Biopolymer composites allow the creation of an optimal environment for the regeneration of chondral and osteochondral defects of articular cartilage, where natural regeneration potential is limited. In this experimental study, we used the sheep animal model for the creation of knee cartilage defects. In the medial part of the trochlea and on the medial condyle of the femur, we created artificial defects (6 × 3 mm²) with microfractures. In four experimental sheep, both defects were subsequently filled with the porous acellular polyhydroxybutyrate/chitosan (PHB/CHIT)-based implant. Two sheep had untreated defects. We evaluated the quality of the newly formed tissue in the femoral trochlea defect site using imaging (X-ray, Computer Tomography (CT), Magnetic Resonance Imaging (MRI)), macroscopic, and histological methods. Macroscopically, the surface of the treated regenerate corresponded to the niveau of the surrounding cartilage. X-ray examination 6 months after the implantation confirmed the restoration of the contour in the subchondral calcified layer and the advanced rate of bone tissue integration. The CT scan revealed a low regenerative potential in the bone zone of the defect compared to the cartilage zone. The percentage change in cartilage density at the defect site was not significantly different to the reference area (0.06–6.4%). MRI examination revealed that the healing osteochondral defect was comparable to the intact cartilage signal on the surface of the defect. Hyaline-like cartilage was observed in most of the treated animals, except for one, where the defect was repaired with fibrocartilage. Thus, the acellular, chitosan-based biomaterial is a promising biopolymer composite for the treatment of chondral and osteochondral defects of traumatic character. It has potential for further clinical testing in the orthopedic field, primarily with the combination of supporting factors.

Novel calcium phosphate cements containing a mixture of four amino acids, glycine, proline, hydroxyproline and either lysine or arginine (CAL, CAK) were characterized and used for treatment of artificial osteochondral defects in knee. It was hypothesized that an enhanced concentration of extracellular collagen amino acids (in complex mixture), in connection with bone cement in defect sites, would support the healing of osteochondral defects with successful formation of hyaline cartilage and subchondral bone. Calcium phosphate cement mixtures were prepared by in situ reaction in a planetary ball mill at aseptic conditions and characterized. It was verified that about 30–60% of amino acids remained adsorbed on hydroxyapatite particles in cements and the addition of amino acids caused around 60% reduction in compressive strength and refinement of hydroxyapatite particles in their microstructure. The significant over-expression of osteogenic genes after the culture of osteoblasts was demonstrated in the cement extracts containing lysine and compared with other cements. The cement pastes were inserted into artificial osteochondral defects in the medial femoral condyle of pigs and, after 3 months post-surgery, tissues were analyzed macroscopically, histologically, immunohistochemically using MRI and X-ray methods. Analysis clearly showed the excellent healing process of artificial osteochondral defects in pigs after treatment with CAL and CAK cements without any inflammation, as well as formation of subchondral bone and hyaline cartilage morphologically and structurally identical to the original tissues. Good integration of the hyaline neocartilage with the surrounding tissue, as well as perfect interconnection between the neocartilage and new subchondral bone tissue, was demonstrated. Tissues were stable after 12 months’ healing.

Dimethyl sulfoxide (DMSO) is widely used as a solvent for small hydrophobic drug molecules. However, the safe volume allowing to avoid its embryotoxic effect has been poorly studied. In this study, we documented the effects of dimethyl sulfoxide (DMSO) in the developing chicken embryo at morphological, histological, and molecular levels. We focused on the developing chicken liver as the main organ involved in the process of detoxification. In our study, 100% DMSO was administered subgerminally onto the eggshell membrane (membrana papyracea) at various volumes (5, 10, 15, 20, 25, 30, 35, and 50 µL) on 4th embryonic day (ED). We focused on histopathological alterations of the liver structure, and noticed the overall impact of DMSO on developing chicken embryos (embryotoxicity, malformation). At the molecular level, we studied cytochrome P450 complex (CYP) isoform’s activities in relation to changes of CYP1A5, CYP3A37, and CYP3A80 gene expression. Total embryotoxicity after application of different doses of DMSO on ED 4, and the embryo lethality increased with increasing DMSO amounts. Overall mortality after DMSO administration ranged below 33%. Mortality was increased with higher amounts of DMSO, mainly from 20 µL. The highest mortality was observed for the highest dose of DMSO over 35 µL. The results also showed a decrease in body weight with increased application volumes of DMSO. At the histological level, we observed mainly the presence of lipid droplets and dilated bile canaliculi and sinusoids in samples over the administration of 25 µL of DMSO. While these findings were not statistically significant, DMSO treatment caused a significant different up-regulation of mRNA expression in all studied genes. For CYP1A5, CYP3A37, and CYP3A80 DMSO volumes needed were 15 µL, 10 µL, and 20 µL, respectively. A significant down-regulation of all studied CYP isoform was detected after application of a DMSO dose of 5 µL. Regarding the morphological results, we can assume that the highest safe dose of DMSO without affecting chicken embryo development and its liver is up to 10 µL. This conclusion is corroborated with the presence of number of malformations and body weight reduction, which correlates with histological findings. Moreover, the gene expression results showed that even the lowest administered DMSO volume could affect hepatocytes at the molecular level causing down-regulation of cytochrome P450 complex (CYP1A5, CYP3A37, CYP3A80).

In this study we observed the effect of Wi-Fi on various fibrous components that form the wall of large blood vessels in the chorioallantoic membrane (CAM) of the chicken embryo. Chicken embryos in the experimental groups (Wi-Fi) were continuously exposed (24 h per day) to Wi-Fi radiation at a frequency of 2.4 GHz and an average power density of 300 µW/m ² for 9 and 14 embryonic days (ED). Subsequently, the CAM samples from the control (CO) and experimental (Wi-Fi) groups were histologically processed and evaluated. The samples stained with haematoxylin and eosin did not reveal any notable differences in the structure of large blood vessels between the CO and Wi-Fi groups. However, the use of special staining methods showed adverse effect of Wi-Fi on the fibrous elements within the blood vessel wall. The optical density (OD) of elastic fibres significantly decreased from 0.294 ± 0.025 (CO 9) to 0.197 ± 0.022 (Wi-Fi 9) at ED 9 and from 0.706 ± 0.028 (CO 14) to 0.271 ± 0.031 (Wi-Fi 14) at ED 14. On the other hand, at ED 9, the OD of collagen fibres exhibited a notable increase from 0.178 ± 0.023 (CO 9) to 0.334 ± 0.020 (Wi-Fi 9). However, at ED 14, there was a significant decline of collagen fibres from 0.418 ± 0.033 (CO 14) to 0.327 ± 0.031 (Wi-Fi 14). Our results support the hypothesis that Wi-Fi could affect the fibrous elements of the large vessel wall and may play a role in the development of different cardiovascular disorders.

The aim of this study was to observe the influence of electromagnetic radiation (EMR) on the structure and ultrastructure of the rat’s liver. The pregnant rats used in the experiment were exposed to a pulsed microwave radiation (frequency of 2.45 GHz; mean power density of 2.8 mW/cm ² ) daily for 2 h, throughout their pregnancy. After delivery, the offspring was not exposed to EMR. Samples of the liver of 5-week-old offspring were subjected to histopathological evaluation. They were processed for light and transmission electron microscopy. Our results indicated that EMR did not cause pronounced changes in the structure of the liver of the investigated offspring. The size and shape of liver lobuli was preserved and the amount of connective tissue in the liver parenchyma did not increase. However, electron microscopy revealed changes in the shape and number of microvilli at the vascular pole of hepatocytes, and formation of vesicles of various shapes and sizes. The endothelial cells were swollen with larger fenestrations compared to the control group. The spaces of Disse were irregular and dilated. Even though these changes were only mild, further studies are needed to determine the effect of EMR and clarify its potential risk during pregnancy.

The functional development of the small intestine was investigated in Japanese quail chicks subjected to simulated microgravity (hypodynamy) on the second day after hatching and reared under these conditions to 63 days of age. On days 5, 7, 14, 21, 28, 35, 42, 56 and 63 the activity of brush-border-bound alkaline phosphatase (AP) in the duodenum and jejunum were determined in experimental animals as well as in control quail chicks housed in a floor box during these periods. As compared with control quails the experimental animals displayed a significantly increased enzyme activity until day 42 in the duodenum and day 35 in the jejunum ( P < 0.001) whereas in older quails no significant enzymatic differences between these groups was found. However, a decrease in food consumption due to a partial physical constraint cannot be excluded. Moreover, the results suggested that the activity of AP in the control birds did not change substantially during all the periods examined. In contrast, in older hypodynamy quail the AP activity significantly decreased in the duodenum on days 56 and 63 and in the jejunum on days 42, 56 and 63, respectively. These results indicate that a) the enhanced intestinal function in early periods of life may reflect the higher sensitivity of small intestine to simulated weightlessness, b) the decrease of the AP activity in older animals to the level of controls might be considered as a part of intestinal mechanisms involved in adaptation of quail chicks to long-term hypodynamy, c) different activity of AP in the small intestine of Japanese quail may not have resulted only from hypodynamy but also due to decreased food intake.