented over 3 years to examine the efficiency of preventive actions aimed at correcting the levels of CHD risk factors. The study was conducted in 6 centers of the country among a non-organized male population aged 40-59 years. The outcome of the preventive measures, largely nonpharmacological ones, made during 3 years suggests that the prevalence of the major CHD risk factors may be reduced in the population. The preventive actions are the most beneficial in arterial hypertension and smoking.

[Detection of ischemic heart disease in a survey of a male population 40-59 years of age (data of cooperative studies performed in Moscow, Kaunas, Kiev and Kharkov)]. / Vyiavlenie ishemicheskoi bolezni serdtsa pri obsledovanii muzhskogo naseleniia 40-59 let (dannye kooperativnogo issledovaniia v Moskve, Kaunase, Kieve, Khar'kove).

The authors analyzed the prevalence of coronary heart disease (CHD) and its various types detected by routine epidemiological methods among the examinees in four cities (Moscow, Kaunas, Kiev, and Kharkov). The results were obtained in the collaboration study, adhering to the rigid standardization of all the procedures applied. Examination of 17, 168 males aged 40-59 years revealed signs of CHD in 13.8%; 3.3% had a history of myocardial infarction; 5.3% suffered from exertional angina without prior myocardial infarction; 5.2% showed a painless course of CHD. The analysis demonstrated that simultaneous use of the standard epidemiological methods is the only way of ensuring a more complete detection of CHD. In different cities, from 36.4 to 60.8% were not aware of the presence of the disease. Secondary preventive measures should involve methods for identifying the major risk factors and eliminating the latter.

[Disorders of regional blood flow in diabetic angiopathy of the lower limbs and their clinical significance]. / Osobennosti narushenii regionarnogo krovotoka pri diabeticheskoi angiopatii nizhnikh konechnostei i ikh klinicheskoe.

Radionuclide methods were used for the examination of 102 patients with diabetic angiopathies of lower extremities. Disturbed blood flow of the muscles was revealed in 100% of the cases, the main blood flow being damaged in 48.6% of the patients. Four forms of diabetic angiopathies of lower extremities were distinguished, each of them requiring individual therapeutic measures.