Luděk Vajner

A cell-free nanofiber composite scaffold regenerated osteochondral defects in miniature pigs

The aim of the study was to evaluate the effect of a cell-free hyaluronate/type I collagen/fibrin composite scaffold containing polyvinyl alcohol (PVA) nanofibers enriched with liposomes, basic fibroblast growth factor (bFGF) and insulin on the regeneration of osteochondral defects. A novel drug delivery system was developed on the basis of the intake effect of liposomes encapsulated in PVA nanofibers. Time-controlled release of insulin and bFGF improved MSC viability in vitro. Nanofibers functionalized with liposomes also improved the mechanical characteristics of the composite gel scaffold. In addition, time-controlled release of insulin and bFGF stimulated MSC recruitment from bone marrow in vivo. Cell-free composite scaffolds containing PVA nanofibers enriched with liposomes, bFGF, and insulin were implanted into seven osteochondral defects of miniature pigs. Control defects were left untreated. After 12 weeks, the composite scaffold had enhanced osteochondral regeneration towards hyaline cartilage and/or fibrocartilage compared with untreated defects that were filled predominantly with fibrous tissue. The cell-free composite scaffold containing PVA nanofibers, liposomes and growth factors enhanced migration of the cells into the defect, and their differentiation into chondrocytes; the scaffold was able to enhance the regeneration of osteochondral defects in minipigs.

Acute and chronic hypoxia as well as 7-day recovery from chronic hypoxia affects the distribution of pulmonary mast cells and their MMP-13 expression in rats

Chronic hypoxia results in pulmonary hypertension due to vasoconstriction and structural remodelling of peripheral lung blood vessels. We hypothesize that vascular remodelling is initiated in the walls of prealveolar pulmonary arteries by collagenolytic metalloproteinases (MMP) released from activated mast cells. Distribution of mast cells and their expression of interstitial collagenase, MMP‐13, in lung conduit, small muscular, and prealveolar arteries was determined quantitatively in rats exposed for 4 and 20 days to hypoxia as well as after 7‐day recovery from 20‐day hypoxia (10% O2). Mast cells were identified using Toluidine Blue staining, and MMP‐13 expression was detected using monoclonal antibody. After 4, but not after 20 days of hypoxia, a significant increase in the number of mast cells and their MMP‐13 expression was found within walls of prealveolar arteries. In rats exposed for 20 days, MMP‐13 positive mast cells accumulated within the walls of conduit arteries and subpleurally. In recovered rats, MMP‐13 positive mast cells gathered at the prealveolar arterial level as well as in the walls of small muscular arteries; these mast cells stayed also in the conduit part of the pulmonary vasculature. These data support the hypothesis that perivascular pulmonary mast cells contribute to the vascular remodelling in hypoxic pulmonary hypertension in rats by releasing interstitial collagenase.

Fibrin/hyaluronic acid composite hydrogels as appropriate scaffolds for in vivo artificial cartilage implantation.

Hydrogels prepared from a mixture of fibrin and high-molecular weight (MW) hyaluronic acid (HA) were found to be suitable scaffolds for chondrocyte seeding and pig knee cartilage regeneration. Collagen in the hydrogels is not necessary for the formation of biomechanically stable tissue. Regenerated cartilage showed very good biomechanical and histological properties only 6 months after implantation. Notably, the quality of the healing process was dependent on the initial chondrocyte concentration of the scaffolds. These experiments were performed according to good laboratory practice (GLP).

Evaluation of different methods detecting intracellular generation of free radicals

Reactive oxygen species (ROS) play several biological roles. We investigated the applicability of fluorescent probes for their detection (i) in rabbit lens epithelial cells during ageing in culture, and (ii) in thin sections of rat heart. We used dihydroethidium (DHE), dichlorofluorescin (DCFH), and dihydrorhodamine 123 (DHR) together with detection of autofluorescence both in cells and in chloroform extracts. Superoxide production was confirmed by a specific histochemical method using Mn2+. All methods demonstrated higher production of ROS in older cells. All probes revealed different sites of ROS production in young and old cells and could be used for investigation of ROS generation during cell ageing. In the thin sections of rat heart DCFH was not suitable for intracellular ROS detection. The results indicate that the potential of fluorescent dyes in ROS detection is not usually fully exploited, and that blue autofluorescence is associated with oxidative damage.

Oxidative Stress in the Developing Rat Brain due to Production of Reactive Oxygen and Nitrogen Species.

Oxidative stress after birth led us to localize reactive oxygen and nitrogen species (RONS) production in the developing rat brain. Brains were assessed a day prenatally and on postnatal days 1, 2, 4, 8, 14, 30, and 60. Oxidation of dihydroethidium detected superoxide; 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate revealed hydrogen peroxide; immunohistochemical proof of nitrotyrosine and carboxyethyllysine detected peroxynitrite formation and lipid peroxidation, respectively. Blue autofluorescence detected protein oxidation. The foetuses showed moderate RONS production, which changed cyclically during further development. The periods and sites of peak production of individual RONS differed, suggesting independent generation. On day 1, neuronal/glial RONS production decreased indicating that increased oxygen concentration after birth did not cause oxidative stress. Dramatic changes in the amount and the sites of RONS production occurred on day 4. Nitrotyrosine detection reached its maximum. Day 14 represented other vast alterations in RONS generation. Superoxide production in arachnoidal membrane reached its peak. From this day on, the internal elastic laminae of blood vessels revealed the blue autofluorescence. The adult animals produced moderate levels of superoxide; all other markers reached their minimum. There was a strong correlation between detection of nitrotyrosine and carboxyethyllysine probably caused by lipid peroxidation initiated with RONS.

Mast cell stabilization with sodium cromoglycate modulates pulmonary vessel wall remodeling during four-day hypoxia in rats

ABSTRACT Aim of the study: In rats, the environment with low content of oxygen induces hypoxic pulmonary hypertension. Remodeling of pulmonary resistance arteries is particularly triggered by the mast cell degranulation products, e.g., rodent-like interstitial collagenase (matrix metalloproteinase 13). Administration of sodium cromoglycate leads to stabilization of mast cell granules, and thus to the modified remodeling process. Materials and Methods: During four-day hypoxia, we treated rats with sodium cromoglycate. Pulmonary vascular remodeling was assessed as well as counts of periarterial pulmonary mast cells, both total and matrix metalloproteinase 13-positive ones. Results: Four-day hypoxia induced remodeling of both resistance arteries and large conduit arteries. We have found increase in the tunica media thickness of resistance arteries. Tunica adventitia thickness of both resistance arteries and large conduit arteries with a diameter of over 300 μm increased as well; the latter ones revealed increase in the number of vasa vasorum in their walls. Mast cell stabilization suppressed hypoxic pulmonary vascular remodeling in resistance pulmonary arteries. Four-day hypoxia led to changes in distribution of toluidine blue-detected and MMP-13 positive periarterial mast cells; this redistribution was also influenced by the administration of sodium cromoglycate. Conclusions: The number of pulmonary periarterial mast cells seemingly decreases during hypoxia due to their degranulation, which disables their identification. Large conduit arteries do not affect final blood pressure in the pulmonary vascular bed; however, their structure changes substantially under hypoxia. Such remodeling changes are not mediated by mast cell products only since they have occurred in spite of stabilization of mast cell granules.

Airway wall remodeling in young and adult rats with experimentally provoked bronchial asthma.

Background: Airway wall remodeling is a typical finding in patients suffering from bronchial asthma. While morphological changes have been thoroughly described in adults, less is known about such changes in children because of the limited accessibility of relevant material. To overcome this constraint, animal asthma models may be used instead of human specimens. This study examined rats with artificially stimulated chronic asthma-like symptoms. Methods: Brown Norway rats of two age categories (young and adult) were sensitized by ovalbumin (OA), and their intrapulmonary airways (IA) were studied using morphometric and histochemical methods. Results: OA administration induced a significant increase in lung resistance in young animals but not in adults. The total IA wall area was significantly increased in both young and adult OA rats. In young animals, thickening of the adventitia played a more crucial role in this increase than it did in adults, in which the mucosa and the submucosa participated to a higher degree. The IA walls of young OA rats had significantly higher levels of infiltrating eosinophils than those of adult OA animals. The multiplication of goblet cells was more pronounced in adult rats, which was associated with a tendency to produce a higher proportion of acidic glycoconjugates. Conclusions: OA stimulation affected the IA of young rats differently than those of adult animals. Changes in the outer IA layer of young rats can be triggered by activated eosinophils; however, stimulated airway epithelium can be a source of factors that influence the inner IA layers in adult rats.

Effect of Inhalation of Single Dose of Beclomethasone on Airway Epithelium

Inhaled corticosteroids are being recommended for the treatment of bronchial asthma for their anti-inflammatory properties and reduction of airway hyperreactivity. The first tissue coming to the contact with all inhaled substances is the airway epithelium. In this experiment, the immediate effect of a single MDI dose of beclomethasone on the ultrastructure of the tracheal and bronchiolar epithelium was studied. Due to the beclomethasone administration, the secretory elements were highly affected. The tracheal goblet cells were damaged, mucus release was significantly accelerated, and the mechanism of secretion was influenced. The bronchiolar Clara cells revealed signs of the pathological alteration. Their secretory granules were usually stored in the cytoplasm. Occasionally, degenerating Clara cells were found after the beclomethasone administration. The injury of ciliated cells in both locations was only mild and this fact was reflected in slight impairment of the tracheal ciliary border. As a morphological sign of impaired self-cleaning ability, inspissated secretion was discovered among cilia. According to this evaluation, the inhalation of the single dose of beclomethasone caused a moderate damage to the tracheal epithelium and a mild one to the epithelium of terminal bronchioles. The results draw attention to the adverse effects of otherwise therapeutically beneficial inhaled glucocorticosteroids.

Local administration of 2% trimecaine affects the content of fucosylated glycoconjugates in goblet cells in rabbit tracheal epithelium

The proportion of fucosylated glycoconjugate‐containing rabbit tracheal goblet cells after intratracheal application of trimecaine was studied to evaluate its possible unfavourable effects. This lapine model is comparable with diagnostic findings in humans because airway epithelia in humans and rabbits are similar; tracheal epithelium is also practically identical to bronchial epithelium in both species. Local trimecaine anaesthesia caused a proportional decrease in percentage of the tracheal goblet cells containing both α(1–2)‐ and α(1–6)‐, α(1–3)‐ and α(1–4)‐fucosylated glycoconjugates as revealed 10 min postexposure using lectin histochemistry. In previous studies, only mild ultrastructural damage to the airways epithelium was revealed, but a conspicuous decrease in sialylated glycoconjugate‐containing tracheal goblet cells and the dominance of acidic sulphated glycoconjugates were observed as after‐effects of the same treatment. Glycoconjugate changes can influence the inner environment of airways (e.g. viscoelastic properties of the airways’ mucus and mucosal barrier functions) and thus the patients defence barriers in airways may be weakened. Concurrently, the histochemical properties of goblet cells can be altered in bronchoscopic specimens. Since trimecaine is widely used as local anaesthesia in airways in bronchoscopy, it is necessary to heed these aforementioned effects.

Reaction of The Goblet Cells to Cholinergic Stimulation

Ivanova I. Ye., T. A. Derendiayeva, G. I. Meleshko, Yeo Ya. Shepelev: Higher Plants in a Biological Life SupportSystemfor Man. Acta vet. Bmo 1996,65: 27-32. A model of human biological life support system (BLSS) was created with a photoautotrophic link including unicellular algae and higher plants having an equal oxygen production. This system model supported the vital activities of two people at the biomass regeneration of 92% from that consumed by them. The plant cultures studied were wheat grown on 11.25 m2 (of a total 15 m2) and several vegetable species. The total photosynthetic productivity of the plants was high, stable over the studied time span, and independent on the composition of the system. However, the grain productivity of wheat decreased periodically, and during certain harvests it decreased to almost zero. A detailed analysis revealed that this decrease was not caused by any of the systems adjacent links as a similar decrease in yield was also found in the control period with an autonomous growth of the plants in a closed volume of a chamber. Inclusion of bigher plants into BLSS led to a considerable improvement od food supply regeneration for man as compared to the previous model with only 26% regeneration effect. This inclusion did not exert any marked effect upon the general closure of the cycle. However, the biological value of the food for man was increased dramatically. This improvement is the main goal of designing and operating such BLSS systems. Regeneration. ecosystem. photosynthesis, photoautotroph link, unicellular algae. higher p.lants The prospect of long-teon existence of man outside the Earths biosphere (interplanetary flights, lunar and planetary bases) is connected with creating an artificial environment with the help of regenerating it from the products of vital activity. This environment must correspond to the evolutionarily conditioned biological needs of the humans and to provide their lives for generations. The fact of adaptive biology shows that the full-value environment for humans and other terrestrial organisms is the natural environment of the Earth. In order to recreate that environment artificially it is necessary to reproduce the mechanisms existing on Earth, i. e. to foon closed ecological systems including man. The lack of our knowledge about the natural environment of the humans prevents us from reproducing it by non-biological means, even if it were possible. The investigation of the problem of creating biological life-support systems (BLSS) of man began as early as in the 60ies. By the present time, experimental BLSS models have been created with different structures and different degrees of the cycle closure. , The creation of the systems was based on the ecological concept according to which strategy and tactics were worked out providing the organisms and populations were studied and then united into biocenoses, including also humans. The main method of solving the problem was the method of experimental modelling from the simple to the complex (Rashevski 1966).