Nitric oxide therapy is beneficial to rehabilitation in professional soccer players: clinical and experimental studies.

Nitric oxide can activate neutrophils and macrophages, facilitate the synthesis of collagen, which allows significantly accelerating the regeneration of traumatized tissues. We studied the effects of nitric oxide-containing gas flow generated by plasma-chemical device "Plason" in a rat model of full-thickness wounds. Histological and morphometric analyses revealed that Plason treated wounds expressed significantly fewer signs of inflammation and contained a more mature granulation tissue on day 4 after the operation. Considering the results of the experimental study, we applied the Plason device in sports medicine for the treatment of lower limb bruises of 34 professional soccer players. Athletes were asked to assess the intensity of pain with the Visual Analogue Scale. Girths of their lower limbs were measured over the course of rehabilitation. Nitric oxide therapy of full-thickness wounds inhibited inflammation and accelerated the regeneration of skin and muscle tissues. Compared with the control, we observed a significant reduction in pain syndrome on days 2-7 after injuries, edema, and hematoma, and shortened treatment duration. This pilot study indicates that the use of nitric oxide is a promising treatment method for sports injuries.

Autologous bone marrow-derived mesenchymal stem cells provide complete regeneration in a rabbit model of the Achilles tendon bundle rupture.

PURPOSE: To ascertain the role of autologous bone marrow-derived mesenchymal stem cells (BM-MSCs) in the tendon regeneration. METHODS: The study was conducted on 58 Achilles tendons from 29 laboratory Chinchilla adult rabbits. The central bundles of 48 tendons were partially removed and substituted with a tissue-engineered construct consisting of a collagen sponge either loaded with BM-MSCs (n = 24) or cell free (n = 24), placed inside a Vicryl mesh tube. The ends of the resected tendon were inserted in the construct to reach a direct contact with the sponge and sutured to the tube. The animals were sacrificed three and six months post-surgery. Ten intact tendons from five rabbits were used as an untreated control. The tissue samples (n = 30) were stained with haematoxylin and eosin, Picrosirius red, primary antibodies to collagen types I and III and studied by bright-field, phase-contrast, polarized light, and scanning electron microscopies followed by semi-quantitative morphometry. RESULTS: Six months results of cell-loaded scaffolds demonstrated parallel collagen fibres, spindle-shaped tenocytes, and neoangiogenesis. In the control cell-free group, the injured areas were filled with a nonspecific fibrotic tissue with minor foci of incomplete regeneration. The biomechanical tests of 28 tendons taken from 14 rabbits showed that the stiffness of the cell-based reconstructed tendons increased to 98% of the value for the intact samples. CONCLUSION: The obtained results support the hypothesis that the application of BM-MSCs in a tissue-engineered tendon construct leads to the restitution of the tendon tissue.

Evaluation of Supercritical CO2-Assisted Protocols in a Model of Ovine Aortic Root Decellularization.

One of the leading trends in the modern tissue engineering is the development of new effective methods of decellularization aimed at the removal of cellular components from a donor tissue, reducing its immunogenicity and the risk of rejection. Supercritical CO2 (scCO2)-assisted processing has been proposed to improve the outcome of decellularization, reduce contamination and time costs. The resulting products can serve as personalized tools for tissue-engineering therapy of various somatic pathologies. However, the decellularization of heterogeneous 3D structures, such as the aortic root, requires optimization of the parameters, including preconditioning medium composition, the type of co-solvent, values of pressure and temperature inside the scCO2 reactor, etc. In our work, using an ovine aortic root model, we performed a comparative analysis of the effectiveness of decellularization approaches based on various combinations of these parameters. The protocols were based on the combinations of treatments in alkaline, ethanol or detergent solutions with scCO2-assisted processing at different modes. Histological analysis demonstrated favorable effects of the preconditioning in a detergent solution. Following processing in scCO2 medium provided a high decellularization degree, reduced cytotoxicity, and increased ultimate tensile strength and Young's modulus of the aortic valve leaflets, while the integrity of the extracellular matrix was preserved.

Chemical cross-linking of xenopericardial biomeshes: A bottom-up study of structural and functional correlations.

Decellularized bovine pericardium (DBP)-based biomeshes are the gold standard in reconstructive surgery. In order to prolong their stability after the transplantation, various chemical cross-linking strategies are employed. However, structural and functional properties of the biomeshes differ in dependence on the cross-linker used. Here, we performed a bottom-up study of structural and functional alterations of DBP-based biomeshes following cross-linking with hexamethylene diisocyanate (HMDC), ethylene glycol diglycidyl ether (EGDE), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and genipin. The in vitro cytotoxicity tests supported their clinical applicability. Their structural differences (eg roughness, fibre thickness, pore morphology) were evaluated using the two-photon confocal laser scanning, atomic force, scanning electron and polarized light microscopies. HMDC and EDC samples appeared to be the roughest. Complex mechanical trials indicated the tendency to reduced Young's Modulus and mechanical anisotropy values of DBP upon cross-linking. The lowest mechanical anisotropy was found in EDC and genipin sample groups. In vitro collagenase susceptibility was the highest for EDC samples and the lowest for EGDE samples. The comparative analysis of the results allowed us to recognize the strengths and weaknesses of each cross-linker in relation to a particular clinical application.

The Inhibiting Effect of Dinitrosyl Iron Complexes with Thiol-containing Ligands on the Growth of Endometrioid Tumours in Rats with Experimental Endometriosis.

The possibility that binuclear dinitrosyl iron complexes with glutathione and cysteine (DNIC-GSН and B-DNIC-Cys) have a strong cytotoxic effect on the growth of endometrioid tumours (EMT) in rats with surgically induced experimental endometriosis established in our previous studies has been supported with experimental data. The increase in the DNIC-GSН or B-DNIC-Cys dose from 10 (in our previous studies) to 20 µmol/kg (after i/p administration to experimental rats) fully suppressed the growth of uterine tissues implanted onto the inner surface of the abdominal wall. At 2 µmol/kg DNIC-GSН, the median value of EMT volume increased from 0 to 15 mm3, while the mean size of EMT-from 55 to 77 mm3 (data from EMT measurements in 10 experimental rats). After treatment of animals with B-DNIC with N-acetyl-L-cysteine (10 µmol/kg) known for its ability to penetrate easily through the cell membrane, the inhibiting effect on EMT growth diminished as could be evidenced from the transformation of ~30% of the implants into large-size EMT. Possible reasons for this phenomenon are discussed.

Collagen fibrillar structures in vocal fold scarring and repair using stem cell therapy: a detailed histological, immunohistochemical and atomic force microscopy study.

Regenerative medicine opens new opportunities in the repair of cicatricial lesions of the vocal folds. Here, we present a thorough morphological study, with the focus on the collagen structures in the mucosa of the vocal folds, dedicated to the effects of stem cells on the vocal folds repair after cicatricial lesions. We used a conventional experimental model of a mature scar of the rabbit vocal folds, which was surgically excised with a simultaneous implantation of autologous bone marrow-derived mesenchymal stem cells (MSC) into the defect. The restoration of the vocal folds was studied 3 months postimplantation of stem cells and 6 months after the first surgery. The collagen structure assessment included histology, immunohistochemistry and atomic force microscopy (AFM) studies. According to the data of optical microscopy and AFM, as well as to immunohistochemical analysis, MSC implantation into the vocal fold defect leads not only to the general reduction of scarring, normal ratio of collagens type I and type III, but also to a more complete restoration of architecture and ultrastructure of collagen fibres in the mucosa, as compared to the control. The collagen structures in the scar tissue in the vocal folds with implanted MSC are more similar to those in the normal mucosa of the vocal folds than to those of the untreated scars. AFM has proven to be an instrumental technique in the assessment of the ultrastructure restoration in such studies. LAY DESCRIPTION: Regenerative medicine opens new opportunities in the repair of the vocal fold scars. Because collagen is a main component in the vocal fold mucosa responsible for the scar formation and repair, we focus on the collagen structures in the mucosa of the vocal folds, using a thorough morphological study based on histology and atomic force microscopy (AFM). Atomic force microscopy is a scanning microscopic technique which allows revealing the internal structure of a tissue with a resolution up to nanometres. We used a conventional experimental model of a mature scar of the rabbit vocal folds, surgically excised and treated with a mesenchymal stem cells transplant. Our morphological study, primarily AFM, explicitly shows that the collagen structures in the scarred vocal folds almost completely restore after the stem cell treatment. Thus, the modern microscopic methods, and especially AFM are instrumental tools for monitoring the repair of the vocal folds scars.

Collagenolytic Enzymes and their Applications in Biomedicine.

Nowadays, enzymatic therapy is a very promising line of treatment for many different diseases. There is a group of disorders and conditions, caused by fibrotic and scar processes and associated with the excessive accumulation of collagen that needs to be catabolized to normalize the connective tissue content. The human body normally synthesizes special extracellular enzymes, matrix metalloproteases (MMPs) by itself. These enzymes can cleave components of extracellular matrix (ECM) and different types of collagen and thus maintain the balance of the connective tissue components. MMPs are multifunctional enzymes and are involved in a variety of organism processes. However, under pathological conditions, the function of MMPs is not sufficient, and these enzymes fail to deal with disease. Thus, medical intervention is required. Enzymatic therapy is a very effective way of treating such collagen-associated conditions. It involves the application of exogenous collagenolytic enzymes that catabolize excessive collagen at the affected site and lead to the successful elimination of disease. Such collagenolytic enzymes are synthesized by many organisms: bacteria, animals (especially marine organisms), plants and fungi. The most studied and commercially available are collagenases from Clostridium histolyticum and from the pancreas of the crab Paralithodes camtschatica, due to their ability to effectively hydrolyse human collagen without affecting other tissues, and their wide pH ranges of collagenolytic activity. In the present review, we summarize not only the data concerning existing collagenase-based medications and their applications in different collagen-related diseases and conditions, but we also propose collagenases from different sources for their potential application in enzymatic therapy.

Medical Applications of Collagen and Collagen-Based Materials.

Collagen and collagen-based materials have been successfully used in medicine for over 50 years. The number of scientific articles about the role of collagen in the construction of scaffolds for tissue engineering has risen precipitously in recent years. The review contains materials about historic and modern applications of collagen in medicine such as soluble collagen injections, solid constructs reconstructed from solution, and decellularized collagen matrices. The analysis of published data proves the efficacy of collagen material in the treatment of chronic wounds, burns, venous and diabetic ulcers, in plastic, reconstructive and general surgery, urology, proctology, gynecology, ophthalmology, otolaryngology, neurosurgery, dentistry, cardiovascular and bone and cartilage surgery, as well as in cosmetology. Further development of collagenoplasty requires addressing the problems of allergic complications, improvement of structure and maximizing therapeutic effects against pathological processes.

Physicochemical parameters of NO-containing gas flow affect wound healing therapy. An experimental study.

Therapy of wounds and inflammatory diseases with NO-containing gas flows (NO-CGF) has proved to be effective in a longterm clinical practice. Plasma-chemical generation of nitric oxide occurs from atmospheric air in Plason device. For the purpose of modification and improvement of NO-therapy, effects of various physicochemical parameters of the NO-CGF on inflammatory and reparative processes in wounds were studied. Treatment of planar full-thickness skin wounds in laboratory rats was analyzed with morphological, immunohistochemical, morphometric and statistical methods. The study showed that the Plason device and the experimental device, which differs from Plason by the NO-CGF temperature, significantly reduce inflammatory and enhance regenerative processes in the wounds. The NO-CGF with an ambient temperature generated by the experimental device has noticeably facilitated the wound healing in direct ration to a nitric oxide content and flow velocity at the area of application. Temperature did not affect the course of wound healing process. The development of a new device for NO-therapy may be of use for both physicians and patients.

Is it possible to combine photodynamic therapy and application of dinitrosyl iron complexes in the wound treatment?

We have studied the effect of interactions between dinitrosyl iron complexes with thiol-containing ligands (DNIC-TL) and diglucamine salt of chlorine e6 (photoditazine, PD) on the rate of photosensitized oxidation of a model organic substrate - tryptophan - in the presence and absence of an amphiphilic polymer, Pluronic F127, as well as on the DNIC-TL and PD photostability. Using EPR and UV spectroscopy, we determined the rate constants for photodegradation of mono- and dinuclear DNIC-TL and PD, respectively. The presence of the photosensitizer and Pluronic F127 has been shown to have a negligible effect on the rate of photodestruction of mono- and dinuclear DNIC-TL, taking into account the changing DNIC-TL and PD concentrations in the photoexcitation conditions. At the same time, in the DNIC-TL presence, the rate of PD photodestruction increases, however, addition of Pluronic F127 leads to a decrease in the rate constant of PD photodestruction. The latter circumstance creates an opportunity for a simultaneous application of DNIC-TL and photodynamic therapy in the wound treatment without losing the PDT efficiency. Indeed, photodynamic therapy in combination with DNIC-TL facilitated skin wound healing in laboratory rats. As shown by a morphological study, application of the DNIC-TL-PD-F127 complex with the subsequent photoactivation was beneficial in reducing inflammation and stimulating regenerative processes.

Rational Surface Design of Upconversion Nanoparticles with Polyethylenimine Coating for Biomedical Applications: Better Safe than Brighter?

Upconversion nanoparticles (UCNPs) coated with polyethylenimine (PEI) are popular background-free optical contrast probes and efficient drug and gene delivery agents attracting attention in science, industry, and medicine. Their unique optical properties are especially useful for subsurface nanotheranostics applications, in particular, in skin. However, high cytotoxicity of PEI limits safe use of UCNP@PEI, and this represents a major barrier for clinical translation of UCNP@PEI-based technologies. Our study aims to address this problem by exploring additional surface modifications to UCNP@PEI to create less toxic and functional nanotheranostic materials. We designed and synthesized six types of layered polymer coatings that envelop the original UCNP@PEI surface, five of which reduced the cytotoxicity to human skin keratinocytes under acute (24 h) and subacute (120 h) exposure. In parallel, we examined the photoluminescence spectra and lifetime of the surface-modified UCNP@PEI. To quantify their brightness, we developed original methodology to precisely measure the colloidal concentration to normalize the photoluminescence signal using a nondigesting mass spectrometry protocol. Our results, specified for the individual coatings, show that, despite decreasing the cytotoxicity, the external polymer coatings of UCNP@PEI quench the upconversion photoluminescence in biologically relevant aqueous environments. This trade-off between cytotoxicity and brightness for surface-coated UCNPs emphasizes the need for the combined assessment of the viability of normal cells exposed to the nanoparticles and the photophysical properties of postmodification UCNPs. We present an optimized methodology for rational surface design of UCNP@PEI in biologically relevant conditions, which is essential to facilitate the translation of such nanoparticles to the clinical applications.

Study of plasma-chemical NO-containing gas flow for treatment of wounds and inflammatory processes.

This work is aimed at exhaustive and detailed study of chemical, physical and physico-chemical characteristics of NO-containing gas flow (NO-CGF) generated by a plasma-chemical generator of Plason device, which has been used in medical practice for more than 15 years for effectively healing wound and inflammatory conditions with exogenous nitric oxide (NO-therapy). Data was obtained on spatial structure of the gas flow, and values of its local parameters in axial and radial directions, such as nitric oxide content, velocity, temperature and mass flow density of nitric oxide, providing altogether the effectiveness of treatment by the exogenous NO-therapy method, were determined experimentally and by computations. It was demonstrated that plasma-chemical synthesis of NO from atmospheric air in a low direct current (DC) arc provides a high mass flow of nitric oxide at the level of 1.6-1.8 mg/s, while in the area of impact of NO-CGF on the biological tissue, on its axis, NO content is 400-600 ppm, flow velocity about 5 m/s, nitric oxide mass flow density 0.25-0.40 mg/(s·cm2), temperature 40-60 °C. Tendencies were determined for designing new devices for further experimental biological and medical research in the field of NO-therapy: lowering the temperature of NO-CGF to ambient temperature will enable variation, in experiments, of the affecting flow parameters in a wide range up to their maximum values: NO content up to 2000 ppm, velocity up to 20 m/s, nitric oxide mass flow density up to 2.5 mg/(s·cm2).

Superoxide Dismutase 1 Nanozyme for Treatment of Eye Inflammation.

Use of antioxidants to mitigate oxidative stress during ocular inflammatory diseases has shown therapeutic potential. This work examines a nanoscale therapeutic modality for the eye on the base of antioxidant enzyme, superoxide dismutase 1 (SOD1), termed "nanozyme." The nanozyme is produced by electrostatic coupling of the SOD1 with a cationic block copolymer, poly(L-lysine)-poly(ethyleneglycol), followed by covalent cross-linking of the complexes with 3,3'-dithiobis(sulfosuccinimidylpropionate) sodium salt. The ability of SOD1 nanozyme as well as the native SOD1 to reduce inflammatory processes in the eye was examined in vivo in rabbits with immunogenic uveitis. Results suggested that topical instillations of both enzyme forms demonstrated anti-inflammatory activity; however, the nanozyme was much more effective compared to the free enzyme in decreasing uveitis manifestations. In particular, we noted statistically significant differences in such inflammatory signs in the eye as the intensities of corneal and iris edema, hyperemia of conjunctiva, lens opacity, fibrin clots, and the protein content in aqueous humor. Clinical findings were confirmed by histological data. Thus, SOD1-containing nanozyme is potentially useful therapeutic agent for the treatment of ocular inflammatory disorders.

Dinitrosyl iron complexes with glutathione incorporated into a collagen matrix as a base for the design of drugs accelerating skin wound healing.

Composites of a collagen matrix and dinitrosyl iron complexes with glutathione (DNIC-GS) (in a dose of 4.0 µmoles per item) in the form of spongy sheets (DNIC-Col) were prepared and then topically applied in rat excisional full-thickness skin wound model. The effects of DNIC-Col were studied in comparison with spontaneously healing wounds (SpWH) and wounds treated with collagen sponges (Col) without DNIC-GS. The composites induced statistically and clinically significant acceleration of complete wound closure (21±1 day versus 23±1 day and 26±1 day for DNIC-Col, Col and SpWH, respectively). Histological examination of wound tissues on days 4, 14, 18 and 21 after surgery demonstrated that this improvement was supported by enhanced growth, maturation and fibrous transformation of granulation tissue and earlier epithelization of the injured area in rats treated with DNIC-Col composites benchmarked against Col and SpWH. It is suggested that the positive effect of the new pharmaceutical material on wound healing is based on the release of NO from decomposing DNIC. This effect is believed to be potentiated by the synergy of DNIC and collagen.

Collagen structure deterioration in the skin of patients with pelvic organ prolapse determined by atomic force microscopy.

We used atomic force microscopy (AFM) to diagnose pathological changes in the extracellular matrix (ECM) of skin connective tissue in patients with pelvic organ prolapse (POP). POP is a common condition affecting women that considerably decreases the patients' quality of life. Deviations from normal morphology of the skin ECM from patients with POP occur including packing and arrangement of individual collagen fibers and arrangement of collagen fibrils. The nanoindentation study revealed significant deterioration of the mechanical properties of collagen fibril bundles in the skin of POP patients as compared with the skin of healthy subjects. Changes in the skin ECM appeared to correlate well with changes in the ECM of the pelvic ligament tissue associated with POP. AFM data on the ECM structure of normal and pathologically altered connective tissue were in agreement with results of the standard histological study on the same clinical specimens. Thus, AFM and related techniques may serve as independent or complementary diagnostic tools for tracking POP-related pathological changes of connective tissue.

Laser radiation effect on chondrocytes and intercellular matrix of costal and articular cartilage impregnated with magnetite nanoparticles.

BACKGROUND AND OBJECTIVE: Magnetic nanoparticles with the ability to absorb laser radiation are the perspective agents for the early diagnostics and laser therapy of degenerative cartilage. The effect of starch stabilized magnetite nanoparticles (SSNPs) on the cartilage structure components has never been studied before. The aim of the work is to establish the Erbium:glass laser effect on costal and articular cartilage impregnated with SSNPs. MATERIALS AND METHODS: Porcine articular and costal cartilage disks (2.0 mm in diameter and 1.5-2 mm in thickness) were impregnated with SSNPs and irradiated using a 1.56 µm laser in therapeutic laser setting. The one sample group underwent the second irradiation after the SSNPs impregnation. The samples were analyzed by the means of histology, histochemistry and transmission electron microscopy (TEM) to reveal the alterations of cells, glycosaminoglycans and collagen network. RESULTS: The irradiated cartilage demonstrates the higher content of cell alterations than the intact one due to the heat and mechanical affection in the course of laser irradiation. However the alterations are localized at the areas near the irradiated surfaces and not dramatic. The impregnation of SSNPs does not cause any additional cell alterations. For both costal and articular cartilage the matrix alterations of irradiated samples are not critical: there is the slight decrease in acid proteoglycan content at the irradiated areas while the collagen network is not altered. Distribution and localization of impregnated SSNPs is described: agglomerates of 150-230 nm are observed located at the borders between matrix and cell lacunas of articular cartilage; SSNPs of 15-45 nm are found in the collagen network of costal cartilage. CONCLUSIONS: It was shown that SSNPs do not appreciably affect the structural components of both articular and costal cartilage and can be safely used for the laser diagnostics and therapy. The area of structural alterations is diffuse and local as the result of the mechanical and heat effect of laser impact. SSNPs reveal the areas of the structural alterations of cartilage matrix and give information about the size of the pores and defects.

Specific features of early stage of the wound healing process occurring against the background of photodynamic therapy using fotoditazin photosensitizer-amphiphilic polymer complexes.

There is a growing demand on the studies of the wound healing potentials of photodynamic therapy. Here we analyze the effects of Fotoditazin, an e6 chlorine derivative, and its complexes with amphiphilic polymers, on the early stage of wound healing in a rat model. A skin excision wound model with prevented contraction was developed in male albino rats divided into eight groups according to the treatment mode. All animals received injections of one of the studied compositions into their wound beds and underwent low-intensity laser irradiation or stayed un-irradiated. The clinical monitoring and histological examination of the wounds were performed. It has been found that all the Fotoditazin formulations have significant effects on the early stage of wound healing. The superposition of the inflammation and regeneration was the main difference between groups. The aqueous solution of Fotoditazin alone induced a significant capillary hemorrhage, while its combinations with amphiphilic polymers did not. The best clinical and morphological results were obtained for the Fotoditazin-Pluronic F127 composition. Compositions of Fotoditazin and amphiphilic polymers, especially Pluronic F127, probably, have a great potential for therapy of wounds. Their effects can be attributed to the increased regeneration and suppressed reactions changes at the early stages of repair.

Scar tissue classification using nonlinear optical microscopy and discriminant analysis.

This paper addresses the scar tissue maturation process that occurs stepwise, and calls for reliable classification. The structure of collagen imaged by nonlinear optical microscopy (NLOM) in post-burn hypertrophic and mature scar, as well as in normal skin, appeared to distinguish these maturation steps. However, it was a discrimination analysis, demonstrated here, that automated and quantified the scar tissue maturation process. The achieved scar classification accuracy was as high as 96%. The combination of NLOM and discrimination analysis is believed to be instrumental in gaining insight into the scar formation, for express diagnosis of scar and surgery planning.

Laser-induced modification of the patellar ligament tissue: comparative study of structural and optical changes.

The effects of non-ablative infrared (IR) laser treatment of collagenous tissue have been commonly interpreted in terms of collagen denaturation spread over the laser-heated tissue area. In this work, the existing model is refined to account for the recently reported laser-treated tissue heterogeneity and complex collagen degradation pattern using comprehensive optical imaging and calorimetry toolkits. Patella ligament (PL) provided a simple model of type I collagen tissue containing its full structural content from triple-helix molecules to gross architecture. PL ex vivo was subjected to IR laser treatments (laser spot, 1.6 mm) of equal dose, where the tissue temperature reached the collagen denaturation temperature of 60 ± 2°C at the laser spot epicenterin the first regime, and was limited to 67 ± 2°C in the second regime. The collagen network was analyzed versus distance from the epicenter. Experimental characterization of the collagenous tissue at all structural levels included cross-polarization optical coherence tomography, nonlinear optical microscopy, light microscopy/histology, and differential scanning calorimetry. Regressive rearrangement of the PL collagen network was found to spread well outside the laser spot epicenter (>2 mm) and was accompanied by multilevel hierarchical reorganization of collagen. Four zones of distinct optical and morphological properties were identified, all elliptical in shape, and elongated in the direction perpendicular to the PL long axis. Although the collagen transformation into a random-coil molecular structure was occasionally observed, it was mechanical integrity of the supramolecular structures that was primarily compromised. We found that the structural rearrangement of the collagen network related primarily to the heat-induced thermo-mechanical effects rather than molecular unfolding. The current body of evidence supports the notion that the supramolecular collagen structure suffered degradation of various degrees, which gave rise to the observed zonal character of the laser-treated lesion.

Beneficial effect of gaseous nitric oxide on the healing of skin wounds.

Intermittent daily exposures (60 s) to NO-containing gas flow (NO dose of 500 ppm) generated by air-plasma unit "Plason" improves healing of skin wounds in rats. The gas flow treatment shortened the recovery time of both aseptic and purulent wounds (300 mm2 area) by nearly a third. The treatment allows to achieve a marked improvement in the histological, histochemical, and electron-microscopic characteristics of the affected tissue. The mechanism of this phenomenon was studied by spin trapping method. The NO status of the wound tissue was investigated with EPR by following the formation of paramagnetic mononitrosyl complexes with iron-diethyldithiocarbamate, or with the heme groups in hemoglobin or myoglobin. For the first 5 min after a gas treatment with the exposure of 60s, detectable NO levels in the affected tissue were slightly lowered with respect to untreated controls. At subsequent times, treated tissues showed the formation of large quantities of nitroso-iron complexes: At 30-40 min after gas exposure, their levels were nearly two orders of magnitude higher than soon after (15 s-5 min) the exposure. The data demonstrate that the accumulation of nitrosyl-iron complexes reflects a sharp rise in endogenous NO production inside the affected tissue. Paradoxically, the beneficial effect of gaseous NO treatment can be mediated by the formation of limited quantities of peroxynitrite due to the reaction between exogenous NO and superoxide anions generated in high amount in wound tissue. This peroxynitrite has a strong prooxidant effect and can activate various antioxidant systems which diminish the amount of superoxide anions in wound tissue. The reduced superoxide levels allow to increase the contents of endogenous NO in gas-treated tissues. Therefore, the beneficial action of the treatment is attributed to enhanced NO bioavailability.